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