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Point mutation of the xylose reductase (XR) gene reduces xylitol accumulation and increases citric acid production in Aspergillus carbonarius.

ABSTRACT: Aspergillus carbonarius accumulates xylitol when it grows on D-xylose. In fungi, D-xylose is reduced to xylitol by the NAD(P)H-dependent xylose reductase (XR). Xylitol is then further oxidized by the NAD(+)-dependent xylitol dehydrogenase (XDH). The cofactor impairment between the XR and XDH can lead to the accumulation of xylitol under oxygen-limiting conditions. Most of the XRs are NADPH dependent and contain a conserved Ile-Pro-Lys-Ser motif. The only known naturally occurring NADH-dependent XR (from Candida parapsilosis) carries an arginine residue instead of the lysine in this motif. In order to overcome xylitol accumulation in A. carbonarius a Lys-274 to Arg point mutation was introduced into the XR with the aim of changing the specificity toward NADH. The effect of the genetic engineering was examined in fermentation for citric acid production and xylitol accumulation by using D-xylose as the sole carbon source. Fermentation with the mutant strain showed a 2.8-fold reduction in xylitol accumulation and 4.5-fold increase in citric acid production compared to the wild-type strain. The fact that the mutant strain shows decreased xylitol levels is assumed to be associated with the capability of the mutated XR to use the NADH generated by the XDH, thus preventing the inhibition of XDH by the high levels of NADH and ensuring the flux of xylose through the pathway. This work shows that enhanced production of citric acid can be achieved using xylose as the sole carbon source by reducing accumulation of other by-products, such as xylitol.

SUBMITTER: Weyda I

PROVIDER: S-EPMC3953602 | biostudies-literature |

REPOSITORIES: biostudies-literature

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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.

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Point mutation of the xylose reductase (XR) gene reduces xylitol accumulation and increases citric acid production in Aspergillus carbonarius.

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