The activity of xylose reductase and xylitol dehydrogenase in yeasts

The activity and the cofactor specificity of xylose reductase and xylitol dehydrogenase were studied in extracts of yeasts from the genera Candida, Kluyveromyces, Pachysolen, Pichia, and Torulopsis grown under microaerobic conditions. It was found that xylitol dehydrogenase in all of the yeast species studied is specific for NAD+; xylose reductase in the xylitol-producing species C. didensiae, C. intermediae, C. parapsilosis, C. silvanorum, C. tropicalis, Kl. fragilis, Kl. marxianus, P. guillermondii, and T. molishiama is specific for NADPH; and xylose reductase in the ethanol-producing species P. stipitis, C. shehatae, and Pa. tannophilus is specific for both NADPH and NADH.     

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.     

Specific features of fermentation of D-xylose and D-glucose by xylose-assimilating yeasts

The ability to assimilate D-glucose and D-xylose was studied in 21 yeast species of the following genera: Candida, Kluyveromyces, Pachysolen, Pichia, and Torulopsis. All the cultures fermented D-glucose with the formation of ethanol. During the assimilation of D-xylose, ethanol was produced by P. stipitis and C. shehatae, whereas xylitol was produced by C. didensiae, C. intermediae, C. parapsilosis, C. silvanorum, C. tropicalis, K. fragilis, K. marxianus, P. guillermondii, and T. molishiama. The yeast P. tannophilus produced comparable amounts of both alcohols. The possible use of xylose-assimilating yeasts for the production of xylitol and ethanol is discussed.     

The Activity of Xylose Reductase and Xylitol Dehydrogenase in Yeasts

The activity and the cofactor specificity of xylose reductase and xylitol dehydrogenase were studied in extracts of yeasts from the genera Candida, Kluyveromyces, Pachysolen, Pichia,and Torulopsis grown under microaerobic conditions. It was found that xylitol dehydrogenase in all of the yeast species studied is specific for NAD⁺; xylose reductase in the xylitol-producing species C. didensiae, C. intermediae, C. parapsilosis, C. silvanorum, C. tropicalis, Kl. fragilis, Kl. marxianus, P. guillermondii, andT. molishiama is specific for NADPH; and xylose reductase in the ethanol-producing species P. stipitis, C. shehatae, and Pa. tannophilus is specific for both NADPH and NADH.     

Induction of Xylose Reductase and Xylitol Dehydrogenase Activities in Pachysolen tannophilus and Pichia stipitis on Mixed Sugars

The induction of xylose reductase and xylitol dehydrogenase activities on mixed sugars was investigated in the yeasts Pachysolen tannophilus and Pichia stipitis. Enzyme activities induced on d-xylose served as the controls. In both yeasts, d-glucose, d-mannose, and 2-deoxyglucose inhibited enzyme induction by d-xylose to various degrees. Cellobiose, l-arabinose, and d-galactose were not inhibitory. In liquid batch culture, P. tannophilus utilized d-glucose and d-mannose rapidly and preferentially over d-xylose, while d-galactose consumption was poor and lagged behind that of the pentose sugar. In P. stipitis, all three hexoses were used preferentially over d-xylose. The results showed that the repressibility of xylose reductase and xylitol dehydrogenase may limit the potential of yeast fermentation of pentose sugars in hydrolysates of lignocellulosic substrates.     

Isolation of a nucleotide activated disaccharide pentapeptide precursor from Methanobacterium thermoautotrophicum

The yeasts Pachysolen tannophilus and Pichia stipitis differed in their ability to utilize D-xylose in the presence of D-fructose. When P. tannophilus was grown aerobically in fructose-xylose mixture, the ketohexose was utilized preferentially over the pentose. However, in P. stipitis cultures, the converse was observed. The effect was associated with the ability of D-fructose to repress the induction of xylose reductase and xylitol dehydrogenase activities in P. tannophilus but not in P. stipitis. Both yeasts grew on D-fructose and fermented it to ethanol when it was supplied as the sole carbon source. The results suggest that there may exist some fundamental difference in the regulation of D-fructose metabolism between P. tannophilus and P. stipitis.     

Repression of xylose-specific enzymes by ethanol in Scheffersomyces (Pichia) stipitis and utility of repitching xylose-grown populations to eliminate diauxic lag

During the fermentation of lignocellulosic hydrolyzates to ethanol by native pentose-fermenting yeasts such as Scheffersomyces (Pichia) stipitis NRRL Y-7124 (CBS 5773) and Pachysolen tannophilus NRRL Y-2460, the switch from glucose to xylose uptake results in a diauxic lag unless process strategies to prevent this are applied. When yeast were grown on glucose and resuspended in mixed sugars, the length of this lag was observed to be a function of the glucose concentration consumed (and consequently, the ethanol concentration accumulated) prior to the switch from glucose to xylose fermentation. At glucose concentrations of 95 g/L, the switch to xylose utilization was severely stalled such that efficient xylose fermentation could not occur. Further investigation focused on the impact of ethanol on cellular xylose transport and the induction and maintenance of xylose reductase and xylitol dehydrogenase activities when large cell populations of S. stipitis NRRL Y-7124 were pre-grown on glucose or xylose and then presented mixtures of glucose and xylose for fermentation. Ethanol concentrations around 50 g/L fully repressed enzyme induction although xylose transport into the cells was observed to be occurring. Increasing degrees of repression were documented between 15 and 45 g/L ethanol. Repitched cell populations grown on xylose resulted in faster fermentation rates, particularly on xylose but also on glucose, and eliminated diauxic lag and stalling during mixed sugar conversion by P. tannophilus or S. stipitis, despite ethanol accumulations in the 60 or 70 g/L range, respectively. The process strategy of priming cells on xylose was key to the successful utilization of high mixed sugar concentrations because specific enzymes for xylose utilization could be induced before ethanol concentration accumulated to an inhibitory level.     

Affinity Purifications of Aldose Reductase and Xylitol Dehydrogenase from the Xylose-Fermenting Yeast Pachysolen tannophilus

Although xylose is a major product of hydrolysis of lignocellulosic materials, few yeasts are able to convert it to ethanol. In Pachysolen tannophilus, one of the few xylose-fermenting yeasts found, aldose reductase and xylitol dehydrogenase were found to be key enzymes in the metabolic pathway for xylose fermentation. This paper presents a method for the rapid and simultaneous purification of both aldose reductase and xylitol dehydrogenase from P. tannophilus. Preliminary studies indicate that this method may be easily adapted to purify similar enzymes from other xylose-fermenting yeasts.     

The expression of a Pichia stipitis xylose reductase mutant with higher K(M) for NADPH increases ethanol production from xylose in recombinant Saccharomyces cerevisiae

Xylose fermentation by Saccharomyces cerevisiae requires the introduction of a xylose pathway, either similar to that found in the natural xylose-utilizing yeasts Pichia stipitis and Candida shehatae or similar to the bacterial pathway. The use of NAD(P)H-dependent XR and NAD(+)-dependent XDH from P. stipitis creates a cofactor imbalance resulting in xylitol formation. The effect of replacing the native P. stipitis XR with a mutated XR with increased K(M) for NADPH was investigated for xylose fermentation to ethanol by recombinant S. cerevisiae strains. Enhanced ethanol yields accompanied by decreased xylitol yields were obtained in strains carrying the mutated XR. Flux analysis showed that strains harboring the mutated XR utilized a larger fraction of NADH for xylose reduction. The overproduction of the mutated XR resulted in an ethanol yield of 0.40 g per gram of sugar and a xylose consumption rate of 0.16 g per gram of biomass per hour in chemostat culture (0.06/h) with 10 g/L glucose and 10 g/L xylose as carbon source.     

Existence of Cyanide-Insensitive Respiration in the Yeast Pichia stipitis and Its Possible Influence on Product Formation during Xylose Utilization

A cyanide-insensitive and salicyl hydroxamic acid-sensitive respiration (CIR) was found in the yeast Pichia stipitis in contrast to Candida utilis, Pachysolen tannophilus, and Saccharomyces cerevisiae. During xylose utilization in the presence of either salicyl hydroxamic acid or cyanide, P. stipitis formed xylitol, arabitol, and ribitol. The existence of CIR is discussed in terms of a redox sink preventing xylitol formation in P. stipitis.     

Construction of a recombinant yeast strain converting xylose and glucose to ethanol

Candida shehatae gene xyll and Pichia stipitis gene xyl2,encoding xylose reductase (XR) and xylitol dehydrogenase (XD) respectively,were amplified by PCR.The genes xyl1 and xyl2 were placed under the control of promoter GAL in vector pYES2 to construct the recombinant expression vector pYES2-PI2.Subsequently the vector pYES2-P12 was transformed into S.cerevisiae YS58 by LiAc to produce the recombinant yeast YS58-12.The alcoholic ferment indicated that the recombinant yeast YS58-12 could convert xylose to ethanol with the xylose consumption rate of 81.3%.     

Alcoholic Fermentation of d-Xylose by Yeasts

Type strains of 200 species of yeasts able to ferment glucose and grow on xylose were screened for fermentation of d-xylose. In most of the strains tested, ethanol production was negligible. Nineteen were found to produce between 0.1 and 1.0 g of ethanol per liter. Strains of the following species produce more than 1 g of ethanol per liter in the fermentation test with 2% xylose: Brettanomyces naardenensis, Candida shehatae, Candida tenuis, Pachysolen tannophilus, Pichia segobiensis, and Pichia stipitis. Subsequent screening of these yeasts for their capacity to ferment d-cellobiose revealed that only Candida tenuis CBS 4435 was a good fermenter of both xylose and cellobiose under the test conditions used.     

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.     

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.     

Control of xylose consumption by xylose transport in recombinant Saccharomyces cerevisiae

Saccharomyces cerevisiae TMB3001 has previously been engineered to utilize xylose by integrating the genes coding for xylose reductase (XR) and xylitol dehydrogenase (XDH) and overexpressing the native xylulokinase (XK) gene. The resulting strain is able to metabolize xylose, but its xylose utilization rate is low compared to that of natural xylose utilizing yeasts, like Pichia stipitis or Candida shehatae. One difference between S. cerevisiae and the latter species is that these possess specific xylose transporters, while S. cerevisiae takes up xylose via the high-affinity hexose transporters. For this reason, in part, it has been suggested that xylose transport in S. cerevisiae may limit the xylose utilization.We investigated the control exercised by the transport over the specific xylose utilization rate in two recombinant S. cerevisiae strains, one with low XR activity, TMB3001, and one with high XR activity, TMB3260. The strains were grown in aerobic sugar-limited chemostat and the specific xylose uptake rate was modulated by changing the xylose concentration in the feed, which allowed determination of the flux response coefficients. Separate measurements of xylose transport kinetics allowed determination of the elasticity coefficients of transport with respect to extracellular xylose concentration. The flux control coefficient, C(J) (transp), for the xylose transport was calculated from the response and elasticity coefficients. The value of C(J) (transp) for both strains was found to be < 0.1 at extracellular xylose concentrations > 7.5 g L(-1). However, for strain TMB3260 the flux control coefficient was higher than 0.5 at xylose concentrations < 0.6 g L(-1), while C(J) (transp) stayed below 0.2 for strain TMB3001 irrespective of xylose concentration.     

瑞氏木霉木糖醇脱氢酶基因的分离与鉴定

将在木聚糖上生长的瑞氏木霉(Trichoderma reesei)RutC-30的cDNA文库全部质粒转化已携带有毕赤氏酵(Pithia stipitis)木糖还原酶基因的重组酿酒酵母(Saccharomycescerevisiae)菌株H475,在H475中构建了瑞氏木霉的cDNA表达亚文库。在以木糖为唯一碳源的选择性酵母合成培养基上,从该亚文库中筛选到瑞氏木霉木糖醇脱氢酶cDNA基因．该基因片段长为1．3kb。Southern、Norhern印迹杂交分析和蛋白质凝胶电泳结果表明该基因确实来源于瑞氏木霉,所编码蛋白质分子量约为40kDa。携带有毕赤氏酵母木糖还原酶和瑞氏木霉木糖醇脱氢酶基因的重组酵母能够在以木糖为唯一碳源的培养基上生长,并能将90％以上的木糖转化为木糖醇、乙醇和其它副产品。     

Ethanol production from xylose by recombinant Saccharomyces cerevisiae expressing protein engineered NADP+-dependent xylitol dehydrogenase

Effects of reversal coenzyme specificity toward NADP+ and thermostabilization of xylitol dehydrogenase (XDH) from Pichia stipitis on fermentation of xylose to ethanol were estimated using a recombinant Saccharomyces cerevisiae expressing together with a native xylose reductase from P. stipitis. The mutated XDHs performed the similar enzyme properties in S. cerevisiae cells, compared with those in vitro. The significant enhancement(s) was found in Y-ARSdR strain, in which NADP+-dependent XDH was expressed; 86% decrease of unfavorable xylitol excretion with 41% increased ethanol production, when compared with the reference strain expressing the wild-type XDH.     

Enhancement of xylitol production in Candida tropicalis by co-expression of two genes involved in pentose phosphate pathway

The yeast Candida tropicalis produces xylitol, a natural, low-calorie sweetener whose metabolism does not require insulin, by catalytic activity of NADPH-dependent xylose reductase. The oxidative pentose phosphate pathway (PPP) is a major basis for NADPH biosynthesis in C. tropicalis. In order to increase xylitol production rate, xylitol dehydrogenase gene (XYL2)disrupted C. tropicalis strain BSXDH-3 was engineered to co-express zwf and gnd genes which, respectively encodes glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6-PGDH), under the control of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter. NADPH-dependent xylitol production was higher in the engineered strain, termed "PP", than in BSXDH-3. In fermentation experiments using glycerol as a co-substrate with xylose, strain PP showed volumetric xylitol productivity of 1.25 g l(-1) h(-1), 21% higher than the rate (1.04 g l(-1) h(-1)) in BSXDH-3. This is the first report of increased metabolic flux toward PPP in C. tropicalis for NADPH regeneration and enhanced xylitol production.     

The positive effect of the decreased NADPH-preferring activity of xylose reductase from Pichia stipitis on ethanol production using xylose-fermenting recombinant Saccharomyces cerevisiae

We focused on the effects of a mutation of xylose reductase from Pichia stipitis (PsXR) on xylose-to-ethanol fermentation using recombinant Saccharomyces cerevisiae transformed with PsXR and PsXDH (xylitol dehydrogenase from P. stipitis) genes. Based on inherent NADH-preferring XR and several site-directed mutagenetic studies using other aldo-keto reductase enzymes, we designed several single PsXR mutants. K270R showing decreased NADPH-preferring activity without a change in NADH-preferring activity was found to be a potent mutant. Strain Y-K270R transformed with K270R PsXR and wild-type PsXDH showed a 31% decrease in unfavorable xylitol excretion with 5.1% increased ethanol production as compared to the control in the fermentation of 15 g l(-1) xylose and 5 g l(-1) glucose.