초록
<P>An advanced strategy of <I>Saccharomyces cerevisiae</I> 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<SUP>+</SUP>-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 <I>in vitro</I> 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 (<I>Y</I><SUB>xylitol</SUB>) 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 <I>Y</I><SUB>xylitol</SUB>. 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 <I>Y</I><SUB>xylitol</SUB> in the current strains harboring XR engineered for utilization of NADH as compared to strains harboring XDH engineered for utilization of NADP<SUP>+</SUP>. The known XDH enzymes all exhibit a relatively high <I>K</I><SUB>m</SUB> for NADP<SUP>+</SUP> so that physiological boundary conditions are somewhat unfavorable for xylitol oxidation by NADP<SUP>+</SUP>. A criterion of physiological fitness is developed for engineered XR working together with wild-type XDH.</P>