A novel strictly NADPH-dependent Pichia stipitis xylose reductase constructed by site-directed mutagenesis

Abstract

Xylose reductase (XR) and xylitol dehydrogenase (XDH) are the key enzymes for xylose fermentation and have been widely used for construction of a recombinant xylose fermenting yeast. The effective recycling of cofactors between XR and XDH has been thought to be important to achieve effective xylose fermentation. Efforts to alter the coenzyme specificity of XR and HDX by site-directed mutagenesis have been widely made for improvement of efficiency of xylose fermentation. We previously succeeded by protein engineering to improve ethanol production by reversing XDH dependency from NAD(+) to NADP(+). In this study, we applied protein engineering to construct a novel strictly NADPH-dependent XR from Pichia stipitis by site-directed mutagenesis, in order to recycle NADPH between XR and XDH effectively. One double mutant, E223A/S271A showing strict NADPH dependency with 106% activity of wild-type was generated. A second double mutant, E223D/S271A, showed a 1.27-fold increased activity compared to the wild-type XR with NADPH and almost negligible activity with NADH.

Cofermentation of Glucose, Xylose, and Cellobiose by the Beetle-Associated Yeast Spathaspora passalidarum

“…The capacity to recycle NADH to NAD ϩ under anaerobic or oxygen-limiting conditions can reduce xylitol production and increase the capacity for xylose assimilation (3,17,19,22,24,29,32). Recently, Hou (15) reported that the crude XR activity of S. passalidarum has a 1.8-fold higher affinity for NADH than for NADPH.…”

“…The xylitol dehydrogenase (XDH) activity of S. passalidarum also has a much higher affinity for xylitol than the corresponding enzyme from S. stipitis does (15). These kinetic characteristics are much more favorable than those achieved with the best aldose reductase genes previously engineered for high selectivity and activity with NADH (19,22,29), and they could account in large part for the high PPP, F6P, and F16P metabolite levels on xylose. The S. passalidarum genome contains two orthologs each for XYL1 and XYL2 (46), so it is not possible to determine from these Fig.…”

Boost in bioethanol production using recombinant Saccharomyces cerevisiae with mutated strictly NADPH-dependent xylose reductase and NADP+-dependent xylitol dehydrogenase

“…Site-directed mutagenesis was applied to construct a novel strictly NADPH-dependent XR from P. stipitis (Khattab et al, 2011a(Khattab et al, , 2011b. Three constructed double mutants E223A/S271A (AA), E223D/S271A (DA), and E223G/S271A (GA) demonstrated strict NADPH dependency without any significant loss of enzyme activity.…”

Generation of Xylose-Fermenting <I>Saccharomyces cerevisiae</I> by Protein-Engineering

“…7) (Kavanagh et al, 2002(Kavanagh et al, , 2003. Therefore, it is very surprisingly that the substitution of Glu 223 →Ala in PsXR leaded to eliminate NADH-dependent activity completely, although the k cat /K m NADPH value was ~25% of WT (Khattab et al, 2011). Furthermore, since only Ser 271 is specific for NADPHbinding, the substitution to alanine may change the coenzyme specificity toward NADH over NADPH.…”