Endogenous NADPH-dependent aldose reductase activity influences product formation during xylose consumption in recombinant Saccharomyces cerevisiae

Introduction of the xylose pathway from Pichia stipitis into Saccharomyces cerevisiae enables xylose utilization in recombinant S. cerevisiae. However, xylitol is a major by-product. An endogenous aldo-keto reductase, encoded by the GRE3 gene, was expressed at different levels in recombinant S. cerevisiae strains to investigate its effect on xylose utilization. In a recombinant S. cerevisiae strain producing only xylitol dehydrogenase (XDH) from P. stipitis and an extra copy of the endogenous xylulokinase (XK), ethanol formation from xylose was mediated by Gre3p, capable of reducing xylose to xylitol. When the GRE3 gene was overexpressed in this strain, the xylose consumption and ethanol formation increased by 29% and 116%, respectively. When the GRE3 gene was deleted in the recombinant xylose-fermenting S. cerevisiae strain TMB3001 (which possesses xylose reductase and XDH from P. stipitis, and an extra copy of endogenous XK), the xylitol yield decreased by 49% and the ethanol yield increased by 19% in anaerobic continuous culture with a glucose/xylose mixture. Biomass was reduced by 31% in strains where GRE3 was deleted, suggesting that fine-tuning of GRE3 expression is the preferred choice rather than deletion.     

Malo-ethanolic fermentation in grape must by recombinant strains of Saccharomyces cerevisiae.

Recombinant strains of Saccharomyces cerevisiae with the ability to reduce wine acidity could have a significant influence on the future production of quality wines, especially in cool climate regions. L-Malic acid and L-tartaric acid contribute largely to the acid content of grapes and wine. The wine yeast S. cerevisiae is unable to effectively degrade L-malic acid, whereas the fission yeast Schizosaccharomyces pombe efficiently degrades high concentrations of L-malic acid by means of a malo-ethanolic fermentation. However, strains of Sz. pombe are not suitable for vinification due to the production of undesirable off-flavours. Heterologous expression of the Sz. pombe malate permease (mae1) and malic enzyme (mae2) genes on plasmids in S. cerevisiae resulted in a recombinant strain of S. cerevisiae that efficiently degraded up to 8 g/l L-malic acid in synthetic grape must and 6.75 g/l L-malic acid in Chardonnay grape must. Furthermore, a strain of S. cerevisiae containing the mae1 and mae2 genes integrated in the genome efficiently degraded 5 g/l of L-malic acid in synthetic and Chenin Blanc grape musts. Furthermore, the malo-alcoholic strains produced higher levels of ethanol during fermentation, which is important for the production of distilled beverages.     

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.     

葡萄糖-6-磷酸脱氢酶基因的Candida tropicalis过量表达及其对木糖醇合成代谢的影响

研究葡萄糖-6-磷酸脱氢酶基因g6pd过量表达对Candida tropicalis木糖醇生物合成代谢的影响。克隆Candidatropicalis CT16的g6pd基因,并将其与表达载体pYES-pgk重组连接,构建重组载体pYES-pgk-g6pd,LiAc/ssDNA/PEG方法转化导入C. tropicalis CT16,筛选阳性转化子,实现g6pd基因的过量表达。结果表明：发酵62 h,阳性转化子C. tropicalis SYG5的葡萄糖-6-磷酸脱氢酶活力提高了300%,发酵液中木糖醇质量浓度达到79.90 g/L,较野生型对照菌株的木糖醇产量提高了12.41%,木糖醇产率提高了44.94%。因此,葡萄糖-6-磷酸脱氢酶在C. tropicalis木糖醇的合成代谢途径中发挥重要作用,增强g6pd基因的表达,可以明显提高菌体NADPH供应量和还原力,有利于木糖醇的生物合成。     

Disruption of URA7 and GAL6 improves the ethanol tolerance and fermentation capacity of Saccharomyces cerevisiae

Screening of the homozygous diploid yeast deletion pool of 4741 non-essential genes identified two null mutants (Deltaura7 and Deltagal6) that grew faster than the wild-type strain in medium containing 8% v/v ethanol. The survival rate of the gal6 disruptant in 10% ethanol was higher than that of the wild-type strain. On the other hand, the glucose consumption rate of the ura7 disruptant was better than that of the wild-type strain in buffer containing ethanol. Both disruptants were more resistant to zymolyase, a yeast lytic enzyme containing mainly beta-1,3-glucanase, indicating that the integrity of the cell wall became more resistance to ethanol stress. The gal6 disruptant was also more resistant to Calcofluor white, but the ura7 disruptant was more sensitive to Calcofluor white than the wild-type strain. Furthermore, the mutant strains had a higher content of oleic acid (C18 : 1) in the presence of ethanol compared to the wild-type strain, suggesting that the disruptants cope with ethanol stress not only by modifying the cell wall integrity but also the membrane fluidity. When the cells were grown in medium containing 5% ethanol at 15 degrees C, the gal6 and ura7 disruptants showed 40% and 14% increases in the glucose consumption rate, respectively.     

响应面法优化酿酒酵母乙醛脱氢酶和乙醇脱氢酶酶活检测方法

从酿酒酵母前处理方式方面对ALD和ADH酶活测定条件进行了优化,采用单因素试验对前处理方式进行了分析研究,结果表明破壁时间、超声波功率、超声时间、反应温度和缓冲液pH值对酶活测定值产生一定的影响.通过Box-Behnken中心组合设计和响应面分析法,确定了测定这2种酶的最理想酵母前处理条件,即超声功率420W,超声时间为11min,破碎时间为12min.     

Modulation of Glycerol and Ethanol Yields During Alcoholic Fermentation in Saccharomyces cerevisiae Strains Overexpressed or Disrupted for GPD1 Encoding Glycerol 3-Phosphate Dehydrogenase

The possibility of the diversion of carbon flux from ethanol towards glycerol in Saccharomyces cerevisiae during alcoholic fermentation was investigated. Variations in the glycerol 3-phosphate dehydrogenase (GPDH) level and similar trends for alcohol dehydrogenase (ADH), pyruvate decarboxylase and glycerol-3-phosphatase were found when low and high glycerol-forming wine yeast strains were compared. GPDH is thus a limiting enzyme for glycerol production. Wine yeast strains with modulated GPD1 (encoding one of the two GPDH isoenzymes) expression were constructed and characterized during fermentation on glucose-rich medium. Engineered strains fermented glucose with a strongly modified [glycerol] : [ethanol] ratio. gpd1Δ mutants exhibited a 50% decrease in glycerol production and increased ethanol yield. Overexpression of GPD1 on synthetic must (200 g/l glucose) resulted in a substantial increase in glycerol production (×4) at the expense of ethanol. Acetaldehyde accumulated through the competitive regeneration of NADH via GPDH. Accumulation of by-products such as pyruvate, acetate, acetoin, 2,3 butane-diol and succinate was observed, with a marked increase in acetoin production. © 1997 John Wiley & Sons, Ltd.     

Functional analysis of structural genes for NAD(+)-dependent formate dehydrogenase in Saccharomyces cerevisiae

Co-consumption of formate by aerobic, glucose-limited chemostat cultures of Saccharomyces cerevisiae CEN.PK 113-7D led to an increased biomass yield relative to cultures grown on glucose as the sole carbon and energy substrate. In this respect, this strain differed from two previously investigated S. cerevisiae strains, in which formate oxidation did not lead to an increased biomass yield on glucose. Enzyme assays confirmed the presence of a formate-inducible, cytosolic and NAD(+)-dependent formate dehydrogenase. To investigate whether this enzyme activity was entirely encoded by the previously reported FDH1 gene, an fdh1Delta null mutant was constructed. This mutant strain still contained formate dehydrogenase activity and remained capable of co-consumption of formate. The formate dehydrogenase activity in the mutant was demonstrated to be encoded by a second structural gene for formate dehydrogenase (FDH2) in S. cerevisiae CEN.PK 113-7D. FDH2 was highly homologous to FDH1 and consisted of a fusion of two open reading frames (ORFs) (YPL275w and YPL276w) reported in the S. cerevisiae genome databases. Sequence analysis confirmed that, in the database genetic background, the presence of two single-nucleotide differences led to two truncated ORFs rather than the full-length FDH2 gene present in strain CEN.PK 113-7D. In the latter strain background an fdh1Deltafdh2Delta double mutant lacked formate dehydrogenase activity and was unable to co-consume formate. Absence of formate dehydrogenase activity did not affect growth on glucose as sole carbon source, but led to a reduced biomass yield on glucose-formate mixtures. These findings are consistent with a role of formate dehydrogenase in the detoxification of exogenous formate.     

糖浓度对酒精酵母GJ2008果糖与葡萄糖利用差异性的影响

在不同糖浓度条件下,以YPDF培养基模拟甘蔗汁进行酒精发酵,测定过程中各参数的变化,并对果糖与葡萄糖消耗过程进行曲线拟合,以拟合方程计算出果糖与葡萄糖代谢一半和代谢完全所需时间.结果表明,糖浓度为90gL～270g/L,酵母GJ2008始终会偏用葡萄糖,果糖利用一直受到葡萄糖的竞争性抑制.糖浓度为90g/L时,细胞生长受糖浓度抑制程度最小,但乙醇产率较低;糖浓度为230g/L,发酵液中葡萄糖含量较低时,果糖受葡萄糖的竞争性抑制得到了解除,果糖的利用急剧加快;糖浓度为250g/L和270g/L,发酵液中葡萄糖含量较低时,果糖受葡萄糖的竞争性抑制得到了解除,但果糖的利用并没有加快,表现为后期酵母数维持值较低.糖浓度对果糖代谢的影响要大于对葡萄糖代谢的影响,较低糖浓度有利于后期果糖与葡萄糖利用差异性的缩小.     

Candida albicans TDH3 gene promotes secretion of internal invertase when expressed in Saccharomyces cerevisiae as a glyceraldehyde-3-phosphate dehydrogenase-invertase fusion protein

We have checked the ability of the Candida albicans GAPDH polypeptide, which lacks a conventional N-terminal signal peptide, to reach the cell wall in Saccharomyces cerevisiae by using an intracellular form of the yeast invertase as a reporter protein. A hybrid TDH3-SUC2 gene containing the C. albicans TDH3 promoter sequences and a coding region encoding a fusion protein formed by the C. albicans GAPDH polypeptide, fused at its C-terminus with the yeast internal invertase, was constructed in a centromer derivative plasmid and transformed into a Suc(-) S. cerevisiae strain. Transformants displayed invertase activity measured in intact whole cells, and were able to grow on sucrose as the sole fermentable carbon source. Northern blot analysis with both TDH3 and SUC2 probes detected a single mRNA species of the expected size (about 2.7 kb), and Western immunoblot analysis of cell-free extracts, using a monoclonal antibody (mAb49) against a C. albicans GAPDH epitope, showed the presence of a 90 kDa polypeptide corresponding to the GAPDH-invertase fusion protein. This indicates that the TDH3 gene is able to direct part of the encoded gene product to the cell wall, and that any putative motifs for this targeting should be within the GAPDH amino acid sequence. Further analysis, using the same approach, of a panel of seven N- and C-terminal GAPDH truncates revealed that the region required for the cell wall targeting is located within the N-terminal half of the protein.     

The Fate of Glucose in Strains S288C and S173-6B of the Yeast Saccharomyces cerevisiae

Intracellular metabolic flux has been investigated in two strains of Saccharomyces cerevisiae grown into stationary phase under both glucose-repressed and glucose-derepressed conditions. By employing a variety of simple methodologies (manometry, enzymatic analysis and colorimetric analysis) we have been able to identify and quantitate carbon flow from glucose without the need for isotopically labelled substrate. We can account for 88–98% (depending on strain and growth conditions) of the carbon products of glucose metabolism under both glycolytic and oxidative conditions as ethanol (27–40%), carbon dioxide (15–26%), acetate (2–3%), glycerol (5–11%), glycogen (5–13%) and trehalose (9–39%).©1997 John Wiley & Sons, Ltd.     

Enzymatic activities of Ura2 and Ura1 proteins (aspartate carbamoyltransferase and dihydro-orotate dehydrogenase) are present in both isolated membranes and cytoplasm of Saccharomyces cerevisiae

Computational analysis predicted three potential hydrophobic transmembrane alpha-helices within the Ura2 multidomain protein of Saccharomyces cerevisiae, the C-terminal subdomain of which catalyses the second step of uridine-monophosphate biosynthesis by its L-aspartate carbamoyltransferase activity (EC 2.1.3.2). The fourth step of pyrimidine biosynthesis is catalysed by dihydro-orotate dehydrogenase (Ura1 protein; EC 1.3.99.11), which was similarly characterized as a peripheral membrane protein. Ex situ, the activities of the investigated enzymes were associated both with isolated yeast membranes, fractionated by differential centrifugation to remove intact nuclei, and with soluble cytoplasmic proteins.