Mutants of Pachysolen tannophilus with Improved Production of Ethanol from d-Xylose

The conversion of d-xylose to ethanol by the yeast Pachysolen tannophilus is relatively inefficient in batch culture. The inefficiency has been attributed in part to concurrent utilization of ethanol in the presence of appreciable concentrations of d-xylose and to the formation of xylitol and other by-products. To increase the concentration of ethanol accumulated in batch cultures, UV-induced mutants of P. tannophilus were selected on the basis of diminished growth on ethanol. Eleven independent mutant loci that conferred the ethanol-defective phenotype were identified. Three led to a greater yield and volumetric rate of production of ethanol than the wild type. One also produced less xylitol and was characterized by a deficiency in activity for malate dehydrogenase.     

Physiological Properties of a Mutant of Pachysolen tannophilus Deficient in NADPH-Dependent d-Xylose Reductase

A d-xylose reductase mutant of Pachysolen tannophilus was isolated on the basis of its poor growth on d-xylose but normal growth on xylitol and d-glucose. Fractionation of cell extracts indicated that the mutant was deficient in d-xylose reductase activity that used NADPH exclusively as a cofactor, but not in activity that used both NADH and NADPH. Mutant cultures grown on d-xylose as the sole carbon source exhibited some properties that would be desired in improved strains. Growth rate, growth yield, and d-xylose consumption rate of the mutant were less sensitive than those of the wild type to changes in aeration rate. d-Xylose was utilized more efficiently in that less of a by-product, xylitol, was produced. In addition, under low aeration conditions, more ethanol was produced. A disadvantage was a relatively slow rate of d-xylose utilization.     

Bioconversion of d-xylose to d-xylonate with Kluyveromyces lactis

d-Xylonate was produced from d-xylose using Kluyveromyces lactis strains which expressed the gene for NADP(+)-dependent d-xylose dehydrogenase from Trichoderma reesei (xyd1). Up to 19 ± 2g d-xylonatel(-1) was produced when K. lactis expressing xyd1 was grown on 10.5 gd-galactosel(-1) and 40 g d-xylosel(-1). Intracellular accumulation of d-xylonate (up to ～70 mg [gbiomass](-1)) was observed. d-Xylose was metabolised to d-xylonate, xylitol and biomass. Oxygen could be reduced to 6mmolO(2)l(-1)h(-1) without loss in titre or production rate, but metabolism of d-xylose and xylitol were more efficient when 12 mmolO(2)l(-1)h(-1) were provided. d-Xylose uptake was not affected by deletion of either the d-xylose reductase (XYL1) or a putative xylitol dehydrogenase encoding gene (XYL2) in xyd1 expressing strains. K. lactis xyd1ΔXYL1 did not produce extracellular xylitol and produced more d-xylonate than the xyd1 strain containing the endogenous XYL1. K. lactis xyd1ΔXYL2 produced high concentrations of xylitol and significantly less d-xylonate than the xyd1 strain with the endogenous XYL2.     

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.     

Decreased xylitol formation during xylose fermentation in Saccharomyces cerevisiae due to overexpression of water-forming NADH oxidase

The recombinant xylose-fermenting Saccharomyces cerevisiae strain harboring xylose reductase (XR) and xylitol dehydrogenase (XDH) from Scheffersomyces stipitis requires NADPH and NAD(+), creates cofactor imbalance, and causes xylitol accumulation during growth on d-xylose. To solve this problem, noxE, encoding a water-forming NADH oxidase from Lactococcus lactis driven by the PGK1 promoter, was introduced into the xylose-utilizing yeast strain KAM-3X. A cofactor microcycle was set up between the utilization of NAD(+) by XDH and the formation of NAD(+) by water-forming NADH oxidase. Overexpression of noxE significantly decreased xylitol formation and increased final ethanol production during xylose fermentation. Under xylose fermentation conditions with an initial d-xylose concentration of 50 g/liter, the xylitol yields for of KAM-3X(pPGK1-noxE) and control strain KAM-3X were 0.058 g/g xylose and 0.191 g/g, respectively, which showed a 69.63% decrease owing to noxE overexpression; the ethanol yields were 0.294 g/g for KAM-3X(pPGK1-noxE) and 0.211 g/g for the control strain KAM-3X, which indicated a 39.33% increase due to noxE overexpression. At the same time, the glycerol yield also was reduced by 53.85% on account of the decrease in the NADH pool caused by overexpression of noxE.     

Induction of aldose reductase and xylitol dehydrogenase activities in Candida tenuis CBS 4435

In this study the ability of various sugars and sugar alcohols to induce aldose reductase (xylose reductase) and xylitol dehydrogenase (xylulose reductase) activities in the yeast Candida tenuis was investigated. Both enzyme activities were induced when the organism was grown on d-xylose or l-arabinose as well as on the structurally related sugars d-arabinose or d-lyxose. Mixtures of d-xylose with the more rapidly metabolizable sugar d-glucose resulted in a decrease in the levels of both enzymes formed. These results show that the utilization of d-xylose by C. tenuis is regulated by induction and catabolite repression. Furthermore, the different patterns of induction on distinct sugars suggest that the synthesis of both enzymes is not under coordinate control.     

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     

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.     

Characterization of xylitol-utilizing mutants of Erwinia uredovora.

Of the four pentitols ribitol, xylitol, D-arabitol, and L-arabitol, Erwinia uredovora was able to utilize only D-arabitol as a carbon and energy source. Although attempts to isolate ribitol- or L-arabitol-utilizing mutants were unsuccessful, mutants able to grow on xylitol were isolated at a frequency of 9 X 10(-8). Xylitol-positive mutants constitutively synthesized both a novel NAD-dependent xylitol-4-dehydrogenase, which oxidized xylitol to L-xylulose, and an L-xylulokinase. The xylitol dehydrogenase had a Km for xylitol of 48 mM and showed best activity with xylitol and D-threitol as substrates. However, D-threitol was not a growth substrate for E. uredovora, and its presence did not induce either dehydrogenase or kinase activity. Attempts to determine the origin of the xylitol catabolic enzymes were unsuccessful; neither enzyme was induced on any growth substrate or in the presence of any polyol tested. Analysis of xylitol-negative mutants isolated after Tn5 mutagenesis suggested that the xylitol dehydrogenase and the L-xylulokinase structural genes were components of two separate operons but were under common regulatory control.     

Metabolic engineering for improved fermentation of pentoses by yeasts

The fermentation of xylose is essential for the bioconversion of lignocellulose to fuels and chemicals, but wild-type strains of Saccharomyces cerevisiae do not metabolize xylose, so researchers have engineered xylose metabolism in this yeast. Glucose transporters mediate xylose uptake, but no transporter specific for xylose has yet been identified. Over-expressing genes for aldose (xylose) reductase, xylitol dehydrogenase and moderate levels of xylulokinase enable xylose assimilation and fermentation, but a balanced supply of NAD(P) and NAD(P)H must be maintained to avoid xylitol production. Reducing production of NADPH by blocking the oxidative pentose phosphate cycle can reduce xylitol formation, but this occurs at the expense of xylose assimilation. Respiration is critical for growth on xylose by both native xylose-fermenting yeasts and recombinant S, cerevisiae. Anaerobic growth by recombinant mutants has been reported. Reducing the respiration capacity of xylose-metabolizing yeasts increases ethanol production. Recently, two routes for arabinose metabolism have been engineered in S. cerevisiae and adapted strains of Pichia stipitis have been shown to ferment hydrolysates with ethanol yields of 0.45 g g^–1 sugar consumed, so commercialization seems feasible for some applications.     

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.     

Mutants of Aerobacter aerogenes capable of utilizing xylitol as a novel carbon

Wild-type Aerobacter aerogenes 1033 is unable to utilize xylitol. A succession of mutants was isolated capable of growth on this compound (0.2%) at progressively faster rates. Whereas the ability to utilize xylitol was achieved in the first-stage mutant (X1) by constitutive production of ribitol dehydrogenase (for which xylitol is a substrate but not an inducer), the basis for enhanced utilization of xylitol in the second-stage mutant (X2) was an alteration of ribitol dehydrogenase. This enzyme was purified from the various mutants. The apparent K(m) for xylitol was 0.12 m with X2 enzyme and 0.29 m with X1 enzyme. The X2 enzyme was also less heat stable and, at 0.05 m substrate concentration, had a higher ratio of activity with xylitol compared to ribitol than did the X1 enzyme. The third mutant (X3), with an even faster growth rate on xylitol, produced a ribitol dehydrogenase indistinguishable physically or kinetically from that of X2. However, X3 produced constitutively an active transport system which accepts xylitol. The usual function of this system is apparently for the transport of d-arabitol since the latter is not only a substrate but also an inducer of the transport system in parental strains of X3. The sequence of mutations described herein illustrates how genes belonging to different metabolic systems can be mobilized to serve a new biochemical pathway.     

D-Arabitol catabolic pathway in Klebsiella aerogenes

Klebsiella aerogenes strain W70 has an inducible pathway for the degradation of d-arabitol which is comparable to the one found in Aerobacter aerogenes strain PRL-R3. The pathway is also similar to the pathway of ribitol catabolism in that it is composed of a pentitol dehydrogenase, d-arabitol dehydrogenase (ADH), and a pentulokinase, d-xylulokinase (DXK). These two enzymes are coordinately controlled and induced in response to d-arabitol, the apparent inducer of synthesis of these enzymes. We obtained mutants which lacked a functional d-xylose pathway and were constitutive for the ribitol catabolic pathway. These mutants were able to grow on the unusual pentitol, xylitol, only if they contained the functional DXK of the d-arabitol pathway. This provided us with a specific selection technique for DXK(+) transductants. As in A. aerogenes, mutants constitutive for ADH were able to use this enzyme to convert the hexitol d-mannitol to d-fructose. With mutants blocked in the normal d-mannitol catabolic pathway, growth on d-mannitol became a test for ADH constitutivity. Growth of such mutants on xylitol, d-arabitol, and d-mannitol was utilized to classify transductants in mapping, by transductional analysis, the loci involved in d-arabitol utilization. Three-point crosses gave the order dalK-dalD-dalC, where dalK is the DXK structural gene, dalD is the ADH structural gene, and dalC is a regulatory site controlling synthesis of both enzymes.     

Effect of enhanced xylose reductase activity on xylose consumption and product distribution in xylose-fermenting recombinant Saccharomyces cerevisiae

Recombinant Saccharomyces cerevisiae TMB3001, harboring the Pichia stipitis genes XYL1 and XYL2 (xylose reductase and xylitol dehydrogenase, respectively) and the endogenous XKS1(xylulokinase), can convert xylose to ethanol. About 30% of the consumed xylose, however, is excreted as xylitol. Enhanced ethanol yield has previously been achieved by disrupting the ZWF1 gene, encoding glucose-6-phosphate dehydrogenase, but at the expense of the xylose consumption. This is probably the result of reduced NADPH-mediated xylose reduction. In the present study, we increased the xylose reductase (XR) activity 4-19 times in both TMB3001 and the ZWF1-disrupted strain TMB3255. The xylose consumption rate increased by 70% in TMB3001 under oxygen-limited conditions. In the ZWF1-disrupted background, the increase in XR activity fully restored the xylose consumption rate. Maximal specific growth rates on glucose were lower in the ZWF1-disrupted strains, and the increased XR activity also negatively affected the growth rate in these strains. Addition of methionine resulted in 70% and 50% enhanced maximal specific growth rates for TMB3255 (zwfl Delta) and TMB3261 (PGK1-XYL1, zwf1 Delta), respectively. Enhanced XR activity did not have any negative effect on the maximal specific growth rate in the control strain. Enhanced glycerol yields were observed in the high-XR-activity strains. These are suggested to result from the observed reductase activity of the purified XR for dihydroxyacetone phosphate.     

l-Xylose and l-lyxose production from xylitol using Alcaligenes 701B strain and immobilized l-rhamnose isomerase enzyme

Xylitol was used as a raw material for production of l-xylose and l-lyxose using Alcaligenes 701B strain and immobilized l-rhamnose isomerase enzyme. Alcaligenes 701B converted xylitol to l-xylulose with a yield of 34% in the bioreactor. l-Xylulose was converted to l-xylose and l-lyxose using immobilized l-rhamnose isomerase enzyme. The final equilibrium between l-xylulose, l-xylose and l-lyxose was 53:26:21. The enzyme assays indicated that Alcaligenes 701B strain has an NAD-dependent xylitol dehydrogenase enzyme responsible for l-xylulose production. Furthermore, NAD(P)H-dependent l-xylulose reductase enzyme was active during conversion of xylitol to l-xylulose. The highest l-xylulose production rate corresponded with the highest growth rate. The Alcaligenes 701B strain used d-xylose for biomass growth, but xylitol was used only for l-xylulose production during conversion phase.     

Acquisition of ability to utilize Xylitol: disadvantages of a constitutive catabolic pathway in Escherichia coli.

Ribitol+ strains of Escherichia coli acquire the ability to utilize xylitol by mutating to constitutive production of the coordinately controlled ribitol catabolic enzymes ribitol dehydrogenase (RDH) and D-ribulokinase (DRK). Such strains concomitantly acquire toxicity to galacitol and L-arabitol, and to D-arabitol if they are unable to utilize it for growth. Strains selected for resistance to these polyols have DRK structural gene mutations or other mutations that eliminate the constitutive production of DRK, consistent with the view that DRK phosphorylates those polyols to toxic substances. Ribitol+ strains selected for growth on 8 mM xylitol fail to grow on 30 mM xylitol. A product of ribitol and xylitol catabolism represses synthesis of RDH, an enzyme required for growth on xylitol. At 30 mM xylitol, greater than 99% of RDH synthesis is repressed. Strains that grow on 8 mM xylitol can mutate to grow on 30 mM xylitol. Such mutants, relieved of this repression, overproduce RDH, resulting in good growth on the poor substrate, xylitol, but poor growth on the normal substrate, ribitol.     

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.