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A novel aldose-aldose oxidoreductase for co-production of D-xylonate and xylitol from D-xylose with Saccharomyces cerevisiae.

ABSTRACT: An open reading frame CC1225 from the Caulobacter crescentus CB15 genome sequence belongs to the Gfo/Idh/MocA protein family and has 47 % amino acid sequence identity with the glucose-fructose oxidoreductase from Zymomonas mobilis (Zm GFOR). We expressed the ORF CC1225 in the yeast Saccharomyces cerevisiae and used a yeast strain expressing the gene coding for Zm GFOR as a reference. Cell extracts of strains overexpressing CC1225 (renamed as Cc aaor) showed some Zm GFOR type of activity, producing D-gluconate and D-sorbitol when a mixture of D-glucose and D-fructose was used as substrate. However, the activity in Cc aaor expressing strain was >100-fold lower compared to strains expressing Zm gfor. Interestingly, C. crescentus AAOR was clearly more efficient than the Zm GFOR in converting in vitro a single sugar substrate D-xylose (10 mM) to xylitol without an added cofactor, whereas this type of activity was very low with Zm GFOR. Furthermore, when cultured in the presence of D-xylose, the S. cerevisiae strain expressing Cc aaor produced nearly equal concentrations of D-xylonate and xylitol (12.5 g D-xylonate l(-1) and 11.5 g D-xylitol l(-1) from 26 g D-xylose l(-1)), whereas the control strain and strain expressing Zm gfor produced only D-xylitol (5 g l(-1)). Deletion of the gene encoding the major aldose reductase, Gre3p, did not affect xylitol production in the strain expressing Cc aaor, but decreased xylitol production in the strain expressing Zm gfor. In addition, expression of Cc aaor together with the D-xylonolactone lactonase encoding the gene xylC from C. crescentus slightly increased the final concentration and initial volumetric production rate of both D-xylonate and D-xylitol. These results suggest that C. crescentus AAOR is a novel type of oxidoreductase able to convert the single aldose substrate D-xylose to both its oxidized and reduced product.

SUBMITTER: Wiebe MG

PROVIDER: S-EPMC4628093 | biostudies-literature | 2015 Nov

REPOSITORIES: biostudies-literature

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A novel aldose-aldose oxidoreductase for co-production of D-xylonate and xylitol from D-xylose with Saccharomyces cerevisiae.

Wiebe Marilyn G MG Nygård Yvonne Y Oja Merja M Andberg Martina M Ruohonen Laura L Koivula Anu A Penttilä Merja M Toivari Mervi M

Applied microbiology and biotechnology 20150812 22

An open reading frame CC1225 from the Caulobacter crescentus CB15 genome sequence belongs to the Gfo/Idh/MocA protein family and has 47 % amino acid sequence identity with the glucose-fructose oxidoreductase from Zymomonas mobilis (Zm GFOR). We expressed the ORF CC1225 in the yeast Saccharomyces cerevisiae and used a yeast strain expressing the gene coding for Zm GFOR as a reference. Cell extracts of strains overexpressing CC1225 (renamed as Cc aaor) showed some Zm GFOR type of activity, produci ...[more]

PMID: 26264136

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Project description:BackgroundProduction of D-xylonate by the yeast S. cerevisiae provides an example of bioprocess development for sustainable production of value-added chemicals from cheap raw materials or side streams. Production of D-xylonate may lead to considerable intracellular accumulation of D-xylonate and to loss of viability during the production process. In order to understand the physiological responses associated with D-xylonate production, we performed transcriptome analyses during D-xylonate production by a robust recombinant strain of S. cerevisiae which produces up to 50 g/L D-xylonate.ResultsComparison of the transcriptomes of the D-xylonate producing and the control strain showed considerably higher expression of the genes controlled by the cell wall integrity (CWI) pathway and of some genes previously identified as up-regulated in response to other organic acids in the D-xylonate producing strain. Increased phosphorylation of Slt2 kinase in the D-xylonate producing strain also indicated that D-xylonate production caused stress to the cell wall. Surprisingly, genes encoding proteins involved in translation, ribosome structure and RNA metabolism, processes which are commonly down-regulated under conditions causing cellular stress, were up-regulated during D-xylonate production, compared to the control. The overall transcriptional responses were, therefore, very dissimilar to those previously reported as being associated with stress, including stress induced by organic acid treatment or production. Quantitative PCR analyses of selected genes supported the observations made in the transcriptomic analysis. In addition, consumption of ethanol was slower and the level of trehalose was lower in the D-xylonate producing strain, compared to the control.ConclusionsThe production of organic acids has a major impact on the physiology of yeast cells, but the transcriptional responses to presence or production of different acids differs considerably, being much more diverse than responses to other stresses. D-Xylonate production apparently imposed considerable stress on the cell wall. Transcriptional data also indicated that activation of the PKA pathway occurred during D-xylonate production, leaving cells unable to adapt normally to stationary phase. This, together with intracellular acidification, probably contributes to cell death.

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.

Dataset Information

A novel aldose-aldose oxidoreductase for co-production of D-xylonate and xylitol from D-xylose with Saccharomyces cerevisiae.

Publications

A novel aldose-aldose oxidoreductase for co-production of D-xylonate and xylitol from D-xylose with Saccharomyces cerevisiae.

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