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Xylulokinase overexpression in two strains of Saccharomyces cerevisiae also expressing xylose reductase and xylitol dehydrogenase and its effect on fermentation of xylose and lignocellulosic hydrolysate.

ABSTRACT: Fermentation of the pentose sugar xylose to ethanol in lignocellulosic biomass would make bioethanol production economically more competitive. Saccharomyces cerevisiae, an efficient ethanol producer, can utilize xylose only when expressing the heterologous genes XYL1 (xylose reductase) and XYL2 (xylitol dehydrogenase). Xylose reductase and xylitol dehydrogenase convert xylose to its isomer xylulose. The gene XKS1 encodes the xylulose-phosphorylating enzyme xylulokinase. In this study, we determined the effect of XKS1 overexpression on two different S. cerevisiae host strains, H158 and CEN.PK, also expressing XYL1 and XYL2. H158 has been previously used as a host strain for the construction of recombinant xylose-utilizing S. cerevisiae strains. CEN.PK is a new strain specifically developed to serve as a host strain for the development of metabolic engineering strategies. Fermentation was carried out in defined and complex media containing a hexose and pentose sugar mixture or a birch wood lignocellulosic hydrolysate. XKS1 overexpression increased the ethanol yield by a factor of 2 and reduced the xylitol yield by 70 to 100% and the final acetate concentrations by 50 to 100%. However, XKS1 overexpression reduced the total xylose consumption by half for CEN.PK and to as little as one-fifth for H158. Yeast extract and peptone partly restored sugar consumption in hydrolysate medium. CEN.PK consumed more xylose but produced more xylitol than H158 and thus gave lower ethanol yields on consumed xylose. The results demonstrate that strain background and modulation of XKS1 expression are important for generating an efficient xylose-fermenting recombinant strain of S. cerevisiae.

SUBMITTER: Johansson B

PROVIDER: S-EPMC93154 | biostudies-literature | 2001 Sep

REPOSITORIES: biostudies-literature

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Xylulokinase overexpression in two strains of Saccharomyces cerevisiae also expressing xylose reductase and xylitol dehydrogenase and its effect on fermentation of xylose and lignocellulosic hydrolysate.

Johansson B B Christensson C C Hobley T T Hahn-Hägerdal B B

Applied and environmental microbiology 20010901 9

Fermentation of the pentose sugar xylose to ethanol in lignocellulosic biomass would make bioethanol production economically more competitive. Saccharomyces cerevisiae, an efficient ethanol producer, can utilize xylose only when expressing the heterologous genes XYL1 (xylose reductase) and XYL2 (xylitol dehydrogenase). Xylose reductase and xylitol dehydrogenase convert xylose to its isomer xylulose. The gene XKS1 encodes the xylulose-phosphorylating enzyme xylulokinase. In this study, we determi ...[more]

PMID: 11526030

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Project description:An advanced strategy of Saccharomyces cerevisiae strain development for fermentation of xylose applies tailored enzymes in the process of metabolic engineering. The coenzyme specificities of the NADPH-preferring xylose reductase (XR) and the NAD?-dependent xylitol dehydrogenase (XDH) have been targeted in previous studies by protein design or evolution with the aim of improving the recycling of NADH or NADPH in their two-step pathway, converting xylose to xylulose. Yeast strains expressing variant pairs of XR and XDH that according to in vitro kinetic data were suggested to be much better matched in coenzyme usage than the corresponding pair of wild-type enzymes, exhibit widely varying capabilities for xylose fermentation. To achieve coherence between enzyme properties and the observed strain performance during fermentation, we explored the published kinetic parameters for wild-type and engineered forms of XR and XDH as possible predictors of xylitol by-product formation (Y(xylitol)) in yeast physiology. We found that the ratio of enzymatic reaction rates using NADP(H) and NAD(H) that was calculated by applying intracellular reactant concentrations to rate equations derived from bi-substrate kinetic analysis, succeeded in giving a statistically reliable forecast of the trend effect on Y(xylitol). Prediction based solely on catalytic efficiencies with or without binding affinities for NADP(H) and NAD(H) were not dependable, and we define a minimum demand on the enzyme kinetic characterization to be performed for this purpose. An immediate explanation is provided for the typically lower Y(xylitol) in the current strains harboring XR engineered for utilization of NADH as compared to strains harboring XDH engineered for utilization of NADP?. The known XDH enzymes all exhibit a relatively high K(m) for NADP? so that physiological boundary conditions are somewhat unfavorable for xylitol oxidation by NADP?. A criterion of physiological fitness is developed for engineered XR working together with wild-type XDH.

Project description:UnlabelledA major obstacle to sustainable lignocellulosic biofuel production is microbe inhibition by the combinatorial stresses in pretreated plant hydrolysate. Chemical biomass pretreatment releases a suite of toxins that interact with other stressors, including high osmolarity and temperature, which together can have poorly understood synergistic effects on cells. Improving tolerance in industrial strains has been hindered, in part because the mechanisms of tolerance reported in the literature often fail to recapitulate in other strain backgrounds. Here, we explored and then exploited variations in stress tolerance, toxin-induced transcriptomic responses, and fitness effects of gene overexpression in different Saccharomyces cerevisiae (yeast) strains to identify genes and processes linked to tolerance of hydrolysate stressors. Using six different S. cerevisiae strains that together maximized phenotypic and genetic diversity, first we explored transcriptomic differences between resistant and sensitive strains to identify common and strain-specific responses. This comparative analysis implicated primary cellular targets of hydrolysate toxins, secondary effects of defective defense strategies, and mechanisms of tolerance. Dissecting the responses to individual hydrolysate components across strains pointed to synergistic interactions between osmolarity, pH, hydrolysate toxins, and nutrient composition. By characterizing the effects of high-copy gene overexpression in three different strains, we revealed the breadth of the background-specific effects of gene fitness contributions in synthetic hydrolysate. Our approach identified new genes for engineering improved stress tolerance in diverse strains while illuminating the effects of genetic background on molecular mechanisms.ImportanceRecent studies on natural variation within Saccharomyces cerevisiae have uncovered substantial phenotypic diversity. Here, we took advantage of this diversity, using it as a tool to infer the effects of combinatorial stress found in lignocellulosic hydrolysate. By comparing sensitive and tolerant strains, we implicated primary cellular targets of hydrolysate toxins and elucidated the physiological states of cells when exposed to this stress. We also explored the strain-specific effects of gene overexpression to further identify strain-specific responses to hydrolysate stresses and to identify genes that improve hydrolysate tolerance independent of strain background. This study underscores the importance of studying multiple strains to understand the effects of hydrolysate stress and provides a method to find genes that improve tolerance across strain backgrounds.

Dataset Information

Xylulokinase overexpression in two strains of Saccharomyces cerevisiae also expressing xylose reductase and xylitol dehydrogenase and its effect on fermentation of xylose and lignocellulosic hydrolysate.

Publications

Xylulokinase overexpression in two strains of Saccharomyces cerevisiae also expressing xylose reductase and xylitol dehydrogenase and its effect on fermentation of xylose and lignocellulosic hydrolysate.

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