Anaerobic and aerobic batch cultivations of Saccharomyces cerevisiae mutants impaired in glycerol synthesis

Glycerol is formed as a by-product in production of ethanol and baker's yeast during fermentation of Saccharomyces cerevisiae under anaerobic and aerobic growth conditions, respectively. One physiological role of glycerol formation by yeast is to reoxidize NADH, formed in synthesis of biomass and secondary fermentation products, to NAD(+). The objective of this study was to evaluate whether introduction of a new pathway for reoxidation of NADH, in a yeast strain where glycerol synthesis had been impaired, would result in elimination of glycerol production and lead to increased yields of ethanol and biomass under anaerobic and aerobic growth conditions, respectively. This was done by deletion of GPD1 and GPD2, encoding two isoenzymes of glycerol 3-phosphate dehydrogenase, and expression of a cytoplasmic transhydrogenase from Azotobacter vinelandii, encoded by cth. In anaerobic batch fermentations of strain TN5 (gpd2-Delta1), formation of glycerol was significantly impaired, which resulted in reduction of the maximum specific growth rate from 0.41/h in the wild-type to 0.08/h. Deletion of GPD2 also resulted in a reduced biomass yield, but did not affect formation of the remaining products. The modest effect of the GPD1 deletion under anaerobic conditions on the maximum specific growth rate and product yields clearly showed that Gdh2p is the important factor in glycerol formation during anaerobic growth. Strain TN6 (gpd1-Delta1 gpd2-Delta1) was unable to grow under anaerobic conditions due to the inability of the strain to reoxidize NADH to NAD(+) by synthesis of glycerol. Also, strain TN23 (gpd1-Delta1 gpd2-Delta1 YEp24-PGKp-cth-PGKt) was unable to grow anaerobically, leading to the conclusion that the NAD(+) pool became limiting in biomass synthesis before the nucleotide levels favoured a transhydrogenase reaction that could convert NADH and NADP(+) to NADPH and NAD(+). Deletion of either GPD1 or GPD2 in the wild-type resulted in a dramatic reduction of the glycerol yields in the aerobic batch cultivations of strains TN4 (gpd1-Delta1) and TN5 (gpd2-Delta1) without serious effects on the maximum specific growth rates or the biomass yields. Deletion of both GPD1 and GPD2 in strain TN6 (gpd1-Delta1 gpd2-Delta1) resulted in a dramatic reduction in the maximum specific growth rate and in biomass formation. Expression of the cytoplasmic transhydrogenase in the double mutant, resulting in TN23, gave a further decrease in micromax from 0.17/h in strain TN6 to 0.09/h in strain TN23, since the transhydrogenase reaction was in the direction from NADPH and NADP(+) to NADH and NADP(+). Thus, it was not possible to introduce an alternative pathway for reoxidation of NADH in the cytoplasm by expression of the transhydrogenase from A. vinelandii in a S. cerevisiae strain with a double deletion in GPD1 and GPD2.     

Glycerol accumulation in the dimorphic yeast Saccharomycopsis fibuligera: cloning of two glycerol 3-phosphate dehydrogenase genes, one of which is markedly induced by osmotic stress

Glycerol plays an important role in the osmoadaptation responses of Saccharomyces cerevisiae. However, there is no detailed investigation about the role of glycerol in the osmoadaptation responses of Saccharomycopsis fibuligera. Here we show that both intra- and extracellular glycerol concentrations in Sm. fibuligera cells responded very quickly when they were subjected to osmotic stress. We then cloned two isogenes encoding putative NAD(+)-dependent glycerol 3-phosphate dehydrogenase (GPD) from Sm. fibuligera PD70 by degenerate PCR and subsequent chromosome walking methods. Those two genes, designated SfGPD1 and SfGPD2, respectively, exhibited 86.6% pairwise identity in their encoding regions, while there was no obvious homology in their non-coding regions. Either SfGPD1 or SfGPD2 could complement the salt tolerance characteristics of the gpd1gpd2 double mutant strain of S. cerevisiae, further demonstrating that both of those genes are functional homologues of S. cerevisiae GPD1 and GPD2. Northern blot analysis revealed that SfGPD1 was induced markedly by osmotic stress, while SfGPD2 was not. In consistency with the observation that there was no obvious glycerol content change when the cells were transferred to anoxic conditions, neither SfGPD1 nor SfGPD2 was induced when the cells were transferred to anoxic conditions, thus suggesting a functional splitting of glycerol 3-phosphate dehydrogenase between S. cerevisiae and Sm. fibuligera.     

酿酒酵母关键节点基因缺损对法尼烯合成的影响

以法尼烯为评价效应物,研究了缺损乙醇合成途径、甘油合成途径、胞质乙酰辅酶A转运途径和法尼基焦磷酸消耗支路关键基因对酿酒酵母WHE4菌株合成法尼烯的影响。通过CRISPR-cas9基因编辑技术,获得8株关键基因缺损菌株。结果表明,与WHE4菌株相比,缺损乙醇脱氢酶基因ADH3-6对乙醇和法尼烯产量没有影响;单独缺损甘油三磷酸脱氢酶基因GPD1和GPD2使甘油积累量分别降低了15%和34%,缺损半乳糖激酶基因GAL1、GAL7、GAL10下调了甲羟戊酸途径所有基因转录水平,它们的缺损均不能提高菌株的法尼烯产量;缺损香叶基香叶基焦磷酸合酶基因BTS1和二酰基甘油二磷酸磷酸酶基因DPP1,法尼烯产量提高了29%,在5 L发酵罐补料分批发酵,菌株WHE4-33(WHE4 Δbts1,Δdpp1)的法尼烯产量达到1 578.91 mg/L。该研究对甲羟戊酸途径上游和下游关键节点基因进行了缺损影响法尼烯合成研究,为构建酿酒酵母萜类化合物高效平台提供了参考价值。     

Isolation of a GPD gene from Debaryomyces hansenii encoding a glycerol 3-phosphate dehydrogenase (NAD+)

A gene homologous to GPD1, coding for glycerol-3-phosphate dehydrogenase (sn-glycerol 3-phosphate: NAD(+) oxidoreductase, EC 1.1.1.8), has been isolated from the halophilic yeast Debaryomyces hansenii by complementation of a Saccharomyces cerevisiae gpd1 Delta mutant. DNA sequencing of the complementing genomic clone indicated the existence of an open reading frame encoding a protein with 369 amino acids. Comparative analysis of the deduced amino acid sequence showed high similarity to homologous genes described for other eukaryotic GPD enzymes. The sequence has been submitted to the GenBank database under Accession No. AY333427.     

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.     

Production of glycerol from glucose by coexpressing glycerol-3-phosphate dehydrogenase and glycerol-3-phosphatase in Klebsiella pneumoniae

As a valuable chemical, 1,3-propanediol (1,3-PD) could be biosynthesized by glycerol fermentation. However, no natural microorganisms that could directly convert glucose into 1,3-PD have been found so far. In this work, genes coding for two enzymes, glycerol-3-phosphate dehydrogenase (GPD, EC 1.1.1.8) and glycerol-3-phosphatase (GPP, EC 3.1.3.21), which were responsible for glycerol production, were organized into the plasmid pUC18K under control of the respective lac promoters. Two recombinant proteins were expressed successfully in wild-type Klebsiella pneumoniae. A glycerol concentration of 6.8 g l(-1) was obtained in flask culture. When glucose was exhausted, dihydroxyacetone was added and medium pH was adjusted to 7.0, and then a 1,3-PD concentration of 0.58 g l(-1) was achieved with engineered K. pneumoniae from glucose.     

高糖胁迫下槲皮素对酿酒酵母胞内损伤的保护作用与机制

以酿酒酵母S288c为模型,分析高糖胁迫下槲皮素对其胞内损伤的保护作用及机制。结果表明:与对照相比,高糖胁迫不影响酵母胞内活性氧(reactive oxygen species,ROS)水平,但显著降低了胞内酶比活力(P0.05);槲皮素处理后,与对照组和高糖组相比,酿酒酵母胞内ROS水平、超氧化物歧化酶和过氧化氢酶活力均显著下降(P0.05),而过氧化物酶(peroxidase,POD)比活力极显著升高(P0.01),说明POD比活力对高糖耐受性反应更为灵敏,可作为衡量高糖胁迫应激机制的重要生理指标,槲皮素可通过调节胞内POD比活力来提高机体的抗氧化能力。另外,实时荧光定量聚合酶链式反应结果表明高质量浓度葡萄糖显著抑制了酵母中GPD2和SUC2的表达水平(P0.05),并极显著提高了HXT1的表达水平(P0.01),而对GUT1的表达影响不显著;槲皮素处理后,高糖胁迫下酵母中GPD2、SUC2和HXT1的表达水平显著提高(P0.05),而GUT1无显著变化,说明槲皮素可能通过高渗透甘油途径、菊糖水解途径和己糖转运途径等来促进葡萄糖的分解代谢,从而达到保护机体细胞免受伤害的作用。结果表明槲皮素对高糖诱导的酿酒酵母胞内损伤具有保护作用,其作用机制可能与自身的抗氧化作用以及利用调节机体内高渗透甘油途径与糖的分解和转运途径存在一定的关联性。     

The importance of the glycerol 3-phosphate shuttle during aerobic growth of Saccharomyces cerevisiae

Maintenance of a cytoplasmic redox balance is a necessity for sustained cellular metabolism. Glycerol formation is the only way by which Saccharomyces cerevisiae can maintain this balance under anaerobic conditions. Aerobically, on the other hand, several different redox adjustment mechanisms exist, one of these being the glycerol 3-phosphate (G3P) shuttle. We have studied the importance of this shuttle under aerobic conditions by comparing growth properties and glycerol formation of a wild-type strain with that of gut2Δ mutants, lacking the FAD-dependent glycerol 3-phosphate dehydrogenase, assuming that the consequent blocking of G3P oxidation is forcing the cells to produce glycerol from G3P. To impose different demands on the redox adjustment capability we used various carbon sources having different degrees of reduction. The results showed that the shuttle was used extensively with reduced substrate such as ethanol, whereas the more oxidized substrates lactate and pyruvate, did not provoke any activity of the shuttle. However, the absence of a functional G3P shuttle did not affect the growth rate or growth yield of the cells, not even during growth on ethanol. Presumably, there must be alternative systems for maintaining a cytoplasmic redox balance, e.g. the so-called external NADH dehydrogenase, located on the outer side of the inner mitochondrial membrane. By comparing the performance of the external NADH dehydrogenase and the G3P shuttle in isolated mitochondria, it was found that the former resulted in high respiratory rates but a comparably low P/O ratio of 1·2, whereas the shuttle gave low rates but a high P/O ratio of 1·7. Our results also demonstrated that of the two isoforms of NAD-dependent glycerol 3-phosphate dehydrogenase, only the enzyme encoded by GPD1 appeared important for the shuttle, since the enhanced glycerol production that occurs in a gut2Δ strain proved dependent on GPD1 but not on GPD2. © 1998 John Wiley & Sons, Ltd.     

Decreasing acetic acid accumulation by a glycerol overproducing strain of Saccharomyces cerevisiae by deleting the ALD6 aldehyde dehydrogenase gene

Glycerol is a major fermentation product of Saccharomyces cerevisiae that contributes to the sensory character of wine. Diverting sugar to glycerol overproduction and away from ethanol production by overexpressing the glycerol 3-phosphate dehydrogenase gene,GPD2, caused S. cerevisiae to produce more than twice as much acetic acid as the wild-type strain (S288C background) in anaerobic cell culture. Deletion of the aldehyde dehydrogenase gene, ALD6, in wild-type and GPD2 overexpressing strains (GPD2-OP) decreased acetic acid production by three- and four-fold, respectively. In conjunction with reduced acetic acid production, the GPD2-OP ald6Delta strain produced more glycerol and less ethanol than the wild-type. The growth rate and fermentation rate were similar for the modified and wild-type strains, although the fermentation rate for the GPD2 ald6Delta strain was slightly less than that of the other strains from 24h onwards. Analysis of the metabolome of the mutants revealed that genetic modification affected the production of some secondary metabolites of fermentation, including acids, esters, aldehydes and higher alcohols, many of which are flavour-active in wine. Modification of GPD2 and ALD6 expression represents an effective strategy to increase the glycerol and decrease the ethanol concentration during fermentation, and alters the chemical composition of the medium such that, potentially, novel flavour diversity is possible. The implications for the use of these modifications in commercial wine production require further investigation in wine yeast strains.     

Expression of the GPD1 and GPP2 orthologues and glycerol retention during growth of Debaryomyces hansenii at high NaCl concentrations

The highly NaCl-tolerant yeast Debaryomyces hansenii produces and obtains high levels of intracellular glycerol as a compatible solute when grown at high NaCl concentrations. The effect of high NaCl concentrations (4%, 8% and 12% w/v) on the glycerol production and the levels of intra- and extracellular glycerol was determined for two D. hansenii strains with different NaCl tolerance and compared to one strain of the moderately NaCl-tolerant yeast Saccharomyces cerevisiae. Initially, high NaCl tolerance seems to be determined by enhanced glycerol production, due to an increased expression of DhGPD1 and DhGPP2 (AL436338) in D. hansenii and GPD1 and GPP2 in S. cerevisiae; however, the ability to obtain high levels of intracellular glycerol seems to be more important. The two D. hansenii strains had higher levels of intracellular glycerol than the S. cerevisiae strain and were able to obtain high levels of intracellular glycerol, even at very high NaCl concentrations, indicating the presence of, for example, a type of closing channel, as previously described for other yeast species.     

CRISPR/Cas9介导的酿酒酵母ADH2基因中断及反义RNA干扰GPD1的表达

CRISPR/Cas9是一个简单、高效的用于靶向目的基因和无标记的基因组工程的工具。本文通过构建酿酒酵母沉默组件PGK-SGPD1-CYC1,使甘油-3-磷酸脱氢酶I(Glycerol-3-phosphate dehydrogenase,GPD1)基因在PGK强启动子、CYC1终止子在特定区域内进行干扰和表达。应用CRISPR/Cas9基因编辑技术,在中断乙醇脱氢酶Ⅱ(alcohol dehydrogenase Ⅱ,ADH2)基因的同时,定点敲入GPD1基因的反义干扰组件,从而特定地干扰GPD1的表达。采用高效的酵母化学转化法将反应组件敲入酿酒酵母Y1H中,CRISPR/Cas9介导的同源重组效率达43.48%,由此获得了ADH2基因中断和GPD1反义干扰的酿酒酵母突变株。发酵实验结果表明,酿酒酵母突变菌株SG1-1与出发菌株Y1H相比,乙醇产率提高了9.07%,甘油产率下降了12.05%,乙酸产率下降了12.30%,结果表明通过中断ADH2基因及插入GPD1反义干扰组件,既能够中断ADH2基因的功能,减少乙醇转化为乙醛,同时也能在一定程度上干扰GPD1基因的表达,提高乙醇产率。     

Glycerol formation during wine fermentation is mainly linked to Gpd1p and is only partially controlled by the HOG pathway

Glycerol 3-phosphate dehydrogenase, a key enzyme in the production of glycerol, is encoded by GPD1 and GPD2. The isoforms encoded by these genes have different functions, in osmoregulation and redox balance, respectively. We investigated the roles of GPD1, GPD2 and HOG1-the kinase involved in the response to osmotic stress-in glycerol production during wine fermentation. We found that the deletion of GPD2 in a wine yeast-derived strain did not affect growth or fermentation performance and reduced glycerol production by only 20%. In contrast, a gpd1delta mutant displayed a prolonged lag phase, and produced 40% less glycerol than the wild-type strain. The deletion of HOG1 resulted in a slight decrease in growth rate and a 20% decrease in glycerol production, indicating that the HOG pathway operates under wine fermentation conditions. However, the hog1delta mutant was not as severely affected as the gpd1delta mutant during the first few hours of fermentation, and continued to express GPD1 strongly. The hog1delta mutant was able to increase glycerol production in response to high sugar concentration (15-28% glucose), to almost the same extent as the wild-type, whereas this response was totally abolished in the gpd1delta mutant. These data show that Gpd1p plays a major role in glycerol formation, particularly during the first few hours of exposure to high sugar concentration, and that GPD2 is only of little significance in anaerobic fermentation by wine yeast. The results also demonstrate that the HOG pathway exerts only limited control over GPD1 expression and glycerol production during wine fermentation.     

Reduced pyruvate decarboxylase and increased glycerol-3-phosphate dehydrogenase [NAD+] levels enhance glycerol production in Saccharomyces cerevisiae

This investigation deals with factors affecting the production of glycerol in Saccharomyces cerevisiae. In particular, the impact of reduced pyruvate-decarboxylase (PDC) and increased NAD-dependent glycerol-3-phosphate dehydrogenase (GPD) levels was studied. The glycerol yield was 4·7 times (a pdc mutant exhibiting 19% of normal PDC activity) and 6·5 times (a strain exhibiting 20-fold increased GPD activity resulting from overexpression of GPD1 gene) that of the wild type. In the strain carrying both enzyme activity alterations, the glycerol yield was 8·1 times higher than that of the wild type. In all cases, the substantial increase in glycerol yield was associated with a reduction in ethanol yield and a higher by-product formation. The rate of glycerol formation in the pdc mutant was, due to a slower rate of glucose catabolism, only twice that of the wild type, and was increased by GPD1 overexpression to three times that of the wild-type level. Overexpression of GPD1 in the wild-type background, however, led to a six- to seven-fold increase in the rate of glycerol formation. The experimental work clearly demonstrates the rate-limiting role of GPD in glycerol formation in S. cerevisiae.     

Isolation and characterization of a high-acetate-producing sake yeast Saccharomyces cerevisiae

The result of sensory evaluation of sake showed that acetic acid imparted desirable acidity when the proportion of acetic acid to lactic acid was about 1/3, even if the concentration of acetic acid was 0.75 g/l. Glycerol balanced the acidity and brought about a harmony between sweetness and acidity in sake. A high-acetate producing sake yeast (MHA-3) was isolated from mutants having low NADH dehydrogenase (NDE) activity. MHA-3 produced 15 times more acetate and 5 times more lactate than the parental strain Kyokai no. 901 (K-901) in a small-scale sake brewing test using 10 kg of rice. In addition, the concentrations of glycerol in sake brewed with MHA-3 were approximately 1.5-fold higher than in that brewed with K-901. The proportion of acetic acid to lactic acid was about 1/3 in sake fermented with MHA-3 and it exhibited a good balance between sweetness and acidity. The activities of glycerol-3-phosphate dehydrogenase (GPD) and aldehyde dehydrogenase (ALD) in MHA-3 were 1.4-fold and 3.1-fold, respectively, higher than those in K-901 while the activity of NDE was 40% that of K-901. MHA-3 accumulated higher amounts of acetate and glycerol than K-901 in static YNB10 medium. The concentrations of acetic acid produced, depending on the quantity of yeast cells added, increased in conjunction with increases in glycerol produced. We suggest that NDE might be linked with GPD and that the nde mutants, which can be used in sake brewing, produced higher amounts of acetate and glycerol.     

Cloning,sequencing and characterization of a gene encoding dihydroxyacetone kinase from Zygosaccharomyces rouxii NRRL2547

The dihydroxyacetone pathway, an alternative pathway for the dissimilation of glycerol via reduction by glycerol dehydrogenase and subsequent phosphorylation by dihydroxyacetone (DHA) kinase, is activated in the yeasts Saccharomyces cerevisiae and Zygosaccharomyces rouxii during osmotic stress. In experiments aimed at investigating the physiological function of the DHA pathway in Z. rouxii, a typical osmotolerant yeast, we cloned and characterized a DAK gene encoding dihydroxyacetone kinase from Z. rouxii NRRL 2547. Sequence analysis revealed a 1761 bp open reading frame, encoding a peptide composed of 587 deduced amino acids with the predicted molecular weight of 61 664 Da. As the amino acid sequence was most closely homologous (68% identity) to the S. cerevisiae Dak1p, we named the gene and protein ZrDAK1 and ZrDak1p, respectively. A putative ATP binding site was also found but no consensus element associated with osmoregulation was found in the upstream region of the ZrDAK1 gene. The ZrDAK1 gene complemented a S. cerevisiae W303-1A dak1delta dak2 delta strain by improving the growth of the mutant on 50 mmol/l dihydroxyacetone and by increasing the tolerance to dihydroxyacetone in a medium containing 5% sodium chloride, suggesting that it is a functional homologue of the S. cerevisiae DAK1. However, expression of the ZrDAK1 gene in the S. cerevisiae dak1delta dak2 delta strain had no significant effect on glycerol levels during osmotic stress. The ZrDAK1 sequence has been deposited in the public data bases under Accession No. AJ294719; regions upstream and downstream of ZrDAK1are deposited as Accession Nos AJ294739 and AJ294720, respectively.     

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.     

Expression of a cytoplasmic transhydrogenase in Saccharomyces cerevisiae results in formation of 2-oxoglutarate due to depletion of the NADPH pool

The intracellular redox state of a cell is to a large extent defined by the concentration ratios of the two pyridine nucleotide systems NADH/NAD(+) and NADPH/NADP(+) and has a significant influence on product formation in microorganisms. The enzyme pyridine nucleotide transhydrogenase, which can catalyse transfer of reducing equivalents between the two nucleotide systems, occurs in several organisms, but not in yeasts. The purpose of this work was to analyse how metabolism during anaerobic growth of Saccharomyces cerevisiae might be altered when transfer of reducing equivalents between the two systems is made possible by expression of a cytoplasmic transhydrogenase from Azotobacter vinelandii. We therefore cloned sth, encoding this enzyme, and expressed it under the control of a S. cerevisiae promoter in a strain derived from the industrial model strain S. cerevisiae CBS8066. Anaerobic batch cultivations in high-performance bioreactors were carried out in order to allow quantitative analysis of the effect of transhydrogenase expression on product formation and on the intracellular concentrations of NADH, NAD(+), NADPH and NADP(+). A specific transhydrogenase activity of 4.53 U/mg protein was measured in the extracts from the strain expressing the sth gene from A. vinelandii, while no transhydrogenase activity could be detected in control strains without the gene. Production of the transhydrogenase caused a significant increase in formation of glycerol and 2-oxoglutarate. Since NADPH is used to convert 2-oxoglutarate to glutamate while glycerol formation increases when excess NADH is formed, this suggested that transhydrogenase converted NADH and NADP(+) to NAD(+) and NADPH. This was further supported by measurements of the intracellular nucleotide concentrations. Thus, the (NADPH/NADP(+)):(NADH/NAD(+)) ratio was reduced from 35 to 17 by the transhydrogenase. The increased formation of 2-oxoglutarate was accompanied by a two-fold decrease in the maximal specific growth rate. Also the biomass and ethanol yields were significantly lowered by the transhydrogenase.