Microbial engineering analysis of xylitol production by recombinant Saccharomyces cerevisiae

Abstract

Xylitol is a well-known five-carbon sugar alcohol with low-calorie and anticariogenic characteristics. It has been used as a sugar substitute in the food industry due to various functional properties. This thesis was concerned with analysis of effects of fermentation strategies and HAP4 gene expression on xylitol production by recombinant Saccharomyces cerevisiae harboring the xylose reductase gene from Pichia stipitis. Various fermentation strategies including batch, fed-batch, repeated fed-batch and fed-batch with cell recycle were tested to increase the yield and rate of xylitol bioconversion from xylose using recombinant Saccharomyces cerevisiae BJ3505:¥äXR. Among them, the fed-batch fermentation with cell recycle was chosen as the most effective fermentation process. Yeast extract was found to be the best nitrogen source for xylitol production and cell viability. The fed-batch fermentation with cell recycle and addition of yeast extract resulted in 2 g/L?hr xylitol productivity and 121 g/L xylitol concentration which are 2 and 5 times higher than the corresponding values of the batch fermentation. As the fraction of viable cells generally plays an important role in bioconversion processes, flow cytometry was used to measure cell viability in a fast and accurate manner. Flow cytometric analysis of the recombinant yeast cells stained with propidium iodide exhibited that cell viability was maintained over 95% throughout the fermentation. The HAP4 gene encoding a transcriptional activator in the respiratory metabolism was overexpressed in S. cerevisiae BJ3505:¥äXR to investigate its effects on xylitol production. In batch fermentation, overexpression of the HAP4 gene in S. cerevisiae BJ3505:¥äXR increased dry cell mass slightly without affecting the performance of xylitol production.