Effects of transhydrogenase expression on xylitol production in recombinant saccharomyces cerevisiae harboring xylose reductase gene

Abstract

Xylitol is a five-carbon sugar alcohol which is widely present in nature. Xylitol has been used as a sugar substitute in the food industry due to a number of functional properties. Xylose conversion to xylitol might be controlled by the supply of NAD(P)H in recombinant Saccharomyces cerevisiae containing the xylose reductase gene. This thesis is concerned with effects of simultaneous expression of Azotobactor vinelandii soluble pyridine nucleotide transhydrogenase (STH) on xylitol production in recombinant S. cerevisiae harboring multi copies of the Pichia stipitis xylose reductase gene (XYL1). The STH gene which encodes an enzyme able to transfer hydrogen ion from NADH to NADPH in a reversible manner, was expressed in the recombinant S. cerevisiae BJ3505:¥äXR. Xylitol productivity was compared between S. cerevisiae BJ3505:¥äXR and S. cerevisiae BJ3505:¥äXR/STH in batch and fed-batch fermentations. The growth profiles and specific XR activity were not significantly different between the two strains in batch culture. The specific STH activity of S. cerevisiae BJ3505:¥äXR/STH was 2.0 unit per mg protein. In fed-batch fermentations, xylitol production patterns were quite different. A final xylitol concentration for S. cerevisiae BJ3505:¥äXR/STH was 59.9 g/L, corresponding to 71% of that obtained with the S. cerevisiae BJ3505:¥äXR (84.2 g/L). Xylitol productivity of S. cerevisiae BJ3505:¥äXR/STH (1.4 g/L hr-1) was only two-thirds than that of S. cerevisiae BJ3505:£¿R (2.1 g/L hr-1). S. cerevisiae BJ3505:¥äXR/STH produced more by-products than the strain without the STH gene. 4.0 g/L acetate and 16.2 g/L ethanol were produced in the S. cerevisiae BJ3505:¥äXR/STH compared with a negligible level of acetate and 7.2 g/L ethanol for S. cerevisiae BJ3505:¥äXR. The specific XR activity was almost the same for the two strains as observed in the batch culture. The specific STH activity was determined approximately 3.0 unit per mg cellular protein in the S. cerevisiae BJ3505:¥äXR/STH. The experimental results suggested that cytoplasmic soluble pyridine nucleotide transhydrogenase directed the reaction toward the formation of NADH and NADP+ from NAD+ and NADPH, which concomitantly reduced the availability of NADPH for xylitol production in the recombinant S. cerevisiae expressing the STH gene.