Kluyveromyces

Biotechnol Bioeng . 2004 Dec 30; {Epub ahead of print}
Synthesis of galacto-oligosaccharide from lactose using beta-galactosidase from Kluyveromyces lactis: Studies on batch and continuous UF membrane-fitted bioreactors; Chockchaisawasdee S et al.; A study of galacto-oligosaccharides (GOS) synthesis from lactose with beta-galactosidase from Kluyveromyces lactis (Maxilact(R) L2000) was carried out . The synthesis was performed using various initial lactose concentrations ranging from 220 to 400 mg/mL and enzyme concentrations ranging from 3 to 9 U/mL, and was investigated at 40 degrees C and pH 7, in a stirred-tank reactor . In the experimental range examined, the results showed the amount of GOS formed depended on lactose concentration but not on enzyme concentration . Galactose was a competitive inhibitor, while glucose was a non-competitive inhibitor . In a further study, a laboratory-scale reactor system, fitted with a 10-kDa NMWCO composite regenerated cellulose membrane, was used in a continuous process . The reactor was operated in cross-flow mode . The effect of operating pressures on flux and productivity was investigated by applying different transmembrane pressures to the system . The continuous process showed better production performance compared to the batch synthesis with the same lactose and enzyme concentrations at 40 degrees C, pH 7 . Comparison of product structures from batch and continuous processes, analyzed by HPAE-PAD and methylation analysis, showed similarities but differed from the structures found in a commercial GOS product (Vivinal(R)GOS) . (c) 2004 Wiley Periodicals, Inc.

FEBS Lett, 2005 Jan 3, 579(1), 30 - 40
The complete mitochondrial genome of the yeast Kluyveromyces thermotolerans; Talla E et al.; We report here the complete nucleotide sequence of the 23.5-kb mitochondrial genome from the yeast Kluyveromyces thermotolerans . It encodes, all on the same DNA strand, three subunits of cytochrome oxidase (COX1, COX2 and COX3), three subunits of ATP synthetase (ATP6, ATP8 and ATP9), the apocytochrome b (COB), the ribosomal protein VAR1, 24 tRNAs, the small and large ribosomal RNAs, and the RNA subunit of RNase P . Three intronic ORFs are present within the COX1 gene group I introns . The K . thermotolerans mitochondrial genome is very similar to the Candida glabrata mitochondrial genome, as judged from clusters of gene order, gene transcription units and sequence similarities . Interestingly, the predicted secondary structure of the abnormal tRNAThr1 contains 10 nucleotides in its anticodon loop . This sequence is available under EMBL Accession No . AJ634268.

Appl Microbiol Biotechnol . 2004 Dec 22; {Epub ahead of print}
Secretory expression of heterologous protein in Kluyveromyces cicerisporus; Cai XP et al.; To explore the potential of heterologous protein expression in Kluyveromyces cicerisporus, three expression plasmids, pUK1-PIT, pUKD-PIT and pUKD-S-PIT, based on the vector pUK1 or pUKD were constructed and transformed, respectively, into yeast strain K . cicerisporus Y179U . Human interferon alpha-2a, used as an example protein, was successfully expressed and secreted by transformant Y179U/pUKD-PIT and Y179U/pUKD-S-PIT . In the flask culture, strain Y179U/pUKD-S-PIT could express interferon at 60 mg/l . The stability of plasmid pUKD-S-PIT in the host was higher than that of pUKD-PIT . This was consistent with their expression levels of interferon . There were two interferon-related bands found by Western blotting analysis . The possible reason for this is discussed.

Curr Genet . 2004 Dec 22; {Epub ahead of print}
A new Hansenula polymorpha HAP4 homologue which contains only the N-terminal conserved domain of the protein is fully functional in Saccharomyces cerevisiae; Sybirna K et al.; In Saccharomyces cerevisiae, the HAP transcriptional complex is involved in the fermentation-respiration shift . This complex is composed of four subunits . Three subunits are necessary for DNA-binding, whereas the Hap4p subunit, glucose-repressed, contains the transcriptional activation domain . Hap4p is the key regulator of the complex activity in response to carbon sources in S . cerevisiae . To date, no HAP4 homologue has been identified, except in Kluyveromyces lactis . Examination of these two HAP4 sequences led to the identification of two very short conserved peptides also identified in other yeasts . In the yeast Hansenula polymorpha, two possible HAP4 homologues have been found . Their deduced amino acid sequences are similar to the ScHap4p and KlHap4p proteins only in the N-terminal 16-amino-acid basic motif . Since molecular genetic tools exist and complete genome sequence is known for this yeast, we expressed one of these putative HpHap4 proteins in S . cerevisiae and showed that this protein is able to restore the growth defect of the S . cerevisiae hap4-deleted strain . A set of experiments was performed to confirm the functional homology of this new gene with ScHAP4 . The discovery of a Hap4-regulatory protein in H . polymorpha with only the N-terminal conserved domain of the S . cerevisiae protein indicates that this domain may play a crucial role during evolution.

Biochimie, 2004 Sep-Oct, 86(9-10), 705 - 12
Kinetic properties of native and mutagenized isoforms of mitochondrial alcohol dehydrogenase III purified from Kluyveromyces lactis; Brisdelli F et al.; By computer modelling and protein engineering we have investigated changes in two amino acid residues located in the coenzyme pocket of the yeast Kluyveromyces lactis mitochondrial alcohol dehydrogenase III . These two residues, Gly 225 and Ala 274, were hypothesized to be involved in the enzyme discrimination between NAD(H) and NADP(H) . Upon changing Gly 225 to Ala we produced an enzyme (mutant G225A) showing very little difference from the wild-type . On the contrary, change at position 274 of Phe instead of Ala (mutant A274F) caused a significant increase of K(m) values for NAD(P) and for NADPH and even a more marked decrease in catalytic activity . The k(cat)/K(m) rates for NADP(H) were also decreased in this mutant . Enzymes with the double changes at 225 and 274 (mutant G225A-A274F) showed, apart the substantial low K(m) value for NADPH and its high catalytic efficiency, kinetic parameters relative to coenzymes which were not additive over the single substitutions . Surprisingly, enzymes with changes at the two positions reduced efficiently acetaldehyde, displaying a K(m) value 10-fold lower and a catalytic efficiency sevenfold higher with respect to parent or singularly mutated enzymes . None of the engineered enzymes would convert formaldehyde, glutaraldehyde or aromatic aldehydes but all enzymes reduced propionaldehyde and butyraldehyde at relative reaction rates approximately half of that exhibited by acetaldehyde . Interestingly only mutant A274F was able to oxidize methanol almost as well as ethanol . In addition, this mutant was capable to convert secondary and cyclic alcohols, at a rate not detected in the other isoforms . These results are in general agreement with the prediction that increasing the size of amino acids in the proximity of the coenzyme pocket would hamper the accommodation of NADP but discord the increased affinity for NADPH as well as for alcoholic or aldehydic substrates with high steric hindrance.

FEMS Yeast Res, 2004 Dec, 5(3), 263 - 9
Molecular-genetic differentiation of the dairy yeast Kluyveromyces lactis and its closest wild relatives; Naumova ES et al.; The currently accepted formal division of the species Kluyveromyces lactis into two taxonomic varieties, Kl . lactis var . lactis and Kl . lactis var . drosophilarum, is based arbitrarily on phenotypic and ecological characters . On the other hand, the genetic hybridisation analysis and molecular karyotyping of its synonyms allowed us {FEMS Yeast Res . 2 (2002) 39} to reinstate them in the genus Zygofabospora Kudriavzev emend G . Naumov (=Kluyveromyces Kurtzman et al., 2001) as the varieties Zf . lactis var . lactis, Zf . lactis var . krassilnikovii, Zf . lactis var . drosophilarum, Zf . lactis var . phaseolospora and Zf . lactis var . vanudenii . In the present work, we studied forty Kl . lactis strains of different geographic and ecological origins by means of restriction analysis of the PCR-amplified non-coding nrDNA regions encompassing the intergenic spacer 2 (IGS2) and the internal transcribed spacers (ITS1 and ITS2) . The results confirmed the complex structure of Kl . lactis . Moreover, four additional genetic populations were identified: three in North America ('aquatic', 'pseudovanudenii' and 'new') and one in Far-East Asia ('oriental') . Comparative sequence analysis of the 5.8S-rRNA gene and the two internal transcribed spacers revealed that the populations 'aquatic' and 'oriental' formed distinct taxa which are phylogenetically separate from the five known populations . However, some discrepancies were observed between the restriction and sequencing data . Genetic hybridisation analysis needs to be done to further elucidate the genetic relationships between the populations of Kl . lactis.

J Dairy Sci, 2004 Dec, 87(12), 4050 - 6
Screening of dairy yeast strains for probiotic applications; Kumura H et al.; To evaluate the potential of yeasts of dairy origin as probiotics, we tested 8 species including Candida humilis, Debaryomyces hansenii, Debaryomyces occidentalis, Kluyveromyces lactis, Kluyveromyces lodderae, Kluyveromyces marxianus, Saccharomyces cerevisiae, and Yarrowia lipolytica, isolated from commercial blue cheese and kefir . Strains were randomly selected from each species and tested for their ability to adhere to human enterocyte-like Caco-2 cells in culture . Among the 8 species, K . lactis showed higher adhesive ability than K . marxianus, K . lodderae, and D . hansenii . The other 4 species were poorly adhesive . All species other than K . marxianus and C . humilis were resistant to acidic conditions . In the presence of bile acid, growth inhibition was undetectable when incubation was carried out at 27 degrees C; however, it was evident for C . humilis and a strain of D . occidentalis when incubated at 37 degrees C . Moreover, the influence of proteinase treatment of living cells of K . lactis and K . lodderae on their adhesion to Caco-2 cells was evaluated . Although a slight reduction was recognized when K . lactis was treated with proteinase K, the influence of intestinal protease treatments of pepsin followed by trypsin was negligible . These results indicated that a proteinaceous factor was unlikely to be involved in adhesion of K . lactis and K . lodderae to Caco-2 cells . No stimulation of IL-8 synthesis by Caco-2 cells was recognized in the presence of K . lactis . In conclusion, K . lactis was the most attractive to continue study for use as probiotic microorganisms.

FEBS Lett, 2004 Nov 5, 577(1-2), 27 - 34
UDP-galactose 4-epimerase from Kluyveromyces fragilis: existence of subunit independent functional site; Brahma A et al.; UDP-galactose 4-epimerase from Kluyveromyces fragilis is a stable homodimer of 75 kDa/subunit with non-covalently bound NAD acting as cofactor . Partial proteolysis with trypsin in the presence of 5'-UMP, a strong competitive inhibitor, led to a degraded product which was purified . Results from SDS-PAGE, size-exclusion (SE)-HPLC and ultracentrifugation indicated its monomeric status and size between 43 and 45 kDa . 'Two-step assay' with UDP-glucose dehydrogenase as coupling enzyme in the presence of NAD ensured epimerase activity of the monomer . The possibility of transient dimerization of monomeric epimerase during catalysis was excluded by SE-HPLC in the presence of excess substrate and NAD . This truncated enzyme retained catalytic site related properties like Km for UDP-galactose, 'NADH-like coenzyme fluorescence' and 'reductive inhibition' similar to its dimeric counterpart . Reversible reactivation of the monomer was achieved up to 95% within 3 min from 8 M urea induced unfolded state, indicating that the catalytic site could form independent of its quaternary structure . Equilibrium unfolding between 0 and 8 M urea indicated that the monomer was less stable compared to the dimer . Chemical modification of amino acids and reconstitution with etheno-NAD suggested that the architecture around the catalytic site of the monomer was conserved . Specific modification reagents further confirmed that the cysteine residues required for catalysis and coenzyme fluorophore reside exclusively on a single subunit negating a 'subunit sharing model' of its catalytic site.

Genetics, 2004 Oct, 168(2), 723 - 31
Enolase and glycolytic flux play a role in the regulation of the glucose permease gene RAG1 of Kluyveromyces lactis; Lemaire M et al.; We isolated a mutant, rag17, which is impaired in glucose induction of expression of the major glucose transporter gene RAG1 . The RAG17 gene encodes a protein 87% identical to S . cerevisiae enolases (Eno1 and Eno2) . The Kleno null mutant showed no detectable enolase enzymatic activity and has severe growth defects on glucose and gluconeogenic carbon sources, indicating that K . lactis has a single enolase gene . In addition to RAG1, the transcription of several glycolytic genes was also strongly reduced in the DeltaKleno mutant . Moreover, the defect in RAG1 expression was observed in other mutants of the glycolytic pathway (hexokinase and phosphoglycerate kinase) . Therefore, it seems that the enolase and a functional glycolytic flux are necessary for induction of expression of the Rag1 glucose permease in K . lactis.

Genome, 2004 Oct, 47(5), 970 - 8
Genome-wide analysis of Kluyveromyces lactis in wild-type and rag2 mutant strains; Becerra M et al.; The use of heterologous DNA arrays from Saccharomyces cerevisiae has been tested and revealed as a suitable tool to compare the transcriptomes of S . cerevisiae and Kluyveromyces lactis, two yeasts with notable differences in their respirofermentative metabolism . The arrays have also been applied to study the changes in the K . lactis transcriptome owing to mutation in the RAG2 gene coding for the glycolytic enzyme phosphoglucose isomerase . Comparison of the rag2 mutant growing in 2% glucose versus 2% fructose has been used as a model to elucidate the importance of transcriptional regulation of metabolic routes, which may be used to reoxidize the NADPH produced in the pentose phosphate pathway . At this transcriptional level, routes related to the oxidative stress response become an interesting alternative for NADPH use.

Yeast, 2004 Oct 15, 21(13), 1067 - 75
Characterization of a gene similar to BIK1 in the yeast Kluyveromyces lactis; Lamas-Maceiras M et al.; In Saccharomyces cerevisiae, Bik1p is a microtubule plus-end-tracking protein that plays several roles in mitosis and ploidy . KlBik1p (from Kluyveromyces lactis) maintains the same structural-domain organization as does S . cerevisiae Bik1p . As part of its characterization, we constructed a stable klbik1 mutant which is sensitive to benomyl only at 14 degrees C and has a higher frequency of crescent-shaped nuclei than S . cerevisiae bik1 mutants . This phenotype is partially rescued by S . cerevisiae BIK1 . Other phenotypes associated with bik1 are not present in the K . lactis mutant . By fusion to GFP we were able to show the functionality of the KlBik1p CAP-Gly domain and found that the fusion protein changes its cellular location during the cell cycle . Copyright (c) 2004 John Wiley & Sons, Ltd.

Can J Microbiol, 2004 Aug, 50(8), 645 - 52
Isolation and transcriptional regulation of the Kluyveromyces lactis FBA1 (fructose-1,6-bisphosphate aldolase) gene; Prado SM et al.; Cloning and transcriptional regulation of the KlFBA1 gene that codes for the class II fructose-1,6-bisphosphate aldolase of the yeast Kluyveromyces lactis are described . KlFBA1 mRNA diminishes transiently during the shift from hypoxic to fully aerobic conditions and increases in the reversal shift . This regulation is mediated by heme since expression was higher in a mutant defective in heme biosynthesis . KlFBA1 transcription is not induced by calcium-shortage, low temperature, or at stationary phase . These data suggest that KlFBA1 plays a role in the balance between oxidative and fermentative metabolism and that this gene is differentially regulated in K . lactis and Saccharomyces cerevisiae, i.e., a respiratory vs . fermentative yeast.

J Biotechnol, 2004 Oct 19, 114(1-2), 69 - 79
Molecular cloning and expression in yeast of caprine prochymosin; Vega-Hernandez MC et al.; We cloned and characterized a preprochymosin cDNA from the abomasum of milk-fed kid goats . This cDNA contained an open reading frame that predicts a polypeptide of 381 amino acid residues, with a signal peptide and a proenzyme region of 16 and 42 amino acids, respectively . Comparison of the caprine preprochymosin sequence with the corresponding sequences of lamb and calf revealed 99 and 94% identity at the amino acid level . The cDNA fragment encoding the mature portion of caprine prochymosin was fused in frame both to the killer toxin signal sequence and to the alpha-factor signal sequence-FLAG in two different yeast expression vectors . The recombinant plasmids were transformed into Kluyveromyces lactis and Saccharomyces cerevisiae cells, respectively . Culture supernatants of both yeast transformants showed milk-clotting activity after activation at acid pH . The FLAG-prochymosin fusion was purified from S . cerevisiae culture supernatants by affinity chromatography . Proteolytic activity assayed toward casein fractions indicated that the recombinant caprine chymosin specifically hydrolysed kappa-casein.

Braz J Biol, 2004 May, 64(2), 317 - 26
Evaluation of biochemical and serological methods to identify and clustering yeast cells of oral Candida species by CHROMagar test, SDS-PAGE and ELISA; Rodrigues JA et al.; The purpose of this work was to evaluate biochemical and serological methods to characterize and identify Candida species from the oral cavity . The strains used were five Candida species previously identified: C . albicans, C . guilliermondii, C . parapsilosis, C . krusei, C . tropicalis, and Kluyveromyces marxianus, as a negative control . The analyses were conducted through the SDS-PAGE associated with statistical analysis using software, chromogenic medium, and CHROMagar Candida (CA), as a differential medium for the isolation and presumptive identification of clinically important yeasts and an enzyme-linked immunoabsorbent assay (ELISA), using antisera produced against antigens from two C . albicans strains . This method enabled the screening of the three Candida species: C . albicans, C . tropicalis, and C . krusei, with 100% of specificity . The ELISA using purified immunoglobulin G showed a high level of cross-reaction against protein extracts of Candida species . The SDS-PAGE method allowed the clustering of species-specific isolates using the Simple Matching coefficient, S(SM) = 1.0 . The protein profile analysis by SDS-PAGE increases what is known about the taxonomic relationships among oral yeasts . This methodology showed good reproducibility and allows collection of useful information for numerical analysis on information relevant to clinical application, and epidemiological and systematical studies.

FEMS Yeast Res, 2004 Sep, 4(8), 833 - 40
Disruption of the MNN10 gene enhances protein secretion in Kluyveromyces lactis and Saccharomyces cerevisiae; Bartkeviciute D et al.; Screening for genes affecting super-secreting phenotype of the over-secreting mutant of Kluyveromyces lactis resulted in isolation of the gene named KlMNN10, sharing high homology with Saccharomyces cerevisiae MNN10 . The disruption of the KlMNN10 in Kluyveromyces lactis, as well as of MNN10 and MNN11 in Saccharomyces cerevisiae, conferred the super-secreting phenotype . MNN10 isolated from Saccharomyces cerevisiae suppressed the super-secretion phenotype in Kluyveromyces lactis klmnn10, as did the homologous KlMNN10 . The genes MNN10 and MNN11 of Saccharomyces cerevisiae encode mannosyltransferases responsible for the majority of the alpha-1,6-polymerizing activity of the mannosyltransferase complex . These data agree with the view that the structure of glycoproteins in a yeast cell wall strongly influences the release of homologous and heterologous proteins in the medium . The set of genes namely the suppressors of the over-secreting phenotype, could be attractive for further analysis of gene functions, over-secreting mechanisms and for construction of new strains optimized for heterologous protein secretion . KlMNN10 has EMBL accession no . AJ575132.

Yeast, 2004 Sep, 21(12), 1045 - 55
Kluyveromyces lactis SSO1 and SEB1 genes are functional in Saccharomyces cerevisiae and enhance production of secreted proteins when overexpressed; Toikkanen JH et al.; The SEB1/SBH1 and the SSO genes encode components of the protein secretory machinery functioning at the opposite ends, ER translocation and exocytosis, respectively, of the secretory pathway in Saccharomyces cerevisiae . Overexpression of these genes can rescue temperature-sensitive (ts) growth defect of many sec mutants impaired in protein secretion . Furthermore, their overexpression in wild-type yeast enhances production of secreted proteins in S . cerevisiae, which suggests that they may be rate-limiting factors in this process . Here we report isolation of Kluyveromyces lactis homologues of these genes . KlSSO1 and KlSEB1 were isolated as clones capable of rescuing growth of ts sso2-1 and seb1Delta seb2Delta sem1Delta strains, respectively, at the restrictive temperature . The encoded Kluyveromyces proteins are up to 70% identical with the S . cerevisiae homologues at the amino acid level and can functionally replace them . Interestingly, KlSSO1 and KlSEB1 show similar enhancing effect on production of a secreted protein as the SSO and SEB1 genes of S . cerevisiae when overexpressed . In accordance with the high homology level of the secretory pathway proteins in different yeast species, the polyclonal antibodies raised against S . cerevisiae Seb1p, Sso2p and Sec4p can detect homologous proteins in cell lysates of K . lactis and Pichia pastoris, the latter also in Candida utilis . The GenBank Accession Nos are AF307983 (K . lactis SSO1) and AF318314 (K . lactis SEB1) . Copyright (c) 2004 John Wiley & Sons, Ltd.

FEMS Yeast Res, 2004 Oct, 5(1), 19 - 27
The KlPGS1 gene encoding phosphatidylglycerolphosphate synthase in Kluyveromyces lactis is essential and assigned to chromosome I; Tyciakova S et al.; The phosphatidylglycerolphosphate synthase (CDP-diacylglycerol:sn-glycerol-3-phosphate 3-phosphatidyltransferase, EC 2.7.8.5) is an essential enzyme in biosynthesis of cardiolipin . In this work we report the isolation, heterological cloning, molecular characterization and physical mapping of the Saccharomyces cerevisiae PEL1/PGS1 homologue from Kluyveromyces lactis . The pel1 mutant strain of S . cerevisiae was used to isolate this homologue by screening a K . lactis genomic library . The novel cloned gene was named KlPGS1 . Its coding region was found to consist of 1623 bp . The corresponding protein exhibits 55% amino acid identity to its S . cerevisiae counterpart . The presence of the mitochondrial presequence indicates its mitochondrial localization . Sporulation and ascus dissection of diploids heterozygous for single-copy disruption of KlPGS1 revealed that the KlPGS1 gene, is essential in K . lactis . Using a DIG-dUTP-labeled DNA probe-originated from the KlPGS1 gene and Southern hybridization of contour-clamped homogeneous electric field (CHEF)-separated K . lactis chromosomal DNA, the KlPGS1 gene was assigned to chromosome I . The nucleotide sequence data reported in this paper were submitted to GenBank and assigned the Accession No . AY176328.

Proc Natl Acad Sci U S A, 2004 Oct 12, 101(41), 14713 - 8 Epub 2004 Sep 15.
A universal telomerase RNA core structure includes structured motifs required for binding the telomerase reverse transcriptase protein; Lin J et al.; Telomerase synthesizes telomeric DNA by copying a short template sequence within its telomerase RNA component . We delineated nucleotides and base-pairings within a previously mapped central domain of the Saccharomyces cerevisiae telomerase RNA (TLC1) that are important for telomerase function and for binding to the telomerase catalytic protein Est2p . Phylogenetic comparison of telomerase RNA sequences from several budding yeasts revealed a core structure common to Saccharomyces and Kluyveromyces yeast species . We show that in this structure three conserved sequences interact to provide a binding site for Est2p positioned near the template . These results, combined with previous studies on telomerase RNAs from other budding yeasts, vertebrates, and ciliates, define a minimal universal core for telomerase RNAs.

Gene, 2004 Sep 15, 339, 111 - 9
Carboxylic acids permeases in yeast: two genes in Kluyveromyces lactis; Lodi T et al.; Two new genes KlJEN1 and KlJEN2 were identified in Kluyveromyces lactis . The deduced structure of their products is typical of membrane-bound carriers and displays high similarity to Jen1p, the monocarboxylate permease of Saccharomyces cerevisiae . Both KlJEN1 and KlJEN2 are under the control of glucose repression mediated by FOG1 and FOG2, corresponding to S . cerevisiae GAL83 and SNF1 respectively, and KlCAT8, proteins involved in glucose signalling cascade in K . lactis . KlJEN1, but not KlJEN2, is induced by lactate . KlJEN2 in contrast is expressed at high level in ethanol and succinate . The physiological characterization of null mutants showed that KlJEN1 is the functional homologue of ScJEN1, whereas KlJEN2 encodes a dicarboxylic acids transporter . In fact, KlJen1p {transporter classification (TC) number: 2.A.1.12.2.} is required for lactate uptake and therefore for growth on lactate . KlJen2p is required for succinate transport, as demonstrated by succinate uptake experiments and by inability of Kljen2 mutant to grow on succinate . This carrier appears to transport also malate and fumarate because the Kljen2 mutant cannot grow on these substrates and the succinate uptake is competed by these carboxylic acids . We conclude that KlJEN2 is the first yeast gene shown to encode a dicarboxylic acids permease.

Appl Microbiol Biotechnol . 2004 Aug 26; {Epub ahead of print}
Robust NADH-regenerator: improved alpha-haloketone-resistant formate dehydrogenase; Yamamoto H et al.; Formate dehydrogenases (FDH) are useful for the regeneration of NADH, which is required for asymmetric reduction by several dehydrogenases and reductases . FDHs have relatively low activity and are labile, especially to alpha-haloketones, thus FDH cannot be applied to the industrial manufacture of optically active alpha-haloalcohols . To stabilize a FDH from Mycobacterium vaccae (McFDH) against the alpha-haloketone ethyl 4-chloroacetoacetate (ECAA), a set of cysteine-mutant enzymes was constructed . Sensitivity to ECAA of mutant C6S was similar to that of the wild-type enzyme, and mutants C249S and C355S showed little activity . In contrast, mutant C256S exhibited remarkable tolerance to ECAA . Surprisingly, mutant C146S was activated by several organic compounds such as ethyl acetate . An optimized mutant, C6A/C146S/C256V (McFDH-26), was obtained by combining several effective mutations . Ethyl ( S)-4-chloro-3-hydroxybutanoate {( S)-ECHB} was synthesized from ECAA to 49.9 g/l with an optical purity of more than 99% e.e . using recombinant Escherichia coli cells coexpressing McFDH-26 and a carbonyl reductase (KaCR1) from Kluyveromyces aestuarii.

FEMS Microbiol Lett, 2004 Sep 1, 238(1), 235 - 40
Pichia anomala and Kluyveromyces wickerhamii killer toxins as new tools against Dekkera/Brettanomyces spoilage yeasts; Comitini F et al.; Two yeast killer toxins active on spoilage yeasts belonging to the genus Dekkera/Brettanomyces are here described for the first time . The two toxins produced by Pichia anomala (DBVPG 3003) and Kluyveromyces wickerhamii (DBVPG 6077), and named Pikt and Kwkt, respectively, differ for molecular weight and biochemical properties . Interestingly, the fungicidal effect exerted by Pikt and Kwkt against Dekkera bruxellensis is stable for at least 10 days in wine . Thus, a potential application for the two toxins as antimicrobial agents active on Dekkera/Brettanomyces during wine ageing and storage can be hypothesised.

Yeast, 2004 Jul 30, 21(10), 813 - 30
Characterization of Kluyveromyces lactis subtelomeric sequences including a distal element with strong purine/pyrimidine strand bias; Nickles K et al.; Telomeres are the specialized structures at the ends of eukaryotic chromosomes and are composed of short T/G-rich DNA repeats and the proteins that interact with them . Internal to telomeres are subtelomeric regions that are species-specific and often repetitive . The yeast Kluyveromyces lactis has telomeric tracts of 10-20 copies of a 25 bp repeat, but the subtelomeric regions have not previously been characterized in detail . Here we have cloned and characterized subtelomeric regions from 10 of the 12 chromosome ends . The amount of sequence examined was 0.7-10 kb for each subtelomeric region . We have identified a K . lactis subtelomeric element, the R element, which has a strong purine/pyrimidine strand bias and extends for about 2 kb . Internal to the R element, we found extensive similarity that is shared among half of the chromosome ends reported here . This similarity appears to include three putative gene families, two of which are also subtelomeric in Saccharomyces cerevisiae.

Biotechnol Prog, 2004 Jul-Aug, 20(4), 1259 - 62
Immobilization of lactase from Kluyveromyces lactis greatly reduces the inhibition promoted by glucose . full hydrolysis of lactose in milk; Mateo C et al.; The kinetic constants (Km, Vmax, and inhibition constants for the different products) of soluble and different immobilized preparations of beta-galactosidase from Kluyveromyces lactis were determined . For the soluble enzyme, the Km was 3.6 mM, while the competitive inhibition constant by galactose was 45 mM and the noncompetitive one by glucose was 758 mM . The immobilized preparations conserved similar values of Km and competitive inhibition, but in some instances much higher values for the noncompetitive inhibition constants were obtained . Thus, when glyoxyl or glutaraldehyde supports were used to immobilize the enzyme, the noncompetitive inhibition was greatly reduced (Ki approximately 15,000 and >40,000 mM, respectively), whereas when using sugar chains to immobilize the enzyme the behavior had an effect very similar to the soluble enzyme . These results presented a great practical relevance . While using the soluble enzyme or the enzyme immobilized via the sugar chain as biocatalysts in the hydrolysis of lactose in milk only around 90% of the substrate was hydrolyzed, by using of these the enzyme immobilized via the glyoxyl or the glutaraldehyde groups, more than 99% of the lactose in milk was hydrolyzed.

Biotechnol Prog, 2004 Jul-Aug, 20(4), 1134 - 9
Reversible and strong immobilization of proteins by ionic exchange on supports coated with sulfate-dextran; Fuentes M et al.; New and strong ionic exchange resins have been prepared by the simple and rapid ionic adsorption of anionic polymers (sulfate-dextran) on porous supports activated with the opposite ionic group (DEAE/MANAE) . Ionic exchange properties of such composites were strongly dependent on the size of the ionic polymers as well as on the conditions of the ionic coating of the solids with the ionic polymers (optimal conditions were 400 mg of sulfate-dextran 5000 kDa per gram of support) . Around 80% of the proteins contained in crude extracts from Escherichia coli and Acetobacter turbidans could be adsorbed on these porous composites even at pH 7 . This interaction was stronger than that using conventional carboxymethyl cellulose (CMC) and even others such as supports coated with aspartic-dextran polymer . By means of the sequential use of the new supports and supports coated with polyethyleneimine (PEI), all proteins from crude extracts could be immobilized . In fact, a large percentage (over 50%) could be immobilized on both supports . Finally, some industrially relevant enzymes (beta-galactosidases from Aspergillus oryzae, Kluyveromyces lactis, and Thermussp . strain T2, lipases from Candida antarctica A and B, Candida rugosa, Rhizomucor miehei, and Rhyzopus oryzae and bovine pancreas trypsin and chymotrypsin) have been immobilized on these supports with very high activity recoveries and immobilization rates . After enzyme inactivation, the protein could be fully desorbed from the support, and then the support could be reused for several cycles . Moreover, in some instances the enzyme stability was significantly improved, mainly in the presence of organic solvents, perhaps as a consequence of the highly hydrophilic microenvironment of the support.

J Dairy Sci, 2004 May, 87(5), 1545 - 50
Comparison of volatile compounds produced in model cheese medium deacidified by Debaryomyces hansenii or Kluyveromyces marxianus; Leclercq-Perlat MN et al.; The aroma of a deacidified cheese medium is the result of the overall perception of a large number of molecules belonging to different classes . The volatile compound composition of (60%) cheese medium (pH 5.8) deacidified by Debaryomyces hansenii (DCM(Dh)) was compared with the one deacidified by Kluyveromyces marxianus (DCM(Km)) . It was determined by dynamic headspace extraction, followed by gas chromatography separation and quantification as well as by mass spectrometry identification . Whatever the media tested, a first class of volatile compounds can be represented by the ones not produced by any of the yeasts, but some of them are affected by K . marxianus or by D . hansenii . A second class of volatile compounds can be represented by the ones produced by K . marxianus, which were essentially esters . Their concentrations were generally higher than their thresholds, explaining the DCM(Km) global fruity odor . A third class can be represented by the ones generated by D . hansenii, which were essentially methyl ketones with fruity, floral (rose), moldy, cheesy, or wine odor plus 2-phenylethanol with a faded-rose odor . The impact of methyl ketones on the DCMDh global flavor was lower than the impact of 2-phenylethanol and even negligible . Therefore, the global faded-rose odor of D . hansenii DCM can be explained by a high concentration of 2-phenylethanol.

Microbiology, 2004 Aug, 150(Pt 8), 2535 - 41
Kluyveromyces phaffii killer toxin active against wine spoilage yeasts: purification and characterization; Comitini F et al.; The killer toxin secreted by Kluyveromyces phaffii (KpKt) is active against spoilage yeast under winemaking conditions and thus has potential applications in the biocontrol of undesired micro-organisms in the wine industry . Biochemical characterization and N-terminal sequencing of the purified toxin show that KpKt is a glycosylated protein with a molecular mass of 33 kDa . Moreover, it shows 93% and 80% identity to a beta-1,3-glucanase of Saccharomyces cerevisiae and a beta-1,3-glucan transferase of Candida albicans, respectively, and it is active on laminarin and glucan, thus showing a beta-glucanase activity . Competitive inhibition of killer activity by cell-wall polysaccharides suggests that glucan (beta-1,3 and beta-1,6 branched glucans) represents the first receptor site of the toxin on the envelope of the sensitive target . Flow cytometry analysis of the sensitive target after treatment with KpKt and K1 toxin of S . cerevisiae, known to cause loss of cell viability via formation of pores in the cell membrane, suggests a different mode of action for KpKt.

Biochemistry, 2004 Aug 10, 43(31), 10212 - 23
UDP-galactose 4-epimerase from Kluyveromyces fragilis: analysis of its hysteretic behavior during catalysis; Nayar S et al.; UDP-galactose 4-epimerase serves as a prototype model of class II oxidoreductases that use bound NAD as a cofactor . This enzyme from Kluyveromyces fragilis is a homodimer with a molecular mass of 75 kDa/subunit . Continuous monitoring of the conversion of UDP-galactose (UDP-gal) to UDP-glucose (UDP-glu) by the epimerase in the presence of the coupling enzyme UDP-glucose dehydrogenase and NAD shows a kinetic lag of up to 80 s before a steady state is reached . The disappearance of the lag follows first-order kinetics (k = 3.22 x 10(-2) s(-1)) at 25 degrees C at enzyme and substrate concentrations of 1.0 nM and 1 mM, respectively . The observed lag is not due to factors such as insufficient activity of the coupling enzyme, association or dissociation or incomplete recruitment of NAD by epimerase, product activation, etc., but was a true expression of the activity of the prepared enzyme . Dissociation of the bound ligand(s) by heat followed by analysis with reverse-phase HPLC, TLC, UV-absorption spectrometry, mass spectrometry, and NMR showed that in addition to 1.78 mol of NAD/dimer, the epimerase also contains 0.77 mol of 5'-UMP/dimer . The latter is a strong competitive inhibitor . Preincubation of the epimerase with the substrate UDP-gal or UDP-glu replaces the inhibitor and also abolishes the lag, which reappeared after the enzyme was treated with 5'-UMP . The lag was not observed as long as the cells were in the growing phase and galactose in the growth medium was limiting, suggesting that association with 5'-UMP is a late log-phase phenomenon . The stoichiometry and conserved amino acid sequence around the NAD binding site of multimeric class I (classical dehydrogenases) and class II oxidoreductases, as reported in the literature, have been compared . It shows that each subunit is independently capable of being associated with one molecule of NAD, suggestive of two NAD binding sites of epimerase per dimer.

Prikl Biokhim Mikrobiol, 2004 May-Jun, 40(3), 332 - 6
{Selection and study of potent lactose-fermenting yeasts}; Golubev VI et al.; Whey-fermenting Kluyveromyces cultures were revealed among 105 yeast strains assimilating lactose . Eighteen most potent strains isolated from milk products fermented galactose, sucrose, and raffinose, in addition to lactose . Many yeast strains fermented inulin . Most strains were resistant to cycloheximide and grew in medium containing glucose, NaCl, and ethanol at concentrations of up to 50, 11-12, and 10-12%, respectively (4 degrees C) . Three strains had mycocinogenic activity . After fermentation of whey with selected yeast strains at 30 degrees C for 2-3 days, ethanol concentration was 4-5%.

Yeast, 2004 Jul 15, 21(9), 781 - 92
Efficient gene targeting in Kluyveromyces lactis; Kooistra R et al.; Integration of a DNA fragment in a host genome requires the action of a double-strand break (DSB) repair mechanism . Homologous recombination (HR) is initiated by binding of Rad52p to DNA ends and results in targeted integration . Binding of the Ku heterodimer (Ku70p/Ku80p) results in random integration via non-homologous end joining (NHEJ) . In contrast to Saccharomyces cerevisiae, the budding yeast Kluyveromyces lactis shows variable, but in general low, gene targeting efficiency . To study and to improve gene targeting efficiency, K . lactis has been used as a model . The KlRAD51, KlRAD52 and KlKU80 genes have been isolated and deletion mutants for these genes have been constructed . Efficiency of gene targeting was determined at the KlADE2 locus using targeting constructs with different lengths of homologous flanking sequences . In wild-type K . lactis, the gene targeting efficiency ranged from 0% with 50 to 88% with 600 bp flanks . The Klku80 mutant, however, showed >97% gene targeting efficiency independently of the size of the homologous flanks . These results demonstrate that deletion of the NHEJ mechanism results in a higher gene targeting efficiency . Furthermore, increased gene targeting efficiency was achieved by the transformation of wild-type K . lactis with the KlADE2 deletion construct in the presence of excess small DNA fragments . Using this method, PCR-generated deletion constructs containing only 50 bp of homologous flanking sequences resulted in efficient targeted gene replacement .

Biotechnol Lett, 2004 May, 26(10), 845 - 8
Use of Zymomonas mobilis and Saccharomyces cerevisiae mixed with Kluyveromyces fragilis for improved ethanol production from Jerusalem artichoke tubers; Szambelan K et al.; Jerusalem artichoke mashed tubers were fermented using single yeasts and a bacterium as well as mixed culture of microorganisms . Kluyveromyces fragilis, a yeast with an active inulinase, was used together with either a commercial distillery yeast, Saccharomyces cerevisiae, or the bacterium Zymomonas mobilis . After batch fermentation the best ethanol concentration of 0.48 g g(-1) for the mixed population and 0.46 g g(-1) for the single population can be obtained . The theoretical yield of the mixed cultures was 2-12% higher than for the single microorganism.

Curr Genet, 2004 Sep, 46(3), 147 - 57 Epub 2004 Jul 15.
Functional characterisation and transcriptional regulation of the KlHEM12 gene from Kluyveromyces lactis; Nunez L et al.; Cloning, sequencing and functional analysis of the Kluyveromyces lactis KlHEM12 gene and its upstream region are reported . The gene encodes for a protein that is highly homologous to uroporphyrinogen decarboxylases from different organisms and complements its mutation in Saccharomyces cerevisiae . Secondary structure prediction allows outlining a topology diagram which is compatible with a (beta/alpha)8-barrel structure . A K . lactis haploid strain carrying a null allele of KlHEM12 showed decreased growth in media not supplemented with hemin (ferriprotoporphyrin IX) and red-fluorescent colonies due to the accumulation of porphyrins . KlHEM12 expression was analysed by Northern blot and promoter fusion to the reporter lacZ gene . Transcription of this gene is not under heme or glucose repression and it is slightly induced by non-fermentable carbon sources through the Hap2/3/4/5 complex .

Int J Food Microbiol, 2004 Aug 15, 95(1), 51 - 9
Occurrence and characterization of yeasts isolated from artisanal Fiore Sardo cheese; Fadda ME et al.; The occurrence of yeast microflora in artisanal Fiore Sardo cheese during ripening was studied . Mean yeast counts ranged from 2.64+/-1 log(10) cfu ml(-1) in milk to 0.65+/-1 log(10) cfu g(-1) in 9 months cheese, with the higher counts observed in 48-h-old cheese . Strains belonging to the prevalent species Debaryomyces hansenii, Kluyveromyces lactis, Geotrichum candidum, Candida zeylanoides and Candida lambica were selected for technological and genotypic characterization . All D . hansenii strains fermented glucose and assimilated lactate, a high percentage assimilated citrate and only a few showed proteolytic and lipolytic activity . All K . lactis strains were able to both assimilate and ferment lactose, to assimilate lactate and to exhibit proteolytic activity on casein . G . candidum assimilated lactate and some strains showed proteolytic and lipolytic activity . C . zeylanoides showed lipolytic activity on tweens and the majority of strains assimilated citrate . C . lambica fermented glucose and assimilated lactate . Considering their diffusion and technological characteristics, an important role for K . lactis and G . candidum in the early stages of the ripening process and for D . hansenii after the first month of ripening can be suggested . RAPD-PCR analysis with M13 primer grouped the isolates in well-separated clusters with their type strains and confirmed the previous phenotypic identification . The high intraspecific homogeneity observed in tested strains could be explained by their isolation from a common substrate and from neighbouring geographical areas . This preliminary study allowed us to isolate autochthon yeast strains showing particular properties which can contribute to the production of typical cheese taste and flavour.

Mol Microbiol, 2004 Jul, 53(1), 263 - 73
Novel yeast killer toxins provoke S-phase arrest and DNA damage checkpoint activation; Klassen R et al.; Certain strains of Pichia acaciae and Wingea robertsiae (synonym Debaryomyces robertsiae) harbour extranuclear genetic elements that confer a killer phenotype to their host . Such killer plasmids (pPac1-2 of P . acaciae and pWR1A of W . robertsiae) were sequenced and compared with the zymocin encoding pGKL1 of Kluyveromyces lactis . Both new elements were found to be closely related to each other, but they are only partly similar to pGKL1 . As for the latter, they encode functions mediating binding of the toxin to the target cell's chitin and a hydrophobic region potentially involved in uptake of a toxin subunit by target cells . Consistently, mutations affecting the target cell's major chitin synthase (Chs3) protect it from toxin action . Heterologous intracellular expression of respective open reading frames identified cell cycle-arresting toxin subunits deviating structurally from the likewise imported gamma-subunit of the K . lactis zymocin . Accordingly, toxicity of both P . acaciae and Wingea toxins was shown to be independent of RNA polymerase II Elongator, which is indispensable for zymocin action . Thus, P . acaciae and Wingea toxins differ in their mode of action from the G1-arresting zymocin . Fluorescence-activated cell sorting analysis and determination of budding indices have proved that such novel toxins mediate cell cycle arrest post-G1 during the S phase . Concomitantly, the DNA damage checkpoint kinase Rad53 is phosphorylated . As a mutant carrying the checkpoint-deficient allele rad53-11 displays toxin hypersensitivity, damage checkpoint activation apparently contributes to coping with toxin stress, rather than being functionally implemented in toxin action.

Lett Appl Microbiol, 2004, 38(4), 257 - 64
In vitro studies on the potential for biological control on Aspergillus section Flavi by Kluyveromyces spp; La Penna M et al.; AIMS: Antagonist activity of Kluyveromyces spp . isolates on Aspergillus section Flavi was studied . METHODS AND RESULTS: The screening of isolates were made through studies of growth at different water activities and temperatures, index of dominance (I(D)), ecological similarity, antifungal activity and impact on aflatoxin B1 accumulation . High optical density was obtained at 25 and 30 degrees C and 48 h of incubation . Cell growth decreases with decrease in water activity . The predominant interaction was mutual intermingling at a(w) = 0.982 and 0.955, while at a(w) = 0.999 and 0.937 mutual inhibition for contact was exhibited . All isolates were catabolically identical to Aspergillus section Flavi and compete by nutritional source . At high water activities yeasts showed inhibitory activity on Aspergillus strains, inhibition percentages varied between 75 and 100% . The isolates Y9, Y14, Y16, Y22, Y25 and Y33 showed antifungal activity and inhibitory activity on aflatoxin B1 accumulation at all water activities assayed from all Aspergillus section Flavi strains . CONCLUSIONS: The data show that the isolates selected in a wide range of environmental conditions could exert their roll like biological control agents for Aspergillus section Flavi in storage maize ecosystem . SIGNIFICANCE AND IMPACT OF THE STUDY: Isolates of Kluyveromyces spp . may have practical value in the postharvest control of storage maize.

Mikrobiologiia, 2004 Mar-Apr, 73(2), 163 - 8
{Activity of the key enzymes in xylose-assimilating yeasts at different rates of oxygen transfer to the fermentation medium}; Iablochkova EN et al.; The activities of xylitol dehydrogenase and xylose reductase in the yeasts Candida shehatae, C . didensiae, C . intermediae, C . tropicalis, Kluyveromyces marxianus, Pichia stipitis, P . guillermondii, Pachysolen tannophilus, and Torulopsis molishiama were studied at different oxygen transfer rates (OTRs) to the fermentation medium (0, 5, and 140 mmol O2/(1 h)) . The activities of these enzymes were maximum in the yeasts P . stipitis and C . shehatae . The xylitol dehydrogenase of all the yeasts was NAD-dependent, irrespective of the intensity of aeration . The xylose reductase of the yeasts C . didensiae, C . intermediae, C . tropicalis, Kl . marxianus, P . guillermondii, and T . molishiama was NADPH-dependent, whereas the xylose reductase of P . stipitis, C . shehatae, and Pa . tannophilus was specific for both NADPH and NADH . The effect of OTR on the activities of the different forms of xylitol dehydrogenase and xylose reductase in the xylose-assimilating yeasts is discussed.

Biotechnol Lett, 2004 May, 26(9), 741 - 6
Kinetics of improved productivity of beta-galactosidase by a cycloheximide-resistant mutant of Kluyveromyces marxianus; Rajoka MI et al.; The maximum volumetric productivity of beta-galactosidase by a Kluyveromyces marxianus mutant, grown on lactose/corn steep liquor medium for 3 d, was 150 IU l(-1) h(-1) which is twice that of the parent organism . During product formation, mutated cells provided more resistance against thermal inactivation.

Eukaryot Cell, 2004 Jun, 3(3), 589 - 97
The deletion of the succinate dehydrogenase gene KlSDH1 in Kluyveromyces lactis does not lead to respiratory deficiency; Saliola M et al.; We have isolated a Kluyveromyces lactis mutant unable to grow on all respiratory carbon sources with the exception of lactate . Functional complementation of this mutant led to the isolation of KlSDH1, the gene encoding the flavoprotein subunit of the succinate dehydrogenase (SDH) complex, which is essential for the aerobic utilization of carbon sources . Despite the high sequence conservation of the SDH genes in Saccharomyces cerevisiae and K . lactis, they do not have the same relevance in the metabolism of the two yeasts . In fact, unlike SDH1, KlSDH1 was highly expressed under both fermentative and nonfermentative conditions . In addition to this, but in contrast with S . cerevisiae, K . lactis strains lacking KlSDH1 were still able to grow in the presence of lactate . In these mutants, oxygen consumption was one-eighth that of the wild type in the presence of lactate and was normal with glucose and ethanol, indicating that the respiratory chain was fully functional . Northern analysis suggested that alternative pathway(s), which involves pyruvate decarboxylase and the glyoxylate cycle, could overcome the absence of SDH and allow (i) lactate utilization and (ii) the accumulation of succinate instead of ethanol during growth on glucose .

FEMS Microbiol Lett, 2004 Jun 15, 235(2), 369 - 75
Purification and characterization of a lysine aminopeptidase from Kluyveromyces marxianus; Ramirez-Zavala B et al.; A lysine aminopeptidase was purified from the yeast Kluyveromyces marxianus . This enzyme was purified 100-fold from a soluble extract obtained at 100,000g . The purification procedure consisted in fractionated precipitation with ammonium sulfate and five chromatography steps . The native enzyme had a molecular mass of 46 kDa assessed through gel filtration . This aminopeptidase depicted an optimal pH of 7.0 and was stable at a pH range of 4-8, its optimal temperature was 45 degrees C and the enzyme became unstable at temperatures above 55 degrees C . The isoelectric point of the purified enzyme was 4.4 . Michaelis constant and Vmax for L-lysine-p-nitroanilide were 0.33 mM and 2.2 mM min(-1) per milligram of protein, respectively . The enzyme was strongly inhibited by bestatin, o-phenanthroline and, to a lesser extent, by EDTA, suggesting that this enzyme is a metalloprotease . Our results suggest that the lysine aminopeptidase from Kluyveromyces marxianus might be of biotechnological relevance.

Biotechnol Prog, 2004 May-Jun, 20(3), 715 - 20
Effect of binary combinations of selected toxic compounds on growth and fermentation of Kluyveromyces marxianus; Oliva JM et al.; The inhibitory effects of various lignocellulose degradation products on glucose fermentation by the thermotolerant yeast Kluyveromyces marxianus were studied in batch cultures . The toxicity of the aromatic alcohol catechol and two aromatic aldehydes (4-hydroxybenzaldehyde and vanillin) was investigated in binary combinations . The aldehyde furfural that usually is present in relatively high concentration in hydrolyzates from pentose degradation was also tested . Experiments were conducted by combining agents at concentrations that individually caused 25% inhibition of growth . Compared to the relative toxicity of the individual compounds, combinations of furfural with catechol and 4-hydroxybenzaldehyde were additive (50% inhibition of growth) . The other binary combinations assayed (catechol with 4-hydroxybenzaldehyde, and vanillin with catechol, furfural, or 4-hydroxybenzaldehyde) showed synergistic effect on toxicity and caused a 60-90% decrease in cell mass production . The presence of aldehydes in the fermentation medium strongly inhibited cell growth and ethanol production . Kluyveromyces marxianus reduces aldehydes to their corresponding alcohols to mitigate the toxicity of these compounds . The total reduction of aldehydes was needed to start ethanol production . Vanillin, in binary combination, was dramatically toxic and was the only compound for which inhibition could not be overcome by yeast strain assimilation, causing a 90% reduction in both cell growth and fermentation.

Yeast, 2004 May, 21(7), 549 - 56
Yeast involved in fermentation of Coffea arabica in East Africa determined by genotyping and by direct denaturating gradient gel electrophoresis; Masoud W et al.; Samples of Coffea arabica were collected during the different stages of the fermentation from two production sites in Tanzania . The yeasts community was identified by genotyping using ITS-PCR and sequence analysis of the D1/D2 domain of the 26S rRNA gene . For confirmation, denaturating gradient gel electrophoresis (DGGE) of PCR-amplified 26S rRNA gene was performed to detect yeast directly from coffee samples without cultivation . Yeast counts were in the range 4.0 x 10(4) - 5.0 x 10(7) CFU/g with an increase during fermentation . Three yeasts species were dominant . The predominant yeast found during fermentation and drying was Pichia kluyveri . Pichia anomala was found in high numbers during drying of coffee beans . Hanseniaspora uvarum was the predominant yeast during fermentation but decreased during drying . Kluyveromyces marxianus, Candida pseudointermedia, Issatchenkia orientalis, Pichia ohmeri and Torulaspora delbrueckii occurred in concentrations of 10(3) CFU/g or below in coffee samples . Saccharomyces cerevisiae and Candida xestobii were not isolated by cultivation, but by the DGGE technique . A good agreement was found between the sequence analysis of the D1/D2 domain of the 26S rRNA gene and sequencing of the DGGE bands .

Appl Biochem Biotechnol, 2004 May, 117(2), 75 - 92
Influence of carbon and nitrogen sources and temperature on hyperproduction of a thermotolerant beta-glucosidase from synthetic medium by Kluyveromyces marxianus; Rajoka MI et al.; The effect of carbon source and its concentration, inoculum size, yeast extract concentration, nitrogen source, pH of the fermentation medium, and fermentation temperature on beta-glucosidase production by Kluyveromyces marxianus in shake-flask culture was investigated . These were the independent variables that directly regulated the specific growth and beta-glucosidase production rate . The highest product yield, specific product yield, and productivity of beta-glucosidase occurred in the medium (pH 5.5) inoculated with 10% (v/v) inoculum of the culture . Cellobiose (20 g/L) significantly improved beta-glucosidase production measured as product yield (YP/S) and volumetric productivity (QP) followed by sucrose, lactose, and xylose . The highest levels of productivity (144 IU/{L.h}) of beta-glucosidase occurred on cellobiose in the presence of CSL at 35 degrees C and are significantly higher than the values reported by other researchers on almost all other organisms . The thermodynamics and kinetics of beta-glucosidase production and its deactivation are also reported . The enzyme was substantially stable at 60 degrees C and may find application in some industrial processes.

Biochim Biophys Acta, 2004 May 25, 1678(2-3), 170 - 5
Isolation and characterization of two nuclear genes encoding glutathione and thioredoxin reductases from the yeast Kluyveromyces lactis; Tarrio N et al.; Response to oxidative stress has been hitherto scarcely studied in the respiratory yeast Kluyveromyces lactis . The genes coding for reductases of glutathione and thioredoxin, KlGLR1 and KlTRR1, respectively, have been cloned and characterized in this work . H(2)O(2) treatment increased transcription and enzyme activity of KlTRR1 but not of KlGLR1, suggesting a different situation from that reported for the fermentative yeast Saccharomyces cerevisiae . A consensus for Yap1p binding is functional in the KlTRR1 promoter.

Biochem Biophys Res Commun, 2004 Jun 11, 318(4), 1031 - 8
Alterations of O-glycosylation, cell wall, and mitochondrial metabolism in Kluyveromyces lactis cells defective in KlPmr1p, the Golgi Ca(2+)-ATPase; Farina F et al.; In yeast the P-type Ca(2+)-ATPase of the Golgi apparatus, Pmr1p, is the most important player in calcium homeostasis . In Kluyveromyces lactis KlPMR1 inactivation leads to pleiotropic phenotypes, including reduced N-glycosylation and altered cell wall morphogenesis . To study the physiology of K . lactis when KlPMR1 was inactivated microarrays containing all Saccharomyces cerevisiae coding sequences were utilized . Alterations in O-glycosylation, consistent with the repression of KlPMT2, were found and a terminal N-acetylglucosamine in the O-glycans was identified . Klpmr1Delta cells showed increased expression of PIRs, proteins involved in cell wall maintenance, suggesting that responses to cell wall weakening take place in K . lactis . We found over-expression of KlPDA1 and KlACS2 genes involved in the Acetyl-CoA synthesis and down-regulation of KlIDP1, KlACO1, and KlSDH2 genes involved in respiratory metabolism . Increases in oxygen consumption and succinate dehydrogenase activity were also observed in mutant cells . The described approach highlighted the unexpected involvement of KlPMR1 in energy-yielding processes.

Appl Environ Microbiol, 2004 May, 70(5), 2632 - 8
Improved production of heterologous proteins by a glucose repression-defective mutant of Kluyveromyces lactis; Donnini C et al.; The secreted production of heterologous proteins in Kluyveromyces lactis was studied . A glucoamylase (GAA) from the yeast Arxula adeninivorans was used as a reporter protein for the study of the secretion efficiencies of several wild-type and mutant strains of K . lactis . The expression of the reporter protein was placed under the control of the strong promoter of the glyceraldehyde-3-phosphate dehydrogenase of Saccharomyces cerevisiae . Among the laboratory strains tested, strain JA6 was the best producer of GAA . Since this strain is known to be highly sensitive to glucose repression and since this is an undesired trait for biomass-oriented applications, we examined heterologous protein production by using glucose repression-defective mutants isolated from this strain . One of them, a mutant carrying a dgr151-1 mutation, showed a significantly improved capability of producing heterologous proteins such as GAA, human serum albumin, and human interleukin-1beta compared to the parent strain . dgr151-1 is an allele of RAG5, the gene encoding the only hexokinase present in K . lactis (a homologue of S . cerevisiae HXK2) . The mutation in this strain was mapped to nucleotide position +527, resulting in a change from glycine to aspartic acid within the highly conserved kinase domain . Cells carrying the dgr151-1 allele also showed a reduction in N- and O-glycosylation . Therefore, the dgr151 strain may be a promising host for the production of heterologous proteins, especially when the hyperglycosylation of recombinant proteins must be avoided.

Appl Biochem Biotechnol, 2004 Apr, 117(1), 49 - 64
Polygalacturonase and ethanol production in Kluyveromyces marxianus: potential use of polygalacturonase in foodstuffs; Serrat M et al.; The coproduction of ethanol and polygalacturonase (PG) in a pilot-scale batch fermentor using yeast extract--glucose (YD)--and sugar beet molasses (SBM)-based media was implemented utilizing a new high-PG-producing strain of Kluyveromyces marxianus . A certain growth inhibition was observed in SBM medium, causing ethanol and PG production to be lower . Ethanol productivity and accumulation values of 1.94 g/(L x h) and 40 g/L, respectively, were attained in YD, whereas the best fermentation efficiency (95.1%) was achieved with SBM medium . Maximal PG synthesis occurred at the end of cell growth, with values of 1.08 and 0.46 U/(mg x h) for the YD and SBM media, respectively . When the cultures reached stationary phase, PG production stopped . The highest accumulation level (17 U/mL) occurred in YD medium, in agreement with previous laboratory-scale studies carried out for this strain . The potential applications of the crude enzyme preparations were evaluated with different fruit juices and vegetable slices . The enzyme was able to increase the filtration rate of orange, pear, and apple juices by twofold . Additionally, complete clarification of apple juice was readily accomplished, whereas cucumber, carrot, and banana tissues were macerated to a lesser extent .

Mol Cell Biol, 2004 May, 24(10), 4083 - 91
Key role of Ser562/661 in Snf1-dependent regulation of Cat8p in Saccharomyces cerevisiae and Kluyveromyces lactis; Charbon G et al.; Utilization of nonfermentable carbon sources by Kluyveromyces lactis and Saccharomyces cerevisiae requires the Snf1p kinase and the Cat8p transcriptional activator, which binds to carbon source-responsive elements of target genes . We demonstrate that KlSnf1p and KlCat8p from K . lactis interact in a two-hybrid system and that the interaction is stronger with a kinase-dead mutant form of KlSnf1p . Of two putative phosphorylation sites in the KlCat8p sequence, serine 661 was identified as a key residue governing KlCat8p regulation . Serine 661 is located in the middle homology region, a regulatory domain conserved among zinc cluster transcription factors, and is part of an Snf1p consensus phosphorylation site . Single mutations at this site are sufficient to completely change the carbon source regulation of the KlCat8p transactivation activity observed . A serine-to-glutamate mutant form mimicking constitutive phosphorylation results in a nearly constitutively active form of KlCat8p, while a serine-to-alanine mutation has the reverse effect . Furthermore, it is shown that KlCat8p phosphorylation depends on KlSNF1 . The Snf1-Cat8 connection is evolutionarily conserved: mutation of corresponding serine 562 of ScCat8p gave similar results in S . cerevisiae . The enhanced capacity of ScCat8S562E to suppress the phenotype caused by snf1 strengthens the hypothesis of direct phosphorylation of Cat8p by Snf1p . Unlike that of S . cerevisiae ScCAT8, KlCAT8 transcription is not carbon source regulated, illustrating the prominent role of posttranscriptional regulation of Cat8p in K . lactis.

Yeast, 2004 Apr 30, 21(6), 511 - 8
The KlSRB10 gene from Kluyveromyces lactis; Nunez L et al.; We report the cloning and sequencing of a gene from Kluyveromyces lactis with high homology to the SRB10 gene (alias UME5, SSN3, GIG2, NUT7, RYE5) from Saccharomyces cerevisiae and other organisms . The KlSRB10 gene is located in a similar configuration to that found in S . cerevisiae, flanked by NOT4 and a gene with high similarity to YPL041c . The translated protein contains 593 amino acids and the characteristic domains of kinases from the CMGC subgroup . The functional relationship to yeast SRB10 is demonstrated by complementation of mutant phenotypes in a haploid S . cerevisiae strain containing a null allele .

Cell Microbiol, 2004 Jun, 6(6), 569 - 80
After chitin docking, toxicity of Kluyveromyces lactis zymocin requires Saccharomyces cerevisiae plasma membrane H+-ATPase; Mehlgarten C et al.; Zymocin, a three-subunit (alpha beta gamma) toxin complex from Kluyveromyces lactis, imposes a cell cycle block on Saccharomyces cerevisiae . Phenotypic analysis of the resistant kti10 mutant implies a membrane defect, suggesting that KTI10 represents a gene involved early in the zymocin response . Consistently, KTI10 is shown here to be allelic to PMA1 encoding H(+)-ATPase, a plasma membrane H(+) pump vital for membrane energization (Delta Psi) . Like pma1 mutants, kti10 cells lose viability at low pH, indicating a pH homeostasis defect, and resist the antibiotic hygromycin B, uptake of which is known to be Pma1 and Delta Psi sensitive . Similar to kti10 cells, pma1 mutants with reported H(+) pump defects survive in the presence of exozymocin but do not resist endogenous expression of its lethal gamma-toxin subunit . Based on DNA sequence data, kti10 cells are predicted to produce a malfunctional Pma1 variant with expression levels that are normal . Intriguingly, zymocin protection of kti10 cells is suppressed by excess H(+), a scenario ineffective in bypassing resistance of chitin or toxin target mutants . Together with unaltered zymocin docking and gamma-toxin import events in kti10 cells, our data suggest that Pma1's role in zymocin action is likely to involve activation of gamma-toxin in a step following its cellular uptake.

FEMS Yeast Res, 2004 May, 4(7), 691 - 8
Ethanol formation and enzyme activities around glucose-6-phosphate in Kluyveromyces marxianus CBS 6556 exposed to glucose or lactose excess; Bellaver LH et al.; The aim of this work was to investigate the physiology of Kluyveromyces marxianus CBS 6556 in terms of its low tendency to form ethanol under exposure to sugar excess, and the split of carbon flux which takes place at the level of glucose-6-phosphate . Measurements were performed in batch cultivations, and after a glucose or a lactose pulse applied to chemostat-grown respiring cells (with a dilution rate of 0.1 h(-1)) . No ethanol formation was observed in batch cultivations or during pulse experiments, unless the oxygen supply was shut down, indicating that this organism is more strictly Crabtree-negative than its close relative K . lactis and other known Crabtree-negative yeasts . During the pulse experiments, activities of phosphoglucoisomerase, glucose-6-phosphate dehydrogenase and phosphoglucomutase in cell-free extracts remained rather constant, at higher levels than those of Saccharomyces cerevisiae grown at similar conditions . When cells were exposed to glucose concentrations as high as 26 gl(-1), the activity of phosphoglucomutase was higher than that in cells exposed to 14 gl(-1) glucose, whereas the activities of phosphoglucoisomerase and glucose-6-phosphate dehydrogenase did not change . Our results suggest that the low tendency for ethanol formation in K . marxianus might be a consequence of this yeast's capacity of keeping the glycolytic flux constant, due at least in part to the diversion of carbon flux towards the biosynthesis of carbohydrates and towards the pentose phosphate pathway.

Eukaryot Cell, 2004 Apr, 3(2), 369 - 84
Genetic dissection of the Kluyveromyces lactis telomere and evidence for telomere capping defects in TER1 mutants with long telomeres; Underwood DH et al.; In the yeast Kluyveromyces lactis, the telomeres are composed of perfect 25-bp repeats copied from a 30-nucleotide RNA template defined by 5-nucleotide terminal repeats . A genetic dissection of the K . lactis telomere was performed by using mutant telomerase RNA (TER1) alleles to incorporate mutated telomeric repeats . This analysis has shown that each telomeric repeat contains several functional regions, some of which may physically overlap . Mutations in the terminal repeats of the template RNA typically lead to telomere shortening, as do mutations in the right side of the Rap1p binding site . Mutations in the left half of the Rap1p binding site, however, lead to the immediate formation of long telomeres . When mutated, the region immediately 3' of the Rap1p binding site on the TG-rich strand of the telomere leads to telomeres that are initially short but eventually undergo extreme telomere elongation . Mutations between this region and the 3' terminal repeat cause elevated recombination despite the presence of telomeres of nearly wild-type length . Mutants with highly elongated telomeres were further characterized and exhibit signs of telomere capping defects, including elevated levels of subtelomeric recombination and the formation of extrachromosomal and single-stranded telomeric DNA . Lengthening caused by some Rap1 binding site mutations can be suppressed by high-copy-number RAP1 . Mutated telomeric repeats from a delayed elongation mutant are shown to be defective at regulating telomere length in cells with wild-type telomerase, indicating that the telomeric repeats are defective at telomere length regulation.

J Biotechnol, 2004 Apr 8, 109(1-2), 139 - 46
How physiological and cultural conditions influence heterologous protein production in Kluyveromyces lactis; Merico A et al.; The optimization and scale-up of a specific protein production process have to take into account cultural conditions as well as cell physiology of growth and influence of foreign protein expression on host cell metabolism . Growth on cheap substrates, efficient secretion ability and a weaker tendency to hypermannosilate proteins than S . cerevisiae, make K . lactis an excellent and well-accepted host for heterologous protein production, even for human use . A fairly good heterologous glucoamylase yield and the setting of the optimal conditions to produce it were obtained expressing the Arxula adeninivorans glucoamylase in a strain of K . lactis and its isogenic mutant, which seems to have higher secretion ability . We performed batch cultures of both strains to analyze the influence of different physiological and environmental parameters on glucoamylase production/secretion . Interestingly, the maintenance of pH in the range of neutrality causes the consumption of a larger amount of carbon source, a longer time of production and a better stability of the active form of the enzyme, thus increasing biomass and glucoamylase production . Furthermore, the enrichment of the culture medium adds up to the action of pH control, forcing the mutant production/secretion to higher levels.

J Biotechnol, 2004 Apr 8, 109(1-2), 131 - 7
Engineered autolytic yeast strains secreting Kluyveromyces lactis beta-galactosidase for production of heterologous proteins in lactose media; Becerra M et al.; Secretion of the heterologous Kluyveromyces lactis beta-galactosidase into culture medium by several Saccharomyces cerevisiae osmotic-remedial thermosensitive-autolytic mutants was assayed and proved that new metabolic abilities were conferred since the constructed strains were able to grow in lactose-containing media . Cell growth became independent of a lactose-uptake mechanism . Higher levels of extra-cellular and intra-cellular beta-galactosidase production, lactose consumption and growth were obtained with the LHDP1 strain, showing a thermosensitive-autolytic phenotype as well as being peptidase-defective . The recombinant strain LHDP1 presented the highest beta-galactosidase yields from biomass and the lowest ethanol levels from lactose . This strain is effective for the heterologous production and release of K . lactis beta-galactosidase into the extra-cellular medium after osmotic shock.

J Biotechnol, 2004 Apr 8, 109(1-2), 93 - 101
KlPMR1 inactivation and calcium addition enhance secretion of non-hyperglycosylated heterologous proteins in Kluyveromyces lactis; Uccelletti D et al.; The Kluyveromyces lactis KlPMR1 gene is the functional homologue of Saccharomyces cerevisiae PMR1 which encodes a Ca(2+)-ATPase localized in the Golgi apparatus . We studied the effects of KlPMR1 inactivation on the glycosylation and secretion of native and heterologous proteins in K . lactis . We used acid phosphatase, recombinant human serum albumin and alpha-glucoamylase from Arxula adeninivorans as reporter proteins . The Klpmr1Delta strain showed enhanced secretion of the heterologous proteins analyzed; the improved rHSA production did not result from enhanced transcription but rather involved increased translation and/or secretion efficiency . The growth rate of mutant cells resulted slower as compared to that of wild-type strain . The addition of 10mM calcium to the culture medium, however, not only completely relieved the growth defect of the mutant cells but also improved the rate of heterologous proteins production . Moreover, the addition of this ion in the culture medium of K . lactis did not suppress the glycosylation defects; this is an important difference with respect to S . cerevisiae where the glycosylation is partially restored by Ca(2+) addition . The Klpmr1Delta strain as a host offers thus an additional advantage for those cases requiring that the produced recombinant protein would not result hyperglycosylated.

J Biotechnol, 2004 Apr 8, 109(1-2), 83 - 92
Kluyveromyces lactis cells entrapped in Ca-alginate beads for the continuous production of a heterologous glucoamylase; de Alteriis E et al.; Viable cells of Kluyveromyces lactis, transformed with the glucoamylase gene from Arxula adeninivorans, were entrapped in beads of Ca-alginate and employed on a lab scale in a continuous stirred and a fluidised bed reactor (FBR), both fed with a rich medium (YEP) containing lactose as carbon source . Experiments with freely suspended cells in batch and chemostat had demonstrated that glucoamylase production was favoured in the presence of lactose and YEP medium . Employing controlled-sized beads having a 2.13 mm diameter, specific glucoamylase productivity was higher in the stirred reactor (CSTR) than in the FBR; in the latter a higher volumetric productivity was achieved, due to the lower void degree . The performance of the immobilised cell systems, in terms of specific glucoamylase productivity, was strongly affected by mass transfer limitations occurring throughout the gel due to the high molecular weight of the product . In the perspective to improve and scale-up the immobilised cell system proposed, a mathematical model, which takes into account substrate transfer limitations throughout the gel, has been developed . The effective lactose diffusivity was related to the bead reactive efficiency by means of the Thiele modulus . The regression of the model parameters on the experimental data of substrate consumption obtained both in the CSTR and in the FBR allowed to estimate lactose diffusivity and the kinetic parameters of the immobilised yeast.

Biosci Biotechnol Biochem, 2004 Mar, 68(3), 638 - 49
A novel NADH-dependent carbonyl reductase from Kluyveromyces aestuarii and comparison of NADH-regeneration system for the synthesis of ethyl (S)-4-chloro-3-hydroxybutanoate; Yamamoto H et al.; To compare NADH-regeneration systems for the synthesis of (S)-4-chloro-3-hydroxybutanoate (ECHB), a novel NADH-dependent carbonyl reductase (KaCR1), which reduced ethyl 4-chloroacetoacetate (ECAA) to form (S)-ECHB, was screened and purified from Kluyveromyces aestuarii and a gene encoding KaCR1 was cloned . Glucose dehydrogenase (GDH) and formate dehydrogenase (FDH) were compared as enzymes for NADH regeneration using Escherichia coli cells coexpressing each enzyme with KaCR1 . E . coli cells coexpressing GDH produced 45.6 g/l of (S)-ECHB from 50 g/l of ECAA and E . coli cells coexpressing FDH, alternatively, produced only 19.0 g/l . The low productivity in the case of FDH was suggested to result from the low activity and instability of FDH.

Yeast, 2004 Mar, 21(4), 325 - 31
Isolation of an acetyl-CoA synthetase gene (ZbACS2) from Zygosaccharomyces bailii; Rodrigues F et al.; A gene homologous to Saccharomyces cerevisiae ACS genes, coding for acetyl-CoA synthetase, has been cloned from the yeast Zygosaccharomyces bailii ISA 1307, by using reverse genetic approaches . A probe obtained by PCR amplification from Z . bailii DNA, using primers derived from two conserved regions of yeast ACS proteins, RIGAIHSVVF (ScAcs1p; 210-219) and RVDDVVNVSG (ScAcs1p; 574-583), was used for screening a Z . bailii genomic library . Nine clones with partially overlapping inserts were isolated . The sequenced DNA fragment contains a complete ORF of 2027 bp (ZbACS2) and the deduced polypeptide shares significant homologies with the products of ACS2 genes from S . cerevisiae and Kluyveromyces lactis (81% and 82% identity and 84% and 89% similarity, respectively) . Phylogenetic analysis shows that the sequence of Zbacs2 is more closely related to the sequences from Acs2 than to those from Acs1 proteins . Moreover, this analysis revealed that the gene duplication producing Acs1 and Acs2 proteins has occurred in the common ancestor of S . cerevisiae, K . lactis, Candida albicans, C . glabrata and Debaryomyces hansenii lineages . Additionally, the cloned gene allowed growth of S . cerevisiae Scacs2 null mutant, in medium containing glucose as the only carbon and energy source, indicating that it encodes a functional acetyl-CoA synthetase . Also, S . cerevisiae cells expressing ZbACS2 have a shorter lag time, in medium containing glucose (2%, w/v) plus acetic acid (0.1-0.35%, v/v) . No differences in cell response to acetic acid stress were detected both by specific growth and death rates . The mode of regulation of ZbACS2 appears to be different from ScACS2 and KlACS2, being subject to repression by a glucose pulse in acetic acid-grown cells .

FEMS Yeast Res, 2004 Mar, 4(6), 573 - 7
A yeast intron as a translational terminator in a plasmid shuttle vector; Gibbs MD et al.; Plasmid shuttle vectors that contain both prokaryotic (Escherichia coli) and eukaryotic origins of replication are routinely used in molecular biology since E . coli is generally the organism of choice for manipulation of recombinant DNA . Initial transformation of the shuttle vector into E . coli allows production of microgram quantities of DNA suitable for transformation of low-transformation-efficiency hosts . A shuttle/expression vector for the yeast Kluyveromyces lactis, pCWK1, allows recombinant protein fused to the killer toxin signal sequence to be secreted to the medium . The heterologous genes are transcribed under the control of the K . lactis LAC4 promoter, which is tightly regulated in K . lactis . However, in E . coli the LAC4 promoter functions constitutively, and as a result, uncontrolled transcription and translation of genes that are toxic in E . coli can result in cell death, and subsequent failure to recover intact E . coli transformants . We have constructed and tested a modified shuttle vector that contains a K . lactis ribosomal intron that acts as a translational terminator in E . coli, preventing or reducing the expression of recombinant proteins and avoiding toxicity . When transcribed in K . lactis, the intron is spliced from the mRNA allowing the translation of intact full-length, active recombinant gene product.

Biochem Biophys Res Commun, 2004 Apr 9, 316(3), 738 - 43
A new kinetic model of recombinant beta-galactosidase from Kluyveromyces lactis for both hydrolysis and transgalactosylation reactions; Kim CS et al.; Previous models based on the Michaelis-Menten kinetic equation, that glucose was not used as an acceptor, did not explain our experimental data for lactose conversion by a recombinant beta-galactosidase from Kluyeromyces lactis . In order to create a new kinetic model based on the data, the effects of galactose and glucose on beta-galactosidase activity were investigated . Galactose acted as an inhibitor at low concentrations of galactose and lactose, but did not inhibit the activity of beta-galactosidase at high concentrations of galactose (above 50mM) and lactose (above 100mM) . The addition of glucose at concentrations below 50mM resulted in an increased reaction rate . A new model of K . lactis beta-galactosidase for both hydrolysis and transgalactosylation reactions with glucose and lactose as acceptors was proposed . The proposed model was fitted well to the experimental data of the time-course reactions for lactose conversion by K . lactis beta-galactosidase at various concentrations of substrate.

Appl Biochem Biotechnol, 2004 Mar, 112(3), 133 - 41
Use of whey ultrafiltrate as a substrate for production of carotenoids by the yeast Rhodotorula rubra; Frengova G et al.; Carotenogenesis of the lactose-negative yeast Rhodotorula rubra GED5 was studied by cocultivation with Kluyveromyces lactis MP11 in whey ultrafiltrate (WU) (35, 50, and 70 g of lactose/L) . Maximum yields of cell mass (24.3 g/L) and carotenoids (10.2 mg/L of culture fluid or 0.421 micro g/g of dry cells) were obtained by growing the microbial association in WU (50 g of lactose/L) in a fermentor with an airflow rate of 0.8 L/(L.min), agitation of 220 rpm, and temperature of 30 degrees C . The identified carotenoid pigments-beta-carotene, torulene, and torularhodin-reached maximum concentrations (133, 26.9, and 222.3 microg/g of dry cells, respectively) on d 5 for torulene and d 6 for beta-carotene and torularhodin.

Nature, 2004 Apr 8, 428(6983), 617 - 24 Epub 2004 Mar 07.
Proof and evolutionary analysis of ancient genome duplication in the yeast Saccharomyces cerevisiae; Kellis M et al.; Whole-genome duplication followed by massive gene loss and specialization has long been postulated as a powerful mechanism of evolutionary innovation . Recently, it has become possible to test this notion by searching complete genome sequence for signs of ancient duplication . Here, we show that the yeast Saccharomyces cerevisiae arose from ancient whole-genome duplication, by sequencing and analysing Kluyveromyces waltii, a related yeast species that diverged before the duplication . The two genomes are related by a 1:2 mapping, with each region of K . waltii corresponding to two regions of S . cerevisiae, as expected for whole-genome duplication . This resolves the long-standing controversy on the ancestry of the yeast genome, and makes it possible to study the fate of duplicated genes directly . Strikingly, 95% of cases of accelerated evolution involve only one member of a gene pair, providing strong support for a specific model of evolution, and allowing us to distinguish ancestral and derived functions.

Int J Food Microbiol, 2004 Mar 15, 91(3), 245 - 52
Purification and characterization of a serine carboxypeptidase from Kluyveromyces marxianus; Ramirez-Zavala B et al.; We purified a carboxypeptidase (CPY) from the yeast of Kluyveromyces marxianus . This enzyme was purified 170 times from a soluble extract of 100000 x g . Purification consisted in a fractionated precipitation with ammonium sulfate and two chromatographic steps consisting of anion exchange chromatography and hydrophobic interactions chromatography . The native enzyme depicted a molecular mass of 67 kDa by gel filtration . This serine carboxypeptidase depicted an optimal pH of 8.5 and was stable at a pH ranging from 6.0 to 9.0, its optimal temperature was of 45 degrees C and was unstable at temperatures above 55 degrees C; Michaelis constant and Vmax for N-benzoyl-l-tyrosine-p-nitroanilide were of 29 microM and 612 microM/min mg of protein, respectively . The enzyme was strongly inhibited by phenylmethylsufonyl fluoride (PMSF) and, to a lesser degree, by trans-epoxysuccinyl-l-leucylamido-(4-guanidine)-butane . This study indicated that K . marxianus carboxypeptidase could be an alternative to other animal-source carboxypeptidases in the industry.

Mol Microbiol, 2004 Mar, 51(5), 1413 - 23
Identification of the sequences required for chromosomal replicator function in Kluyveromyces lactis; Irene C et al.; The analysis of replication intermediates of a Kluyveromyces lactis chromosomal autonomous replicating sequence (ARS), KARS101, has shown that it is active as a chromosomal replicator . KARS101 contains a 50 bp sequence conserved in two other K . lactis ARS elements . The deletion of the conserved sequence in KARS101 completely abolished replicator activity, in both the plasmids and the chromosome . Gel shift assays indicated that this sequence binds proteins present in K . lactis nuclear extracts, and a 40 bp sequence, previously defined as the core essential for K . lactis ARS function, is required for efficient binding . Reminiscent of the origin replication complex (ORC), the binding appears to be ATP dependent . A similar pattern of protection of the core was seen with in vitro footprinting . KARS101 also functions as an ARS sequence in Saccharomyces cerevisiae . A comparative study using S . cerevisiae nuclear extracts revealed that the sequence required for binding is a dodecanucleotide related to the S . cerevisiae ARS consensus sequence and essential for S . cerevisiae ARS activity.

Mol Cell Biochem, 2004 Jan-Feb, 256-257(1-2), 319 - 27
Trehalose-enzyme interactions result in structure stabilization and activity inhibition . The role of viscosity; Sampedro JG et al.; Stress resistance is essential for survival . The mechanisms of molecule stabilization during stress are of interest for biotechnology, where many enzymes and other biomolecules are increasingly used at high temperatures and/or salt concentrations . Diverse organisms, exhibit rapid synthesis and accumulation of the disaccharide trehalose in response to stress . Trehalose is also rapidly hydrolyzed as soon as stress ends . In isolated enzymes, trehalose stabilizes both, structure and activity . In contrast, at optimal assay conditions, trehalose inhibits enzyme activity . A general mechanism underlying the trehalose effects observed at all temperatures probably is the trehalose-mediated increase in solution viscosity that leads to protein domain motion inhibition . This may be analyzed using Kramer's theory . The role of viscosity in the effects of trehalose is analyzed in examples from the literature and in studies on the plasma membrane H(+)-ATPase from Kluyveromyces lactis . In the cell, it may be proposed that the large concentration of trehalose reached during stress stabilizes structures through viscosity . However, once stress ends trehalose has to be rapidly hydrolyzed in order to avoid the viscosity-mediated inhibition of enzymes.

Mol Microbiol, 2004 Feb, 51(4), 1015 - 25
The galactokinase of Hypocrea jecorina is essential for cellulase induction by lactose but dispensable for growth on d-galactose; Seiboth B et al.; Lactose is the only soluble carbon source which can be used economically for the production of cellulases or heterologous proteins under cellulase expression signals by Hypocrea jecorina (=Trichoderma reesei) . Towards an understanding of lactose metabolism and its role in cellulase formation, we have cloned and characterized the gal1 (galactokinase) gene of H . jecorina, which catalyses the first step in d-galactose catabolism . It exhibits a calculated Mr of 57 kDa, and shows moderate identity (about 40%) to its putative homologues of Saccharomyces cerevisiae and Kluyveromyces lactis . Gal1 is a member of the GHMP family, shows conservation of a Gly/Ser rich region involved in ATP binding and of amino acids (Arg 51, Glu 57, Asp 60, Asp 214, Tyr 270) responsible for galactose binding . A single transcript was formed constitutively during the rapid growth phase on all carbon sources investigated and accumulated to about twice this level during growth on d-galactose, l-arabinose and their corresponding polyols . Deletion of gal1 reduces growth on d-galactose but does only slightly affect growth on lactose . This is the result of the operation of a second pathway for d-galactose catabolism, which involves galactitol as an intermediate, and whose transient concentration is strongly enhanced in the delta-gal1 strain . In this pathway, galactitol is catabolised by the lad1-encoded l-arabinitol-4-dehydrogenase, because a gal1/lad1 double delta-mutant failed to grow on d-galactose . In the delta-gal1 strain, induction of the Leloir pathway gene gal7 (encoding galactose-1-phosphate uridylyltransferase) by d-galactose, but not by l-arabinose, is impaired . Induction of cellulase gene expression by lactose is also impaired in a gal1 deleted strain, whereas their induction by sophorose (the putative cellulose-derived inducer) was shown to be normal, thus demonstrating that galactokinase is a key enzyme for cellulase induction during growth on lactose, and that induction by lactose and sophorose involves different mechanisms.

J Ind Microbiol Biotechnol, 2004 Jan, 31(1), 35 - 40 Epub 2004 Feb 03.
A growth kinetic model of Kluyveromyces marxianus cultures on cheese whey as substrate; Longhi LG et al.; This work presents a multi-route, non-structured kinetic model for determination of microbial growth and substrate consumption in an experimental batch bioreactor in which beta-galactosidase is produced by Kluyveromyces marxianus growing on cheese whey . The main metabolic routes for lactose, and oxygen consumption, cell growth, and ethanol production are derived based on experimental data . When these individual rates are combined into a single growth rate, by rewriting the model equations, the model re-interpretation has a complexity similar to that of the usual variations of the Monod kinetic model, available in the literature . Furthermore, the proposed model is in good agreement with the experimental data for different growth temperatures, being acceptable for dynamic simulations, processes optimization, and implementations of model-based control technologies.

Yeast, 2004 Jan 15, 21(1), 41 - 51
KlSEC53 is an essential Kluyveromyces lactis gene and is homologous with the SEC53 gene of Saccharomyces cerevisiae; Staneva D et al.; Phosphomannomutase (PMM) is a key enzyme, which catalyses one of the first steps in the glycosylation pathway, the conversion of D-mannose-6-phosphate to D-mannose-1-phosphate . The latter is the substrate for the synthesis of GDP-mannose, which serves as the mannosyl donor for the glycosylation reactions in eukaryotic cells . In the yeast Saccharomyces cerevisiae PMM is encoded by the gene SEC53 (ScSEC53) and the deficiency of PMM activity leads to severe defects in both protein glycosylation and secretion . We report here on the isolation of the Kluyveromyces lactis SEC53 (KlSEC53) gene from a genomic library by virtue of its ability to complement a Saccharomyces cerevisiae sec53 mutation . The sequenced DNA fragment contained an open reading frame of 765 bp, coding for a predicted polypeptide, KlSec53p, of 254 amino acids . The KlSec53p displays a high degree of homology with phosphomannomutases from other yeast species, protozoans, plants and humans . Our results have demonstrated that KlSEC53 is the functional homologue of the ScSEC53 gene . Like ScSEC53, the KlSEC53 gene is essential for K . lactis cell viability . Phenotypic analysis of a K . lactis strain overexpressing the KlSEC53 gene revealed defects expected for impaired cell wall integrity .

Proc Natl Acad Sci U S A, 2004 Feb 10, 101(6), 1632 - 7 Epub 2004 Jan 26.
Evolution of the MAT locus and its Ho endonuclease in yeast species; Butler G et al.; The genetics of the mating-type (MAT) locus have been studied extensively in Saccharomyces cerevisiae, but relatively little is known about how this complex system evolved . We compared the organization of MAT and mating-type-like (MTL) loci in nine species spanning the hemiascomycete phylogenetic tree . We inferred that the system evolved in a two-step process in which silent HMR/HML cassettes appeared, followed by acquisition of the Ho endonuclease from a mobile genetic element . Ho-mediated switching between an active MAT locus and silent cassettes exists only in the Saccharomyces sensu stricto group and their closest relatives: Candida glabrata, Kluyveromyces delphensis, and Saccharomyces castellii . We identified C . glabrata MTL1 as the ortholog of the MAT locus of K . delphensis and show that switching between C . glabrata MTL1a and MTL1alpha genotypes occurs in vivo . The more distantly related species Kluyveromyces lactis has silent cassettes but switches mating type without the aid of Ho endonuclease . Very distantly related species such as Candida albicans and Yarrowia lipolytica do not have silent cassettes . In Pichia angusta, a homothallic species, we found MATalpha2, MATalpha1, and MATa1 genes adjacent to each other on the same chromosome . Although some continuity in the chromosomal location of the MAT locus can be traced throughout hemiascomycete evolution and even to Neurospora, the gene content of the locus has changed with the loss of an HMG domain gene (MATa2) from the MATa idiomorph shortly after HO was recruited.

Appl Biochem Biotechnol, 2004 Jan, 112(1), 25 - 35
High-temperature wine making using the thermotolerant yeast strain Kluyveromyces marxianus IMB3; Kourkoutas Y et al.; Kluyveromyces marxianus IMB3 yeast cells were immobilized on delignified cellulosic material, apple, and quince separately . Both immobilized and free cells were used in high-temperature wine making, and their fermented grape must contained 3 to 4% alcohol . Semisweet wines were produced by the addition of potable alcohol to the fermented must . Preliminary sensory evaluation of the produced semisweet wines showed good flavor and aroma . The final product contained extremely low levels of higher and amyl alcohols while ethyl acetate was at levels usually present in wines . The ferment produced may be blended with other products to improve their quality.

FEMS Microbiol Lett, 2004 Jan 15, 230(1), 19 - 25
Cu/Zn superoxide dismutase in yeast mitochondria - a general phenomenon; Nedeva TS et al.; Fermentative and respiratory yeast strains of genera Saccharomyces, Kluyveromyces, Pichia, Candida and Hansenula have been investigated for mitochondrial localization of Cu/Zn superoxide dismutase (SOD) . Pure mitochondrial fractions were obtained and the specific activities of Cu/Zn and Mn SODs were measured in comparison with those in the corresponding cell-free extracts . The Cu/Zn SOD: Mn SOD ratio in mitochondria and crude extracts was calculated and was considered a specific characteristic of all tested strains . Electrophoretical visualization of SOD patterns provided evidence for possible migration of cytosolic Cu/Zn SOD to mitochondria . The characteristic Cu/Zn SOD profile in mitochondria of all tested strains suggested its ubiquity within the fermentative and respiratory yeasts.

Mol Biol Cell, 2004 Mar, 15(3), 1459 - 69 Epub 2004 Jan 12.
The yeast elongator histone acetylase requires Sit4-dependent dephosphorylation for toxin-target capacity; Jablonowski D et al.; Kluyveromyces lactis zymocin, a heterotrimeric toxin complex, imposes a G1 cell cycle block on Saccharomyces cerevisiae that requires the toxin-target (TOT) function of holo-Elongator, a six-subunit histone acetylase . Here, we demonstrate that Elongator is a phospho-complex . Phosphorylation of its largest subunit Tot1 (Elp1) is supported by Kti11, an Elongator-interactor essential for zymocin action . Tot1 dephosphorylation depends on the Sit4 phosphatase and its associators Sap185 and Sap190 . Zymocin-resistant cells lacking or overproducing Elongator-associator Tot4 (Kti12), respectively, abolish or intensify Tot1 phosphorylation . Excess Sit4.Sap190 antagonizes the latter scenario to reinstate zymocin sensitivity in multicopy TOT4 cells, suggesting physical competition between Sit4 and Tot4 . Consistently, Sit4 and Tot4 mutually oppose Tot1 de-/phosphorylation, which is dispensable for integrity of holo-Elongator but crucial for the TOT-dependent G1 block by zymocin . Moreover, Sit4, Tot4, and Tot1 cofractionate, Sit4 is nucleocytoplasmically localized, and sit4Delta-nuclei retain Tot4 . Together with the findings that sit4Delta and totDelta cells phenocopy protection against zymocin and the ceramide-induced G1 block, Sit4 is functionally linked to Elongator in cell cycle events targetable by antizymotics.

Mol Cell Biol, 2004 Jan, 24(2), 912 - 23
Template requirements for telomerase translocation in Kluyveromyces lactis; Underwood DH et al.; Telomeres are synthesized by telomerase, a specialized reverse transcriptase, which contains a template in its intrinsic RNA component . In Kluyveromyces lactis, the repeats synthesized by the wild-type telomerase are 25 nucleotides (nt) in length and uniform in sequence . To determine the role of the 5-nt repeats defining the ends of the K . lactis telomerase RNA template in telomerase translocation, we have made mutations in and around them and observed their effects on telomere length and the sequence of newly made telomeric repeats . These template mutations typically result in telomeres that are shorter than those of wild-type cells . The mismatches between the telomerase template and the telomeric tip that occur after telomerase-mediated incorporation of the mutations are normally not removed . Instead, the mutations lead to the synthesis of aberrant repeats that range in size from 31 to 13 bp . Therefore, the specificity with which the telomeric tip aligns with the telomere is critical for the production of the uniform repeats seen in K . lactis . In addition, the region immediately 3' of the template may play an important role in translocation of the enzyme.

J Ind Microbiol Biotechnol, 2003 Dec, 30(12), 715 - 20 Epub 2003 Dec 19.
Evaluation of Kluyveromyces marxianus FII 510700 grown on a lactose-based medium as a source of a natural bioemulsifier; Lukondeh T et al.; Mannoprotein with emulsification properties was extracted from the cell walls of Kluyveromyces marxianus grown on a lactose-based medium by autoclaving cells in a citrate buffer at pH 7.The purified product was evaluated for chemical and physical stability to establish its potential use as a natural emulsifier in processed foods . The yield of purified bioemulsifier from this strain of K . marxianus was 4-7% of the original dry cell weight . The purified product, at a concentration of 12 g l(-1), formed emulsions that were stable for 3 months when subjected to a range of pH (3-11) and NaCl concentrations (2-50 g l(-1)) . The composition of this mannoprotein was 90% carbohydrate (mannan) and 4-6% protein . These values are similar to mannoprotein extracted from cells of Saccharomyces cerevisiae, which is the traditional source . Consequently K . marxianus cultivated on a low-cost lactose-based medium such as whey, a lactose-rich clean waste of the dairy industry, could be developed as a source of bioemulsifier for use in the food industry.

Appl Microbiol Biotechnol, 2004 May, 64(4), 543 - 50 Epub 2003 Dec 20.
The relative glucose uptake abilities of non-Saccharomyces yeasts play a role in their coexistence with Saccharomyces cerevisiae in mixed cultures; Nissen P et al.; The growth and glucose uptake of single cultures of the wine-related yeasts Kluyveromyces thermotolerans, Torulaspora delbrueckii, and Saccharomyces cerevisiae were investigated . The yeasts had different specific glucose uptake rates (qs) that depended on the residual glucose concentration and the oxygen availability . In mixed cultures, the qs values of the yeasts were not subject to any interaction effects over a wide range of glucose concentrations . Our results strongly indicate that the relative glucose uptake abilities of both non-Saccharomyces yeasts, i.e . the qs(non-Saccharomyces)/qs(S . cerevisiae) ratios, regulated their abilities to compete for space in mixed cultures with S . cerevisiae, which, in turn, regulated their early deaths . This hypothesis enabled us to explain why K . thermotolerans was less able than T . delbrueckii to coexist with S . cerevisiae in mixed cultures . Furthermore, it enabled us to explain why oxygen increased the abilities of K . thermotolerans and T . delbrueckii to coexist with S . cerevisiae in the mixed cultures .

Eur J Biochem, 2004 Jan, 271(1), 58 - 68
UDP-galactose 4-epimerase from Kluyveromyces fragilis . Evidence for independent mutarotation site; Brahma A et al.; UDP-galactose 4-epimerases from the yeast Kluyvero-myces fragilis and Escherichia coli are both homodimers but the molecular mass of the former (75 kDa/subunit) is nearly double that of the latter (39 kDa/subunit) . Protein databank sequence homology revealed the possibility of mutarotase activity in the excess mass of the yeast enzyme . This was confirmed by three independent assay protocols . With the help of specific inhibitors and chemical modification reagents, the catalytic sites of epimerase and mutarotase were shown to be distinct and independent . Partial proteolysis with trypsin in the presence of specific inhibitors, 5'-UMP for epimerase and galactose for mutarotase, protected the respective activities . Similar digestion with double inhibitors cleaved the molecule into two fragments of 45 and 30 kDa . After separation by size-exclusion HPLC, they manifested exclusively epimerase and mutarotase activities, respectively . Epimerases from Kluyveromyces lactis var lactis, Pachysolen tannophilus and Schizosaccharomyces pombi also showed associated mutarotase activity distinct from the constitutively formed mutarotase activity . Thus, the bifunctionality of homodimeric yeast epimerases of 65-75 kDa/subunit appears to be universal . In addition to the inducible bifunctional epimerase/mutarotase, K . fragilis contained a smaller constitutive monomeric mutarotase of approximately 35 kDa.

Curr Genet, 2004 Mar, 45(3), 129 - 39 Epub 2003 Dec 19.
Regulation of glycolysis in Kluyveromyces lactis: role of KlGCR1 and KlGCR2 in glucose uptake and catabolism; Neil H et al.; In Kluyveromyces lactis, the casein kinase I (Rag8p) regulates the transcription of glycolytic genes and the expression of the low-affinity glucose transporter gene RAG1 . This control involves the transcription factor Sck1p, a homologue of Sgc1p of Saccharomyces cerevisiae . SGC1 is known to interact genetically with ScGCR1 and ScGCR2, which code for regulators of glycolytic gene expression . Therefore, we studied the role of KlGCR1 and KlGCR2 genes in K . lactis . The Klgcr1 null mutant could not grow on glucose when respiration was blocked by antimycin A (Rag(- )phenotype) . In contrast, the Klgcr2 null mutant could grow under the same conditions, although at a reduced rate . In both mutants, the transcription of glycolytic genes was affected, while that of ribosomal protein genes was not modified . Furthermore, the transcription of the glucose permease genes was also found to be affected in the two mutants, although dissimilarly . While RAG1 transcription decreased at high glucose concentrations, the expression of the high-affinity glucose permease gene HGT1 was unexpectedly impaired under gluconeogenic conditions, in the absence of glucose . Gel mobility shift assays performed with purified maltose-binding protein-KlGcr1p showed that KlGcr1p could interact directly with the promoters of the glycolytic genes, but not with the promoters of the glucose permease genes . Thus, the control exerted by KlGcr1p and KlGcr2p upon glucose transporter genes is probably indirect.

Nucleic Acids Res, 2004 Jan 1, 32 Database issue, D315 - 8
Génolevures: comparative genomics and molecular evolution of hemiascomycetous yeasts; Sherman D et al.; The Genolevures online database provides data and tools to facilitate comparative genomic studies on hemiascomycetous yeasts . Now, four complete genome sequences recently determined (Candida glabrata, Kluyveromyces lactis, Debaryomyces hansenii, Yarrowia lipolytica) have been added to the partial sequences of 13 species previously analysed by a random approach . The database also includes the reference genome Saccharomyces cerevisiae . Data are presented with a focus on relations between genes and genomes: conservation of genes and gene families, speciation, chromosomal reorganization and synteny . The Genolevures site includes a community area for specific studies by members of the international community.

Appl Microbiol Biotechnol, 2004 Jun, 64(6), 787 - 93 Epub 2003 Dec 13.
Lactulose production by beta-galactosidase in permeabilized cells of Kluyveromyces lactis; Lee YJ et al.; Lactulose production from lactose and fructose was investigated with several commercial beta-galactosidases . The enzyme from Kluyveromyces lactis exhibited the highest lactulose productivity among the beta-galactosidases tested . The reaction conditions for lactulose production were optimized using cells that had been permeabilized by treatment with 50% (v/v) ethanol: cell concentration, 10.4 g l(-1); concentration of substrates, 40% (w/v) lactose and 20% (w/v) fructose; temperature, 60( degrees )C; pH 7.0 . Under these conditions, the permeabilized cells produced approximately 20 g l(-1) lactulose in 3 h with a lactulose productivity of 6.8 g l(-1) h(-1) . These results represent 1.3- and 2.1-fold increases in lactulose concentration and productivity compared with untreated washed cells . This is the first reported trial of enzymatic synthesis of lactulose using permeabilized yeast cells.

Gene, 2003 Dec 24, 323, 43 - 55
Functional characterization of CaCBF1, the Candida albicans homolog of centromere binding factor 1; Biswas K et al.; The centromere binding factor 1 (Cbf1) is necessary for proper chromosome segregation and transcriptional activation of methionine biosynthesis genes in the yeast Saccharomyces cerevisiae and is essential for viability in the related yeasts Kluyveromyces lactis and Candida glabrata . To study the function of Cbf1p in Candida albicans, the major human fungal pathogen, we constructed strains in which both alleles of the CaCBF1 gene were deleted . The Deltacbf1 mutants exhibited a slow growth phenotype and were temperature-sensitive at 42 degrees C . In addition, the mutants were auxotrophic for sulfur amino acids and could grow on minimal medium only when it was supplemented with either methionine or cysteine, suggesting that CaCBF1 is necessary for the expression of genes involved in assimilation of inorganic sulfate . Deletion of CaCBF1 also resulted in morphological abnormalities, many cells being unusually large . All mutant phenotypes were complemented by reintroduction of a functional CaCBF1 copy . The Deltacbf1 mutants neither showed enhanced sensitivity to the microtubule destabilizing agent thiabendazole nor did they exhibit an increased frequency of chromosome loss . These results suggest that Cbf1p is not necessary for efficient chromosome segregation in C . albicans.

FEMS Yeast Res, 2003 Dec, 4(3), 233 - 45
Phylogenetic circumscription of Saccharomyces, Kluyveromyces and other members of the Saccharomycetaceae, and the proposal of the new genera Lachancea, Nakaseomyces, Naumovia, Vanderwaltozyma and Zygotorulaspora; Kurtzman CP; Genera currently assigned to the Saccharomycetaceae have been defined from phenotype, but this classification does not fully correspond with species groupings determined from phylogenetic analysis of gene sequences . The multigene sequence analysis of Kurtzman and Robnett {FEMS Yeast Res . 3 (2003) 417-432} resolved the family Saccharomycetaceae into 11 well-supported clades . In the present study, the taxonomy of the Saccharomyctaceae is evaluated from the perspective of the multigene sequence analysis, which has resulted in reassignment of some species among currently accepted genera, and the proposal of the following five new genera: Lachancea, Nakaseomyces, Naumovia, Vanderwaltozyma and Zygotorulaspora.

Mol Genet Genomics, 2004 Jan, 270(6), 558 - 68 Epub 2003 Nov 29.
Pyruvate decarboxylases from the petite-negative yeast Saccharomyces kluyveri; Moller K et al.; Saccharomyces kluyveri is a petite-negative yeast, which is less prone to form ethanol under aerobic conditions than is S . cerevisiae . The first reaction on the route from pyruvate to ethanol is catalysed by pyruvate decarboxylase, and the differences observed between S . kluyveri and S . cerevisiae with respect to ethanol formation under aerobic conditions could be caused by differences in the regulation of this enzyme activity . We have identified and cloned three genes encoding functional pyruvate decarboxylase enzymes (PDCgenes) from the type strain of S . kluyveri (Sk- PDC11, Sk- PDC12 and Sk- PDC13) . The regulation of pyruvate decarboxylase in S . kluyveri was studied by measuring the total level of Sk- PDC mRNA and the overall enzyme activity under various growth conditions . It was found that the level of Sk- PDC mRNA was enhanced by glucose and oxygen limitation, and that the level of enzyme activity was controlled by variations in the amount of mRNA . The mRNA level and the pyruvate decarboxylase activity responded to anaerobiosis and growth on different carbon sources in essentially the same fashion as in S . cerevisiae . This indicates that the difference in ethanol formation between these two yeasts is not due to differences in the regulation of pyruvate decarboxylase(s), but rather to differences in the regulation of the TCA cycle and the respiratory machinery . However, the PDC genes of Saccharomyces/ Kluyveromyces yeasts differ in their genetic organization and phylogenetic origin . While S . cerevisiae and S . kluyveri each have three PDC genes, these have apparently arisen by independent duplications and specializations in each of the two yeast lineages.

Lett Appl Microbiol, 2003, 37(6), 438 - 42
Growth and beta-galactosidase activity in cultures of Kluyveromyces marxianus under increased air pressure; Pinheiro R et al.; AIMS: To investigate the effect of total air pressure raise on cell growth and intracellular beta-galactosidase activity in batch cultures of Kluyveromyces marxianus CBS 7894 . METHODS AND RESULTS: A pressurized bioreactor was used for K . marxianus batch cultivation under increased air pressure from 1.2 to 6 bar . Under these conditions no inhibition of cell growth was observed . Moreover, the improvement of the oxygen transfer rate (OTR) from the gas to the culture medium by pressurization led to an enhancement of the cell growth rate obtained at atmospheric pressure without aeration . The specific beta-galactosidase productivity increased from 5.8 to 17.0 U gCD-1 h-1 using a 6-bar air pressure instead of air at atmospheric pressure . The antioxidant enzyme superoxide dismutase (SOD) was slightly induced by the air pressure raise, which indicates that the defensive mechanisms of the cells can cope with an air pressure up to 6 bar . CONCLUSIONS: These experiments showed that the increase of air pressure up to 6 bar is an alternative to other methods of preventing the oxygen limitation and can be applied in the beta-galactosidase production by K . marxianus . SIGNIFICANCE AND IMPACT OF THE STUDY: The results here reported proved that, in what biological aspects are concerned, it is possible to use the air pressure increase as an optimization parameter of beta-galactosidase production in high-density cell cultures of K . marxianus strains.

Biotechnol Lett, 2003 Oct, 25(20), 1769 - 74
Expression and characterization of Kluyveromyces lactis beta-galactosidase in Escherichia coli; Kim CS et al.; Kluyveromyces lactis beta-galactosidase gene, LAC4, was expressed in Escherichia coli as a soluble His-tagged recombinant enzyme under the optimized culture conditions . The expressed protein was multimeric with a subunit molecular mass of 118 kDa . The dimeric form of the beta-galactosidase was the major fraction but had a lower activity than those of the multimeric forms . The purified enzyme required Mn2+ for activity and was inactivated irreversibly by imidazole above 50 mM . The activity was optimal at 37 and 40 degrees C for o-nitrophenyl-beta-D-galactopyranoside (oNPG) and lactose, respectively . The optimum pH value is 7 . The Km and Vmax values of the purified enzyme for oNPG were 1.5 mM and 560 micromol min(-1) mg(-1), and for lactose 20 mM and 570 micromol min(-1) mg(-1), respectively.

Mol Cell Biol, 2003 Dec, 23(23), 8729 - 39
The Rap1p-telomere complex does not determine the replicative capacity of telomerase-deficient yeast; Smolikov S et al.; Telomeres are nucleoprotein structures that cap the ends of chromosomes and thereby protect their stability and integrity . In the presence of telomerase, the enzyme that synthesizes telomeric repeats, telomere length is controlled primarily by Rap1p, the budding yeast telomeric DNA binding protein which, through its C-terminal domain, nucleates a protein complex that limits telomere lengthening . In the absence of telomerase, telomeres shorten with every cell division, and eventually, cells enter replicative senescence . We have set out to identify the telomeric property that determines the replicative capacity of telomerase-deficient budding yeast . We show that in cells deficient for both telomerase and homologous recombination, replicative capacity is dependent on telomere length but not on the binding of Rap1p to the telomeric repeats . Strikingly, inhibition of Rap1p binding or truncation of the C-terminal tail of Rap1p in Kluyveromyces lactis and deletion of the Rap1p-recruited complex in Saccharomyces cerevisiae lead to a dramatic increase in replicative capacity . The study of the role of telomere binding proteins and telomere length on replicative capacity in yeast may have significant implications for our understanding of cellular senescence in higher organisms.

Biomacromolecules, 2003 Nov-Dec, 4(6), 1763 - 72
Fluorescence and infrared spectrometric study of cell walls from Candida, kluyveromyces, Rhodotorula and schizosaccharomyces yeasts in relation with their chemical composition; Bahmed K et al.; Composition, level, and arrangement of the structural polysaccharides determine biophysical properties of fungal cell walls . A small amount of a beta(1-->4) linear homopolymer of GlcNAc in the cell wall forms chitin . To study the components of the cell walls and to estimate the quantity of chitin for different strains, two spectroscopic methods were applied . Because chemical and enzymatic methods are destructive, long, and complex, fluorescence and infrared (IR) spectroscopies were applied on cell walls and on chitin enriched fractions . The results were compared to chemical assays . IR spectra allow identifying the structural types of polysaccharides in yeast walls . Fluorescence spectroscopy was not appropriated for a full and accurate quantitative determination of the polymers but revealed level variations similar to results obtained by chemical analytical methods . The infrared spectra, using a chemometric approach (PLS1), allowed a fairly good estimation of chitin in enriched fractions with respect to the chemical assays.

Curr Genet, 2004 Feb, 45(1), 1 - 8 Epub 2003 Nov 01.
KNQ1, a Kluyveromyces lactis gene encoding a drug efflux permease; Takacova M et al.; Several transport systems play an important role in conferring multiple drug resistance, presumably due to their catalysis of the energy-dependent extrusion of a large number of structurally and functionally unrelated compounds out of the cells . In the present work, the gene named KNQ1 (encoding Kluyveromyces lactis membrane permease) was cloned by functional complementation of the cycloheximide-hypersensitivity phenotype of the Saccharomyces cerevisiae mutant strain lacking a functional PDR5 gene . The isolated gene exhibited 48.9% identity with the S . cerevisiae ATR1 gene conferring resistance to aminotriazole and 4-nitroquinoline- N-oxide and encoded a protein of 553 amino acids . When present in multicopy, it efficiently complemented the phenotype associated with the Delta pdr5 or Delta pdr1Delta pdr3 mutations in S . cerevisiae . Overexpression of the KNQ1 gene in K . lactis wild-type strains led to resistance against several cytotoxic compounds, like 4-nitroquinoline- N-oxide, 3-aminotriazole, bifonazole and ketoconazole . The gene was assigned to K . lactis chromosome III and its expression was found to be responsive to oxidative stress induced by hydrogen peroxide . Based on the phenotype of homologous and heterologous transformants, we propose that the gene encodes a membrane-associated component of the machinery responsible for decreasing the concentration of several toxic compounds in the cytoplasm of yeast cells.

Yeast, 2003 Oct 30, 20(14), 1171 - 5
LYS2 gene and its mutation in Kluyveromyces lactis; Alberti A et al.; The KlLYS2 gene, encoding the alpha-aminoadipate reductase of Kluyveromyces lactis, was isolated by complementation of a lysA1 mutant . The deduced amino acid sequence shared an identity of 73% with the LYS2 product of Saccharomyces cerevisiae . Despite the high sequence homology of the alpha-aminoadipate reductase genes, the two yeast species differently responded to the presence of alpha-aminoadipate in the medium . Wild-type S . cerevisiae is known to be sensitive to alpha-aminoadipate, but becomes resistant when mutated to lys2 . In contrast, K . lactis strains were found to be naturally resistant to alpha-aminoadipate . Therefore, the positive selection procedure for the isolation of lys2 mutants on alpha-aminoadipate, as practised in S . cerevisiae, cannot be applied to K . lactis . A possible reason of this difference may be that the catalytic rate of the alpha-aminoadipate reductase differs in the two yeasts . The EMBL/Genbank Accession No . for the KlLYS2 gene is AJ504405 .

Biol Proced Online, 2003, 5, 108 - 115 Epub 2003 May 1.
Measuring Solution Viscosity and its Effect on Enzyme Activity; Uribe S et al.; In proteins, some processes require conformational changes involving structural domain diffusion . Among these processes are protein folding, unfolding and enzyme catalysis . During catalysis some enzymes undergo large conformational changes as they progress through the catalytic cycle . According to Kramers theory, solvent viscosity results in friction against proteins in solution, and this should result in decreased motion, inhibiting catalysis in motile enzymes . Solution viscosity was increased by adding increasing concentrations of glycerol, sucrose and trehalose, resulting in a decrease in the reaction rate of the H(+)-ATPase from the plasma membrane of Kluyveromyces lactis . A direct correlation was found between viscosity (eta) and the inhibition of the maximum rate of catalysis (V(max)) . The protocol used to measure viscosity by means of a falling ball type viscometer is described, together with the determination of enzyme kinetics and the application of Kramers' equation to evaluate the effect of viscosity on the rate of ATP hydrolysis by the H(+)-ATPase.

Curr Genet, 2004 Jan, 44(6), 305 - 16 Epub 2003 Oct 21.
Isocitrate lyase of the yeast Kluyveromyces lactis is subject to glucose repression but not to catabolite inactivation; Lopez ML et al.; KlICL1, encoding the isocitrate lyase of Kluyveromyces lactis, was isolated by complementation of the Saccharomyces cerevisiae icl1 deletion mutant . Sequence analysis revealed an open reading frame of 1626 nucleotides encoding a protein with 542 amino acids . The deduced protein shows extensive homologies to isocitrate lyases from various organisms, with an overall identity of 69% to the enzyme from S . cerevisiae . The KlICL1 gene has two major transcription start-points, located at -113 bp and -95 bp relative to the ATG translation start codon . The gene is expressed on ethanol medium only in respiratory-competent cells . Transcription is repressed by glucose . Mutants carrying a Klcat8 deletion lack the ability to derepress KlICL1 transcription . A Klicl1 deletion mutant does not grow on ethanol medium and lacks any isocitrate lyase activity . A strain lacking the gene KlFBP1, which encodes the gluconeogenic enzyme fructose 1,6-bisphosphatase, lacks the ability to grow on non-fermentable carbon sources . This implies that K . lactis does not contain additional isoenzymes catalyzing either of the reactions . Enzyme assays revealed that neither KlIcl1p nor KlFbp1p are subject to catabolite inactivation . However, the respective enzymes from S . cerevisiae are efficiently inactivated when expressed in K . lactis . Thus, despite the extensive sequence similarities of the enzymes involved, non-fermentative carbohydrate metabolism in the two yeasts displays distinct regulatory properties.

RNA, 2003 Nov, 9(11), 1323 - 32
A template-proximal RNA paired element contributes to Saccharomyces cerevisiae telomerase activity; Seto AG et al.; The ribonucleoprotein complex telomerase is critical for replenishing chromosome-end sequence during eukaryotic DNA replication . The template for the addition of telomeric repeats is provided by the RNA component of telomerase . However, in budding yeast, little is known about the structure and function of most of the remainder of the telomerase RNA . Here, we report the identification of a paired element located immediately 5' of the template region in the Saccharomyces cerevisiae telomerase RNA . Mutations disrupting or replacing the helical element showed that this structure, but not its exact nucleotide sequence, is important for telomerase function in vivo and in vitro . Biochemical characterization of a paired element mutant showed that the mutant generated longer products and incorporated noncognate nucleotides . Sequencing of in vivo synthesized telomeres from this mutant showed that DNA synthesis proceeded beyond the normal template . Thus, the S . cerevisiae element resembles a similar element found in Kluyveromyces budding yeasts with respect to a function in template boundary specification . In addition, the in vitro activity of the paired element mutant indicates that the RNA element has additional functions in enzyme processivity and in directing template usage by telomerase.

J Mol Biol, 2003 Oct 24, 333(3), 479 - 92
Sites for interaction between Gal80p and Gal1p in Kluyveromyces lactis: structural model of galactokinase based on homology to the GHMP protein family; Menezes RA et al.; The induction of transcription of the galactose genes in yeast involves the galactose-dependent binding of ScGal3p (in Saccharomyces cerevisiae) or KlGal1p (in Kluyveromyces lactis) to Gal80p . This binding abrogates Gal80's inhibitory effect on the activation domain of Gal4p, which can then activate transcription . Here, we describe the isolation and characterization of new interaction mutants of K.lactis GAL1 and GAL80 using a two-hybrid screen . We present the first structural model for Gal1p to be based on the published crystal structures of other proteins belonging to the GHMP (galactokinase, homoserine kinase, mevalonate kinase and phosphomevalonate kinase) kinase family and our own X-ray diffraction data of Gal1p crystals at 3A resolution . The locations of the various mutations in the modelled Gal1p structure identify domains involved in the interaction with Gal80p and provide a structural explanation for the phenotype of constitutive GAL1 mutations.

Eukaryot Cell, 2003 Oct, 2(5), 1115 - 27
Factors influencing the recombinational expansion and spread of telomeric tandem arrays in Kluyveromyces lactis; Natarajan S et al.; We have previously shown that DNA circles containing telomeric repeats and a marker gene can promote the recombinational elongation of telomeres in Kluyveromyces lactis by a mechanism proposed to involve rolling-circle DNA synthesis . Wild-type cells acquire a long tandem array at a single telomere, while telomerase deletion (ter1-delta) cells, acquire an array and also spread it to multiple telomeres . In this study, we further examine the factors that affect the formation and spread of telomeric tandem arrays . We show that a telomerase(+) strain with short telomeres and high levels of subtelomeric gene conversion can efficiently form and spread arrays, while a telomere fusion mutant is not efficient at either process . This indicates that an elevated level of gene conversion near telomeres is required for spreading but that growth senescence and a tendency to elongate telomeres in the absence of exogenously added circles are not . Surprisingly, telomeric repeats are frequently deleted from a transforming URA3-telomere circle at or prior to the time of array formation by a mechanism dependent upon the presence of subtelomeric DNA in the circle . We further show that in a ter1-delta strain, long tandem arrays can arise from telomeres initially containing a single-copy insert of the URA3-telomere sequence . However, the reduced rate of array formation in such strains suggests that single-copy inserts are not typical intermediates in arrays formed from URA3-telomere circles . Using heteroduplex circles, we have demonstrated that either strand of a URA3-telomere circle can be utilized to form telomeric tandem arrays . Consistent with this, we demonstrate that 100-nucleotide single-stranded telomeric circles of either strand can promote recombinational telomere elongation.

FEMS Yeast Res, 2003 Oct, 4(1), 29 - 35
A Kluyveromyces lactis mutant in the essential gene KlLSM4 shows phenotypic markers of apoptosis; Mazzoni C et al.; We report the study of Kluyveromyces lactis cells expressing a truncated form of KlLSM4, a gene ortholog to LSM4 of Saccharomyces cerevisiae which encodes an essential protein involved in both pre-mRNA splicing and mRNA decapping . We had previously demonstrated that the first 72 amino acids of the K . lactis Lsm4p (KlLsm4Deltap) can restore cell growth in both K . lactis and S . cerevisiae cells not expressing the endogenous protein . However, cells showed a remarkable loss of viability in stationary phase . Here we report that cells expressing KlLsm4Deltap presented clear apoptotic markers such as chromatin condensation, DNA fragmentation, accumulation of reactive oxygen species, and showed increased sensitivity to different drugs . RNA analysis revealed that pre-mRNA splicing was almost normal while mRNA degradation was significantly delayed, pointing to this as the possible step responsible for the observed phenotypes.

J Biochem Mol Biol, 2003 Sep 30, 36(5), 442 - 9
Molecular cloning and characterization of an NADPH quinone oxidoreductase from Kluyveromyces marxianus; Kim WH et al.; NAD(P)H quinone oxidoreductase is a ubiquitous enzyme that is known to directly reduce quinone substrates to hydroquinones by a two-electron reaction . We report the identification of NADPH quinone oxidoreductase from Kluyveromyces marxianus (KmQOR), which reduces quinone substrates directly to hydroquinones . The KmQOR gene was sequenced, expressed in Escherichia coli, purified, and characterized . The open-reading frame of the KmQOR gene consists of 1143 nucleotides, encoding a 380 amino acid polypeptide . The nucleotide sequence of the KmQOR gene was assigned to EMBL under accession number AY040868 . The M(r) that was determined by SDS-PAGE for the protein subunit was about 42 kDa, and the molecular mass of the native KmQOR was 84 kDa, as determined by column calibration, indicating that the native protein is a homodimer . The KmQOR protein efficiently reduced 1,4-benzoquinone, whereas no activities were found for menadiones and methoxyquinones . These observations, and the result of an extended sequence analysis of known NADPH quinone oxidoreductase, suggest that KmQOR possesses a different action mechanism.

Curr Genet, 2003 Dec, 44(5), 268 - 76 Epub 2003 Oct 03.
Cloning and biochemical characterization of hexokinase from the methylotrophic yeast Hansenula polymorpha; Karp H et al.; We previously showed that, unlike other yeasts, Hansenula polymorpha possesses a glucokinase HPGLK1 that can mediate glucose repression in this yeast, although it cannot replace the regulatory function of hexokinase 2 in Saccharomyces cerevisiae . In the present study, the H . polymorpha hexokinase gene HPHXK1 was cloned by complementation of the glucose growth deficiency of the H . polymorpha double kinase-negative mutant A31-10 with a genomic library . The sequence of the 483-amino acid hexokinase protein deduced from the HPHXK1 gene showed the highest degree of identity (56%) with hexokinase from Schwanniomyces occidentalis, whereas the identity with hexokinase from Kluyveromyces lactis and both hexokinases from Sac . cerevisiae was 55% . The hexokinase protein was purified from crude extracts of H . polymorpha, using ion exchange chromatography and gel filtration . The K(m) values of the purified enzyme for glucose, fructose and ATP were 0.26 mM, 1.1 mM and 0.32 mM, respectively . H . polymorpha hexokinase was inhibited by trehalose-6-phosphate ( K(i)=12 microM) and ADP ( K(i)=1.6 mM), but not by glucose-6-phosphate . Transformation of a H . polymorpha hexokinase-negative mutant with a plasmid carrying the HPHXK1 gene restored the ability of the mutant to phosphorylate fructose and to repress the synthesis of alcohol oxidase and catalase by fructose . Therefore, hexokinase is specifically needed for the establishment of fructose repression in H . polymorpha.

J Microbiol Methods, 2003 Nov, 55(2), 433 - 40
Acquisition of flocculation phenotype by Kluyveromyces marxianus when overexpressing GAP1 gene encoding an isoform of glyceraldehyde-3-phosphate dehydrogenase; Almeida C et al.; The use of flocculating yeast strains has been considered as a convenient approach to obtain high cell densities in bioreactors with increasing productivity in continuous operations . In Kluyveromyces marxianus ATTC 10022, the GAP1 gene encodes an isoform of glyceraldehyde-3-phosphate dehydrogenase-p37-that is accumulated in the cell wall and is involved in flocculation . To test the use of p37 as a tool for engineering Kluyveromyces cells to display a flocculation phenotype, K . marxianus CCT 3172 was transformed with an expression vector containing GAP1 . This vector is based on the pY37 previously described, harbouring a S11 Kluyveromyces origin of replication, and the expression of GAP1 is under the control of GAL1 . Kluyveromyces cells overexpressing GAP1 acquired a flocculent phenotype together with the accumulation of p37 in the cell wall . The results support the use of GAP1 gene as a molecular tool for inducing flocculation.

Arch Latinoam Nutr, 2003 Jun, 53(2), 194 - 201
{Production and partial characterization of beta-galactosidase from Kluyveromyces lactis grown in deproteinized whey}; Ramirez Matheus AO et al.; The purpose of this work was to optimize the beta-galactosidase production by Kluyveromyces lactis, applying the Surface Response Methodology (SRM) and using deproteinized whey as fermentation medium . An Orthogonal Central Compound Design (OCCD) was used without repetition, with four factors: temperature, pH, agitation speed and fermentation time . Then, enzyme activity (U/ml) as response variable was used . Thirty trials in twenty-five treatments, with six repetitions at the central point, were carried out, in a New Brunswick Bioflo 2000 fermentor with a volume of 2 liters . The deproteinized whey obtained by thermocoagulation was chemically analyzed . The results were: moisture 93.83%, total solids 6.17%, protein 0.44%, lactose 4.85%, acidity 0.43% and pH 4.58 . The best conditions in the enzyme production were: temperature 30.3 degrees C, pH 4.68, agitation speed 191 r.p.m . and fermentation time 18.5 h . with an enzyme production of 8.3 U/ml . The degree of purification obtained was 7.4 times and the yield was 50.8% . The purified enzyme had an optimum temperature of 60 degrees C and a pH of 6.2 . This work shows that the yeast Kluyveromyces lactis grown in deproteinized whey is able to produce the enzyme beta-galactosidase and SRM can be used in the fermentology processes, specifically in determining the best suitable operation conditions.

Mikrobiologiia, 2003 Jul-Aug, 72(4), 466 - 9
{The activity of xylose reductase and xylitol dehydrogenase in yeasts}; Iablochkova EN et al.; The activity and the cofactor specificity of xylose reductase and xylitol dehydrogenase were studied in extracts of yeasts from the genera Candida, Kluyveromyces, Pachysolen, Pichia, and Torulopsis grown under microaerobic conditions . It was found that xylitol dehydrogenase in all of the yeast species studied is specific for NAD+; xylose reductase in the xylitol-producing species C . didensiae, C . intermediae, C . parapsilosis, C . silvanorum, C . tropicalis, Kl . fragilis, Kl . marxianus, P . guillermondii, and T . molishiama is specific for NADPH; and xylose reductase in the ethanol-producing species P . stipitis, C . shehatae, and Pa . tannophilus is specific for both NADPH and NADH.

Biotechnol Lett, 2003 Sep, 25(17), 1403 - 6
Production of a thermostable extracellular lipase by Kluyveromyces marxianus; Deive FJ et al.; Kluyveromyces marxianus was grown in submerged culture in a complex medium with several potential inducers of lipolytic activity (triacylglycerols, fatty acids) . The highest extracellular lipolytic enzyme production (about 80 U ml(-1) in 3 d) was obtained when the medium was supplemented with 2 g urea l(-1) plus 5 g tributyrin l(-1) . Addition of surfactants (1 g l(-1)) did not improve production . The lipase had a high thermal stability in aqueous solution (73% residual activity after 9 d at 50 degrees C, 16 min half-life time at 100 degrees C) . It was also stable at acidic pH and showed good tolerance to organic solvents (70% residual activity after 2 d in n-hexane of cyclohexane).

J Dairy Sci, 2003 Sep, 86(9), 2783 - 9
Configuration of a bioreactor for milk lactose hydrolysis; Genari AN et al.; Permeabilized microbial cells can be used as a crude enzyme preparation for industrial applications . Immobilization and process recycling can compensate for the low specific activity of this preparation . For biomass immobilization, the common support is alginate beads; however, its low surface area and the low biomass concentration limit the activity . We here describe a biocatalyst consisting of a paste of permeabilized Kluyveromyces lactis cells gelled with manganese alginate over a semicircular stainless steel screen . A ratio of wet permeabilized biomass to alginate of 50:4 (wt/wt) resulted in a paste with maximum immobilized beta-galactosidase activity and maximum gel biomass retention . The biocatalysts retained activity better when stored in milk at 4 degrees C than in 50% glycerol . The unused biocatalysts stored in milk did not lose activity after 50 d . However, repeated use of the same biocatalyst 40 times resulted in almost 50% loss of activity . A bioreactor design with two different conditions of operation were tested for milk lactose hydrolysis using this biocatalyst . The bioreactor was operated at 40 degrees C as packed bed or with recirculation, similar to a continuous stirred tank reactor . The continuous system with recirculation resulted in 82.9% lactose hydrolysis at a residence time of 285.5 min (flow of 2.0 ml/min), indicating the potential of this system for processing low lactose milk, or even in processing other substrates, using an appropriate biocatalyst.

Mol Genet Genomics, 2003 Nov, 270(2), 190 - 9 Epub 2003 Sep 09.
Structural and functional analysis of the killer element pPin1-3 from Pichia inositovora; Klassen R et al.; Strains of the yeast Pichia inositovora that carry the linear plasmids pPin1-1 (18 kb) and pPin1-3 (10 kb) display a killer activity towards Saccharomyces cerevisiae . Cloning and sequencing of the smaller plasmid, pPin1-3, revealed that it is 9683 bp long and has 154-bp terminal inverted repeats . Comparison of pPin1-3 with the only other completely sequenced killer plasmid, pGKL1 of Kluyveromyces lactis, revealed differences in genome organization . The Pichia element has four ORFs that account for 95% of the sequence . ORF1 is homologous to the putative immunity gene of the K . lactis system . A viral B-type DNA polymerase is encoded by ORF2 . The predicted product of ORF3 displays similarities to the alpha- and beta-subunits of the heterotrimeric K . lactis killer toxin, also known as zymocin . A cysteine-rich chitin-binding site and a chitinase signature, characteristic for the alpha-subunit of zymocin were identified in Orf3p . Chitin affinity chromatography and Western analysis confirmed the plasmid specific expression and secretion of a protein that cross-reacts with an antibody raised against the alpha-subunit of K . lactis zymocin . Disruption of the major chitin synthase-gene ( CHS3) renders S . cerevisiae resistant to the toxin, providing further evidence that chitin is the cellular receptor for the P . inositovora toxin . Orf4p of pPin1-3 displays only weak similarities to the gamma-subunit of zymocin, which causes a G1 cell-cycle arrest in S . cerevisiae . However, disruption of the S . cerevisiae gene ELP3/TOT3, which encodes a histone-acetyltransferase that is essential for zymocin action, resulted in reduced sensitivity to the P . inositovora toxin also . Thus, despite obvious differences in genome organization and protein architecture, both killer systems very probably have similar modes of action.

Mol Microbiol, 2003 Sep, 49(5), 1297 - 307
Elongator's toxin-target (TOT) function is nuclear localization sequence dependent and suppressed by post-translational modification; Fichtner L et al.; The toxin target (TOT) function of the Saccharomyces cerevisiae Elongator complex enables Kluyveromyces lactis zymocin to induce a G1 cell cycle arrest . Loss of a ubiquitin-related system (URM1-UBA4 ) and KTI11 enhances post-translational modification/proteolysis of Elongator subunit Tot1p (Elp1p) and abrogates its TOT function . Using TAP tagging, Kti11p contacts Elongator and translational proteins (Rps7Ap, Rps19Ap Eft2p, Yil103wp, Dph2p) . Loss of YIL103w and DPH2 (involved in diphtheria toxicity) suppresses zymocicity implying that both toxins overlap in a manner mediated by Kti11p . Among the pool that co-fractionates with RNA polymerase II (pol II) and nucleolin, Nop1p, unmodified Tot1p dominates . Thus, modification/proteolysis may affect association of Elongator with pol II or its localization . Consistently, an Elongator-nuclear localization sequence (NLS) targets green fluorescent protein (GFP) to the nucleus, and its truncation yields TOT deficiency . Similarly, KAP120 deletion rescues cells from zymocin, suggesting that Elongator's TOT function requires NLS- and karyopherin-dependent nuclear import.

Mol Biol Cell, 2003 Aug, 14(8), 3449 - 58 Epub 2003 May 18.
Conservation of the prion properties of Ure2p through evolution; Baudin-Baillieu A et al.; The yeast inheritable {URE3} element corresponds to a prion form of the nitrogen catabolism regulator Ure2p . We have isolated several orthologous URE2 genes in different yeast species: Saccharomyces paradoxus, S . uvarum, Kluyveromyces lactis, Candida albicans, and Schizosaccharomyces pombe . We show here by in silico analysis that the GST-like functional domain and the prion domain of the Ure2 proteins have diverged separately, the functional domain being more conserved through the evolution . The more extreme situation is found in the two S . pombe genes, in which the prion domain is absent . The functional analysis demonstrates that all the homologous genes except for the two S . pombe genes are able to complement the URE2 gene deletion in a S . cerevisiae strain . We show that in the two most closely related yeast species to S . cerevisiae, i.e., S . paradoxus and S . uvarum, the prion domains of the proteins have retained the capability to induce {URE3} in a S . cerevisiae strain . However, only the S . uvarum full-length Ure2p is able to behave as a prion . We also show that the prion inactivation mechanisms can be cross-transmitted between the S . cerevisiae and S . uvarum prions.

Appl Biochem Biotechnol, 2003 Jul, 110(1), 23 - 32
Solid-phase reducing agents as alternative for reducing disulfide bonds in proteins; Grazu V et al.; Disulfide reduction of Kluyveromyces lactis and Aspergillus oryzae beta-galactosidases and beta-lactoglobulin was assessed . Reduction was performed using one of two thiol-containing agents: dithiothreitol (DTT) or thiopropyl-agarose with a high degree of substitution (1000 micromol of SH groups/g of dried gel) . Both reductants allowed an increase of three- (for K . lactis beta-galactosidase) and fourfold (for A . oryzae beta-galactosidase) in the initial content of SH groups in the lactases . Nearly sevenfold fewer micromoles of SH groups per milligram of protein were needed to perform the reduction of K . lactis beta-galactosidase with thiopropyl-agarose than for the same reduction with DTT . However, for A . oryzae beta-galactosidase, nearly twice as many micromoles of SH groups per milligram of protein were needed with thiopropylagarose than with DTT . Disulfide bonds in beta-lactoglobulin were not accessible to thiopropyl-agarose, since this reduction was only possible in the presence of 6 M urea . These results proved that highly substituted thiopropyl-agarose is as good a reducing agent as DTT, for the reduction of disulfide bonds in proteins . Moreover, excess reducing agent was very simply separated from the reduced protein by filtration, making it easier to control the reaction and providing reduced protein solutions free of reductant . All these advantages substantially cut down the time required and therefore the cost of the overall process.

Int Microbiol, 2003 Sep, 6(3), 201 - 5 Epub 2003 Jul 30.
Molecular evolution in yeast of biotechnological interest; Querol A et al.; The importance of yeast in the food and beverage industries was only realized about 1860, when the role of these organisms in food manufacture became evident . Since they grow on a wide range of substrates and can tolerate extreme physicochemical conditions, yeasts, especially the genera Saccharomyces and Kluyveromyces, have been applied to many industrial processes, Industrial strains of these genera are highly specialized organisms that have evolved to utilize a range of environments and ecological niches to their full potential . This adaptation is called "domestication" . This review describes the phylogenetic relationships among Saccharomyces and Kluyveromyces species and the different mechanisms involved in the adaptive evolution of industrial yeast strains.

Arch Microbiol, 2003 Oct, 180(4), 257 - 63 Epub 2003 Jul 31.
Characterization of early deaths of non- Saccharomyces yeasts in mixed cultures with Saccharomyces cerevisiae; Nissen P et al.; The survival of Kluyveromyces thermotolerans and Torulaspora delbrueckii in mixed cultures with Saccharomyces cerevisiae was examined at low oxygen availability in a defined grape juice medium . In these fermentations, K . thermotolerans and T . delbrueckii died off earlier than S . cerevisiae, and K . thermotolerans and T . delbrueckii exhibited parabolic death kinetics . Furthermore, the early deaths seemed to be non-apoptotic in nature . In order to understand the mechanism causing the early deaths, various single- and mixed-culture fermentations were carried out . The early deaths could not be explained by nutrient depletion or the presence of toxic compounds . Rather, they seemed to be mediated by a cell-to-cell contact mechanism at high cell densities of S . cerevisiae, and to a lesser ability of K . thermotolerans and T . delbrueckii to compete for space, as compared to S . cerevisiae . These results contribute to an increased understanding of why K . thermotolerans and T . delbrueckii die off before S . cerevisiae in wine fermentations.

Int J Food Microbiol, 2003 Sep 1, 86(1-2), 123 - 30
Characterization of yeast isolates originating from Hungarian dairy products using traditional and molecular identification techniques; Vasdinyei R et al.; Yeast isolates collected from various Hungarian dairy products were identified using simplified identification system (SIM) and restriction fragment analysis of PCR-amplified 18S rDNA with the neighbouring ITS1 region (ITS-PCR; ribotyping) . The isolates were grouped into 26 species; Geotrichum candidum, Debaryomyces hansenii, Yarrowia lipolytica, Kluyveromyces lactis and Candida catenulata were found as the predominant species . SIM and ITS-PCR proved to be useful and convenient taxonomic tools for rapid identification at species level . Two most frequent species, G . candidum and D . hansenii, were further characterized by randomly amplified polymorphic DNA (RAPD-PCR) analysis . RAPD-PCR using M13 primer resulted in discrimination between most strains of the same species and allowed a certain degree of intraspecific typing.

J Biol Chem, 2003 Oct 10, 278(41), 39280 - 6 Epub 2003 Jul 25.
The unique hexokinase of Kluyveromyces lactis . Molecular and functional characterization and evaluation of a role in glucose signaling; Bar D et al.; The Crabtree-negative yeast Kluyveromyces lactis is capable of adjusting its glycolytic flux to the requirements of respiration by tightly regulating glucose uptake . RAG5 encoding the only glucose and fructose phosphorylating enzyme present in K . lactis is required for the up-regulation of glucose transport and also for glucose repression . To understand the significance of the molecular identity and specific function(s) of the corresponding kinase to glucose signaling, RAG5 was overexpressed and its gene product KlHxk1 (Rag5p) isolated and characterized . Stopped-flow kinetics and sedimentation analysis indicated a monomer-homodimer equilibrium of KlHxk1 in a condition of catalysis, i.e . in the presence of substrates and products . The kinetic constants of ATP-dependent glucose phosphorylation identified a 53-kDa monomer as the high affinity/high activity form of the novel enzyme for both glycolytic substrates suggesting a control of glucose phosphorylation at the level of dimer formation and dissociation . In contrast to the highly homologous hexokinase isoenzyme 2 of Saccharomyces cerevisiae (ScHxk2), KlHxk1 was not inhibited by free ATP in a physiological range of nucleotide concentration . Mass spectrometric sequencing of tryptic peptides of KlHxk1 identified unmodified serine at amino acid position 156 . The corresponding amino acid in ScHxk2 is serine 157, which represents the autophosphorylation-inactivation site . KlHxk1 did not display, however, the typical pattern of inactivation under the respective in vitro conditions and maintained a high residual glucose phosphorylating activity . The biophysical and functional data are discussed with respect to a possible regulatory role of KlHxk1 in glucose metabolism and signaling in K . lactis.

Biotechnol Lett, 2003 Apr, 25(8), 623 - 9
Microwave-assisted synthesis of galacto-oligosaccharides from lactose with immobilized beta-galactosidase from Kluyveromyces lactis; Maugard T et al.; Galacto-oligosaccharides (GOS) were synthesized from lactose by immobilized and free beta-galactosidase from Kluyveromyces lactis (Lactozym 3000 L HP-G) using either focused microwave irradiation or conventional heating . Immobilization of the beta-galactosidase on to Duolite A-568 increased the synthesis of GOS . GOS selectivity (GOS synthesis/lactose hydrolysis ratio) increased when the water activity of the media was reduced, notably with a high initial lactose concentration but also by using co-solvents in the media . The advantage of microwave heating on GOS formation was also examined . Addition of solvent and carrying out the reaction under microwave irradiation resulted an increase in the production of GOS . The selectivity for GOS synthesis can be increased by 217-fold under microwave irradiation, using immobilized beta-glucosidase and with added co-solvents such as hexanol.

Biotechnol Lett, 2003 Apr, 25(7), 531 - 6
Screening of yeasts for the production of the aroma compound 2-phenylethanol in a molasses-based medium; Etschmann MM et al.; Fourteen yeast strains were screened for production of 2-phenylethanol from L-phenylalanine with molasses as carbon source . Up to 1 g 2-phenylethanol l-1 was obtained . Using oleyl alcohol as a second phase for in situ product removal to enhance the production of 2-phenylethanol increased the yield to about 3 g 2-phenylethanol l-1 at 35 degrees C . The most productive strains were Kluyveromyces marxianus CBS 600 and CBS 397.

J Gen Appl Microbiol, 2003 Apr, 49(2), 85 - 93
Isolation of the mitochondrial nucleoids from yeast Kluyveromyces lactis and analyses of the nucleoid proteins; Miyakawa I et al.; Mitochondrial (mt) nucleoids were isolated from yeast Kluyveromyces lactis with morphological intactness . SDS-polyacrylamide gel electrophoresis (SDS-PAGE) revealed more than 20 proteins that are associated with the mt-nucleoids . However, the protein profile of the mt-nucleoids of K . lactis was significantly different from that of the mt-nucleoid proteins from Saccharomyces cerevisiae . SDS-DNA PAGE, which detected an Abf2p, a major mitochondrial DNA-binding protein, among the mt-nucleoid proteins of S . cerevisiae on a gel, detected only a 17-kDa protein in the K . lactis mt-nucleoid proteins . The 17-kDa protein was purified as homogeneous from the mt-nucleoids by a combination of acid extraction, hydroxyapatite chromatography and DNA-cellulose chromatography . The 17-kDa protein introduced a negative supercoil into circular plasmid DNA in the presence of topoisomerase I, as does S . cerevisiae Abf2p, and it packed K . lactis mtDNA into nucleoid-like particles in vitro . These results, together with the determination of the N-terminal amino acid sequence, suggested that the 17-kDa protein is an Abf2p homologue of K . lactis and plays structural roles in compacting mtDNA in cooperation with other nucleoid proteins.

Genes Dev, 2003 Jul 15, 17(14), 1779 - 88 Epub 2003 Jun 27.
A novel pseudoknot element is essential for the action of a yeast telomerase; Tzfati Y et al.; Telomerase contains an essential RNA, which includes the template sequence copied by the reverse transcription action of telomerase into telomeric DNA . Using phylogenetic comparison, we identified seven conserved sequences in telomerase RNAs from Kluyveromyces budding yeasts . We show that two of these sequences, CS3 and CS4, are essential for normal telomerase function and can base-pair to form a putative long-range pseudoknot . Disrupting this base-pairing was deleterious to cell growth, telomere maintenance, and telomerase activity . Restoration of the base-pairing potential alleviated these phenotypes . Mutating this pseudoknot caused a novel mode of shifting of the boundaries of the RNA template sequence copied by telomerase . A phylogenetically derived model of yeast TER structure indicates that these RNAs can form two alternative predicted core conformations of similar stability: one brings the CS3/CS4 pseudoknot spatially close to the template; in the other, CS3 and CS4 move apart and the conformation of the template is altered . We propose that such disruption of the pseudoknot, and potentially the predicted telomerase RNA conformation, affects polymerization to cause the observed shifts in template usage.

Mycol Res, 2003 Apr, 107(Pt 4), 401 - 7
Efficient isolation of genes differentially expressed on cellulose by suppression subtractive hybridization in Agaricus bisporus; Morales P et al.; The production of cellulases on minimal medium in the edible mushroom Agaricus bisporus is regulated by the carbon source: induced by cellulose and repressed by glucose . In order to isolate cellulose-growth specific sequences, a cDNA library from A . bisporus using suppression subtractive hybridization (SSH) was constructed . Northern blot analysis indicated that a high level of enrichment was achieved; 183 clones were isolated . A preliminary screen with cellulose-specific genes of A . bisporus (cel1, cel2, cel3 and cel4) using Southern hybridization resulted in 28 clones to be cel3, and 5 clones were cel2 . The remaining 144 clones were sequenced . Partial sequences of the following genes were found: a beta-glucosidase homologue of the blvk gene of Kluyveromyces marxianus; a cellulase homologue of an endoglucanase (avicellase III) of Aspergillus aculeatus, four different xylanases homologue of the xyn genes of different fungi, and one hexose transporter homologue to the hxtA gene of Aspergillus parasiticus . The apparent full-length of two hydrophobins homologue to the abh3 gene of A . bisporus and one histone homologue to the h2a gene of Aspergillus niger were also found . The remaining sequences did not have homology to any known genes.

Int J Food Microbiol, 2003 Aug 15, 85(1-2), 87 - 100
Optimising growth conditions for the pectinolytic activity of Kluyveromyces wickerhamii by using response surface methodology; Moyo S et al.; This present study was undertaken to find optimum conditions of pH, temperature and, period of incubation for the pectinolytic activity of Kluyveromyces wickerhamii isolated from rotting fruits and to assess the effect of these factors by use of response surface methodology (RSM) . A central composite rotatable design was used as an experimental design for the analysis of the allocation of treatment combinations . A second order polynomial regression model was fitted and was found adequate, with an R(2) of 0.94469 (P<0.001) . The effects of temperature and pH were the most significant factors in influencing enzyme production . Estimated optimum conditions were as follows: pH 5.0, temperature, 32 degrees C and an incubation period of 91 h . Pectinesterase (PE), pectin lyase (PL), and cellulase activities were not detected . Pectinase production was partially constitutive . Pectin was degraded by the isolated strain of K . wickerhamii in the current study, and the pectinolytic activity is referred to as polygalacturonase (PG) activity . Crude enzyme extract was thermostable at various temperatures and, stimulated by the presence of Ca(2+) ions but inhibited by other ions like Mg(2+), Zn(2+), Co(2+), Mn(2+) and Na(+).

Appl Microbiol Biotechnol, 2004 Jan, 63(4), 418 - 21 Epub 2003 Jun 12.
Development of a bisphenol A-adsorbing yeast by surface display of the Kluyveromyces yellow enzyme on Pichia pastoris; Mergler M et al.; A novel surface-engineered strain of yeast Pichia pastoris was constructed that displays at its surface Kluyveromyces lactis Yellow Enzyme (KYE) fused to the C-terminal half of Saccharomyces cerevisiae alpha-agglutinin . The expression of the fusion protein was controlled by the AOX1-promoter . The new strain showed an increased sorption of the xenoestrogen Bisphenol A (BPA) . It was shown that sorption of BPA depended on the presence of methanol in the growth medium and on the pH of the binding assays . The binding kinetics were typical for binding at a surface . The present results demonstrate that the alpha-agglutinin surface display system can be used in the yeast P . pastoris.

Appl Microbiol Biotechnol, 2003 Oct, 62(5-6), 523 - 7 Epub 2003 May 21.
Sequential cloned gene integration in the yeast Kluyveromyces lactis; Wang YC et al.; Two integrating vectors developed for use in Saccharomyces cerevisiae were successfully employed for cloned gene integration in the yeast Kluyveromyces lactis . A delta-integrating vector carrying the dominant selection marker neo allowed tandem integrations of a CUP1p-lacZ cassette into one or two chromosomal sites . A delta/UB-integrating vector, which contains a reusable selection cassette, enabled multiple rounds of integration and the sequential insertion of stable, dispersed copies of CUP1p-lacZ . Subsequent gene expression was closely correlated with integrated copy number illustrating the promise of this method for metabolic engineering in K . lactis . While both vectors contain an S . cerevisiae delta target sequence, the presence of delta-like elements in K . lactis has not been confirmed . Given the degree of illegitimate recombination in this yeast species, the insertions likely occurred at random locations in the chromosomes.

Mol Genet Genomics, 2003 May, 269(2), 188 - 96 Epub 2003 Feb 13.
Mutant casein kinase I (Hrr25p/Kti14p) abrogates the G1 cell cycle arrest induced by Kluyveromyces lactiszymocin in budding yeast; Mehlgarten C et al.; Zymocin, a toxic protein complex produced by Kluyveromyces lactis, inhibits cell cycle progression in Saccharomyces cerevisiae . In studying its action, a resistant mutant ( kti14-1) was found to express the tot-phenotype typical of totDelta cells, toxin target (TOT) mutants that are impaired in RNA polymerase II Elongator function . Phenotypic analysis of a kti14-1 tot3Delta double mutant revealed a functional link between KTI14 and TOT/Elongator . Unlike totDelta cells, the kti14-1 mutant is sensitive to the drug methylmethane sulfonate (MMS), indicating that, besides being affected in TOT function, kti14-1 cells are also compromised in DNA repair . Single-copy complementation identified HRR25, which codes for casein kinase I (CKI), as KTI14 . Kinase-minus hrr25 mutations (K38A and T176I) conferred zymocin resistance, while deletion of the other yeast CKI genes ( YCK1-3) had no effect . A mutation in KTI14 that truncates the P/Q-rich C-terminus of Hrr25p also dissociates MMS sensitivity from zymocin resistance; this mutant is resistant to the toxin, but shows normal sensitivity to MMS . Thus, although kinase-minus mutations are sufficient to protect yeast cells from zymocin, toxicity is also dependent on the integrity of the C-terminal region of Hrr25p, which has been implicated in determining the substrate specificity or localization of Hrr25p.

Prikl Biokhim Mikrobiol, 2003 May-Jun, 39(3), 302 - 6
{Specific features of fermentation of D-xylose and D-glucose by xylose-assimilating yeasts}; Iablochkova EN et al.; The ability to assimilate D-glucose and D-xylose was studied in 21 yeast species of the following genera: Candida, Kluyveromyces, Pachysolen, Pichia, and Torulopsis . All the cultures fermented D-glucose with the formation of ethanol . During the assimilation of D-xylose, ethanol was produced by P . stipitis and C . shehatae, whereas xylitol was produced by C . didensiae, C . intermediae, C . parapsilosis, C . silvanorum, C . tropicalis, K . fragilis, K . marxianus, P . guillermondii, and T . molishiama . The yeast P . tannophilus produced comparable amounts of both alcohols . The possible use of xylose-assimilating yeasts for the production of xylitol and ethanol is discussed.

FEMS Yeast Res, 2003 Jun, 3(4), 417 - 32
Phylogenetic relationships among yeasts of the 'Saccharomyces complex' determined from multigene sequence analyses; Kurtzman CP et al.; Species of Saccharomyces, Arxiozyma, Eremothecium, Hanseniaspora (anamorph Kloeckera), Kazachstania, Kluyveromyces, Pachytichospora, Saccharomycodes, Tetrapisispora, Torulaspora, and Zygosaccharomyces, as well as three related anamorphic species assigned to Candida (C . castellii, C . glabrata, C . humilis), were phylogenetically analyzed from divergence in genes of the rDNA repeat (18S, 26S, ITS), single copy nuclear genes (translation elongation factor 1alpha, actin-1, RNA polymerase II) and mitochondrially encoded genes (small-subunit rDNA, cytochrome oxidase II) . Single-gene phylogenies were congruent for well-supported terminal lineages but deeper branches were not well resolved . Analysis of combined gene sequences resolved the 75 species compared into 14 clades, many of which differ from currently circumscribed genera.

FEMS Yeast Res, 2003 Jun, 3(4), 327 - 31
The Kluyver effect revisited; Fukuhara H; Yeast species can grow on various sugars . However, in many cases the growth on certain sugars (especially oligosaccharides) occurs only under aerobic conditions, and not in anaerobiosis or in the absence of respiration . Fermentation is blocked under these conditions . This apparent dependence of sugar utilization on the respiration has been called Kluyver effect, and such 'respiration-dependent' species are called Kluyver effect positive . A yeast may be Kluyver effect positive for some sugars and not for others . The physiological meaning and the molecular basis of the phenomenon are not clear . It has recently been reported that Kluyveromyces lactis, which is Kluyver effect positive for galactose and a few other sugars, could be converted into a Kluyver effect-negative form by introduction of relevant sugar transporter genes . Such results offer for the first time a direct support to the hypothesis that the immediate cause of the Kluyver effect may be the low level of sugar transporter activities which is not sufficient to sustain the high substrate flow necessary for fermentative growth, whereas the energy-efficient respiratory growth does not require a high rate of sugar uptake . We examined to what extent this sugar transporter theory of the Kluyver effect can be generalized.

Yeast, 2003 May, 20(7), 611 - 24
KlROM2 encodes an essential GEF homologue in Kluyveromyces lactis; Lorberg A et al.; Cellular integrity in yeasts is ensured by a rigid cell wall whose synthesis is controlled by a MAP kinase signal transduction cascade . In Saccharomyces cerevisiae upstream regulatory components of this MAP kinase pathway involve a single protein kinase C, which is regulated in part by interaction with the small GTPase Rho1p . This small G protein is in turn rendered inactive (GDP-bound) or is activated (GTP-bound) by the influence of GTPase activating proteins (GAPs) and the GDP/GTP exchange factors (GEFs), respectively . We report here on the isolation of a gene from Kluyveromyces lactis, KlROM2, which encodes a member of the latter protein family . The nucleotide sequence contains an open reading frame of 1227 amino acids, with an overall identity of 57% to the Rom2 protein of S . cerevisiae . Four conserved sequence motifs could be identified: a RhoGEF domain, a DEP sequence, a CNH domain and a less conserved pleckstrin homology (PH) sequence . Klrom2 null mutants show a lethal phenotype, which indicates that the gene may encode the only functional GEF regulating the cellular integrity pathway in K . lactis . Conditional genomic expression of KlROM2 resulted in sensitivity towards caffeine and Calcofluor white as typical phenotypes of mutants defective in this pathway . Overexpression of KIROM2 from multicopy plasmids under the control of the ScGAL1 promoter severely impaired growth in both S . cerevisiae and in K . lactis . The fact that the lethal phenotype was not prevented in mpk1 deletion mutants indicates that growth inhibition is not simply caused by hyperactivation of the Pkc1p signal transduction pathway .

Yeast, 2003 May, 20(7), 563 - 73
Occurrence, horizontal transfer and degeneration of VDE intein family in Saccharomycete yeasts; Okuda Y et al.; VDE is a homing endonuclease gene originally discovered as an intervening element in VMA1s of Saccharomyces cerevisiae . There have been two independent subfamilies of VDE, one from S . cerevisiae strain X2180-1A and the other from Saccharomyces sp . DH1-1A in the host VMA1 gene, and they share the identity of 96.3% . In order to search the occurrence, intra/interspecies transfer and molecular degeneration of VDE, complete sequences of VMA1 in 10 strains of S . cerevisiae, eight species of saccharomycete yeasts, Candida glabrata and Kluyveromyces lactis were determined . We found that six of 10 S . cerevisiae strains contain VDEs 99.7-100% identical to that of the strain X2180-1A, one has no VDE, whereas the other three harbour VDEs 100% identical to that of the strain DH1-1A . S . carlsbergensis has two VMA1s, one being 99.8% identical to that of the strain X2180-1A with VDE 100% identical to that of the strain DH1-1A and the other containing the same VMA1 in S . pastorianus with no VDE . This and other evidence indicates that intra/interspecies transmissions of VDEs have occurred among saccharomycete yeasts . Phylogenetic analyses of VMA1 and VDE suggest that the S . cerevisiae VDEs had branched earlier than other VDEs from an ancestral VDE and had invaded into the host loci as relatively late events . The two VDEs seemed to degenerate in individual host loci, retaining their splicing capacity intact . The degeneration of the endonuclease domains was distinct and, if compared, its apparent rate was much faster than that of the protein-splicing domains .

Biol Proced Online, 1998 May 14, 1, 48 - 58
Dealing with different methods for Kluyveromyces lactis beta-galactosidase purification; Becerra M et al.; Several micro-scale chromatography-based procedures for purification of the beta-galactosidase from the yeast Kluyveromyces lactis were assayed . Purified enzyme was suitable to be used as antigen to induce polyclonal antibodies production . Specific staining of non-denaturing PAGE gels with chromogenic substrates allowed the determination of the number of subunits forming the native enzyme.

Appl Biochem Biotechnol, 2003 Spring, 105 -108, 141 - 53
Effect of lignocellulosic degradation compounds from steam explosion pretreatment on ethanol fermentation by thermotolerant yeast Kluyveromyces marxianus; Oliva JM et al.; The filtrate from steam-pretreated poplar was analyzed to identify degradation compounds . The effect of selected compounds on growth and ethanolic fermentation of the thermotolerant yeast strain Kluyveromyces marxianus CECT 10875 was tested . Several fermentations on glucose medium, containing individual inhibitory compounds found in the hydrolysate, were carried out . The degree of inhibition on yeast strain growth and ethanolic fermentation was determined . At concentrations found in the prehy-drolysate, none of the individual compounds significantly affected the fermentation . For all tested compounds, growth was inhibited to a lesser extent than ethanol production . Lower concentrations of catechol (0.96 g/L) and 4-hydroxybenzaldehyde (1.02 g/L) were required to produce the 50% reduction in cell mass in comparison to other tested compounds.

Appl Biochem Biotechnol, 2003 Spring, 105 -108, 265 - 76
Molecular characterization of a gene for aldose reductase ( CbXYL1) from Candida boidinii and its expression in Saccharomyces cerevisiae; Kang MH et al.; Candida boidinii produces significant amounts of xylitol from xylose, and assays of crude homogenates for aldose (xylose) reductase (XYL1p) have been reported to show relatively high activity with NADH as a cofactor even though XYL1p purified from this yeast does not have such activity . A gene coding for XYL1p from C . boidinii (CbXYL1) was isolated by amplifying the central region using primers to conserved domains and by genome walking . CbXYL1 has an open reading frame of 966 bp encoding 321 amino acids . The C . boidinii XYL1p is highly similar to other known yeast aldose reductases and is most closely related to the NAD(P)H-linked XYL1p of Kluyveromyces lactis . Cell homogenates from C . boidinii and recombinant Saccharomyces cerevisiae were tested for XYL1p activity to confirm the previously reported high ratio of NADH:NADPH linked activity . C . boidinii grown under fully aerobic conditions showed an NADH:NADPH activity ratio of 0.76, which was similar to that observed with the XYL1p from Pichia stipitis XYL1, but which is much lower than what was previously reported . Cells grown under low aeration showed an NADH:NADPH activity ratio of 2.13 . Recombinant S . cerevisiae expressing CbXYL1 showed only NADH-linked activity in cell homogenates . Southern hybridization did not reveal additional bands . These results imply that a second, unrelated gene for XYL1p is present in C . boidinii.

Appl Biochem Biotechnol, 2003 Spring, 105 -108, 255 - 63
D-Xylose transport by Candida succiphila and Kluyveromyces marxianus; Stambuk BU et al.; The kinetics and regulation of D-xylose uptake were investigated in the efficient pentose fermentor Candida succiphila, and in Kluyveromyces marxianus, which assimilate but do not ferment pentose sugars . Active high affinity (Km approximately 3.8 mM; Vmax approximately 15 nmol/{mg min}) and putative facilitated diffusion low-affinity (Km approximately 140 mM; Vmax approximately 130 nmol/{mg min}) transport activities were found in C . succiphila grown, respectively, on xylose or glucose . K . marxianus showed facilitated diffusion low-affinity (Km approximately 103 mM; Vmax approximately 190 nmol/{mg min}) transport activity when grown on xylose under microaerobic conditions, and both a low-affinity and an active high-affinity (Km approximately 0.2 mM; Vmax approximately 10 nmol/{mg min}) transport activity when grown on xylose under fully aerobic conditions.

Res Microbiol, 2003 Apr, 154(3), 215 - 22
Relation between cell wall chitin content and susceptibility to amphotericin B in Kluyveromyces, Candida and Schizosaccharomyces species; Bahmed K et al.; Yeast strains belonging to the genera Candida, Kluyveromyces and Schizosaccharomyces were tested for their susceptibility (or resistance) to amphotericin B (AmB) in relation to their cell wall chitin content . Results showed that membrane sterol contents did not enable us to explain resistance or susceptibility of these yeasts to AmB . Indeed, we noted that resistant strains were as rich in ergosterol as sensitive strains . The suppression of the wall of yeasts induced an increase in susceptibility to AmB . Strains with high cell wall chitin content were more sensitive to this polyenic antifungal agent than strains with low chitin content . Growth of the yeasts in the presence of chitin induced a resistance of the yeasts to AmB . Similar results were obtained after treatment of the cells by chitinase . In contrast, growth of the yeasts in the presence of chitin synthase activators induced high susceptibility to AmB . Yeast cell wall chitin is an aminopolysaccharide, usually at low concentrations . In Schizosaccharomyces pombe its presence was not established . This polymer is associated with glucans in the wall matrix of the lateral wall and in the budding scars . Even at low content, this polymer seems to play an essential role in the sensitivity (or resistance) of yeast cells to AmB.

FEMS Yeast Res, 2002 Mar, 2(1), 39 - 46
Five new combinations in the yeast genus Zygofabospora Kudriavzev emend . G . Naumov (pro parte Kluyveromyces) based on genetic data; Naumov GI et al.; The rDNA sequencing data obtained during the last 5 years in several laboratories clearly demonstrate that within the heterogeneous genus Kluyveromyces there is a group of highly related species, which we refer to as the genus Zygofabospora Kudriavzev 1960 emend . G . Naumov 2002 . This genus includes four hybridizing species, Zf . marxiana, Zf . dobzhanskii, Zf . lactis, Zf . wickerhamii (Zygofabospora sensu stricto), and two taxonomically related species, Zf . aestuarii, Zf . nonfermentans (Nagahama et al.) G . Naumov comb . nov . (Zygofabospora sensu lato) . We studied the relationships of the yeasts composing the Zf . lactis complex . Genetic hybridization analysis and molecular karyotyping revealed partial genetic-isolation varieties, Zf . lactis var . drosophilarum (Shehata et al.) G . Naumov comb . nov . and Zf . lactis var . phaseolospora (Shehata et al.) G . Naumov comb . nov . from North America, and Zf . lactis var . krassilnikovii (Kudriavzev) G . Naumov comb . nov . from Europe . The dairy yeast Zf . lactis var . lactis G . Naumov comb . nov . yields highly fertile hybrids with its wild ancestor Zf . lactis var . krassilnikovii and semi-sterile hybrids with North American taxa . Besides, Zf . lactis var . lactis and Zf . lactis var . krassilnikovii formed fertile hybrids with the South African yeast Zf . lactis var . vanudenii (van der Walt et Nel) G . Naumov comb . nov . The reinstatement of the latter yeast at the variety level has been done taking into account the results of the present study and the literature data on its geographic isolation, high divergence of the karyotype and mitochondrial DNA.

FEMS Yeast Res, 2002 May, 2(2), 233 - 44
Steady-state and transient-state analyses of aerobic fermentation in Saccharomyces kluyveri; Moller K et al.; Some yeasts, such as Saccharomyces cerevisiae, produce ethanol at fully aerobic conditions, whereas other yeasts, such as Kluyveromyces lactis, do not . In this study we investigated the occurrence of aerobic alcoholic fermentation in the petite-negative yeast Saccharomyces kluyveri that is only distantly related to S . cerevisiae . In aerobic glucose-limited continuous cultures of S . kluyveri, two growth regimens were observed: at dilution rates below 0.5 h(-1) the metabolism was purely respiratory, and at dilution rates above 0.5 h(-1) the metabolism was respiro-fermentative . The dilution rate at which the switch in metabolism occurred, i.e . the critical dilution rate, was 66% higher than the typical critical dilution rate of S . cerevisiae . The maximum specific oxygen consumption rate around the critical dilution rate was found to 13.6 mmol (g dry weight)(-1) h(-1) and the capacity of the pyruvate dehydrogenase-bypass pathway was estimated to be high from in vitro enzyme activities; especially the specific activity of acetyl-CoA synthetase was much higher than in S . cerevisiae at all tested conditions . Addition of glucose to respiring cells of S . kluyveri led to ethanol formation after a delay of 20-50 min (depending on culture conditions prior to the pulse), which is in contrast to S . cerevisiae that ferments immediately after glucose addition.

FEMS Yeast Res, 2002 Dec, 2(4), 533 - 8
Existence of cerebroside in Saccharomyces kluyveri and its related species; Takakuwa N et al.; Sphingolipids are ubiquitous compounds derived from ceramide that consist of a sphingoid long-chain base with a 2-amino group amide linked to fatty acid and are present in the membranes of many organisms . As a principal sphingolipid, Saccharomyces cerevisiae contains a free ceramide and its inositol-phosphorylated derivatives (acidic types) but not a neutral glycosylated ceramide, glucosylceramide (cerebroside), which usually appears in eukaryotic cells . When 31 strains accepted in the genera Saccharomyces, Torulaspora, Zygosaccharomyces, and Kluyveromyces were analyzed for sphingolipids, cerebrosides were found in S . kluyveri, Z . cidri, Z . fermentati, K . lactis, K . thermotolerans, and K . waltii . The cerebrosides of S . kluyveri and K . lactis included 9-methyl 4-trans, 8-trans-sphingadienine and its putative metabolic intermediates . A unique characteristic of S . kluyveri was the presence of a trihydroxy sphingoid base, which rarely occurs in fungal cerebrosides . A polymerase chain reaction with primers targeted to the glucosylceramide synthase gene of other microorganisms amplified the fragments of the expected size from S . kluyveri and K . lactis and further extended to the adjacent regions . The presumed protein of S . kluyveri had 54.4% similarity to that of K . lactis, higher than the glucosylceramide synthases from Candida albicans, Pichia pastoris, and other organisms . From these observations, the divergence of S . kluyveri from the lineage of K . lactis in their evolution is discussed.

Bioresour Technol, 2003 Feb, 86(3), 305 - 7
Utilization of corn silage juice by Klyuveromyces marxianus; Hang YD et al.; Corn silage juice was found to be a favorable substrate for production of fodder yeasts . Kluyveromyces marxianus NRRL Y-610 yielded significantly more cell dry weight than other cultures examined . In shake-flask experiments, the yeast produced over 13 g of cell dry weight per liter of corn silage juice and completely consumed the organic pollutants (lactic acid, acetic acid, and ethanol) . The yeast settled rapidly and had a yeast volume index of 21 ml/g . The results indicate that K . marxianus NRRL Y-610 could be used to efficiently remove lactic acid and other organic compounds from corn silage juice with the concomitant production of fodder yeast.

Appl Microbiol Biotechnol, 2003 Sep, 62(4), 387 - 91 Epub 2003 Apr 03.
Cloning of the KcURA3 gene and development of a transformation system for Kluyveromyces cicerisporus; Zhang J et al.; KcURA3 was cloned from Kluyveromyces cicerisporus CBS4857 by complementation of the ura3 mutation in Saccharomyces cerevisiae . KcURA3 encodes a 267-amino-acid protein with 80% sequence identity to that of S . cerevisiae . An ura3 mutant strain from K . cicerisporus CBS4857, named Y179U, was obtained by selection on 5-fluoroorotic acid plates . Sequence analysis of this mutated gene revealed that it contained a point mutation at nucleotide position +277 . Two vectors, pUK1 and pUKD, bearing KcURA3 were constructed . Either the lithium acetate method or electroporation could be used to transform pUK1 and pUKD intoY179U . The transformation efficiency using electroporation was higher than that using the lithium acetate method.

Curr Genet, 2003 Jul, 43(4), 281 - 8 Epub 2003 Apr 04.
Functional characterization of the Frt1 sugar transporter and of fructose uptake in Kluyveromyces lactis; Diezemann A et al.; Most yeast hexose transporters studied so far at the molecular level mediate facilitated diffusion of glucose and fructose . Here, we report that a novel Kluyveromyces lactis gene, FRT1, encodes a proton-coupled fructose-uptake transporter . Frt1, when expressed in a Saccharomyces cerevisiae hxt null mutant strain that is unable to take up monosaccharides, restored growth on fructose . Determination of substrate specificities and kinetic parameters revealed Frt1 as a fructose transporter with a K(m) of 0.16+/-0.02 mM . Uptake of fructose was accompanied by an initial alkalization of the medium, indicating a proton-coupled uptake mechanism . Deletion of the FRT1 gene in a K . lactis strain already deleted for its RAG1 and HGT1 hexose transporter genes completely prevented uptake of and growth with fructose but not with glucose . Kinetic parameters of Frt1 in K . lactis, as assessed in a rag1 hgt1 mutant strain, were comparable with those obtained after heterologous expression in S . cerevisiae . Transcription of the FRT1 gene, which was undetectable when cells were grown in ethanol, was induced by various sugars . Our results indicate that, unlike S . cerevisiae, K . lactis exhibits proton symport systems for the uptake of hexoses, in addition to their facilitated diffusion.

Yeast, 2003 Apr 15, 20(5), 369 - 79
Induction and characterization of a novel amine oxidase from the yeast Kluyveromyces marxianus; Corpillo D et al.; An amine oxidase from the yeast Kluyveromyces marxianus was induced, purified and completely characterized; it was shown to belong to the class of copper-containing amine oxidases (E.C . 1.4.3.6) . The enzyme was induced by putrescine and, very strongly, by copper(II); structural-functional characterization of the enzyme was performed, including determination of molecular weight, glycosylation, copper and TPQ content, isoelectric point, K(M) and k(CAT) (with benzylamine as substrate), pH, temperature and ionic strength effect on catalysis, substrate and inhibitor specificity . A 700 bp clone was isolated containing the cDNA that encodes for the C-terminus of the enzyme; the amino acid sequence deduced (the first available for a benzylamine oxidase from yeast) was compared to that of other copper amine oxidases from microorganisms and higher organisms . From the results obtained, the putrescine/benzylamine oxidase from Kluyveromyces marxianus was found to have a good homology with other enzymes of this class from microorganisms, and particularly with AO I from Aspergillus niger . Nonetheless, some features resulted closer to those of animal amine oxidases and histaminases . Some potential biotechnological applications are proposed . The cDNA Accession No . is AJ320485 .

Anal Biochem, 2003 Apr 1, 315(1), 77 - 84
High yield electroextraction of proteins from yeast by a flow process; Ganeva V et al.; High yields of intracellular enzymes from yeast can be obtained by application of a series of electric field pulses with a flow process . Up to 80-90% of the total activity can be liberated without any further or previous treatment of cells . The method is based on electroinduced changes in the cell envelope leading to a leakage of part of the intracellular proteins without formation of debris and permits treatment of large volumes . Field parameters require a limited electrical power . Treatment of at least 20% wet weight suspensions is possible . The optimal field conditions must be adjusted to the suspension concentration . Maximal yield is obtained within 4h at 30 degrees C for enzymes from Saccharomyces cerevisiae such as hexokinase, 3-phosphoglycerate kinase, and glyceraldehyde-3-phosphate dehydrogenase . The extraction of beta-D-galactosidase from Kluyveromyces lactis lasts 10h but can be accelerated by adding dithiothreitol in the postpulse medium . The specific activities of the electroextracted enzymes are higher than those obtained by mechanical disintegration or enzymatic lysis.

Biochem Biophys Res Commun, 2003 Apr 4, 303(2), 427 - 32
The influence of glycosylation on secretion, stability, and immunogenicity of recombinant HBV pre-S antigen synthesized in Saccharomyces cerevisiae; Lee J et al.; Three types of recombinant pre-S antigens (i.e., pre-S1S2) of hepatitis B virus (HBV) were synthesized in Saccharomyces cerevisiae and secreted into extracellular medium: wild type (pre-S1S2) and two mutant antigens, pre-S1 degrees S2 (Asn15Gln) and pre-S1 degrees S2 degrees (Asn15Gln and Asn123Gln) . An N-terminus sequence (Ser5-Ala28) of human interleukin 1 beta (hIL-1 beta) was used as synthetic prosequence of recombinant HBV surface antigen (pre-S), secreted from S . cerevisiae . The expression cassette comprised the signal peptide of the killer toxin of Kluyveromyces lactis, the synthetic prosequence above, KEX2 dibasic endopeptidase cleavage site (-Lys-Arg-), and the surface antigen . The recombinant pre-S1S2 and pre-S1 degrees S2 were secreted in the hyper-mannosylated form, while the recombinant pre-S1 degrees S2 degrees was produced without N-glycosylation . It has been demonstrated that the two particular N-linked glycans at Asn15 and Asn123 interfered with the B-cell response to the HBV-derived pre-S1S2, resulting in low titers of pre-S1S2-neutralizing antibodies . This problem was overcome by eliminating both of the N-glycosylation signals . Despite enhanced immunogenicity, the recombinant pre-S1 degrees S2 degrees showed two major problems: (1) inefficient Kex2 cleavage process in the secretory pathway and (2) the severe proteolytic degradation by yeast proteases . The efficiency of Kex2 cleavage increased dramatically by removing N-glycosylation signal in the synthetic prosequence, but the proteolysis of pre-S1 degrees S2 degrees was somewhat inevitable . Further systematic approaches including modulation of degree of N-glycosylation or relocation of N-glycosylation sites in the recombinant pre-S1S2 may make it possible to achieve both enhanced immunogenicity and resistance towards proteolytic degradation of the secreted pre-S antigen.

Mol Genet Genomics, 2003 Mar, 268(6), 729 - 38 Epub 2003 Feb 11.
Mutant telomeres inhibit transcriptional silencing at native telomeres of the yeast Kluyveromyces lactis; Gurevich R et al.; We report the identification and characterization of transcriptional silencing at native telomeres in the budding yeast Kluyveromyces lactis . We show that K . lactis telomeres are able to repress the transcription of a gene located at the junction between the telomeric repeat tract and the subtelomeric domain . As in Saccharomyces cerevisiae, switching between the repressed and derepressed transcriptional states occurs . C-terminal truncation of the telomere binding protein Rap1p, which leads to a regulated alteration in telomere length, reduces telomeric silencing . In addition, telomeric silencing is reduced dramatically in telomerase RNA mutants in which telomere length control has been lost . This is consistent with the possibility that the structure of the entire telomere affects the silencing functions exhibited by its internal domain.

Yeast, 2003 Mar, 20(4), 331 - 41
Viable Saccharomyces cerevisiae cells at high concentrations cause early growth arrest of non-Saccharomyces yeasts in mixed cultures by a cell-cell contact-mediated mechanism; Nissen P et al.; The growth of Kluyveromyces thermotolerans and Torulaspora delbrueckii was examined in mixed cultures with Saccharomyces cerevisiae in YPD modified for wine fermentations . Although the three yeasts had similar maximum specific growth rates in these fermentations, K . thermotolerans and T . delbrueckii arrested growth earlier than S . cerevisiae, thereby obtaining lower stationary phase cell concentrations than S . cerevisiae . Various single and mixed culture fermentations with the three yeasts were carried out in order to find an explanation for this phenomenon . The early growth arrests of K . thermotolerans and T . delbrueckii were absent in single cultures of the two yeasts, and they seemed to be due neither to nutrient limitations nor to the presence of growth-inhibitory compounds . Rather, they seemed to be due to a cell-cell contact mechanism dependent on the presence of viable S . cerevisiae cells at high concentrations . These results contribute to an increased understanding of why K . thermotolerans and T . delbrueckii arrest growth before S . cerevisiae during wine fermentations .

Genome Biol . 2003;4(2):R10 . Epub 2003 Jan 23.
Evidence from comparative genomics for a complete sexual cycle in the 'asexual' pathogenic yeast Candida glabrata; Wong S et al.; BACKGROUND: Candida glabrata is a pathogenic yeast of increasing medical concern . It has been regarded as asexual since it was first described in 1917, yet phylogenetic analyses have revealed that it is more closely related to sexual yeasts than other Candida species . We show here that the C . glabrata genome contains many genes apparently involved in sexual reproduction . RESULTS: By genome survey sequencing, we find that genes involved in mating and meiosis are as numerous in C . glabrata as in the sexual species Kluyveromyces delphensis, which is its closest known relative . C . glabrata has a putative mating-type (MAT) locus and a pheromone gene (MFALPHA2), as well as orthologs of at least 31 other Saccharomyces cerevisiae genes that have no known roles apart from mating or meiosis, including FUS3, IME1 and SMK1 . CONCLUSIONS: We infer that C . glabrata is likely to have an undiscovered sexual stage in its life cycle, similar to that recently proposed for C . albicans . The two Candida species represent two distantly related yeast lineages that have independently become both pathogenic and 'asexual' . Parallel evolution in the two lineages as they adopted mammalian hosts resulted in separate but analogous switches from overtly sexual to cryptically sexual life cycles, possibly in response to defense by the host immune system.

FEMS Microbiol Lett, 2003 Feb 14, 219(1), 129 - 35
Broad and complex antifungal activity among environmental isolates of lactic acid bacteria; Magnusson J et al.; More than 1200 isolates of lactic acid bacteria isolated from different environments were screened for antifungal activity in a dual-culture agar plate assay . Approximately 10% of the isolates showed inhibitory activity and 4% showed strong activity against the indicator mould Aspergillus fumigatus . The antifungal spectra for 37 isolates with strong activity and five isolates with low or no activity were determined . Several of the strains showed strong inhibitory activity against the moulds A . fumigatus, Aspergillus nidulans, Penicillium commune and Fusarium sporotrichioides, and also against the yeast Rhodotorula mucilaginosa . Penicillium roqueforti and the yeasts Pichia anomala and Kluyveromyces marxianus were not inhibited . Several isolates showed reduced antifungal activity after storage and handling . The majority of the fungal inhibitory isolates were identified by 16S rDNA sequencing as Lactobacillus coryniformis . Lactobacillus plantarum and Pediococcus pentosaceus were also frequently identified among the active isolates . The degree of fungal inhibition was not only related to production of lactic or acetic acid . In addition, antifungal cyclic dipeptides were identified after HPLC separation and several other active fractions were found suggesting a highly complex nature of the antifungal activity.

FEMS Microbiol Lett, 2003 Feb 14, 219(1), 105 - 13
The KlFUS1 gene is required for proper haploid mating and its expression is enhanced by the active form of the Galpha (Gpa1) subunit involved in the pheromone response pathway of the yeast Kluyveromyces lactis; Lloret A et al.; The Kluyveromyces lactis FUS1 gene was cloned, physically characterized and its role in the mating response pathway was determined . The gene encodes a putative membrane protein, whose structure shows a single membrane-spanning segment, a short extracellular amino-terminus and a long carboxy-terminus, located in the cytoplasmic side . The predicted primary structure of the protein shows a number of serine and threonine residues in the amino-terminus, which in analogy to Fus1p of Saccharomyces cerevisiae might be O-glycosylated . A fus1-GFP hybrid protein was tentatively located in the plasma membrane of dividing cells and upon activation of the pheromone response pathway, the protein seems to be relocated at the tip of elongated cells . KlFus1p is required for optimal conjugation of sexual partners and its expression is significantly enhanced by overexpression of both a constitutively active form of KlGpa1p, the G protein alpha subunit that triggers the mating response in this strain, and the KlSte12p transcription factor . Inactivation of the KlSte12 protein strongly reduces mating and affects KlFUS1 gene expression . The KlFUS1 gene has been deposited in the GenBank under accession number AF519444.

Mol Biol Cell, 2003 Feb, 14(2), 721 - 9
A truncated form of KlLsm4p and the absence of factors involved in mRNA decapping trigger apoptosis in yeast; Mazzoni C et al.; The LSM4 gene of Saccharomyces cerevisiae codes for an essential protein involved in pre-mRNA splicing and also in mRNA decapping, a crucial step for mRNA degradation . We previously demonstrated that the first 72 amino acids of the Kluyveromyces lactis Lsm4p (KlLsm4p), which contain the Sm-like domains, can restore cell viability in both K . lactis and S . cerevisiae cells not expressing the endogenous protein . However, the absence of the carboxy-terminal region resulted in a remarkable loss of viability in stationary phase cells () . Herein, we demonstrate that S . cerevisiae cells expressing the truncated LSM4 protein of K . lactis showed the phenotypic markers of yeast apoptosis such as chromatin condensation, DNA fragmentation, and accumulation of reactive oxygen species . The study of deletion mutants revealed that apoptotic markers were clearly evident also in strains lacking genes involved in mRNA decapping, such as LSM1, DCP1, and DCP2, whereas a slight effect was observed in strains lacking the genes DHH1 and PAT1 . This is the first time that a connection between mRNA stability and apoptosis is reported in yeast, pointing to mRNA decapping as the crucial step responsible of the observed apoptotic phenotypes.

Wei Sheng Wu Xue Bao, 2002 Aug, 42(4), 431 - 5
{Construction of a set of secreting expression vectors for Saccharomyces cerevisiea}; Zhao Y et al.; The DNA fragment ecoding the Signal peptide of inulinase of Kluyveromyces smarxianu was synthesized chemically . This fragment was cloned in-frame in the expression vector pYES2 of Saccharomyces cerevisiae, resulting in a set of new secreting expression vectors pYES2 I, pYES2 II, pYES2 III . The L-Asparaginase gene (ASN) of E . coli and alpha-acetylactate decarboxylase gene (ALDC) of B . brevis which were amplified by PCR and cloned into the new vectors respectively were transformed into Saccharomyces cerevisia, and most of enzyme activities were secreted into the medium . The new secreting expression vectors still have excellent segregational stability even after growth for 100 h in the absence of selective pressure.

Biotechnol Bioeng, 2003 Mar 30, 81(7), 837 - 47
Quantitative comparison of transient growth of Saccharomyces cerevisiae, Saccharomyces kluyveri, and Kluyveromyces lactis; Herwig C et al.; A multitude of metabolic regulations occur in yeast, particularly under dynamic process conditions, such as under sudden glucose excess . However, quantification of regulations and classification of yeast strains under these conditions have yet to be elucidated, which requires high-frequency and consistent quantification of the metabolic response . The present study aimed at quantifying the dynamic regulation of the central metabolism of strains Saccharomyces cerevisiae, S . kluyveri, and Kluyveromyces lactis upon sudden glucose excess, accomplished by a shift-up in dilution rate inside of the oxidative region using a small metabolic flux model . It was found that, under transient growth conditions, S . kluyveri behaved like K . lactis, while classification using steady-state conditions would position S . kluyveri close to S . cerevisiae . For transient conditions and based on the observation whether excess glucose is initially used for catabolism (energy) or anabolism (carbon), we propose to classify strains into energy-driven, such as S . cerevisiae, and carbon-driven, such as S . kluyveri and K . lactis, strains . Furthermore, it was found that the delayed onset of fermentative catabolism in carbon-driven strains is a consequence of low catabolic flux and the initial shunt of glucose in non-nitrogen-containing biomass constituents . The MFA model suggests that energy limitation forced the cell to ultimately increase catabolic flux, while the capacity of oxidative catabolism is not sufficient to process this flux oxidatively . The combination of transient experiments and its exploitation with reconciled intrinsic rates using a small metabolic model could corroborate earlier findings of metabolic regulations, such as tight glucose control in carbon-driven strains and transient changes in biomass composition, as well as explore new regulations, such as assimilation of ethanol before glucose . The benefit from using small metabolic flux models is the richness of information and the enhanced insight into intrinsic metabolic pathways without a priori knowledge of adaptation kinetics . Used in an online context, this approach serves as an efficient tool for strain characterization and physiological studies .

Wei Sheng Wu Xue Bao, 1999 Apr, 39(2), 141 - 7
{Construction of thermotolerant ethanol-producing yeast by protoplast fusion}; Wen T et al.; Protoplast fusion between Saccharomyces cerevisiae A001 and Kluyveromyces Y034 has been studied . A001 is a high alcohol yield yeast and can ferment maltose into ethanol, but can not grow at 45 degrees C . However, Y034 can grow above 45 degrees C but has a lower alcohol yield and can not ferment maltose . We obtained the fusants which were tested in several aspects such as cell morphology, physiological and biochemical feature, isozyme patterns analysis, alcohol fermentation at high temperature(45 degrees C) and so on . The fusant AY023 with the final ethanol concentration of 7.4% has been selected, which is thermotolerant yeast with the highest alcohol yield at 45 degrees C until now.

Wei Sheng Wu Xue Bao, 1998 Jun, 38(3), 208 - 12
{Studies on optimal conditions for inulinase production by Kluyveromyces sp . Y-85}; Wei W et al.; The application of response surface method to optimal medium-screeing for inulinase production by Kluyveromyces sp . Y-85 has been investigated . The experiments of condition control for the enzyme production was carried out in a 15 L fermenter . A large scale fermentation (1000 L fermenter) was performed five batches, and average inulinase activity of 68.9 u/ml achieved.

J Ind Microbiol Biotechnol, 2003 Jan, 30(1), 52 - 6 Epub 2003 Jan 03.
Evaluation of Kluyveromyces marxianus as a source of yeast autolysates; Lukondeh T et al.; Cells of Kluyveromyces marxianus FII 510700 and Saccharomyces cerevisiae CBS 1907 were autolysed in phosphate buffer, pH 4.5, for a maximum of 10 days to compare chemical changes that occur in the carbohydrate, protein, amino acid and nucleic acid content . Approximately 2.2-3% carbohydrate, 9.5-12% protein, 0.6-1.0% DNA and 6-7% RNA were recovered in the autolysates . The main amino acids were beta-alanine, phenylalanine, cysteine, methionine, glutamic acid and isoleucine . No significant differences in the yeast autolysates of K . marxianus and S . cerevisiae were observed . Consequently, K . marxianus produced from lactose-based media has potential as a source of yeast autolysates used in the food industry.

Chemosphere, 2003 Mar, 50(8), 1075 - 83
A comparative study on the biosorption characteristics of some yeasts for Remazol Blue reactive dye; Aksu Z et al.; Biosorption capacities and rates of different kinds of dried yeasts (Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces marxianus, Candida sp., C . tropicalis, C . lipolytica, C . utilis, C . quilliermendii and C . membranaefaciens) for Remazol Blue reactive dye from aqueous solutions were compared under laboratory conditions as a function of initial pH and initial dye concentration . Optimum initial biosorption pH was determined as 2 for all the yeasts . All the yeast species showed comparable and very high dye sorption at 100 mg/l initial dye concentration . The equilibrium sorption capacity of the biomass increased with increasing initial dye concentration up to 400 mg/l for Candida sp . C . lipolytica and C . tropicalis; up to 300 mg/l for C . quilliermendii and C . utilis and up to 200 mg/l for S . cerevisiae, S . pombe, K . marxianus and C . membranaefaciens while the adsorption yield of dye showed the opposite trend for all the yeasts . Among the nine yeast species, C . lipolytica exhibited the highest dye uptake capacity (Q(0) = 250 mg/g) . Both the Freundlich and Langmuir adsorption models were found suitable for describing the biosorption of the dye by all the Candida yeasts (except C . membranaefaciens) . The results indicated that the dye uptake process followed the pseudo-second-order kinetics for each dye-yeast system .

Mol Immunol, 2003 Jan, 39(12), 729 - 38
Comparison of three microbial hosts for the expression of an active catalytic scFv; Robin S et al.; Antibodies represent an interesting protein framework on which catalytic functions can be grafted . In previous studies, we have reported the characterization of the catalytic antibody 4B2 obtained on the basis of the "bait and switch" strategy which catalyzes two different chemical reactions: the allylic isomerization of beta,gamma-unsaturated ketones and the Kemp elimination . We have cloned the antibody 4B2 and expressed it as a single-chain Fv (scFv) fragment in different expression systems, Escherichia coli and two yeasts species, in order to elicit the most suitable system to study its catalytic activity . The scFv4B2 was secreted as an active form in the culture medium of Pichia pastoris and Kluyveromyces lactis, which led respectively to 4 and 1.3mg/l after purification . In E . coli, different strategies were investigated to increase the cytoplasmic soluble fraction, which resulted, in all cases, in the expression of a low amount of functional antibodies . By contrast, substantial amount of scFv4B2 could be purified when it was expressed as inclusion bodies (12mg/l) and submitted to an in vitro refolding process . Its catalytic activity was measured and proved to be comparable to that of the whole IgG . However, the instability of the scFv4B2 in solution prevented from an exhaustive characterization of its activity and stabilization of this protein appears to be essential before designing strategies to improve its catalytic activity.

Biotechnol Bioeng, 2003 Mar 20, 81(6), 712 - 8
Application of a gratuitous induction system in Kluyveromyces lactis for the expression of intracellular and secreted proteins during fed-batch culture; Panuwatsuk W et al.; A gratuitous induction system in the yeast Kluyveromyces lactis was evaluated for the expression of intracellular and extracellular products during fed-batch culture . The Escherichia coli lacZ gene (beta-galactosidase; intracellular) and MFalpha1 leader-BPTI cassette (bovine pancreatic trypsin inhibitor; extracellular) were placed under the control of the inducible K . lactis LAC4 promotor, inserted into partial-pKD1 plasmids, and transformed into a ga1-209 K . lactis strain . To obtain a high level of production, culture conditions for growth and expression were initially evaluated in tube cultures . A selective medium containing 5 g/L glucose (as carbon source) and 0.5 g/L galactose (as inducer) demonstrated the maximum activity of both beta-galactosidase and secreted BPTI . This level of expression had no significant effect on the growth of the recombinant cells; growth rate dropped by approximately 11%, whereas final biomass concentrations remained the same . In shake-flask culture, biomass concentration, beta-galactosidase activity, and BPTI secreted activity were 4 g/L, 7664 U/g dry cell, and 0.32 mg/L, respectively . Fed-batch culture (with a high glucose concentration and a low galactose {inducer} concentration feed) resulted in a 6.5-fold increase in biomass, a 23-fold increase in beta-galactosidase activity, and a 3-fold increase in BPTI secreted activity . The results demonstrate the success of gratuitous induction during high-cell-density fed-batch culture of K . lactis . A very low concentration of galactose feed was sufficient for a high production level .

Acta Microbiol Immunol Hung, 2002, 49(4), 483 - 91
From yeast genetics to biotechnology; Maraz A; Roots of classical yeast genetics go back to the early work of Lindegreen in the 1930s, who studied thallism, sporulation and inheritance of wine yeast strains belonging to S . cerevisiae . Consequent mutation and hybridization of heterothallic S . cerevisae strains resulted in the discovery of life cycle and mating type system, as well as construction of the genetic map . Elaboration of induced mutation and controlled hybridization of yeast strains opened up new possibilities for the genetic analysis of technologically important properties and for the production of improved industrial strains, but a big drawback was the widely different genetic properties of laboratory and industrial yeast strains . Genetic analysis and mapping of industrial strains were generally hindered because of homothallism, poor sporulation and/or low spore viability of brewing and wine yeast strains {1, 2} . In spite of this, there are a few examples of the application of sexual hybridization in the study of genetic control of important technological properties, e.g . sugar utilization, flocculation and flavor production in brewing yeast strains {3} or in the improvement of ethanol producing S . cerevisiae strains {4} . Rare mating and application of karyogamy deficient (kar-) mutants also proved useful in strain improvement {5} . Importance of yeasts in biotechnology is enormous . This includes food and beverage fermentation processes where a wide range of yeast species are playing role, but S . cerevisiae is undoubtedly the most important species among them . New biotechnology is aiming to improve these technologies, but besides this, a completely new area of yeast utilization has been emerged, especially in the pharmaceutical and medical areas . Without decreasing the importance of S . cerevisiae, numerous other yeast species, e.g . Kluyveromyces lactis, Hansenula polymorpha, Pichia pastoris, Schizosaccharomyces pombe and Yarrowia lipolytica have gained increasing potentialities in the modern fermentation biotechnology {6} . Developments in yeast genetics, biochemistry, physiology and process engineering provided bases of rapid development in modern biotechnology, but elaboration of the recombinant DNA technique is far the most important milestone in this field . Other molecular genetic techniques, as molecular genotyping of yeast strains proved also very beneficial in yeast fermentation technologies, because dynamics of both the natural and inoculated yeast biota could be followed by these versatile DNA-based techniques.

Yeast, 2003 Jan 15, 20(1), 13 - 23
The acetyl co-enzyme A synthetase genes of Kluyveromyces lactis; Zeeman AM et al.; Two Kluyveromyces lactis genes encoding acetyl co-enzyme A synthetase isoenzymes were isolated . One we named KlACS1, as it has high similarity to the ACS1 gene of Saccharomyces cerevisiae . The other gene, KlACS2, showed more similarity to S . cerevisiae ACS2 than to KlACS1 or ScACS1 . This suggests that divergence of the two isogenes occurred before the evolutionary separation of the species and that the different functions have been conserved . In line with this idea is the regulation of transcription of the genes . The mode of regulation appeared to be maintained between ScACS1 and KlACS1 and between ScACS2 and KlACS2 . The KlACS1 transcript was absent in glucose-grown cells, whereas transcription levels in ethanol- and acetate-grown cells were high . Disruption of the KlACS1 gene did not result in growth defects on glucose or ethanol . The growth rate on acetate, however, was reduced by a factor of two . KlACS2 was expressed at similar levels during growth on glucose and acetate, whereas expression on ethanol was slightly higher . A null mutant in this gene showed a reduced growth rate on all three carbon sources . Taken together, these data suggest that KlACS2 is used during growth on glucose and that KlACS1 is most dominant during growth on acetate . Strains in which both ACS genes are deleted could only be retrieved when a plasmid containing the ACS2 gene was present, suggesting that the double mutant is lethal . Tetrad analysis confirmed that non-viable spores with a deduced Klacs1Klacs2 genotype germinated but could not divide further . It therefore appears that, as in S . cerevisiae, the pyruvate dehydrogenase bypass formed by the enzymes pyruvate decarboxylase, acetaldehyde dehydrogenase and acetyl co-enzyme A synthetase is essential for growth . These results are in apparent contradiction with the growth on glucose of a strain with a disruption in the only structural pyruvate decarboxylase gene of K . lactis . Residual enzyme activity might, however, account for this discrepancy, or Acs fulfils an additional as yet unknown function, separate from its enzymatic activity .

Yeast, 2003 Jan 15, 20(1), 1 - 11
Studies of yeast Kluyveromyces lactis mutations conferring super-secretion of recombinant proteins; Bartkeviciute D et al.; We have isolated mutants responsible for a super-secretion phenotype in Kluyveromyces lactis using the gene coding for a Bacillus amyloliquefaciens alpha-amylase as a marker for secretion . These mutations defined two groups, dominant and recessive . The recessive mutant strain, which secreted the heterologous protein in five-fold excess compared to the wild-type strain, was used for the cloning of genes, restraining the super-secreting phenotype . In screening for genes affecting super-secreting phenotype, we found that multiple copies of 10 different independently isolated DNA sequences suppressed the super-secreting phenotype . The first among the genes characterized, named KlSEL1 ('secretion lowering') showed homology to Saccharomyces cerevisiae ORF YML013w . The KlSEL1 gene is predicted to encode a polypeptide of 620 amino acid residues containing a putative transmembrane domain and UBX domain, characteristic for the ubiquitin-regulatory proteins . We demonstrated that the disruption of the SEL1 orthologues in K . lactis and S . cerevisiae conferred the super-secreting phenotype . SEL1 isolated from S . cerevisiae suppressed the super-secretion phenotype in K . lactis klsel1 strain, likewise homologous KlSEL1 . No other phenotypic features for strains lacking the SEL1 gene were noticed except for the S . cerevisiae mutant growth being notably slower than in a wt strain . No growth changes were observed in the K . lactis klsel1 mutant . The set of genes (suppressors of over-secreting phenotype) could be attractive for further analysis of gene functions, super-secreting mechanisms and construction of new strains . This collection could be useful for the expedient construction of reduced yeast genomes, optimized for heterologous protein secretion .

J Gen Appl Microbiol, 2001 Feb, 47(1), 1 - 19
Regulation of the amylolytic and (hemi-)cellulolytic genes in aspergilli; Tsukagoshi N et al.; Filamentous fungi produce high levels of polysaccharide-degrading enzymes and are frequently used for the production of industrial enzymes . Because of the high secretory capacity for enzymes, filamentous fungi are effective hosts for the production of foreign proteins . Genetic studies with Aspergillus nidulans have shown pathway-specific regulatory systems that control a set of genes that must be expressed to catabolize particular substrates . Besides the pathway-specific regulation, wide domain regulatory systems exist that affect a great many individual genes in different pathways . A molecular analysis of various regulated systems has confirmed the formal models derived from purely genetic data . In general, many genes are subject to more than one regulatory system . In this article, we describe two transcriptional activators, AmyR and XlnR, and an enhancer, Hap complex, in view of their regulatory roles in the expression of the amylolytic and (hemi-)cellulolytic genes mainly in aspergilli . The amyR gene has been isolated as a transcriptional activator involved in the expression of amylolytic genes from A . oryzae, A . niger, and A . nidulans, and the xlnR gene, which has been isolated from A . niger and A . oryzae, activates the expression of xylanolytic genes as well as some cellulolytic genes in aspergilli . Both AmyR and XlnR have a typical zinc binuclear cluster DNA-binding domain at their N-terminal regions . Hap complex, a CCAAT-binding complex, enhances the overall promoter activity and increases the expression levels of many fungal genes, including the Taka-amylase A gene . Hap complex comprises three subunits, HapB, HapC, and HapE, in A . nidulans and A . oryzae as well as higher eukaryotes, whereas HAP complex in Saccharomyces cerevisiae and Kluyveromyces lactis has the additional subunit, Hap4p, which is responsible for the transcriptional activation . Hap complex is suggested to enhance transcription by remodeling the chromatin structure . The regulation of gene expression in filamentous fungi of industrial interest could follow basically the same general principles as those discovered in A . nidulans . The knowledge of regulation of gene expression in combination with traditional genetic techniques is expected to be increasingly utilized for strain breeding . Furthermore, this knowledge provides a basis for the rational application of transcriptional regulators for biotechnological processes in filamentous fungi.

Eukaryot Cell, 2002 Aug, 1(4), 548 - 57
Functional diversity of silencers in budding yeasts; Sjostrand JO et al.; We studied the silencing of the cryptic mating-type loci HMLa and HMRa in the budding yeast Kluyveromyces lactis . A 102-bp minimal silencer fragment was defined that was both necessary and sufficient for silencing of HMLalpha . Mutagenesis of the silencer revealed three distinct regions (A, B, and C) that were important for silencing . Recombinant K . lactis ribosomal DNA enhancer binding protein 1 (Reb1p) could bind the silencer in vitro, and point mutations in the B box abolished both Reb1p binding and silencer function . Furthermore, strains carrying temperature-sensitive alleles of the REBI gene derepressed the transcription of the HMLalpha1 gene at the nonpermissive temperature . A functional silencer element from the K . lactis cryptic HMRa locus was also identified, which contained both Reb1p binding sites and A boxes, strongly suggesting a general role for these sequences in K lactis silencing . Our data indicate that different proteins bind to Kluyveromyces silencers than to Saccharomyces silencers . We suggest that the evolution of silencers is rapid in budding yeasts and discuss the similarities and differences between silencers in Saccharomyces and Kluyveromyces.

Res Microbiol, 2002 Nov, 153(9), 593 - 8
Effects of the loss of triose phosphate isomerase activity on carbon metabolism in Kluyveromyces lactis; Capitanio D et al.; The effect of the loss of triose phosphate isomerase activity on carbon metabolism in Kluyveromyces lactis was studied in batch and in continuous cultures . The Kltpi1 mutant was able to grow on media containing glucose as the sole carbon source both in batch and in continuous culture, unlike the corresponding S . cerevisiae mutant . In K . lactis tpi1 mutant no glycerol production was detected in chemostat cultivations . DHAP accumulation triggers glycerol production only when glucose is the sole carbon source in excess . The analysis of the activities of some key enzymes of carbon metabolism shows that in chemostat cultivations on mixed-substrates the activities of enzymes involved in ethanol assimilation are higher both in K . lactis wild type and mutant strains than in S . cerevisiae.

Biotechnol Bioeng, 2003 Jan 20, 81(2), 127 - 33
Kinetics of lactose hydrolysis by beta-galactosidase of Kluyveromyces lactis immobilized on cotton fabric; Zhou QZ et al.; A mathematic model for describing the Michaelis-Menten-type reaction kinetics with product competitive inhibition and side-reaction is proposed . A multiresponse nonlinear simulation program was employed to determine the coefficients of a four-parameter rate expression . The rate expression was compared with the conventional Michaelis-Menten reaction rate models with and without product inhibition . Experimental data were obtained using beta-galactosidase of Kluyveromyces lactis immobilized on cotton fabric in a batch system at a temperature of 37 degrees C and at various initial concentrations of dissolved lactose ranging from 3-12.5% (w/v) . The reaction is followed by concentration changes with time in the tank . Samples were obtained after the outlet stream of the packed bed reactor is mixed in a well-stirred tank . High-performance liquid chromatography (HPLC) was applied to monitor the concentrations of all the sugars (reactants as well as products) . The four-parameter rate model is featured with a term to describe the formation of trisaccharides, a side-reaction of the enzymatic hydrolysis . The proposed model simulates the process of lactose hydrolysis and the formation of glucose and galactose, giving better accuracy compared with the previous models .

Carbohydr Res, 2002 Nov 5, 337(20), 1873 - 7
Effect of sodium orthovanadate on glycosylation of the phosphopeptidomannans involved in the cell-cell aggregation of the yeast Kluyveromyces bulgaricus; Thiebault F et al.; In studies of yeast flocculation it has been found that low concentrations of vanadium contained in sodium orthovanadate do not affect the growth and the cell-cell adhesion of the yeast Kluyveromyces bulgaricus, whereas high concentrations delay the growth of the yeasts and strongly inhibit flocculation . Moreover, higher sensitivity to Hygromycin B and calcofluor white was taken to imply altered cell wall integrity which is supported by compositional analysis of the extracted phosphopeptidomannans . Yeasts grown on sodium orthovanadate show a decrease in the percentage of phosphopeptidomannans and their compositions . It is proposed that the vanadium contained in sodium orthovanadate has a similar conformation to phosphorus and competes with phosphorus in phosphorylated compounds . The decrease of carbohydrate components and phosphorus linking to phosphopeptidomannans detected may alter their structure and modify ligand binding properties.

Nahrung, 2002 Oct, 46(5), 321 - 6
A recombinant Saccharomyces cerevisiae strain for efficient conversion of lactose in salted and unsalted cheese whey into ethanol; Tahoun MK et al.; For utilization of lactose in salted and unsalted cheese whey, intergeneric protoplast fusion between lactose nonfermenting, salt-tolerant Saccharomyces cerevisiae ATCC4126 and lactose fermenting Kluyveromyces lactis CBS683 was carried out . The fusion process gave rise to new hybrid yeast strains that revealed higher significant DNA contents than parental strains . The recombinants showed growth on either lactose or sucrose . The ethanol yields by some recombinants were 5.55% from sweet whey and 4.66% from salted whey containing up to 6% sodium chloride compared to 4.15 and 2.86% for parental K . lactis CBS683, respectively.

Biochemistry, 2002 Nov 19, 41(46), 13833 - 8
Identification of the first fungal NADP-GAPDH from Kluyveromyces lactis; Verho R et al.; Deletion of the phosphoglucose isomerase gene, PGI1, in Saccharomyces cerevisiae leads to a phenotype for which glucose is toxic . This is related to overproduction of NADPH through the oxidative part of the pentose phosphate pathway and the incompetence of S . cerevisiae to deal with this overproduction . A similar deletion (rag2) in Kluyveromyces lactis does not lead to such a phenotype . We transformed a genomic library of K . lactis in a yeast vector to a S . cerevisiae strain with a pgi1 deletion and screened for growth on glucose . We found a gene (GDP1) which encodes a phosphorylating glyceraldehyde-3-phosphate dehydrogenase, NADP-GAPDH (EC 1.2.1.13), that accepts both NADP and NAD . This is the first report of a eukaryotic, nonplant, NADP-linked GAPDH . Presumably, operation of this enzyme in the reverse direction enabled the transformed S . cerevisiae pgi1 deletion mutant to reoxidize the excess NADPH produced when glucose catabolism was forced through the pentose pathway . On the other hand, transcription of the gene in K . lactis was upregulated during growth on D-xylose, which suggests that in K . lactis the enzyme is involved in regeneration of NADPH needed for xylose assimilation, but transcription was not detected in a rag2 mutant grown on glucose . The presence of an asparagine (Asn46 in NADP-GAPDH) instead of the conserved aspartate found in related but NAD-specific enzymes may explain the ability of NADP-GAPDH to work with NADP as well as NAD.

J Biol Chem, 2003 Jan 10, 278(2), 956 - 61 Epub 2002 Nov 06.
Subunit communications crucial for the functional integrity of the yeast RNA polymerase II elongator (gamma-toxin target (TOT)) complex; Frohloff F et al.; In response to the Kluyveromyces lactis zymocin, the gamma-toxin target (TOT) function of the Saccharomyces cerevisiae RNA polymerase II (pol II) Elongator complex prevents sensitive strains from cell cycle progression . Studying Elongator subunit communications, Tot1p (Elp1p), the yeast homologue of human IKK-associated protein, was found to be essentially involved in maintaining the structural integrity of Elongator . Thus, the ability of Tot2p (Elp2p) to interact with the HAT subunit Tot3p (Elp3p) of Elongator and with subunit Tot5p (Elp5p) is dependent on Tot1p (Elp1p) . Also, the association of core-Elongator (Tot1-3p/Elp1-3p) with HAP (Elp4-6p/Tot5-7p), the second three-subunit subcomplex of Elongator, was found to be sensitive to loss of TOT1 (ELP1) gene function . Structural integrity of the HAP complex itself requires the ELP4/TOT7, ELP5/TOT5, and ELP6/TOT6 genes, and elp6Delta/tot6Delta as well as elp4Delta/tot7Delta cells can no longer promote interaction between Tot5p (Elp5p) and Tot2p (Elp2p) . The association between Elongator and Tot4p (Kti12p), a factor that may modulate the TOT activity of Elongator, requires Tot1-3p (Elp1-3p) and Tot5p (Elp5p), indicating that this contact requires a preassembled holo-Elongator complex . Tot4p also binds pol II hyperphosphorylated at its C-terminal domain Ser(5) raising the possibility that Tot4p bridges the contact between Elongator and pol II.

FEMS Microbiol Lett, 2002 Oct 29, 216(1), 99 - 103
Change of cell wall chitin content in amphotericin B resistant Kluyveromyces strains; Bahmed K et al.; The culture of two Kluyveromyces species, Kluyveromyces lactis (ATCC 96897) and Kluyveromyces bulgaricus (ATCC 96631), in the presence of subinhibitory doses of amphotericin B leads to the selection of mutants which are resistant to this polyene . The mutants show an alteration of their cell wall composition with the main change corresponding to an increase of chitin . The enzyme activities involved in the metabolism of this polymer, i.e . chitin synthases and chitinase, were measured . The results demonstrate that in both mutants the activity of chitinase was drastically decreased by 99% in comparison with the activity measured in the corresponding wild-type strain while no significant change of the chitin synthase I, II and III activities could be detected.

Curr Genet, 2002 Oct, 42(1), 21 - 6 Epub 2002 Sep 27.
Isolation, heterological cloning and sequencing of the RPL28 gene in Kluyveromyces lactis; Takacova M et al.; By virtue of heterologous functional complementation of the Saccharomyces cerevisiae Delta pdr5 mutant strain, using a Kluyveromyces lactis genomic library, three different K . lactis chromosomal inserts were obtained . Transformation of the S . cerevisiae Delta pdr1 Delta pdr3 mutant strain, hypersensitive to drugs, with isolated plasmids resulted in resistance to cycloheximide and fluconazole . Transformation of K . lactis host strains, using the cloned chromosomal fragments, led to an increased level of resistance to some mitochondrial inhibitors and azole antifungals . The nucleotide sequence of the cloned inserts revealed that two of them contain the drug efflux transporter gene Kl-PDR5 and the third contains a DNA segment homologous to chromosome VII of S . cerevisiae . Along with three novel ORFs, encoding two proteins of unknown molecular function and one putative hexose transporter, this segment also contained the Kl-RPL28 gene, found to be responsible for the cycloheximide resistance of heterologous transformants . This gene codes for the large subunit ribosomal protein (149 amino acids) that shares 89.9% identity with its S . cerevisiae counterpart . The coding region of Kl-RPL28 was found to be interrupted with one intron near the 5' end . The nucleotide sequence data reported in this paper were submitted to GenBank and assigned the accession number AF493565.

J Dairy Sci, 2002 Oct, 85(10), 2497 - 502
Enhancement of lactase activity in milk by reactive sulfhydryl groups induced by heat treatment; Jimenez-Guzman J et al.; The effects of heat treatments of milk and whey prior to lactose hydrolysis with Kluyveromyces lactis beta-galactosidase were studied . It was observed that heat treatment of milk significantly increases lactase activity, with a maximum activity increase found when milk was heated at 55 degrees C . In whey from 55 up to 75 degrees C, beta-galactosidase activity decreased slightly . Nevertheless, heating whey at 85 degrees C for 30 min raised the rate of hydrolysis significantly . Electrophoretic patterns and UV spectra proved that the activity change correlated with milk protein denaturation, particularly that of beta-lactoglobulin . Heating whey permeate did not increase the enzyme activity as heating whole whey; but heating whey prior to ultrafiltration also resulted in enzyme activation . Measurement of free sulfhydryl (SH) groups in both whey and heated whey permeate showed that the liberation of free SH is highly correlated to the change of the activity . Furthermore, this activation can be reversed by oxidizing the reactive sulfhydryl groups, proving that the observed effect may be related to the release of free SH to the medium, rather than to the denaturation of a thermolabile protein inhibitor.

Genes Dev, 2002 Nov 1, 16(21), 2800 - 12
A bulged stem tethers Est1p to telomerase RNA in budding yeast; Seto AG et al.; It is well established that the template for telomeric DNA synthesis is provided by the RNA subunit of telomerase; however, the additional functions provided by most of the rest of the RNA (>1000 nucleotides in budding yeast) are largely unknown . By alignment of telomerase RNAs of Saccharomyces cerevisiae and six Kluyveromyces species followed by mutagenesis of the S . cerevisiae RNA, we found a conserved region that is essential for telomere maintenance . Phylogenetic analysis and computer folding revealed that this region is conserved not only in primary nucleotide sequence but also in secondary structure . A common bulged-stem structure was predicted in all seven yeast species . Mutational analysis showed the structure to be essential for telomerase function . Suppression of bulged-stem mutant phenotypes by overexpression of Est1p and loss of co-immunoprecipitation of the mutant RNAs with Est1p indicated that this bulged stem is necessary for association of Est1p, a telomerase regulatory subunit . Est1p in yeast extract bound specifically to a small RNA containing the bulged stem, suggesting a direct interaction . We propose that this RNA structure links the enzymatic core of telomerase with Est1p, thereby allowing Est1p to recruit or activate telomerase at the telomere.

Biochem Biophys Res Commun, 2002 Nov 8, 298(4), 559 - 65
Cell surface expression of a GPI-anchored form of mouse acetylcholinesterase in Klpmr1Delta cells of Kluyveromyces lactis; Uccelletti D et al.; The mouse acetylcholinesterase AChE(H) was expressed in the yeast Kluyveromyces lactis . The AChE(H) activity was detectable in intact cells whereas it was absent in the culture media . Glucanase treatment and immunoelectron microscopy data indicated that AChE(H) is anchored to plasma membrane and that the mouse GPI-signaling is compatible with the K . lactis targeting machinery . The AChE(H) was also expressed in a K . lactis strain carrying an inactivated allele of KlPMR1, the gene coding for a P-type Ca(2+)-ATPase of the Golgi apparatus . This mutant displays changes in protein glycosylation and cell wall structure . The AChE(H) activity detected in Klpmr1Delta cells was more than twofold higher than that observed in wild-type cells . The combination of AChE expression and anchoring with the characteristics of Klpmr1Delta strain of K . lactis resulted in yeast cells displaying high AChE activity . This could be regarded as a novel sensing unit to be employed for detecting AChE inhibitors as pesticides.

Yeast, 2002 Nov, 19(15), 1351 - 63
Metabolic physiology of aroma-producing Kluyveromyces marxianus; Wittmann C et al.; Kluyveromyces marxianus has a high potential for industrial production of aroma compounds, such as 2-phenylethanol, which is derived in a bioconversion from L-phenylalanine . In the present work the product yield of K . marxianus in batch cultivation was estimated as 0.65 mol 2-phenylethanol/mol L-phenylalanine and thus significantly below the theoretical optimum of 1 mol/mol . By a comprehensive approach of stoichiometric balancing and GC-MS analysis of various substrates and products of K . marxianus a detailed insight into its metabolism was gained . For this purpose ring-labelled ((13)C(6)) L-phenylalanine and naturally labelled glucose were applied as substrates in tracer studies in batch culture . The produced aroma compounds 2-phenylethanol and 2-phenylethylacetate stem exclusively from the supplied L-phenylalanine, whereas glucose was not converted into these products because of efficient feed-back inhibition of prephenate dehydratase in the L-phenylalanine biosynthetic pathway . It could be further shown that the supplied L-phenylalanine completely covers the anabolic cellular demand for this amino acid . Quantification of (13)CO(2) in the exhaust gas provided clear evidence for catabolic breakdown of L-phenylalanine during cultivation . Metabolic balancing around the pool of free intracellular L-phenylalanine revealed a significant loss of L-phenylalanine into catabolic and anabolic pathways . While 73.3% of L-phenylalanine was converted into 2-phenylethanol or 2-phenylethylacetate, 22.4% was catabolized through the cinnamate pathway and 4.3% was directed towards protein biosynthesis . Catabolic breakdown of L-phenylalanine via hydroxylation to L-tyrosine could be excluded . In addition to an insight into metabolic functioning and regulation of 2-phenylethanol-producing K . marxianus, the approach presented here provides important information on potential targets for genetic optimization of 2-phenylethanol-producing yeasts .

Curr Microbiol, 2002 Dec, 45(6), 459 - 61
Isolation and characterization of the genes encoding delta(8)-sphingolipid desaturase from Saccharomyces kluyveri and Kluyveromyces lactis; Takakuwa N et al.; Saccharomyces kluyveri IFO 1685 and Kluyveromyces lactis IFO 1090 synthesize cerebroside containing 9-methyl- trans-4, trans-8-sphingadienine as a sphingoid base . From the genome of the two strains, the regions encompassing Delta(8)-sphingolipid desaturase were amplified and sequenced . The nucleotide sequences of these regions revealed single open reading frames of 1707 bp for S . kluyveri and 1722 bp for K . lactis, encoding polypeptides of 568 and 573 amino acids with molecular weights of 66.5 and 67.1 kDa, respectively . Conversion of 4-hydroxysphinganine to 4-hydroxy- trans-8-sphingenine in the cells of Saccharomyces cerevisiae was observed by the expressed gene from K . lactis and not by that from S . kluyveri . These findings may be explained by the difference in substrate specificity for the sphingoid base moiety between Delta(8)-sphingolipid desaturases of S . kluyveri and K . lactis.

Plasmid, 2002 Sep, 48(2), 142 - 8
Linear plasmids pWR1A and pWR1B of the yeast Wingea robertsiae are associated with a killer phenotype; Klassen R et al.; Wingea robertsiae CBS6693 (synonym Debaryomyces robertsiae) was previously reported to harbor two cryptic linear plasmids, designated pWR1A (8.3 kb) and pWR1B (14.6 kb) . Reexamination of a putative plasmid encoded killer phenotype involved UV-curing as well as a highly sensitive toxin assay . Killer activities of concentrated culture supernatants prepared from both, a plasmid carrying and a cured plasmid-free strain, were examined in liquid media . Supernatants collected from plasmid carrying strains subjected to cultures of the plasmid-free derivative had clear concentration-dependent inhibitory effects, whereas plasmid harboring cells were not affected . Incubation at 65 degrees C for 10 min totally destroyed the toxin . Since supernatants prepared from the plasmid-free strain did not possess such killer activity and the presence of the plasmids confered resistance, toxin as well as immunity functions appear plasmid encoded . Beyond this, chitin affinity chromatography and Western blot analysis proved plasmid specific expression and secretion of a protein displaying similarities to the alpha-subunit of the Kluyveromyces lactis killer toxin . The assay applied in this study will most probably allow disclosure of other hidden killer phenomena, which may have escaped detection by conventionally applied plate assays.

Mol Microbiol, 2002 Oct, 46(1), 169 - 77
Suppression of a nuclear frameshift mutation by a mitochondrial tRNA in the yeast Kluyveromyces lactis; Fiori A et al.; A fragment of mitochondrial DNA containing the Kluyveromyces lactis gene for valine-tRNA (tRNAVAL) was isolated as a multicopy suppressor of a respiratory-deficient mutant of this yeast . The mutant produced a truncated Cox14p because of a +1 frameshift mutation in COX14, a nuclear gene encoding a protein imported into mitochondria which is necessary for respiration (Fiori et al . 2000 Yeast 16: 307-314) . We report here that the mitochondrial tRNAVAL gene, when transformed into K . lactis cells, is transcribed outside mitochondria and suppresses the frameshift mutation in COX14 restoring the correct reading frame during translation of its mRNA . In fact, using histidine tagging, the existence of a suppressed Cox14p of normal length was demonstrated in mutants expressing the mt-tRNAVAL from the nucleus . Suppression could occur through a non-canonical four base pairing between the tRNAVAL and the mutated mRNA or through slippage of ribosomes during translation . This is a new case of informational suppression in that the suppression of a chromosomal mutation is not caused by a second mutation but to a mislocalization/expression of a mt-tRNA.

Biosci Biotechnol Biochem, 2002 Aug, 66(8), 1775 - 8
Purification and properties of a carbonyl reductase useful for production of ethyl (S)-4-chloro-3-hydroxybutanoate from Kluyveromyces lactis; Yamamoto H et al.; A novel carbonyl reductase (KLCR1) that reduced ethyl 4-chloroacetoacetate (ECAA) to synthesize ethyl (S)-4-chloro-3-hydroxybutanoate ((S)-ECHB) was purified from Kluyveromyces lactis . KLCR1 catalyzed the NADPH-dependent reduction of ECAA enantioselectively but not the oxidation of (S)-ECHB . From partial amino acid sequences, KLCR1 was suggested to be an alpha subunit of fatty acid synthase (FAS) but did not have FAS activity.

Prikl Biokhim Mikrobiol, 2002 Jul-Aug, 38(4), 389 - 92
{Isolation of S-adenosyl-L-methionine from an enriched Kluyveromyces lactis mutant, grown in whey media}; Mincheva KP et al.; The ability of six lactose-digesting yeast strains to accumulate S-adenosyl-L-methionine (AdoMet) when grown on whey medium was studied . Kluyveromyces lactis ATCC 8585 accumulated the greatest amount of AdoMet (15.2 mg per gram biomass) and was chosen for the development of an Ado-Met-enriched mutant . The Ado-Met-enriched mutant AM-65 was selected from mutants induced with N-methyl-N-nitro-N'-nitrosoguanidine . The strain accumulated 61.6 mg AdoMet per gram biomass . The effect of concentrations of medium components on AdoMet biosynthesis was studied . The ability of the mutant to accumulate AdoMet remained stable after multiple reinoculations.

Appl Environ Microbiol, 2002 Oct, 68(10), 4884 - 93
Yeast diversity and persistence in botrytis-affected wine fermentations; Mills DA et al.; Culture-dependent and -independent methods were used to examine the yeast diversity present in botrytis-affected ("botrytized") wine fermentations carried out at high ( approximately 30 degrees C) and ambient ( approximately 20 degrees C) temperatures . Fermentations at both temperatures possessed similar populations of Saccharomyces, Hanseniaspora, Pichia, Metschnikowia, Kluyveromyces, and Candida species . However, higher populations of non-Saccharomyces yeasts persisted in ambient-temperature fermentations, with Candida and, to a lesser extent, Kluyveromyces species remaining long after the fermentation was dominated by SACCHAROMYCES: In general, denaturing gradient gel electrophoresis profiles of yeast ribosomal DNA or rRNA amplified from the fermentation samples correlated well with the plating data . The direct molecular methods also revealed a Hanseniaspora osmophila population not identified in the plating analysis . rRNA analysis also indicated a large population (>10(6) cells per ml) of a nonculturable Candida strain in the high-temperature fermentation . Monoculture analysis of the Candida isolate indicated an extreme fructophilic phenotype and correlated with an increased glucose/fructose ratio in fermentations containing higher populations of CANDIDA: Analysis of wine fermentation microbial ecology by using both culture-dependent and -independent methods reveals the complexity of yeast interactions enriched during spontaneous fermentations.

Mol Genet Genomics, 2002 Sep, 268(1), 49 - 55 Epub 2002 Jul 12.
Defects in yeast RNA polymerase II transcription elicit hypersensitivity to G1 arrest induced by Kluyveromyces lactis zymocin; Kitamoto HK et al.; The G1 cell cycle arrest imposed by Kluyveromyces lactis zymocin on Saccharomyces cerevisiae requires a functional RNA polymerase II (pol II) TOT/Elongator complex . In a study of zymocin's mode of action, genetic scenarios known to impair transcription or affect the pol II machinery itself were found to elicit hypersensitivity to zymocin . Thus, mutations in components of SAGA, SWI/SNF, Mediator and Ccr4-Not, complexes involved in transcriptionally relevant functions such as nucleosome modification, chromatin remodelling and formation of the preinitiation complex, make yeast cells hypersensitive to the lethal effects of zymocin . The defects at the level of transcriptional elongation displayed by rtf1Delta, ctk1, fcp1 and rpb2 mutants also result in zymocin hypersensitivity . Intriguingly, inactivation of histone deacetylase (HDAC) activity, which is expected to reduce the demand for the histone acetyltransferase (HAT) function of TOT/Elongator, also reduces sensitivity to zymocin . Thus, zymocin interferes with pol II-dependent transcription, and this effect requires the HAT function of TOT, presumably while the Elongator complex is associated with pol II.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 1996, 28(5), 540 - 546
The Construction and Application of the Multi-copy Integration Vector in K . lactis; Tang NY et al.; The vector pIRK was constructed by using a 2.2 kb EcoRI fragment of rDNA from Kluyveromyces lactis for targeted homologous recombination, with the URA3 gene from Saccharomyces cerevisiae acting as a selection marker . By the examination of the copy number, stability and chromosomal location of the vector in K . lactis transformants the results demonstrated that: (1) of the different transformants, the average copy number of the plasmid pIRK was 120 per cell; (2) after 50 generations of growth in rich medium, the vector displayed high stability . (3) all integration events occurred in the chromosome IV where genomic rDNA located . Using this vector, the LAC4 gene cloned from K . fragilis was expressed . The yield of beta-galactosidase related directly to the vector's copy number . The highest activity of beta-galactosidase produced by transformants was 8.6 times higher than that produced by the wild type strain of K . fragilis under the same conditions.

Plasmid, 2002 Jul, 48(1), 13 - 23
Replication of a linear mini-chromosome with terminal inverted repeats from the Kluyveromyces lactis linear DNA plasmid k2 in the cytoplasm of Saccharomyces cerevisiae; Bennett AM et al.; The k1 and k2 linear DNA plasmids of Kluveromyces lactis replicate in the cytoplasm under the control of plasmid-encoded genes . These plasmids can also replicate autonomously in the cytoplasm of mitochondrial DNA-deficient strains of Saccharomyces cerevisiae . Essential for replication are plasmid-specific terminal inverted repeats (TIRs) to which a terminal protein (TP) is attached at the 5' ends . A plasmid was constructed with k2 TIRs in opposite orientations and with a selectable marker (URA3) under the control of k1UCS2 (upstream conserved sequence 2, the promoter of k1 open reading frame 2) in between the TIRs . Transformation of k1- and k2-containing S . cerevisiae with a fragment generated by releasing the TIR-flanked fragment from the plasmid by restriction digestion was very efficient, despite the absence of a TP . Transformation was also achieved with a fragment generated by PCR . Southern blotting demonstrated that transformants contained multiple copies of DNA fragments with the same size as the transforming DNA, supporting the hypothesis that these were replicating linear mini-chromosomes . The high frequency of transformation strongly suggests that these mini-chromosomes readily replicate supported by k2 . Derivatives with a heterologous gene, firefly luciferase (LUC), expressed luciferase at high levels provided the gene was adjacent to a cytoplasmic plasmid promoter (k2UCS5).

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 1997, 29(2), 129 - 134
Secretory Expression of Porcine Insulin Precursor in Kluyveromyces lactis and Its Conversion into Human Insulin; Feng YM et al.; Porcine insulin precursor (PIP) was cloned to vectors derived from plasmid pKD1 and expressed in Kluyveromyces lactis . The secretory expression level of PIP was 20 to 30 mg per liter of the culture medium . Human insulin obtained from PIP through tryptic transpeptidation was characterized . Its amino acid composition, crystalline shape and biological activity are identical with those of native insulin.

Microbiology, 2002 Sep, 148(Pt 9), 2789 - 95
The antiapoptotic protein Bcl-x(L) prevents the cytotoxic effect of Bax, but not Bax-induced formation of reactive oxygen species, in Kluyveromyces lactis; Poliakova D et al.; The murine proapoptotic protein Bax was expressed in Kluyveromyces lactis to investigate its effect on cell survival and production of reactive oxygen species (ROS) . Bax expression decreased the number of cells capable of growing and forming colonies, and it increased the number of cells producing ROS, as detected by both dihydrorhodamine 123 fluorescence and the intracellular content of SH groups . Mutation in the beta-subunit of F(1)-ATPase, or mitochondrial deficiency resulting from deletion of mtDNA (rho(0) mutant), increased the sensitivity to Bax, indicating that Bax cytotoxicity does not require mitochondrial respiratory-chain functions . The antiapoptotic protein Bcl-x(L), when co-expressed with Bax, localized to the mitochondria and prevented Bax cytotoxicity . However, this co-expression did not prevent the production of ROS . These data suggest that in K . lactis cells expressing Bax, ROS are not the sine qua non of cell death and that the antiapoptotic function of Bcl-x(L) is not limited to its antioxidant property.

Appl Environ Microbiol, 2002 Sep, 68(9), 4322 - 7
Lactobacillus plantarum MiLAB 393 produces the antifungal cyclic dipeptides cyclo(L-Phe-L-Pro) and cyclo(L-Phe-trans-4-OH-L-Pro) and 3-phenyllactic acid; Strom K et al.; We have isolated a Lactobacillus plantarum strain (MiLAB 393) from grass silage that produces broad-spectrum antifungal compounds, active against food- and feed-borne filamentous fungi and yeasts in a dual-culture agar plate assay . Fusarium sporotrichioides and Aspergillus fumigatus were the most sensitive among the molds, and Kluyveromyces marxianus was the most sensitive yeast species . No inhibitory activity could be detected against the mold Penicillium roqueforti or the yeast Zygosaccharomyces bailii . An isolation procedure, employing a microtiter well spore germination bioassay, was devised to isolate active compounds from culture filtrate . Cell-free supernatant was fractionated on a C(18) SPE column, and the 95% aqueous acetonitrile fraction was further separated on a preparative HPLC C(18) column . Fractions active in the bioassay were then fractionated on a porous graphitic carbon column . The structures of the antifungal compounds cyclo(L-Phe-L-Pro), cyclo(L-Phe-trans-4-OH-L-Pro) and 3-phenyllactic acid (L/D isomer ratio, 9:1), were determined by nuclear magnetic resonance spectroscopy, mass spectrometry, and gas chromatography . MIC values against A . fumigatus and P . roqueforti were 20 mg ml(-1) for cyclo(L-Phe-L-Pro) and 7.5 mg ml(-1) for phenyllactic acid . Combinations of the antifungal compounds revealed weak synergistic effects . The production of the antifungal cyclic dipeptides cyclo(L-Phe-L-Pro) and cyclo(L-Phe-trans-4-OH-L-Pro) by lactic acid bacteria is reported here for the first time.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 1998, 30(1), 35 - 40
Cloning of DNA Fragments Possessing Promoter Function from Kluyveromyces cicerisporus; Xu GZ et al.; Using three yeast promoter-probe plasmids pSK-kan401, pSK-kan1105, pSK-kan1238 with different reading frames, eight DNA fragments possessing high efficient promoter function have been cloned from K . cicerisporus . Their 3'-DNA sequences have been analyzed . The relative strength of their promoter function was studied by comparing their ability to promote the expression of the reporter gene APHI with that of glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) promoter in K . lactis . The results showed that the insertion fragments in pSK-kan401-41 and pSK-kan1105-51 have stronger promoter function and they are also functional in K . cicerisporus Y179.

FEMS Microbiol Lett, 2002 Aug 6, 213(2), 239 - 44
Characterization of PG2, an early endoPG produced by Sclerotinia sclerotiorum, expressed in yeast; Cotton P et al.; Sclerotinia sclerotiorum, a plant pathogenic ascomycete, contains a neutral endopolygalacturonase (endoPG) subfamily of genes that was previously isolated . We report here that pg2, a member of this subfamily, is early and strongly expressed during the first steps of pathogenesis of sunflower cotyledons . The corresponding protein, PG2, was produced in the heterologous Kluyveromyces lactis system and purified . Characterization of the recombinant enzyme revealed a narrow pH activity curve with an optimal pH of 4.5 . Hydrolysis of polygalacturonic acid by PG2 resulted in the accumulation of oligomers ranging from 2- to 9-mer . This degradation profile indicates a random attack on the polymer and demonstrates an endo-mode of action . These results provide evidence that pg2 contributes to the infection process during the early phase of host colonization.

Plasmid, 2002 May, 47(3), 224 - 33
Genome organization of the linear Pichia etchellsii plasmid pPE1A: evidence for expression of an extracellular chitin-binding protein homologous to the alpha-subunit of the Kluyveromyces lactis killer toxin; Klassen R et al.; Pichia etchellsii CBS2011 (synonym Debaryomyces etchellsii) is a non-killer yeast harbouring two cryptic linear cytoplasmic DNA-elements, pPE1A (6.7 kb) and pPE1B (12.8 kb) . Cloning and complete sequencing of pPE1A revealed a 6749-bp element with a remarkably high A+T content of 77.6% . The termini of pPE1A were found to consist of inversely orientated identical nucleotide repetitions of 178bp, to which proteins are linked at the 5'-ends . It is only the second small, non-autonomous cytoplasmic yeast linear plasmid for which the complete nucleotide sequence is known . Five open reading frames (ORFs) were identified preceded by upstream conserved sequence motifs (UCS) characteristic for cytoplasmic promoters and perfectly matching the UCS consensus (ATNTGA) . As none of the putative genes encodes a DNA-polymerase, pPE1A is the first yeast linear plasmid known that does not possess its own element-specific replication machinery . No function could be attributed to ORF1, 3, 4, and 5; the predicted ORF2 gene product is similar to chitin-binding proteins and chitinases, highest homologies were found to the precursor of the alpha- and beta-subunits of the secreted Kluyveromyces lactis zymocin . Consistently, the Orf2p could be isolated from the culture fluid by chitin-Sepharose affinity chromatography and characterized by immuno-probing with an antibody specific for the K . lactis killer toxin alpha-subunit . Production of the protein was found to be plasmid-dependent . The sequence of pPE1A has been submitted to the EMBL data library, Accession No . AJ409097.

J Bacteriol, 2002 Aug, 184(16), 4384 - 91
Trehalose-mediated inhibition of the plasma membrane H+-ATPase from Kluyveromyces lactis: dependence on viscosity and temperature; Sampedro JG et al.; The effect of increasing trehalose concentrations on the kinetics of the plasma membrane H+-ATPase from Kluyveromyces lactis was studied at different temperatures . At 20 degrees C, increasing concentrations of trehalose (0.2 to 0.8 M) decreased V(max) and increased S(0.5) (substrate concentration when initial velocity equals 0.5 V(max)), mainly at high trehalose concentrations (0.6 to 0.8 M) . The quotient V(max)/S(0.5) decreased from 5.76 micromol of ATP mg of protein(-1) x min(-1) x mM(-1) in the absence of trehalose to 1.63 micromol of ATP mg of protein(-1) x min(-1) x mM(-1) in the presence of 0.8 M trehalose . The decrease in V(max) was linearly dependent on solution viscosity (eta), suggesting that inhibition was due to hindering of protein domain diffusional motion during catalysis and in accordance with Kramer's theory for reactions in solution . In this regard, two other viscosity-increasing agents, sucrose and glycerol, behaved similarly, exhibiting the same viscosity-enzyme inhibition correlation predicted . In the absence of trehalose, increasing the temperature up to 40 degrees C resulted in an exponential increase in V(max) and a decrease in enzyme cooperativity (n), while S(0.5) was not modified . As temperature increased, the effect of trehalose on V(max) decreased to become negligible at 40 degrees C, in good correlation with the temperature-mediated decrease in viscosity . The trehalose-mediated increase in S(0.5) was similar at all temperatures tested, and thus, trehalose effects on V(max)/S(0.5) were always observed . Trehalose increased the activation energy for ATP hydrolysis . Trehalose-mediated inhibition of enzymes may explain why yeast rapidly hydrolyzes trehalose when exiting heat shock.

Mol Microbiol, 2002 Aug, 45(3), 817 - 26
Protein interactions within Saccharomyces cerevisiae Elongator, a complex essential for Kluyveromyces lactis zymocicity; Fichtner L et al.; mTn3-tagging identified Kluyveromyces lactis zymocin target genes from Saccharomyces cerevisiae as TOT1-3/ELP1-3 coding for the RNA polymerase II (pol II) Elongator histone acetyltransferase (HAT) complex . tot phenotypes resulting from mTn3 tagging were similar to totDelta null alleles, suggesting loss of Elongator's integrity . Consistently, the Tot1-3/Elp1-3 proteins expressed from the mTn3-tagged genes were all predicted to be C-terminally truncated, lacking approximately 80% of Tot1p, five WD40 Tot2p repeats and two HAT motifs of Tot3p . Besides its role as a HAT, Tot3p assists subunit communication within Elongator by mediating Tot2-Tot4, Tot2-Tot5, Tot2-Tot1 and Tot4-Tot5 protein-protein interactions . TOT1 and TOT2 are essential for Tot4-Tot2 and Tot4-Tot3 interactions respectively . The latter was lost with a C-terminal Tot2p truncation; the former was affected by progressively truncating TOT1 . Despite being dispensable for Tot4-Tot2 interaction, the extreme C-terminus of Tot1p may play a role in TOT/Elongator function, as its truncation confers zymocin resistance . Tot4p/Kti12p, an Elongator-associated factor, also interacted with pol II and could be immunoprecipitated while being bound to the ADH1 promoter . Two-hybrid analysis showed that Tot4p also interacts with Cdc19p, suggesting that Tot4p plays an additional role in concert with Cdc19p, perhaps co-ordinating cell growth with carbon source metabolism.

Yeast, 2002 Aug, 19(11), 933 - 47
6-phosphofructokinase from Pichia pastoris: purification, kinetic and molecular characterization of the enzyme; Kirchberger J et al.; 6-Phosphofructokinase from Pichia pastoris was purified for the first time to homogeneity applying seven steps, including pseudo-affinity dye-ligand chromatography on Procion Blue H-5R-Sepharose . The specific activity of the purified enzyme was about 80 U/mg . It behaves as a typically allosteric 6-phosphofructokinase exhibiting activation by AMP and fructose 2,6-bis(phosphate), inhibition by ATP and cooperativity to fructose 6-phosphate . However, in comparison with the enzymes from Saccharomyces cerevisiae and Kluyveromyces lactis, the activation ratio of 6-phosphofructokinase from Pichia pastoris by AMP is several times higher, the ATP inhibition is stronger and the apparent affinity to fructose 6-phosphate is significantly lower . Aqueous two-phase affinity partitioning with Cibacron Blue F3G-A did not reflect remarkable structural differences of the nucleotide binding sites of the Pfks from Pichia pastoris and Saccharomyces cerevisiae . The structural organisation of the active enzyme seems to be different in comparison with hetero-octameric 6-phosphofructokinases from other yeast species . The enzyme was found to be a hetero-oligomer with an molecular mass of 975 kDa (sedimentation equilibrium measurements) consisting of two distinct types of subunits in an equimolar ratio with molecular masses of 113 kDa and 98 kDa (SDS-PAGE), respectively, and a third non-covalently complexed protein component (34 kDa, SDS-PAGE) . The latter seems to be necessary for the catalytic activity of the enzyme . Sequencing of the N-terminus (VTKDSIXRDLEXENXGXXFF) and of peptide fragments by applying MALDI-TOF PSD, m/z 1517.3 (DAMNVVNH) and m/z 2177.2 {AQNCNVC(L/I)SVHEAHTM} gave no relevant information about the identity of this protein .

J Food Prot, 2002 Jul, 65(7), 1179 - 82
Degradation of natural phosphorylated compounds and added polyphosphates in milk by Pseudomonas fluorescens CECT378, Lactococcus lactis CECT539, and Kluyveromyces marxianus CECT10584; Belloque J et al.; The degradation of natural phosphorylated compounds (galactose-1-phosphate, N-acetyl-glucosamine-1-phosphate, glycerophosphoethanolamine, and glycerophosphocholine) and added phosphorylated compounds (diphosphate) in milk was investigated by phosphorus 31 nuclear magnetic resonance on the incubation of a sterile milk with Pseudomonas fluorescens CECT381, Lactococcus lactis CECT539, and Kluyveromyces marxianus CECT10584 . This preliminary study showed that the degradation of these compounds was dependent on the compound, microorganism, and temperature of incubation . K . marxianus CECT10584 did not show any capability to degrade these compounds, and L . lactis CECT539 was only able to degrade diphosphate at its optimum growth temperature . P . fluorescens CECT381 was the most active strain and possessed more hydrolytic capabilities at 10 degrees C than at its optimum growth temperature . It is suggested that cold-induced enzymes are involved in the ability of P . fluorescens CECT381 to hydrolyze the natural phosphorylated compounds in milk . Consequent potential alterations of dairy products are discussed.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 1999, 31(4), 477 - 478
Analysis of Phylogenetic Relationships Among Twelve Yeast Species; Zhang PW et al.; Partial sequences of 25 S ribosomal DNA for nine Candida yeast and one Kluyveromyces yeast strains were cloned and determined . We compared them with two published 25 S rDNA sequences of Saccharomyces cerevisiae and Candida albicans . An evolutionary tree of the twelve species was inferred from about 370 sites of 5' end of 25 S ribosomal DNA using the methods of neighbor-joining and bootstrap . The molecular data indicate a very close affinity between Candida kefyr CBS834 and Kluyveromyces cicerisporus CBS4857.

Yeast, 2002 Jul, 19(10), 813 - 24
Two mechanisms for oxidation of cytosolic NADPH by Kluyveromyces lactis mitochondria; Overkamp KM et al.; Null mutations in the structural gene encoding phosphoglucose isomerase completely abolish activity of this glycolytic enzyme in Kluyveromyces lactis and Saccharomyces cerevisiae . In S . cerevisiae, the pgi1 null mutation abolishes growth on glucose, whereas K.lactis rag2 null mutants still grow on glucose . It has been proposed that, in the latter case, growth on glucose is made possible by an ability of K . lactis mitochondria to oxidize cytosolic NADPH . This would allow for a re-routing of glucose dissimilation via the pentose-phosphate pathway . Consistent with this hypothesis, mitochondria of S . cerevisiae cannot oxidize NADPH . In the present study, the ability of K . lactis mitochondria to oxidize cytosolic NADPH was experimentally investigated . Respiration-competent mitochondria were isolated from aerobic, glucose-limited chemostat cultures of the wild-type K . lactis strain CBS 2359 and from an isogenic rag2Delta strain . Oxygen-uptake experiments confirmed the presence of a mitochondrial NADPH dehydrogenase in K.lactis . This activity was ca . 2.5-fold higher in the rag2Delta mutant than in the wild-type strain . In contrast to mitochondria from wild-type K . lactis, mitochondria from the rag2Delta mutant exhibited high rates of ethanol-dependent oxygen uptake . Subcellular fractionation studies demonstrated that, in the rag2Delta mutant, a mitochondrial alcohol dehydrogenase was present and that activity of a cytosolic NADPH-dependent 'acetaldehyde reductase' was also increased . These observations indicate that two mechanisms may participate in mitochondrial oxidation of cytosolic NADPH by K . lactis mitochondria: (a) direct oxidation of cytosolic NADPH by a mitochondrial NADPH dehydrogenase; and (b) a two-compartment transhydrogenase cycle involving NADP(+)- and NAD(+)-dependent alcohol dehydrogenases .

Int Immunopharmacol, 2002 May, 2(6), 767 - 74
Resveratrol modulates rat macrophage functions; Leiro J et al.; This study investigated the effects of trans-resveratrol (trans-3,4',5-trihydroxystilbene, RESV), a natural polyphenol from grapes with known antioxidant activity, on the respiratory-burst responses and phagocytic activity of rat macrophages . RESV at concentrations of 1-10 microM significantly and dose-dependently inhibited (a) the extracellular production of reactive oxygen intermediates (ROls) by resident peritoneal macrophages stimulated with phorbol 12-myristate 13-acetate (PMA) (a potent activator of protein kinase C, PKC) and (b) intracellular production of ROIs after opsonin-independent phagocytosis of Kluyveromyces lactis cells . Over the 10-100 microM concentration ranges, RESV likewise inhibited the production of reactive nitrogen intermediates (RNIs) by macrophages stimulated with thioglycollate . RESV concentrations above 10 microM also dose-dependently inhibited the phagocytosis of K . lactis cells . The results obtained demonstrate that RESV is a potent inhibitor of the antipathogen responses of rat macrophages and, thus, suggest that this agent may have applications in the treatment of diseases involving macrophage hyperresponsiveness.

Bioresour Technol, 2002 Jul, 83(3), 251 - 3
Use of waste Chinese cabbage as a substrate for yeast biomass production; Choi MH et al.; The possibility of using waste Chinese cabbage (Brassica campestris) as a substrate for microbial biomass production was investigated . The juice from waste Chinese cabbage contains relatively high amounts of reducing sugars suitable for yeast culture . The cell mass and protein content of four species of yeast, Candida utilis, Pichia stipitis, Kluyveromyces marxianus, and Saccharomyces cerevisiae, were determined when cultured in juice extracted from cabbage waste . Compared to YM broth containing the same level of sugar, all the strains except C . utilis showed higher total protein production in cabbage juice medium (CJM).

Proc Natl Acad Sci U S A, 2002 Jul 9, 99(14), 9272 - 7 Epub 2002 Jul 01.
Gene order evolution and paleopolyploidy in hemiascomycete yeasts; Wong S et al.; The wealth of comparative genomics data from yeast species allows the molecular evolution of these eukaryotes to be studied in great detail . We used "proximity plots" to visually compare chromosomal gene order information from 14 hemiascomycetes, including the recent Genolevures survey, to Saccharomyces cerevisiae . Contrary to the original reports, we find that the Genolevures data strongly support the hypothesis that S . cerevisiae is a degenerate polyploid . Using gene order information alone, 70% of the S . cerevisiae genome can be mapped into "sister" regions that tile together with almost no overlap . This map confirms and extends the map of sister regions that we constructed previously by using duplicated genes, an independent source of information . Combining gene order and gene duplication data assigns essentially the whole genome into sister regions, the largest gap being only 36 genes long . The 16 centromere regions of S . cerevisiae form eight pairs, indicating that an ancestor with eight chromosomes underwent complete doubling; alternatives such as segmental duplications can be ruled out . Gene arrangements in Kluyveromyces lactis and four other species agree quantitatively with what would be expected if they diverged from S . cerevisiae before its polyploidization . In contrast, Saccharomyces exiguus, Saccharomyces servazzii, and Candida glabrata show higher levels of gene adjacency conservation, and more cases of imperfect conservation, suggesting that they split from the S . cerevisiae lineage after polyploidization . This finding is confirmed by sequences around the C . glabrata TRP1 and IPP1 loci, which show that it contains sister regions derived from the same duplication event as that of S . cerevisiae.

Eur J Biochem, 2002 Jul, 269(13), 3256 - 63
Pyruvate decarboxylase from Kluyveromyces lactis . An enzyme with an extraordinary substrate activation behaviour; Krieger F et al.; Pyruvate decarboxylase (EC 4.1.1.1) was isolated and purified from the yeast Kluyveromyces lactis . The properties of this enzyme relating to the native oligomeric state, the subunit size, the nucleotide sequence of the coding gene(s), the catalytic activity, and protein fluorescence as well as circular dichroism are very similar to those of the well characterized pyruvate decarboxylase species from yeast . Remarkable differences were found in the substrate activation behaviour of the two pyruvate decarboxylases using three independent methods: steady-state kinetics, stopped-flow measurements, and kinetic dilution experiments . The dependence of the observed activation rate constant on the substrate concentration of pyruvate decarboxylase from K . lactis showed a minimum at a pyruvate concentration of 1.5 mm . According to the mechanism of substrate activation suggested this local minimum occurs due to the big ratio of the dissociation constants for the binding of the first (regulatory) and the second (catalytic) substrate molecule . The microscopic rate constants of the substrate activation could be determined by a refined fit procedure . The influence of the artificial activator pyruvamide on the activation of the enzyme was studied.

J Mol Biol, 2002 May 17, 318(5), 1237 - 49
Structural mimicry of proline kinks: tertiary packing interactions support local structural distortions; Ceruso MA et al.; Proline residues in the helical segments of soluble and transmembrane proteins have received special attention from both a structural and functional perspective . A feature of these helices is the structural distortion termed "proline-kink", which has been associated with the presence of the proline residue . However, a recent report on the yeast heat-shock transcription factor of Kluyveromyces lactis (HSF_KL) suggests that these proline-associated deformations can be achieved in the absence of proline residues, thus raising the question of the mechanisms responsible for the structural mimicry of proline-related features . In this study, the specific interactions responsible for the distortion were characterized by comparative analysis of the atomic details of the packing interactions that surround the evolutionarily conserved proline-kink in the alpha2 helix of HSF_KL and a set of 39 structurally related proteins that lacked the distortion . The mechanistic details inferred from this analysis were confirmed with molecular dynamics simulations . The study shows that the packing interactions between the alpha2 and alpha1 helices in HSF_KL are responsible for the stabilization of the conserved kink, whether a proline residue that divides the helix into segments is present or not . The proline-kink can facilitate the formation of tertiary packing interactions that would otherwise not be possible . However, it is the ability to establish differential packing interactions for the helix segments, rather than the structural properties of the proline-kink itself, that emerges as the key factor for the characteristic distortion.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2000, 32(5), 516 - 520
Using Kl LAC4 as a Reporter Gene in Candida albicans; Chen X et al.; The lacZ gene which codes for beta-galactosidase from E.coli does not work in Candida albicans . In this work a reporter system with Kl LAC4 gene was contructed, coding for beta-galactosidase from Kluyveromyces lactis . Kl LAC4 gene was fused to the promoter of ADH1 of Candida albicans with the terminator sequence of ADH1 at the tail . The constructed plasmid, named pYPB1-LAC4, was used to transform Candida albicans . beta-galactosidase activity was measured by plate assay, when cultured on solid medium, and by photometric assay, when cultured in liquid medium . The results suggest that LAC4 of Kluyveromyces lactis can serve as a reporter gene in Candida albicans.

Mol Cell Biol, 2002 Jul, 22(13), 4512 - 21
Recombinational telomere elongation promoted by DNA circles; Natarajan S et al.; Yeast mutants lacking telomerase are capable of maintaining telomeres by an alternate mechanism that depends on homologous recombination . We show here, by using Kluyveromyces lactis cells containing two types of telomeric repeats, that recombinational telomere elongation generates a repeating pattern common in most or all telomeres in survivors that retain both repeat types . We propose that these patterns arise from small circles of telomeric DNA being used as templates for rolling-circle gene conversion and that the sequence from the lengthened telomere is spread to other telomeres by additional, more typical gene conversion events . Consistent with this, artificially constructed circles of DNA containing telomeric repeats form long tandem arrays at telomeres when transformed into K . lactis cells . Mixing experiments done with two species of telomeric circles indicated that all of the integrated copies of the transforming sequence arise from a single original circular molecule.

Biophys Chem, 2002 Jun 19, 97(2-3), 173 - 87
Nucleosome organization on Kluyveromyces lactis centromeric DNAs; Mattei S et al.; The preferential assembly of specialized nucleosomes on budding yeast centromeres can be due either to the higher stability of specialized centromeric nucleosomes and/or to the lower stability of canonical centromeric nucleosomes with respect to bulk nucleosomes . We have evaluated the thermodynamic stability of canonical nucleosomes, assembled on Kluyveromyces lactis centromeric DNAs, with a competitive reconstitution assay and a theoretical method recently developed by us . The results, obtained by both methods, show that all five known centromeric DNAs from K . lactis are able to organize canonical nucleosomes, characterized by higher stability with respect those of bulk DNA . With 'footprinting' and theoretical prediction, based on sequence-dependent DNA elasticity, we have found that centromeric canonical nucleosomes are characterized by nucleosome dyad axis multiple positioning, rotationally phased . The isoenergetic nucleosome multiple positions are relevant in understanding the transition from canonical to specialized nucleosomes in interacting with centromere protein complexes . The satisfactory agreement between the results obtained from theoretical and experimental methods shows that sequence-dependent centromeric DNA elasticity has a main role in nucleosome thermodynamic stability and positioning.

Genome Res, 2002 Jun, 12(6), 930 - 43
Genomic evolution of the long terminal repeat retrotransposons in hemiascomycetous yeasts; Neuveglise C et al.; We identified putative long terminal repeat- (LTR) retrotransposon sequences among the 50,000 random sequence tags (RSTs) obtained by the Genolevures project from genomic libraries of 13 Hemiascomycetes species . In most cases additional sequencing enabled us to assemble the whole sequences of these retrotransposons . These approaches identified 17 distinct families, 10 of which are defined by full-length elements . We also identified five families of solo LTRs that were not associated with retrotransposons . Ty1-like retrotransposons were found in four of five species that are phylogenetically related to Saccharomyces cerevisiae (S . uvarum, S . exiguus, S . servazzii, and S . kluyveri but not Zygosaccharomyces rouxii), and in two of three Kluyveromyces species (K . lactis and K . marxianus but not K . thermotolerans) . Only multiply crippled elements could be identified in the K . lactis and S . servazzii strains analyzed, and only solo LTRs could be identified in S . uvarum . Ty4-like elements were only detected in S . uvarum, indicating that these elements appeared recently before speciation of the Saccharomyces sensu stricto species . Ty5-like elements were detected in S . exiguus, Pichia angusta, and Debaryomyces hansenii . A retrotransposon homologous with Tca2 from Candida albicans, an element absent from S . cerevisiae, was detected in the closely related species D . hansenii . A complete Ty3/gypsy element was present in S . exiguus, whereas only partial, often degenerate, sequences resembling this element were found in S . servazzii, Z . rouxii, S . kluyveri, C . tropicalis, and Yarrowica lipolytica . P . farinosa (syn . P . sorbitophila) is currently the only yeast species in which no LTR retrotransposons or remnants have been found . Thorough analysis of protein sequences, structural characteristics of the elements, and phylogenetic relationships deduced from these data allowed us to propose a classification for the Ty1/copia elements of hemiascomycetous yeasts and a model of LTR-retrotransposon evolution in yeasts.

J Biotechnol, 2002 Jun 26, 96(2), 155 - 68
Evaluation of a recombinant Klebsiella oxytoca strain for ethanol production from cellulose by simultaneous saccharification and fermentation: comparison with native cellobiose-utilising yeast strains and performance in co-culture with thermotolerant yeast and Zymomonas mobilis; Golias H et al.; In the simultaneous saccharification and fermentation to ethanol of 100 g l(-1) microcrystalline cellulose, the cellobiose-fermenting recombinant Klebsiella oxytoca P2 outperformed a range of cellobiose-fermenting yeasts used in earlier work, despite producing less ethanol than reported earlier for this organism under similar conditions . The time taken by K . oxytoca P2 to produce up to about 33 g l(-1) ethanol was much less than for any other organism investigated, including ethanol-tolerant strains of Saccharomyces pastorianus, Kluyveromyces marxianus and Zymomonas mobilis . Ultimately, it produced slightly less ethanol (maximum 36 g l(-1)) than these organisms, reflecting its lower ethanol tolerance . Significant advantages were obtained by co-culturing K . oxytoca P2 with S . pastorianus, K . marxianus or Z . mobilis, either isothermally, or in conjunction with temperature-profiling to raise the cellulase activity . Co-cultures produced significantly more ethanol, more rapidly, than either of the constituent strains in pure culture at the same inoculum density . K . oxytoca P2 dominated the early stages of the co-cultures, with ethanol production in the later stages due principally to the more ethanol tolerant strain . The usefulness of K . oxytoca P2 in cellulose simultaneous saccharification and fermentation should be improved by mutation of the strain to increase its ethanol tolerance.

Biochim Biophys Acta, 2002 Jun 7, 1576(1-2), 176 - 82
Isolation of genes encoding novel transcription factors which interact with the Hap complex from Aspergillus species; Tanaka A et al.; The Saccharomyces cerevisiae CCAAT-binding factor is composed of four subunits Hap2p, Hap3p, Hap4p and Hap5p . Three subunits, Hap2/3/5p, are required for DNA-binding and Hap4p is involved in transcriptional activation . Although homologues of Hap2/3/5p (in the case of Aspergillus nidulans; HapB/C/E, respectively) were found in many eukaryotes, no Hap4p homologues have been found except for the other yeast, Kluyveromyces lactis . With the lexA-hap2, -hapB, -hapC, or -hapE fusion gene, we evaluated the ability of interaction between Aspergillus Hap subunits and S . cerevisiae Hap4p subunit in S . cerevisiae . Using the system with lexA-hapB, a gene encoding a novel transcriptional activator, which interacted with the Hap complex, was isolated from A . nidulans and designated hapX.

Appl Microbiol Biotechnol, 2002 May, 58(6), 842 - 7 Epub 2002 Feb 08.
Oxidative stress response of Kluyveromyces marxianus to hydrogen peroxide, paraquat and pressure; Pinheiro R et al.; The aim of this work was to study the oxidative stress response of Kluyveromyces marxianus to hydrogen peroxide (50 mM), paraquat (1 mM), an increase in air pressure (120 kPa, 600 kPa) and pure oxygen pressure (120-600 kPa) in a pressurized bioreactor . The effect of these oxidants on metabolism and on the induction of antioxidant enzymes was investigated . The exposure for 1 h of K . marxianus at exponential growth phase with either H(2)O(2) or paraquat, under air pressure of 120 kPa or 600 kPa, induced an increase in both superoxide dismutase (SOD) and glutathione reductase (GR) content . SOD induction by the chemical oxidants was independent of the air pressure values used . A 2-fold increase in SOD activity was observed after 1 h of exposure to H(2)O(2) and a 3-fold increase was obtained by the presence of paraquat, with both air pressures studied . In contrast, GR activity was raised 1.7-fold by the exposure to both chemicals with 120 kPa, but a 2.4-fold GR induction was obtained with 600 kPa . As opposed to Saccharomyces cerevisiae, catalase was not induced and was even lower than the normal basal levels . This antioxidant enzyme seemed to be inhibited under increasing oxygen partial pressure . The cells showed a significant increase in SOD and GR activity levels, 4.7-fold and 4.4-fold, when exposed for 24 h to 120 kPa pure oxygen pressure . This behaviour was even more patent with 400 kPa . However, whenever cells were previously exposed to low air pressures, low enzymatic activity levels were measured after subsequent exposure to pure oxygen pressure.

J Biol Chem, 2002 Jul 19, 277(29), 26276 - 80 Epub 2002 May 15.
Saccharomyces cerevisiae RNA polymerase II is affected by Kluyveromyces lactis zymocin; Jablonowski D et al.; The G(1) arrest imposed by Kluyveromyces lactis zymocin on Saccharomyces cerevisiae cells requires a functional RNA polymerase II (pol II) Elongator complex . In studying a link between zymocin and pol II, progressively truncating the carboxyl-terminal domain (CTD) of pol II was found to result in zymocin hypersensitivity as did mutations in four different CTD kinase genes . Consistent with the notion that Elongator preferentially associates with hyperphosphorylated (II0) rather than hypophosphorylated (IIA) pol II, the II0/IIA ratio was imbalanced toward II0 on zymocin treatment and suggests zymocin affects pol II function, presumably in an Elongator-dependent manner . As judged from chromatin immunoprecipitations, zymocin-arrested cells were affected with regards to pol II binding to the ADH1 promotor and pol II transcription of the ADH1 gene . Thus, zymocin may interfere with pol II recycling, a scenario assumed to lead to down-regulation of pol II transcription and eventually causing the observed G(1) arrest.

Bioresour Technol, 2002 Apr, 82(2), 177 - 81
High-temperature alcoholic fermentation of whey using Kluyveromyces marxianus IMB3 yeast immobilized on delignified cellulosic material; Kourkoutas Y et al.; A novel system for high-temperature alcoholic fermentation of whey is described . This system consists of Kluyveromyces marxianus yeast immobilized on delignified cellulosic material (DCM) . The effect of pH, initial lactose concentration and temperature on the fermentation of a synthetic medium containing lactose was studied . Batch fermentations of whey were also carried out and the formation of volatile by-products was examined . The concentrations of higher alcohols were found to be in very low levels leading to a product of improved quality . The fermented whey had an improved characteristic aroma compared to unfermented whey . The possibility to use fermented whey as raw material for the production of a novel, low alcohol content drink was also investigated.

Appl Biochem Biotechnol, 2002 Mar, 97(3), 209 - 18
Nutritional profile of food yeast Kluyveromyces fragilis biomass grown on whey; Paul D et al.; Biomass of food yeast Kluyveromyces fragilis (MTCC 188) grown on deproteinized whey supplemented with 0.8% diammonium hydrogen phosphate and 10 ppm indole-3-acetic acid, had a crude protein content of 37% . The true protein content based on nitrogen fractionation procedure was 28.1% . Total nucleic acid content was 4.82% . This amount does not appear to be toxicologically offensive . Crude fiber, ash, and lipid content of K.fragilis dry cells were found to be 4.9%, 16%, and 7.8%, respectively . Essential fatty acids of both omega-3 and omega-6 series were found present in the fat of the yeast and represented 21.5% of the total fatty acids . All the essential amino acids were present in the proteins of K . fragilis; however, sulfur containing amino acids were found in lower amounts . Calculated protein scores indicate moderate biological value . B vitamins in the biomass were present as expected, but folic acid and pyridoxine were present in high concentration.

Appl Biochem Biotechnol, 2002 Mar, 97(3), 193 - 208
Production, purification, and characterization of a polygalacturonase from a new strain of Kluyveromyces marxianus isolated from coffee wet-processing wastewater; Serrat M et al.; A new high polygalacturonase (PG)-producing Kluyveromyces marxianus strain was isolated from coffee wet-processing wastewater . PG production in this strain is not repressed in the presence of 100 g/L of glucose and, being growth-associated, reached its maximum accumulation in the culture medium at the beginning of the stationary phase . Oxygen and galacturonic acid negatively regulated enzyme synthesis, and glucose as the carbon source afforded better enzyme yields than lactose . The data reported here show that this strain exhibits the highest index of PG production among the wild-type strains reported so far (18.8 U/mL) . PG was readily purified by ion-exchange chromatography on SP-Sepharose FF . The activity corresponded to a single protein with an M(r) of 41.7kDa according to sodium dodecyl sulfatepolyacrylamide gel electrophoresis . The enzyme was stable in the pH range of 3.0-5.0 and displayed an optimal temperature of 55 degrees C; it showed a typical endosplitting way of substrate hydrolysis and exhibited a fair degree of activity on pectin with a high degree of esterification.

Mol Microbiol, 2002 May, 44(3), 865 - 75
KTI11 and KTI13, Saccharomyces cerevisiae genes controlling sensitivity to G1 arrest induced by Kluyveromyces lactis zymocin; Fichtner L et al.; The Kluyveromyces lactis zymocin and its gamma-toxin subunit inhibit cell cycle progression of Saccharomyces cerevisiae . To identify S . cerevisiae genes conferring zymocin sensitivity, we complemented the unclassified zymocin-resistant kti11 and kti13 mutations using a single-copy yeast library . Thus, we identified yeast open reading frames (ORFs) YBL071w-A and YAL020c/ATS1 as KTI11 and KTI13 respectively . Disruption of KTI11 and KTI13 results in the complex tot phenotype observed for the gamma-toxin target site mutants, tot1-7, and includes zymocin resistance, thermosensitivity, hypersensitivity to drugs and slow growth . Both loci, KTI11 and KTI13, are actively transcribed protein-encoding genes as determined by reverse transcriptase-polymerase chain reaction (RT-PCR) and in vivo HA epitope tagging . Kti11p is highly conserved from yeast to man, and Kti13p/Ats1p is related to yeast Prp20p and mammalian RCC1, components of the Ran-GTP/GDP cycle . Combining disruptions in KTI11 or KTI13 with a deletion in TOT3/ELP3 coding for the RNA polymerase II (RNAPII) Elongator histone acetyltransferase (HAT) yielded synthetic effects on slow growth phenotype expression . This suggests genetic interaction and possibly links KTI11 and KTI13 to Elongator function.

Yeast, 2002 May, 19(7), 601 - 9
The KlTrk1 gene encodes a low affinity transporter of the K+ uptake system in the budding yeast Kluyveromyces lactis; Miranda M et al.; Potassium uptake in Saccharomyces cerevisiae is mediated by at least two proteins, known as Trk1p and Trk2p . Direct involvement in cation movements has been demonstrated for Trk1p, which is the high affinity transporter . S . cerevisiae cells also show low affinity potassium uptake, perhaps mediated by Trk2p . Mutants lacking Trk1p, lose high affinity system, but when grown with moderate potassium concentrations, Trk2p seems to replace it . Mutants lacking both proteins are viable but require at least 10 mM K(+) in the medium to sustain growth . Here we report the cloning and characterization of a gene from Kluyveromyces lactis encoding a homologue of these two proteins . KlTrkp is a 1070 amino acid peptide that shows, overall, higher homology with Trk2p than with Trk1p, and its disruption gives rise to cells with deficient potassium transport and with an increased K(+) requirement for normal growth . Determination of kinetic parameters in the K . lactis wild-type and Kltrk1Delta strains, as well as in Sctrk1Delta Sctrk2Delta S . cerevisiae cells expressing KlTrk1, indicated that this is a low affinity component of a major potassium uptake system in K . lactis .

Appl Microbiol Biotechnol, 2002 Mar, 58(4), 503 - 10 Epub 2002 Feb 01.
Production of volatile compounds by cheese-ripening yeasts: requirement for a methanethiol donor for S-methyl thioacetate synthesis by Kluyveromyces lactis; Arfi K et al.; Five cheese-ripening yeasts (Geotrichum candidum, Saccharomyces cerevisiae, Kluyveromyces lactis, Yarrowia lipolytica and Debaryomyces hansenii) were compared with respect to their ability to generate volatile aroma compounds . K . lactis produced a variety of esters - ethylacetate (EA) being the major one - and relatively limited amounts of volatile sulphur compounds (VSCs) . Conversely, G . candidum produced significant amounts of VSCs {with the thioester S-methyl thioacetate (MTA) being the most prevalent} and lower quantities of non-sulphur volatile compounds than K . lactis . We suspect that K . lactis is able to produce and/or accumulate acetyl CoA - a common precursor of MTA and EA - but that it produces limited amounts of methanethiol (MTL); both acetyl CoA and MTL are precursors for MTA synthesis . When supplemented with exogenous MTL, MTA production greatly increased in K . lactis cultures whereas it was unchanged in G . candidum cultures, suggesting that MTL is a limiting factor for MTA synthesis in K . lactis but not in G . candidum . Our results are discussed with respect to L-methionine catabolism.

Bioorg Med Chem, 2002 Jun, 10(6), 1767 - 75
Molecular recognition by Kluyveromyces of amphotericin B-loaded, galactose-tagged, poly (lactic acid) microspheres; Kassab R et al.; In an effort to develop a new way of drug delivery, especially for polyenic antifungal molecules, we have incorporated amphotericin B (AmB) into biodegradable galactosylated poly (L-lactic acid) (L-PLA) and poly (L-lactic-co-glycolic acid) (PLGA) microspheres . These drug carriers were prepared by solvent evaporation using an oil/water (o/w) emulsion . The ratio of galactosyl spacers with different chain lengths was 1.74-2.78% . The maximal quantity of AmB encapsulated reported to 100 mg of the galactosylated microspheres was 7.14 mg for L-PLA (encapsulation rate 45% of mole) and 6.42 mg for PLGA derivatives (encapsulation rate 81% of mole) . In our yeast model, drug release depended on three factors: (i) presence of galactosylated antennae, (ii) length of galactosyl antenna and (iii) nature of the polymer . More of the AmB trapped in PLGA microspheres was released than from PLA microspheres . These novel functionalised microspheres could be required for the delivering of therapeutic agents according to their recognition to specific cells.

Mol Microbiol, 2002 Feb, 43(3), 783 - 91
Molecular analysis of KTI12/TOT4, a Saccharomyces cerevisiae gene required for Kluyveromyces lactis zymocin action; Fichtner L et al.; TOT, the putative Kluyveromyces lactis zymocin target complex from Saccharomyces cerevisiae, is encoded by TOT1-7, six loci of which are isoallelic to RNA polymerase II (RNAPII) Elongator genes (ELP1-6) . Unlike TOT1-3 (ELP1-3) and TOT5-7 (ELP5, ELP6 and ELP4 respectively), which display zymocin resistance when deleted, TOT4 (KTI12) also renders cells refractory to zymocin when maintained in multicopy or overexpressed from the GAL10 promoter . Elevated TOT4 copy number results in an intermediate tot phenotype, which includes mild sensitivities towards caffeine, Calcofluor white and elevated growth temperature, suggesting that TOT4 influences TOT/Elongator function . Tot4p interacts with Elongator, as shown by co-immunoprecipitation, and cell fractionation studies demonstrate partial co-migration with RNAPII and Elongator . As Elongator subunit interaction is not affected by either deletion of TOT4 or multicopy TOT4, Tot4p may not be a structural Elongator subunit but, rather, may regulate TOT/Elongator in a fashion that requires transient physical contact with TOT/Elongator . Consistent with a regulatory role, the presence of a potential P-loop motif conserved between yeast and human TOT4 homologues suggests capability of ATP or GTP binding and P-loop deletion renders Tot4p biologically inactive.

J Dairy Res, 2001 Nov, 68(4), 639 - 52
Chemical and microbiological characterisation of kefir grains; Garrote GL et al.; Chemical and microbiological composition of four Argentinean kefir grains from different sources as well as characteristics of the corresponding fermented milk were studied . Kefir grains CIDCA AGK1, AGK2 and AGK4 did not show significant differences in their chemical and microbiological composition . In contrast, protein and yeast content of AGK3 was higher than in the other grains . Although grain microflora comprised lactobacilli, lactococcus, acetic acid bacteria and yeast, we found an important difference regarding species . Lactococcus lactis subsp . lactis, Lactobacillus kefir, Lactobacillus plantarum, Acetobacter and Saccharomyces were present in all types of kefir grain . While Leuconostoc mesenteroides was only isolated from grains CIDCA AGK1 and Lactococcus lactis subsp . lactis biovar diacetylactis, Lactobacillus parakefir and Kluyveromyces marxianus were only isolated from CIDCA AGK2 grains . All grains produced acid products with pH between 3.5 and 4.0 . The apparent viscosity of AGK1 fermented milk was greater than the product obtained with AGK4 . All fermented milks had inhibitory power towards Escherichia coli but AGK1 and AGK2 supernatants were able to halt the bacterial growth for at least 25 h . Grain weight increment in AGK1, AGK2 and AGK3 during growth in milk did not show significant differences . Despite their fermenting activity, AGK4 grains did not increase their weight.

Curr Microbiol, 2002 May, 44(5), 379 - 82
The beta-galactosidase activity in Kluyveromyces marxianus CBS6556 decreases by high concentrations of galactose; Martins DB et al.; In this paper we report on the effect of different concentrations of lactose and galactose in the production of beta-galactosidase by Kluyveromyces marxianus CBS6556 . The results clearly demonstrate a decrease in enzyme specific activity during cultivation at high concentrations of L-lactose or D-galactose, despite the fact that these carbohydrates are normally used for induction of the beta-galactosidase activity . Therefore, maximum induction of beta-galactosidase in K . marxianus batch cultures was obtained at low concentrations of the inducer carbohydrates, in the range between 0.5 to 15 mM . Those informations can help to design low cost medium with higher beta-galactosidase productivity by K . marxianus cells.

Mol Genet Genomics, 2002 Mar, 267(1), 124 - 32 Epub 2002 Feb 22.
Lactose metabolism and cellulase production in Hypocrea jecorina: the gal7 gene, encoding galactose-1-phosphate uridylyltransferase, is essential for growth on galactose but not for cellulase induction; Seiboth B et al.; Lactose is at present the only soluble carbon source which can be used economically for the production by Hypocrea jecorina (= Trichoderma reesei) of cellulases or heterologous proteins under the control of cellulase expression signals . However, the mechanism by which lactose triggers the formation of cellulases is unknown . To enhance our understanding of lactose metabolism and its relationship to cellulase formation, we have cloned and characterized the gal7 gene (for galactose-1-phosphate uridylyltransferase) of H . jecorina . The gene encodes a polypeptide of 43.8 kDa, the sequence of which exhibits a moderate level of identity (about 50%) to that of the Gal7 proteins of Saccharomyces cerevisiae and Kluyveromyces lactis, and contains an active-site signature typical for galactose-1-phosphate uridylyltransferase family 1 . H . jecorina gal7 is not clustered with other genes of galactose metabolism . A single 1.7-kb transcript is synthesized constitutively during the rapid growth phase and accumulated to twice this level during incubation in the presence of D-galactose and L-arabinose and the corresponding polyols (dulcitol, arabitol) . A gal7 deletion mutant, constructed by replacing the gal7 reading frame by the H . jecorina pyr4 gene, was unable to grow on D-galactose between pH 4.5 and 7.5, thus proving that in H . jecorina gal7 is essential for metabolism of D-galactose, whereas the growth rate of the mutant on lactose was only reduced by about 50% . The rate of formation of cellobiohydrolase Cel7A and the abundance of the corresponding (cbh1) transcript during growth on lactose was only slightly lower in the absence of gal7, but a significant delay in decay of the cbh1 transcript was noted during later stages of growth . The results suggest that H . jecorina uses only the Leloir pathway for metabolism of D-galactose and lactose . Furthermore, we conclude that metabolism of lactose past the galactose-1-phosphate step is not essential for cellulase formation.

Curr Genet, 2002 Mar, 40(6), 355 - 64 Epub 2002 Feb 28.
Regulation of glycolysis by casein kinase I (Rag8p) in Kluyveromyces lactis involves a DNA-binding protein, Sck1p, a homologue of Sgc1p of Saccharomyces cerevisiae; Lemaire M et al.; The casein kinase I (Rag8p) of Kluyveromyces lactis has previously been shown to regulate the transcription of the low-affinity glucose transporter gene RAG1 . To study this regulation, we have isolated multicopy suppressors of the rag8 mutation . One of them, SCK1 (suppressor of casein kinase), was characterised . The predicted product of the gene has a DNA-binding signature of the basic-helix-loop-helix type . It has an overall identity of 38% with Sgc1p (Tye7p) of Saccharomyces cerevisiae . The sck1 null mutant exhibited a Rag- phenotype (which indicates a reduced flux of glycolysis) that can be complemented by the SGC1 gene of S . cerevisiae . The level of transcription of several glycolytic genes, including RAG1, was reduced about twofold in glucose media in the sck1 null mutant . Moreover, in a rag8 mutant, the expression of SCK1 was strongly affected . Altogether, the results suggest that the regulation of glycolysis by casein kinase I involves, at least in part, Sck1p in K . lactis.

Nucleic Acids Res . 2002 Mar 15;30(6):e23.
A second set of loxP marker cassettes for Cre-mediated multiple gene knockouts in budding yeast; Gueldener U et al.; Heterologous markers are important tools required for the molecular dissection of gene function in many organisms, including Saccharomyces cerevisiae . Moreover, the presence of gene families and isoenzymes often makes it necessary to delete more than one gene . We recently introduced a new and efficient gene disruption cassette for repeated use in budding yeast, which combines the heterologous dominant kan(r) resistance marker with a Cre/loxP-mediated marker removal procedure . Here we describe an additional set of four completely heterologous loxP-flanked marker cassettes carrying the genes URA3 and LEU2 from Kluyveromyces lactis, his5(+) from Schizosaccharomyces pombe and the dominant resistance marker ble(r) from the bacterial transposon Tn5, which confers resistance to the antibiotic phleomycin . All five loxP--marker gene--loxP gene disruption cassettes can be generated using the same pair of oligonucleotides and all can be used for gene disruption with high efficiency . For marker rescue we have created three additional Cre expression vectors carrying HIS3, TRP1 or ble(r) as the yeast selection marker . The set of disruption cassettes and Cre expression plasmids described here represents a significant further development of the marker rescue system, which is ideally suited to functional analysis of the yeast genome.

Appl Microbiol Biotechnol, 2002 Feb, 58(2), 195 - 201
Evaluation of pKD1-based plasmid systems for heterologous protein production in Kluyveromyces lactis; Panuwatsuk W et al.; The stability of pKD1-based vectors was evaluated during the synthesis of intracellular and extracellular gene products in the yeast Kluyveromyces lactis . The Escherichia coli lacZ and MFalpha1 leader-BPTI (bovine pancreatic trypsin inhibitor) cassettes were placed under the control of the inducible K . lactis LAC4 promoter and inserted into the pKD1-based plasmids . To induce gene expression while maintaining inducer level, a gratuitous gal1-209 K . lactis strain was employed . Selective medium containing 5 g glucose/l and 0.5 g galactose (inducer)/l allowed optimum expression and secretion of heterologous products without a significant effect on the growth of the recombinant cells . During long-term sequential batch cultures (60 generations), plasmid instability was mainly the result of structural instability . The expression and secretion of BPTI resulted in greater structural instability relative to the intracellular beta-galactosidase . For both products, vectors carrying the pKD1 replication origin and the cis-acting stability locus (partial-pKD1 vectors) were more stable than vectors carrying the full pKD1 sequence (full-pKD1 vectors) . However, after 55 generations, the beta-galactosidase and BPTI activities were still higher with the full-pKD1 vectors . This was due to the significantly higher initial beta-galactosidase and BPTI activities for the full-pKD1 vectors (approximately 85% and 47% higher, respectively) relative to the partial-pKDI vectors . Southern blots confirmed that these increases were due to the higher copy number of the vectors carrying the full pKD1 sequence . In contrast to our previously reported results for the secretion of invertase, full-pKD1 vectors were preferred for the expression/secretion of beta-galactosidase and BPTI for at least 55 generations . Due to their structural stability, partial-pKD1 vectors will be advantageous for very long cultivation times.

Yeast, 2002 Mar 15, 19(4), 319 - 28
Efficient PCR-based gene disruption in Saccharomyces strains using intergenic primers; Reid RJ et al.; Gene disruptions are a vital tool for understanding Saccharomyces cerevisiae gene function . An arrayed library of gene disruption strains has been produced by a consortium of yeast laboratories; however their use is limited to a single genetic background . Since the yeast research community works with several different strain backgrounds, disruption libraries in other common laboratory strains are desirable . We have developed simple PCR-based methods that allow transfer of gene disruptions from the S288C-derived strain library into any Saccharomyces strain . One method transfers the unique sequence tags that flank each of the disrupted genes and replaces the kanamycin resistance marker with a recyclable URA3 gene from Kluyveromyces lactis . All gene-specific PCR amplifications for this method are performed using a pre-existing set of primers that are commercially available . We have also extended this PCR technique to develop a second general gene disruption method suitable for any transformable strain of Saccharomyces .

Antonie Van Leeuwenhoek, 2001 Dec, 80(3-4), 225 - 36
Isolation and biochemical characterization of cell wall tight protein complex involved in self-flocculation of Kluyveromyces bulgaricus; Gehin G et al.; Flocculation of yeasts is a cell-cell aggregation phenomenon which is driven by interactions between cell wall lectins and cell wall heteropolysaccharides . In Sabouraud medium, Kluyveromyces bulgaricus was highly flocculent . Incubation of flocculent K . bulgaricus cells with EDTA or Hecameg led to extracts showing hemagglutinating and flocculating properties . Purification of the extracts by native PAGE gave two bands which allowed flocculation of deflocculated K . bulgaricus . Both bands with specific reflocculating activity were composed of five subunits, of which only three possessed weak reflocculating activity upon deflocculated yeast . The mixture of these three proteins allow the recovery of initial specific reflocculating activity of the complex . These three proteins, denoted p28, p36 and p48, presented, in their first 15 amino acids, homologies with glycolysis enzymes, i.e., 3-phosphoglycerate mutase, glyceraldehyde-3-phosphate dehydrogenase and enolase, respectively . However, no such enzymatic activity could be detected in the crude extract issued from treatment with EDTA and Hecameg of flocculent yeast cells . When yeasts had grown in glucose poor medium, flocculation was drastically affected . The EDTA and Hecameg crude extracts showed weak reflocculating activity . After PAGE, the protein complexes did not appear in the EDTA extract, but they did appear in the Hecameg crude extract . These results suggest that: (i) self-flocculation of K . bulgaricus depends on the expression of different floc-forming protein complex, (ii) these proteins are galactose specific lectins showing homologies in their primary structure with glycolysis enzymes.

Yeast, 2002 Feb, 19(3), 257 - 68
An analysis of inter- and intraspecific genetic variabilities in the Kluyveromyces marxianus group of yeast species for the reconsideration of the K . lactis taxon; Belloch C et al.; In the present work, we analyse the sequences of the 5.8S rRNA gene and the two internal transcribed spacers 1 and 2 (5.8S-ITS region), obtained from 39 strains belonging to the species Kluyveromyces aestuarii, K . dobzhanskii, K . lactis and K . marxianus, K . nonfermentans and K . wickerhamii, to solve the phylogenetic relationships among these species and also to determine the possible genetic basis for the delimitation of the two currently accepted K . lactis varieties: lactis, including lactose-positive strains isolated from dairy products, and drosophilarum, comprising lactose-negative strains isolated from insects and plant exudates . The determination of the phylogenetic relationships within the species K . lactis, together with the examination of the electrophoretic karyotypes and phenotypic characterization of strains representatives of K . lactis var . lactis and var . drosophilarum, allowed differentiation of two groups of strains . The first, and ancestral, group comprises lactose-negative strains isolated from natural habitats in North America . The second, and derived, group includes both lactose-negative strains isolated from natural habitats in Europe and wine fermentation in South Africa, and lactose-positive strains associated with dairy products . These results suggest that the present taxon K . lactis is a complex of different species, subspecies or, at least, genetically structured populations .

Genetics, 2002 Jan, 160(1), 63 - 73
Dynamics of telomeric DNA turnover in yeast; McEachern MJ et al.; Telomerase adds telomeric DNA repeats to telomeric termini using a sequence within its RNA subunit as a template . We characterized two mutations in the Kluyveromyces lactis telomerase RNA gene (TER1) template . Each initially produced normally regulated telomeres . One mutation, ter1-AA, had a cryptic defect in length regulation that was apparent only if the mutant gene was transformed into a TER1 deletion strain to permit extensive replacement of basal wild-type repeats with mutant repeats . This mutant differs from previously studied delayed elongation mutants in a number of properties . The second mutation, TER1-Bcl, which generates a BclI restriction site in newly synthesized telomeric repeats, was indistinguishable from wild type in all phenotypes assayed: cell growth, telomere length, and in vivo telomerase fidelity . TER1-Bcl cells demonstrated that the outer halves of the telomeric repeat tracts turn over within a few hundred cell divisions, while the innermost few repeats typically resisted turnover for at least 3000 cell divisions . Similarly deep but incomplete turnover was also observed in two other TER1 template mutants with highly elongated telomeres . These results indicate that most DNA turnover in functionally normal telomeres is due to gradual replicative sequence loss and additions by telomerase but that there are other processes that also contribute to turnover.

Biochem Biophys Res Commun, 2002 Jan 18, 290(2), 802 - 5
Iminodiacetate and nitrilotriacetate degradation by Kluyveromyces marxianus IMB3; Ternan NG et al.; The thermotolerant yeast Kluyveromyces marxianus IMB3 was capable of utilising either iminodiacetate or nitrilotriacetate as a sole source of nitrogen for growth . Cell extracts contained iminodiacetate dehydrogenase and nitrilotriacetate monooxygenase activities, suggesting the presence in the yeast of orthologues of these bacterial enzymes . The activities were not detectable in complete medium-growth cells, nor in nitrogen-starved cells, suggesting an inducible biodedgradation pathway for biodegradation of these xenobiotics, which has not been previously reported in a eukaryotic cell system . This observation emphasises the hitherto unrealised importance of yeast strains in the biodegradation of xenobiotics in the environment.

Mikrobiol Z, 2001 Sep-Oct, 63(5), 44 - 8
{Search for lectin producers among some yeast species}; Kovalenko EA et al.; Hemagglutinating properties of culture liquid and cells of 142 yeast strains of species Saccharomyces cerevisiae, Kluyveromyces marxianus, K . dobzhanskii, K . lactis var . lactis and K . lactis var . drosophilarum, isolated from various sources have been studied . A capacity of yeast (44% of the studied strains) to synthesize both lectins bound to a cell and extracellular lectins has been established; the latter have been found in yeast for the first time.

Microbiology, 2002 Jan, 148(Pt 1), 41 - 50
Secretion of active anti-Ras single-chain Fv antibody by the yeasts Yarrowia lipolytica and Kluyveromyces lactis; Swennen D et al.; Yarrowia lipolytica and Kluyveromyces lactis secretion vectors were constructed and assessed for the expression of heterologous proteins . An anti-Ras single-chain antibody fragment (scFv) coding sequence was fused in-frame to different pre- or prepro-regions, or downstream from a reporter secretory gene (Arxula adeninivorans glucoamylase), separated by a Kex2 protease (Kex2p)-like processing sequence . Both organisms are able to secrete soluble scFv, with yields depending on the nature of the expression cassette, up to levels ranging from 10 to 20 mg l(-1) . N-terminal sequence analysis of the purified scFv showed that fusions are correctly processed to the mature scFv by a signal peptidase or a Kex2p-type endoprotease present in Y . lipolytica and K . lactis . The scFv protein also retains the capacity to bind to a glutathioneS-transferase (GST)-Harvey-Ras(Val12) fusion, indicating that the antibody is functional . These results indicate that the yeasts Y . lipolytica and K . lactis have potential for industrial production of soluble and active scFv.

Genetics, 2001 Dec, 159(4), 1479 - 89
Sit4p protein phosphatase is required for sensitivity of Saccharomyces cerevisiae to Kluyveromyces lactis zymocin; Jablonowski D et al.; We have identified two Saccharomyces cerevisiae genes that, in high copy, confer resistance to Kluyveromyces lactis zymocin, an inhibitor that blocks cells in the G(1) phase of the cell cycle prior to budding and DNA replication . One gene (GRX3) encodes a glutaredoxin and is likely to act at the level of zymocin entry into sensitive cells, while the other encodes Sap155p, one of a family of four related proteins that function positively and interdependently with the Sit4p protein phosphatase . Increased SAP155 dosage protects cells by influencing the sensitivity of the intracellular target and is unique among the four SAP genes in conferring zymocin resistance in high copy, but is antagonized by high-copy SAP185 or SAP190 . Since cells lacking SIT4 or deleted for both SAP185 and SAP190 are also zymocin resistant, our data support a model whereby high-copy SAP155 promotes resistance by competition with the endogenous levels of SAP185 and SAP190 expression . Zymocin sensitivity therefore requires a Sap185p/Sap190p-dependent function of Sit4p protein phosphatase . Mutations affecting the RNA polymerase II Elongator complex also confer K . lactis zymocin resistance . Since sit4Delta and SAP-deficient strains share in common several other phenotypes associated with Elongator mutants, Elongator function may be a Sit4p-dependent process.

Mycopathologia, 2001, 152(2), 85 - 9
Genetic and physiological variants of yeast selected from palm wine; Ezeronye OU et al.; Genetic screening of 1200-palm wine yeasts lead to the selection of fourteen isolates with various genetic and physiological properties . Nine of the isolates were identified as Saccharamyces species, three as Candida species, one as Schizosaccharomyces species and one as Kluyveromyces species . Five of the isolates were wild type parents, two were respiratory deficient mutants (rho) and nine were auxotrophic mutants . Four isolates were heterozygous diploid (alphaa) and two were homozygous diploid (aa/alphaalpha) for the mating a mating types were further identified on mating with type loci . Four Mat alpha and four Mat a types were further identified on mating with standard haploid yeast strains . Forty-five percent sporulated on starvation medium producing tetrads . Fifty-two percent of the four-spored asci contained four viable spores . Maximum specific growth rate {micromax} of the fourteen isolates range from 0.13-0.26, five isolates were able to utilize exogenous nitrate for growth . Percentage alcohol production range between 5.8-8.8% for palm wine yeast, 8.5% for bakers' yeast and 10.4% for brewers yeast . The palm wine yeast were more tolerant to exogenous alcohol but had a low alcohol productivity . Hybridization enhanced alcohol productivity and tolerance in the palm wine yeasts.

Int J Syst Evol Microbiol, 2001 Nov, 51(Pt 6), 2189 - 98
Three new species of Saccharomyces sensu lato van der Walt from Yaku Island in Japan: Saccharomyces naganishii sp . nov., Saccharomyces humaticus sp . nov . and Saccharomyces yakushimaensis sp . nov; Mikata K et al.; Three new yeast species were isolated from soil and partially decayed leaves in Yaku Island and Iriomote Island in the Nansei Islands of Japan . Based on DNA hybridization and physiological characters, these represent novel taxa . These are designated Saccharomyces naganishii sp . nov . (type strain IFO 10181T = CBS 8797T), Saccharomyces humaticus sp . nov . (type strain IFO 10673T = CBS 8893T) and Saccharomyces yakushimaensis sp . nov . (type strain IFO 1889T = CBS 8894T) . The phylogenetic relationships of the three new species with members of other ascomycetous genera (e.g . Kluyveromyces, Saccharomyces, Torulaspora and Zygosaccharomyces) were estimated by 18S rDNA gene sequence analysis.

J Bacteriol, 2002 Jan, 184(2), 427 - 32
Respiration-dependent utilization of sugars in yeasts: a determinant role for sugar transporters; Goffrini P et al.; In many yeast species, including Kluyveromyces lactis, growth on certain sugars (such as galactose, raffinose, and maltose) occurs only under respiratory conditions . If respiration is blocked by inhibitors, mutation, or anaerobiosis, growth does not take place . This apparent dependence on respiration for the utilization of certain sugars has often been suspected to be associated with the mechanism of the sugar uptake step . We hypothesized that in many yeast species, the permease activities for these sugars are not sufficient to ensure the high substrate flow that is necessary for fermentative growth . By introducing additional sugar permease genes, we have obtained K . lactis strains that were capable of growing on galactose and raffinose in the absence of respiration . High dosages of both the permease and maltase genes were indeed necessary for K . lactis cells to grow on maltose in the absence of respiration . These results strongly suggest that the sugar uptake step is the major bottleneck in the fermentative assimilation of certain sugars in K . lactis and probably in many other yeasts.

Mol Microbiol, 2001 Nov, 42(4), 1095 - 105
Kluyveromyces lactis zymocin mode of action is linked to RNA polymerase II function via Elongator; Jablonowski D et al.; The putative Kluyveromyces lactis zymocin target complex, TOT, from Saccharomyces cerevisiae comprises five Tot proteins, four of which are RNA polymerase II (RNAP II) Elongator subunits . Recently, two more Elongator subunit genes, ELP6 (TOT6) and ELP4 (TOT7), have been identified . Deletions of both TOT6 and TOT7 result in the complex tot phenotype, including resistance to zymocin, thermosensitivity, slow growth and hypersensitivity towards drugs, thus reinforcing the notion that TOT/Elongator may be crucial in signalling zymocicity . Mutagenesis of ELP3/TOT3, the Elongator histone acetyltransferase (HAT) gene, revealed that zymocin sensitivity could be uncoupled from Elongator wild-type function, indicating that TOT interacts genetically with zymocin . To test the possibility that zymocin functions by affecting RNAP II activity in a TOT/Elongator-dependent manner, global poly(A)+ mRNA levels were found to decline drastically on zymocin treatment . Moreover, cells overexpressing Fcp1p, the RNAP II carboxy-terminal domain phosphatase, acquired partial zymocin resistance, whereas cells underproducing RNAP II became zymocin hypersensitive . This suggests that zymocin may convert TOT/Elongator into a cellular poison toxic for RNAP II function and eventually leading to the observed G1 cell cycle arrest.

Genetics, 2001 Nov, 159(3), 929 - 38
Dual mutations reveal interactions between components of oxidative phosphorylation in Kluyveromyces lactis; Clark-Walker GD et al.; Loss of mtDNA or mitochondrial protein synthesis cannot be tolerated by wild-type Kluyveromyces lactis . The mitochondrial function responsible for rho(0)-lethality has been identified by disruption of nuclear genes encoding electron transport and F(0)-ATP synthase components of oxidative phosphorylation . Sporulation of diploid strains heterozygous for disruptions in genes for the two components of oxidative phosphorylation results in the formation of nonviable spores inferred to contain both disruptions . Lethality of spores is thought to result from absence of a transmembrane potential, Delta Psi, across the mitochondrial inner membrane due to lack of proton pumping by the electron transport chain or reversal of F(1)F(0)-ATP synthase . Synergistic lethality, caused by disruption of nuclear genes, or rho(0)-lethality can be suppressed by the atp2.1 mutation in the beta-subunit of F(1)-ATPase . Suppression is viewed as occurring by an increased hydrolysis of ATP by mutant F(1), allowing sufficient electrogenic exchange by the translocase of ADP in the matrix for ATP in the cytosol to maintain Delta Psi . In addition, lethality of haploid strains with a disruption of AAC encoding the ADP/ATP translocase can be suppressed by atp2.1 . In this case suppression is considered to occur by mutant F(1) acting in the forward direction to partially uncouple ATP production, thereby stimulating respiration and relieving detrimental hyperpolarization of the inner membrane . Participation of the ADP/ATP translocase in suppression of rho(0)-lethality is supported by the observation that disruption of AAC abolishes suppressor activity of atp2.1.

Appl Environ Microbiol, 2001 Dec, 67(12), 5621 - 5
Efficient homolactic fermentation by Kluyveromyces lactis strains defective in pyruvate utilization and transformed with the heterologous LDH gene; Bianchi MM et al.; A high yield of lactic acid per gram of glucose consumed and the absence of additional metabolites in the fermentation broth are two important goals of lactic acid production by microrganisms . Both purposes have been previously approached by using a Kluyveromyces lactis yeast strain lacking the single pyruvate decarboxylase gene (KlPDC1) and transformed with the heterologous lactate dehydrogenase gene (LDH) . The LDH gene was placed under the control the KlPDC1 promoter, which has allowed very high levels of lactate dehydrogenase (LDH) activity, due to the absence of autoregulation by KlPdc1p . The maximal yield obtained was 0.58 g g(-1), suggesting that a large fraction of the glucose consumed was not converted into pyruvate . In a different attempt to redirect pyruvate flux toward homolactic fermentation, we used K . lactis LDH transformant strains deleted of the pyruvate dehydrogenase (PDH) E1alpha subunit gene . A great process improvement was obtained by the use of producing strains lacking both PDH and pyruvate decarboxylase activities, which showed yield levels of as high as 0.85 g g(-1) (maximum theoretical yield, 1 g g(-1)), and with high LDH activity.

Can J Microbiol, 2001 Sep, 47(9), 861 - 70
Characterization of major protein phosphatases from selected species of Kluyveromyces . Comparison with protein phosphatases from Yarrowia lipolytica; Jolivet P et al.; Casein phosphatase activities have been identified in five yeast strains grown on Pi-deficient medium . Maximal endocellular activities appeared in the exponential phase . Exocellular phosphatases were significantly produced from Yarrowia lipolytica W-29 and Kluyveromyces marxianus, in the early stationary phase . Major phosphatases from K . marxianus were one heavy acid phosphatase composed of 64-67 kDa subunits, which could be secreted in the medium, and one type 2A protein phosphatase with an apparent molecular mass of 147 kDa and a 52 kDa catalytic subunit dissociated by 80% ethanol treatment . The characteristics of phosphatases purified from K . marxianus were compared with those previously purified from Y . lipolytica.

Mol Genet Genomics, 2001 Oct, 266(2), 326 - 35
A DNA replication origin and a replication fork barrier used in vivo in the circular plasmid pKD1; Fabiani L et al.; As in other yeasts, ARS-containing plasmids can be maintained extrachromosomally in Kluyveromyces lactis . Although some fragments of K . lactis DNA have ARS activity in both K . lactis and Saccharomyces cerevisiae, it appears that the sequences required for ARS activity in the two yeasts are different . As an approach to a better understanding of ARS structure and function in K . lactis, we analyzed the replication of the circular plasmid pKD1 . We identified a 159-bp sequence able to promote autonomous replication of pKD1 in both yeasts; this fragments contains both a sequence related to the S . cerevisiae ARS consensus sequence and a region of 53% identity to the 40-bp sequence essential for K . lactis KARS101 function . By the analysis of in vivo replication intermediates we provide the first direct evidence that DNA replication initiates at or near the K . lactis ARS element . Replication terminates at the cisacting stability locus of pKD1, which functions as a replication fork barrier (RFB) and is necessary for proper plasmid segregation . RFB activity requires the pKDI gene products that are important for plasmid segregation, suggesting a link between DNA replication termination and plasmid segregation in a eukaryotic organism.

Crit Rev Biotechnol, 2001, 21(3), 177 - 218
Transformation systems of non-Saccharomyces yeasts; Wang TT et al.; This review describes the transformation systems including vectors, replicons, genetic markers, transformation methods, vector stability, and copy numbers of 13 genera and 31 species of non-Saccharomyces yeasts . Schizosaccharomyces pombe was the first non-Saccharomyces yeast studied for transformation and genetics . The replicons of non-Saccharomyces yeast vectors are from native plasmids, chromosomal DNA, and mitochondrial DNA of Saccharomyces cerevisiae, non-Saccharomyces yeasts, protozoan, plant, and animal . Vectors such as YAC, YCp, YEp, YIp, and YRp were developed for non-Saccharomyces yeasts . Forty-two types of genes from bacteria, yeasts, fungi, and plant were used as genetic markers that could be classified into biosynthetic, dominant, and colored groups to construct non-Saccharomyces yeasts vectors . The LEU2 gene and G418 resistance gene are the two most popular markers used in the yeast transformation . All known transformation methods such as spheroplast-mediating method, alkaline ion treatment method, electroporation, trans-kingdom conjugation, and biolistics have been developed successfully for non-Saccharomyces yeasts, among which the first three are most widely used . The highest copy number detected from non-Saccharomyces yeasts is 60 copies in Kluyveromyces lactis . No general rule is known to illustrate the transformation efficiency, vector stability, and copy number, although factors such as vector composition, host strain, transformation method, and selective pressure might influence them.

Mol Biol Cell, 2001 Oct, 12(10), 3191 - 203
Role for telomere cap structure in meiosis; Maddar H et al.; Telomeres, the natural ends of eukaryotic chromosomes, are essential for the protection of chromosomes from end-to-end fusions, recombination, and shortening . Here we explore their role in the process of meiotic division in the budding yeast, Kluyveromyces lactis . Telomerase RNA mutants that cause unusually long telomeres with deregulated structure led to severely defective meiosis . The severity of the meiotic phenotype of two mutants correlated with the degree of loss of binding of the telomere binding protein Rap1p . We show that telomere size and the extent of potential Rap1p binding to the entire telomere are irrelevant to the process of meiosis . Moreover, we demonstrate that extreme difference in telomere size between two homologous chromosomes is compatible with the normal function of telomeres during meiosis . In contrast, the structure of the most terminal telomeric repeats is critical for normal meiosis . Our results demonstrate that telomeres play a critical role during meiotic division and that their terminal cap structure is essential for this role.

Biotechnol Appl Biochem, 2001 Oct, 34(Pt 2), 103 - 7
Nisin production from Lactococcus lactis A.T.C.C . 7962 using supplemented whey permeate; Flores SH et al.; The influence of pH control and aeration (20% dissolved oxygen) on nisin production in a supplemented cheese whey permeate was examined during batch fermentation with Lactococcus lactis subsp . lactis A.T.C.C . 7962 . A maximum nisin activity of 5280 i.u./ml of medium was observed in the raw extract of nisin after 9 h of fermentation with a constant pH at 4.9 . However, the fermentation was continued until 24 h, when a decrease in the nisin activity was observed . The pH control did not influence the nisin production and aeration of the culture medium increased cell growth (biomass) but not nisin activity . The yeast Kluyveromyces marxianus, used as an alternative method to control pH, has not been efficient.

Int J Food Microbiol, 2001 Sep 19, 69(1-2), 53 - 8
Yeasts associated with Sardinian ewe's dairy products; Cosentino S et al.; In the present work, the occurrence of yeasts in different types of typical Sardinian ewe's cheeses (32 samples of pecorino, 32 of caciotta, 40 of feta, 56 of ricotta) was determined . For the strains isolated the following properties were studied: proteolytic and lipolytic activities, the ability to grow at different temperatures, different concentrations of salt, and to assimilate and/or ferment compounds like lactate, citrate, lactose, glucose, galactose, lactic acid . Of 160 samples analysed, 76.2% yielded growth of yeasts . Yeast counts showed a certain variability among the samples . The highest levels were observed in caciotta and feta cheeses . A total of 281 strains belonging to 16 genera and 25 species were identified . In general, Debaryomyces hansenii was the dominant species, representing 28.8% of the total isolates . Other frequently appearing species were Geotrichum candidum, Kluyveromyces lactis and K . marxianus . Other genera encountered were Pichia, Candida, Dekkera, Yarrowia and Rhodotorula . With regard to the biochemical and technological properties of the yeasts, only K . lactis, K . marxianus and Dek . anomala assimilated and fermented lactose, whereas the majority of the species assimilated lactic acid . The assimilation of citrate was a characteristic of D . hansenii, R . rubra and Y . lipolytica . On the whole, the yeasts were weakly proteolytic while lipolytic activity was present in several species . A high percentage of strains showed a certain tolerance to low temperatures while only some strains of D . hansenii and K . lactis were able to grow at a 10% NaCl concentration.

Int J Food Microbiol, 2001 Sep 19, 69(1-2), 45 - 51
Yeasts from Water Buffalo Mozzarella, a traditional cheese of the Mediterranean area; Romano P et al.; Countries of the Mediterranean area are characterized by production of artisanal cheeses, obtained from goat, sheep, cow and buffalo raw milk . The numbers and species of yeasts in the different cheeses are variable, but some species are more frequently detected than others . Kluyveromyces marxianus, K . lactis with their anamorph, Candida kefir, Debaryomyces hansenii and C . famata, C . colliculosa and C . catenulata are dominant species in several cheeses . However, Saccharomyces cerevisiae is often detected in pasta filata cheeses, such as Water Buffalo Mozzarella (WBM) or Cacio Cavallo Podolico . Recently, a comprehensive study of yeasts isolated from Mozzarella cheese produced in Basilicata (Southern Italy) has been carried out . The study has focused on lactose and/or galactose fermenting species (Kluyveromyces and Saccharomyces) to evaluate their role on the functional and sensory properties of the product . End products in milk were evaluated and the biodiversity in terms of production of sulphur dioxide, higher alcohols, ethyl acetate, and acetaldehyde was studied . In particular, S . cerevisiae strains from Water Buffalo Mozzarella cheese, compared to strains isolated from different habitats, such as wine, exhibited considerable difference in the production of some volatile compounds . The diversity observed could be related to the particular microhabitat of S . cerevisiae occurring in whey cheese of water buffalo milk.

Int J Food Microbiol, 2001 Sep 19, 69(1-2), 153 - 6
Yeast populations in Sardinian feta cheese; Fadda ME et al.; In this study, the yeast populations in feta cheese from two different Sardinian dairies were examined . Samples of good quality feta (32) and samples of feta with a slimy surface defect (10) were examined from Dairy A . Similar, samples of good quality feta (23), feta with slimy surface defects (14) and samples with swelling defects (6) were examined from Dairy B . Kluyveromyces lactis was the dominating species in feta from Dairy A (95.2% of samples) followed by Debaryomyces hansenii (76.2%), Dekkera anomala (28.6%) and Dek . bruxellensis (19%) . D . hansenii was dominant in samples from Dairy B (93%), followed by K . lactis (23.3%), Geotrichum candidum (23.3%) and Dek . anomala (18.6%) . No significant difference was observed between the occurrence of yeast species in feta of good quality and in feta with slimy surface defects, thus confirming that slimy production is not associated with yeast contaminations . The swelling of samples observed in Dairy B seems to be caused by Dek . anomala . In fact, this strong fermenting species was present in all swelled samples in numbers exceeding 10(6) CFU g(-1), while it was isolated in very low concentration in only 5.4% of good samples.

Int J Food Microbiol, 2001 Sep 19, 69(1-2), 11 - 24
DNA typing methods for differentiation of Debaryomyces hansenii strains and other yeasts related to surface ripened cheeses; Petersen KM et al.; The discriminative power of ITS-PCR, ITS-PCR RFLP and mitochondrial (mt)-DNA RFLP were evaluated for differentiation of yeasts of importance for surface ripened cheeses . In total 60 isolates were included . Of these, 40 strains of the following species, Debaryomyces hansenii var . hansenii, D . hansenii var . fabryi, Saccharomyces cerevisiae, Candida zeylanoides, Kluyveromyces lactis and Yarrowia lipolytica, were obtained from culture collections and 20 isolates of D . hansenii representing six different phenotypes were collected from seven Danish producers of surface ripened cheeses . ITS-PCR was evaluated for differentiation at species level on the 40 strains obtained from culture collections . Ten strains of each variety of D . hansenii and five strains of each of the above mentioned species were analysed . For each of the investigated species, a specific ITS1-5.8S rDNA-ITS2 region size was observed . Accordingly ITS-PCR was found valuable for differentiation at species level of yeasts of importance for surface ripened cheeses . ITS-PCR RFLP was investigated for the purpose of strain typing of D . hansenii . Ten CBS strains of each variety of D . hansenii were analysed . Only one enzyme (TaqI) out of several investigated (BamHI, DpnI, Fnu4HI, HaeIII, HindIII, HpaII, NlaII, Sau3AI, TaqI) demonstrated genetic diversity within the strains . This enzyme divided the 20 strains in three groups . Sequence analysis of the ITS1-5.8S rDNA-ITS2 region for the type strains of each variety of D . hansenii showed an identity of 99.84%, corresponding to a difference in one basepair . Based on these results, ITS-PCR RFLP was found ineffective for strain typing of D . hansenii . MtDNA RFLP using HaeIII and HpaII was evaluated for strain typing of D . hansenii on the 20 CBS strains of D . hansenii . The CBS strains were divided into 16 groups according to their restriction profiles, which proved the method useful for typing of D . hansenii at subspecies level . The 20 dairy isolates showed a lower genetic variability than the CBS strains as they were divided into eight groups . Cluster analysis of the 20 CBS strains and the 20 dairy isolates based on their mtDNA restriction profiles showed (max . similarity level = 52%) that the dairy isolates only clustered with the CBS strains of D . hansenii var . hansenii . For some of the dairies more than one strain of D . hansenii were found to be involved in the ripening process, indicating that the method could be useful for subspecies typing and investigation of the microbial succession between strains of D . hansenii during the ripening process of surface ripened cheeses.

FEMS Microbiol Lett, 2001 Sep 25, 203(2), 229 - 33
The role of glucose in the Kluyveromyces bulgaricus flocculation phenomenon: transduction by cAMP-dependent protein kinase pathway?
Gehin G, Bonaly R, Coulon J.
Yeast flocculation appears to be dependent on several culture conditions such as nitrogen or carbon sources . In 0.2% glucose medium Kluyveromyces bulgaricus flocculation intensity is weak (10% at maximum) by comparison with flocculation in 2% glucose medium (85% maximum) . Addition of glucose to K . bulgaricus in exponential growth phase in 0.2% glucose medium produced a rapid increase of the flocculation percentage during the 30 min following the addition of glucose . cAMP and 2,4-dinitrophenol showed similar effects while cAMP-dependent protein kinase (PKA) inhibitors exhibited an antagonist effect . Moreover, the induction of flocculation did not seem to imply translation of new proteins: cycloheximide had no effect, although growth was inhibited . The induction of flocculation mainly implies ATP hydrolysis for activation or secretion of galactose-specific receptors as demonstrated by treatment with NaN(3) . We propose a hypothesis that involves a PKA transduction signal leading to flocculation.

Yeast, 2001 Oct, 18(14), 1347 - 55
The KlCYC1 gene, a downstream region for two differentially regulated transcripts; Freire-Picos MA et al.; KlCYC1 encodes for cytochrome c in the yeast Kluyveromyces lactis and is transcribed in two mRNAs with different 3'-processing points . This is an uncommon transcription mechanism in yeast mRNAs . The 3' sequence encompassing the whole region that is needed to produce both mRNAs is analysed . We have determined identical processing points in K.lactis and in Saccharomyces cerevisiae cells transformed with KlCYC1; positions 698 and 1092 (with respect to the TAA) are the major polyadenylation points . This shows that the cis-elements present in the KlCYC1 3'-untranslated region (3'-UTR) direct a processing mechanism that has been conserved in yeast . In K . lactis there is a high predominance of the shorter transcript (1.14 kb) only at the initial logarithmic growth phase . Interestingly, this growth phase-dependent regulation of 3'-UTR processing is lost when the gene is expressed in S . cerevisiae .

Yeast, 2001 Oct, 18(14), 1285 - 99
Saccharomyces cerevisiae cell wall chitin, the Kluyveromyces lactis zymocin receptor; Jablonowski D et al.; The exozymocin secreted by Kluyveromyces lactis causes sensitive yeast cells, including Saccharomyces cerevisiae, to arrest growth in the G(1) phase of the cell cycle . Despite its heterotrimeric (alpha beta gamma) structure, intracellular expression of its smallest subunit, the gamma-toxin, is alone responsible for the G(1) arrest . The alpha subunit, however, has a chitinase activity that is essential for holozymocin action from the cell exterior . Here we show that sensitive yeast cells can be rescued from zymocin treatment by exogenously applying crude chitin preparations, supporting the idea that chitin polymers can compete for binding to zymocin with chitin present on the surface of sensitive yeast cells . Consistent with this, holozymocin can be purified by way of affinity chromatography using an immobilized chitin matrix . PCR-mediated deletions of chitin synthesis (CHS) genes show that most, if not all, genetic scenarios that lead to complete loss (chs3 Delta), blocked export (chs7 Delta) or reduced activation (chs4 Delta), combined with mislocalization (chs4 Delta chs5 Delta; chs4 Delta chs6 Delta; chs4 Delta chs5 Delta chs6 Delta) of chitin synthase III activity (CSIII), render cells refractory to the inhibitory effects of exozymocin . In contrast, deletions in CHS1 and CHS2, which code for CSI and CSII, respectively, have no effect on zymocin sensitivity . Thus, CSIII-polymerized chitin, which amounts to almost 90% of the cell's chitin resources, appears to be the carbohydrate receptor required for the initial interaction of zymocin with sensitive cells .

Mol Cell Biol, 2001 Oct, 21(20), 7054 - 64
Hpr1 is preferentially required for transcription of either long or G+C-rich DNA sequences in Saccharomyces cerevisiae; Chavez S et al.; Hpr1 forms, together with Tho2, Mft1, and Thp2, the THO complex, which controls transcription elongation and genome stability in Saccharomyces cerevisiae . Mutations in genes encoding the THO complex confer strong transcription-impairment and hyperrecombination phenotypes in the bacterial lacZ gene . In this work we demonstrate that Hpr1 is a factor required for transcription of long as well as G+C-rich DNA sequences . Using different lacZ segments fused to the GAL1 promoter, we show that the negative effect of lacZ sequences on transcription depends on their distance from the promoter . In parallel, we show that transcription of either a long LYS2 fragment or the S . cerevisiae YAT1 G+C-rich open reading frame fused to the GAL1 promoter is severely impaired in hpr1 mutants, whereas transcription of LAC4, the Kluyveromyces lactis ortholog of lacZ but with a lower G+C content, is only slightly affected . The hyperrecombination behavior of the DNA sequences studied is consistent with the transcriptional defects observed in hpr1 cells . These results indicate that both length and G+C content are important elements influencing transcription in vivo . We discuss their relevance for the understanding of the functional role of Hpr1 and, by extension, the THO complex.

Appl Biochem Biotechnol, 2001 Jun, 94(3), 257 - 64
Optimization of inulinase production by Kluyveromyces marxianus using factorial design; Kalil SJ et al.; Factorial design and response surface techniques were used to optimize the culture medium for the production of inulinase by Kluyveromyces marxianus . Sucrose was used as the carbon source instead of inulin . Initially, a fractional factorial design (2(5-1)) was used in order to determine the most relevant variables for enzyme production . Five parameters were studied (sucrose, peptone, yeast extract, pH, and K2HPO4), and all were shown to be significant . Sucrose concentration and pH had negative effects on inulinase production, whereas peptone, yeast extract, and K2HPO4 had positive ones . The pH was shown to be the most significant variable and should be preferentially maintained at 3.5 . According to the results from the first factorial design, sucrose, peptone, and yeast extract concentrations were selected to be utilized in a full factorial design . The optimum conditions for a higher enzymatic activity were then determined: 14 g/L of sucrose, 10 g/L of yeast extract, 20 g/L of peptone, 1 g/L of K2HPO4 . The enzymatic activity in the culture conditions was 127 U/mL, about six times higher than before the optimization.

Yeast, 2001 Sep 30, 18(13), 1249 - 56
Isolation and study of KlLSM4, a Kluyveromyces lactis gene homologous to the essential gene LSM4 of Saccharomyces cerevisiae; Mazzoni C et al.; We have isolated the KlLSM4 gene as a multicopy suppressor of a Kluyveromyces lactis mutant which shows a rag(-) phenotype (resistance to antimycin A on glucose) . This gene is homologous to the ScLSM4 of Saccharomyces cerevisiae, which codes for an essential 187 amino acid protein containing Sm-like domains . These motifs are present in the evolutionarily conserved family of the Sm-like proteins, which are involved in a large number of cellular processes, including pre-mRNA splicing and mRNA decapping . We demonstrated that the first 72 amino acids of KlLsm4p, which contain the Sm-like domains, can restore cell viability in both K . lactis and S . cerevisiae cells lacking the wild-type protein . However, the absence of the carboxy-terminal region resulted in a remarkable loss of cell viability in the stationary phase . The KlLSM4 sequence has been deposited in the EMBL Data library under Accession No . AJ311719 .

Yeast, 2001 Sep 30, 18(13), 1239 - 47
An SSB encoded by and operating on linear killer plasmids from Kluyveromyces lactis; Schaffrath R et al.; The Kluyveromyces lactis linear plasmids k1 and k2 belong to the family of protein-primed linear DNA genomes, which includes adenoviruses . Here we identify the 18 kDa gene product of k2ORF5 as a novel putative single-stranded DNA binding protein, SSB . As judged from Western analysis using an epitope-tagged fusion protein and ssDNA-agarose affinity chromatography, the Orf5 protein preferentially binds to ssDNA in vitro . Consistently, electrophoretic mobility shift assays demonstrate that ssDNA plasmid probes from k1 and k2 are retarded by this Orf5-associated SSB activity . ORF5 gene shuffle-mediated mutagenesis in vivo results in k1/k2 plasmid instability, pointing towards a role for the Orf5 protein in plasmid replication . Consistently, the Orf5 protein protects ssDNA from exonuclease digestion and stimulates Klenow enzyme . Our findings suggest a functional role for the Orf5 protein as a putative SSB probably required during k1/k2 plasmid DNA synthesis .

J Appl Microbiol, 2001 Sep, 91(3), 541 - 7
The effect of oxygen on the survival of non-Saccharomyces yeasts during mixed culture fermentations of grape juice with Saccharomyces cerevisiae; Holm Hansen E et al.; AIMS: The effect of oxygen on the survival of Torulaspora delbrueckii and Kluyveromyces thermotolerans during mixed culture fermentations in grape juice with Saccharomyces cerevisiae was investigated . METHODS AND RESULTS: Fermentations were carried out in two simple fermentation systems differing in the availability of oxygen . At low available oxygen conditions, T . delbrueckii and K . thermotolerans began to die off after two days of mixed culture fermentation . In filtrates from 2-day-old mixed cultures, single cultures of T . delbrueckii and K . thermotolerans survived and actively produced ethanol to concentrations of approx . 65 and 70 g l-1, respectively, at low available oxygen conditions . Oxygen clearly increased the survival time and decreased the death rate of T . delbrueckii and K . thermotolerans in mixed cultures, whereas it did not affect the growth and survival of S . cerevisiae . CONCLUSION: Our results show that the deaths of T . delbrueckii and K . thermotolerans in mixed cultures at low available oxygen conditions are not due to toxic metabolites produced by the yeasts but rather to the lack of oxygen . Furthermore, they indicate that T . delbrueckii and K . thermotolerans are less tolerant to low available oxygen conditions than S . cerevisiae . SIGNIFICANCE AND IMPACT OF THE STUDY: Our study reveals new knowledge on the mechanisms underlying the succession of yeasts during wine fermentations . This knowledge may be of importance when creating defined, mixed starter cultures for the controlled production of wines with a wide range of flavour compositions.

Appl Microbiol Biotechnol, 2001 Aug, 56(3-4), 411 - 3
Electroinduced extraction of beta-galactosidase from Kluyveromyces lactis; Ganeva V et al.; A new methodology for the extraction of beta-galactosidase from the yeast Kluyveromyces lactis was obtained by electropulsation . The application of a series of electric pulses (2 ms duration, 1 Hz frequency, and 4-4.5 kV/cm field strength) to fresh cells suspended in deionized water, followed by incubation in PBS, led to a spontaneous slow release of enzyme at a yield of 75-80% without any further treatment . Most of the enzyme was extracted within 8 h after electropulsation . This release was dependent on the growth phase . The specific activity of beta-galactosidase in the supernatant of pulsed cells was higher by a factor of 1.5-1.7 in comparison with crude extract.

Curr Genet, 2001 Jul, 39(5-6), 311 - 8
Carbon source-dependent transcriptional regulation of the QCR8 gene in Kluyveromyces lactis . Identification fo cis-acting regions and trans-acting factors in the KlQCR8 upstream region; Brons JF et al.; The QCR8 gene of the yeast K1uyveromyces lactis is transcriptionally regulated by the carbon source in the growth medium . Deletion analysis of the KlQCR8 promoter shows that an element located between -144 bp and -113 bp specifically controls induction of QCR8 gene expression on non-fermentable carbon sources . Specific and differential protein-binding to the activating sequence was observed with extracts from glucose- and ethanol/glycerol-grown cells . Induction of the reporter gene and protein-binding was dependent on the presence of a functional KlCAT8 gene, suggesting that, in K . lactis, K1Cat8p acts in the transcriptional regulation of respiratory function . The activating element contains no other known regulatory sites but two elements required for RNA holoenzyme functioning, raising the intriguing possibility of carbon source-dependent regulation by a subunit of the RNA polymerase holoenzyme in K . lactis.

Curr Genet, 2001 Jul, 39(5-6), 305 - 10
Isolation and characterization of the RAD59 homologue of Kluyveromyces lactis; van den Bosch M et al.; Homologous recombination in the yeast Saccharomyces cerevisiae is under the control of the RAD52 epistasis group . Genes belonging to this group show strong conservation during evolution and homologues of most members have been identified in other eukaryotic organisms such as Schizosaccharomyces pombe, Drosophila and mammals . A homologue of the ScRAD59 gene, which shows structural and functional overlap with ScRAD52, has not been identified in other organisms until now . Previous assessment of the ScRAD59 function revealed that the product of this gene is required for certain types of ScRAD51-independent recombination and single-strand annealing . Also, in the distantly related fission yeast, Sch . pombe, a second RAD52 homologue has been identified (rad/22B+), but this gene more closely resembles ScRAD52 than ScRAD59 at the amino-acid level . In this study, the isolation of a homologue of ScRAD59 in Kluyveromyces lactis, KlRAD59, is described . A Klrad159 null allele results in moderate sensitivity to X-rays, indicating that the KlRAD59 gene is involved in the repair of X-ray-induced DNA damage . The amino acids in the putative K1Rad59 protein share 53% identity and 11% similarity with ScRad59 . The KlRAD59 gene fully complements both the X-ray-sensitive phenotype and defects in recombination of the Scrad59 mutant strain . Our results underscore the evolutionary conservation of the RAD52 group of genes and provide evidence that the presence of additional RAD52 homologues is not limited to Sac . cerevisiae and Sch . pombe and might be a general phenomenon.

Acta Microbiol Pol, 2001, 50(1), 45 - 51
Optimization of process parameters for the continuous ethanol production by Kluyveromyces lactis immobilized cells in hydrogel copolymer carrier; Deriase SF et al.; In the present study the optimized parameters for highest ethanol productivity by Kluyveromyces lactis immobilized cells bioreactor were obtained using the method of Lagrange multipliers . Immobilized growing yeast cells in PVA: HEMA (7%: 10%, w/w) hydrogel copolymer carrier produced by radiation polymerization were used in a packed-bed column reactor for the continuous production of ethanol from lactose at different levels of concentrations (50, 100 and 150) gL(-1) . The results indicate that volumetric ethanol productivity is influenced by substrate concentration and dilution rate . The highest value 7.17 gL(-1) h(-1) is obtained at higher lactose concentration (150 gL(-1)) in feed medium and 0.3 h(-1) dilution rate . The same results have been obtained through the application of "LINGO" software for mathematical optimization.

J Bacteriol, 2001 Sep, 183(18), 5257 - 61
Three target genes for the transcriptional activator Cat8p of Kluyveromyces lactis: acetyl coenzyme A synthetase genes KlACS1 and KlACS2 and lactate permease gene KlJEN1; Lodi T et al.; The aerobic yeast Kluyveromyces lactis and the predominantly fermentative Saccharomyces cerevisiae share many of the genes encoding the enzymes of carbon and energy metabolism . The physiological features that distinguish the two yeasts appear to result essentially from different organization of regulatory circuits, in particular glucose repression and gluconeogenesis . We have isolated the KlCAT8 gene (a homologue of S . cerevisiae CAT8, encoding a DNA binding protein) as a multicopy suppressor of a fog1 mutation . The Fog1 protein is a homologue of the Snf1 complex components Gal83p, Sip1p, and Sip2p of S . cerevisiae . While CAT8 controls the key enzymes of gluconeogenesis in S . cerevisiae, KlCAT8 of K . lactis does not (I . Georis, J . J . Krijger, K . D . Breunig, and J . Vandenhaute, Mol . Gen . Genet . 264:193-203, 2000) . We therefore examined possible targets of KlCat8p . We found that the acetyl coenzyme A synthetase genes, KlACS1 and KlACS2, were specifically regulated by KlCAT8, but very differently from the S . cerevisiae counterparts . KlACS1 was induced by acetate and lactate, while KlACS2 was induced by ethanol, both under the control of KlCAT8 . Also, KlJEN1, encoding the lactate-inducible and glucose-repressible lactate permease, was found under a tight control of KlCAT8.

J Bacteriol, 2001 Sep, 183(18), 5223 - 9
Feedback regulation of glucose transporter gene transcription in Kluyveromyces lactis by glucose uptake; Milkowski C et al.; In the respirofermentative yeast Kluyveromyces lactis, only a single genetic locus encodes glucose transporters that can support fermentative growth . This locus is polymorphic in wild-type isolates carrying either KHT1 and KHT2, two tandemly arranged HXT-like genes, or RAG1, a low-affinity transporter gene that arose by recombination between KHT1 and KHT2 . Here we show that KHT1 is a glucose-induced gene encoding a low-affinity transporter very similar to Rag1p . Kht2p has a lower K(m) (3.7 mM) and a more complex regulation . Transcription is high in the absence of glucose, further induced by low glucose concentrations, and repressed at higher glucose concentrations . The response of KHT1 and KHT2 gene regulation to high but not to low concentrations of glucose depends on glucose transport . The function of either Kht1p or Kht2p is sufficient to mediate the characteristic response to high glucose, which is impaired in a kht1 kht2 deletion mutant . Thus, the KHT genes are subject to mutual feedback regulation . Moreover, glucose repression of the endogenous beta-galactosidase (LAC4) promoter and glucose induction of pyruvate decarboxylase were abolished in the kht1 kht2 mutant . These phenotypes could be partially restored by HXT gene family members from Saccharomyces cerevisiae . The results indicate that the specific responses to high but not to low glucose concentrations require a high rate of glucose uptake.

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