Reduced Oxidative Pentose Phosphate Pathway Flux in Recombinant Xylose-Utilizing Saccharomyces cerevisiae Strains Improves the Ethanol Yield from Xylose

In recombinant, xylose-fermenting Saccharomyces cerevisiae, about 30% of the consumed xylose is converted to xylitol. Xylitol production results from a cofactor imbalance, since xylose reductase uses both NADPH and NADH, while xylitol dehydrogenase uses only NAD⁺. In this study we increased the ethanol yield and decreased the xylitol yield by lowering the flux through the NADPH-producing pentose phosphate pathway. The pentose phosphate pathway was blocked either by disruption of the GND1 gene, one of the isogenes of 6-phosphogluconate dehydrogenase, or by disruption of the ZWF1 gene, which encodes glucose 6-phosphate dehydrogenase. Decreasing the phosphoglucose isomerase activity by 90% also lowered the pentose phosphate pathway flux. These modifications all resulted in lower xylitol yield and higher ethanol yield than in the control strains. TMB3255, carrying a disruption of ZWF1, gave the highest ethanol yield (0.41 g g⁻¹) and the lowest xylitol yield (0.05 g g⁻¹) reported for a xylose-fermenting recombinant S. cerevisiae strain, but also an 84% lower xylose consumption rate. The low xylose fermentation rate is probably due to limited NADPH-mediated xylose reduction. Metabolic flux modeling of TMB3255 confirmed that the NADPH-producing pentose phosphate pathway was blocked and that xylose reduction was mediated only by NADH, leading to a lower rate of xylose consumption. These results indicate that xylitol production is strongly connected to the flux through the oxidative part of the pentose phosphate pathway.     

Comparing the xylose reductase/xylitol dehydrogenase and xylose isomerase pathways in arabinose and xylose fermenting Saccharomyces cerevisiae strains

Background

Ethanolic fermentation of lignocellulosic biomass is a sustainable option for the production of bioethanol. This process would greatly benefit from recombinant Saccharomyces cerevisiae strains also able to ferment, besides the hexose sugar fraction, the pentose sugars, arabinose and xylose. Different pathways can be introduced in S. cerevisiae to provide arabinose and xylose utilisation. In this study, the bacterial arabinose isomerase pathway was combined with two different xylose utilisation pathways: the xylose reductase/xylitol dehydrogenase and xylose isomerase pathways, respectively, in genetically identical strains. The strains were compared with respect to aerobic growth in arabinose and xylose batch culture and in anaerobic batch fermentation of a mixture of glucose, arabinose and xylose.

Results

The specific aerobic arabinose growth rate was identical, 0.03 h^-1, for the xylose reductase/xylitol dehydrogenase and xylose isomerase strain. The xylose reductase/xylitol dehydrogenase strain displayed higher aerobic growth rate on xylose, 0.14 h^-1, and higher specific xylose consumption rate in anaerobic batch fermentation, 0.09 g (g cells)^-1 h^-1 than the xylose isomerase strain, which only reached 0.03 h^-1 and 0.02 g (g cells)^-1h^-1, respectively. Whereas the xylose reductase/xylitol dehydrogenase strain produced higher ethanol yield on total sugars, 0.23 g g^-1 compared with 0.18 g g^-1 for the xylose isomerase strain, the xylose isomerase strain achieved higher ethanol yield on consumed sugars, 0.41 g g^-1 compared with 0.32 g g^-1 for the xylose reductase/xylitol dehydrogenase strain. Anaerobic fermentation of a mixture of glucose, arabinose and xylose resulted in higher final ethanol concentration, 14.7 g l^-1 for the xylose reductase/xylitol dehydrogenase strain compared with 11.8 g l^-1 for the xylose isomerase strain, and in higher specific ethanol productivity, 0.024 g (g cells)^-1 h^-1 compared with 0.01 g (g cells)^-1 h^-1 for the xylose reductase/xylitol dehydrogenase strain and the xylose isomerase strain, respectively.

Conclusion

The combination of the xylose reductase/xylitol dehydrogenase pathway and the bacterial arabinose isomerase pathway resulted in both higher pentose sugar uptake and higher overall ethanol production than the combination of the xylose isomerase pathway and the bacterial arabinose isomerase pathway. Moreover, the flux through the bacterial arabinose pathway did not increase when combined with the xylose isomerase pathway. This suggests that the low activity of the bacterial arabinose pathway cannot be ascribed to arabitol formation via the xylose reductase enzyme.     

Implementation of a transhydrogenase-like shunt to counter redox imbalance during xylose fermentation in Saccharomyces cerevisiae

Three enzymes responsible for the transhydrogenase-like shunt, including malic enzyme (encoded by MAE1), malate dehydrogenase (MDH2), and pyruvate carboxylase (PYC2), were overexpressed to regulate the redox state in xylose-fermenting recombinant Saccharomyces cerevisiae. The YPH499XU/MAE1 strain was constructed by overexpressing native Mae1p in the YPH499XU strain expressing xylose reductase and xylitol dehydrogenase from Scheffersomyces stipitis, and native xylulokinase. Analysis of the xylose fermentation profile under semi-anaerobic conditions revealed that the ethanol yield in the YPH499XU/MAE1 strain (0.38?±?0.01 g g^?1 xylose consumed) was improved from that of the control strain (0.31?±?0.01 g g^?1 xylose consumed). Reduced xylitol production was also observed in YPH499XU/MAE1, suggesting that the redox balance was altered by Mae1p overexpression. Analysis of intracellular metabolites showed that the redox imbalance during xylose fermentation was partly relieved in the transformant. The specific ethanol production rate in the YPH499XU/MAE1–MDH2 strain was 1.25-fold higher than that of YPH499XU/MAE1 due to the additional overexpression of Mdh2p, whereas the ethanol yield was identical to that of YPH499XU/MAE1. The specific xylose consumption rate was drastically increased in the YPH499XU/MAE1–MDH2–PYC2 strain. However, poor ethanol yield as well as increased production of xylitol was observed. These results demonstrate that the transhydrogenase function implemented in S. cerevisiae can regulate the redox state of yeast cells.     

Direct evidence for a xylose metabolic pathway in Saccharomyces cerevisiae

Xylose transport, xylose reductase, and xylitol dehydrogenase activities are demonstrated in Saccharomyces cerevisiae. The enzymes in the xylose catabolic pathway necessary for the conversion of xylose to xylulose are present, although S. cerevisiae cannot grow on xylose as a sole carbon source. Xylose transport is less efficient than glucose transport, and its rate is dependent upon aeration. Xylose reductase appears to be a xylose inducible enzyme and xylitol dehydrogenase activity is constitutive, although both are repressed by glucose. Both xylose reductase and xylitol dehydrogenase activities are five- to tenfold lower in S. cerevisiae as compared to Candida utilis. In vivo conversion of (14)C-xylose in S. cerevisiae is demonstrated and xylitol is detected, although no significant levels of any other (14)C-labeled metabolites (e. g., ethanol) are observed.     

Intermediary Metabolite Concentrations in Xylulose- and Glucose-Fermenting Saccharomyces cerevisiae Cells

Glucose and xylulose fermentation and product formation by Saccharomyces cerevisiae were compared in batch culture under anaerobic conditions. In both cases the main product was ethanol, with glycerol, xylitol, and arabitol produced as by-products. During glucose and xylulose fermentation, 0.74 and 0.37 g of cell mass liter⁻¹, respectively, were formed. In glucose-fermenting cells, the carbon balance could be closed, whereas in xylulose-fermenting cells, about 25% of the consumed sugar carbon could not be accounted for. The rate of sugar consumption was 3.94 mmol g of initial biomass⁻¹ h⁻¹ for glucose and 0.39 mmol g of initial biomass⁻¹ h⁻¹ for xylulose. Concentrations of the intermediary metabolites fructose-1,6-diphosphate (FDP), pyruvate (PYR), sedoheptulose 7-phosphate (S7P), erytrose 4-phosphate, citrate (CIT), fumarate, and malate were compared for both types of cells. Levels of FDP, PYR, and CIT were lower, and levels of S7P were higher in xylulose-fermenting cells. After normalization to the carbon consumption rate, the levels of FDP were approximately the same, whereas there was a significant accumulation of S7P, PYR, CIT, and malate, especially of S7P, in xylulose-fermenting cells compared with in glucose-fermenting cells. In the presence of 15 μM iodoacetate, an inhibitor of the enzyme glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12), FDP levels increased and S7P levels decreased in xylulose-assimilating cells compared with in the absence of the inhibitor, whereas fermentation was slightly slowed down. The specific activity of transaldolase (EC 2.2.1.2), the pentose phosphate pathway enzyme reacting with S7P and glyceraldehyde-3-phosphate, was essentially the same for both glucose- and xylulose-fermenting cells. It was, however, several orders of magnitude lower than that reported for a Torula yeast and Candida utilis. The presence of iodoacetate did not influence the activity of transaldolase in xylulose-fermenting cells. The results are discussed in terms of a competition between the pentose phosphate pathway and glycolysis for the common metabolite, glyceraldehyde-3-phosphate, which would explain the low rates of xylulose assimilation and ethanol production from xylulose by S. cerevisiae.     

Fed-batch fermentation of xylose by a fast-growing mutant of xylose-assimilating recombinant Saccharomyces cerevisiae

Mutants of xylose-assimilating recombinant Saccharomyces cerevisiae carrying the xylose reductase and xylitol dehydrogenase genes on plasmid pEXGD8 were selected, after ethyl methanesulfonate treatment, for their rapid growth on xylose medium. The fastest growing strain (strain IM2) showed a lower activity of xylose reductase but a higher ratio of xylitol dehydrogenase to xylose reductase activities than the parent strain, as well as high xylulokinase activity. Southern hybridization of the chromosomal DNA indicated that plasmid pEXGD8 was integrated into the chromosome of mutant IM2, resulting in an increase in the stability of the cloned genes. In batch fermentation under O₂ limitation, the yield and production rate of ethanol were improved 1.6 and 2.7 times, respectively, compared to the parent strain. In fed-batch culture with slow feeding of xylose and appropriate O₂ supply at a low level, xylitol excreted from the cells was limited and the ethanol yield increased 1.5 times over that in the batch culture, with a high initial concentration of xylose, although the production rate was reduced. The results suggested that slow conversion of xylose to xylitol led to a lower level of intracellular xylitol, resulting in less excretion of xylitol, and an increase in the ethanol yield. It was also observed that the oxidation of xylitol was strongly affected by the O₂ supply.Correspondence to: T. Yoshida     

Conversion of xylose to ethanol by recombinant Saccharomyces cerevisiae: importance of xylulokinase (XKS1) and oxygen availability.

The yeast Saccharomyces cerevisiae efficiently ferments hexose sugars to ethanol, but it is unable to utilize xylose, a pentose sugar abundant in lignocellulosic materials. Recombinant strains containing genes coding for xylose reductase (XR) and xylitol dehydrogenase (XDH) from the xylose-utilizing yeast Pichia stipitis have been reported; however, such strains ferment xylose to ethanol poorly. One reason for this may be the low capacity of xylulokinase, the third enzyme in the xylose pathway. To investigate the potential limitation of the xylulokinase step, we have overexpressed the endogenous gene for this enzyme (XKS1) in S. cerevisiae that also expresses the P. stipitis genes for XR and XDH. The metabolism of this recombinant yeast was further investigated in pure xylose bioreactor cultivation at various oxygen levels. The results clearly indicated that overexpression of XKS1 significantly enhances the specific rate of xylose utilization. In addition, the XK-overexpressing strain can more efficiently convert xylose to ethanol under all aeration conditions studied. One of the important illustrations is the significant anaerobic and aerobic xylose conversion to ethanol by the recombinant Saccharomyces; moreover, this was achieved on pure xylose as a carbon. Under microaerobic conditions, 5.4 g L(-1) ethanol was produced from 47 g L(-1) xylose during 100 h. In fed-batch cultivations using a mixture of xylose and glucose as carbon sources, the specific ethanol production rate was highest at the highest aeration rate tested and declined by almost one order of magnitude at lower aeration levels. Intracellular metabolite analyses and in vitro enzyme activities suggest the following: the control of flux in a strain that overexpresses XKS1 has shifted to the nonoxidative steps of the pentose phosphate pathway (i.e., downstream of xylose 5-phosphate), and enzymatic steps in the lower part of glycolysis and ethanol formation pathways (pyruvate kinase, pyruvate decarboxylase, and alcohol dehydrogenase) do not have a high flux control in this recombinant strain. Furthermore, the intracellular ATP levels were found to be significantly lower for the XK strain compared with either the control strain under similar conditions or glucose-grown Saccharomyces. The ATP : ADP ratios were also lower for the XK strain, especially under microaerobic conditions (0.9 vs 6.4).     

Reduced oxidative pentose phosphate pathway flux in recombinant xylose-utilizing Saccharomyces cerevisiae strains improves the ethanol yield from xylose

In recombinant, xylose-fermenting Saccharomyces cerevisiae, about 30% of the consumed xylose is converted to xylitol. Xylitol production results from a cofactor imbalance, since xylose reductase uses both NADPH and NADH, while xylitol dehydrogenase uses only NAD(+). In this study we increased the ethanol yield and decreased the xylitol yield by lowering the flux through the NADPH-producing pentose phosphate pathway. The pentose phosphate pathway was blocked either by disruption of the GND1 gene, one of the isogenes of 6-phosphogluconate dehydrogenase, or by disruption of the ZWF1 gene, which encodes glucose 6-phosphate dehydrogenase. Decreasing the phosphoglucose isomerase activity by 90% also lowered the pentose phosphate pathway flux. These modifications all resulted in lower xylitol yield and higher ethanol yield than in the control strains. TMB3255, carrying a disruption of ZWF1, gave the highest ethanol yield (0.41 g g(-1)) and the lowest xylitol yield (0.05 g g(-1)) reported for a xylose-fermenting recombinant S. cerevisiae strain, but also an 84% lower xylose consumption rate. The low xylose fermentation rate is probably due to limited NADPH-mediated xylose reduction. Metabolic flux modeling of TMB3255 confirmed that the NADPH-producing pentose phosphate pathway was blocked and that xylose reduction was mediated only by NADH, leading to a lower rate of xylose consumption. These results indicate that xylitol production is strongly connected to the flux through the oxidative part of the pentose phosphate pathway.     

The non-oxidative pentose phosphate pathway controls the fermentation rate of xylulose but not of xylose in Saccharomyces cerevisiae TMB3001

Saccharomyces cerevisiae is able to ferment xylose, when engineered with the enzymes xylose reductase (XYL1) and xylitol dehydrogenase (XYL2). However, xylose fermentation is one to two orders of magnitude slower than glucose fermentation. S. cerevisiae has been proposed to have an insufficient capacity of the non-oxidative pentose phosphate pathway (PPP) for rapid xylose fermentation. Strains overproducing the non-oxidative PPP enzymes ribulose 5-phosphate epimerase (EC 5.1.3.1), ribose 5-phosphate ketol isomerase (EC 5.3.1.6), transaldolase (EC 2.2.1.2) and transketolase (EC 2.2.1.1), as well as all four enzymes simultaneously, were compared with respect to xylose and xylulose fermentation with their xylose-fermenting predecessor S. cerevisiae TMB3001, expressing XYL1, XYL2 and only overexpressing XKS1 (xylulokinase). The level of overproduction in S. cerevisiae TMB3026, overproducing all four non-oxidative PPP enzymes, ranged between 4 and 23 times the level in TMB3001. Overproduction of the non-oxidative PPP enzymes did not influence the xylose fermentation rate in either batch cultures of 50 g l(-1) xylose or chemostat cultures of 20 g l(-1) glucose and 20 g l(-1) xylose. The low specific growth rate on xylose was also unaffected. The results suggest that neither of the non-oxidative PPP enzymes has any significant control of the xylose fermentation rate in S. cerevisiae TMB3001. However, the specific growth rate on xylulose increased from 0.02-0.03 for TMB3001 to 0.12 for the strain overproducing only transaldolase (TAL1) and to 0.23 for TMB3026, suggesting that overproducing all four enzymes has a synergistic effect. TMB3026 consumed xylulose about two times faster than TMB30001 in batch culture of 50 g l(-1) xylulose. The results indicate that growth on xylulose and the xylulose fermentation rate are partly controlled by the non-oxidative PPP, whereas control of the xylose fermentation rate is situated upstream of xylulokinase, in xylose transport, in xylose reductase, and/or in the xylitol dehydrogenase.     

Oxygen-dependent xylitol metabolism in Pichia stipitis

Pichia stipitis CBS 6054 was cultivated in chemostat cultures under aerobic and oxygen-limited conditions with xylitol alone, a mixture of xylitol and glucose and a mixture of xylitol and xylose. Xylitol metabolism was strictly respiratory and no ethanol was formed. Simultaneous feeding of xylitol and glucose and xylitol and xylose to oxygen-limited xylitol-pregrown cells resulted in ethanol formation. In vitro both pyruvate decarboxylase activity and alcohol dehydrogenase activity were present in cells metabolising xylitol under oxygen-limited conditions; however, this did not result in ethanol formation. Glucose, xylose and xylitol utilisation, respectively, were compared under anaerobic conditions with regard to growth rate, carbon source and oxygenation level during pre-cultivation. Irrespective of pre-growth conditions, xylitol was not metabolised under anaerobic conditions, whereas ethanol was formed from both xylose and glucose. Anaerobic xylose utilisation required induction of a xylose-utilising metabolic pathway during pre-cultivation. Received: 23 February 1999 / Received last revision: 20 July 1999 / Accepted: 1 August 1999     

Characterization of Bioethanol Production from Hexoses and Xylose by the White Rot Fungus Trametes versicolor

Bioethanol production by white rot fungus (Trametes versicolor), identified from fungal mixture in naturally decomposing wood samples, from hexoses and xylose was characterized. Results showed that T. versicolor can grow in culture, under hypoxic conditions, with various mixtures of hexoses and xylose and only xylose. Xylose was efficiently fermented to ethanol in media containing mixtures of hexoses and xylose, such as MBMC and G11XY11 media (Table?1), yielding ethanol concentrations of 20.0 and 9.02?g/l, respectively, after 354?h of hypoxic culture. Very strong correlations were found between ethanolic fermentation (alcohol dehydrogenase activity and ethanol production), sugar consumption and xylose catabolism (xylose reductase, xylitol dehydrogenase and xylulokinase activities) after 354?h in culture in MBMC medium. In a medium (G11XY11) containing a 1:1 glucose/xylose ratio, fermentation efficiency of total sugars into ethanol was 80% after 354?h.     

Anaerobic Xylose Fermentation by Recombinant Saccharomyces cerevisiae Carrying XYL1, XYL2, and XKS1 in Mineral Medium Chemostat Cultures

For ethanol production from lignocellulose, the fermentation of xylose is an economic necessity. Saccharomyces cerevisiae has been metabolically engineered with a xylose-utilizing pathway. However, the high ethanol yield and productivity seen with glucose have not yet been achieved. To quantitatively analyze metabolic fluxes in recombinant S. cerevisiae during metabolism of xylose-glucose mixtures, we constructed a stable xylose-utilizing recombinant strain, TMB 3001. The XYL1 and XYL2 genes from Pichia stipitis, encoding xylose reductase (XR) and xylitol dehydrogenase (XDH), respectively, and the endogenous XKS1 gene, encoding xylulokinase (XK), under control of the PGK1 promoter were integrated into the chromosomal HIS3 locus of S. cerevisiae CEN.PK 113-7A. The strain expressed XR, XDH, and XK activities of 0.4 to 0.5, 2.7 to 3.4, and 1.5 to 1.7 U/mg, respectively, and was stable for more than 40 generations in continuous fermentations. Anaerobic ethanol formation from xylose by recombinant S. cerevisiae was demonstrated for the first time. However, the strain grew on xylose only in the presence of oxygen. Ethanol yields of 0.45 to 0.50 mmol of C/mmol of C (0.35 to 0.38 g/g) and productivities of 9.7 to 13.2 mmol of C h⁻¹ g (dry weight) of cells⁻¹ (0.24 to 0.30 g h⁻¹ g [dry weight] of cells⁻¹) were obtained from xylose-glucose mixtures in anaerobic chemostat cultures, with a dilution rate of 0.06 h⁻¹. The anaerobic ethanol yield on xylose was estimated at 0.27 mol of C/(mol of C of xylose) (0.21 g/g), assuming a constant ethanol yield on glucose. The xylose uptake rate increased with increasing xylose concentration in the feed, from 3.3 mmol of C h⁻¹ g (dry weight) of cells⁻¹ when the xylose-to-glucose ratio in the feed was 1:3 to 6.8 mmol of C h⁻¹ g (dry weight) of cells⁻¹ when the feed ratio was 3:1. With a feed content of 15 g of xylose/liter and 5 g of glucose/liter, the xylose flux was 2.2 times lower than the glucose flux, indicating that transport limits the xylose flux.     

Dynamic control over feedback regulatory mechanisms improves NADPH flux and xylitol biosynthesis in engineered E. coli

We report improved NADPH flux and xylitol biosynthesis in engineered E. coli. Xylitol is produced from xylose via an NADPH dependent reductase. We utilize 2-stage dynamic metabolic control to compare two approaches to optimize xylitol biosynthesis, a stoichiometric approach, wherein competitive fluxes are decreased, and a regulatory approach wherein the levels of key regulatory metabolites are reduced. The stoichiometric and regulatory approaches lead to a 20-fold and 90-fold improvement in xylitol production, respectively. Strains with reduced levels of enoyl-ACP reductase and glucose-6-phosphate dehydrogenase, led to altered metabolite pools resulting in the activation of the membrane bound transhydrogenase and an NADPH generation pathway, consisting of pyruvate ferredoxin oxidoreductase coupled with NADPH dependent ferredoxin reductase, leading to increased NADPH fluxes, despite a reduction in NADPH pools. These strains produced titers of 200 g/L of xylitol from xylose at 86% of theoretical yield in instrumented bioreactors. We expect dynamic control over the regulation of the membrane bound transhydrogenase as well as NADPH production through pyruvate ferredoxin oxidoreductase to broadly enable improved NADPH dependent bioconversions or production via NADPH dependent metabolic pathways.     

Acetone stimulation of ethanol production from d-xylose by Pachysolen tannophilus

Summary Pachysolen tannophilus, a homothallic yeast, converts xylose to ethanol at a yield of 0.3 (g/g xylose). Concomitant with ethanol production, xylitol accumulates in the culture medium at similar yields (0.3 g/g xylose). The addition of the hydrogen-accepting compound, acetone, increases the amount of ethanol produced by this organism by 50–70%. The increase in ethanol is directly correlated with a decrease in xylitol secreted. The results indicate that conversion of acetone to 2-propanol by the cells provides the NAD⁺ used as a cofactor by xylitol dehydrogenase, the enzyme responsible for converting xylitol to xylulose.The mention of firm names or trade products does not imply that they are endorsed or recommended by the U. S. Department of Agriculture over other firms or similar products not mentioned.     

一种基因工程改造的NADH高亲和力木糖还原酶突变基因可促进酿酒酵母发酵木糖生成乙醇

在导入表达毕赤酵母(Pichia stipitis)木糖还原酶(xylose reductase,XR)和木糖醇脱氢酶(xylitol dehydrogenase,XDH)基因的重组酿酒酵母中,木糖还原酶活性主要依赖辅酶NADPH,木糖醇脱氢酶活性依赖辅酶 NAD+,两者的辅助因子不同导致细胞内电子氧化还原的不平衡,是造成木糖醇积累,影响木糖代谢和乙醇产量的主要原因之一.将经过基因工程改造获得的NADH高亲和力的木糖还原酶突变基因m1,与毕赤酵母木糖醇脱氢酶(PsXDH)基因xyl2共转染酿酒酵母AH109,以转染毕赤酵母木糖还原酶(PsXR)基因xyl1和xyl2重组质粒的酵母细胞为对照菌株,在SC/-Leu/-Trp营养缺陷型培养基中进行筛选,获得的阳性转化子分别命名为AH-M-XDH和AH-XR-XDH.重组酵母在限制氧通气条件下对木糖和葡萄糖进行共发酵摇瓶培养,HPLC检测发酵底物的消耗和代谢产物的产出情况.结果显示,与对照菌株AH-XR-XDH相比,AH-M-XDH的木糖利用率明显提高,乙醇得率增加了16%,木糖醇产生下降了41.4%.结果证实,通过基因工程改造的木糖代谢关键酶,可用于酿酒酵母发酵木糖生产乙醇,其能通过改善酿酒酵母细胞内氧化还原失衡的问题,提高木糖利用率和乙醇产率.     

Xylose fermentation by Saccharomyces cerevisiae

We have performed a comparative study of xylose utilization in Saccharomyces cerevisiae transformants expressing two key enzymes in xylose metabolism, xylose reductase (XR) and xylitol dehydrogenase (XDH), and in a prototypic xylose-utilizing yeast, Pichia stipitis. In the absence of respiration (see text), baker's yeast cells convert half of the xylose to xylitol and ethanol, whereas P. stipilis cells display rather a homofermentative conversion of xylose to ethanol. Xylitol production by baker's yeast is interpreted as a result of the dual cofactor dependence of the XR and the generation of NADPH by the pentose phosphate pathway. Further limitations of xylose utilization in S. cerevisiae cells are very likely caused by an insufficient capacity of the non-oxidative pentose phosphate pathway, as indicated by accumulation of sedoheptulose-7-phosphate and the absence of fructose-1,6-bisphosphate and pyruvate accumulation. By contrast, uptake at high substrate concentrations probably does not limit xylose conversion in S. cerevisiae XYL1/XYL2 transformants. Correspondence to: M. Ciriacy     

The Activity of Key Enzymes in Xylose-Assimilating Yeasts at Different Rates of Oxygen Transfer to the Fermentation Medium

The activities of xylitol dehydrogenase and xylose reductase in the yeasts Candida shehatae, C. didensiae, C. intermediae, C. tropicalis, Kluyveromyces marxianus, Pichia stipitis, P. guillermondii, Pachysolen tannophilus, and Torulopsis molishiama were studied at different oxygen transfer rates (OTRs) to the fermentation medium (0, 5, and 140 mmol O₂/(l h)). The activities of these enzymes were maximum in the yeasts P. stipitis and C. shehatae. The xylitol dehydrogenase of all the yeasts was NAD⁺-dependent, irrespective of the intensity of aeration. The xylose reductase of the yeasts C. didensiae, C. intermediae, C. tropicalis, Kl. marxianus, P. guillermondii, and T. molishiama was NADPH-dependent, whereas the xylose reductase of P. stipitis, C. shehatae, and Pa. tannophilus was specific for both NADPH and NADH. The effect of OTR on the activities of the different forms of xylitol dehydrogenase and xylose reductase in xylose-assimilating yeasts is discussed.     

Effect of oxygen transfer rate on levels of key enzymes of xylose metabolism in Debaryomyces hansenii

The titers of key enzymes of xylose metabolism were measured and correlated with the kinetics of xylitol production by Debaryomyces hansenii under different oxygen transfer rates (OTR) in a batch reactor. An OTR change from 2.72 to 4.22 mmol O₂ l⁻¹ min⁻¹ resulted in a decrease in NADPH-dependent xylose reductase (XR) and NAD ± -dependent xylitol dehydrogenase (XDH) activities. For higher values of OTR (12.93 mmol O₂ l⁻¹ min⁻¹, the XDH titer increased twofold whereas the XR titer did not show a significant change. At the lowest OTR (2.72 mmol O₂ l⁻¹ min⁻¹), xylitol (and ethanol) production rates showed the highest values. However, xylitol specific productivity was twice as high as ethanol specific productivity. The titer of the NADPH-forming enzyme, glucose-6-phosphate dehydrogenase (GPDH), increased from 333 to 412 mU mg⁻¹ when the OTR was increased. However, 6-phosphogluconate dehydrogenase (PGDH) activity remained unchanged and at a lower level, which indicates that this enzyme is responsible for the carbon flux control of the oxidative branch of the pentose phosphate pathway. The activity of the alcohol-forming enzyme was repressed at the higher amount of oxygen, decreasing its activity more than 50%. The changes in ADH suggested that two different metabolic regions under oxygen-limited conditions can be hypothesized for xylose metabolism by D. hansenii. For low OTR values (up to 4.22 mmol O₂ l⁻¹ min⁻¹), a fermentative-type activity is displayed. At higher OTR values (above 4.22 mmol O₂ l⁻¹ min⁻¹), no significant fermentative activity is reported.     

Effects of NADH-preferring xylose reductase expression on ethanol production from xylose in xylose-metabolizing recombinant Saccharomyces cerevisiae

Efficient conversion of xylose to ethanol is an essential factor for commercialization of lignocellulosic ethanol. To minimize production of xylitol, a major by-product in xylose metabolism and concomitantly improve ethanol production, Saccharomyces cerevisiae D452-2 was engineered to overexpress NADH-preferable xylose reductase mutant (XR(MUT)) and NAD?-dependent xylitol dehydrogenase (XDH) from Pichia stipitis and endogenous xylulokinase (XK). In vitro enzyme assay confirmed the functional expression of XR(MUT), XDH and XK in recombinant S. cerevisiae strains. The change of wild type XR to XR(MUT) along with XK overexpression led to reduction of xylitol accumulation in microaerobic culture. More modulation of the xylose metabolism including overexpression of XR(MUT) and transaldolase, and disruption of the chromosomal ALD6 gene encoding aldehyde dehydrogenase (SX6(MUT)) improved the performance of ethanol production from xylose remarkably. Finally, oxygen-limited fermentation of S. cerevisiae SX6(MUT) resulted in 0.64 g l?1 h?1 xylose consumption rate, 0.25 g l?1 h?1 ethanol productivity and 39% ethanol yield based on the xylose consumed, which were 1.8, 4.2 and 2.2 times higher than the corresponding values of recombinant S. cerevisiae expressing XR(MUT), XDH and XK only.     

Minimal metabolic engineering of Saccharomyces cerevisiae for efficient anaerobic xylose fermentation: a proof of principle

When xylose metabolism in yeasts proceeds exclusively via NADPH-specific xylose reductase and NAD-specific xylitol dehydrogenase, anaerobic conversion of the pentose to ethanol is intrinsically impossible. When xylose reductase has a dual specificity for both NADPH and NADH, anaerobic alcoholic fermentation is feasible but requires the formation of large amounts of polyols (e.g., xylitol) to maintain a closed redox balance. As a result, the ethanol yield on xylose will be sub-optimal. This paper demonstrates that anaerobic conversion of xylose to ethanol, without substantial by-product formation, is possible in Saccharomyces cerevisiae when a heterologous xylose isomerase (EC 5.3.1.5) is functionally expressed. Transformants expressing the XylA gene from the anaerobic fungus Piromyces sp. E2 (ATCC 76762) grew in synthetic medium in shake-flask cultures on xylose with a specific growth rate of 0.005 h(-1). After prolonged cultivation on xylose, a mutant strain was obtained that grew aerobically and anaerobically on xylose, at specific growth rates of 0.18 and 0.03 h(-1), respectively. The anaerobic ethanol yield was 0.42 g ethanol x g xylose(-1) and also by-product formation was comparable to that of glucose-grown anaerobic cultures. These results illustrate that only minimal genetic engineering is required to recruit a functional xylose metabolic pathway in Saccharomyces cerevisiae. Activities and/or regulatory properties of native S. cerevisiae gene products can subsequently be optimised via evolutionary engineering. These results provide a gateway towards commercially viable ethanol production from xylose with S. cerevisiae.