Pichia

Mutat Res, 1996 Oct 25, 357(1-2), 67 - 74
Radiosensitivity of haplont yeast cells irradiated with sparsely and densely ionizing radiations; Korogodin VI et al.; Five haploid and three diploid yeast strains of various species (Yarrowia lipolytica, Pichia pinus and Pichia guilliermondii) were irradiated with alpha-particles from 239Pu and gamma-rays from 137Cs or 60Co in the stationary phase of growth . A common feature of these species is that they exhibit a haploid state as a normal vegetative state in natural conditions . It was shown that the transition from the haploid to the diploid state is not accompanied by increased radioresistance, and diploid strains were unable to perform liquid-holding recovery . The absence of diploid-specific recovery in diploid strains was also supported by the fact that the RBE of alpha-particles was almost identical for haploid and the corresponding diploid strains being much smaller than that observed in typical wild-type diploid strains capable of diploid-specific recovery . The results suggest that haplont yeast may have evolved to diplont yeast via the development of a specific repair system conferring specific resistance in the diploid state.

Gene, 1996 Oct 24, 177(1-2), 163 - 7
Inducible expression of a heterologous protein in Hansenula polymorpha using the alcohol oxidase 1 promoter of Pichia pastoris; Raschke WC et al.; Pichia pastoris (Pp) and Hansenula polymorpha (Hp) are methylotrophic yeasts commonly used for industrial purposes . Growth of either of these yeasts in the presence of methanol as the carbon source results in high-level induction of alcohol oxidase expression . The respective alcohol oxidase genes, AOX1 in Pp and MOX in Hp, have similar regulatory characteristics . Our studies show that the Pp AOX1 promoter (AOX1p) can be used for methanol-induced expression of a heterologous gene in Hp . Furthermore, the size of an AOX1p-heterologous gene-AOX1 terminator cassette transcript synthesized in Hp is indistinguishable from that synthesized in Pp suggesting that transcription both initiates and terminates at the same sites in both yeast species . Induction of AOX1p in Hp demonstrates that the methanol-inducible regulatory mechanism in Hp is able to recognize and activate the Pp promoter in spite of extensive sequence variations between AOX1p and MOXp.

Gene, 1996 Oct 24, 177(1-2), 69 - 76
Secretory production of recombinant urokinase-type plasminogen activator-annexin V chimeras in Pichia pastoris; Okabayashi K et al.; To produce a thrombi-targeting plasminogen activator, we expressed a fused gene that contains a modified pre-sequence of Mucor pussilus rennin (MPR) followed by a chimeric gene of single-chain urokinase-type plasminogen activator (scu-PA)::annexin V (AV) . The fused gene was ligated into an integrative vector, under the control of the alcohol oxidase 1 (AOX1) promoter (p), and transformed into Pichia pastoris . Transformants were monitored for the secretion of fibrinolytic activity . The highest expressing clone, HB225, secreted as much as 600 international units (IU) of fibrinolytic activity per ml of culture medium under optimal conditions . It contained three tandem copies of the full-size vector disruptively integrated into the AOX1 sequence . Western blot analysis revealed that the secreted chimera was highly susceptible to proteolysis . Addition of excess amino acids (aa) to the culture medium minimized the degree of proteolysis . Two major species of chimera, 85 and 65 kDa, were then isolated from the culture medium . The former was the intact form consisting of a single-chain and showing full enzyme activity after activation by plasmin . The latter was an enzymatically processed form consisting of two chains held by a disulfide bond, having full enzyme activity without activation . Both chimeras exhibited calcium-dependent phospholipid (PL)-binding affinities similar to the parent AV.

Carbohydr Res, 1996 Oct 23, 293(1), 101 - 17
The extracellular polysaccharide of Pichia (Hansenula) holstii NRRL Y-2448: the structure of the phosphomannan backbone; Parolis LA et al.; The phosphomannan core of the exopolysaccharide of Pichia (Hansenula) holstii NRRL Y-2448 was isolated after hydrolytic removal of the oligosaccharide phosphate side-chains . The core polysaccharide and its dephosphorylated derivative were subjected to extensive 1D and 2D NMR spectroscopy which yielded information on the linkage sites and on the sequence of the mannosyl residues in the major oligosaccharide repeating unit . The most probable structure for the repeating unit was -{6-O-PO3H2-alpha-D-Man-(1-->3)-alpha-D-Man-(1-->2)-alpha-D-Man-(1 -->2)}-alpha-D-Man-(1-->6)-{alpha-D-Man-(1-->2)}-alpha-D-Man-(1-->6)- . A semiquantitative conformational analysis was performed by Monte Carlo simulations and the result was confirmed by comparison with the experimentally determined NMR data . The distance distribution for the phosphate groups was determined from the modeling and was found to cover the expected range of distances for phosphorylated high-mannose oligosaccharides.

Gene, 1996 Oct 17, 176(1-2), 197 - 201
Intracellular production of a major cytomegalovirus antigenic protein in the methylotrophic yeast Pichia pastoris; Battista MC et al.; We have previously shown that single or multiple epitopes of the major human cytomegalovirus (HCMV) antigens, produced as fusion proteins in prokaryotes can be valuable diagnostic material in the serology of HCMV infection . In this work we moved to a eukaryotic system, to produce one of the most immunogenic HCMV antigens, ppUL44 (also called pp52 due to its apparent molecular size on acrylamide gels), as a non-fusion protein, in an attempt to eliminate some non-specific reactivity of human sera with bacterial carrier proteins . We expressed the DNA encoding ppUL44 in a highly efficient expression system based on the methylotrophic yeast, Pichia pastoris . Good levels of intracellular, soluble pp52 were produced . We observed an indistinguishable pattern of the yeast pp52 from the viral native protein in immunoblotting and a good reactivity with human sera.

FEBS Lett, 1996 Oct 7, 394(3), 268 - 72
Expression and pharmacological characterization of the human mu-opioid receptor in the methylotrophic yeast Pichia pastoris; Talmont F et al.; The human mu-opioid receptor cDNA from which the 32 amino-terminal codons were substituted by the Saccharomyces cerevisiae alpha-mating factor signal sequence has been expressed in the methylotrophic yeast Pichia pastoris using the host promoter of the alcohol oxidase-1 gene . Cell membranes exhibited specific and saturable binding of the opioid antagonist {3H}diprenorphine (Kd = 0.2 nM and Bmax = 400 fmol/mg protein or 800 sites/cell) . Competition studies with non-selective, and mu-, delta- and kappa-selective opioid agonists and antagonists revealed a typical mu-opioid receptor binding profile, suggesting proper folding of the protein in yeast membranes.

Curr Opin Biotechnol, 1996 Oct, 7(5), 525 - 30
Quality and authenticity of heterologous proteins synthesized in yeast; Eckart MR et al.; Yeast, especially Saccharomyces cerevisiae and Pichia pastoris, are major hosts employed in the expression of authentic heterologous proteins of high quality in the biopharmaceutical, industrial and academic environments . There has been recent progress in characterizing and controlling the factors involved in determining authenticity.

Curr Opin Biotechnol, 1996 Oct, 7(5), 517 - 24
The expression of recombinant proteins in yeasts; Sudbery PE; The methylotrophic yeasts Hansenula polymorpha and Pichia pastoris are rapidly becoming the systems of choice for the expression of recombinant proteins in yeast . However, the powerful genetic techniques available in Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe are still exploited to establish models to study medically important cell processes and screen for pharmacologically active compounds.

Appl Environ Microbiol, 1996 Oct, 62(10), 3894 - 6
A glycerol-3-phosphate dehydrogenase-deficient mutant of Saccharomyces cerevisiae expressing the heterologous XYL1 gene; Liden G et al.; The gene XYL1, encoding a xylose reductase, from Pichia stipitis was transformed into a mutant of Saccharomyces cerevisiae incapable of glycerol production because of deletion of the genes GPD1 and GPD2 . The transformed strain was capable of anaerobic glucose conversion in the presence of added xylose, indicating that the xylose reductase reaction can fulfill the role of the glycerol-3-phosphate dehydrogenase reaction as a redox sink . The specific xylitol production rate obtained was 0.38 g g-1 h-1.

Appl Environ Microbiol, 1996 Oct, 62(10), 3864 - 7
Involvement of carnitine acyltransferases in peroxisomal fatty acid metabolism by the yeast Pichia guilliermondii; Pagot Y et al.; This article provides information about peroxisomal fatty acid metabolism in the yeast Pichia guilliermondii . The existence of inducible mitochondrial carnitine palmitoyltransferase and peroxisomal carnitine octanoyl-transferase activities was demonstrated after culture of this yeast in a medium containing methyl oleate . The subcellular sites and induction patterns were studied . The inhibition of carnitine octanoyl- and palmitoyl-transferases by chlorpromazine to a large extent prevented the otherwise observed metabolism-dependent inactivation of thiolase by 2-bromofatty acids in vivo . We concluded that the metabolism of long- and medium-chain fatty acids in the peroxisome of this yeast involved carnitine intermediates.

Curr Microbiol, 1996 Oct, 33(4), 237 - 42
Characterization of the Genetic System of the Xylose-Fermenting Yeast Pichia stipitis
Melake T, Passoth V V, Klinner U.
High mutant frequencies indicated that the wild-type strains of Pichia stipitis are haploid . Sporulation ability of these clones pointed to a homothallic life cycle . Mating was induced by cultivation under nutritionally poor conditions on malt extract medium . Conjugation was followed immediately by sporulation . However, hybrids could be rescued by transferring the nascent zygotes to complete medium before meiosis had started . Under rich nutritional conditions, hybrids were mitotically stable and did not sporulate . The segregation pattern of auxotrophic markers of diploid zygotes indicated regular meiosis, although asci contained preferentially spore dyads.

Yeast, 1996 Sep 30, 12(12), 1187 - 200
Cloning of the Penicillium minioluteum gene encoding dextranase and its expression in Pichia pastoris; Roca H et al.; The DEX gene encoding an extracellular dextranase was isolated from the genomic DNA library of Penicillium minioluteum by hybridization using the dextranase cDNA as a probe . Comparison of the gene and cDNA sequences revealed that the DEX gene does not contain introns . Amino acid sequences comparison of P . minioluteum dextranase with other reported dextranases reveals a significant homology (29% identity) with a dextranase from Arthrobacter sp . CB-8 . The DEX gene fragment encoding a mature protein of 574 amino acids was expressed in the methylotrophic yeast Pichia pastoris by using the SUC2 gene signal sequence from Saccharomyces cerevisiae under control of the alcohol oxidase-1 (AOX1) promoter . Over 3.2 g/l of enzymatically active dextranase was secreted into the medium after induction by methanol . The yeast product was indistinguishable from the native enzyme in specific activity and the N-terminus of both proteins were identical.

Biochim Biophys Acta, 1996 Sep 13, 1297(1), 99 - 104
Trp-16 is essential for the activity of alpha-galactosidase and alpha-N-acetylgalactosaminidase; Zhu A et al.; By expressing site-directed mutants in the methylotrophic yeast strain Pichia pastoris, the role of a tryptophan residue at position 16 in the activity of alpha-galactosidase and alpha-N-acetylgalactosaminidase, two closely related exoglycosidases, was studied . A substitution of Trp-16 with an arginine residue in alpha-N-acetylgalactosaminidase abolished the enzyme activity, which was confirmed by replacing a 600 bp fragment containing the mutation with the corresponding wild-type sequence . The same tryptophan residue was then substituted with an alanine in both enzymes by site-directed mutagenesis to reveal a possible relationship between their active sites . The purified alpha-N-acetylgalactosaminidase mutant demonstrated a specific activity of 2.8 x 10(-2) U/mg and a Vmax/K(m) of 4.3 x 10(-2), which were both more than a thousandfold lower than corresponding values for the wild-type enzyme . Furthermore, the mutant failed to bind to an affinity resin, suggesting the involvement of Trp-16 in substrate-binding . In addition, the purified alpha-galactosidase mutant resulted in more than a 10(4)-fold decrease in specific activity . Thus our data suggest that Trp-16 in both alpha-galactosidase and alpha-N-acetylgalactosaminidase is critical for enzymatic activity, which in turn supports the hypothesis that these two enzymes may share a catalytic mechanism involving similar residues in their active sites.

Plant Cell Physiol, 1996 Sep, 37(6), 816 - 24
Isolation and characterization of mitochondrial nucleoids from the yeast Pichia jadinii; Miyakawa I et al.; Mitochondrial (mt) nucleoids were isolated with a high degree of purity from the yeast Pichia jadinii, in which the mitochondrial DNA (mtDNA) is linear . Field-inversion gel electrophoresis (FIGE) revealed that significant amounts of mtDNA could be isolated intact, as linear molecules of 41 kbp, from the isolated mt-nucleoids . Fifteen different proteins were detected in the mt-nucleoid fraction and, eight of these proteins bound to DNA . The patterns of mt-nucleoid proteins and of the DNA-binding proteins after gel electrophoresis in the presence of SDS were somewhat different from those of such proteins from Saccharomyces cerevisiae . The corresponding proteins isolated from the mt-nucleoids of four other species of yeast in the genera Pichia and Williopsis also differed from one another in terms of electrophoretic mobility in the presence of SDS . In immunoblotting experiments, antibodies that had been raised against the 67-kDa protein of mt-nucleoids from S.cerevisiae and the YMN-1 monoclonal antibody that is specific for a 48-kDa protein in the mt-nucleoids from S . cerevisiae did not recognize any proteins in the mt-nucleoids from Pichia jadinii and four other species of yeast . The results suggest the considerable diversity of the proteins in the mt-nucleoids of yeasts.

Protein Expr Purif, 1996 Sep, 8(2), 254 - 61
Overexpression in Pichia pastoris and crystallization of an elicitor protein secreted by the phytopathogenic fungus, Phytophthora cryptogea; O'Donohue MJ et al.; A synthetic gene encoding beta-cryptogein, a member of the elicitin family, has been cloned into a vector for expression by the methylotrophic yeast, Pichia pastoris . Having first optimized the gene construction for secretion, we have overexpressed a modified beta-cryptogein in a secreted form . A purification scheme suited to this expression system has been developed and highly pure, biologically active protein has been obtained . For structural analysis of this recombinant beta-cryptogein, and new mutated forms thereof, optimal conditions for the crystallization of this protein have been determined and crystals that diffract to 2.2 A have been obtained.

Protein Expr Purif, 1996 Sep, 8(2), 204 - 14
Overexpression, purification, and characterization of recombinant barley alpha-amylases 1 and 2 secreted by the methylotrophic yeast Pichia pastoris; Juge N et al.; Recombinant barley alpha-amylase isozymes 1 and 2 were secreted by Pichia pastoris at up to 50 and 1 mg/liter, respectively, representing approximately a 50-fold increase compared to the levels of the heterologous expression by Saccharomyces cerevisiae . The cDNA clones E or pM/C encoding isozymes 1 and 2, respectively, were placed under the control of regulatory sequences from the Pichia AOX1 gene in the vector pHIL-D2 . Both isozymes were effectively secreted to the medium as directed by their own signal sequences and easily purified to homogeneity in quantitative yield by affinity chromatography on beta-cyclodextrin-Sepharose . The N-terminal sequence, pI, and Mr indicated that native-like processing took place . Electrospray ionization mass spectrometry, however, revealed microheterogeneity for recombinant isozyme 1 . While Mr of one recombinant isozyme 1 form of 45,452 was in excellent agreement with a value of 45,447 calculated from the sequence, liquid chromatography/mass spectrometry of endo Lys C-generated peptides followed by tandem mass spectrometry on a nanoelectrospray ionization/mass spectrometry/mass spectrometry system identified additional recombinant isozyme 1 forms to be glycosylated on Thr410, N-acetylated on His1, S-glutathionylated on Cys95, or C-terminally truncated of -412RS, -411QRS, and -410LQRS . The recombinant enzymes and the alpha-amylases from barley malt closely resembled each other in enzymatic activity on insoluble Blue Starch, amylose of degree of polymerization 17, and 2-chloro-4-nitrophenyl beta-D-maltoheptaoside as well as in Ca2+ dependency of activity . Pichia pastoris thus produced in high yields recombinant alpha-amylase that is similar with respect to structure and function to the enzyme purified from malt extracts . This greatly facilitates future mutational analysis of barley alpha-amylase in order to probe structure/function relationships.

J Gen Virol, 1996 Sep, 77 ( Pt 9), 2001 - 8
Immunogenic presentation of a conserved gp41 epitope of human immunodeficiency virus type 1 on recombinant surface antigen of hepatitis B virus; Eckhart L et al.; In view of the high antigenic variability of human immunodeficiency virus type 1 (HIV-1), a vaccine against AIDS must induce an immune response to epitopes as invariable as possible among the various virus strains and clones . Previously the highly conserved six amino acid sequence Glu-Leu-Asp-Lys-Trp-Ala (ELDKWA) from gp41, defining the epitope of the human MAb 2F5, was shown to elicit HIV-1-neutralizing antibodies when presented on haemagglutinin of influenza virus . We investigated the immunogenic potential of the MAb 2F5 epitope and two of its major escape epitopes as internal fusions to the hepatitis B virus (HBV) surface antigen (HBsAg) . Recombinant HBsAg-HIV proteins produced in the methylotrophic yeast Pichia pastoris self-assembled into 22 nm lipoprotein particles . Mice immunized with these particles developed an anti-HBsAg immune response in a range that is considered to be protective against HBV infection in humans . More importantly, antisera had extremely high titres of antibodies reactive with a structurally flexible form of the HIV-1 epitope, whereby strong cross-reactivity with the escape variants of the epitope was observed . Although HIV-1 gp 160 and the ectodomain of gp41 containing the epitope were significantly recognized, the antisera failed to neutralize HIV-1 in vitro . These data, together with those on the haemagglutinin-ELDKWAS fusion suggest that the ability of the MAb 2F5 epitope to induce neutralizing antibodies depends on the molecular context in which it is presented . Therefore, further characterization of secondary and tertiary structure requirements of the epitope is indispensable for the full exploitation of its potential as a vaccine component.

FEMS Microbiol Lett, 1996 Sep 1, 142(2-3), 147 - 53
Isolation and characterisation of mutants from the halotolerant yeast Pichia sorbitophila defective in H+/glycerol symport activity; Oliveira RP et al.; Pichia sorbitophila, a yeast species that is highly resistant to osmotic stress in general and to salt stress in particular, was subjected to a mutagenesis strategy in order to obtain mutants deficient in the glycerol active uptake previously described . Density centrifugation was used for enrichment of NaCl sensitive mutants in either glucose or glycerol media . Several phenotypic classes of mutants were identified, to which physiological tests were applied concerning the activity of the symporter, its accumulation capacity and the detection of the activity of glycerol pathway specific enzymes . From these, two mutant strains were selected, presenting a clearly deficient phenotype on H+/glycerol symport activity.

J Biol Chem, 1996 Aug 23, 271(34), 20594 - 602
Characterization of the reductase domain of rat neuronal nitric oxide synthase generated in the methylotrophic yeast Pichia pastoris . Calmodulin response is complete within the reductase domain itself; Gachhui R et al.; Rat neuronal NO synthase (nNOS) is comprised of a flavin-containing reductase domain and a heme-containing oxygenase domain . Calmodulin binding to nNOS increases the rate of electron transfer from NADPH into its flavins, triggers electron transfer from flavins to the heme, activates NO synthesis, and increases reduction of artificial electron acceptors such as cytochrome c . To investigate what role the reductase domain plays in calmodulin's activation of these functions, we overexpressed a form of the nNOS reductase domain (amino acids 724-1429) in the yeast Pichia pastoris that for the first time exhibits a complete calmodulin response . The reductase domain was purified by 2',5'-ADP affinity chromatography yielding 25 mg of pure protein per liter of culture . It contained 1 FAD and 0.8 FMN per molecule . Most of the protein as isolated contained an air-stable flavin semiquinone radical that was sensitive to FeCN6 oxidation . Anaerobic titration of the FeCN6-oxidized reductase domain with NADPH indicated the flavin semiquinone re-formed after addition of 1-electron equivalent and the flavins could accept up to 3 electrons from NADPH . Calmodulin binding to the recombinant reductase protein increased its rate of NADPH-dependent flavin reduction and its rate of electron transfer to cytochrome c, FeCN6, or dichlorophenolindophenol to fully match the rate increases achieved when calmodulin bound to native full-length nNOS . Calmodulin's activation of the reductase protein was associated with an increase in domain tryptophan and flavin fluorescence . We conclude that many of calmodulin's actions on native nNOS can be fully accounted for through its interaction with the nNOS reductase domain itself.

Proc Natl Acad Sci U S A, 1996 Aug 20, 93(17), 8989 - 94
The methylotrophic yeast Pichia pastoris synthesizes a functionally active chromophore precursor of the plant photoreceptor phytochrome; Wu SH et al.; Induction of the expression of an algal phytochrome cDNA in the methylotrophic yeast Pichia pastoris led to time-dependent formation of photoactive holophytochrome without the addition of exogenous bilins . Both in vivo and in vitro difference spectra of this phytochromic species are very similar to those of higher plant phytochrome A, supporting the conclusion that this species possesses a phytochromobilin prosthetic group . Zinc blot analyses confirm that a bilin chromophore is covalently bound to the algal phytochrome apoprotein . The hypothesis that P . pastoris contains phytochromobilin synthase, the enzyme that converts biliverdin IX alpha to phytochromobilin, was also addressed in this study . Soluble extracts from P . pastoris were able to convert biliverdin to a bilin pigment, which produced a native difference spectrum upon assembly with oat apophytochrome A . HPLC analyses confirm that biliverdin is converted to both 3E- and 3Z-isomers of phytochromobilin . These investigations demonstrate that the ability to synthesize phytochromobilin is not restricted to photosynthetic organisms and support the hypothesis of a more widespread distribution of the phytochrome photoreceptor.

J Biol Chem, 1996 Aug 16, 271(33), 20156 - 62
Expression of recombinant HLA-DR2 molecules . Replacement of the hydrophobic transmembrane region by a leucine zipper dimerization motif allows the assembly and secretion of soluble DR alpha beta heterodimers; Kalandadze A et al.; Major histocompatibility complex (MHC) class II molecules are membrane-anchored heterodimers that present peptides on the surface of antigen presenting cells to T cells . Soluble HLA-DR2 molecules were expressed for structural and functional characterization of the MHC/peptide/T cell receptor recognition unit . The alpha and beta chains of DR2 (encoded by the DRA, DRB1*1501 genes) did not assemble in mammalian or insect cell lines when the transmembrane regions of one or both chains were truncated . The hydrophobic transmembrane regions of DRalpha and DRbeta facilitate assembly of the heterodimer and were therefore replaced by the leucine zipper dimerization motifs from the transcription factors Fos and Jun, which assemble as a soluble, tightly packed coiled coil structure . The DRalpha-Fos and DRbeta-Jun constructs were expressed in a methyltrophic yeast, Pichia pastoris, using the alpha-mating factor secretion signal to direct expression to the secretory pathway . DR alphabeta heterodimers were purified from supernatants using an antibody specific for the DR alphabeta heterodimer . Kinetic and quantitative peptide binding experiments demonstrated that recombinant DR2 molecules were efficiently loaded with an antigenic peptide . Soluble DR2 molecules can be used to define structural aspects of the MHC/peptide/T cell receptor interaction and to study the signals induced by T cell receptor recognition of soluble DR2.peptide complexes.

Arch Biochem Biophys, 1996 Aug 15, 332(2), 305 - 12
Identification of a novel pelD gene expressed uniquely in planta by Fusarium solani f . sp . pisi (Nectria haematococca, mating type VI) and characterization of its protein product as an endo-pectate lyase; Guo W et al.; Antibodies prepared against a pectin-inducible pectate lyase (PLA) produced by a phytopathogenic fungus Fusarium solani f . sp . pisi (Nectria haematococca, mating type VI) were previously found to protect the host from infection . The gene (pelA) and two of its homologs were cloned and sequenced . Here we report the isolation of a new pectate lyase gene, pelD, from a genomic library of F . solani pisi . A 1.5-kb DNA fragment containing pelD and its flanking regions was sequenced . The nucleotide sequence of pelD would encode a protein of 24.5 kDa which shares 49, 44, and 65% amino acid sequence identity with PLA, PLB, and PLC, respectively, from the same fungus . Because the first 19 amino acid residues appeared to be a signal peptide, the mature enzyme could be a 22.7-kDa protein . pelD transcripts and PLD protein could not be detected in fungus cultured in glucose, pectin, pea epicotyl extract, or a pea cell wall preparation . However, pelD transcripts were readily found by RT-PCR with RNA isolated from infected pea tissues . The cDNA of pelD, thus obtained, was expressed in Pichia pastoris with the putative pelD signal sequence . The secreted PLD was purified and characterized to be an endopectate lyase, and its lyase activity could be inhibited by anti-PLA IgG . Thus, protection of the host observed with the anti-PLA antibodies could reflect inhibition of immunologically related pectate lyases including PLD which is expressed uniquely in planta.

Biochem J, 1996 Aug 15, 318 ( Pt 1), 125 - 31
Drosophila melanogaster angiotensin I-converting enzyme expressed in Pichia pastoris resembles the C domain of the mammalian homologue and does not require glycosylation for secretion and enzymic activity; Williams TA et al.; Drosophila melanogaster angiotensin I-converting enzyme (AnCE) is a secreted single-domain homologue of mammalian angiotensin I-converting enzyme (ACE) which comprises two domains (N and C domains) . In order to characterize in detail the enzymic properties of AnCE and to study the influence of glycosylation on the secretion and enzymic activity of this enzyme, we overexpressed AnCE (expression level, 160 mg/l) and an unglycosylated mutant (expression level, 43 mg/l) in the yeast Pichia pastoris . The recombinant enzyme was apparently homogeneous on SDS/PAGE without purification and partial deglycosylation demonstrated that all three potential sites for N-linked glycosylation were occupied by oligosaccharide chains . Each N-glycosylation sequence (Asn-Xaa-Ser/Thr) was disrupted by substituting a glutamine for the asparagine residue at amino acid positions 53, 196 and 311 by site-directed mutagenesis to produce a single mutant . Expression of the unglycosylated mutant in Pichia produced a secreted catalytically active enzyme (AnCE delta CHO) . This mutant displayed unaltered kinetics for the hydrolyses of hippuryl-His-Leu, angiotensin 1 and N-acetyl-Ser-Asp-Lys-Pro (AcSDKP) and was equally sensitive to ACE inhibitors compared with wild-type AnCE . However, AnCE delta CHO was less stable, displaying a half-life of 4.94 h at 37 degrees C, compared with AnCE which retained full activity under the same conditions . Two catalytic criteria demonstrate the functional resemblance of AnCE with the human ACE C domain: first, the kcat/Km of AcSDKP hydrolysis and secondly, the kcat/Km and optimal chloride concentration for hippuryl-His-Leu hydrolysis . A range of ACE inhibitors were far less potent towards AnCE compared with the human ACE domains, except for captopril which suggests an alternative structure in AnCE corresponding to the region of the S1 subsite in the human ACE active sites.

J Biol Chem, 1996 Aug 2, 271(31), 18973 - 80
Isolation and characterization of Pas2p, a peroxisomal membrane protein essential for peroxisome biogenesis in the methylotrophic yeast Pichia pastoris; Wiemer EA et al.; The pas2 mutant of the methylotrophic yeast Pichia pastoris is characterized by a deficiency in peroxisome biogenesis . We have cloned the PpPAS2 gene by functional complementation and show that it encodes a protein of 455 amino acids with a molecular mass of 52 kDa . In a Pppas2 null mutant, import of both peroxisomal targeting signal 1 (PTS1)- and PTS2-containing proteins is impaired as shown by biochemical fractionation and fluorescence microscopy . No morphologically distinguishable peroxisomal structures could be detected by electron microscopy in Pppas2 null cells induced on methanol and oleate, suggesting that PpPas2p is involved in the early stages of peroxisome biogenesis . PpPas2p is a peroxisomal membrane protein (PMP) and is resistant to extraction by 1 M NaCl or alkaline sodium carbonate, suggesting that it is a peroxisomal integral membrane protein . Two hydrophobic domains can be distinguished which may be involved in anchoring PpPas2p to the peroxisomal membrane . PpPas2p is homologous to the Saccharomyces cerevisiae Pas3p . The first 40 amino acids of PpPas2p, devoid of the hydrophobic domains, are sufficient to target a soluble fluorescent reporter protein to the peroxisomal membrane, with which it associates tightly . A comparison with the membrane peroxisomal targeting signal of PMP47 of Candida boidinii revealed a stretch of positively charged amino acids common to both sequences . The role of peroxisomal membrane targeting signals and transmembrane domains in anchoring PMPs to the peroxisomal membrane is discussed.

J Biol Chem, 1996 Aug 2, 271(31), 18310 - 3
Synthesis and cleavage- secretion of enzymatically active rabbit angiotensin-converting enzyme in Pichia pastoris; Sadhukhan R et al.; Many biologically important ectoproteins that are anchored in the plasma membrane via a hydrophobic domain undergo a proteolytic cleavage process, which releases the ectodomain to the extracellular milieu in a regulated fashion . Angiotensin-converting enzyme (ACE) is one such protein that is secreted from human and mouse cells by its cleavage at one of two alternative sites in the ectodomain . Here, we report similar cleavage-secretion of ACE in the yeast Pichia pastoris . The cleavage site used in yeasts was identical to one of the two sites used in mouse cells . Moreover, as in mammalian cells, ACE secretion in yeast was inhibited by compound 3, a potent inhibitor of the metzincin family of metalloproteases . ACE proteins cleavage-secreted from yeast and from mammalian cells had identical enzymatic properties . These results demonstrate the existence of a secretase activity in yeast whose properties closely resemble those of the mammalian ACE secretase.

Lett Appl Microbiol, 1996 Aug, 23(2), 79 - 84
Fatty acid patterns of film-forming yeasts and new evidence for the heterogeneity of Pichia membranaefaciens; Noronha-da-Costa P et al.; Total fatty acids (C14:0 to C18:3) of 50 strains assigned by classical identification methods to Pichia membranaefaciens (28), P . anomala (15), Dekkera anomala (2), D . bruxellensis (2) and Candida vini (3) were determined and data analysed by multivariate statistical procedures . Principal components cluster analysis defined six groups of strains . Thirteen strains of P . anomala formed a well-defined cluster, whereas P . membranaefaciens was split into three groups . The taxonomic status of the P . membranaefaciens strains was evaluated by determination of the G + C content and DNA-DNA hybridization . The results provided evidence in support of P . membranaefaciens encompassing distinct species . Only eight strains were certified as P . membranaefaciens and six were included in the same cluster, indicating that numerical analysis of fatty acid profiles points to genetic differences which remain undetectable by conventional phenotypic tests.

J Biochem (Tokyo), 1996 Aug, 120(2), 229 - 32
Construction and expression of bi-functional proteins of single-chain Fv with effector domains; Luo D et al.; We fused various polypeptide extensions to the C-termini of single chain Fv (scFv) and disulfide-stabilized Fv (dsFv) fragments to facilitate detection of bi-functional proteins or to add biological effector domains, which included the human metallothionein (HMT) motif and biotin mimetic sequence . These bi-functional proteins were expressed and secreted in a recombinant Pichia pastoris system and showed specific anti-idiotype binding activity, as determined by competitive radioimmunoassaying . However, the fusion protein constructed with dsFv- HMT, but not scFv-HMT, had lost this binding activity . The interruption of the structural conformation as a result in dsFv-HMT may be explained by the interactions between the cysteines engineered in dsFv domains and the cysteines in the HMT region.

Yakugaku Zasshi, 1996 Aug, 116(8), 622 - 9
{Ligand binding properties and esterase-like activity of recombinant human serum albumin}; Takeshima K et al.; The ligand binding properties and esterase-like activity of recombinant human serum albumin (rHSA) expressed by Pichia pastoris were compared with those of plasma derived human albumin (pHSA) . The binding of long fatty acid ions was determined by the equilibrium partition method using radiolabeled palmitate . The association constants and the number of binding sites of diazepam, salicylate and warfarin were determined by specific and nonspecific binding models . The high affinity binding of bilirubin was kinetically determined from the oxidation rate of free bililubin in the binding mixture . The binding parameters of these five ligands obtained with rHSA were within the same range observed with pHSA preparations . The kinetic parameters for hydrolytic activity of rHSA toward p-nitrophenyl acetate was also similar to pHSA . These results indicate that rHSA and pHSA have the same functional property.

Protein Expr Purif, 1996 Aug, 8(1), 119 - 25
Production and isolation of the recombinant N-lobe of human serum transferrin from the methylotrophic yeast Pichia pastoris; Mason AB et al.; The N-lobe of human serum transferrin has been expressed in the methylotrophic yeast Pichia pastoris by placing the hTF/2N cDNA under the control of the methanol-inducible alcohol oxidase promoter . Following induction with methanol, the N-lobe was efficiently secreted into a basal salt medium in shake flasks at a level of 150-240 mg/liter . As judged by mobility on SDS-PAGE, immunoreactivity with two domain-specific monoclonal antibodies, and both thermal stability and spectral properties (indictative of correct folding and ability to bind iron), the recombinant N-lobe produced by the yeast cells appears to be identical to that produced in a mammalian expression system . Electrospray-mass spectrometry and a third domain specific antibody, however, show that approximately 80% of the protein from the yeast cells contains one or two hexose residues.

FEMS Microbiol Lett, 1996 Aug 1, 141(2-3), 227 - 31
Biochemical characterization of a mutant of the yeast Pichia anomala derepressed for malic acid utilization in the presence of glucose; Amador P et al.; The mutant IGC 40 x 1001 of the yeast Pichia anomala IGC 4380, which displays inverse diauxic growth in a medium with glucose and malic acid, was studied to elucidate the biochemical mechanisms underlying that behavior . Time course changes of enzyme activities during growth of the mutant in that mixture of substrates indicated that the gluconeogenic enzymes remained active during the first phase of diauxic growth, while glycolytic enzyme activities were significantly reduced . This reduction was essentially due to an alteration in the maximum velocity and not in substrate affinity . Malate, citrate, and adenosine triphosphate did not affect significantly the activities of the glucose phosphorylating enzymes in cell extracts of either the mutant or the wild strain . In P . anomala, unlike Saccharomyces cerevisiae, the fructose/glucose phosphorylating ratio was not associated with repression/derepression conditions.

J Allergy Clin Immunol, 1996 Aug, 98(2), 331 - 43
Cloning and expression in yeast Pichia pastoris of a biologically active form of Cyn d 1, the major allergen of Bermuda grass pollen; Smith PM et al.; BACKGROUND: Pollen of grasses, such as Bermuda grass (Cynodon dactylon), represent a major cause of type I allergy . OBJECTIVE: In this report we attempted to clone and express a biologically active form of recombinant Cyn d 1, the major allergen of Bermuda grass pollen, in the yeast Pichia pastoris . METHODS: Clones encoding Cyn d 1 were isolated by screening a Bermuda grass pollen complementary DNA library with specific monoclonal antibodies and by polymerase chain reaction amplification . Recombinant Cyn d 1 was expressed in Escherichia coli and yeast . The expressed proteins were analyzed by Western blotting to assess binding to Cyn d 1-specific monoclonal antibodies and IgE from sera of patients allergic to Bermuda grass pollen . RESULTS: Two isoforms of Cyn d 1 were cloned . Recombinant Cyn d 1 expressed in bacteria bound two monoclonal antibodies raised against Cyn d 1 but was not recognized by IgE from sera of patients allergic to Bermuda grass pollen . Cyn d 1 expressed in yeast bound both the monoclonal antibodies and human IgE . CONCLUSION: An IgE-reactive Cyn d 1 was expressed in yeast but not in bacteria, suggesting that posttranslational modifications (e.g., glycosylation), which occur in eukaryotic cells such as yeast, are necessary for the production of a biologically active allergen.

Appl Environ Microbiol, 1996 Aug, 62(8), 2832 - 8
Phosphorus-31 and carbon-13 nuclear magnetic resonance studies of glucose and xylose metabolism in cell suspensions and agarose-immobilized cultures of Pichia stipitis and Saccharomyces cerevisiae; Lohmeier-Vogel EM et al.; The metabolism of glucose and xylose as a function of oxygenation in Pichia stipitis and Saccharomyces cerevisiae cell suspensions was studied by 31P and 13C nuclear magnetic resonance spectroscopy . The rate of both glucose and xylose metabolism was slightly higher and the production of ethanol was slightly lower in aerobic than in anoxic cell suspensions of P . stipitis . As well, the cytoplasmic pH of oxygenated cells was more alkaline than that of nonoxygenated cells . In contrast, in S . cerevisiae, the intracellular pH and the rate of glucose metabolism and ethanol production were the same under aerobic and anoxic conditions . Agarose-immobilized Pichia stipitis was able to metabolize xylose or glucose for 24 to 60 h at rates and with theoretical yields of ethanol similar to those obtained with anoxic cell suspensions . Cell growth within the beads, however, was severely compromised . The intracellular pH {pH(int)} of the entrapped cells fell to more acidic pH values in the course of the perfusions relative to corresponding cell suspensions . Of importance was the observation that no enhancement in the rate of carbohydrate metabolism occurred in response to changes in the pH(int) value . In contrast to P . stipitis, agarose-immobilized Saccharomyces cerevisiae showed a dramatic twofold increase in its ability to metabolize glucose in the immobilized state relative to cell suspensions . This strain was also able to grow within the beads, although the doubling time for the entrapped cells was longer, by a factor of 2, than the value obtained for log-phase batch cultures . Initially, the pH(int) of the immobilized cells was more alkaline than was observed with the corresponding S . cerevisiae cell suspensions; however, over time, the intracellular pH became increasingly acidic . As with immobilized P . stipitis, however, the pH(int) did not play a key role in controlling the rate of glucose metabolism.

Proteins, 1996 Jul, 25(3), 398 - 400
Crystallization and preliminary X-ray diffraction studies of human procathepsin L; Coulombe R et al.; Human procathepsin L has been expressed in the yeast Pichia pastoris and its inactive (Cys25Ser) and unglycosylated (Thr110Ala) mutant purified, concentrated to 4 mg/ml, and crystallized by vapor diffusion against solution containing 1.4 M (Na,K)PO4 buffer, pH 7.8 . Crystal size was increased by multiple macroseeding . The crystals are orthorhombic, of space group P2 1 2 1 2 1, with cell dimensions of a = 40.2 A, b = 88.4 A, and c = 94.9 A . A 2.2 A native data set was collected using synchrotron radiation . Although molecular replacement solution for the mature portion of the enzyme was easily found, the resulting maps could not be interpreted in the proregion . Heavy-atom derivative search is in progress.

Yeast, 1996 Jul, 12(9), 815 - 22
Invertase secretion in Hansenula polymorpha under the AOX1 promoter from Pichia pastoris; Rodriguez L et al.; A DNA fragment containing a transcription regulating region of the alcohol oxidase (AOX1) gene from the methylotrophic yeast Pichia pastoris was used in the construction of a vector for the expression of heterologous proteins in the methylotrophic yeast Hansenula polymorpha . We used this vector to clone the SUC2 gene from Saccharomyces cerevisiae into H . polymorpha yeast . The culture conditions for invertase production using a fed-batch culture were studied . More than 1.5 x 10(3) U/ml of biologically active invertase (1 g/l) were secreted to the cellular periplasmic space . The fermentative process was scaled up to 50 l . Invertase produced from H . polymorpha was glycosylated, but it contained significantly less carbohydrate than protein produced by S . cerevisiae . Using the Western-blot technique, it was observed that invertase secreted from H . polymorpha and invertase secreted from S . cerevisiae showed common antigenic determinants.

EMBO J, 1996 Jul 1, 15(13), 3275 - 85
Characterization of a novel component of the peroxisomal protein import apparatus using fluorescent peroxisomal proteins; Kalish JE et al.; Fluorescent peroxisomal probes were developed by fusing green fluorescent protein (GFP) to the matrix peroxisomal targeting signals PTS1 and PTS2, as well as to an integral peroxisomal membrane protein (IPMP) . These proteins were used to identify and characterize novel peroxisome assembly (pas) mutants in the yeast Pichia pastoris . Mutant cells lacking the PAS10 gene mislocalized both PTS1-GFP and PTS2-GFP to the cytoplasm but did incorporate IPMP-GFP into peroxisome membranes . Similar distributions were observed for endogenous peroxisomal matrix and membrane proteins . While peroxisomes from translocation-competent pas mutants sediment in sucrose gradients at the density of normal peroxisomes, >98% of peroxisomes from pas10 cells migrated to a much lower density and had an extremely low ratio of matrix:membrane protein . These data indicate that Pas10p plays an important role in protein translocation across the peroxisome membrane . Consistent with this hypothesis, we find that Pas10p is an integral protein of the peroxisome membrane . In addition, Pas10p contains a cytoplasmically-oriented C3HC4 zinc binding domain that is essential for its biological activity.

Biochemistry, 1996 Jun 25, 35(25), 8149 - 57
Potency and selectivity of the cathepsin L propeptide as an inhibitor of cysteine proteases; Carmona E et al.; The cathepsin L propeptide (phcl-2) was expressed in Saccharomyces cerevisiae using a human procathepsin L/alpha-factor fusion construct containing a stop codon at position -1 (the C-terminal amino acid of the proregion) . Since the yield after purification was very low, the cathepsin L propeptide was also obtained by an alternate procedure through controlled processing of an inactive mutant of procathepsin L (Cys25Ser/Thrl10Ala) expressed in Pichia pastoris, by small amounts of cathepsin L . The peptide resulting from the cleavage of the proenzyme (phcl-1) was then purified by HPLC . The purified propeptides were characterized by N-terminal sequencing and mass spectrometry and correspond to incomplete forms of the proregion (87 and 81 aa for phcl-1 and phcl-2 respectively, compared to 96 aa for the complete cathepsin L propeptide) . The two peptides were found to be potent and selective inhibitors of cathepsin L at pH 5.5, with Ki values of 0.088 nM for phcl-1 and 0.66 nM for phcl-2 . The Ki for inhibition of cathepsin S was much higher (44.6 nM with phcl-1), and no inhibition of cathepsin B or papain could be detected at up to 1 microM of the propeptide . The inhibitory activity was also found to be strongly pH-dependent . Two synthetic peptides of 75 and 44 aa corresponding to N-terminal truncated versions of the propeptide were also prepared by solid phase synthesis and displayed Ki values of 11 nM and 2900 nM, respectively, against cathepsin L . The data obtained for the 4 propeptide derivatives of various lengths indicate that the first 20 residues in the N-terminal region of the propeptide are more important for inhibition than the C-terminal region which contributes little to the overall inhibitory activity.

EMBO J, 1996 Jun 17, 15(12), 2914 - 23
The peroxisome biogenesis disorder group 4 gene, PXAAA1, encodes a cytoplasmic ATPase required for stability of the PTS1 receptor; Yahraus T et al.; In humans, defects in peroxisome assembly result in the peroxisome biogenesis disorders (PBDs), a group of genetically heterogeneous, lethal recessive diseases . We have identified the human gene PXAAA1 based upon its similarity to PpPAS5, a gene required for peroxisome assembly in the yeast Pichia pastoris . Expression of PXAAA1 restored peroxisomal protein import in fibroblasts from 16 unrelated members of complementation group 4 (CG4) of the PBD . Consistent with this observation, CG4 patients carry mutations in PXAAA1 . The product of this gene, Pxaaa1p, belongs to the AAA family of ATPases and appears to be a predominantly cytoplasmic protein . Substitution of an arginine for the conserved lysine residue in the ATPase domain of Pxaaa1p abolished its biological activity, suggesting that Pxaaa1p is an ATPase . Furthermore, Pxaaa1p is required for stability of the predominantly cytoplasmic PTS1 receptor, Pxr1p . We conclude that Pxaaa1p plays a direct role in peroxisomal protein import and is required for PTS1 receptor activity.

Biochem Mol Biol Int, 1996 Jun, 39(3), 471 - 85
Cloning, expression and characterization of a blood group B active recombinant alpha-D-galactosidase from soybean (Glycine max); Davis MO et al.; A cDNA encoding soybean alpha-D-galactosidase {E.C . 3.2.1.22} was obtained by screening a soybean library with Phaseolus alpha-D-galactosidase cDNA . The Glycine max alpha-D-galactosidase cDNA is 1.75 kb long and contains untranslated 5' and 3' sequences . The deduced amino acid sequence of the soybean gene has a high degree of homology with other eucaryotic alpha-D-galactosidases . Recombinant alpha-D-galactosidase (rGal) was expressed in Pichia pastoris and purified by affinity chromatography . Purified rGal was homogeneous as judged by SDS-PAGE analysis with the relative molecular mass under reducing conditions of 39.8, and under nonreducing conditions 38.0 kDa . The expressed protein contained the sequence NGLGHTPPMG at the N-terminus, corresponding to the deduced amino acid sequence of the soybean gene . The relative native molecular mass by Sephacryl S-200 chromatography was determined to be 33.1 kDa . The specific activity was 295.6 mumoles of PNP-alpha-D-galactopyranoside hydrolyzed per mg pure rGal per min . rGal was highly specific for alpha-D-galactosyl residues . No detectable hemagglutinin or protease activity was present in the preparations . Furthermore, rGal was active against the blood group B antigen in native human erythrocyte cell suspension assays . The only detectable erythrocyte phenotypic change was loss of the B and P1 epitopes . Consequently, recombinant Glycine max alpha-D-galactosidase may have useful biotechnical applications in the potential mass production of universally transfusable type O erythrocytes by enzymatic conversion.

Protein Expr Purif, 1996 Jun, 7(4), 423 - 30
Production and purification of human fibroblast collagenase (MMP-1) expressed in the methylotrophic yeast Pichia pastoris; Rosenfeld SA et al.; The cDNA that encodes the proenzyme form of human fibroblast collagenase (proMMP-1) was expressed in the methylotrophic yeast Pichia pastoris . The proMMP-1 encoding DNA was fused to the Saccharomyces cerevisiae pre-pro alpha-mating factor secretion signal in the P . pastoris pPIC9 expression plasmid, transformed into strain GS115 (His-), and His+ Muts (slow methanol utilization) transformants were selected . Full-length proenzyme and processed forms of the protein could be detected in yeast culture supernatants following shake flask and 10-liter fermentations . The protein was purified to greater than 95% homogeneity . The recombinant proMMP-1 was comparable to the native fibroblast material based on (i) migration of the full-length molecule as a 52-kDa protein on reducing SDS-PAGE, (ii) correct N-terminal amino acid sequence, (iii) activation of the full-length molecule by 4-amino-phenylmercuric acetate to yield processed protein species, (iv) degradation of gelatin as monitored by zymogram gels, and (v) enzymatic activity . These data suggest that the P . pastoris expression system offers a convenient and efficient means to produce and purify MMP-1.

J Histochem Cytochem, 1996 Jun, 44(6), 581 - 9
Labeling of peroxisomes with green fluorescent protein in living P . pastoris cells; Monosov EZ et al.; We exploited the light-activated fluorescent properties of the green fluorescent protein (GFP) of the jellyfish Aequorea victoria for studies on the peroxisomal sorting of polypeptides . GFP and GFP-SKL (containing a C-terminal, tripeptide peroxisomal targeting signal, SKL) were expressed from a methanol-inducible, alcohol oxidase (AOX1) promoter in the methylotrophic yeast Pichia pastoris . GFP was cytosolic, whereas the GFP-SKL fusion protein was targeted to peroxisomes, as demonstrated by biochemical fractionation of organelles on Nycodenz gradients . Neither GFP nor GFP-SKL affected the viability of yeast cells but both were fluorescent on excitation with 395-nm UV light . The subcellular locations of GFP and GFP-SKL in living yeast cells were monitored by fluorescence microscopy and their fluorescence was coupled to photo-oxidation of diaminobenzidine (DAB), resulting in the deposition of electron-dense oxidized DAB at intracellular locations of GFP derivatives . This photooxidation procedure permitted facile ultrastructural localization of GFP in cells by electron microscopy, and provided further evidence that GFP produced in P . pastoris is cytosolic, whereas GFP-SKL is peroxisomal . The GFP-SKL fusion protein is therefore a versatile reporter for the peroxisomal compartment, with many applications for studies involving peroxisomal import and biogenesis.

Curr Genet, 1996 Jun, 30(1), 3 - 11
Moving pictures and pulsed-field gel electrophoresis show only linear mitochondrial DNA molecules from yeasts with linear-mapping and circular-mapping mitochondrial genomes; Jacobs MA et al.; The mobility of mitochondrial DNA (mtDNA) in pulsed-field gel electrophoresis (PFGE) and its appearance in moving pictures from fluorescence microscopy were used to investigate the mitochondrial genome structure for five Pichia and Williopsis strains of yeast . An apocytochrome b-gene hybridization probe identified only linear mtDNA molecules for each strain when total cellular DNA was fractionated by PFGE . Most of the mass of DNA isolated from mitochondria for one linear-mapping and one circular-mapping mitochondrial genome was found in linear molecules much larger than the genome size of 50 kb; some molecules were as long as 1500 kb, but only a trace amount of apparently circular mtDNA was found for the strain with the circular-mapping genome . Probes for both the apocytochrome-b and mitochondrial small rRNA subunit genes hybridized strongly to mtDNA of approximately 50-100 kb, but weakly to the larger DNA from mitochondria of these two strains . For the four linear-mapping strains, PFGE revealed two or three distinct bands of linear mtDNA, larger than the genome size, within a smear of approximately 50-100 kb, but a smear without bands was found for the circular-mapping strain.

Proc Natl Acad Sci U S A, 1996 May 28, 93(11), 5357 - 62
Three-dimensional structure of human protein kinase C interacting protein 1, a member of the HIT family of proteins; Lima CD et al.; The three-dimensional structure of protein kinase C interacting protein 1 (PKCI-1) has been solved to high resolution by x-ray crystallography using single isomorphous replacement with anomalous scattering . The gene encoding human PKCI-1 was cloned from a cDNA library by using a partial sequence obtained from interactions identified in the yeast two-hybrid system between PKCI-1 and the regulatory domain of protein kinase C-beta . The PKCI-1 protein was expressed in Pichia pastoris as a dimer of two 13.7-kDa polypeptides . PKCI-1 is a member of the HIT family of proteins, shown by sequence identity to be conserved in a broad range of organisms including mycoplasma, plants, and humans . Despite the ubiquity of this protein sequence in nature, no distinct function has been shown for the protein product in vitro or in vivo . The PKCI-1 protomer has an alpha+beta meander fold containing a five-stranded antiparallel sheet and two helices . Two protomers come together to form a 10-stranded antiparallel sheet with extensive contacts between a helix and carboxy terminal amino acids of a protomer with the corresponding amino acids in the other protomer . PKCI-1 has been shown to interact specifically with zinc . The three-dimensional structure has been solved in the presence and absence of zinc and in two crystal forms . The structure of human PKCI-1 provides a model of this family of proteins which suggests a stable fold conserved throughout nature.

Ann N Y Acad Sci, 1996 May 15, 782, 555 - 65
Molecular cloning of a lytic beta-1,3-glucanase gene from Oerskovia xanthineolytica LLG109 . A beta-1,3-glucanase able to selectively permeabilize the yeast cell wall; Ferrer P et al.; Molecular cloning of the beta gIII gene encoding for an endo-beta-1,3-glucanase (beta gl II) from Oerskovia xanthineolytica LLG109, a yeast-lytic gram-positive bacterium, has been conducted in order to elucidate its primary sequence and subsequently express it into B . subtilis . This endo-beta-1,3-glucanase exhibits low yeast-lytic activity toward viable S . cerevisiae cells, and it has shown ability to selectively permeabilize the yeast cell wall and release intracellular proteins produced by yeast . Highly degenerate oligonucleotides have been used to PCR-amplify a region of the beta-1,3-glucanase II encoding gene from O . xanthineolytica LLG109 . The amplified fragment has been cloned and sequenced . The deduced amino acid sequence contains regions identical to the amino acid sequences previously determined by direct sequencing of the purified enzyme from O . xanthineolytica LLG109 . By using the 180-bp PCR product as a homologous probe, we have been able to isolate four positive clones harboring plasmids pPF1A, pPF1B, pPF8A, and pPF9A, respectively, from a partial genomic library from O . xanthineolytica LLG109 . All four plasmids contained a 2.7-kb BamHI insert that hybridized to the PCR probe under high stringency conditions . The 2.7-kb fragment seemed to be identical in all four cases regarding preliminary partial restriction mapping analysis done on the four plasmids . The 1.5-kb BamHI/KpnI restriction fragment from pPF8A and pPF9A hybridizing with the 180-bp PCR probe is presently being sequenced . The cloning of the lytic beta-1,3-glucanase from O . xanthineolytica LLG109 expands the number of yeast lytic beta-glucanases so far cloned . The availability of the nucleotide sequences of such a family of genes will allow further understanding of the role and mode of action of these enzymes in yeast cell wall degradation . In addition, a more extensive study on the structure and functional relationships of these enzymes will allow us to engineer "tailor-made" lytic beta-1,3-glucanases for use in new and improved large-scale selective cell permeabilization (SCP) and selective protein recovery (SPR) from yeast cells, not only from S . cerevisiae but also from alternative yeast expression systems such as Hansenula polymorpha, Pichia pastoris, and others, which are becoming of increasing importance in biotechnology.

Yeast, 1996 May, 12(6), 541 - 53
Secretion of a variant of human single-chain urokinase-type plasminogen activator without an N-glycosylation site in the methylotrophic yeast, Pichia pastoris and characterization of the secreted product; Tsujikawa M et al.; Human single-chain urokinase-type plasminogen activator without an N-glycosylation site (scu-PA-Q302) was produced in the methylotrophic yeast, Pichia pastoris using the shortened prepeptide sequence of a fungal aspartic proteinase, Mucor pusillus rennin (MPR) . The level of urokinase-type plasminogen activator (u-PA) immunoreactive material in YPM medium was 0.47 mg/l; however, most of the secreted product had been processed to smaller polypeptides . The N-terminal amino acid sequence of major species was identical to that of the low molecular weight two-chain u-PA . Some approaches to minimizing the proteolysis of scu-PA-Q302 were attempted . Addition of Triton X-100, L-arginine and ammonium phosphate to the YPM medium minimized the proteolysis of scu-PA-Q302 and increased the yield of immunoreactive material to approximately 5 mg/l . Use of proteinase A- or proteinase B-deficient strains of yeast did not reduce the degradation . Co-expression of scu-PA-Q302 and urinary trypsin inhibitor resulted in partial reduction of the major species of proteolysis . Scu-PA-Q302 was purified from the culture supernatant of the improved medium by two successive chromatographies on Phenyl-Sepharose and S-Sepharose . The purified protein had a molecular weight of 47 kDa . It did not contain detectable N-linked oligosaccharides, but contained O-linked oligosaccharides attached to the light chain . N-terminal amino acid sequencing of the purified preparation showed that the shortened prepeptide sequence of MPR was correctly processed by the Pichia yeast . Scu-PA-Q302 closely resembles natural scu-PA with respect to its enzymatic activity against the chromogenic substrate S-2444 and its in vitro fibrinolytic properties.

Biosci Biotechnol Biochem, 1996 May, 60(5), 818 - 22
Should Petasospora BOIDIN et ABADIE (Saccharomycetaceae) be retained?--the phylogeny based on the partial sequences of 18S and 26S ribosomal RNAs; Yamada Y et al.; The six strains of the Pichia species, once classified in the genus Petasospora, were examined for their 18S (positions 1451-1618, 168 bases) and 26S (positions 1611-1835, 225 bases and 493-622, 130 bases) rRNA partial base sequencings . In the 18S rRNA partial base sequencings, the type species of the genus Petasospora (Petasospora rhodanensis) was found to be closely related to Pichia anomala (identical to Hansenula anomala, type species of genus Hansenula) with base differences of two, but not to Pichia membranaefaciens, the type species of the genus Pichia, with base differences of ten . However, the species was not so closely related to P . anomala in the 26S rRNA partial base sequencings with base differences of twelve and 68 percent similarity . The genus Petasospora was known to be a very heterogeneous taxon phylogenetically with base differences of thirty-one to three and ninety-two to eight and with 40 to 83 percent similarities . The sequence data obtained here and the phenotypic features described previously indicate that the type species of the genus Petasospora (Pet . rhodanensis, identical to Pichia rhodanensis) is adequate to be accommodated temporarily in the genus Pichia, a heterogeneous taxon, until the precise taxonomic position of the type species is defined.

Appl Environ Microbiol, 1996 May, 62(5), 1839 - 41
Identification of multiple plasmids released from recombinant genomes of Hansenula polymorpha by transformation of Escherichia coli; Graupner S et al.; Total DNAs isolated from two Hansenula polymorpha (Pichia angusta) strains having chromosomal single or tandem multiple integrations of a pUC18-derived expression plasmid produced Escherichia coli transformants which contained plasmids of different size and/or organization than that of the expression plasmid . Evidence that plasmid-like structures are formed in H . polymorpha and that their formation is stimulated by DNA damage is presented in this study.

Mol Cell Biol, 1996 May, 16(5), 2527 - 36
The Pichia pastoris PER6 gene product is a peroxisomal integral membrane protein essential for peroxisome biogenesis and has sequence similarity to the Zellweger syndrome protein PAF-1; Waterham HR et al.; We report the cloning of PER6, a gene essential for peroxisome biogenesis in the methylotrophic yeast Pichia pastoris . The PER6 sequence predicts that its product Per6p is a 52-kDa polypeptide with the cysteine-rich C3HC4 motif . Per6p has significant overall sequence similarity with the human peroxisome assembly factor PAF-1, a protein that is defective in certain patients suffering from the peroxisomal disorder Zellweger syndrome, and with car1, a protein required for peroxisome biogenesis and caryogamy in the filamentous fungus Podospora anserina . In addition, the C3HC4 motif and two of the three membrane-spanning segments predicted for Per6p align with the C3HC4 motifs and the two membrane-spanning segments predicted for PAF-1 and car1 . Like PAF-1, Per6p is a peroxisomal integral membrane protein . In methanol- or oleic acid-induced cells of per6 mutants, morphologically recognizable peroxisomes are absent . Instead, peroxisomal remnants are observed . In addition, peroxisomal matrix proteins are synthesized but located in the cytosol . The similarities between Per6p and PAF-1 in amino acid sequence and biochemical properties, and between mutants defective in their respective genes, suggest that Per6p is the putative yeast homolog of PAF-1.

Int J Food Microbiol, 1996 Apr, 29(2-3), 167 - 75
Media for preservative resistant yeasts: a collaborative study; Hocking AD; An international collaborative study was carried out to determine the most effective medium for selective isolation and enumeration of preservative resistant yeasts . Such a medium should prevent the growth of other yeasts such as Saccharomyces cerevisiae that are tolerant to lower levels of commonly used food preservatives, and sensitive yeasts such as Rhodotorula species . The study compared two non-selective media that are in common use for cultivation of yeasts from foods, Malt Extract agar (MEA) and Tryptone Glucose Yeast extract agar (TGY) with media made selective for preservative resistant yeasts by addition of 0.5% acetic acid to these two basal media (MEAA and TGYA) . A fifth medium, Zygosaccharomyces bailii medium (ZBM) was also included in the study . These media were compared for their efficacy in selective isolation and enumeration of the preservative resistant yeasts Zygosaccharomyces bailii, Schizosaccharomyces pombe and Pichia membranaefaciens . MEA and TGY without acetic acid were used as control, non-selective media, and Rhodotorula glutinis was the preservative sensitive control culture . Seven laboratories in six countries took part in the study . Of the non-selective media, TGY generally gave the highest counts, and TGY amended with 0.5% acetic acid (TGYA) was the best medium for recovery of all three preservative-resistant yeasts . ZBM was found to be selective for Z . bailii, but counts of this yeast on ZBM were significantly lower than on TGYA . R . glutinis did not grow on any of the selective media.

Biochem Biophys Res Commun, 1996 Mar 18, 220(2), 372 - 6
Inhibition of Aspergillus serine proteinase by Streptomyces subtilisin inhibitor and high-level expression of this inhibitor in Pichia pastoris; Markaryan A et al.; Aspergillus fumigatus encodes an extracellular serine proteinase of the subtilisin family that is thought to be involved in invasive aspergillus infection of immunocompromised patients . When the structure of proteinase K was used to model this Aspergillus serine proteinase, Streptomyces subtilisin inhibitor (SSI) was predicted to be capable of binding to the serine proteinase . SSI purified from S . albogriseolus inhibited the serine proteinase with Ki of 1 x 10(-9)M . This value is higher than that for subtilisin probably because the serine proteinase lacks the S(4-6) site that interacts with the P(4-6) site of SSI . A high level expression for SSI, established in Pichia pastoris, yielded 0.5g of SSI per liter of culture medium . The secreted product was easily purified to homogeneity and biochemically characterized . The recombinant SSI showed a Ki of 1.1 x 10(-9) and 1 x 10(-8) for the serine proteinases from A . fumigatus and A . flavus, respectively.

Eur J Biochem, 1996 Mar 15, 236(3), 978 - 83
Influence of expression system on chromophore binding and preservation of spectral properties in recombinant phytochrome A; Gartner W et al.; N-Terminal deletion mutants of the plant photoreceptor phytochrome, additionally truncated at two different positions at their C-terminal ends, were expressed both in Escherichia coli and in yeast (Pichia pastoris) and converted into chromoproteins upon chromophore incorporation . The start and end positions of the cDNA employed (phyA from oat) mimic the positions of tryptic cleavage (deletion of the first 64 amino acids, and stop codons after amino acid positions 425 or 595, generating 39-kDa and 59-kDa peptides, respectively . The absorption properties and photochromicity upon red/far-red irradiation of these mutants were compared with their tryptic counterparts derived from native oat phytochrome and with recombinant products possessing intact N-termini, but C-terminal positions identical to those of the corresponding tryptic fragments (45-kDa and 65-kDa peptides) . All recombinant 65-kDa and 59kDa peptides bound the chromophore after expression and showed the appropriate absorption spectra of the Pr and the Pfr forms . The smaller chromopeptides (45-kDa and 39-kDa) behaved differently depending on the expression system employed . E . coli-derived peptides exhibited a phytochrome-like difference spectrum only when the intact N-terminus was present (45-kDa product) . The recombinant 39-kDa peptide from E . coli was incapable of chromophore binding whereas the identical peptide sequence expressed by P . pastoris formed a chromoprotein with phycocyanobilin . This recombinant phytochrome fragment exhibited a difference spectrum (Pr-Pfr) with an even larger Pfr absorption band than the comparable tryptic 39-kDa fragment . Selectivity of chromophore incorporation and spectral properties suggest that interactions between protein domains of phytochrome control the protein folding and the Pr/Pfr absorption characteristics . Evidently, trypsin digestion down to the 39-kDa fragment affects protein conformation also in terms of Pfr conservation.

J Biol Chem, 1996 Mar 15, 271(11), 6490 - 6
Efficient expression of the gene for spinach phosphoribulokinase in Pichia pastoris and utilization of the recombinant enzyme to explore the role of regulatory cysteinyl residues by site-directed mutagenesis; Brandes HK et al.; Phosphoribulokinase (PRK), unique to photosynthetic organisms, is regulated in higher plants by thioredoxin-mediated thiol-disulfide exchange in a light-dependent manner . Prior attempts to overexpress the higher plant PRK gene in Escherichia coli for structure-function studies have been hampered by sensitivity of the recombinant protein to proteolysis as well as toxic effects of the protein on the host . To overcome these impediments, we have spliced the spinach PRK coding sequence immediately downstream from the AOX1 (alcohol oxidase) promoter of Pichia pastoris, displacing the chromosomal AOX1 gene . The PRK gene is now expressed, in response to methanol, at 4-6% of total soluble protein, without significant in vivo degradation of the recombinant enzyme . This recombinant spinach PRK is purified to homogeneity by successive anion-exchange and dye-affinity chromatography and is shown to be electrophoretically and kinetically indistinguishable from the authentic spinach counterpart . Site-specific replacement of all of PRK's cysteinyl residues (both individually and in combination) demonstrates a modest catalytically facilitative role for Cys-55 (one of the regulatory residues) and the lack of any catalytic role for Cys-16 (the other regulatory residue), Cys-244, or Cys-250 . Mutants with seryl substitutions at position 55 display non-hyperbolic kinetics relative to the concentration of ribulose 5-phosphate . Sulfate restores hyperbolic kinetics and enhances kinase activity, presumably reflecting conformational differences between the position 55 mutants and wild-type enzyme . Catalytic competence of the C16S-C55S double mutant proves that mere loss of free sulfhydryl groups by oxidative regulation cannot account entirely for the accompanying total inactivation.

J Biol Chem, 1996 Mar 15, 271(11), 6298 - 305
Developmental expression of a tandemly repeated, proline-and glutamine-rich amino acid motif on hyphal surfaces on Candida albicans; Staab JF et al.; cDNA sequences encoding a cell wall protein have been isolated from the opportunistic pathogen, Candida albicans, an organism that can cause serious disease in immunocompromised patients such as those with AIDS . The cDNA encodes a peptide that is largely composed of an acidic, repeated motif 10 amino acids in length that is rich in proline and glutamine residues . The cDNA gene product was found to be present on hyphal surfaces by immunofluorescence assays using monospecific antisera raised to the recombinant protein produced in Pichia pastoris . The hyphae-specific surface location was also seen on organisms colonizing the gastrointestinal mucosa of mice, indicating that the antigen is produced and developmentally regulated during growth in host tissues . The cDNA clone hybridized to an abundant messenger RNA 2.3 kilobases in size that was present in hyphal but not yeast forms . These studies demonstrate that the bud-hypha transition is accompanied by the de novo synthesis of proteins that are targeted to hyphal surfaces . The primary sequence of the unique amino acid motif shares features with surface proteins of other lower eukaryotic microorganisms and with host acidic salivary proline-rich proteins.

Arch Biochem Biophys, 1996 Mar 15, 327(2), 324 - 9
Characterization of recombinant alpha-galactosidase for use in seroconversion from blood group B to O of human erythrocytes; Zhu A et al.; Alpha-Galactosidase (alpha-GAL) purified from green coffee bean cleaves the terminal galactose residues from the surface of group B erythrocytes, thereby converting these cells serologically to group O cells . Such enzymatically converted red cells have been transfused into group A and O recipients as part of the first phase of FDA-approved clinical trials . Recently we expressed the recombinant alpha-GAL (r)alpha-GAL) in large quantities in a methylotrophic yeast strain Pichia pastoris and purified the protein to apparent homogeneity by chromatography on a macro prep S50 column . Purified (r)alpha-GAL, migrating as a single band of 41 kDa on a SDS-PAGE, appears to be identical to its native counterpart in specific activity (32 U/mg) and kinetic parameters (K(m) =0.363 mM and V(max) = 46.9 U/mg) . Both enzymes demonstrate the same pH profile in the pH range from 2 to 9, with an optimal pH at 6.4 when tested with the substrate p-nitrophenol-alpha-D-galactopyranoside . Furthermore, as with its native counterpart, (r)alpha-GAL specifically cleaves alpha-linked terminal galactose residues from group B red cells without affecting other major antigens on the red cell surface . In addition, we developed a method for using RT-PCR to detect possible DNA contamination in the purified protein preparation, which is one of the concerns for in vivo studies . Thus, with a simple procedure for over-expression and purification of (r)alpha-GAL from P . pastoris culture, one can readily obtain the enzyme needed for large-scale sero-conversion of red cells.

J Lipid Res, 1996 Mar, 37(3), 599 - 605
Expression and secretion of rabbit plasma cholesteryl ester transfer protein by Pichia pastoris; Kotake H et al.; The rabbit cholesteryl ester transfer protein (CETP) was expressed in the methylotrophic yeast Pichia pastoris by introducing the CETP cDNA under the control of the methanol-inducible alcohol oxidase promoter . The cDNA was cloned from in vitro amplified cDNA of rabbit liver mRNA . The nucleotide sequence of the cloned cDNA differed slightly from the previously published sequence that changed the amino acid sequence in six residues . Interestingly, five of these replacements are identical to the corresponding residues in human CEPT . In addition, the encoded mature N-terminal sequence was changed from Cys- to Arg-Glu-Phe- to link the CETP sequence to the yeast acid phosphatase signal peptide . The culture medium of the transformed cells induced with 1% methanol contained both cholesteryl ester and triglyceride transfer activity comparable to that of rabbit plasma . Like rabbit plasma, the lipid transfer activity in the medium could be inhibited by monoclonal antibodies that block CE/TG transfer or TG transfer alone . Immunoblot analysis of M(r) = 80 K and minor species of M(r) = 60-100 K . In spite of these differences, the specific transfer activity of the recombinant CETP was indistinguishable from that of rabbit plasma CETP of M(r) = 74 K . N-Glycosidase F treatment converted both the recombinant and plasma CETP to a single species of M(r) = 55 K . Both the plasma and recombinant CETP lost their activity after removal of N-linked carbohydrate and sialic acid . A single 55 K component was found in the cell-lysates . The intracellular form of the recombinant CETP was not modified by N-glycosidase F treatment . In conclusion, the recombinant CETP is synthesized as an inactive polypeptide that is processed and secreted as a functional glycoprotein . In addition, the N-terminal Cys residue of the plasma CETP is not required for its activity.

Plant Cell, 1996 Mar, 8(3), 519 - 27
Identification in vitro of a post-translational regulatory site in the hinge 1 region of Arabidopsis nitrate reductase; Su W et al.; Nitrate reductase (NR) is rapidly inactivated by phosphorylation of serine residues in response to loss of light or reduction in CO2 levels . To identify sites within NR protein that play a role in this post-translational regulation, a heterologous expression system and an in vitro inactivation assay for Arabidopsis NR were developed . Protein extracts containing NR kinases and inhibitor proteins were prepared from an NR-defective mutant that had lesions in both the NIA1 and NIA2 NR genes of Arabidopsis . Active NR protein was produced in a Pichia pastoris expression system . Incubation of these two preparations resulted in a Mg-ATP-dependent inactivation of NR that was reversed with EDTA . Mutant forms of NR were constructed, produced in P . pastoris, and tested in the in vitro inactivation assay . Six conserved serine residues in the hinge 1 region of NR, which separates the molybdenum cofactor and heme domains, were specifically targeted for mutagenesis because they are located in a potential regulatory region identified as a target for NR kinases in spinach . A change in Ser-534 to aspartate was found to block NR inactivation; changes in the other five serines had no effect . The aspartate that replaced Ser-534 did not appear to mimic a phosphorylated serine but simply prevented the NR from being inactivated . These results identify Ser-534, located in the hinge 1 of NR and conserved among higher plants NRs, as an essential site for post-translational regulation in vitro.

J Immunol, 1996 Mar 1, 156(5), 1880 - 5
Human natural yeast killer toxin-like candidacidal antibodies; Polonelli L et al.; A murine mAb (mAbKT4, IgG1) that neutralized in vitro the anti-Candida activity of a killer toxic (KT) from the yeast Pichia anomala acted as an idiotypic (Id) vaccine in eliciting anti-Id Abs with toxin-like activity (KT-IdAb) in a rat vaginitis model . In this study, we demonstrate that intravaginal or intragastric inoculations of Candida albicans bearing a receptor for the toxin was able to recall KT-IdAb production in the vagina of the animals primarily immunized with mAbKT4 and also to elicit by themselves an Ab that functionally mimicked the KT (KTAb) . Anti-Id-like, KT-like Abs were also consistently found in the vaginal fluid of human vaginitis patients who were infected by Candida but who had never been exposed to the Id vaccine . These Abs were as candidacidal in vitro as those raised in rat vagina by the Id vaccination, and, likewise, their cytocidal effect was totally neutralized by previous reaction with mAbKT4 . Importantly, they were also able to confer a significant anticandidal protection in the rat vaginitis model, comparable to that achievable by KT-IdAb passively transferred to naive rats from Id-vaccinated animals . Thus, candidacidal Abs representing the internal image of a yeast KT are part of the Ab repertoire that follows infection or immunization with Candida . It is speculated that the host's immune system response may exploit the KT receptor of microbial pathogens to produce microbicidal Abs, possibly mirroring competition events among microorganisms in natural habitats.

Biotechnol Appl Biochem, 1996 Feb, 23 ( Pt 1), 23 - 8
Biochemical characterization of the recombinant Boophilus microplus Bm86 antigen expressed by transformed Pichia pastoris cells; Montesino R et al.; In the present paper we report the biochemical characteristics of the recombinant tick (Boophilus microplus) gut antigen Bm86 that previously has been cloned, expressed and recovered at high levels in the methylotrophic yeast Pichia pastoris . The results demonstrate that rBm86 had a modification at position 92 (Thr replaced by Ile) and aggregated, forming particles ranging between 17 and 40 nm . The rBm86 was N-glycosylated, having at least two non-glycosylated sequons (Asn-329 and Asn-363) and a ratio of only 0.4/65 (free Cys/total Cys)/mol of protein.

J Interferon Cytokine Res, 1996 Feb, 16(2), 119 - 26
High yield expression and secretion of the ovine pregnancy recognition hormone interferon-tau by Pichia pastoris; Van Heeke G et al.; The early conceptus (embryo and associated membranes) of domestic ruminats signals its presence to the maternal uterus through production of interferon-tau (IFN-tau) . Production of IFN-tau ensures continued production of progesterone, the hormone of pregnancy, by the ovarian corpus luteum . This paper reports the high-level expression and efficient secretion of biologically active recombinant ovine IFN-tau (rOvIFN-tau) by Pichia pastoris . The developed method produces more than 80% pure recombinant ovine IFN-tau, obviating the need for further purification for many purposes . Initial fermentation studies produced IFN-tau at 280 mg/liter and demonstrate the potential of this system for large-scale production of IFN-tau.

Toxicon, 1996 Feb, 34(2), 213 - 24
Facile production of native-like kappa-bungarotoxin in yeast: an enhanced system for the production of a neuronal nicotinic acetylcholine receptor probe; Fiordalisi JJ et al.; Research on the mammalian central nervous system had been hindered by the limited number and meager supply of naturally occurring toxins that can be used as pharmacological reagents . The kappa-neurotoxins in particular are not found abundantly in nature and are difficult to obtain and isolate in quantities sufficient for research purposes . Here we report the expression and isolation of relatively large quantities of the kappa-neurotoxin, kappa-bungarotoxin, in an active form using a yeast, Pichia pastoris, expression system . The resultant product of the expression system has a short amino-terminal amino acid extension relative to venom-derived kappa-bungarotoxin, but is equivalent to the native toxin in physical and biological properties, as judged by the CD spectra, the ability to form dimers in solution, and the activity on chick ciliary ganglia . The yeast system produces approximately 0.2 mg from a 2 liter culture and the purification takes approximately 2 days . In contrast, E . coli, the only other available expression system for this toxin, produces one-fifth to one-half as much active material from a 5 liter high-density fermentation and the resulting protein takes over a week to purify . No high mol . wt disulfide-bonded aggregates were found in the yeast expression system product, indicating that the product is that of a biologically assisted folding process . This has significant implications not only for the efficient production of native toxin but also for the production of mutant proteins to study the structure-function relationship in these proteins.

Arch Biochem Biophys, 1996 Feb 1, 326(1), 8 - 14
Functional expression of recombinant spiny dogfish shark (Squalus acanthias) cytochrome P450c17 (17 alpha-hydroxylase/C17,20-lyase) in yeast (Pichia pastoris); Trant JM; The cDNA encoding the spiny dogfish shark (Squalus acanthias) testicular form of cytochrome P450c17 (CYP17) was used to direct the heterologous expression of a functional enzyme in yeast (Pichia pastoris) . This protein possesses two enzymatic activities: 17 alpha-hydroxylase and C17,20-lyase reactions . Cytochrome P450c17 is a key steroidogenic enzyme for the production of sex steroids in gonadal tissue and for cortisol production in adrenal tissue . This study describes the culture conditions and the enzymatic activity of recombinant shark cytochrome P450c17 . The shark enzyme was compatible with the endogenous yeast NADPH-cytochrome P450 reductase and was bioactive within the living yeast cell . Progesterone (at 15 microM) was metabolized (51 pmol/min/10(9) cells) faster than pregnenolone (36 pmol/min/10(9) cells) . Both progesterone and pregnenolone were completely metabolized to their respective androgens (androstenedione and dehydroepiandrosterone) . Although 11 beta-hydroxy-progesterone was readily 17 alpha-hydroxylated by the shark P450, the lyase reaction was not evident . Alterations to the 2-carbon sidechain of progesterone (21-hydroxylation or 20 beta-reduction) prevented metabolism . High-density cultures (> 1.5 x 10(9) cells/ml) yielded the greatest quantity of recombinant protein but cultures of lower density produced more recombinant protein per cell . This is the first report of heterologous expression in yeast of a steroidogenic cytochrome P450 from a lower vertebrate.

Adv Exp Med Biol, 1996, 409, 147 - 55
Recombinant expression and epitope mapping of grass pollen allergens; Suphioglu C et al.; We have studied the expression of recombinant forms of Group 1 allergens from rye-grass and Bermuda grass pollens . Recombinant Lol p 1 expressed in bacteria bound serum IgE from allergic patients . Based on analysis of fragments of the Lol p 1 cDNA clone, the major IgE-reactive epitope has been mapped to the C-terminus . However, although SDS-denatured natural Cyn d 1 (from Bermuda grass) bound IgE, the full or partial recombinant proteins expressed in bacteria did not bind IgE . We have since expressed Cyn d 1 in the yeast Pichia pastoris and restored IgE binding . cDNA clones encoding two isoforms of Lol p 5, Lol p 5A and Lol p 5B, have been expressed in bacteria and resulting polypeptides show IgE-binding . Random fragments of these clones have been generated and when expressed as partial recombinant proteins in bacteria, allowed us to identify the major IgE-binding epitopes . The allergenic epitopes were localised towards the C-terminal half of the molecule . Although both isoforms shared similar IgE-reactive epitopes, Lol p 5B did not recognise the Lol p 5A-specific monoclonal antibody A7 . At sequence level, there appear to be several amino acid differences between the antigenic epitopes of these two isoallergens . These results aid in the design of diagnostics and in grass pollen immunotherapy.

Mycopathologia, 1996, 135(1), 1 - 8
Killer factor interference in mixed opportunistic yeast cultures; Conti S et al.; The interaction of the killer yeast Pichia anomala UP 25F with the killer toxin-sensitive clinical isolate Candida albicans UCSC 10S and its natural toxin-resistant mutant derivative C . albicans UCSC 10R were studied under various conditions . A differential inhibition was shown to occur in vitro at pH and temperature values, which are not encountered in vivo, only by using preformed killer toxin, since antagonism due to yeast growth proved to be predominant on the killer effect . Under adverse growth conditions, the P . anomala killer yeast proved to be able to produce an anatoxin antigenically related to the active or heat inactivated killer toxin . These findings suggest that killer toxins may not function as potential virulence factors in the competition between the opportunistic killer yeast P . anomala and sensitive microorganisms for colonization in the course of natural human infections.

Parasitol Res, 1996, 82(2), 114 - 6
Inhibitory effect of a Pichia anomala killer toxin on Pneumocystis carinii infectivity to the SCID mouse; Seguy N et al.; A Pichia anomala killer toxin has been demonstrated to have a specific inhibitory effect on the in vitro attachment of Pneumocystis carinii . The results presented herein show that this yeast toxin is also effective against P . carinii infectivity in reducing parasite colonization in the lungs of SCID mice . The specificity of this inhibitory effect was controlled using a monoclonal antibody neutralizing the killer properties of the yeast toxin.

Yeast, 1996 Jan, 12(1), 31 - 40
Cloning and characterization of the Pichia pastoris PRC1 gene encoding carboxypeptidase Y; Ohi H et al.; We purified a 58 kDa serine protease from culture-supernatant of Pichia pastoris and found that the NH2-terminal amino acid sequence of this protease is closely homologous to that of mature protein of Saccharomyces cerevisiae carboxypeptidase Y (CPY), which is encoded by the PRC1 gene . Using the S . cerevisiae PRC1 gene as a hybridization probe, a cross-hybridizing fragment of P . pastoris genomic DNA was identified and the gene, PRC1, encoding CPY, was cloned . The open reading frame of the P . pastoris PRC1 gene consists of 1569 bp encoding a protein of 523 amino acids . The molecular mass of the protein is calculated to be 59.44 kDa without sugar chains . The protein comprises 20 amino acids of pre (signal)-peptide, 87 amino acids of pro-peptide and 416 amino acids of mature peptide, and has four N-glycosylation sites . The NH2-terminal amino acid sequence of mature peptide is completely identical with that of the protease purified from the culture-supernatant . There is 61% identity between the amino acid sequences of P . pastoris Prc1p and S . cerevisiae Prc1p . Chromosomal disruption of the PRC1 gene resulted in the loss of CPY activity . Over-expression of the PRC1 gene under regulation of the P . pastoris AOX1 promoter resulted in accumulation of a large amount of active CPY in the intracellular fraction, and secretion of a slightly larger molecule that is thought to be pro-CPY . The nucleotide sequence data reported in this paper will appear in the EMBL Nucleotide Sequence Databases under the Accession Number X87987.

Appl Biochem Biotechnol, 1996 Spring, 57-58, 267 - 76
Increased xylose reductase activity in the xylose-fermenting yeast Pichia stipitis by overexpression of XYL1; Dahn KM et al.; The Pichia stipitis xylose reductase gene (XYL1) was inserted into an autonomous plasmid that P . stipitis maintains in multicopy . The plasmid pXOR with the XYL1 insert or a control plasmid pJM6 without XYL1 was introduced into P . stipitis . When grown on xylose under aerobic conditions, the strain with pXOR had up to 1.8-fold higher xylose reductase (XOR) activity than the control strain . Oxygen limitation led to higher XOR activity in both experimental and control strains grown on xylose . However, the XOR activities of the two strains grown on xylose were similar under oxygen limitation . When grown on glucose under aerobic or oxygen-limited conditions, the experimental strain had XOR activity up to 10 times higher than that of the control strain . Ethanol production was not improved, but rather it decreased with the introduction of pXOR compared to the control, and this was attributed to nonspecific effects of the plasmid.

Appl Biochem Biotechnol, 1996 Spring, 57-58, 201 - 12
Peculiarities of the regulation of fermentation and respiration in the crabtree-negative, xylose-fermenting yeast Pichia stipitis; Passoth V et al.; The respiration of Pichia stipitis was not repressed by either high concentrations of fermentable sugars or oxygen limitation . Fermentation was not induced by high sugar concentrations, but was inactivated by aerobic conditions . The activity of pyruvate dehydrogenase was constitutive . In contrast, pyruvate decarboxylase, alcohol dehydrogenase, and aldehyde dehydrogenase were induced by a reduction in the oxygen tension . It was demonstrated that in P . stipitis, the pyruvate decarboxylase is not induced by a signal from glycolysis . Contrary to Saccharomyces cerevisiae, the pyruvate decarboxylase was not inhibited by phosphate.

Microbiology, 1996 Jan, 142 ( Pt 1), 165 - 72
A heterologous reductase affects the redox balance of recombinant Saccharomyces cerevisiae; Meinander N et al.; Recombinant Saccharomyces cerevisiae harbouring the xylose reductase (XR) gene XYL1 from Pichia stipitis was grown in anoxic chemostat culture at two different dilution rates . At each dilution rate a transient experiment, encompassing a shift in the sugar content of the medium from glucose to glucose plus xylose was performed . The steady states at the beginning and the end of the transients were compared in terms of specific product fluxes from glucose metabolism . At both dilution rates, the specific glycerol flux decreased and the specific acetate and CO2 fluxes increased . The specific ethanol flux was not affected . At the lower dilution rate, the production of biomass decreased during the transient, but at the higher dilution rate it increased . The changes in product pattern can be explained as being due to the redox perturbation caused by the consumption of reduced cofactors in the XR-catalysed reaction . Regeneration of NAD partly through xylose reduction instead of glycerol production decreased the formation of glycerol . Additionally, xylose reduction activated those pathways which produce reduced cofactors, such as acetate formation and the pentose phosphate pathway, indicated by increased acetate and CO2 production . The dual cofactor specificity of XR, with a preference for NADPH over NADH, was evident from the effects of xylose reduction on product fluxes . Comparison of the xylose reduction rates at low and high glucose flux indicated that the supply of reduced cofactors partly controlled the reaction rate . At the higher dilution rate, control by some other factor such as xylose transport or XR activity increased . Calculation of carbon balances at the steady states showed that all substrate carbon was recovered in biomass or products . Based on the specific product fluxes, calculations of quantitative cofactor balances at the steady states was attempted . However, sensitivity calculations showed that analysis errors in the range of 5% caused substantial errors in the cofactor balance, without affecting the carbon balance.

Gene, 1995 Dec 29, 167(1-2), 215 - 9
High-level production of spinach glycolate oxidase in the methylotrophic yeast Pichia pastoris: engineering a biocatalyst; Payne MS et al.; Glycolate oxidase (GO) is a flavo-enzyme that catalyzes the oxidation of glycolate, and is useful for the biocatalytic production of glyoxylate . We have produced high levels of spinach GO in the methylotrophic yeast Pichia pastoris (Pp), by chromosomal integration of multiple copies of an expression cassette containing the GO coding sequence under control of the methanol-inducible alcohol oxidase I promoter . Under fermentation conditions, greater than 250 units of GO per gram of cells (wet weight) was obtained, corresponding to roughly 20-30% of soluble cell protein . This recombinant Pp strain was used as a whole-cell biocatalyst for conversion of glycolic acid to glyoxylic acid.

FEBS Lett, 1995 Dec 27, 377(3), 451 - 6
Expression of functional mouse 5-HT5A serotonin receptor in the methylotrophic yeast Pichia pastoris: pharmacological characterization and localization; Weiss HM et al.; The methylotrophic yeast Pichia pastoris was tested for heterologous expression of the mouse 5-HT5A receptor . Three different expression plasmids were constructed where the cDNA of the receptor was cloned under the transcriptional control of the highly inducible promoter of the P . pastoris alcohol oxidase 1 (AOX1) gen . The expression plasmids differed with respect to the signal sequences used for N-terminal fusion . In two cases the coding region was additionally fused to the c-myc tag to permit immunological detection of the receptor . Expression of functional receptor after transformation of strain GS115 was detected by radioligand binding using {3H}LSD . The construct with the best expression levels in strain GS115 was used for transformation of the protease deficient strain SMD1163 . Here, the expression level was 2-8 times higher . Whole cells as well as crude membrane preparations of recombinant clones showed saturable binding of {3H}LSD with a Kd of approximately 1.9 nM . Receptor concentrations of approximately 22 pmol/mg membrane protein revealed the potential of the P . pastoris expression system for high level expression of membrane proteins . The pharmacological properties were comparable to those reported for the receptor expressed in mammalian systems.

Protein Expr Purif, 1995 Dec, 6(6), 813 - 20
Expression of a synthetic gene encoding the anticoagulant-antimetastatic protein ghilanten by the methylotropic yeast Pichia pastoris; Brankamp RG et al.; Ghilantens are a family of cysteine-rich inhibitors of the clotting enzyme, factor Xa, that are produced in the salivary glands of the South American leech Haementeria ghilianii . In this study, a gene, designed from the amino acid sequence of a specific ghilanten isoform, was assembled from eight double-stranded oligonucleotide fragments . A yeast expression plasmid, pPIC9HG-2, was constructed by making an inframe fusion of the ghilanten-coding sequences with the region encoding the pre-pro alpha-mating factor signal sequence for secretion . The expression of ghilanten in pPIC9HG-2 was under the control of the methanol-inducible, alcohol oxidase (AOX1) promoter . Pichia pastoris yeast strains KM 71 and SMD 1168 were transformed with linearized pPIC9HG-2 to target integration of the plasmid to the chromosomal 5'-AOX1 locus via homologous recombination . Both strains yielded His+ transformants that secreted a potent anticoagulant activity into the medium . Product yield was improved by using buffered media (pH 6.0) supplemented with either casamino acids or a mixture of yeast extract and peptone . The protease-deficient strain, SMD 1168, secreted about a twofold higher level of r-ghilanten than KM 71 . Significant clonal variation in the expression of r-ghilanten was found among the His+ transformants . A high producing clone was selected for production at the 2-liter shake flask and 10-liter bioreactor scales . r-Ghilanten was recovered from the fermentation broths in a single step by heparin Sepharose affinity chromatography . Protein sequence analysis of the amino terminus showed that the correct processing to yield mature ghilanten varied with the fermentation conditions.

Appl Microbiol Biotechnol, 1995 Dec, 44(3-4), 314 - 20
Xylulose fermentation by Saccharomyces cerevisiae and xylose-fermenting yeast strains; Yu S et al.; Xylulose fermentation by four strains of Saccharomyces cerevisiae and two strains of xylose-fermenting yeasts, Pichia stipitis CBS 6054 and Candida shehatae NJ 23, was compared using a mineral medium at a cell concentration of 10 g (dry weight)/l . When xylulose was the sole carbon source and fermentation was anaerobic, S . cerevisiae ATCC 24860 and CBS 8066 showed a substrate consumption rate of 0.035 g g cells-1 h-1 compared with 0.833 gg cells-1 h-1 for glucose . Bakers' yeast and S . cerevisiae isolate 3 consumed xylulose at a much lower rate although they fermented glucose as rapidly as the ATCC and the CBS strains . While P . stipitis CBS 6054 consumed both xylulose and glucose very slowly under anaerobic conditions, C . shehatae NJ 23 fermented xylulose at a rate of 0.345 gg cells-1 h-1, compared with 0.575 gg cells-1 h-1 for glucose . For all six strains, the addition of glucose to the xylulose medium did not enhance the consumption of xylulose, but increased the cell biomass concentrations . When fermentation was performed under oxygen-limited conditions, less xylulose was consumed by S . cerevisiae ATCC 24860 and C . shehatae NJ 23, and 50%- 65% of the assimilated carbon could not be accounted for in the products determined.

Appl Environ Microbiol, 1995 Dec, 61(12), 4251 - 7
Yeast succession in the Amazon fruit Parahancornia amapa as resource partitioning among Drosophila spp; Morais PB et al.; The succession of yeasts colonizing the fallen ripe amapa fruit, from Parahancornia amapa, was examined . The occupation of the substrate depended on both the competitive interactions of yeast species, such as the production of killer toxins, and the selective dispersion by the drosophilid guild of the amapa fruit . The yeast community associated with this Amazon fruit differed from those isolated from other fruits in the same forest . The physiological profile of these yeasts was mostly restricted to the assimilation of a few simple carbon sources, mainly L-sorbose, D-glycerol, DL-lactate, cellobiose, and salicin . Common fruit-associated yeasts of the genera Kloeckera and Hanseniaspora, Candida guilliermondii, and Candida krusei colonized fruits during the first three days after the fruit fell . These yeasts were dispersed and served as food for the invader Drosophila malerkotliana . The resident flies of the Drosophila willistoni group fed selectively on patches of yeasts colonizing fruits 3 to 10 days after the fruit fell . The killer toxin-producing yeasts Pichia kluyveri var . kluyveri and Candida fructus were probably involved in the exclusion of some species during the intermediate stages of fruit deterioration . An increase in pH, inhibiting toxin activity and the depletion of simple sugars, may have promoted an increase in yeast diversity in the later stages of decomposition . The yeast succession provided a patchy environment for the drosophilids sharing this ephemeral substrate.

Appl Environ Microbiol, 1995 Dec, 61(12), 4184 - 90
Xylose-metabolizing Saccharomyces cerevisiae strains overexpressing the TKL1 and TAL1 genes encoding the pentose phosphate pathway enzymes transketolase and transaldolase; Walfridsson M et al.; Saccharomyces cerevisiae was metabolically engineered for xylose utilization . The Pichia stipitis CBS 6054 genes XYL1 and XYL2 encoding xylose reductase and xylitol dehydrogenase were cloned into S . cerevisiae . The gene products catalyze the two initial steps in xylose utilization which S . cerevisiae lacks . In order to increase the flux through the pentose phosphate pathway, the S . cerevisiae TKL1 and TAL1 genes encoding transketolase and transaldolase were overexpressed . A XYL1- and XYL2-containing S . cerevisiae strain overexpressing TAL1 (S104-TAL) showed considerably enhanced growth on xylose compared with a strain containing only XYL1 and XYL2 . Overexpression of only TKL1 did not influence growth . The results indicate that the transaldolase level in S . cerevisiae is insufficient for the efficient utilization of pentose phosphate pathway metabolites . Mixtures of xylose and glucose were simultaneously consumed with the recombinant strain S104-TAL . The rate of xylose consumption was higher in the presence of glucose . Xylose was used for growth and xylitol formation, but not for ethanol production . Decreased oxygenation resulted in impaired growth and increased xylitol formation . Fermentation with strain S103-TAL, having a xylose reductase/xylitol dehydrogenase ratio of 0.5:30 compared with 4.2:5.8 for S104-TAL, did not prevent xylitol formation.

J Bacteriol, 1995 Dec, 177(24), 7070 - 7
Cloning of a novel constitutively expressed pectate lyase gene pelB from Fusarium solani f . sp . pisi (Nectria haematococca, mating type VI) and characterization of the gene product expressed in Pichia pastoris; Guo W et al.; Since plant-pathogenic fungi must penetrate through pectinaceous layers of the host cell wall, pectin-degrading enzymes are thought to be important for pathogenesis . Antibodies prepared against a pectin-inducible pectate lyase (pectate lyase A {PLA}) produced by a phytopathogenic fungus, Fusarium solani f . sp . pisi (Nectria haematococca, mating type VI), was previously found to protect the host from infection . The gene (pelA) and its cDNA were cloned and sequenced . Here we report the isolation of a new pectate lyase gene, pelB, from a genomic library of F . solani f . sp . pisi with the pelA cDNA as the probe . A 2.6-kb DNA fragment containing pelB and its flanking regions was sequenced . The coding region of pelB was amplified by reverse transcription-mediated PCR, using total RNA isolated from F . solani pisi culture grown in the presence of glucose as the sole carbon source . The predicted open reading frame of pelB would encode a 25.6-kDa protein of 244 amino acids which has 65% amino acid sequence identity with PLA from F . solani f . sp . pisi but no significant homology with other pectinolytic enzymes . The first 16 amino acid residues at the N terminus appeared to be a signal peptide . The pelB cDNA was expressed in Pichia pastoris, yielding a pectate lyase B (PLB) which was found to be a glycoprotein of 29 kDa . PLB was purified to homogeneity by using a two-step procedure involving ammonium sulfate precipitation followed by Superdex G75 gel filtration chromatography . Purified PLB showed optimal lyase activity at pH 10.0 . A rapid drop in the viscosity of the substrate and Mono Q anion-exchange chromatography of the products generated by the lyase showed that PLB cleaved polygalacturonate chains in an endo fashion . Western blotting (immunoblotting) with antibodies raised against PLA showed that PLB and PLA are immunologically related to each other . The 5' flanking regions of both pelA and pelB were translationally fused to the beta-glucuronidase gene and introduced into F . solani f . sp . pisi, and beta-glucuronidase activities of the transformants were measured . Expression of the marker gene by the transformants showed that pelA expression is induced by pectin and repressed by glucose, whereas expression of pelB is constitutive and is not subject to glucose repression . Reverse transcription-mediated PCR showed that both pelA and pelB are expressed when F . solani f . sp . pisi infects pea epicotyl.

Arch Biochem Biophys, 1995 Dec 1, 324(1), 65 - 70
High-level expression and purification of coffee bean alpha-galactosidase produced in the yeast Pichia pastoris; Zhu A et al.; alpha-Galactosidase isolated from coffee beans cleaves the terminal alpha-galactose residues from oligosaccharide chains on blood group B red cells, thus generating group O cells . Such enzymatically converted red cells not only maintain full erythrocyte integrity and viability in vitro, but also demonstrate immune tolerance and a normal life span in vivo . In order to produce large quantities of recombinant alpha-galactosidase for use in the study of blood-type conversion, we subcloned the cDNA coding for coffee bean alpha-galactosidase into the EcoRI site of the vector pPIC9 in order to express the enzyme in Pichia pastoris, a methylotrophic yeast strain . After P . pastoris transformation, colonies were screened for high-level expression of alpha-galactosidase, based on enzyme activity . In order to increase enzyme production, the growth conditions in the shake flask culture and fermentor culture were optimized . Under the conditions applied, biologically active alpha-galactosidase was produced and secreted into the culture medium at a level of approximately 0.4 g per liter of the fermentor culture . The protein was purified to apparent homogeneity by a simple chromatography procedure, as suggested by a single band of 41 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis . Its homogeneity was further confirmed by chromatofocusing and N-terminal sequencing . P . pastoris appears to be the choice as host for the large-scale production of recombinant alpha-galactosidase used for blood type conversion.

Arch Biochem Biophys, 1995 Nov 10, 323(2), 352 - 60
Cloning of a new pectate lyase gene pelC from Fusarium solani f . sp . pisi (Nectria haematococca, mating type VI) and characterization of the gene product expressed in Pichia pastoris; Guo W et al.; Antibodies prepared against a pectate lyase (PLA) produced by a phytopathogenic fungus Fusarium solani f . sp . pisi (Nectria haematococca, mating type VI) were previously found to protect the host against infection . The cDNA and gene (pelA) for PLA were cloned and sequenced . A new pectate lyase gene, pelC, was isolated from a genomic library of F . solani pisi with pelA cDNA as a probe . A 1.3-kb DNA fragment containing the pelC gene and its flanking regions was identified and sequenced . The coding region of pelC was amplified by reverse transcription-polymerase chain reaction using total RNA isolated from a pectin-induced F . solani pisi culture as template . The open reading frame of pelC was predicted to encode a 23.3-kDa protein of 219 amino acid residues, which shares 51% identity with PLA from F . solani pisi . No typical fungal leader peptide sequence could be identified at the N-terminus of the predicted protein sequence . The amplified pelC cDNA was expressed in Pichia pastoris yielding a pectate lyase C (PLC) with a molecular mass of 26.0 kDa and containing carbohydrates . PLC was purified to homogeneity using Mono Q anion-exchange chromatography . Purified PLC required Ca2+ for its activity and showed optimal lyase activity at pH 9.5 and 55 degrees C . Rapid drop in the viscosity of the substrate and Mono Q anion-exchange chromatography of the products generated by the lyase showed that PLC cleaved polygalacturonate chains in an endo fashion . Western blot using antibodies raised against PLA and PLC showed that PLC and PLA are immunologically related to each other.

Protein Eng, 1995 Nov, 8(11), 1177 - 87
Large-scale expression, purification and characterization of small fragments of thrombomodulin: the roles of the sixth domain and of methionine 388; White CE et al.; Fragments of human thrombomodulin (TM) have been expressed in large quantities in the Pichia pastoris yeast expression system and purified to homogeneity . Fermentation of P . pastoris resulted in yields of 170 mg/l TM . Purification to homogeneity resulted in an overall 10% yield, so that quantities of approximately 20 mg purified fragments can be readily obtained . Smaller fragments of TM, such as the individual fourth or fifth domains, were not active, nor were equimolar mixtures of the two domains . These results demonstrate that the fourth and fifth epidermal growth factor (EGF)-like domains together comprise the smallest active fragment of TM . The fragment containing the fourth and fifth EGF-like domains {TMEGF(4-5)} had 10% the specific activity of rabbit TM . Comparison of the M388L mutant TMEGF(4-5) fragment with the same mutant TMEGF(4-5-6) fragment showed that the fragment with the sixth domain had a 10-fold better Km value for thrombin than the fragment that did not contain the sixth domain; this factor completely accounts for the higher specific activity of the fragments containing the sixth domain . Comparison of the wild-type and M388L mutants showed that the M388L mutation resulted in a 2-fold increase in kcat for the activation of protein C by the thrombin-TM fragment complex, completely accounting for the 2-fold increase in specific activity of these mutant fragments.

Anal Biochem, 1995 Nov 1, 231(2), 342 - 8
Concanavalin A- and wheat germ agglutinin-conjugated lectins as a tool for the identification of multiple N-glycosylation sites in heterologous protein expressed in yeast; Garcia R et al.; We report here a methodology that allows the identification of glycosylation sites by a combination of protein enzymatic digestion, glycopeptide separation on a reverse-phase HPLC column, and further recognition in a dot-blot system using concanavalin A-horseradish peroxidase . Wheat germ agglutinin-horseradish peroxidase is used as the recognition system for peptides generated after proteolytic digestion of endoglycosidase H deglycosylated protein . Glycosylation sites were confirmed by automatic Edman degradation and fast atom bombardment mass spectrometry . This methodology was applied to a model glycoprotein, alpha-amylase from Bacillus licheniformis, which is unglycosylated in its natural host and appears highly glycosylated when expressed in the methylotrophic yeast Pichia pastoris.

J Interferon Cytokine Res, 1995 Nov, 15(11), 955 - 63
Expression of a human mutant monocyte chemotactic protein 3 in Pichia pastoris and characterization as an MCP-3 receptor antagonist; Masure S et al.; The cDNA encoding human monocyte chemotactic protein 3 (hMCP-3) was cloned in pHIL-S1, a vector designed for inducible secreted heterologous expression in the methylotrophic yeast Pichia pastoris . After transformation of P . pastoris by electroporation, several clones with the human MCP-3 gene integrated at the alcohol oxidase (AOX-1) locus were isolated . One of these clones (M30) expressed the mature MCP-3 protein with three additional amino acids at its NH2 terminus as a secretion product in the supernatant . The recombinant protein comigrated on SDS-PAGE and cross-reacted immunologically with synthetic hMCP-3 . Intermediate-scale production in shake flasks was obtained at expression levels of approximately 1 mg per liter . The recombinant mutant MCP-3 was purified to homogeneity by adsorption on silicic acid, affinity chromatography on heparin-Sepharose, and reversed-phase HPLC . At the amino terminus of the purified recombinant protein, the presence of the additional sequence Arg-Glu-Phe was confirmed by direct protein sequence analysis . The recombinant hMCP-3 mutein was not glycosylated, as evidenced by deglycosylation experiments and by mass spectrometry . In analogy with MCP-1, the amino terminus of MCP-3 is crucial for its agonistic effect on receptive cells . At concentrations up to 3.5 micrograms/ml, the recombinant mutein was not active in vitro as a chemotactic factor for monocytes . However, the mutant MCP-3 acted as an MCP-3 receptor antagonist in a competition chemotaxis assay at 100- to 1000-fold excess over the synthetic MCP-3 agonist . It might thus be a useful tool to study antagonism of MCP-3 action in vitro and in disease models of cancer and inflammation.

Mol Cell Biol, 1995 Nov, 15(11), 6406 - 19
Formation of the peroxisome lumen is abolished by loss of Pichia pastoris Pas7p, a zinc-binding integral membrane protein of the peroxisome; Kalish JE et al.; We have cloned and sequenced PAS7, a gene required for peroxisome assembly in the yeast Pichia pastoris . The product of this gene, Pas7p, is a member of the C3HC4 superfamily of zinc-binding proteins . Point mutations that alter conserved residues of the C3HC4 motif abolish PAS7 activity and reduce zinc binding, suggesting that Pas7p binds zinc in vivo and that zinc binding is essential for PAS7 function . As with most pas mutants, pas7 cells exhibit a pronounced deficiency in import of peroxisomal matrix proteins that contain either the type 1 peroxisomal targeting signal (PTS1) or the type 2 PTS (PTS2) . However, while other yeast and mammalian pas mutants accumulate ovoid, vesicular peroxisomal intermediates, loss of Pas7p leads to accumulation of membrane sheets and vesicles which lack a recognizable lumen . Thus, Pas7p appears to be essential for protein translocation into peroxisomes as well as formation of the lumen of the organelle . Consistent with these data, we find that Pas7p is an integral peroxisomal membrane protein which is entirely resistant to exogenous protease and thus appears to reside completely within the peroxisome . Our observations suggest that the function of Pas7p defines a previously unrecognized step in peroxisome assembly: formation of the peroxisome lumen . Furthermore, because the peroxisomal intermediates in the pas7 delta mutant proliferate in response to peroxisome-inducing environmental conditions, we conclude that Pas7p is not required for peroxisome proliferation.

J Biochem (Tokyo), 1995 Oct, 118(4), 825 - 31
Vl-linker-Vh orientation-dependent expression of single chain Fv-containing an engineered disulfide-stabilized bond in the framework regions; Luo D et al.; Single chain Fv fragments (scFv) derived from an antibody, MAb 174H.64 (Tru-ScintRSQ kit, Biomira), were constructed in both orientations, i.e . Vh-linker-Vl and Vl-linker-Vh, but only the latter form could be expressed and secreted in the recombinant Pichia pastoris system . The secreted scFv protein showed specific anti-idiotype binding activity . Additionally, the molecular graphic modeling has been used to identify a possible site for the introduction of an interchain disulfide bond in the framework region of Fv . These Cys-modifications of the sites were done using a method of PCR-mediated mutagenesis . The engineered protein (disulfide-stabilized Fv: dsFv) was expressed and tested for its binding activity . It was found that dsFv was as active as the corresponding scFv and more stable as determined by competitive radioimmunoassay.

Protein Expr Purif, 1995 Oct, 6(5), 619 - 24
Production and purification of N-terminal half-transferrin in Pichia pastoris; Steinlein LM et al.; Human serum transferrin, the major iron transport protein in humans, is a monomeric glycoprotein that is composed of two homologous domains; the N-terminal domain is formed by amino acids 1-331 and the C-terminal domain is formed by amino acids 338-679 . Each domain is capable of binding one iron atom concomittantly with a carbonate anion; however, the two homologous iron binding sites are not chemically equivalent . The cDNA sequence coding for the N-terminal domain has been cloned and overexpressed in the methylotrophic yeast, Pichia pastoris . The transformants secrete a protein of approximately 38 kDa (the size expected for N-terminal half-transferrin), its N-terminal sequence agrees with the predicted sequence, and the protein reacts with anti-human serum transferrin antibodies . The purified protein appears to be properly folded and can bind iron as demonstrated by its spectral properties and urea-PAGE mobility . It is estimated that N-terminal half-transferrin represents approximately 90% of all protein secreted into the culture medium and that it is expressed at levels exceeding 50 mg/l . This study demonstrates that N-terminal half-transferrin can easily be expressed in the simple host system, Pichia pastoris, and that the purified protein is capable of reversibly binding iron.

FEMS Microbiol Lett, 1995 Oct 1, 132(1-2), 39 - 43
A hydroxymethylglutaryl-CoA reductase inhibitor synthesized by yeasts; Gunde-Cimerman N et al.; Screening of different yeast species showed that they are able to synthesize hydroxymethylglutaryl-CoA (HMGCoA) reductase inhibitors . Crude methanol extracts and the purified inhibitors from Pichia labacensis and Candida cariosilignicola were tested for their biological activity on the solubilized microsomal HMGCoA reductase from Chinese hamster ovary cells . Identification of the inhibitors was studied by thin layer chromatography, high pressure liquid chromatography and mass spectroscopy.

Biochim Biophys Acta, 1995 Sep 27, 1252(1), 28 - 34
Production and characterization of recombinant human proteinase inhibitor 6 expressed in Pichia pastoris; Sun J et al.; The human intracellular serine proteinase inhibitor, proteinase inhibitor 6 (PI-6), was expressed in the methylotropic yeast Pichia pastoris . The PI-6 cDNA was modified to encode six histidine residues immediately after the initiation codon, and was placed under the control of the P . pastoris alcohol oxidase promoter in the vector pHIL-D2 . On the methanol induction, active recombinant PI-6 was produced within the yeast cells, and following cell lysis, was separated from yeast proteins by affinity chromatography using nickel nitrilo-tri-acetic acid (NTA) resin . The interaction of recombinant PI-6 with a range of serine proteinases was studied . Second order association rate constants (ka) were derived for the interaction with trypsin (1.8 x 10(6) M-1 s-1), thrombin (1.2 x 10(5) M-1 s-1), urokinase plasminogen activator (4.0 x 10(4) M-1 s-1), plasmin (1.3 x 10(6) M-1 s-1), and activated protein C (7.5 x 10(3) M-1 s-1) . By monitoring complex formation, recombinant PI-6 was also shown to interact with factor Xa . No complex formation was observed with chymotrypsin, human leukocyte elastase, cathepsin G and tissue plasminogen activator, although PI-6 is apparently a substrate for chymotrypsin, leukocyte elastase and cathepsin G.

Gene, 1995 Sep 22, 163(1), 19 - 26
An inducible acid phosphatase from the yeast Pichia pastoris: characterization of the gene and its product; Payne WE et al.; To develop the budding yeast Pichia pastoris (Pp) as a model system for the study of protein secretion, we have characterized a secreted acid phosphatase (Pho1p) from this yeast . Pho1p can be induced 100-fold by starvation for phosphate . The enzyme was purified to homogeneity from a cell-wall extract by DEAE-Sepharose chromatography . We selected mutants that lacked extracellular phosphatase activity and the gene (PHO1) encoding Pho1p was isolated from a recombinant plasmid library of Pp DNA by complementation of the mutant defect . PHO1 encodes a protein of 468 amino acids (aa) with homology to repressible acid phosphatases from other yeast species . The sequence contains a 15-aa N-terminal signal sequence and six potential N-linked glycosylation sites . Antiserum to Pho1p was used to show that Pho1p transits the Pp secretory pathway in less than 5 min.

Biochem J, 1995 Sep 1, 310 ( Pt 2), 601 - 4
Flavin-dependent alcohol oxidase from the yeast Pichia pinus . Spatial localization of the coenzyme FAD in the protein structure: hot-tritium bombardment and ESR experiments; Averbakh AZ et al.; The spatial localization of the coenzyme FAD in the quaternary structure of the alcohol oxidase from the yeast Pichia pinus was studied by tritium planigraphy and ESR methods . In the present paper we measured the specific radioactivity of FAD labelled as a part of the alcohol oxidase complex . The specific-radioactivity ratio for two FAD portions (FMN and AMP) was calculated . ESR experiments show 4 A (0.4 nm) to be the depth of immersion of paramagnetic isoalloxazines into alcohol oxidase octamer molecules . It is suggested that FAD molecules are bound to the surface of the octamer, rather than to the subunit interfaces . The orientation of the prosthetic group FAD in the alcohol oxidase protein is discussed.

Biochemistry, 1995 Aug 22, 34(33), 10340 - 9
Human alanyl-tRNA synthetase: conservation in evolution of catalytic core and microhelix recognition; Shiba K et al.; The class II Escherichia coli and human alanyl-tRNA synthetases cross-acylate their respective tRNAs and require, for aminoacylation, an acceptor helix G3:U70 base pair that is conserved in evolution . We report here the primary structure and expression in the yeast Pichia of an active human alanyl-tRNA synthetase . The N-terminal 498 amino acids of the 968-residue polypeptide have substantial (41%) identity with the E . coli protein . A closely related region encompasses the class-defining domain of the E . coli enzyme and includes the part needed for recognition of the acceptor helix . As a result, previously reported mutagenesis, modeling, domain organization, and biochemical characterization on the E . coli protein appear valid as a template for the human protein . In particular, we show that both the E . coli enzyme and the human enzyme purified from Pichia aminoacylate 9-base pair RNA duplexes whose sequences are based on the acceptor stems of either E . coli or human alanine tRNAs . In contrast, the sequences of the two enzymes completely diverge in an internal portion of the C-terminal half that is essential for tetramer formation by the E . coli enzyme, but that is dispensable for microhelix aminoacylation . This divergence correlates with the expressed human enzyme behaving as a monomer . Thus, the region of close sequence similarity may be a consequence of strong selective pressure to conserve the acceptor helix G3:U70 base pair as an RNA signal for alanine.

Biochem Biophys Res Commun, 1995 Aug 4, 213(1), 52 - 8
Expression, purification, and neurotrophic activity of amyloid precursor protein-secreted forms produced by yeast; Ohsawa I et al.; The secreted form of amyloid precursor protein (APPs) including most of the extracellular domain of APP is released from the cell surface, suggesting physiological significance of APPs in vivo . We used the methylotrophic yeast Pichia pastoris as a host system for the production of recombinant APPs (rAPPs) . Two rAPPss derived from isoforms of APP (APP695 and APP770) were secreted into the culture medium from the yeast, which carried cDNA encoding the N-terminal portion of APP under the control of a P . pastoris alcohol oxidase promoter . Like APPss produced by the transfected COS-1 cells, the purified rAPPss from yeast were shown to be biologically active in terms of neurite outgrowth of embryonic rat neocortical explants . These rAPPss could be valuable tools for investigating the biological functions of APPss.

Yeast, 1995 Aug, 11(10), 945 - 52
Molecular cloning of the GTP-cyclohydrolase structural gene RIB1 of Pichia guilliermondii involved in riboflavin biosynthesis; Liauta-Teglivets O et al.; The structural gene of GTP-cyclohydrolase, involved in riboflavin biosynthesis, was cloned from a Pichia guilliermondii genomic library . A 1855 bp genomic DNA fragment complementing the riboflavin auxotrophies of an Escherichia coli ribA mutant, defective in GTP-cyclohydrolase II, and a P . guilliermondii rib1 mutant was isolated and sequenced . An open reading frame with the potential to encode a protein of 344 amino acids with a predicted molecular mass of 38,711 Da was detected . The P . guilliermondii enzyme shows a high degree of homology to GTP-cyclohydrolases type II from E . coli and Baccillus subtilis and to GTP-cyclohydrolase from Saccharomyces cerevisiae . Functional GTP-cyclohydrolase from P . guilliermondii may consist of four identical subunits.

EMBO J, 1995 Aug 1, 14(15), 3627 - 34
The Pichia pastoris peroxisomal protein PAS8p is the receptor for the C-terminal tripeptide peroxisomal targeting signal; Terlecky SR et al.; The peroxisomal targeting signal 1 (PTS1), consisting of a C-terminal tripeptide (SKL and variants), directs polypeptides to the peroxisome matrix in evolutionarily diverse organisms . Previous studies in the methylotrophic yeast Pichia pastoris identified a 68 kDa protein, PAS8p, as a potential component of the PTS1 import machinery . We now report several new properties of this molecule which, taken together, show that it is the peroxisomal PTS1 receptor . (i) PAS8p is localized to and tightly associated with the cytoplasmic side of the peroxisomal membrane, (ii) peroxisomes of wild-type, but not of pas8 delta (null) mutant, P.pastoris cells bind a PTS1-containing peptide (CRYHLKPLQSKL), (iii) CRYHLKPLQSKL can be cross-linked to PAS8p after binding at the peroxisome membrane and (iv) purified PAS8p binds CRYHLKPLQSKL with high affinity (nanomolar dissociation constant) . In addition, the tetratricopeptide repeat (TPR) domain of PAS8p is identified as the PTS1 binding region.

Microbiology, 1995 Aug, 141 ( Pt 8), 2003 - 12
Anti-Candida activity of a novel killer toxin from the yeast Williopsis mrakii; Hodgson VJ et al.; A screening of putative killer yeast strains showed that spore-forming ascomycetous yeasts of the genera Pichia and Williopsis displayed the broadest range of activity against sensitive strains of Candida spp . and Saccharomyces cerevisiae . Williopsis mrakii (NCYC 500) showed extensive anti-Candida activity against strains isolated from clinical specimens . W . mrakii killer factor was produced in minimal media as a function of growth and its activity reached constant levels as cells entered stationary phase . The proteinaceous killer toxin was found to be unstable without a specific range of temperature and pH (above 30 degrees C and pH 4.0), and further analysis showed that the active toxin molecule was an acidic polypeptide with a relative molecular mass between 1.8-5.0 kDa . At critical concentrations the killer factor exerted a greater effect on stationary phase cells of Candida than cells from an exponential phase of growth . At low concentrations, the killer toxin produced a fungistatic effect on sensitive yeasts but at higher concentrations there was evidence to suggest that membrane damage accounted for the zymocidal effects of the killer factor . the cidal nature of the toxin was reflected in a rapid decrease in sensitive cell viability . Findings presented suggest that W . mrakii killer toxin has potential as a novel antimycotic agent in combatting medically important strains of Candida.

J Biol Chem, 1995 Jul 21, 270(29), 17229 - 36
The Hansenula polymorpha PER3 gene is essential for the import of PTS1 proteins into the peroxisomal matrix; van der Klei IJ et al.; PER genes are essential for the assembly of peroxisomes in Hansenula polymorpha . Here we describe the PER3 gene which was cloned by functional complementation of a H . polymorpha per3 mutant . The complementing PER3 gene encodes a protein of 569 amino acids (Per3p) with a calculated mass of 63.9 kDa; Per3p belongs to the tetratricopeptide repeat protein family and is located in both the cytosol and the peroxisomal matrix . Remarkably, Per3p does not contain a known targeting signal (PTS1 or PTS2) . The PER3 gene product shows similarity to the Saccharomyces cerevisiae Pas10p (40% identity) and the Pichia pastoris Pas8p (55% identity) . However, their function apparently cannot be interchanged since the P . pastoris PAS8 gene failed to functionally complement a H . polymorpha per3 disruption mutant . The per3 disruption mutant contained normal but small peroxisomes in which PTS2 proteins (both homologous and heterologous) were imported . Other matrix proteins (in particular PTS1 proteins) resided in the cytosol where they were normally assembled and active . We argue that Per3p is a component of the peroxisomal import machinery and most probably shuttles matrix proteins from the cytosol to the organellar matrix.

FEBS Lett, 1995 Jul 17, 368(2), 293 - 6
In vitro dissociation and re-assembly of peroxisomal alcohol oxidases of Hansenula polymorpha and Pichia pastoris; Evers ME et al.; We have studied the in vitro inactivation/dissociation and subsequent reactivation/re-assembly of peroxisomal alcohol oxidases (AO) from the yeasts Hansenula polymorpha and Pichia pastoris . Both proteins are homo-oligomers consisting of eight identical subunits, each containing one FAD as the prosthetic group . They were both rapidly inactivated upon incubation in 80% glycerol, due to their dissociation into the constituting subunits, which however still contained FAD . Dilution of dissociated AO in neutral buffer lead to reactivation of the protein due to AO re-assembly, as was demonstrated by non-denaturing PAGE . After use of mixtures of purified AO from H . polymorpha and P . pastoris active hybrid AO oligomers were formed . When prior to dissociation FAD was chemically removed from AO, reactivation or re-assembly did not occur independent of externally added FAD.

Gene, 1995 Jul 4, 160(1), 33 - 9
The PAH2 gene is required for peroxisome assembly in the methylotrophic yeast Hansenula polymorpha and encodes a member of the tetratricopeptide repeat family of proteins; Nuttley WM et al.; Peroxisome assembly mutants in the methylotrophic yeast, Hansenula polymorpha, were selected by a novel procedure involving the inability of mutants to use both oleic acid and methanol as carbon sources . These compounds are both metabolized within peroxisomes through two different enzymatic pathways . 15 mutant strains called mut (methanol non-utilizing) were isolated . These strains were assigned to ten genetic complementation groups . Subcellular fractionation analysis showed that peroxisomal matrix enzymes were mislocalized to the cytoplasm in mut strains . Electron microscopy confirmed that the inability of mut strains to grow on oleic acid and methanol was due to defects in peroxisome assembly . Functional complementation of a mutant strain, mut2, with a plasmid library of H . polymorpha genomic DNA sequences has identified a gene, PAH2, that restores growth on methanol and the correct localization of matrix enzymes to the peroxisome . PAH2 encodes Pah2p, a polypeptide of 569 amino acids that is a member of the tetratricopeptide repeat (TPR) family of proteins . Pah2p shows identity with Pas8p and Pas10p, two proteins required for peroxisome assembly in the yeasts Pichia pastoris and Saccharomyces cerevisiae, respectively, and which have been suggested to be receptors that recognize peroxisomal targeting signal-1 (PTS1) motifs.

J Cell Biol, 1995 Jul, 130(1), 51 - 65
Human peroxisomal targeting signal-1 receptor restores peroxisomal protein import in cells from patients with fatal peroxisomal disorders; Wiemer EA et al.; Two peroxisomal targeting signals, PTS1 and PTS2, are involved in the import of proteins into the peroxisome matrix . Human patients with fatal generalized peroxisomal deficiency disorders fall into at least nine genetic complementation groups . Cells from many of these patients are deficient in the import of PTS1-containing proteins, but the causes of the protein-import defect in these patients are unknown . We have cloned and sequenced the human cDNA homologue (PTS1R) of the Pichia pastoris PAS8 gene, the PTS1 receptor (McCollum, D., E . Monosov, and S . Subramani . 1993 . J . Cell Biol . 121:761-774) . The PTS1R mRNA is expressed in all human tissues examined . Antibodies to the human PTS1R recognize this protein in human, monkey, rat, and hamster cells . The protein is localized mainly in the cytosol but is also found to be associated with peroxisomes . Part of the peroxisomal PTS1R protein is tightly bound to the peroxisomal membrane . Antibodies to PTS1R inhibit peroxisomal protein-import of PTS1-containing proteins in a permeabilized CHO cell system . In vitro-translated PTS1R protein specifically binds a serine-lysine-leucine-peptide . A PAS8-PTS1R fusion protein complements the P . pastoris pas8 mutant . The PTS1R cDNA also complements the PTS1 protein-import defect in skin fibroblasts from patients--belonging to complementation group two--diagnosed as having neonatal adrenoleukodystrophy or Zellweger syndrome . The PTS1R gene has been localized to a chromosomal location where no other peroxisomal disorder genes are known to map . Our findings represent the only case in which the molecular basis of the protein-import deficiency in human peroxisomal disorders is understood.

Yeast, 1995 Jul, 11(9), 839 - 47
A short-chain dehydrogenase gene from Pichia stipitis having D-arabinitol dehydrogenase activity; Hallborn J et al.; An NAD(+)-dependent D-arabinitol dehydrogenase (polyol dehydrogenase) gene was isolated from Pichia stipitis CBS 6054 and cloned in Saccharomyces cerevisiae . The gene was isolated by screening of a lambda-cDNA library with a zymogram technique . D-Arabinitol, xylitol, D-glucitol and galactitol are substrates for the recombinant protein . With D-arabinitol as substrate the reaction product is D-ribulose . The molecular weight of the native tetramer enzyme is 110,000 Da and the monomer is 30,000 Da . The amino acid sequence is homologous to the short-chain dehydrogenase family . It is 85.5% identical to a D-arabinitol dehydrogenase from Candida albicans . The gene in P . stipitis was induced by D-arabinitol and P . stipitis was able to grow on D-arabinitol . The physiological role of D-arabinitol metabolism is discussed.

Protein Expr Purif, 1995 Jun, 6(3), 329 - 36
Functional expression of soluble forms of human CD38 in Escherichia coli and Pichia pastoris; Fryxell KB et al.; Cyclic adenosine diphosphate (ADP)-ribose (cADPR), a metabolite of nicotinamide adenine dinucleotide (NAD+), mobilizes calcium from intracellular stores in many cells . The synthesis of cADPR from NAD+ and its subsequent hydrolysis to ADPR is catalyzed by an ADP-ribosyl cyclase and a cADPR hydrolase, respectively . The ADP-ribosyl cyclase cloned from the ovotestis of the marine invertebrate Aplysia californica has amino acid sequence homology to the human lymphocyte surface antigen CD38 . CD38 has been shown to catalyze both the formation and the hydrolysis of cADPR . In this study, we produced soluble, enzymatically active CD38 using recombinant expression techniques in bacteria and yeast . We engineered a gene coding for a soluble form of CD38 by excision of the region of the gene coding for the N-terminal amino acids representing the putative membrane spanning sequence and short putative intracellular sequence . For expression in bacteria (Escherichia coli), this construct was cloned into the pFlag-1 plasmid which allows induced, periplasmic expression and relatively simple purification of the soluble CD38 . For expression in yeast (Pichia pastoris) the CD38 sequence was further modified to eliminate four putative N-linked glycosylation sites and the resulting construct was expressed as a secreted protein . Both systems produce soluble enzymes of approximately 30 kDa and both recombinant enzymes display similar cyclase and hydrolase activities.

Biosci Biotechnol Biochem, 1995 Jun, 59(6), 1172 - 4
The phylogeny of Yamadazyma ohmeri (Etchells et Bell) Billon-Grand based on the partial sequences of 18S and 26S ribosomal RNAs: the proposal of Kodamaea gen . nov . (Saccharomycetaceae); Yamada Y et al.; Two strains of Yamadazyma ohmeri were examined for their 18S and 26S rRNA partial base sequences . The two strains had identical or very similar partial base sequences to those of the type strain . All the three strains of Y . ohmeri were phylogenetically separated from any other accepted species of the genera Yamadazyma, Debaryomyces, Pichia, Williopsis, Metschnikowia, and Clavispora . Based on the sequence data obtained and the phenotypic characteristics, Kodamaea gen . nov . was proposed for the species.

J Biol Chem, 1995 May 5, 270(18), 10940 - 51
PER3, a gene required for peroxisome biogenesis in Pichia pastoris, encodes a peroxisomal membrane protein involved in protein import; Liu H et al.; PER genes are essential for the biogenesis of peroxisomes in the yeast Pichia pastoris . Here we describe the cloning of PER3 and functional characterization of its product Per3p . The PER3 sequence predicts that Per3p is a 713-amino acid (81-kDa) hydrophobic protein with at least three potential membrane-spanning domains . We show that Per3p is a membrane protein of the peroxisome . Methanol- or oleate-induced cells of per3-1, a mutant strain generated by chemical mutagenesis, lack normal peroxisomes but contain numerous abnormal vesicular structures . The vesicles contain thiolase, a PTS2 protein, but only a small portion of several other peroxisomal enzymes, including heterologously expressed luciferase, a PTS1 protein . These results suggest that the vesicles in per3-1 cells are peroxisomal remnants similar to those observed in cells of patients with the peroxisomal disorder Zellweger syndrome, and that the mutant is deficient in PTS1 but not PTS2 import . In a strain in which most of PER3 was deleted, peroxisomes as well as peroxisomal remnants appeared to be completely absent, and both PTS1- and PTS2-containing enzymes were located in the cytosol . We propose that Per3p is an essential component of the machinery required for import of all peroxisomal matrix proteins and is composed of independent domains involved in the import of specific PTS groups.

Biosci Biotechnol Biochem, 1995 May, 59(5), 945 - 8
The phylogeny of Williopsis salicorniae Hinzelin, Kurtzman et Smith based on the partial sequences of 18S and 26S ribosomal RNAs (Saccharomycetaceae)
Yamada Y, Yano J, Matsuda M, Higashi T, Mikata K.
Williopsis salicorniae IFO 10733 (type strain), which is characterized by the formation of saturn-shaped ascospores, by the incapability of assimilating nitrate, and by a lower DNA base composition (36.7 mol% G + C), was examined for its partial base sequences of 18S and 26S rRNAs . In the 18S rRNA partial base sequencings, it had an identical base sequence with the type strain of Ogataea glucozyma (identical to Pichia glucozyma, identical to Hansenula glucozyma), which produces hat-shaped ascospores and has the ability to assimilate nitrate and methanol and a higher DNA base composition (45.1 mol% G + C) . In the 26S rRNA partial base sequencings, the base differences were four, and the percent similarity was 87 between the type strains of the two species . The data obtained are discussed phylogenetically and taxonomically.

Mol Gen Genet, 1995 Apr 10, 247(1), 61 - 72
Linear mitochondrial DNAs from yeasts: telomeres with large tandem repetitions; Nosek J et al.; The terminal structure of the linear mitochondrial DNA (mtDNA) from the yeast Candida parapsilosis was investigated . This mtDNA, 30 kb long, has symmetrical ends forming inverted terminal repeats . These repeats are made up of a variable number of tandemly repeating units of 738 bp each; the terminal nucleotide corresponds to a precise position within the last repeat unit sequence . The ends had an open structure accessible to enzymes, with a 5' single-stranded extension of about 110 nucleotides . No circular forms were detected in the DNA preparations . Two other unrelated species, Pichia philodendra and Candida salmanticensis also appear to have a linear mtDNA of similar organization . These linear DNAs (which we name Type 2 linear mtDNAs) are distinct from the previously described linear mtDNAs of yeasts whose termini are formed by a closed hairpin loop (Type 1 linear mtDNA) . The terminal structure of C . parapsilosis mtDNA is reminiscent of the linear mitochondrial genomes of the ciliate Tetrahymena although, in the latter, the telomeric tandem repeat unit is considerably shorter.

FEMS Microbiol Lett, 1995 Apr 1, 127(3), 229 - 34
A novel formaldehyde oxidation pathway in methylotrophic yeasts: methylformate as a possible intermediate; Sakai Y et al.; A considerable amount of methylformate accumulated in the culture medium of methanol-grown methylotrophic yeasts . Methylformate is considered as an intermediate in a novel formaldehyde oxidation pathway . Through investigations with Pichia methanolica, methylformate formation was found to be catalysed by a new type of alcohol dehydrogenase, which was named methylformate synthase . When cells were grown on a relatively high concentration of methanol or exposed to a high concentration of formaldehyde, formation of methylformate was enhanced and the level of methylformate synthase in the cells increased . How methylformate synthase is involved in formaldehyde oxidation and formaldehyde detoxification is discussed.

FEMS Microbiol Lett, 1995 Apr 1, 127(3), 213 - 22
Interactions between killer yeasts and pathogenic fungi; Walker GM et al.; A total of 17 presumptive killer yeast strains were tested in vitro for growth inhibitory and killing activity against a range of fungal pathogens of agronomic, environmental and clinical significance . Several yeasts were identified which displayed significant activity against important pathogenic fungi . For example, isolates of the opportunistic human pathogen, Candida albicans, were generally very sensitive to Williopsis mrakii killer yeast activity, whilst killer strains of Saccharomyces cerevisiae and Pichia anomala markedly inhibited the growth of certain wood decay basidiomycetes and plant pathogenic fungi . Results indicate that such yeasts, together with their killer toxins, may have potential as novel antimycotic biocontrol agents.

Int J Syst Bacteriol, 1995 Apr, 45(2), 386 - 9
A novel approach for discovering retrotransposons: characterization of a long terminal repeat element in the spoilage yeast Pichia membranaefaciens and its use in strain identification; Pearson BM et al.; A novel PCR-based approach designed to detect retrotransposon long terminal repeat (LTR) elements via their association with tRNA genes was applied to Pichia membranaefaciens, an industrially important food spoilage yeast . A single primer based on tRNA gene sequences was used to amplify DNA fragments from different strains, and an observed fragment size difference among strains was found to correspond to the expected size of an integrated LTR . A 289-bp element was cloned as part of the larger fragment and shown to be present in a high copy number and variable genomic location in all strains examined . Sequence analysis revealed the element to be bounded by nucleotides TG at the 5' end and CA at the 3' end and to exhibit target site duplication and other sequence motifs diagnostic of retrotransposon LTRs . LTR sequence data enabled the development of a rapid identification method which distinguished among different strains . The novel method for LTR isolation and the strain identification system are both likely to prove generally applicable for a wide range of other organisms.

Appl Environ Microbiol, 1995 Mar, 61(3), 1027 - 32
Biocontrol of mold growth in high-moisture wheat stored under airtight conditions by Pichia anomala, Pichia guilliermondii, and Saccharomyces cerevisiae; Petersson S et al.; Pichia anomala inhibits the growth of Penicillium roqueforti and Aspergillus candidus on agar . In this investigation, antagonistic activity on agar against 17 mold species was determined . The abilities of Pichia anomala, Pichia guilliermondii, and Saccharomyces cerevisiae to inhibit the growth of the mold Penicillium roqueforti in nonsterile high-moisture wheat were compared by adding 10(3) Penicillium roqueforti spores and different amounts of yeast cells per gram of wheat . Inoculated grain was packed in glass tubes, incubated at 25 degrees C with a restricted air supply, and the numbers of yeast and mold CFU were determined on selective media after 7 and 14 days . Pichia anomala reduced growth on agar plates for all of the mold species tested in a dose-dependent manner . Aspergillus fumigatus and Eurotium amstelodami were the most sensitive, while Penicillium italicum and Penicillium digitatum were the most resistant . Pichia anomala had the strongest antagonistic activity in wheat, with 10(5) and 10(6) CFU/g completely inhibiting the growth of Penicillium roqueforti . Inhibition was least pronounced at the optimum temperature (21 degrees C) and water activity (0.95) for the growth of Penicillium roqueforti . Pichia guilliermondii slightly reduced the growth of Penicillium roqueforti in wheat inoculated with 10(5) and 10(6) yeast CFU/g . S . cerevisiae inhibited mold growth only weakly at the highest inoculum level . Pichia anomala grew from 10(3) to 10(7) CFU/g of wheat in 1 week . To reach the same level, Pichia guilliermondii had to be inoculated at 10(4) CFU while S . cerevisiae required an inoculum of 10(5) CFU to reach 10(7) CFU/g of wheat.

Biosci Biotechnol Biochem, 1995 Mar, 59(3), 518 - 20
The phylogenetic relationships of Pichia jadinii, formerly classified in the genus Hansenula, and related species based on the partial sequences of 18S and 26S ribosomal RNAs (Saccharomycetaceae); Yamada Y et al.; We analyzed 18S and 26S rRNA partial base sequences {positions 1451-1618 (168 bases) of 18S rRNA and positions 1611-1835 (225 bases) and 493-622 (130 bases) of 26S rRNA} of a total of three strains of Pichia jadinii and Candida utilis . The three strains had identical base sequences with the type strain of P . jadinii (IFO 0987) in the 18S rRNA partial base sequencings . In the 26S rRNA partial base sequencings, there were partial base sequences similar to each other (1-0 base difference and 87-95 percent similarities) . The sequence data obtained are discussed taxonomically and phylogenetically, especially in connection with Williopsis saturnus, the type species of the genus Williopsis Zender.

Biosci Biotechnol Biochem, 1995 Mar, 59(3), 445 - 50
The phylogenetic relationships of the Q9-equipped, hat-shaped ascospore-forming species of the genus Yamadazyma Billon-Grand (Saccharomycetaceae) based on the partial sequences of 18S and 26S ribosomal RNAs; Yamada Y et al.; Sixteen strains of the sixteen species, including the type species, Y . philogaea (CBS 6696, type strain), of the genus Yamadazyma were examined for their partial base sequences of 18S and 26S rRNAs . The genus Yamadazyma Billon-Grand was found to have a heterogeneous nature phylogenetically . In the partial base sequences in positions 1451-1618 (168 bases) of 18S rRNA, the number of base differences was 4-0 within the genus except for Y . spartinae, Y . inositovora, Y . ohmeri, and Y . besseyi . The base differences numbered 6-1, 11-8, and 8-4 with D . hansenii, P . membranaefaciens, and S . cerevisiae, respectively . In the partial base sequences in positions 1611-1835 (225 bases) of 26S rRNA, the number of base differences was 14-0 within the genus . The base differences numbered 19-0, 31-24, and 25-17 with D . hansenii, P . membranaefaciens, and S . cerevisiae, respectively . In the partial base sequences in positions 493-622 (130 bases) of 26S rRNA, the percent similarities were 73-93 . The percent similarities were 77-90, 64-71, and 68-79 with D . hansenii, P . membranaefaciens, and S . cerevisiae, respectively . Yamadazyma inositovora, Y . spartinae, and Y . ohmeri were not closely related phylogenetically . Yamadazyma besseyi (Q-7) was separate phylogenetically from the species mentioned above of the genera Yamadazyma, Debaryomyces, Pichia, and Saccharomyces (base differences, 13-7 and 62-17; percent similarities, 48-63) . The discussion was made phylogenetically and taxonomically, especially on transferring Y . besseyi to a separate taxon.

Biosci Biotechnol Biochem, 1995 Mar, 59(3), 439 - 44
The phylogenetic relationships of methanol-assimilating yeasts based on the partial sequences of 18S and 26S ribosomal RNAs: the proposal of Komagataella gen . nov . (Saccharomycetaceae); Yamada Y et al.; Twelve strains of methanol-assimilating yeast species were examined for their partial base sequences of 18S and 26S rRNAs . In the partial base sequencings of 18S rRNA (positions 1451-1618, 168 bases), P . kodamae had the same partial base sequence as Ogataea minuta, and P . finlandica, P . pini, P . trehalophila, C . maris, and C . methanolovescens were the same in the partial base sequences as O . glucozyma . Candida boidinii and C . methylica were similar to O . glucozyma, but somewhat different from O . minuta (the base differences, one and four, respectively) . Pichia naganishii had seven, four, six, and nine base differences with O . minuta, O . glucozyma, P . anomala, and P . membranaefaciens, respectively, and C . methanosorbosa did four, three, five, and twelve base differences . Pichia pastoris was quite different (the base differences among the methanol-assimilating yeasts, 32-31), and no bases were found in the fingerprint segment . In the partial base sequencings of 26S rRNA (positions 1611-1835, 225 bases; positions 493-622, 130 bases), P . finlandica, P . kodamae, P . pini, P . trehalophila, C . maris, C . methanolovescens, and C . methanosorbosa were similar to O . minuta and O . glucozyma . Pichia naganishii, C . boidinii, and C . methylica had somewhat different partial base sequences . The base differences and the percent similarities of P . pastoris were quite high (101-89) and quite low (40-47) . Based on the sequence data obtained, the methanol-assimilating yeast species are discussed taxonomically and phylogenetically . A new genus, Komagataella was proposed for P . pastoris with a new combination, Komagataella pastoris.

Microbiologia, 1995 Mar, 11(1), 51 - 8
{Sherry wine microorganisms}; Garcia Maiquez E; Sherry wine presents, during all its wine-making and aging process, a great diversity of yeast and bacteria, as well as in the wine itself; its particular wine-making system, with traditional and legal additions to correct the acidity and to get a final alcoholic content of 15%, originates a selection of accompanying microorganisms . Species of the genera Kloeckera, Candida, Saccharomyces, Pichia, Hansenula and Saccharomycodes, have been isolated during the fermentation process in different proportions . This fact confirms that, besides S . cerevisiae, strains of S . chevalieri and S . fermentati have an important role in the fermentative process, and that the film-forming Saccharomyces have great activity in the fermentation . The biological aging of the Sherry wine, carried out by S . cheresiensis, S . beticus, S . feduchii and S . rouxii, has been studied in "finos" and "manzanillas" . Different species and percentages in both wines have been described.

Eur J Biochem, 1995 Feb 15, 228(1), 50 - 4
Amino acid substitutions in the yeast Pichia stipitis xylitol dehydrogenase coenzyme-binding domain affect the coenzyme specificity; Metzger MH et al.; Directed mutagenesis has been used to identify a set of amino acids in the Pichia stipitis xylitol dehydrogenase, encoded by the xylitol dehydrogenase gene XYL2, which is involved in specific NAD binding . Within the binding domain, a characteristic beta alpha beta-fold is centered around a glycine motif GXGXXG also containing conserved aspartate and lysine/arginine residues . The mutation D207-->G and the double mutation D207-->G and D210-->G increased the apparent Km for NAD ninefold and decreased the xylitol dehydrogenase activity to 47% and 35%, respectively, as compared to the unaltered enzyme . The introduction of the potential NADP-recognition sequence (GSRPVC) of the alcohol dehydrogenase from Thermoanaerobium brockii into the xylitol dehydrogenase allowed the mutant enzyme to use both NAD and NADP as cofactor with equal apparent Km values . Although this mutant enzyme displayed an unaltered NADP acceptance, the reduction of the NAD specificity in the stably expressed enzyme variant is an important first step towards the long-term goal to reverse the coenzyme specificity from NAD to NADP . The mutagenized XYL2 gene could still mediate growth of Saccharomyces cerevisiae transformants on xylose minimal-medium plates when expressed together with the xylose reductase gene (XYL1).

Biochem J, 1995 Feb 15, 306 ( Pt 1), 235 - 9
Characterization of the invertase from Pichia anomala; Rodriguez J et al.; Synthesis of invertase (EC 3.2.1.26) in Pichia anomala is controlled by the carbon source in the culture medium . The enzyme was purified to homogeneity from P . anomala cells fully derepressed for invertase synthesis and shown to be a multimeric glycoprotein composed of identical subunits with an apparent molecular mass of 86.5 kDa . The carbohydrate moiety accounts for approx . 30% of the total mass of the molecule and consists of manno-oligosaccharides N-linked to the polypeptide . Most of the characteristics of the enzyme analysed in this study were similar to those previously reported for other yeast invertases, with the remarkable exception of its thermal sensitivity which appears after 15 min incubation at temperatures above 32 degrees C.

Yeast, 1995 Feb, 11(2), 111 - 9
Characterization of a glycerol/H+ symport in the halotolerant yeast Pichia sorbitophila; Lages F et al.; Pichia sorbitophila is a halotolerant yeast capable of surviving to extracellular NaCl concentrations up to 4 M in mineral medium when glucose or glycerol are the only carbon and energy sources . Evidence is presented here that glycerol, the main compatible solute this yeast accumulates so as to maintain osmotic balance, is actively co-transported with protons . This transport system was shown to be constitutive, not needing induction by either glycerol or salt, and was not repressible by glucose . In glucose- or glycerol-grown cells, a simple diffusion was detectable, and iterative calculations were performed to calculate kinetic parameters, in the presence and in the absence of NaCl . At 25 degrees C, pH 5.0, in glucose-grown cells these were: Km = 0.81 +/- 0.11 mM and Vmax = 634.2 +/- 164.8 mumol h-1 per g (glycerol); Km = 1.28 +/- 0.60 mM and Vmax = 558.6 +/- 100.6 mumol h-1 per g (protons) . Correspondent stoichiometry was approximately 1, either for these conditions or in the presence of 1 M-NaCl . An increase in accumulation capacity was evident when different concentrations of NaCl were present . This capacity was shown to be dependent on delta pH and membrane potential, consistently with an electrogenic character . We suggest that the main role of this system is in osmoregulation, by keeping glycerol accumulated inside the cells, compensating for leakage, due to its liposoluble character.

Biochem Biophys Res Commun, 1995 Jan 5, 206(1), 335 - 40
Peroxisomal activation of long- and very long-chain fatty acids in the yeast Pichia pastoris; Kalish JE et al.; In mammals, beta-oxidation of very long-chain fatty acids (VLCFA) takes place in peroxisomes . This process is impaired in X-linked adrenoleukodystrophy (XALD) patients as a result of decreased activity of peroxisomal very long-chain acyl-CoA synthetase (VLCS) . We investigated VLCFA and long chain fatty acid (LCFA) activation in the yeast Pichia pastoris . Both VLCFA and LCFA were activated to their CoA derivatives in an organelle fraction . When organelles were fractionated on a sucrose gradient, VLCS activity co-localized with peroxisomes while long chain acyl-CoA synthetase activity associated primarily with mitochondria . Consistent with these findings, only VLCS activity was reduced in organelle fractions from peroxisome assembly (pas) mutants . Furthermore, no VLCS activity was detected in pas mutants at the density of normal peroxisomes . Thus, we conclude that VLCS is a peroxisomal enzyme in P . pastoris and this organism may serve as an excellent model system to investigate the molecular basis of XALD.

Nucleic Acids Symp Ser, 1995, (34), 23 - 4
The distribution of ND genes in yeast mitochondrial genomes and the mitochondrial DNA structure of Pichia membranaefacens; Kitano H et al.; For long time, it has been believed that the yeast mitochondrial (mt) genome lacks NADH dehydrogenase subunit genes which are designated ND genes . However, our complete mtDNA sequencing of yeast Hansenula wingei led us to the first finding of seven mitochondrial ND genes . We investigated the distribution of ND genes in mtDNAs of other yeasts including Pichia membranaefaciens, Yarrowia lypolitica, Candida maltosa, Saccharomyces kluyveri and Saccharomyces exiguus . By Southern hybridization with probes of H . wingei's ND1, 2 and 5 genes, we detected positive signals on mtDNAs in P . membranaefaciens, Y . lypolitica, and C . maltosa . To confirm this, we cloned and sequenced DNA fragment of ND5 gene in P . membranaefaciens . We have discussed the sequence homology and genome structure.

Prog Growth Factor Res, 1995, 6(2-4), 209 - 14
A 3-dimensional model for the insulin-like growth factor binding proteins (IGFBPs); supporting evidence using the structural determinants of the IGF binding site on IGFBP-3; Spencer EM et al.; It is generally accepted but not established that the insulin-like growth factor (IGF) binding site on the IGF binding proteins (IGFBPs) is in the N-terminal region . However, other workers have reported C-terminal fragments with IGF binding determinants . Therefore, we tested the hypothesis that both the N- and C-terminal regions of IGFBPs are involved in binding . Using a protein A gene fusion system, a cDNA encoding residues 1-147 (N147) was cloned into the plasmid pRIT2T and expressed in E . coli as a fusion protein . Since an Asn N-terminal to the gly of IGFBP-3 had been engineered into the cDNA construct, protein A was cleaved from N147 by hydroxylamine . Purified N147 was refolded in a DTT/cystamine redox system at pH 8.4 under nitrogen atmosphere . Both ligand binding Westerns and solution binding assays demonstrated that the recombinant derived N147 bound IGFs . The 147 and 176 residue N-terminal fragments, including a C-terminal fragment (residues 151-263) of IGFBP-3 were also expressed in pichia (yeast) as glycosylated proteins . Solution binding assays showed that they all bound labelled IGF-1 . In conclusion, IGFBP-3 contains at least two binding determinants, one on the N- and one on the C-terminal domain . There may also be a possible contribution from the intermediate (I) domain . Our molecular genetic approach to mapping the binding region for IGFs on IGFBP-3 can now be tested on the other mutants we have prepared . Subsequently, site directed mutagenesis can be used to pinpoint key functional residues.

Ukr Biokhim Zh, 1995 Jan-Feb, 67(1), 32 - 7
{Ferrireductase from Pichia guilliermondii: properties and regulation of activity and synthesis}; Fedorovych DV et al.; Properties of Pichia guilliermondii ferrireductase of the crude extracts and ammonium sulfate preparations were studied . NADH and Cu (II) ions are necessary for ferrireductase activity . Mg (II) also stimulates this reaction while oxygen acts as a slight inhibitor . Ferrireductase reduces Fe (III) in complex with citrate, iron-binding peptide from the culture medium of P . guilliermondii, rhodotorulic acid, coprogen, desferrioxamine B and EDTA . Mutants rib80, rib81 and hit of Pichia guilliermondii that have damaged system of riboflavin biosynthesis and iron transport regulation are characterized by high ferrireductase activity . Iron through negative feed-back mechanism regulates activity of ferrireductase synthesis in P . guilliermondii, Candida famata, Candida krusei, Candida boidinii, but not in Pichia pinus and Hansenula polymorpha.

Adv Exp Med Biol, 1995, 362, 325 - 30
Purification and characterization of recombinant human cathepsin E; Iida H et al.; The human cathepsin E was purified from the culture supernatant of Pichia pastoris strain transformed with a human cathepsin E expression plasmid . Purification was performed by a three-step procedure, TSKgel Phenyl-5PW, Toyopearl HW55S and TSKgel DEAE-5PW column chromatographies . The purified recombinant cathepsin E had the molecular mass of around 82-kDa with the amino-terminal sequence started with Ile37 of the predicted amino acid sequence, suggesting the human cathepsin E was accumulated in the culture suparnatant as the mature dimer enzyme . The result of endoglycosidase-H digestion followed by Western blot analysis of the purified recombinant cathepsin E suggested that the human cathepsin E expressed in Pichia pastoris received N-linked high-mannose type glycosylation.

Adv Exp Med Biol, 1995, 362, 319 - 24
Expression of human cathepsin E in methylotrophic yeast, Pichia pastoris; Yamada M et al.; The human gastric cathepsin E (CTSE) was expressed in the methylotrophic yeast Pichia pastoris by placing the CTSE cDNA under the control of the methanol-inducible alcohol oxidase promoter . The human CTSE expressed in P . pastoris was efficiently secreted into the culture medium as an active enzyme directed by its native signal sequence, whereas CTSE has been shown to be retained in mammalian tissue cells . The recombinant human CTSE was secreted as a 90-kDa molecule and then converted via an 84-kDa intermediate to an 82-kDa molecule . The 90-kDa molecule and the 82-kDa molecule were considered to be the proenzyme and the mature enzyme as dimeric forms, respectively.

J Basic Microbiol, 1995, 35(6), 367 - 73
The acidic ribosomal proteins of different yeast species . Phosphorylation by ribosome-associated protein kinases; Cytrynska M et al.; Two major ribosomal proteins of Mr 13 kDa and 38 kDa were identified as phosphorylation substrates for ribosome-bound protein kinases from different yeast species . The phosphorylation level was much higher in ribosomes from the cells collected at the exponential growth phase, than in those from the stationary phase . Isoelectrofocusing of the protein band of 13 kDa and subsequent silver staining allowed to identify from four in the case of Pichia stipitis up to ten in Saccharomyces cerevisiae . Saccharomycodes Ludwigii, Torulopsis utilis and Kloeckera apiculata individual peptides . In the most of the yeast species studies five phosphorylated peptides were observed . However, only one or two such phosphopeptides were detected in Pichia stipitis and Trichosporon cutaneum ribosomes, respectively . The same phosphoprotein forms were identified in the in vivo 32P-labelling experiments.

J Appl Bacteriol, 1995 Jan, 78(1), 82 - 7
The effect of hydroxycinnamic acids and potassium sorbate on the growth of 11 strains of spoilage yeasts; Stead D; Hydroxycinnamic acids and their derivatives occur widely in plants, fruits and wine . The effect of the common hydroxycinnamic acids (caffeic, coumaric and ferulic acids), at concentrations of 100 and 500 mg l-1, on growth of 11 strains of spoilage yeasts was measured spectrophotometrically and compared with that of potassium sorbate . Ferulic acid was the most generally inhibitory hydroxycinnamic acid . At 500 mg l-1 it appreciably inhibited Pichia anomala, Debaryomyces hansenii and Saccharomyces cerevisiae and prevented detectable growth of one strain each of P . anomala and D . hansenii . Caffeic acid was the least inhibitory compound and coumaric acid had an intermediate effect . The more resistant strains of yeast were P . membranaefaciens, Saccharomycodes ludwigii and Zygosaccharomyces bailii . Sensitivity to hydroxycinnamic acid was, in general, associated with sensitivity to potassium sorbate; at a given concentration potassium sorbate was more inhibitory than were any of the hydroxycinnamic acids.

Scand J Infect Dis, 1995, 27(1), 85 - 7
Catheter-related infections by Hansenula anomala in children; Yamada S et al.; During August and September, 1992, we experienced 4 cases of Hansenula anomala (H . anomala, synonym Pichia anomala) fungemia in immunocompromised patients . Two patients had been suffering from a malignant disease, 3 of them had received broad-spectrum antibiotics and a central venous catheter (CVC) had been inserted in all of them . H . anomala was isolated as the sole pathogen from all 4 patients . Three of them responded favorably to fluconazole after withdrawal of the catheter, but one failed . H . anomala should be considered as a possible cause of catheter-related infections.

J Cell Sci, 1995 Jan, 108 ( Pt 1), 25 - 35
Divergent modes of autophagy in the methylotrophic yeast Pichia pastoris; Tuttle DL et al.; The budding yeast Pichia pastoris responds to methanolic media by synthesizing high levels of cytosolic enzymes (e.g . formate dehydrogenase) and peroxisomal enzymes (e.g . alcohol oxidase), which are necessary to assimilate this carbon source . Major alterations in cellular metabolism are initiated upon a shift in carbon source to ethanol or glucose . These alterations require the synthesis of new proteins and the rapid degradation of those enzymes no longer needed for methanol utilization . In this study, we have measured cytosolic and peroxisomal enzyme activities and examined the fate of morphologically distinct peroxisomes to assess the degradative response of this yeast during nutrient adaptation . Utilizing biochemical, morphological and genetic approaches, we have shown that there exist in P . pastoris at least two pathways for the sequestration of peroxisomes into the vacuole for degradation . The ethanol-induced pathway is independent of protein synthesis and includes an intermediate stage in which individual peroxisomes are sequestered into autophagosomes by wrapping membranes, which then fuse with the vacuole . This process is analogous to macroautophagy . The glucose-induced pathway invokes the engulfment of clusters of peroxisomes by finger-like protrusions of the vacuole by a process analogous to microautophagy . Unlike ethanol adaptation, glucose stimulated the degradation of formate dehydrogenase as well . Peroxisomes remained outside the vacuoles of glucose-adapted cycloheximide-treated normal cells, suggesting that protein synthesis is required for peroxisome entry into the yeast vacuole . Two complementary mutants (gsa1 and gsa2) that are unable to degrade peroxisomes or formate dehydrogenase during glucose adaptation were isolated . The mutated gene products appear to function in one or more events upstream of degradation within the vacuole, since ethanol-induced peroxisome degradation proceeded normally in these mutants and peroxisomes were found outside the vacuoles of glucose-adapted gsa2 cells . Mutants lacking vacuolar proteinases A and B were unable to degrade alcohol oxidase or formate dehydrogenase during ethanol or glucose adaptation . Peroxisomes were found to accumulate within the vacuoles of these proteinase mutants during adaptation . Combined, the results suggest that there exist in Pichia pastoris two independent pathways for the sequestration of peroxisomes into the vacuole, the site of degradation.

J Clin Microbiol, 1995 Jan, 33(1), 8 - 10
Use of yeast killer system to identify species of the Nocardia asteroides complex; Provost F et al.; We evaluated the ability of the yeast killer system to differentiate members belonging to the Nocardia asteroides complex (Nocardia asteroides, Nocardia farcinica, and Nocardia nova) . Nocardia strains were selected randomly from clinical isolates . Type strains of each Nocardia species and recognized killer yeasts were taken from different collections . A clear area of inhibition surrounding the yeast cells demonstrated a positive killer effect on Nocardia spp . Two yeast strains, Pichia mrakii (K9) and Pichia lynferdii (K76), showed different killer activities against each Nocardia species . The group N . asteroides was identified as K9+ K76-, the group N . farcinica was identified as K9- K76-, and the group N . nova was identified as K9+ K76+ . The three killer identifications correlated with specific taxonomic groups determined by using classical methods . The yeast killer system may be a useful means for identifying organisms within the N . asteroides complex.

Biochim Biophys Acta, 1994 Dec 14, 1209(2), 165 - 70
Expression, purification, and characterization of the Kunitz-type proteinase inhibitor domain of the amyloid beta-protein precursor-like protein-2; Van Nostrand WE et al.; In this report we describe the use of the methylotrophic industrial yeast Pichia pastoris as a host system for the large scale production of the Kunitz-type proteinase inhibitor (KPI) domain of the amyloid beta-protein precursor-like protein-2 (APLP-2) . The expression plasmid for the KPI domain of APLP-2 encoded amino acids 305-364 of the APLP-2 cDNA (Slunt et al . (1994) J . Biol . Chem . 269, 2637-2644) . The secreted 60 amino-acid product was purified to homogeneity and biochemically characterized . Amino-acid sequencing of the expressed KPI domain of APLP-2 verified its integrity . The proteinase inhibitory properties of the KPI domain of APLP-2 were compared to those of the KPI domain of proteinase nexin-2/amyloid beta-protein precursor (PN-2/A beta PP) . Both KPI domains potently inhibited trypsin and, to a lesser extent, chymotrypsin, plasmin, and coagulation factors XIa and IXa . However, the KPI domain of APLP-2 was a approximately 20-fold less effective inhibitor of coagulation factor XIa compared to the KPI domain of PN-2/A beta PP . Similarly, the KPI domain of APLP-2 was a less effective anticoagulant in coagulation based assays than the KPI domain of PN-2/A beta PP . These studies indicate that the KPI domains of PN-2/A beta PP and APLP-2 form a family of proteinase inhibitors although the former is a better inhibitor of factor XIa and a more potent anticoagulant than the latter.

J Cell Biol, 1994 Dec, 127(5), 1259 - 73
Role of the PAS1 gene of Pichia pastoris in peroxisome biogenesis; Heyman JA et al.; Several groups have reported the cloning and sequencing of genes involved in the biogenesis of yeast peroxisomes . Yeast strains bearing mutations in these genes are unable to grow on carbon sources whose metabolism requires peroxisomes, and these strains lack morphologically normal peroxisomes . We report the cloning of Pichia pastoris PAS1, the homologue (based on a high level of protein sequence similarity) of the Saccharomyces cerevisiae PAS1 . We also describe the creation and characterization of P . pastoris pas1 strains . Electron microscopy on the P . pastoris pas1 cells revealed that they lack morphologically normal peroxisomes, and instead contain membrane-bound structures that appear to be small, mutant peroxisomes, or "peroxisome ghosts." These "ghosts" proliferated in response to induction on peroxisome-requiring carbon sources (oleic acid and methanol), and they were distributed to daughter cells . Biochemical analysis of cell lysates revealed that peroxisomal proteins are induced normally in pas1 cells . Peroxisome ghosts from pas1 cells were purified on sucrose gradients, and biochemical analysis showed that these ghosts, while lacking several peroxisomal proteins, did import varying amounts of several other peroxisomal proteins . The existence of detectable peroxisome ghosts in P . pastoris pas1 cells, and their ability to import some proteins, stands in contrast with the results reported by Erdmann et al . (1991) for the S . cerevisiae pas1 mutant, in which they were unable to detect peroxisome-like structures . We discuss the role of PAS1 in peroxisome biogenesis in light of the new information regarding peroxisome ghosts in pas1 cells.

Appl Environ Microbiol, 1994 Dec, 60(12), 4245 - 54
High-efficiency transformation of Pichia stipitis based on its URA3 gene and a homologous autonomous replication sequence, ARS2; Yang VW et al.; This paper describes the first high-efficiency transformation system for the xylose-fermenting yeast Pichia stipitis . The system includes integrating and autonomously replicating plasmids based on the gene for orotidine-5'-phosphate decarboxylase (URA3) and an autonomous replicating sequence (ARS) element (ARS2) isolated from P . stipitis CBS 6054 . Ura- auxotrophs were obtained by selecting for resistance to 5-fluoroorotic acid and were identified as ura3 mutants by transformation with P . stipitis URA3 . P . stipitis URA3 was cloned by its homology to Saccharomyces cerevisiae URA3, with which it is 69% identical in the coding region . P . stipitis ARS elements were cloned functionally through plasmid rescue . These sequences confer autonomous replication when cloned into vectors bearing the P . stipitis URA3 gene . P . stipitis ARS2 has features similar to those of the consensus ARS of S . cerevisiae and other ARS elements . Circular plasmids bearing the P . stipitis URA3 gene with various amounts of flanking sequences produced 600 to 8,600 Ura+ transformants per micrograms of DNA by electroporation . Most transformants obtained with circular vectors arose without integration of vector sequences . One vector yielded 5,200 to 12,500 Ura+ transformants per micrograms of DNA after it was linearized at various restriction enzyme sites within the P . stipitis URA3 insert . Transformants arising from linearized vectors produced stable integrants, and integration events were site specific for the genomic ura3 in 20% of the transformants examined . Plasmids bearing the P . stipitis URA3 gene and ARS2 element produced more than 30,000 transformants per micrograms of plasmid DNA . Autonomously replicating plasmids were stable for at least 50 generations in selection medium and were present at an average of 10 copies per nucleus.

Yeast, 1994 Dec, 10(12), 1601 - 12
Development of a transformation system for the yeast Yamadazyma (Pichia) ohmeri; Piredda S et al.; This communication describes the development of genetic tools for the yeast Yamadazyma ohmeri . Nystatin enrichment proved highly effective for isolating various auxotrophic strains, which were classified by complementation analysis . Biosynthetic genes encoding known biochemical functions were isolated by polymerase chain reaction, including YoLEU2 and YoURA3 that were sequenced . Using these homologous genes as selective markers, DNA transformation was accomplished by electroporation . Transformation with pBR322-based plasmids, cut within the coding region of the homologous marker gene, yielded 20 to 50 stable transformants per microgram of DNA . In about 80% of the cases, integration of plasmid DNA sequence occurred by homologous recombination of a single plasmid into the chromosome . Excision of the plasmid permitted gene replacement, as illustrated by the substitution of a wild-type URA3 gene by an in vitro generated deletion . Sequences conferring extrachromosomal replication were isolated from Y . ohmeri DNA . Plasmids based on pBR322 carrying such an ARS and either selective markers transformed at 10(4)/microgram and were shown to replicate freely in Y . ohmeri at an approximate copy number of 40 . Unexpectedly, we observed that BS-SKR derivatives carrying either YoLEU2 or YoURA3 but no Y . ohmeri ARS also replicated extrachromosomally . Linearization of transforming plasmids within regions homologous or not to chromosomal sequences stimulated transformation frequencies up to four-fold.

Biochem Biophys Res Commun, 1994 Nov 30, 205(1), 381 - 8
Expression, purification, and characterization of porcine leukocyte 12-lipoxygenase produced in the methylotrophic yeast, Pichia pastoris; Reddy RG et al.; A cDNA coding for porcine leukocyte 12-lipoxygenase was expressed intracellularly in the methylotrophic yeast Pichia pastoris under the regulatory control of the alcohol oxidase promoter . The recombinant 12-lipoxygenase contained in the yeast cell lysate was soluble, displayed the catalytic properties of the native enzyme, and was recognized by antibodies prepared against native 12-lipoxygenase derived from porcine leukocytes . The catalytically active enzyme of the 100,000 x g supernatant obtained from the yeast lysate was readily purified by immunoaffinity chromatography to near homogeneity . Porcine leukocyte 12-lipoxygenase is the first arachidonic acid oxygenase to be expressed in yeast, an easy, inexpensive, and rapid method of expressing native and site-directed mutants of recombinant proteins.

Curr Genet, 1994 Nov-Dec, 26(5-6), 443 - 50
The Pichia pastoris HIS4 gene: nucleotide sequence, creation of a non-reverting his4 deletion mutant, and development of HIS4-based replicating and integrating plasmids; Crane DI et al.; We have obtained a clone of the Pichia pastoris HIS4 gene and have determined its nucleotide sequence . Based upon its deduced amino-acid sequence, the product of the P . pastoris HIS4 gene has the same structural organization as the Saccharomyces cerevisiae His4 protein and appears to encode a trifunctional enzyme catalyzing the second (phosphoribosyl-ATP pyrophosphohydrolase), third (phosphoribosyl-AMP cyclohydrolase), and tenth (histidinol dehydrogenase) steps in histidine biosynthesis . The chromosomal copy of the HIS4 gene was disrupted by homologous recombination, creating the strain SGY58 . The his4 delta deletion mutation in this strain lacks the entire coding region of this gene and has a reversion rate that is undetectable . A set of complementary plasmids that carry the HIS4 gene was also developed . Among these are nine E . coli-P . pastoris shuttle vectors that transform the his4 delta deletion mutant at high efficiency and an integration vector for creating site-specific alterations of the P . pastoris genome.

Yeast, 1994 Nov, 10(11), 1459 - 66
Isolation and preliminary characterization of Pichia pinus mutants insensitive to glucose repression; Alamae T et al.; A new method for the isolation of glucose repression-insensitive mutants in the methylotrophic yeast Pichia pinus was developed . The method is based on screening of small suspension samples derived from 2-deoxyglucose-resistant colonies for alcohol oxidase activity . Alcohol oxidase activity was evaluated by determination of formaldehyde excreted by cells . Mutants with glucose non-repressible alcohol oxidase and catalase synthesis were obtained . All mutants grew poorly on D-xylose compared to the wild type, whereas growth on L-arabinose was similar to the wild type . Changes in the glucose transport system were suggested to be responsible for altered growth characteristics and defective glucose repression.

Appl Microbiol Biotechnol, 1994 Nov, 42(2-3), 326 - 33
The influence of cosubstrate and aeration on xylitol formation by recombinant Saccharomyces cerevisiae expressing the XYL1 gene; Hallborn J et al.; Xylitol formation by a recombinant Saccharomyces cerevisiae strain containing the XYL1 gene from Pichia stipitis CBS 6054 was investigated under three sets of conditions: (a) with glucose, ethanol, acetate, or glycerol as cosubstrates, (b) with different oxygenation levels, and (c) with different ratios of xylose to cosubstrate . With both glucose and ethanol the conversion yields were close to 1 g xylitol/g consumed xylose . Decreased aeration increased the xylitol yield on the basis of consumed cosubstrate, while the rate of xylitol formation decreased . The xylitol yield based on consumed cosubstrate also increased with increased-xylose:cosubstrate ratios . The transformant utilized the cosubstrate more efficiently than did a reference strain in terms of utilization rate and growth rate, implying that the regeneration of NAD(P)+ during xylitol formation by the transformant balanced the intracellular redox potential.

Appl Microbiol Biotechnol, 1994 Nov, 42(2-3), 319 - 25
Isolation and characterization of the Pichia stipitis transketolase gene and expression in a xylose-utilising Saccharomyces cerevisiae transformant; Metzger MH et al.; The gene TKT from P . stipitis, encoding the enzyme transketolase (EC 2.2.1.1), was cloned from a genomic library by hybridization with a S . cerevisiae TKT1-gene-specific probe . The nucleotide sequence determined contains an open-reading frame of 2085 base pairs (bp) encoding a protein of 695 amino acids with a predicted molecular mass of 75113 Da . The TKT gene was actively expressed in S . cerevisiae when placed under the control of the homologous PDC1(-15) promoter and could complement a S . cerevisiae tkt deletion . The TKT protein was immunologically detectable using S . cerevisiae transketolase-specific antiserum . Overexpression of the P . stipitis TKT gene in a xylose-utilizing S . cerevisiae XYL1/XYL2 integrant led to a drastically extended generation time during growth on xylose minimal medium under aerobic conditions.

Biotechnology (N Y), 1994 Nov, 12(11), 1119 - 24
High-level secretion and very efficient isotopic labeling of tick anticoagulant peptide (TAP) expressed in the methylotrophic yeast, Pichia pastoris; Laroche Y et al.; Tick anticoagulant peptide (TAP) is a potent and specific inhibitor of the blood coagulation protease Factor Xa . We designed and assembled a synthetic TAP-encoding gene (tapo) based on codons preferentially observed in the highly expressed Pichia pastoris alcohol oxidase 1 gene (AOX1), and fused it to a novel hybrid secretory prepro leader sequence . Expression from this gene yielded biologically active rTAP, which was correctly processed at the amino-terminal fusion site, and accumulated in the medium to approximately 1.7 g/l . This corresponds to a molar concentration of 0.24 mM, and is the highest yet described for a recombinant product secreted from P . pastoris . It also represents a seven-fold improvement in productivity compared to rTAP secretion from Saccharomyces cerevisiae, making P . pastoris an attractive host for the industrial-scale production of this potential therapeutic agent . This system was also used to prepare 21 mg 15N-rTAP, 11 mg 13C-rTAP and 27 mg 15N/13C-rTAP, with isotope incorporation levels higher than 98%, and purities sufficient to allow their use in determining the solution structure of the tick anticoagulant peptide using high field NMR.

Yeast, 1994 Nov, 10(11), 1415 - 9
Efficient expression and secretion of recombinant alpha amylase in Pichia pastoris using two different signal sequences; Paifer E et al.; We have cloned and expressed a bacterial thermostable alpha amylase gene in Pichia pastoris using the methanol-controlled alcohol oxidase (AOX1) promoter . Two integrative vectors were constructed with two different secretion signal sequences in order to obtain efficient secretion of the protein . One vector contains the structural gene encoding the mature alpha amylase fused to the SUC2 gene signal sequence from Saccharomyces cerevisiae . In the other vector, the alpha amylase is expressed with its own signal sequence . In both cases, the alpha amylase were secreted into the culture medium with high efficiency, around 2.5 and 0.9 g/l respectively.

J Biol Chem, 1994 Sep 30, 269(39), 23981 - 7
Intermediates in the folic acid biosynthetic pathway are incorporated into molybdopterin the yeast, Pichia canadensis; Irby RB et al.; Biosynthesis of molybdopterin was followed in the yeast, Pichia canadensis, using labeled precursors . High performance liquid chromatography analysis of extracts from cells labeled with {U-14C}guanosine showed that the label was incorporated into the molybdopterin oxidation product, dephospho Form A . Dephospho Form A isolated from cells labeled with {U-14C,5'-3H}guanosine was devoid of tritium, indicating partial loss of the ribose moiety of guanosine during the synthesis of molybdopterin . In vivo labeling of P . canadensis using {7-14C}neopterin and {6,7,1-14C}hydroxymethylpterin led to label from both compounds appearing in dephospho Form A as well as in folic acid in wild type cells . When these labeled precursors were incubated with P . canadensis mutants blocked in molybdopterin synthesis, only folic acid was labeled . These results suggest a shared pathway in the biosyntheses of molybdopterin and folic acid . {6-14C}Glucose labeling experiments led to exclusive incorporation into the 4'-position of dephospho Form A but not in folic acid . It is proposed that molybdopterin synthesis branches from the folic acid biosynthetic pathway at dihydrohydroxymethylpterin and that a 3-carbon phosphorylated compound such as glyceraldehyde 3-phosphate may condense with dihydrohydroxymethylpterin to form the 4-carbon side chain precursor to molybdopterin.

Med Hypotheses, 1994 Sep, 43(3), 167 - 71
The yeast killer phenomenon: a hypothetical way to control Pneumocystis carinii pneumonia; Cailliez JC et al.; Pneumocystis carinii is an important agent of pneumonia in immunocompromised individuals, especially in acquired immunodeficiency syndrome AIDS patients P . carinii attaches specifically to type 1 pneumocytes . Although this phenomenon must play a marked role in pneumocystosis pathophysiology, no therapeutic molecules able to inhibit specifically the parasite attachment were found . A killer toxin, secreted by the yeast Pichia anomala, induced a significant decrease in P . carinii in vitro attachment and inhibited the parasite infectivity in SCID mice . Killer toxins cannot be used as systemic antibiotics . However, it was possible to produce antiidiotypic antibodies against a monoclonal antibody specific of the toxin active site . These antilds were shown to mimic the in vitro killer effect for the toxin and were called 'antibiobodies' . The susceptibility of P . carinii to the antimicrobial activity of the killer toxin made it possible to hypothesize that the killer phenomenon could constitute a new way for the treatment and prophylaxis of P . carinii infections.

J Bacteriol, 1994 Sep, 176(18), 5622 - 30
NADH dehydrogenase subunit genes in the mitochondrial DNA of yeasts; Nosek J et al.; The genes encoding the NADH dehydrogenase subunits of respiratory complex I have not been identified so far in the mitochondrial DNA (mtDNA) of yeasts . In the linear mtDNA of Candida parapsilosis, we found six new open reading frames whose sequences were unambiguously homologous to those of the genes known to code for NADH dehydrogenase subunit proteins of different organisms, i.e., ND1, ND2, ND3, ND4L, ND5, and ND6 . The gene for ND4 also appears to be present, as judged from hybridization experiments with a Podospora gene probe . Specific transcripts from these open reading frames (ND genes) could be detected in the mitochondria . Hybridization experiments using C . parapsilosis genes as probes suggested that ND genes are present in the mtDNAs of a wide range of yeast species including Candida catenulata, Pichia guilliermondii, Clavispora lusitaniae, Debaryomyces hansenii, Hansenula polymorpha, and others.

J Biol Chem, 1994 Aug 26, 269(34), 21835 - 44
The Pichia pastoris PAS4 gene encodes a ubiquitin-conjugating enzyme required for peroxisome assembly; Crane DI et al.; We report here the cloning and initial characterization of PAS4, a gene required for peroxisome assembly in the yeast Pichia pastoris . The PAS4 gene encodes a 24-kDa protein (Pas4p) that is located on the cytoplasmic surface of peroxisomes and is induced during peroxisome proliferation . Analysis of the Pas4p sequence revealed a high degree of similarity to ubiquitin-conjugating enzymes, particularly in the region surrounding the putative active-site cysteine residue with which ubiquitin forms a thioester bond . As expected for a ubiquitin-conjugating enzyme, substitution of alanine or serine for the conserved active-site cysteine residue abolished PAS4 function . In addition, a small amount of a 32 kDa form of Pas4p (the predicted size of a Pas4p-ubiquitin conjugate) was detected both in vivo and in vitro . This species was eliminated by reducing agents and was not detected in the cysteine to alanine substitution mutant, suggesting that it is a Pas4p-ubiquitin conjugate . Using a yeast strain that overexpresses a Myc-ubiquitin fusion protein, we demonstrate directly that this conjugate contains ubiquitin . We conclude from these observations that PAS4 is a member of the ubiquitin-conjugating enzyme gene family and that one or more ubiquitination reactions are required for peroxisome assembly.

Genetika, 1994 Aug, 30(8), 1123 - 9
{The Petergof genetic collection of microorganisms}; Samsonova MG et al.; The Petergof Genetic Collection (PGC) of microalgae was created in the 1960s during study of the regularities of mutational processes . A collection of yeasts has been maintained at the Department of Genetics and Selection of St . Petersburg State University since 1977 . This collection contains some 1000 genetically marked strains of the yeasts Saccharomyces cerevisiae and Pichia methanolica, and the algae collection comprises about 600 strains of Chlamydomonas reinhardtii, Chlorella vulgaris, and Scenedesmus obliquus . The structure of the collection and the employment of strains in basic and applied research, as well as for educational purposes, are discussed . On the basis of the original software GENESTRAIN, a yeast PGC database (DB) was developed . A visual interface that contains information about selection of Ch . reinhardtii strains and crosses made was created in the HyperCard operational system.

J Clin Microbiol, 1994 Aug, 32(8), 1923 - 9
CHROMagar Candida, a new differential isolation medium for presumptive identification of clinically important Candida species; Odds FC et al.; CHROMagar Candida is a novel, differential culture medium that is claimed to facilitate the isolation and presumptive identification of some clinically important yeast species . We evaluated the use of this medium with 726 yeast isolates, including 82 isolated directly on the medium from clinical material . After 2 days of incubation at 37 degrees C, 285 C . albicans isolates gave distinctive green colonies that were not seen with any of 441 other yeast isolates representing 21 different species . A total of 54 C . tropicalis isolates also developed distinctive dark blue-gray colonies with a halo of dark brownish purple in the surrounding agar . C . krusei isolates (n = 43) also formed highly characteristic rough, spreading colonies with pale pink centers and a white edge that was otherwise encountered only rarely with isolates of C . norvegensis . Trichosporon spp . (n = 34) formed small, pale colonies that became larger and characteristically rough with prolonged incubation . Most of the other 310 yeasts studied formed colonies with a color that ranged from white to pink to purple with a brownish tint . The only exceptions were found among isolates identified as Geotrichum sp . or Pichia sp., some of which formed colonies with a gray to blue color and which in two instances formed a green pigment or a dark halo in the agar . The specificity and sensitivity of the new medium for the presumptive identification of C . albicans, C . krusei, and C . tropicalis exceeded 99% for all three species . A blinded reading test involving four personnel and 57 yeast isolates representing nine clinically important species confirmed that colonial appearance after 48 h of incubation on CHROMagar Candida afforded the correct presumptive recognition of C . albicans, C . tropicalis, C, krusei, and Trichosporon spp . None of nine bacterial isolates grew on CHROMagar Candida within 72 h, and bacteria (Escherichia coli) grew from only 4 of 104 vaginal, 100 oral, and 99 anorectal swabs . The new medium supported the growth of 19 of 23 dermatophyte fungi tested and 41 of 43 other molds representing a broad range of fungal pathogens and contaminants . In parallel cultures of 348 clinical specimens set up on Sabourand agar and CHROMagar Candida, both media grew yeasts in the same 78 instances . CHROMagar Candida is recommended as a useful isolation medium capable of the presumptive identification of the yeast species most commonly isolated from clinical material and facilitating recognition of mixed yeast cultures.

Biosci Biotechnol Biochem, 1994 Jul, 58(7), 1245 - 57
The phylogenetic relationships of the hat-shaped ascospore-forming, nitrate-assimilating Pichia species, formerly classified in the genus Hansenula Sydow et Sydow, based on the partial sequences of 18S and 26S ribosomal RNAs (Saccharomycetaceae): the proposals of three new genera, Ogataea, Kuraishia, and Nakazawaea; Yamada Y et al.; The twenty-seven strains of the hat-shaped ascospore-forming, nitrate-assimilating species, formerly classified in the genus Hansenula, of the genus Pichia were examined for their 18S and 26S rRNA partial base sequencings . All the strains examined were separate phylogenetically from the type strain of P . membranaefaciens (type species of genus Pichia) . Based on the sequence data obtained {by number of base differences (five or more) with P . anomala and base sequences on fingerprint segment} in the 18S rRNA partial base sequences, these species were divided into seven groups . Group I, including P . anomala (identical to H . anomala, type species of genus Hansenula), P . canadensis, P . muscicola, P . silvicola, P . subpelliculosa, P . americana, P . bimundalis, P . ciferrii, P . syndowiorum, P . bispora, and P . fabianii, corresponded to the genus Hansenula Sydow et Sydow . Groups II and III were comprised of P . capsulata and P . holstii, respectively . Group IV included P . angusta, P . minuta var . minuta, P . minuta var . nonfermentans, P . philodendra, P . glucozyma, and P . henricii . Groups V, VI, and VII included P . jadinii, P . petersonii, and P . dryadoides, respectively . The nitrate assimilation-negative species, P . wickerhamii was phylogenetically distant from P . membranaefaciens . The seven groupings are discussed phylogenetically and taxonomically . For Groups IV, II, and III, the three new genera were proposed as Ogataea, Kuraishia, and Nakazawaea, respectively, with the type species, O . minuta (identical to P . minuta), K . capsulata (identical to P . capsulata), and N . holstii (identical to P . holstii).

Biosci Biotechnol Biochem, 1994 Jul, 58(7), 1236 - 44
The phylogenetic relationships of the saturn-shaped ascospore-forming species of the genus Williopsis Zender and related genera based on the partial sequences of 18S and 26S ribosomal RNAs (Saccharomycetaceae): the proposal of Komagataea Gen . Nov; Yamada Y et al.; The partial base sequences of 18S and 26S rRNAs of strains of Williopsis and Saturnospora species were analyzed . In the three regions partially sequenced, the higher base differences were observed in the strains examined of the three species, W . californica, W . mucosa, and W . pratensis, compared with those of W . saturnus var . saturnus (type species of genus Williopsis), W . beijerinckii, W . mrakii, W . saturnus var . subsufficiens, W . suaveolens, P . membranaefaciens (type species of genus Pichia), C . matritensis (type species of genus Citeromyces), and S'spora dispora (type species of genus Saturnospora): the percent similarities were 52-82 in positions 493-622, 130 bases, of 26S rRNA, and the number of base differences was 28-6 in positions 1611-1835, 225 bases, of 26S rRNA, and the number of base differences was 25-4 in positions 1451-1618, 168 bases, of 18S rRNA . In the 18S rRNA partial base sequencings, W . mucosa had an identical base sequence with P . anomala (identical to H . anomala, type species of genus Hansenula) . Based on the sequence data obtained, the taxonomic positions of the three Williopsis species mentioned above are discussed . The genus Zygowilliopsis Kudriavzev was postulated to be retained and emended, and a new genus, Komagataea was proposed for W . pratensis with a new combination, Komagataea pratensis.

Biochim Biophys Acta, 1994 Jun 12, 1206(2), 279 - 85
Secretion of human intracellular aspartic proteinase cathepsin E expressed in the methylotrophic yeast, Pichia pastoris and characterization of produced recombinant cathepsin E; Yamada M et al.; The human gastric cathepsin E (CTSE), a dimeric aspartic proteinase, was expressed in the methylotrophic yeast Pichia pastoris by placing the CTSE cDNA under the control of the methanol inducible alcohol oxidase promoter . The human CTSE expressed in P . pastoris was secreted into the culture medium as an active enzyme directed by its native signal sequence despite its intracellular localization in mammalian cells . The time course analysis of the culture supernatant of the P . pastoris transformant expressing human CTSE revealed that the recombinant human CTSE was secreted as a 90 kDa molecule and then converted via an 84 kDa intermediate to an 82 kDa mature molecule . A large-scale culture of the transformant was performed in a high cell density fermentor and the recombinant human CTSE was highly purified from the culture supernatant . The purified recombinant cathepsin E had the molecular mass of 82 kDa with the amino-terminal sequence starting with Ile37 of the sequence deduced from its cDNA sequence, suggesting that the human cathepsin E was accumulated in the culture supernatant as mature dimeric enzyme . The result of endoglycosidase-H digestion followed by Western blot analysis of the purified recombinant cathepsin E suggested that the human cathepsin E expressed in P . pastoris received N-linked high-mannose type glycosylation . The enzymatic properties of the recombinant enzyme were comparable to those of natural human CTSE.

Mol Gen Genet, 1994 Jun 3, 243(5), 489 - 99
The positive and negative cis-acting elements for methanol regulation in the Pichia pastoris AOX2 gene; Ohi H et al.; The methylotrophic yeast Pichia pastoris has two alcohol oxidase genes, AOX1 and AOX2 . The AOX2 gene is transcribed at a much lower level than the AOX1 gene . Apart from this difference in expression levels, the two genes are regulated similarly . To study the role of cis-acting elements in the promoter region of the AOX2 gene, we constructed expression plasmids in which the human serum albumin (HSA) gene was placed under the control of various deleted or mutated AOX2 promoter derivatives . By analyzing the expression of HSA in P . pastoris transformants, we have identified three cis-acting regulatory elements in the AOX2 promoter . The positive cis-acting element AOX2-UAS, located between positions -337 and -313 (relative to the transcription initiation codon), is required for response to transcriptional induction by methanol in an orientation-independent manner, and artificial amplification of the AOX2-UAS resulted in an increase in the transcriptional activity of the promoter . A sequence homologous to AOX2-UAS was also found in the AOX1 promoter, and in methanol-regulated promoters in other methylotrophic yeast . Two negative cis-acting elements, AOX2-URS1 and AOX2-URS2 play a role in repressing transcription from the AOX2 promoter . The function of AOX2-UAS is completely repressed by this unique repression system when both the AOX2-URS1 and AOX2-URS2 are functional.

Genetika, 1994 Jun, 30(6), 783 - 90
{Development of a method for vector transformation of the methyltrophic yeast Pichia methanolica}; Tarutina MG et al.; A method for transformation of the methylotrophic yeast Pichia methanolica (formerly P . pinus MH4) was developed . Mutants leu1 were shown to be transformed with different efficiency using 2.2-10.7-kb linear and circular DNA molecules containing the LEU2 gene of the yeast Saccharomyces cerevisiae, which complemented the leu1 mutation of the recipient . Efficiency of transformation with short molecules was higher than with long molecules . Transformation with linear DNA was more efficient than with circular DNA of equal size . Transformants contained both replication-unstable and integration forms in different proportions . Significant rearrangements in the episome and integration forms of transforming DNA were found in transformants obtained using the 10.7-kb circular YEp13 plasmid . Integration of linear DNA was not accompanied by rearrangements in DNA molecules . Certain clones isolated after transformation with linear DNA contained autonomously replicating circular vector molecules formed as a result of a ligase reaction in vivo . The LEU2 gene of S . cerevisiae contains an unknown sequence acting as the ARS replicon in cells of both P . methanolica and of another species of the methylotrophic yeasts, Hansenula polymorpha.

Mycopathologia, 1994 Jun, 126(3), 173 - 7
Killer toxin secretion through the cell wall of the yeast Pichia anomala; Cailliez JC et al.; A secreted killer toxin was detected through the cell wall of Pichia anomala cells by ultrastructural immunodetection with a specific monoclonal antibody (MAb KT4) . MAb KT4 was successively detected by colloidal gold labeled streptavidin and biotinylated anti-mouse F(ab')2 antibodies . The antigenic determinants of the toxin were localized throughout the cytoplasm and the cell wall of killer yeast cells . The Lowicryl K4M-immunogold method gave very satisfactory results and showed that the killer toxin was somewhat concentrated in the yeast cell wall layers before being exported into the medium . In agreement with previous reports, the binding of MAb KT4 suggested that the P . anomala killer toxin secretion did not result from a homogeneous diffusion across the yeast cell wall.

Yeast, 1994 May, 10(5), 625 - 36
Transfer RNA profiling: a new method for the identification of pathogenic Candida species; Santos MA et al.; A new molecular taxonomic method applicable to the identification of medically important Candida species and other yeast species has been developed . It is based on the electrophoretic pattern of total tRNA samples (a 'tRNA profile') isolated from Candida species and generated using high-resolution semi-denaturing urea-polyacrylamide gel electrophoresis and methylene blue staining . Species-specific tRNA profiles for the species C . albicans, C . tropicalis, C . parapsilosis, C . guilliermondii, C . glabrata and Pichia guilliermondii were obtained . Detailed studies with the major human pathogen of the Candida genus, C . albicans, demonstrated that the tRNA profile for a given species was both reproducible and strain-independent; seven different C . albicans strains generated identical tRNA profiles . Minor strain-specific heterogeneities in the tRNA profiles of C . guilliermondii and C . parapsilosis were detected, but in neither case did they significantly alter the species-specific diagnostic tRNA profile . The potential of this method in clarifying taxonomic anomalies was demonstrated by the finding that Type I and Type II strains of C . stellatoidea generate very different tRNA profiles, with that of a Type II strain being identical to the C . albicans tRNA profile . This method offers a number of advantages over current electrophoretic karyotype methods for species identification, both within the Candida genus and with yeast species in general.

J Biotechnol, 1994 Mar 31, 33(2), 135 - 46
High level expression of the B . microplus Bm86 antigen in the yeast Pichia pastoris forming highly immunogenic particles for cattle; Rodriguez M et al.; Recently, a gene coding for the Bm86 tick gut glycoprotein was cloned, expressed in Escherichia coli and shown to induce an immunological response in cattle to damage ticks engorging on these animals (Rand et al., 1989) . We report here the increased expression of the Bm86 antigen from the cattle tick Boophilus microplus in the methylotrophic yeast Pichia pastoris . The recombinant protein was obtained with a purity higher than 95% by a procedure with a high yield . The conducted biochemical studies demonstrated the antigen to be glycosylated and found to form particles of around 17 to 45 nm in diameter with enhanced immunogenic properties . Ticks engorging on vaccinated cattle were significantly damaged as a result of the immune response against the recombinant antigen . This system permits the obtainment in a high yield of the tick Bm86 antigen, in a glycosylated and particulated form.

Biotechnology (N Y), 1994 Feb, 12(2), 181 - 4
Rapid selection using G418 of high copy number transformants of Pichia pastoris for high-level foreign gene expression; Scorer CA et al.; Pichia pastoris is a methylotrophic yeast increasingly important in the production of therapeutic proteins . Expression vectors are based on the methanol-inducible AOX1 promoter and are integrated into the host chromosome . In most cases high copy number integration has been shown to be important for high-level expression . Since this occurs at low frequency during transformation, we previously used DNA dot blot screens to identify suitable clones . In this paper we report the use of vectors containing the Tn903 kanr gene conferring G418-resistance . Initial experiments demonstrated that copy number showed a tight correlation with drug-resistance . Using a G418 growth inhibition screen, we readily isolated a series of transformants, containing progressively increasing numbers (1 to 12) of a vector expressing HIV-1 ENV, which we used to examine the relationship between copy number and foreign mRNA levels . Northern blot analysis indicated that ENV mRNA levels from a single-copy clone were nearly as high as AOX1 mRNA, and increased progressively with increasing copy number so as to greatly exceed AOX1 mRNA . We have also developed protocols for the selection, using G418, of high copy number transformants following spheroplast transformation or electroporation . We anticipate that these protocols will simplify the use of Pichia as a biotechnological tool.

J Biol Chem, 1994 Jan 28, 269(4), 3041 - 6
The primary and subunit structure of a novel type killer toxin produced by a halotolerant yeast, Pichia farinosa; Suzuki C et al.; A halotolerant yeast, Pichia farinosa KK1 strain, produces a unique killer toxin termed SMK toxin (salt-mediated killer toxin) which shows its maximum killer activity in the presence of 2 M NaCl . The toxin consists of two distinct subunits, alpha and beta, which are tightly linked without a disulfide bond under acidic conditions, even in the presence of 6 M urea . Under neutral conditions, however, the alpha subunit precipitates, resulting in the dissociation of the subunits and the loss of killer activity . The nucleotide sequence of the SMK1 gene predicts a 222 amino acid preprotoxin with a typical signal sequence, the hydrophobic alpha, an interstitial gamma polypeptide with a putative glycosylation site, and the hydrophilic beta . Amino acid sequence analyses of peptide fragments including the carboxyl-terminal peptides fragments including the carboxyl-terminal peptides from each subunit suggest that the alpha and beta subunits consist of amino acid residues 19-81 and 146-222 of the preprotoxin, respectively, and the molecular weight of the mature alpha beta dimer is 14,214 . The KEX2-like endopeptidase and KEX1-like carboxypeptidase may be involved in the stepwise processing of the SMK preprotoxin . The maturation process and the functions of the SMK toxin are compared with the K1 toxin of Saccharomyces cerevisiae.

J Biol Chem, 1994 Jan 7, 269(1), 556 - 66
PAY4, a gene required for peroxisome assembly in the yeast Yarrowia lipolytica, encodes a novel member of a family of putative ATPases; Nuttley WM et al.; PAY genes are required for peroxisome assembly in the yeast Yarrowia lipolytica . Here we characterize one mutant, pay4, and describe the cloning and sequencing of the PAY4 gene . The pay4 mutant shows no identifiable peroxisomes by biochemical and morphological criteria . The complementing PAY4 gene encodes a polypeptide, Pay4p, 1025 amino acids in length and having a predicted molecular mass of 112,258 Da . The predicted Pay4p sequence contains two putative ATP-binding domains and shows structural relationships to other potential ATP-binding proteins involved in biological processes as diverse as peroxisome biogenesis, vesicle-mediated protein transport, cell cycle control, and transcriptional regulation . These proteins all share a highly conserved stretch of approximately 175 amino acids that contains a consensus sequence for ATP binding . Pay4p shows sequence conservation with Pas1p and Pas5p, putative ATPases required for peroxisomal assembly in the yeasts Saccharomyces cerevisiae and Pichia pastoris, respectively . Pay4p, Pas1p, and Pas5p are presumably related members of a family of putative ATPases involved in peroxisome biogenesis . Pay4p is synthesized in low amounts in Y . lipolytica cells grown in glucose, and there is a rapid and pronounced increase in the levels of Pay4p upon transfer of the cells to a medium containing oleic acid as the sole carbon source.

Antonie Van Leeuwenhoek, 1994, 66(4), 313 - 7
Yeast communities of the cactus Pilosocereus arrabidae as resources for larval and adult stages of Drosophila serido; Morais PB et al.; The feeding behavior of Drosophila serido on the yeast communities of necrotic stem tissue of Pilosocereus arrabidae were studied in a sand dune ecosystem of Rio de Janeiro, Brazil . The prevalence of cactophilic yeasts including Pichia barkeri, Candida sonorensis and Geotrichum sp . in the crops and external surfaces of D . serido reflected its association with the cactus habitat . The effective number of yeasts vectored on the surface of flies was higher than that in the crops . Also overlap between the yeasts from stems and from crops was partial suggesting selective feeding by the flies in the substrates visited . The females had a higher effective number of yeast species and a lower similarity than males with the yeast community of P . arrabidae . This was probably related to the search for oviposition sites by females . The presence of Pichia thermotolerans-like and Pichia amethionina var pachycereana in the flies, but not in P . arrabidae stems, indicated that D . serido was not limited to this cactus species . The larvae and adults lived in different patches with the adults feeding in patches with higher yeast species richness . The larvae had a narrower feeding niche and higher overlap with P . arrabidae, and preferred P . barkeri and Pichia cactophila as food . Adult flies fed on patches with the most frequent yeasts except for P . cactophila . Pichia caribaea was found in higher frequency in the adult crops than in the stems . Our data suggested that there was food selection and diet partitioning between adult and larval stages of D . serido.

Gene, 1993 Dec 22, 136(1-2), 111 - 9
The intracellular production and secretion of HIV-1 envelope protein in the methylotrophic yeast Pichia pastoris; Scorer CA et al.; The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein, gp120 (ENV), is required in large quantities for immunological studies and as a potential vaccine component . We have expressed the DNA encoding gp120 in a highly efficient expression system based on the methylotrophic yeast, Pichia pastoris . The native gene was found to contain a sequence which resembled a Saccharomyces cerevisiae polyadenylation consensus and acted as a premature polyadenylation site in P . pastoris, resulting in the production of truncated mRNA . As full-length mRNA was produced in S . cerevisiae, this indicates differences in mRNA 3'-end formation between the two yeasts . Inactivation of this site by site-directed mutagenesis revealed several additional fortuitous polyadenylation sites within the gene . We have designed and constructed a 69%-synthetic gene with increased G + C content which overcomes this transcriptional problem, giving rise to full-length mRNA . High levels of intracellular, insoluble, unglycosylated ENV were produced {1.25 mg/ml in high-density (2 x 10(10) cells per ml) fermentations} . ENV also was secreted from P . pastoris using the S . cerevisiae alpha-factor prepro secretion leader and the S . cerevisiae invertase signal sequence . However, a high proportion of the secreted product was found to be hyperglycosylated, in contrast to other foreign proteins secreted from P . pastoris . There also was substantial proteolytic degradation, but this was minimized by maintaining a low pH on induction . Insoluble, yeast-derived ENV proteins are being considered as vaccine antigens and the P . pastoris system offers an efficient method of production.

J Cell Biol, 1993 Nov, 123(3), 535 - 48
Cloning and characterization of PAS5: a gene required for peroxisome biogenesis in the methylotrophic yeast Pichia pastoris; Spong AP et al.; The biogenesis and maintenance of cellular organelles is of fundamental importance in all eukaryotic cells . One such organelle is the peroxisome . The establishment of a genetic system to study peroxisome biogenesis in the methylotrophic yeast Pichia pastoris has yielded many different complementation groups of peroxisomal assembly (pas) or peroxisome-deficient (per) mutants . Each appears to be deficient in functional peroxisomes . One of these mutants, pas5, has been characterized, complemented, and the gene sequenced . Ultrastructural studies show that normal peroxisomes are not present in pas5, but aberrant peroxisomal structures resembling "membranous ghosts" are frequently observed . The "peroxisome ghosts" appear to be induced and segregated to daughter cells normally . Biochemical fractionation analysis of organelles of the pas5 mutant reveals that peroxisomal matrix enzymes are induced normally but are found mostly in the cytosol . However, purification of peroxisome ghosts from the mutant shows that small amounts (< 5%) of matrix enzymes are imported . The PAS5 gene was cloned and found to encode a 127-kD protein, which contains a 200-amino acid-long region of homology with PAS1, NEM-sensitive factor (NSF), and other related ATPases . Weak homology to a yeast myosin was also observed . The gene is not essential for growth on glucose but is essential for growth on oleic acid and methanol . The role of PAS5 in peroxisome biogenesis is discussed.

Yeast, 1993 Nov, 9(11), 1251 - 8
The 5-aminoimidazole ribonucleotide-carboxylase structural gene of the methylotrophic yeast Pichia methanolica: cloning, sequencing and homology analysis; Hiep TT et al.; The ADE1 gene of the yeast Pichia methanolica encodes phosphoribosyl-5-aminoimidazole-carboxylase (AIRC, EC 4.1.1.21), which is involved in purine biosynthesis . The gene was cloned by complementation of an ade2 mutation in Saccharomyces cerevisiae and a 3077 nucleotide DNA fragment was sequenced . The sequence possessed a single open reading frame, corresponding to a 543 amino acid sequence . The sequence of this putative protein has been compared to the proteins of homologous genes from S . cerevisiae, Schizosaccharomyces pombe, Escherichia coli, chicken and man . The analysis revealed remarkable homology between yeast AIRCs, while for other proteins homology was limited to defined regions.

Yeast, 1993 Nov, 9(11), 1189 - 97
Transformation in the methylotrophic yeast Pichia methanolica utilizing homologous ADE1 and heterologous Saccharomyces cerevisiae ADE2 and LEU2 genes as genetic markers; Hiep TT et al.; Development of transformation systems for methylotrophic yeasts is the starting point for research aimed at developing molecular genetics of these genera and will be the key to their further successful use in biotechnology . We transformed Pichia methanolica using selector genes ADE2 and LEU2 from Saccharomyces cerevisiae and ADE1 (homologue of S . cerevisiae ADE2 gene) from P . methanolica which was cloned and sequenced in our laboratory (Hiep et al., 1991) . Lithium transformation of P . methanolica strains was inefficient with intact plasmids . Linearization of plasmids at a unique restriction site within the ADE1 gene prior to transformation substantially increased its frequency . Transformation with linear ADE1, ADE2 or LEU2 gene fragments was even more effective . Introduced DNA fragments either circularized in vivo, irrespective of the structures of their ends, giving unstable transformants; or integrated at different sites of the host genome . Using this transformation system, we obtained a disruption of the ADE1 gene on the chromosome by inserting the S . cerevisiae LEU2 gene . The disruption mutation ade1::LEU2 was used to study the mechanism of intragenic recombination in P . methanolica.

Biochemistry, 1993 Oct 12, 32(40), 10848 - 65
Cytochrome c peroxidase binds two molecules of cytochrome c: evidence for a low-affinity, electron-transfer-active site on cytochrome c peroxidase; Stemp ED et al.; We have studied the affinity and stoichiometry of binding of cytochrome c (Cc) to zinc-substituted cytochrome c peroxidase {(ZnP)CcP}, which is structurally and electrostatically equivalent to ferrous CcP . Transient absorption spectroscopy has been used to measure both the total quenching of the triplet-state (ZnP)CcP {3(ZnP)CcP} by Fe3+Cc and the fraction of that quenching that is due to electron transfer (et) . This redox quenching results in the formation of an intermediate (I) containing the zinc porphyrin pi-cation radical {(ZnP)+CcP} and Fe2+Cc . In titrations of (ZnP)CcP with Fe3+Cc(F) at low ionic strength, where F represents the fungal cytochromes c from Candida krusei, Pichia membranefaciens, or the yeast protein iso-1, the appearance of the et intermediate lags behind the total quenching, with appreciable formation of I occurring only for Cc to CcP ratios > 1 . This behavior results from the formation of a 2:1 complex, where one Fe3+Cc(F) binds to a high-affinity domain that exhibits strong quenching yet is et-inactive, while the second Fe3+Cc(F) binds to a low-affinity domain that allows efficient et quenching . At constant concentrations of both proteins, raising the ionic strength eliminates most of the et quenching but reduces the total quenching only minimally, confirming that et occurs preferentially at the low-affinity binding domain, which is the more sensitive to ionic strength . Analogous experiments also favor a 2:1 binding stoichiometry for horse Cc {Cc(horse)} at low ionic strength, with et quenching again proceeding much more favorably in the 2:1 complex than in the 1:1 complex, as with Cc(F) . However, the Fe3+Cc(horse) quenches only by electron transfer, unlike the Cc(F) . The decay of the triplet-state (ZnP)CcP or magnesium-substituted CcP {(MgP)CcP} was examined during titrations with Fe3+Cc to determine limits for the dissociation rate constant (koff) for the complex . Fe3+Cc(horse) bound to the high-affinity domain in a 1:1 complex at low ionic strength is in rapid exchange, with koff > 50 S-1, whereas Fe3+Cc(F) has koff < 200 s-1 . Both types of Fe3+Cc have koff > 10(4)S-1 when they are bound to the low-affinity domain in a 2:1 complex, at both low and high ionic strengths . In contrast, when in the ferrous form, both types of Cc have much lower values of koff (< 10 S-1) at low ionic strength when bound to the low-affinity domain.(ABSTRACT TRUNCATED AT 400 WORDS)

Gene, 1993 Sep 6, 131(1), 35 - 41
The dacA gene of Bacillus stearothermophilus coding for D-alanine carboxypeptidase: cloning, structure and expression in Escherichia coli and Pichia pastoris; Despreaux CW et al.; The bacterial D-alanine carboxypeptidases (CPases) remove C-terminal D-alanyl residues from sugar-peptide cell wall precursors . The CPases have many characteristics in common with the high-M(r) penicillin-binding proteins (PBPs) whose inhibition by beta-lactam antibiotics is lethal . The CPases are attractive as model PBPs, because of their relatively lower M(r) and higher activity in vitro . We have cloned and sequenced the Bacillus stearothermophilus gene (dacA) coding for a membrane-bound CPase . The nucleotide (nt) sequence of the gene is homologous to that of the Escherichia coli and Bacillus subtilis dacA loci, which also code for membrane-bound CPases . E . coli host cells lysed when expression of B . stearothermophilus dacA was induced . The same coding sequence was expressed in the methylotrophic yeast, Pichia pastoris, using the alcohol oxidase-1 (AOX1) promoter . Over 100 micrograms/ml of CPase was efficiently secreted into the medium after induction by methanol, without adversely affecting this host . The yeast product is indistinguishable from the native enzyme in structure and activity . The ability to secrete large amounts of heterologous protein and the lack of endogenous peptidoglycan metabolism makes P . pastoris an attractive candidate for the production of PBPs.

Biotechnology (N Y), 1993 Aug, 11(8), 905 - 10
Recent advances in the expression of foreign genes in Pichia pastoris; Cregg JM et al.; The Pichia pastoris heterologous gene expression system has been utilized to produce attractive levels of a variety of intracellular and extracellular proteins of interest . Recent advances in our understanding and application of the system have improved its utility even further . These advances include: (1) methods for the construction of P . pastoris strains with multiple copies of AOX1-promoter-driven expression cassettes; (2) mixed-feed culture strategies for high foreign protein volumetric productivity rates; (3) methods to reduce proteolysis of some products in high cell-density culture media; (4) tested procedures for purification of secreted products; and (5) detailed information on the structures of N-linked oligosaccharides on P . pastoris secreted proteins . In this review, these advances along with basic features of the P . pastoris system are described and discussed.

Bioorg Med Chem, 1993 Jul, 1(1), 71 - 5
A synthesis of (R)-recifeiolide by the aid of biochemical reaction as the key-step; Mochizuki N et al.; (R)-Recifeiolide, a naturally occurring macrolactone, was synthesized in optically pure form by the aid of biocatalysts . Lipase-catalyzed lactonization of the racemic precursor afforded the desired compound with a concomitant kinetic resolution . The optically active acyclic precursor could be synthesized by the reduction of corresponding ketone with Pichia farinosa IAM 4682 . The yeast reduction proceeded with the anti-Prelog rule, selecting si-face attack on the carbonyl group to give (R)-alcohol with > 95% e.e.

FEBS Lett, 1993 Jun 7, 324(1), 9 - 14
Dual relationships of xylitol and alcohol dehydrogenases in families of two protein types; Persson B et al.; Xylitol dehydrogenase encoded by gene XYL2 from Pichia stipitis is a member of the medium-chain alcohol dehydrogenase family, as evidenced by the domain organization and a distant homology (24% residue identity with the human class I gamma 1 alcohol dehydrogenase) . Much of a loop structure is missing, like in mammalian sorbitol and prokaryotic threonine dehydrogenases, many additional differences occur, and relationships are closest with the sorbitol dehydrogenase, the equivalence of which in P . stipitis may actually be the xylitol dehydrogenase . A second P . stipitis gene, also cloned and corresponding to a xylitol dehydrogenase, is highly different from XYL2, but encodes an enzyme with structural properties typical of the short-chain dehydrogenase family, which also contains an alcohol dehydrogenase (from Drosophila) . Thus, yeast xylitol dehydrogenases, like alcohol and polyol dehydrogenases from other sources, have dual derivations, combining similar enzyme activities in separate protein families . In contrast to the situation with the other enzymes, both forms of xylitol dehydrogenase are present in one organism.

Genetika, 1993 Jun, 29(6), 922 - 7
{Cloning of the RIB7 gene encoding the riboflavin synthase of the yeast Pichia guilliermondii}; Logvinenko EM et al.; The RIB7 gene encoding the enzyme of the final stage of riboflavin biosynthesis in Pichia guilliermondii--riboflavin synthase was cloned on the pFL38 shuttle vector as the Sau3A fragment of the chromosomal DNA of about 4 kb . The HindIII fragment of 1.4 kb was subcloned from the hybrid plasmid pFR7 obtained onto the pUC18 plasmid . The plasmid pR7 thus constructed transform Escherichia coli ribB-45 mutant cells with a blocked riboflavin synthase approximately at the same frequency as pFR7 . High riboflavin synthase activity was discovered in the E . coli transformants carrying pR7 but not pFR7 . Using both plasmids we also complemented rib17 mutant of P . guilliermondii.

J Clin Microbiol, 1993 Jun, 31(6), 1644 - 5
Resolutive Candida utilis fungemia in a nonneutropenic patient; Bougnoux ME et al.; We report here the second case of Candida utilis infection in humans . The patient was apparently immunocompetent, had no central catheter, and survived an 8-day fungemia . Genomic analysis confirmed the conspecificity of medical and industrial strains of C . utilis and that of the anamorphic yeast C . utilis with the teleomorphic yeast Pichia jadinii.

Curr Genet, 1993 May-Jun, 23(5-6), 468 - 71
Genomic comparisons among parental and fusant strains of Candida shehatae and Pichia stipitis; Selebano ET et al.; DNA-DNA binding experiments on selected fusants of Candida shehatae and Pichia stipitis showed that the nucleus of these strains was composed predominantly of Pichia DNA . Electrophoretic karyotyping revealed that the fusants contained four chromosomes, similar to those found in the Pichia parental strain . In addition, the fusants showed only marginal increases in cell DNA content when compared with the parents . Karyogamy was confirmed, however, by the isolation of recombinant phenotypic segregants, induced by meiotic and mitotic segregation . The results suggest that the fusion led to integration of Candida genes, rather than whole chromosomes, with the entire genome of P . stipitis.

Lipids, 1993 May, 28(5), 397 - 401
2-Hydroxyhexadecanoic and 8,9,13-trihydroxydocosanoic acid accumulation by yeasts treated with fumonisin B1; Kaneshiro T et al.; Fumonisin B1 is a sphingolipid-like compound that enhances the accumulation of yeast sphingolipids and 2-hydroxy fatty acids . These lipids occur both as freely extractable and cell bound components in yeast fermentations . Both free and bound 2-hydroxy fatty acids produced by Pichia sydowiorum NRRL Y-7130 were increased when fumonisin B1 (50 mg/L) was added to the usual growth medium containing yeast extract/malt extract/peptone/glucose . Fumonisin-treated cultures contained 38 mg/L more 2-hydroxyhexadecanoic and 15 mg/L more 2-hydroxyoctadecanoic acids than did untreated cultures . By contrast, fumonisin inhibited the accumulation of free 8,9,13-trihydroxydocosanoic acid in Rhodotorula sp . YB-2501 cultures, leading to 240 mg/L lower trihydroxy acid production than by untreated cultures.

J Cell Biol, 1993 May, 121(4), 761 - 74
The pas8 mutant of Pichia pastoris exhibits the peroxisomal protein import deficiencies of Zellweger syndrome cells--the PAS8 protein binds to the COOH-terminal tripeptide peroxisomal targeting signal, and is a member of the TPR protein family; McCollum D et al.; We previously described the isolation of mutants of the yeast Pichia pastoris that are deficient in peroxisome assembly (pas mutants) . We describe the characterization of one of these mutants, pas8, and the cloning of the PAS8 gene . The pas8 mutant is deficient for growth, but not for division or segregation of peroxisomes, or for induction of peroxisomal proteins . Two distinct peroxisomal targeting signals, PTS1 and PTS2, have been identified that are sufficient to direct proteins to the peroxisomal matrix . We show that the pas8 mutant is deficient in the import of proteins with the PTS1, but not the PTS2, targeting signal . This is the same import deficiency as that found in cells from patients with the lethal human peroxisomal disorder Zellweger syndrome . Cloning and sequencing of the PAS8 gene reveals that it is a novel member of the tetratricopeptide repeat gene family . Antibodies raised against bacterially expressed PAS8 are used to show that PAS8 is a peroxisomal, membrane-associated protein . Also, we have found that in vitro translated PAS8 protein is capable of binding the PTS1 targeting signal specifically, raising the possibility that PAS8 is a PTS1 receptor.

Biotechnology (N Y), 1993 May, 11(5), 606 - 10
Conversion of starch to ethanol in a recombinant Saccharomyces cerevisiae strain expressing rice alpha-amylase from a novel Pichia pastoris alcohol oxidase promoter; Kumagai MH et al.; A recombinant Saccharomyces cerevisiae, expressing and secreting rice alpha-amylase, converts starch to ethanol . The rice alpha-amylase gene (OS103) was placed under the transcriptional control of the promoter from a newly described Pichia pastoris alcohol oxidase genomic clone . The nucleotide sequences of ZZA1 and other methanol-regulated promoters were analyzed . A highly conserved sequence (TTG-N3-GCTTCCAA-N5-TGGT) was found in the 5' flanking regions of alcohol oxidase, methanol oxidase, and dihydroxyacetone synthase genes in Pichia pastoris, Hansenula polymorpha, and Candida boidinii S2 . The yeast strain containing the ZZA1-OS103 fusion secreted biologically active enzyme into the culture media while fermenting soluble starch.

Yeast, 1993 Apr, 9(4), 315 - 30
Phylogenetic study of ribosomal DNA of cactophilic Pichia species by restriction mapping; Shen R et al.; The rDNAs of strains of the cactophilic Pichia species P . amethionina, P . antillensis, P . barkeri, P . cactophila, P . caribaea, P . deserticola, P . heedii, P . kluyveri, P . norvegenesis, P . opuntiae, P . pseudocactophila, P . thermotolerans and their varieties and anamorphs were mapped with 15 restriction endonucleases, and compared to P . membranaefaciens and P . salictaria as possible non-cactophilic relatives . The existence of species complexes among those taxa was confirmed . P . membranaefaciens was a plausible ancestral species, and its closest relative in the cactophilic group was P . deserticola . These two species appeared to be moderately related to P . heedii and to P . barkeri, but the latter was shown clearly to belong to the P . kluyveri complex, in spite of a 6 mol% G+C difference in their nuclear DNAs . P . cactophila and P . pseudocactophila ostensibly emerged from P . norvegensis, a facultatively cactophilic yeast . The P . amethionina, P . cactophila and P . opuntiae species complexes appeared independent from one another and from all other species studied . P . salictaria did not appear to be related to P . amethionina.

Mol Cell Biol, 1993 Apr, 13(4), 2315 - 23
Linear mitochondrial DNAs of yeasts: closed-loop structure of the termini and possible linear-circular conversion mechanisms; Dinouel N et al.; The terminal structure of the linear mitochondrial DNA (mtDNA) from three yeast species has been examined . By enzymatic digestion, alkali denaturation, and sequencing of cloned termini, it was shown that in Pichia pijperi and P . jadinii, both termini of the linear mtDNA were made of a single-stranded loop covalently joining the two strands, as in the case of vaccinia virus DNA . The left and right loop sequences were in either of two orientations, suggesting the existence of a flip-flop inversion mechanism . Contiguous to the terminal loops, inverted terminal repeats were present . The mtDNA from Williopsis mrakii seems to have an analogous structure, although terminal loops could not be directly demonstrated . Electron microscopy revealed the presence, among linear molecules, of a small number of circular DNAs, mostly of monomer length . Linear and circular models of replication are considered, and possible conversion mechanisms between linear and circular forms are discussed . A flip-flop inversion mechanism between the inverted repeat sequences within a circular intermediate may be involved in the generation of the linear form of mtDNA.

Mol Cell Biol, 1993 Apr, 13(4), 2309 - 14
Linear mitochondrial DNAs of yeasts: frequency of occurrence and general features; Fukuhara H et al.; In most yeast species, the mitochondrial DNA (mtDNA) has been reported to be a circular molecule . However, two cases of linear mtDNA with specific termini have previously been described . We examined the frequency of occurrence of linear forms of mtDNA among yeasts by pulsed-field gel electrophoresis . Among the 58 species from the genera Pichia and Williopsis that we examined, linear mtDNA was found with unexpectedly high frequency . Thirteen species contained a linear mtDNA, as confirmed by restriction mapping, and labeling, and electron microscopy . The mtDNAs from Pichia pijperi, Williopsis mrakii, and P . jadinii were studied in detail . In each case, the left and right terminal fragments shared homologous sequences . Between the terminal repeats, the order of mitochondrial genes was the same in all of the linear mtDNAs examined, despite a large variation of the genome size . This constancy of gene order is in contrast with the great variation of gene arrangement in circular mitochondrial genomes of yeasts . The coding sequences determined on several genes were highly homologous to those of the circular mtDNAs, suggesting that these two forms of mtDNA are not of distant origins.

Eur J Cell Biol, 1993 Apr, 60(2), 283 - 90
Selective autophagy of peroxisomes in methylotrophic yeasts; Tuttle DL et al.; The methylotrophic yeasts Pichia pastoris and Hansenula polymorpha respond to a methanol substrate by synthesizing peroxisomal enzymes resulting in the formation of large peroxisomes . When the carbon source was changed from methanol to glucose, we observed a rapid loss of peroxisomes . In this comparative study, we utilized biochemical and morphological techniques to characterize the loss of peroxisomes in these yeasts . We used metabolic labeling and chase procedures to evaluate whether this loss was due to suppressed synthesis or enhanced degradation . The synthesis of alcohol oxidase was depressed 10-fold when cultures grown in methanol attained stationary growth . However, no further reduction of synthesis was observed upon transfer of these cultures to glucose medium . In stationary phase cultures maintained in methanol, two peroxisomal proteins, alcohol oxidase and dihydroxyacetone synthase, were degraded with a half-life of over 3 h . However, within 3 h of glucose repression, as much as 80% of the radiolabeled peroxisomal proteins were lost from both yeasts . This glucose-mediated degradative event appeared to be specific for peroxisomal proteins, since mitochondrial proteins were stable . Ultrastructural examination of both yeasts revealed that glucose induced the sequestration of peroxisomes into the yeast vacuole, the presumed site of degradation . These results suggest that peroxisome loss during glucose repression is due to a selective, enhanced degradation of whole peroxisomes by autophagic mechanisms.

Int J Food Microbiol, 1993 Feb, 17(4), 329 - 41
Survey of the physiological properties of the most frequent yeasts associated with commercial chilled foods; Guerzoni ME et al.; A comparative analysis of the initial and final population of yeasts, lactic acid bacteria and psychrotrophic bacteria, in a number of chilled foods, varying in ingredients, physico-chemical characters and origin, gave evidence that yeasts could play a significant role in the spoilage . The yeast populations appeared to be unexpectedly uniform and comprised principally strains of Yarrowia lipolytica, Debaryomyces hansenii and Pichia membranaefaciens . A survey of 62 isolates, comprising physiological characteristics such as growth temperatures, proteolytic and lipolytic activities, hydrophobicity, aw and preservative tolerance, in addition to organic acid production, indicated that these dominant species have very few common characters and that they are endowed with a spoilage potential probably linked to different physiological properties . The isolates of Y . lipolytica exhibited the strongest proteolytic and lipolytic activities and a pronounced hydrophobicity while D . hansenii isolates were characterized by a high growth rate at low temperature and at intermediate aw . P . membranaefaciens isolates showed a remarkable tolerance to acetic acid as a sole selective factor . A hypothesis of separate growth loci, in multicomponent or polyphasic food systems, was formulated.

Scand J Immunol, 1993 Jan, 37(1), 105 - 10
Idiotypic vaccination: immunoprotection mediated by anti-idiotypic antibodies with antibiotic activity; Polonelli L et al.; Anti-Id antibodies were raised in mice against a monoclonal antibody (MoAb KT4) that neutralized the in vitro activity of a Pichia anomala yeast killer toxin . Monoclonal antibody was administered to BALB/C syngeneic mice with different schedules of immunization before intravenous challenge with increasing amounts of yeast killer toxin-sensitive Candida albicans cells . The course of candidosis was studied in comparison with mice non-immunized and immunized with an isotype-matched unrelated MoAb subdivided into control groups . Protection was reflected by statistically significant increases in survival rate of mice immunized with MoAb KT4 which showed variable serum levels of yeast killer toxin-like anti-Id antibodies . MoAb KT4 affinity chromatography purified mouse anti-Id antibodies were capable of killing in vitro the yeast cells of the Candida albicans strain used for the experimental infection . This is the first report of antimicrobial protection that exploits the role of anti-idiotypic antibodies presumably acting in vivo as antibiotics (idiotypic vaccination).

Appl Biochem Biotechnol, 1993 Spring, 39-40, 135 - 47
Cloning and improving the expression of Pichia stipitis xylose reductase gene in Saccharomyces cerevisiae; Chen Z et al.; The intact Pichia stipitis xylose reductase gene (XR) has been cloned and expressed in Saccharomyces cerevisiae . The possible further improvement of the expression of the Pichia gene in the new host was studied . To improve the expression of the XR gene in yeast (Saccharomyces cerevisiae), its 5'noncoding sequence containing the genetic elements for transcription and translation was systematically replaced by that from the yeast genes . It was found that the Pichia genetic signal for transcription of XR is more effective than the yeast TRP5 promoter, but is about half as effective as the yeast strong promoter of the alcohol dehydrogenase gene (ADC1) . However, the nucleotide sequence immediately adjacent to the initiation codon of XR, which controls the translation of the gene product, seemed to be five times less effective than the corresponding sequence of the ADC1 gene . By totally replacing its 5'-noncoding sequence with that of the yeast ADC1 gene, the expression of XR in yeast was found to be nearly ten times higher . Furthermore, the cloned Pichia XR described in this article contains very little of its 3'-noncoding sequence . In order to study whether the 3'-noncoding sequence is important to its expression in S . cerevisiae, the intact 3'-noncoding sequences of the yeast xylulokinase gene was spliced to the 3' end of the PADC1-XR structural gene . This latter modification has resulted in a twofold further increase in the expression of the Pichia XR in yeast.

Microbios, 1993, 76(306), 29 - 33
Formate dehydrogenase activity in methanol-utilizing yeasts; Illeova V et al.; The growth and activity of formate dehydrogenase (FDH) in five methanol-utilizing yeasts at various methanol concentrations were investigated . The parameters observed were inhibited at 4% methanol concentration in the medium . For Candida boidinii and Pichia trehalophila FDH activity was not found . The highest value was detected for Pichia lindneri (0.14 U/mg protein).

Acta Microbiol Pol, 1993, 42(1), 35 - 9
Candida valida--a yeast lacking the reverse transsulphuration pathway; Piotrowska M; A new yeast strain PGR-13 was classified as the variant of Candida valida . It is unique in its ability to ferment well and ready formation of respiratory deficient mutants . The lack of the reverse transsulphuration pathway was found to be a common trait of both PGR-13 and the type strain of Pichia membranaefaciens.

Biochem Int, 1992 Dec, 28(4), 651 - 7
Characterisation of genes encoding two novel members of the aldo-keto reductase superfamily; Dalrymple BP et al.; The predicted amino acid sequence of the protein encoded by a cDNA clone isolated from the protozoan haemoparasite Babesia bovis has approximately 22% amino acid identity with the Pichia stipitis xylose reductase . There are similar levels of amino acid identity with other members of the aldo-keto reductase superfamily . The identities include many residues highly conserved in the superfamily . However, the amino acid sequence of the B . bovis protein (AKR1) clearly lies outside the cluster of the previously characterized members of the superfamily . A putative protein encoded by a previously undescribed partially characterized open reading frame at the igrA (increased glyphosate resistance) locus of Pseudomonas sp . strain PG2982 also exhibits similarity to AKR1 and the aldo-keto reductases.

Can J Microbiol, 1992 Dec, 38(12), 1233 - 7
Segregation of altered parental properties in fusions between Saccharomyces cerevisiae and the D-xylose fermenting yeasts Candida shehatae and Pichia stipitis; Gupthar AS; A prototrophic strain of Saccharomyces cerevisiae CSIR Y190 MATa xyl-, resistant to high levels of ethanol, was hybridized with xylose-fermenting, auxotrophic mutants of Candida shehatae and Pichia stipitis through polyethylene glycol-induced protoplast fusion in an attempt to produce ethanol-tolerant, xylose-fermenting hybrids . Mononucleate fusants were obtained, but these dissociated into a mixture of parental-type segregants . Purified Candida- and Pichia-resembling segregants failed to acquire improved ethanol tolerance but expressed other novel properties of S . cerevisiae, suggesting that karyogamy was impaired after internuclear gene transfer.

Curr Genet, 1992 Nov, 22(5), 429 - 31
The electrophoretic banding pattern of the chromosomes of Pichia stipitis and Candida shehatae; Passoth V et al.; The electrophoretic karyotype of fu1ur strains of P . stipitis and five strains of C . shehatae were compared by means of OFAGE and TAFE techniques . Although the number of chromosomal bands was six in all cases except one, P . stipitis revealed a clearly distinct pattern in comparison to C . shehatae . Both yeasts showed remarkable chromosome length polymorphism.

Antonie Van Leeuwenhoek, 1992 Nov, 62(4), 267 - 72
Clavispora opuntiae and other yeasts associated with the moth Sigelgaita sp . in the cactus Pilosocereus arrabidae of Rio de Janeiro, Brazil; Rosa CA et al.; Clavispora opuntiae was the prevalent yeast associated with the feeding sites of Sigelgaita sp . larvae in the cactus Pilosocereus arrabidae . Also associated with this habitat were Candida sonorensis, Pichia cactophila, Pichia barkeri, Candida sp . A, Geotrichum sp., Geotrichum sericeum and the yeast like organisms Prototheca zopfii and Acremonium sp . Atypical yeast biotypes that may represent new species of Pichia, Sporopachydermia and Candida were isolated . Mating types of Clavispora opuntiae were at a ratio 70 h- to 3 h- and reduced levels of sporulation suggested low pressure for sexual reproduction in this habitat . Sigelgaita sp . probably was not an important vector for Clavispora opuntiae because it was not isolated from an adult or eggs of this moth.

Antonie Van Leeuwenhoek, 1992 Oct, 62(3), 189 - 99
Formation of antigenic extracellular polysaccharides by selected strains of Mucor spp., Rhizopus spp., Rhizomucor spp., Absidia corymbifera and Syncephalastrum racemosum; De Ruiter GA et al.; In this study, polyclonal IgG antibodies raised against extracellular polysaccharides (EPS) of Mucor racemosus were characterised as almost specific for moulds belonging to the order of Mucorales . Cross-reactivity in the ELISA could be observed only towards the yeast Pichia membranaefaciens . EPS were isolated from various cultures of M . hiemalis growing on six different carbon sources and two nitrogen sources, with ratios varying from 0.13 to 0.44 relative to the amount of biomass . Other strains including Mucor spp., Rhizopus spp., Rhizomucor spp., Absidia corymbifera and Syncephalastrum racemosum also excreted EPS, with ratios varying from 0.05 to 0.23 . In all cases, the excreted EPS had similar antigenic properties as determined by ELISA . No enzymatic degradation of the antigenic parts of the polysaccharides could be observed upon prolonged incubation . Considering that all tested strains formed similar amounts of antigenic EPS there might be scope for the specific detection of biomass of Mucoralean moulds using ELISA techniques for example in food.

Mol Gen Genet, 1992 Sep, 234(3), 481 - 8
Molecular cloning of the imidazoleglycerolphosphate dehydratase gene of Trichoderma harzianum by genetic complementation in Saccharomyces cerevisiae using a direct expression vector; Goldman GH et al.; The Trichoderma harzianum imidazoleglycerolphosphate dehydratase gene (igh) has been isolated by complementation of a Saccharomyces cerevisiae his3 mutant using a direct expression vector . This Escherichia coli-yeast shuttle vector was developed to allow efficient cloning and expression of cDNA libraries . The cDNA is 627 nucleotides long and codes for a protein of 209 amino acids with an apparent molecular mass of 22,466 daltons . The predicted protein sequence showed 63.6%, 58.7%, and 38.4% identity respectively to the corresponding enzymes from S . cerevisiae, Pichia pastoris and E . coli . Northern analysis showed that the expression of the igh gene in T . harzianum is not inhibited by external histidine and the level of igh mRNA was about threefold higher in cells starved of histidine.

J Cell Biol, 1992 Aug, 118(3), 499 - 508
Transport of microinjected alcohol oxidase from Pichia pastoris into vesicles in mammalian cells: involvement of the peroxisomal targeting signal; Walton PA et al.; This report describes the microinjection of a purified peroxisomal protein, alcohol oxidase, from Pichia pastoris into mammalian tissue culture cells and the subsequent transport of this protein into vesicular structures . Transport was into membrane-enclosed vesicles as judged by digitonin-permeabilization experiments . The transport was time and temperature dependent . Vesicles containing alcohol oxidase could be detected as long as 6 d after injection . Coinjection of synthetic peptides containing a consensus carboxyterminal tripeptide peroxisomal targeting signal resulted in abolition of alcohol oxidase transport into vesicles in all cell lines examined . Double-label experiments indicated that, although some of the alcohol oxidase was transported into vesicles that contained other peroxisomal proteins, the bulk of the alcohol oxidase did not appear to be transported to preexisting peroxisomes . While the inhibition of transport of alcohol oxidase by peptides containing the peroxisomal targeting signal suggests a competition for some limiting component of the machinery involved in the sorting of proteins into peroxisomes, the organelles into which the majority of the protein is targeted appear to be unusual and distinct from endogenous peroxisomes by several criteria . Microinjected alcohol oxidase was transported into vesicles in normal fibroblasts and also in cell lines derived from patients with Zellweger syndrome, which are unable to transport proteins containing the ser-lys-leu-COOH peroxisomal targeting signal into peroxisomes (Walton et al., 1992) . The implications of this result for the mechanism of peroxisomal protein transport are discussed.

J Bacteriol, 1992 Aug, 174(15), 4943 - 51
An efficient screen for peroxisome-deficient mutants of Pichia pastoris; Liu H et al.; We describe a rapid and efficient screen for peroxisome-deficient (per) mutants in the yeast Pichia pastoris . The screen relies on the unusual ability of P . pastoris to grow on two carbon sources, methanol and oleic acid, both of which absolutely require peroxisomes to be metabolized . A collection of 280 methanol utilization-defective (Mut-) P . pastoris mutants was isolated, organized into 46 complementation groups, and tested for those that were also oleate-utilization defective (Out-) but still capable of growth on ethanol and glucose . Mutants in 10 groups met this phenotypic description, and 8 of these were observed by electron microscopy to be peroxisome deficient (Per-) . In each per mutant, Mut-, Out-, and Per- phenotypes were tightly linked and therefore were most likely due to a mutation at a single locus . Subcellular fractionation experiments indicated that the peroxisomal marker enzyme catalase was mislocalized to the cytosol in both methanol- and oleate-induced cultures of the mutants . In contrast, alcohol oxidase, a peroxisomal methanol utilization pathway enzyme, was virtually absent from per mutant cells . The relative ease of per mutant isolation in P . pastoris, in conjunction with well-developed procedures for its molecular and genetic manipulation, makes this organism an attractive system for studies on peroxisome biogenesis.

Yeast, 1992 Aug, 8(8), 613 - 28
Development of the yeast Pichia pastoris as a model organism for a genetic and molecular analysis of peroxisome assembly; Gould SJ et al.; We describe the isolation of mutants of the yeast Pichia pastoris that are deficient in peroxisome assembly (pas) . These mutants of P . pastoris can be identified solely by their inability to grow on methanol and oleic acid, the utilization of which requires peroxisomal enzymes, and are defined by the absence of normal peroxisomes as judged by electron microscopy and biochemical fractionation experiments . These mutants are the result of genetic defects at single loci and represent at least eight different complementation groups . The isolation of pas mutants of P . pastoris by a simple screen for mutants unable to use methanol and oleic acid represents a significantly more efficient method for identification of pas mutants than is possible in other organisms . To exploit this advantage fully we also developed new reagents for the genetic and molecular manipulation of P . pastoris . These include a set of auxotrophic strains with an essentially wild-type genetic background, plasmids that act as Escherichia coli-P . pastoris shuttle vectors, and genomic DNA libraries for isolation of P . pastoris genes by functional complementation of mutants or by nucleic acid hybridization . The availability of numerous pas mutants and the reagents necessary for their molecular analysis should lead to the isolation and characterization of genes involved in peroxisome assembly.

Biochem Biophys Res Commun, 1992 Jul 31, 186(2), 1138 - 45
High level expression, purification, and characterization of the Kunitz-type protease inhibitor domain of protease nexin-2/amyloid beta-protein precursor; Wagner SL et al.; The protease inhibitor, protease nexin-2 (PN-2), is the secreted isoform of the Alzheimer's amyloid beta-protein precursor (A beta PP) that contains the Kunitz-type protease inhibitor (KPI) domain . Here we describe the use of the methylotrophic industrial yeast Pichia pastoris as a host system for the large scale production of the KPI domain of PN-2/A beta PP . In addition to the 57 amino acid KPI domain, the expression product contained an additional four amino acid residues at its amino terminus that correspond to amino acids 285-288 of A beta PP (Ponte et al . 1988 Nature 311:525-527) . This expression system generated yields of greater than 1.0 gram of KPI domain per liter of fermentation media . The secreted 61 amino acid product was purified to homogeneity and biochemically characterized . Amino acid analysis and sequencing of the entire expressed KPI domain verified its integrity . Similar to native PN-2/A beta PP, the purified KPI domain potently inhibited trypsin, chymotrypsin, and coagulation factor XIa . Although heparin augments the inhibition of factor XIa by native PN-2/A beta PP it had no effect on the inhibition of factor XIa by expressed KPI domain suggesting that heparin binds to regions on native PN-2/A beta PP outside of the protease inhibitory domain . This KPI domain expression product should be useful in studying the physiologic and pathophysiologic functions of PN-2/A beta PP.

Can J Microbiol, 1992 Jul, 38(7), 654 - 8
Alcoholic glucose and xylose fermentations by the coculture process: compatibility and typing of associated strains; Laplace JM et al.; As part of the simultaneous fermentation of both glucose and xylose to ethanol by a coculture process, compatibilities between xylose-fermenting yeasts and glucose-fermenting species were investigated . Among the Saccharomyces species tested, none inhibited growth of the xylose-fermenting yeasts . By contrast, many xylose-fermenting yeasts, among the 11 tested, exerted an inhibitory effect on growth of the selected Saccharomyces species . Killer character was demonstrated in three strains of Pichia stipitis . Such strains, despite their high fermentative performances, cannot be used to ferment D-xylose in association with the selected Saccharomyces species . From compatibility tests between xylose-fermenting yeasts and Saccharomyces species, pairs of microorganisms suitable for simultaneous xylose and glucose fermentations by coculture are proposed . Strains associated in the coculture process are distinguished by their resistance to mitochondrial inhibitors . The xylose-fermenting yeasts are able to grow on media containing erythromycin (1 g/L) or diuron (50 mg/L), whereas the Saccharomyces species are inhibited by these mitochondrial inhibitors.

Biochimie, 1992 May, 74(5), 455 - 61
Conservative system for dosage-dependent modulation of translational fidelity in eukaryotes; Chernoff YO et al.; Variations in dosage of some genes can alter the level of translational fidelity . The Saccharomyces cerevisiae genes that act as dosage-dependent suppressors and/or modulators of suppression, are the following: some tRNA genes (for example, tRNA(Gln)) inducing readthrough by mispairing; genes coding for either translational elongation factor or other proteins taking part in translation; and some genes of unknown function . We suggest that the SUP35 protein is a factor which may play a major role in balance-dependent regulation of translational fidelity . Homologues of this genes have been identified in other yeast genera (Pichia), green algae (Chlamydomonas) and various animals including man . No homologies have been found in the polychaeta (Nereis) or in insects (Drosophila) . Rates of evolution differ for two separate parts of the genes; the N-terminal part, which is important for ambiguous translation in Saccharomyces, is markedly variable in the organisms tested . However, the C-terminal part which is required for yeast viability has a common origin but a separate evolution from that of the EF-Tu protein family.

Appl Environ Microbiol, 1992 May, 58(5), 1577 - 82
Styrene formation by the decomposition by Pichia carsonii of trans-cinnamic acid added to a ground fish product; Shimada K et al.; It is not well known how the formation of styrene by microorganisms can occur in foods . In this study, we described and characterized the production of styrene by a yeast isolated from chikuwa fish paste . The styrene was not detected in fresh and normal food products nor in the food package's plastic film . The food containing styrene contained cinnamic acid as an antimicrobial agent and spice, and it was contaminated by 5.4 x 10(6) CFU of a yeast per gram . On the basis of morphological and biochemical features, the yeast isolated was determined to be a strain of Pichia carsonii, now designated strain CHI . Strain CHI, which was able to grow on cinnamic acid, had the ability to form styrene from trans-cinnamic acid via trans-p-coumaric and caffeic acids . The MIC of trans-cinnamic acid against strain CHI was 230 micrograms/ml . Strain CHI thrived well at pH 5.0 and 26.0 degrees C and was tolerant to 20% NaCl . Styrene was subsequently produced in ground fish meat containing cinnamic acid into which strain CHI had been inoculated . The yeast was found to be an environmental contaminant in food processing plants of the chikuwa manufacturer.

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