Deletion of the GRE3 aldose reductase gene and its influence on xylose metabolism in recombinant strains of Saccharomyces cerevisiae expressing the xylA and XKS1 genes.

Saccharomyces cerevisiae ferments hexoses efficiently but is unable to ferment xylose. When the bacterial enzyme xylose isomerase (XI) from Thermus thermophilus was produced in S. cerevisiae, xylose utilization and ethanol formation were demonstrated. In addition, xylitol and acetate were formed. An unspecific aldose reductase (AR) capable of reducing xylose to xylitol has been identified in S. cerevisiae. The GRE3 gene, encoding the AR enzyme, was deleted in S. cerevisiae CEN.PK2-1C, yielding YUSM1009a. XI from T. thermophilus was produced, and endogenous xylulokinase from S. cerevisiae was overproduced in S. cerevisiae CEN.PK2-1C and YUSM1009a. In recombinant strains from which the GRE3 gene was deleted, xylitol formation decreased twofold. Deletion of the GRE3 gene combined with expression of the xylA gene from T. thermophilus on a replicative plasmid generated recombinant xylose utilizing S. cerevisiae strain TMB3102, which produced ethanol from xylose with a yield of 0.28 mmol of C from ethanol/mmol of C from xylose. None of the recombinant strains grew on xylose.     

Metabolic engineering of a xylose-isomerase-expressing Saccharomyces cerevisiae strain for rapid anaerobic xylose fermentation

After an extensive selection procedure, Saccharomyces cerevisiae strains that express the xylose isomerase gene from the fungus Piromyces sp. E2 can grow anaerobically on xylose with a mu(max) of 0.03 h(-1). In order to investigate whether reactions downstream of the isomerase control the rate of xylose consumption, we overexpressed structural genes for all enzymes involved in the conversion of xylulose to glycolytic intermediates, in a xylose-isomerase-expressing S. cerevisiae strain. The overexpressed enzymes were xylulokinase (EC 2.7.1.17), ribulose 5-phosphate isomerase (EC 5.3.1.6), ribulose 5-phosphate epimerase (EC 5.3.1.1), transketolase (EC 2.2.1.1) and transaldolase (EC 2.2.1.2). In addition, the GRE3 gene encoding aldose reductase was deleted to further minimise xylitol production. Surprisingly the resulting strain grew anaerobically on xylose in synthetic media with a mu(max) as high as 0.09 h(-1) without any non-defined mutagenesis or selection. During growth on xylose, xylulose formation was absent and xylitol production was negligible. The specific xylose consumption rate in anaerobic xylose cultures was 1.1 g xylose (g biomass)(-1) h(-1). Mixtures of glucose and xylose were sequentially but completely consumed by anaerobic batch cultures, with glucose as the preferred substrate.     

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.     

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.     

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.     

High-level functional expression of a fungal xylose isomerase: the key to efficient ethanolic fermentation of xylose by Saccharomyces cerevisiae?

Evidence is presented that xylose metabolism in the anaerobic cellulolytic fungus Piromyces sp. E2 proceeds via a xylose isomerase rather than via the xylose reductase/xylitol-dehydrogenase pathway found in xylose-metabolising yeasts. The XylA gene encoding the Piromyces xylose isomerase was functionally expressed in Saccharomyces cerevisiae. Heterologous isomerase activities in cell extracts, assayed at 30 degrees C, were 0.3-1.1 micromol min(-1) (mg protein)(-1), with a Km for xylose of 20 mM. The engineered S. cerevisiae strain grew very slowly on xylose. It co-consumed xylose in aerobic and anaerobic glucose-limited chemostat cultures at rates of 0.33 and 0.73 mmol (g biomass)(-1) h(-1), respectively.     

Metabolic engineering of a phosphoketolase pathway for pentose catabolism in Saccharomyces cerevisiae

Low ethanol yields on xylose hamper economically viable ethanol production from hemicellulose-rich plant material with Saccharomyces cerevisiae. A major obstacle is the limited capacity of yeast for anaerobic reoxidation of NADH. Net reoxidation of NADH could potentially be achieved by channeling carbon fluxes through a recombinant phosphoketolase pathway. By heterologous expression of phosphotransacetylase and acetaldehyde dehydrogenase in combination with the native phosphoketolase, we installed a functional phosphoketolase pathway in the xylose-fermenting Saccharomyces cerevisiae strain TMB3001c. Consequently the ethanol yield was increased by 25% because less of the by-product xylitol was formed. The flux through the recombinant phosphoketolase pathway was about 30% of the optimum flux that would be required to completely eliminate xylitol and glycerol accumulation. Further overexpression of phosphoketolase, however, increased acetate accumulation and reduced the fermentation rate. By combining the phosphoketolase pathway with the ald6 mutation, which reduced acetate formation, a strain with an ethanol yield 20% higher and a xylose fermentation rate 40% higher than those of its parent was engineered.     

Generation of the improved recombinant xylose-utilizing Saccharomyces cerevisiae TMB 3400 by random mutagenesis and physiological comparison with Pichia stipitis CBS 6054

The recombinant xylose-utilizing Saccharomyces cerevisiae TMB 3399 was constructed by chromosomal integration of the genes encoding D-xylose reductase (XR), xylitol dehydrogenase (XDH), and xylulokinase (XK). S. cerevisiae TMB 3399 was subjected to chemical mutagenesis with ethyl methanesulfonate and, after enrichment, 33 mutants were selected for improved growth on D-xylose and carbon dioxide formation in Durham tubes. The best-performing mutant was called S. cerevisiae TMB 3400. The novel, recombinant S. cerevisiae strains were compared with Pichia stipitis CBS 6054 through cultivation under aerobic, oxygen-limited, and anaerobic conditions in a defined mineral medium using only D-xylose as carbon and energy source. The mutation led to a more than five-fold increase in maximum specific growth rate, from 0.0255 h(-1) for S. cerevisiae TMB 3399 to 0.14 h(-1) for S. cerevisiae TMB 3400, whereas P. stipitis grew at a maximum specific growth rate of 0.44 h(-1). All yeast strains formed ethanol only under oxygen-limited and anaerobic conditions. The ethanol yields and maximum specific ethanol productivities during oxygen limitation were 0.21, 0.25, and 0.30 g ethanol g xylose(-1) and 0.001, 0.10, and 0.16 g ethanol g biomass(-1) h(-1) for S. cerevisiae TMB 3399, TMB 3400, and P. stipitis CBS 6054, respectively. The xylitol yield under oxygen-limited and anaerobic conditions was two-fold higher for S. cerevisiae TMB 3399 than for TMB 3400, but the glycerol yield was higher for TMB 3400. The specific activity, in U mg protein(-1), was higher for XDH than for XR in both S. cerevisiae TMB 3399 and TMB 3400, while P. stipitis CBS 6054 showed the opposite relation. S. cerevisiae TMB 3400 displayed higher specific XR, XDH and XK activities than TMB 3399. Hence, we have demonstrated that a combination of metabolic engineering and random mutagenesis was successful to generate a superior, xylose-utilizing S. cerevisiae, and uncovered distinctive physiological properties of the mutant.     

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.     

Expression of bifunctional enzymes with xylose reductase and xylitol dehydrogenase activity in Saccharomyces cerevisiae alters product formation during xylose fermentation.

To enhance metabolite transfer in the two initial sequential steps of xylose metabolism in yeast, two structural genes of Pichia stipitis, XYL1 and XYL2 encoding xylose reductase (XR) and xylitol dehydrogenase (XDH), respectively, were fused in frame. Four chimeric genes were constructed, encoding fusion proteins with different orders of the enzymes and different linker lengths. These genes were expressed in Saccharomyces cerevisiae. The fusion proteins exhibited both XR and XDH activity when XYL1 was fused downstream of XYL2. The specific activity of the XDH part of the complexes increased when longer peptide linkers were used. Bifunctional enzyme complexes, analyzed by gel filtration, were found to be tetramers, hexamers, and octamers. No degradation products were detected by Western blot analysis. S. cerevisiae strains harboring the bifunctional enzymes grew on minimal-medium xylose plates, and oxygen-limited xylose fermentation resulted in xylose consumption and ethanol formation. When a fusion protein, containing a linker of three amino acids, was coexpressed with native XR and XDH monomers in S. cerevisiae, enzyme complexes consisting of chimerical and native subunits were formed. The total activity of these complexes showed XR and XDH activities similar to the activities obtained when the monomers were expressed individually. Strains which coexpressed chimerical subunits together with native XR and XDH monomers consumed less xylose and produced less xylitol. However, the xylitol yield was lower in these strains than in strains expressing only native XR and XDH monomers, 0.55 and 0.62, respectively, and the ethanol yield was higher. The reduced xylitol yield was accompanied by reduced glycerol and acetate formation suggesting enhanced utilization of NADH in the XR reaction.     

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.     

Heterologous expression of Aspergillus oryzae xylose reductase and xylitol dehydrogenase genes facilitated xylose utilization in the yeast Saccharomyces cerevisiae

The cDNA encoding a putative xylose reductase (xyrA) from Aspergillus oryzae was cloned and coexpressed in the yeast Saccharomyces cerevisiae with A. oryzae xylitol dehydrogenase cDNA (xdhA). XyrA exhibited NADPH-dependent xylose reductase activity. The S. cerevisiae strain, overexpressing the xyrA, xdhA, endogenous XKS1, and TAL1 genes, grew on xylose as sole carbon source, and produced ethanol.     

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.     

Comparative study on a series of recombinant flocculent Saccharomyces cerevisiae strains with different expression levels of xylose reductase and xylulokinase

Ethanol production from xylose is important for the utilization of lignocellulosic biomass as raw materials. Recently, we reported the development of an industrial xylose-fermenting Saccharomyces cerevisiae strain, MA-R4, which was engineered by chromosomal integration to express the genes encoding xylose reductase and xylitol dehydrogenase from Pichia stipitis along with S. cerevisiae xylulokinase gene constitutively using the alcohol-fermenting flocculent yeast strain, IR-2. IR-2 has the highest xylulose-fermenting ability of the industrial diploid strains, making it a useful host strain for genetically engineering xylose-utilizing S. cerevisiae. To optimize the activities of xylose metabolizing enzymes in the metabolic engineering of IR-2 for further improvement of ethanol production from xylose, we constructed a set of recombinant isogenic strains harboring different combinations of genetic modifications present in MA-R4, and investigated the effect of constitutive expression of xylulokinase and of different levels of xylulokinase and xylose reductase activity on xylose fermentation. This strain comparison showed that constitutive expression of xylulokinase increased ethanol production from xylose at the expense of xylitol excretion, and that high activity of xylose reductase resulted in an increased rate of xylose consumption and an increased glycerol yield. Moreover, strain MA-R6, which has moderate xylulokinase activity, grew slightly better but accumulated more xylitol than strain MA-R4. These results suggest that fine-tuning of introduced enzyme activity in S. cerevisiae is important for improving xylose fermentation to ethanol.     

Fermentation of corn fibre sugars by an engineered xylose utilizing Saccharomyces yeast strain

The ability of a recombinant Saccharomyces yeast strain to ferment the sugars glucose, xylose, arabinose and galactose which are the predominant monosaccharides found in corn fibre hydrolysates has been examined. Saccharomyces strain 1400 (pLNH32) was genetically engineered to ferment xylose by expressing genes encoding a xylose reductase, a xylitol dehydrogenase and a xylulose kinase. The recombinant efficiently fermented xylose alone or in the presence of glucose. Xylose-grown cultures had very little difference in xylitol accumulation, with only 4 to 5g/l accumulating, in aerobic, micro-aerated and anaerobic conditions. Highest production of ethanol with all sugars was achieved under anaerobic conditions. From a mixture of glucose (80g/l) and xylose (40g/l), this strain produced 52g/l ethanol, equivalent to 85% of theoretical yield, in less than 24h. Using a mixture of glucose (31g/l), xylose (15.2g/l), arabinose (10.5g/l) and galactose (2g/l), all of the sugars except arabinose were consumed in 24h with an accumulation of 22g ethanol/l, a 90% yield (excluding the arabinose in the calculation since it is not fermented). Approximately 98% theoretical yield, or 21g ethanol/l, was achieved using an enzymatic hydrolysate of ammonia fibre exploded corn fibre containing an estimated 47.0g mixed sugars/l. In all mixed sugar fermentations, less than 25% arabinose was consumed and converted into arabitol.     

Improvement of xylose fermentation in respiratory-deficient xylose-fermenting Saccharomyces cerevisiae

Effective conversion of xylose in lignocelluloses is expected to reduce the production cost of second-generation biofuels significantly. The factors affecting xylose fermentation in Saccharomyces cerevisiae that express xylose reductase-xylitol dehydrogenase (XR-XDH) are studied. Although overproduction of non-oxidative pentose phosphate pathway significantly increased the aerobic-specific growth rate on xylose and slightly improved conversion of xylose to ethanol under oxygen-limited conditions, the elimination of respiration by deleting cytochrome C oxidase subunit IV gene impeded aerobic growth on xylose. However, the adaptive evolution of the respiratory-deficient strain with an NADP(+)-preferring XDH mutant in xylose media dramatically improved its xylose-fermenting ability. The specific growth rate, ethanol yield, and xylitol yield of the evolved strain on xylose were 0.06h(-1), 0.39gg(-1), and 0.13gg(-1) consumed xylose, respectively. Similar to anaerobic fermentation, the evolved strain exhibited accumulated ethanol rather than recycled it under aerobic conditions.     

A glycerol-3-phosphate dehydrogenase-deficient mutant of Saccharomyces cerevisiae expressing the heterologous XYL1 gene.

The gene XYL1, encoding a xylose reductase, from Pichia stipitis was transformed into a mutant of Saccharomyces cerevisiae incapable of glycerol production because of deletion of the genes GPD1 and GPD2. The transformed strain was capable of anaerobic glucose conversion in the presence of added xylose, indicating that the xylose reductase reaction can fulfill the role of the glycerol-3-phosphate dehydrogenase reaction as a redox sink. The specific xylitol production rate obtained was 0.38 g g-1 h-1.     

Endogenous xylose pathway in Saccharomyces cerevisiae

The baker's yeast Saccharomyces cerevisiae is generally classified as a non-xylose-utilizing organism. We found that S. cerevisiae can grow on D-xylose when only the endogenous genes GRE3 (YHR104w), coding for a nonspecific aldose reductase, and XYL2 (YLR070c, ScXYL2), coding for a xylitol dehydrogenase (XDH), are overexpressed under endogenous promoters. In nontransformed S. cerevisiae strains, XDH activity was significantly higher in the presence of xylose, but xylose reductase (XR) activity was not affected by the choice of carbon source. The expression of SOR1, encoding a sorbitol dehydrogenase, was elevated in the presence of xylose as were the genes encoding transketolase and transaldolase. An S. cerevisiae strain carrying the XR and XDH enzymes from the xylose-utilizing yeast Pichia stipitis grew more quickly and accumulated less xylitol than did the strain overexpressing the endogenous enzymes. Overexpression of the GRE3 and ScXYL2 genes in the S. cerevisiae CEN.PK2 strain resulted in a growth rate of 0.01 g of cell dry mass liter(-1) h(-1) and a xylitol yield of 55% when xylose was the main carbon source.     

Effect of the reversal of coenzyme specificity by expression of mutated Pichia stipitis xylitol dehydrogenase in recombinant Saccharomyces cerevisiae

AIMS: To determine the effects on xylitol accumulation and ethanol yield of expression of mutated Pichia stipitis xylitol dehydrogenase (XDH) with reversal of coenzyme specificity in recombinant Saccharomyces cerevisiae. METHODS AND RESULTS: The genes XYL2 (D207A/I208R/F209S) and XYL2 (S96C/S99C/Y102C/D207A/I208R/F209S) were introduced into S. cerevisiae, which already contained the P. stipitis XYL1 gene (encoding xylose reductase, XR) and the endogenously overexpressed XKS1 gene (encoding xylulokinase, XK). The specific activities of mutated XDH in both strains showed a distinct increase in NADP(+)-dependent activity in both strains with mutated XDH, reaching 0.782 and 0.698 U mg(-1). In xylose fermentation, the strain with XDH (D207A/I208R/F209S) had a large decrease in xylitol and glycerol yield, while the xylose consumption and ethanol yield were decreased. In the strain with XDH (S96C/S99C/Y102C/D207A/I208R/F209S), the xylose consumption and ethanol yield were also decreased, and the xylitol yield was increased, because of low XDH activity. CONCLUSIONS: Changing XDH coenzyme specificity was a sufficient method for reducing the production of xylitol, but high activity of XDH was also required for improved ethanol formation. SIGNIFICANCE AND IMPACT OF THE STUDY: The difference in coenzyme specificity was a vital parameter controlling ethanolic xylose fermentation but the XDH/XR ratio was also important.     

Xylose-metabolizing Saccharomyces cerevisiae strains overexpressing the TKL1 and TAL1 genes encoding the pentose phosphate pathway enzymes transketolase and transaldolase.

Saccharomyces cerevisiae was metabolically engineered for xylose utilization. The Pichia stipitis CBS 6054 genes XYL1 and XYL2 encoding xylose reductase and xylitol dehydrogenase were cloned into S. cerevisiae. The gene products catalyze the two initial steps in xylose utilization which S. cerevisiae lacks. In order to increase the flux through the pentose phosphate pathway, the S. cerevisiae TKL1 and TAL1 genes encoding transketolase and transaldolase were overexpressed. A XYL1- and XYL2-containing S. cerevisiae strain overexpressing TAL1 (S104-TAL) showed considerably enhanced growth on xylose compared with a strain containing only XYL1 and XYL2. Overexpression of only TKL1 did not influence growth. The results indicate that the transaldolase level in S. cerevisiae is insufficient for the efficient utilization of pentose phosphate pathway metabolites. Mixtures of xylose and glucose were simultaneously consumed with the recombinant strain S104-TAL. The rate of xylose consumption was higher in the presence of glucose. Xylose was used for growth and xylitol formation, but not for ethanol production. Decreased oxygenation resulted in impaired growth and increased xylitol formation. Fermentation with strain S103-TAL, having a xylose reductase/xylitol dehydrogenase ratio of 0.5:30 compared with 4.2:5.8 for S104-TAL, did not prevent xylitol formation.