Pichia Pinus Research Articles

Microbial reduction of α-chloroketone to α-chlorohydrin

Microbial reduction of α-chloroketone to α-chlorohydrin was studied as one of the approaches for construction of the chiral center of the corresponding epoxide. About 100 microorganisms covering many species of Candida, Pichia, Hansenula, Geotrichum, Rhodococcus and Aureobasidium were screened to reduce the α-chloroketone stereospecifically. Many strains provided the R-α-chlorohydrin with 100% enantiomeric excess (ee), e.g., Candida sonorensis SC 16117, Geotrichum candidum SC 5469, Rhodotorula glutinis SC 16293, Sphingomonas paucimobilis SC 16113, Pichia silvicola SC 16159 and Rhodococcus equi SC 15835. Few microorganisms showed preferential formation of S-α-chlorohydrin after reduction. Among them, Pichia pinus SC 13864 and two Pichia methanolica strains SC 16116 and SC 13860 were the best, providing the S-α-chlorohydrin with ee of 88%, 79% and 78%, respectively. The enantiospecificity of the reduction by these Pichia species can be modified by changing the pH or prior heat treatment of the cells and S-α-chlorohydrin with ≥95% ee was obtained by appropriate modification of reaction conditions. Journal of Industrial Microbiology & Biotechnology (2001) 26, 259–262.

Microbial reduction of alpha-chloroketone to alpha-chlorohydrin.

Microbial reduction of alpha-chloroketone to alpha-chlorohydrin was studied as one of the approaches for construction of the chiral center of the corresponding epoxide. About 100 microorganisms covering many species of Candida, Pichia, Hansenula, Geotrichum, Rhodococcus and Aureobasidium were screened to reduce the alpha-chloroketone stereospecifically. Many strains provided the R-alpha-chlorohydrin with 100% enantiomeric excess (ee), e.g., Candida sonorensis SC 16117, Geotrichum candidum SC 5469, Rhodotorula glutinis SC 16293, Sphingomonas paucimobilis SC 16113, Pichia silvicola SC 16159 and Rhodococcus equi SC 15835. Few microorganisms showed preferential formation of S-alpha-chlorohydrin after reduction. Among them, Pichia pinus SC 13864 and two Pichia methanolica strains SC 16116 and SC 13860 were the best, providing the S-alpha-chlorohydrin with ee of 88%, 79% and 78%, respectively. The enantiospecificity of the reduction by these Pichia species can be modified by changing the pH or prior heat treatment of the cells and S-alpha-chlorohydrin with > or =95% ee was obtained by appropriate modification of reaction conditions.

Microbial reduction of alpha-chloroketone to alpha-chlorohydrin.

Microbial reduction of alpha-chloroketone to alpha-chlorohydrin was studied as one of the approaches for construction of the chiral center of the corresponding epoxide. About 100 microorganisms covering many species of Candida, Pichia, Hansenula, Geotrichum, Rhodococcus and Aureobasidium were screened to reduce the alpha-chloroketone stereospecifically. Many strains provided the R-alpha-chlorohydrin with 100% enantiomeric excess (ee), e.g., Candida sonorensis SC 16117, Geotrichum candidum SC 5469, Rhodotorula glutinis SC 16293, Sphingomonas paucimobilis SC 16113, Pichia silvicola SC 16159 and Rhodococcus equi SC 15835. Few microorganisms showed preferential formation of S-alpha-chlorohydrin after reduction. Among them, Pichia pinus SC 13864 and two Pichia methanolica strains SC 16116 and SC 13860 were the best, providing the S-alpha-chlorohydrin with ee of 88%, 79% and 78%, respectively. The enantiospecificity of the reduction by these Pichia species can be modified by changing the pH or prior heat treatment of the cells and S-alpha-chlorohydrin with > or =95% ee was obtained by appropriate modification of reaction conditions.

Polyol production by culture of methanol-utilizing yeast

Four methanol-utilizing yeasts, Candida boidinii, Hansenula polymorpha, Hansenula ofunaensis, and Pichia pinus, produced polyols from corresponding sugars in a methanol medium. H. polymorpha produced larger amounts of xylitol than the other yeasts. Productivity was the highest at pH 8 when 5 g (dry)/ l cultured cells were incubated with 2.5 g/ l urea as the nitrogen source in a medium containing 1% (v/v) methanol and 1 g/ l MgSO 4·7H 2O. Under these conditions, 57 g/ l xylitol was obtained from 110 g/ l d-xylose after 3 d of cultivation. The largest amount of xylitol (58 g/ l; yield, 0.62 g/g) was produced from 125 g/ l, d-xylose and 5% (w/v) glycerol instead of methanol after 4 d of cultivation.

Evaluation of enzymes produced from yeast

Pichia pinus was found to be capable of growing on mango wastes, producing pectinase (pectin lyase, EC-4.2.2.10) and lactase (beta-galactosidase, EC-3.2.1.23) enzymes. The two enzymes were successively purified by precipitation with ammonium sulfate followed by chromatography on Sephadex G-120. The purification procedure provided 1,846 and 929 fold purification with 20.6 and 24% yield recovery of pectinase and lactase, respectively. the km value of pectinase was 0.33% for pectin at pH 4.5 and that for lactase was 0.166% for lactose at pH 7.0. The purified enzymes, pectinase and lactase are stable up to 50 degrees C for 60 and 45 min, respectively, with 20 and 35% loss of their activity. Gel filtration on Sephadex G-200 indicated that the molecular weights of the purified pectinase was 90 x 10(3) Dalton and of lactase 115 x 10(3) Dalton. On the basis of the evaluation tests done, the enzymes were considered to have a potential technological interest as treating mango pastes (residues left after mango juice preparation) with the two prepared enzymes resulted in an increase of the colour intensity, total carbohydrate content and juice yield. Treating milk with the purified lactase also showed an increase in the total carbohydrate and reducing sugar produced.

Radiosensitivity of haplont yeast cells irradiated with sparsely and densely ionizing radiations

Five haploid and three diploid yeast strains of various species ( Yarrowia lipolytica, Pichia pinus and Pichia guilliermondii) were irradiated with α-particles from 239Pu and γ-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 α-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.

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

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.

Permeabilization of the methylotrophic yeast Pichia pinus for intracellular enzyme analysis: a quantitative study

A quantitative in situ assay of methanol oxidizing enzymes in permeabilized suspensions of the methylotrophic yeast Pichia pinus is described. Activities of the intracellular enzymes alcohol oxidase, formaldehyde dehydrogenase and formate dehydrogenase were readily measured in cell suspensions treated with 0.1% surfactant, digitonin or cetyltrimethylammoniumbromide (CTAB) for 15 min. Recovery of enzyme activities from the permeabilized cell suspension was higher than from disrupted cells. The permeabilizing ability of surfactant was dependent on its concentration, being highest at 0.1%. Cell permeabilization caused a marked decrease in dry weight and protein content of cells whereas no significant immidiate leakage of the intracellular enzymes alcohol oxidase, formaldehyde dehydrogenase and formate dehydrogenase was observed. However, 20-h storage of permeabilized suspension at 8°C resulted in marked leakage of dehydrogenases but not of alcohol oxidase from the cells. Addition of CTAB at 0.01% and digitonin at 0.01% and 0.1% to the cell-free extract for 1 h did not inhibit formaldehyde dehydrogenase and alcohol oxidase, whereas incubation with CTAB at 0.1% for the same time reduced activity of these enzymes to 62% and 37% from the initial, respectively. Properties of alcohol oxidase (K m towards methanol and substrate specificity) in permeabilized cell suspensions were similar to these measured in cell-free extract. Treatment of cell suspension with surfactants reduced the viability of cells dependent on the type of surfactant and its concentration.

Isolation and preliminary characterization of Pichia pinus mutants insensitive to glucose repression.

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.

Methylotrophic yeast microbiosensor based on ion-sensitive field effect transistors for methanol and ethanol determination

Bioassays for methanol and ethanol based on disposable biosensors were developed using mutant cells of methylotrophic yeasts Hansenula Polymorpha 34-19 and Pichia pinus 2468, respectively. Cells were immobilized in calcium alginate gel on the gate of pH-sensitive field effect transistors and the local acidification of the medium due to specific consumption of alcohols was recorded. The assay time was a few minutes. As the washing time exceeded 1 h, after a single use of the biosensor a new membrane was formed on the transducer or another biosensor was used. Both developed assays showed in unbuffered medium a detection limit near to 0.5 mM and linear calibration graphs over the alcohol concentration range 5–100 mM. Specificity studies showed no cross-sensitivity and no response to similar alcohols such as propanols and butanols, although a response to formaldehyde was observed in both instances. The effects of the cell contents in the membrane, buffer concentration and sample pH on the response of the biosensors were studied.

Methanol and ethanol utilization in methylotrophic yeast Pichia pinus wild-type and mutant strains

In the wild-type strain of methylotrophic yeast Pichia pinus diauxic growth is observed during cultivation in medium containing a mixture of methanol and ethanol: firstly, “slow” phase of ethanol utilization is revealed and, secondly, a “fast” phase of methanol consumption is shown. Diauxic growth is observed also in ecr1 mutant, impaired in ethanol-induced catabolite repression of methylotrophic metabolism enzymes, but the order of utilization of the alcohols is inverted in this mutant. Such succession of alcohols utilization in both strains correlates well with the sequence of synthesis of microbody enzymes which catalyze key reactions of C1- and C2-metabolism. On the contrary, simultaneous utilization of methanol and ethanol from the mixture, as well as synchronous synthesis of both peroxisomal and glyoxisomal enzymes is observed in adh1 mutant which has reduced alcohol dehydrogenase activity. The strong differences between the wild-type strain and adh1 mutant were observed also in the kinetics of specific activity changes for C1-metabolizing enzymes, localized in cytosol. In the wild-type strain during growth on methanol and ethanol mixture such changes correlate with the sequence of alcohol utilization. At the same time, in adh1 mutant the activities of formaldehyde dehydrogenase and formate dehydrogenase during the growth on the alcohols mixture are as high as during growth on methanol only, but the activity of dihydroxyacetone kinase is as low as under the growth on ethanol and is lower than on methanol.

Structure comparison and evolutionary relations between elongation factors EF-Tu (EF-1?) and SUP 2 proteins

On the basis of high homology and structural similarity, three genes, SUP2 Saccharomyces cerevisiae, SUP2 Pichia pinus and GST1 Homo sapiens, might be considered as members of one family named SUP2. Comparison of the primary structure of SUP2 proteins and elongation factors EF-Tu(EF-1 alpha) from 19 different species was performed. It was found that SUP2 proteins bear more homology to eukaryotic elongation factor than to procaryotic EF-Tu, though the degree of sequence conservation in SUP2 proteins is smaller than in EF-1 alpha factors. The extensive phylogenetic analysis of SUP2 and EF-Tu(EF-1 alpha) genes was performed by means of 3 methods, 2 phenetic and one cladystic (maximal parsimony). The data support the close relation of SUP2 genes to other elongation factor genes.

Divergence and conservation of SUP2(SUP35) gene of yeasts Pichia pinus and Saccharomyces cerevisiae

SUP2 (SUP35) is an omnipotent suppressor gene, coding for an EF-1 alpha-like protein factor, intimately involved in the control of translational accuracy in yeast Saccharomyces cerevisiae. In the present study a SUP2 gene analogue from yeast Pichia pinus was isolated by complementation of the temperature-sensitive sup2 mutation of S. cerevisiae. The nucleotide sequence of the SUP2 gene of P. pinus codes for a protein of 82.4 kDa, exceeding the Sup2 protein of S. cerevisiae by 6 kDa. Like the SUP2 gene product of S. cerevisiae, the Sup2 protein of P. pinus represents a fusion of a unique N-terminal part and a region homologous to EF-1 alpha. The comparison of amino acid sequences of the Sup2 proteins reveals high conservation (76%) of the C-terminal region and low conservation (36%) of the N-terminal part where, in addition, the homologous correspondence is ambiguous. Proteins related to the Sup2 of S. cerevisiae were found in P. pinus and some other yeast species by the immunoblotting technique. The relation between the evolutionary conservation of different regions of the Sup2 protein and their functional significance is discussed.

Yeast conversion of mango waste or methanol to single cell protein and other metabolites

The utilization of methanol or mango-waste hydrolyzate by Pichia pinus was studied with respect to the biomass, single cell protein and other metabolites. Optimum cultural conditions on both substrates were 30°C and pH 4·8–5. The maximum yield was obtained at the third day of growth (3·6 g/litre in methanol medium, 6·2 g/litre in mango-peel extract medium). At the end of the logarithmic phase, methanol yeast cells contained 52·2% crude protein, 36% true protein and 14·2% nucleic acids, while cells grown on mango-peel extract contained 62·2% crude protein, 39% true protein and 12·9% nucleic acids. The amino acid composition of the protein, especially of the essential acids, was comparable to FAO standards. α-Ketoglutaric and pyruvic acids were found in cells from both substrates but dihydroxyacetone was found in high quantity in methanol medium (976·9 μg%) while acetoacetic acid was found only in mango-peel medium (244·4 μg%).

Production and evaluation of a yeast utilizing an industrial waste

Pichia pinus was grown in a semi-continuous process on mango-peel extract medium. The yield was 8 g/litre after 12 h at the end of the logarithmic phase. The biomass of Pichia pinus contained 53·7% protein. Amino acid analyses of the protein revealed the presence of all amino acids in substantial amounts except for those containing sulphur, which were the limiting amino acids. When fed to rats the protein biomass exhibited good nutritional values (protein efficiency ratio, 2·77 ± 0·126; apparent digestibility, 71·54 ± 1·02) as compared to the casein diet (protein efficiency ratio, 2·913 ± 0·127; apparent digestibility, 90·156 ± 0·64). Alkaline phosphatase levels were insignificantly different from the control during the experimental period (21, 28, 35 and 42 feeding days). The transaminases, γ-glutamyl transferase activities and creatinine levels in the serum were significantly varied at the 35th and 42nd feeding days. The glutamate oxaloacetate transaminase, urea and uric acid levels in serum were significantly increased only after 35 feeding days. No histological changes were observed in either liver or kidney tissues.

Mutants of the methylotrophic yeast Pichia pinus defective in C2 metabolism

AbstractA collection of mutants of Pichia pinus which are unable to grow on ethanol but retain the ability to grow on glucose and methanol, was obtained. Genetic and biochemical analysis of these strains revealed mutations in seven nuclear genes affecting activities of isocitrate lyase (icl1), malate synthase (mls1), phosphoenolpyruvate carboxykinase (pck1), ‘malic’ enzyme (mdd1) and acetyl‐CoA synthetase (acs1, acs2 and acs3). All mutations except acs1‐acs3 have no effect on the activities of other enzymes involved in C2 metabolism. Mutations acs1, acs2 and acs3 have a pleiotropic action, leading to partial reduction in activities of isocitrate lyase and malate synthase. Ethanol‐induced repression of the synthesis of the methanol oxidative enzymes, alcohol oxidase and catalase, is not impaired in these seven mutant classes. On the other hand, C2 compound‐induced inactivation of alcohol oxidase and catalase is impaired in mutants acs1, acs2, acs3 and icl1. It was suggested that glyoxylate and acetate (or acetate precursors) act as low molecular weight effectors, ‘switching on’ inactivation and repression, respectively, of alcohol oxidase and catalase in the medium containing ethanol or acetate.

Species-specific extracellular antigen production by ascomycetous yeasts, detected by ELISA.

In this study many yeasts produced heat-stable immunogenic extracellular polysaccharides (EPS). These antigens could be easily detected by competitive enzyme-linked immunosorbent assay (ELISA) or by sandwich ELISA. The antigens of most ascomycetous yeast species tested were almost all species-specific, but those of some basidiomycetous species gave cross reactions with many yeast species, ascomycetes included. Antigens related or similar to those produced by Saccharomyces cerevisiae were detected in culture liquids of 11 strains of the latter, of Kluyveromyces marxianus, of Zygosaccharomyces florentinus and in several samples of beer and wine, but not in culture liquids of 63 other ascomycetous yeast species, 5 Saccharomyces species included. The EPS ELISA of Saccharomyces exiguus gave positive reactions with culture liquids of Candida milleri, C. tropicalis, Hanseniaspora vineae, S. dairensis, S. exiguus, S. unisporus and Saccharomycodes ludwigii. The EPS ELISA of Hansenula wickerhamii gave positive reactions with H. henricii, H. wickerhamii and Pichia pinus, thus showing the relationship of these two genera. The EPS ELISA of Geotrichum candidum appeared to be genus-specific like that of filamentous moulds. In addition to several G. candidum strains, G. fermentans, G. klebahnii responded positively, as well as Candida blankii and C. hydrocarbofumarica. The EPS ELISA of Stephanoascus ciferrii was very specific. Except for 10 strains of this species, only Trichosporon terrestre gave a strong positive reaction in the competitive ELISA.

Genetic control of methanol utilization in yeasts

Considered are our own data and those found in literature on the properties of yeast mutants impaired in their ability to utilize methanol as sole carbon and energy source; hypotheses about the role of alcohol oxidase and citrate synthase in biogenesis of peroxisomes are proposed. It has been proved that formaldehyde reductase participates in the control of the formaldehyde level in the cell. Properties of mutants defective in the catabolite repression and inactivation of enzymes of methanol metabolism are described. The existence of several autonomous mechanisms of the catabolite repression of alcohol oxidase has been shown. It has been found, that the induction of glyoxysomal enzymes of C2-metabolism is repressed by methanol in the ecr1 mutant of Pichia pinus with the affected repression of alcohol oxidase by ethanol. Data are presented on the regulatory properties of the recently discovered acidification system of the medium induced by methanol. Such acidification occurs due to symport extrusion of protons and formate anions from the cells.

Life cycle, meiotic recombination and unilateral gene transfer in methanolotrophic yeasts of the genus Pichia

In order to perform strain improvement of the methanol utilising yeasts Pichia pinus and Pichia lindneri their life cycles were elucidated. Both yeasts are monoecious and self-fertile. Karyogamy by autodiploidization is followed by meiosis leading to Mendelian segregation within the offspring. In Pichia pinus crosses via cell or protoplast fusion is feasible as proven by tetrad analysis. This is not the case in Pichia lindneri. The only possiblity to obtain recombinants in this yeast is brought about by a yet unknown mechanism, leading to a unilateral transfer of genetic material, found after cocultivation of mutants. This is revealed by the evaluation of F1 offspring from meiotic divisions after autodiploidisation. Although analysis of nuclear and mitochondrial DNA revealed rather gross differences not only between the species but also within different strains of Pichia pinus, only a limited exchange of genetic material is possible in interstrain crosses.

Multiplicity of mechanisms of carbon catabolite repression involved in the synthesis of alcohol oxidase in the methylotrophic yeast Pichia pinus

AbstractThe effect of various carbon compounds on the synthesis of alcohol oxidase in a medium with methanol was studied in the wild type strain of Pichia pinus as well as in gcr1 and ecr1 mutants defective in glucose and ethanol repression of methanol metabolic enzymes, respectively. Compounds repressing the synthesis of alcohol oxidase in the wild type strain were divided into four groups. Repression of alcohol oxidase by compounds of the first group (glucose, fructose, mannose, galactose, L‐sorbose and xylose) was impaired only in the gcr1 mutant and that by compounds of the second group (ethanol, acetate, 2‐oxoglutarate and erythritol) only in the ecr1 mutant. Repression by compounds of the third group (malate, dihydroxyacetone) was not impaired in both these regulatory mutants and that by compounds of the fourth group (succinate, fumarate, L‐arabinose, sorbitol, salicin, xylitol and cellobiose) was partially reduced in both gcr1 and ecr1 strains.Mutation gcr1 causes a significant decrease in phosphofructokinase activity. It also led to a six‐ to seven‐fold increase in intracellular pools of glucose‐6‐phosphate and fructose‐6‐phosphate and to a two‐fold decrase in the intracellular pool of fructose‐1,6‐bisphosphate. In ecr1 strains, a decrese in 2‐oxoglutarate dehydrogenase activity accompanied by an increae in activities of NAD‐ and NADP‐dependent isocitrate dehydrogenases and NAD‐ and NADP‐dependent glutamate dehydrogenases was demonstrated. The intracellular pool of 2‐oxoglutarate was increased 2·5‐fold in ecr1 strains. Genes GCR1 and ECR1 are not linked.The mechanisms of catabolite repression of alcohol oxidase in methylotrophic yeasts are discussed.