Genetic analysis in the methylotrophic yeast Hansenula polymorpha

Techniques are described for the induction, isolation, and characterization of mutants of Hansenula polymorpha. In addition, techniques for controlled passage through the life cycle and genetic analyses, including complementation, tetrad and random spore analysis, have been developed and used to assign mutants to 62 complementation groups. We report that organism conforms to the expected genetics of a homothallic yeast and displays a Mendelian segregation of genes through meiosis. Preliminary mapping data are presented indicating linkage of three genes on a single linkage fragment. Enymatic analysis of methanol-non-utilizing mutants identified one class which is totally deficient in the key assimilatroy enzyme, dihydroxyacetone synthase.     

Carbon source requirements for mating and mating-type switching in the methylotrophic yeasts Ogataea (Hansenula) polymorpha and Komagataella phaffii (Pichia pastoris)

The methylotrophic yeasts Ogataea (Hansenula) polymorpha and Komagataella phaffii (Pichia pastoris) have important industrial applications and are models for several biological processes including peroxisome biology and methanol metabolism. We examined the carbon source requirements for mating-type (MAT) switching and mating in both species. Haploid strains of O. polymorpha and K. phaffii are homothallic, and switch MAT by a flip/flop mechanism in which a chromosomal region containing the MAT genes undergoes an inversion. MAT switching is induced by nitrogen starvation in both species and can be detected 4–6 hr after induction. Both switching and mating require a utilizable carbon source that can be either fermentable or nonfermentable. We further observed that although methanol can be used as a sole carbon source in both species, it does not support the induction of MAT switching or mating. Our results provide insight into the nutritional cues that influence entry into sexual processes in methylotrophic yeasts that undergo flip/flop MAT switching.     

D-lactate-selective amperometric biosensor based on the mitochondrial fraction of Ogataea polymorpha recombinant cells

During the recent decades, a lot of data about the significance of D-lactate determination in food technology and quality control have been accumulated. Nowadays, the development of new methods for the determination of D-lactate is very relevant, especially with regard to biosensors. To construct a D-lactate-selective biosensor, we suggest using the mitochondria of recombinant yeast cells of Ogataea (Hansenula) polymorpha “tr6” (gcr1 catX/Δcyb2, prAOX_DLDH) overproducing D-lactate: cytochrome c-oxidoreductase (DLDH, EC 1.1.2.4) and lacking an L-lactate-specific enzyme (flavocytochrome b₂, E.C. 1.1.2.3). The usage of the pure enzyme is problematic due to the complexity of its isolation and stabilization because of the intramembranous localization of DLDH. The enzyme catalyzes D-lactate oxidation to pyruvate coupled with ferricytochrome c reduction to ferrocytochrome c. The constructed biosensor is characterized by high sensitivity (18.5 А·М⁻¹·m⁻²), a low detection limit (3 μM of D-lactate), wide linear ranges, good selectivity, and sufficient stability. The real samples' analysis of D-lactate in dairy products was performed, and high correlation of the obtained results with the reference approach (0.7 < r < 1) and literature data was demonstrated.     

Development of new dominant selectable markers for the nonconventional yeasts Ogataea polymorpha and Candida famata

Nonconventional yeast Candida famata and Ogataea polymorpha are interesting organisms for basic and applied studies. O. polymorpha is methylotrophic thermotolerant yeast capable of xylose alcoholic fermentation whereas C. famata is capable of riboflavin overproduction. Still, the new tools for molecular research of these species are needed. The aim of this study was to develop the new dominant selective markers for C. famata and O. polymorpha usable in metabolic engineering experiments. In this work, the BSD gene from Aspergillus terreus coding for blasticidin S deaminase, O. polymorpha AUR1 gene required for sphingolipid synthesis and IMH3 gene, which encodes IMP dehydrogenase, were tested as the new dominant selective marker genes. Our results showed that AUR1 and IMH3 genes could be used as dominant selective markers for O. polymorpha with frequencies of transformation of 40 and 20 transformants per microgram of DNA, respectively. The IMH3 gene was successfully used as the marker for construction of O. polymorpha strains with increased ethanol production from xylose due to overexpression of TAL1, TKL1 and AOX1 genes. The BSD gene from A. terreus, conferring resistance to blasticidin, was found to be efficient for selection of C. famata transformants.     

PER1, GUP1 and CWH43 of methylotrophic yeast Ogataea minuta are involved in cell wall integrity

In eukaryotes, the glycosylphosphatidylinositol (GPI) modification of many glycoproteins on the cell surface is highly conserved. The lipid moieties of GPI‐anchored proteins undergo remodelling processes during their maturation. To date, the products of the PER1, GUP1 and CWH43 genes of the yeast Saccharomyces cerevisiae have been shown to be involved in the lipid remodelling. Here, we focus on the putative GPI remodelling pathway in the methylotrophic yeast Ogataea minuta. We found that the O. minuta homologues of PER1, GUP1 and CWH43 are functionally compatible with those of S. cerevisiae. Disruption of GUP1 or CWH43 in O. minuta caused a growth defect under non‐permissive conditions. The O. minuta per1Δ mutant exhibited a more fragile phenotype than the gup1Δ or cwh43Δ mutants. To address the role of GPI modification in O. minuta, we assessed the effect of these mutations on the processing and localization of the O. minuta homologues of the Gas1 protein; in S. cerevisiae, Gas1p is an abundant and well‐characterized GPI‐anchored protein. We found that O. minuta possesses two copies of the GAS1 gene, which we designate GAS1A and GAS1B. Microscopy and western blotting analysis showed mislocalization and/or lower retention of Gas1Ap and Gas1Bp within the membrane fraction in per1Δ or gup1Δ mutant cells, suggesting the significance of lipid remodelling for GPI‐anchored proteins in O. minuta. Localization behaviour of Gas1Bp differed from that of Gas1Ap. Our data reveals, for the first time (to our knowledge), the existence of genes related to GPI anchor remodelling in O. minuta cells.     

HpYPS1 and HpYPS7 encode functional aspartyl proteases localized at the cell surface in the thermotolerant methylotrophic yeast Hansenula polymorpha

In the present study, we functionally analysed two yapsin genes of the thermotolerant methylotrophic yeast Hansenula polymorpha, HpYPS1 and HpYPS7, for their roles in maintaining cell wall integrity and proteolytic processing. Both HpYPS1 and HpYPS7 proteins were shown to largely localize on the cell wall via glycosylphosphatidylinositol anchor. Heterologous expression of HpYPS1 completely restored all of the growth defects of the Saccharomyces cerevisiae yps1-deletion strains, while HpYPS7 expression exhibited a limited complementation effect on the S. cerevisiae yps7-deletion strain. However, different from S. cerevisiae, deletion of the HpYPS genes generated only minor influence on the sensitivity to cell wall stress. Likewise, HpYPS1 expression was significantly induced only by a subset of stressor agents, such as sodium dodecyl sulphate and tunicamycin. HpYps1p was shown to consist of two subunits, whereas HpYps7p comprises a single long polypeptide chain. Biochemical analysis revealed that HpYps1p has much stronger proteolytic cleavage activity at basic amino acids, compared to HpYps7p. Consistent with the much higher proteolytic activity and expression level of HpYps1p compared to HpYps7p, the sole disruption of HpYPS1 was sufficient in eliminating the aberrant proteolytic cleavage of recombinant proteins secreted by H. polymorpha. The results indicate that, although their roles in the maintenance of cell wall integrity are not critical, HpYps1p and HpYps7p are functional aspartic proteases at the cell surface of H. polymorpha. Furthermore, our data present the high biotechnological potential of H. polymorpha yps1-mutant strains as hosts useful for the production of secretory recombinant proteins.     

Excessive membrane development following exposure of the methylotrophic yeast Hansenula polymorpha to oleic acid-containing media

We studied the physiological responses of Hansenula polymorpha during adaptation of cells to oleic acid-containing media. Growth experiments indicated that the organism was unable to use oleic acid as the sole source of carbon and energy. However, upon incubation of glucose-grown cells in mineral media containing oleic acid, activities of various enzymes of the β-oxidation pathway were induced. These enzymes were localized in microbodies together with alcohol oxidase. Furthermore, a drastic increase in phospholipid content of the cells was observed; this was due to a rapid proliferation of membranes. These consisted of a variable number of membranous layers which were continuous with the peroxisomal membrane. Upon continued incubation, the membrane proliferations extended and large compartments were formed. This process was dependent on the presence of peroxisomes in the cells since it was not observed in peroxisome-deficient mutant strains of H. polymorpha. The newly formed membranous compartments differed from peroxisomes since they did not contain peroxisomal matrix proteins; these were confined to the single enlarged organelle which was incorporated in the membranous structure and characterized by a large alcohol oxidase crystalloid. The membranous compartments are considered to be whole entities since they could not be separated from the peroxisomes by common cell fraction methods; also they were degraded entirely after a shift of cells to glucose-excess condition. Freeze fracturing reveled that the substructure of the membranes greatly resembled that of normal peroxisomal membranes. Since a distinct enhancement of different peroxisomal membrane proteins was observed during the initial hours after the shift, we assume that exposure of H. polymorpha to acid lead to a drastic overproduction of peroxisomal membranes.     

The glycerol and ethanol production kinetics in low-temperature wine fermentation using Saccharomyces cerevisiae yeast strains

Increasing glycerol production in low-temperature wine fermentation is of concern for winemakers to improve the quality of wines. The objective of this study was to investigate the effect of 10 different Saccharomyces cerevisiae on the kinetics of production of glycerol, ethanol and the activities of glycerol-3-phosphate dehydrogenase (GPD) and alcohol dehydrogenase (ADH) in low-temperature fermentation. Ethanol production was influenced by temperature, and it was slightly higher at 13 °C than at 25 °C. Glycerol yields were significantly affected by both temperature and strains. More glycerol was produced at 25 °C than at 13 °C because the activity of GPD was higher at 25 °C than at 13 °C. Glycerol production of the different yeast strains was up to 3.19 and 3.18 g L⁻¹ at 25 and 13 °C, respectively. Therefore, isolating the yeast strains with high glycerol production and adaptation to low-temperature fermentation is still the best method in winemaking.     

Fermentation properties of a wine yeast over-expressing the Saccharomyces cerevisiae glycerol 3-phosphate dehydrogenase gene (GPD2)

Wine yeasts efficiently convert sugar into ethanol. The possibility of diverting some of the sugar into compounds other than ethanol by using molecular genetic methods was tested. Over-expression of the yeast glycerol 3-phosphate dehydrogenase gene ( GPD2 ) in a laboratory strain of Saccharomyces cerevisiae led to an approximate two-fold increase in the extracellular glycerol concentration. In the medium fermented with the modified strain, acetic acid concentration also increased approximately two-fold when respiration was blocked. A strain deleted for the GPD2 gene had the opposite phenotype, producing lower amounts of glycerol and acetic acid, with the latter compound only reduced during non-respiratory growth. A commercial wine yeast over-expressing GPD2 produced 16.5 g/L glycerol in a wine fermentation, compared to 7.9 g/L obtained with the parent strain. As seen for the laboratory strain, acetic acid concentrations were also increased when using the genetically modified wine yeast. A panel of wine judges confirmed the increase in volatile acidity of these wines. The altered glycerol biosynthetic pathway sequestered carbon from glycolysis and reduced the production of ethanol by 6 g/L.     

Metabolic regulation in the yeast Hansenula polymorpha. Growth of dihydroxyacetone kinase/glycerol kinase-negative mutants on mixtures of methanol and xylose in continuous cultures

The physiological responses of Hansenula polymorpha wild-type and mutant strains 17B (dihydroxyacetone kinase-negative) and 17BG51 (dihydroxyacetone kinase- and glycerol kinase-negative) to growth on mixtures of xylose and methanol in chemostats were investigated. Increasing methanol concentrations (0–110 mM ) in the feed of the wild-type culture resulted in increasing cell densities and a gradual switch towards methanol metabolism. At the lower methanol feed concentrations the mutant cultures used methanol and xylose to completion and changes in enzyme patterns comparable to the wild type were observed. This was not reflected in significant changes in cell densities. Instead, formaldehyde assimilation resulted in dihydroxyacetone (DHA) production, which was proportional to the amount of methanol added. At intermediate methanol concentration the cultures showed a strong variation in DHA levels and cell densities. Further increased in the methanol feed concentrations resulted in a drop in DHA accumulation rates, repression of alcohol oxidase synthesis and accumulation of residual methanol. The phenomena were studied in more detail in transition experiments and with gradients of methanol. The results indicate that xylulose-5-phosphate (Xu5P) generated in xylose metabolism served as acceptor molecule for formaldehyde assimilation by the peroxisomal enzyme DHA synthase. Accumulation of DHA in the mutant cultures, however, further diminished the availability of carbon for growth. The data suggest that with increasing methanol concentrations Xu5P eventually became growth rate limiting. This resulted in an unstable situation but wash-out of the culture did not occur to a significant extent. Instead, DHA accumulation ceased and cell densities, and enzymes specifically involved in xylose metabolism increase, indicating that the organism resumed its xylose metabolism. The molecular mechanisms controlling the partitioning of Xu5P over xylose (pentose phosphate pathway) and methanol (peroxisome) metabolism under these conditions remain to be elucidated.     

环境对酒精酵母产生乙醇耐性的影响

乙醇对细胞的抑制主要表现在对生长率、发酵率、糖分解酶、膜势能的抑制及膜磷脂的裂解,主要阐述了渗透压和温度对酒精酵母产生乙醇耐性的影响。     

Mutants of the methylotrophic yeast Pichia pinus defective in C2 metabolism

A 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.     

葡萄酒酒精发酵过程中酵母菌种类、代谢产物的变化及其关键控制工艺研究进展

酒精发酵是葡萄酒酿造的主要过程。本文介绍了酒精发酵过程中酵母菌种类的变化,不同酵母菌种类发酵对葡萄酒品质的影响,并对酒精发酵过程中代谢产物及其种类进行了综合概述,同时,还阐述了影响酒精发酵的因素及其关键控制工艺等。      

Simultaneous expression of the S and L surface antigens of hepatitis B, and formation of mixed particles in the methylotrophic yeast, Hansenula polymorpha

An expression system has been developed for the methylotrophic yeast Hansenula polymorpha and used to co-express both the L (preS1-S2-S) and S hepatitis B surface antigens (HBsAg) under the control of strong methanol-inducible promoters derived from the methanol oxidase and from the formate dehydrogenase genes. A unique feature of this H. polymorpha expression system is the possibility of integrating up to 100 copies of an expression cassette via a multimeric integration mechanism. Several multimeric integrants containing various numbers of L and S expression cassettes were constructed to give a spectrum of strains characterized by different L to S ratios. The expression level of S antigen was 5-8% of the total soluble cell protein. Analysis by sucrose and CsCl density gradient centrifugation and by particle-specific immunoassays demonstrated that the synthesized HBsAg spontaneously assembled into composite subviral particles containing both S and L proteins. Only a minor portion of the L protein was found to be glycosylated. These H polymorpha-derived composite particles can be used for the production of a hepatitis B virus vaccine with the potential for improved immunogenicity due to the presence of a wider spectrum of epitopes and negligible glycosylation.     

Overexpression of vacuolar H+‐ATPase‐related genes in bottom‐fermenting yeast enhances ethanol tolerance and fermentation rates during high‐gravity fermentation

Vacuolar H⁺‐ATPase (V‐ATPase) is thought to play a role in stress tolerance. In this study it was found that bottom‐fermenting yeast strains, in which the V‐ATPase‐related genes DBF2, VMA41/CYS4/NHS5 and RAV2 were overexpressed, exhibited stronger ethanol tolerance than the parent strain and showed increased fermentation rates in a high‐sugar medium simulating high‐gravity fermentation. Among the strains examined, the DBF2‐overexpressing bottom‐fermenting yeast strain exhibited the highest ethanol tolerance and fermentation rate in YPM20 medium. Using this strain, high‐gravity fermentation was performed by adding sugar to the wort, which led to increased fermentation rates and yeast viability compared with the parent strain. These findings indicate that V‐ATPase is a stress target in high‐gravity fermentation and suggests that enhancing the V‐ATPase activity increases the ethanol tolerance of bottom‐fermenting yeast, thereby improving the fermentation rate and cell viability under high‐gravity conditions. Copyright © 2012 The Institute of Brewing & Distilling     

Effect of the respiro-fermentative balance during yeast propagation on fermentation and wort attenuation

Breweries use different yeast strains to create beers with different flavours and aromas. Yeast propagation must produce yeast that performs consistently from the first fermentation to harvesting and re-pitching in subsequent fermentations. Breweries propagate yeast in wort leading to low efficiency fermentative growth in Crabtree-positive yeast. There is limited knowledge on the impact on beer production when fermenting with yeast propagated in sugar limited and nutrient supplemented wort. It was hypothesised that propagating yeast in this way would have a positive impact on subsequent fermentation performance. Saccharomyces cerevisiae was propagated at the laboratory scale in standard wort with a high carbon to nitrogen (C:N) ratio (850) or in modified wort supplemented with yeast extract to achieve a low C:N ratio (100) and at varying sugar concentrations. Propagation in low C:N wort with 2°P sugar yielded a 27% decrease in fermentation efficiency and a 46% increase in cell production compared to 2°P high C:N wort. This suggests nitrogen is critical to the respiro-fermentative balance during growth. Yeast propagated in standard wort resulted in slower fermentations and significant under-attenuation compared to yeast grown in the modified wort with low sugar and high nitrogen. The results of this study suggest the nitrogen and sugar content drive the respiro-fermentative balance during yeast propagation. The metabolism of yeast during propagation induces significant downstream impacts on the subsequent fermentation performance and wort attenuation. © 2020 The Institute of Brewing & Distilling     

Biosynthesis of higher alcohol flavour compounds by the yeast Saccharomyces cerevisiae: impact of oxygen availability and responses to glucose pulse in minimal growth medium with leucine as sole nitrogen source

Higher alcohol formation by yeast is of great interest in the field of fermented beverages. Among them, medium‐chain alcohols impact greatly the final flavour profile of alcoholic beverages, even at low concentrations. It is widely accepted that amino acid metabolism in yeasts directly influences higher alcohol formation, especially the catabolism of aromatic and branched‐chain amino acids. However, it is not clear how the availability of oxygen and glucose metabolism influence the final higher alcohol levels in fermented beverages. Here, using an industrial Brazilian cachaça strain of Saccharomyces cerevisiae, we investigated the effect of oxygen limitation and glucose pulse on the accumulation of higher alcohol compounds in batch cultures, with glucose (20 g/l) and leucine (9.8 g/l) as the carbon and nitrogen sources, respectively. Fermentative metabolites and CO₂/O₂ balance were analysed in order to correlate the results with physiological data. Our results show that the accumulation of isoamyl alcohol by yeast is independent of oxygen availability in the medium, depending mainly on leucine, α‐keto‐acids and/or NADH pools. High‐availability leucine experiments showed a novel and unexpected accumulation of isobutanol, active amyl alcohol and 2‐phenylethanol, which could be attributed to de novo biosynthesis of valine, isoleucine and phenylalanine and subsequent outflow of these pathways. In carbon‐exhausted conditions, our results also describe, for the first time, the metabolization of isoamyl alcohol, isobutanol, active amyl alcohol but not of 2‐phenylethanol, by yeast strains in stationary phase, suggesting a role for these higher alcohols as carbon source for cell maintenance and/or redox homeostasis during this physiological phase. Copyright © 2014 John Wiley & Sons, Ltd.