Genetic Traits Beneficial for Xylose Utilization by Recombinant Saccharomyces cerevisiae

Saccharomyces cerevisiae ferments hexoses in lignocellulosic hydrolysates under anaerobic conditions with high rates and ethanol yields. However, S. cerevisiae is naturally unable to utilize the pentose fraction of the hydrolysates. Xylose is the most abundant pentose sugar, and although recombinant S. cerevisiae strains capable of ethanolic xylose fermentation have been obtained through metabolic engineering, there still remains the challenge of transferring the acquired knowledge into an industrial context. The present thesis describes an enhancement of the xylose utilization capability of recombinant S. cerevisiae through several approaches. A series of integrating plasmids was constructed in order to facilitate improvements of the XR-XDH xylose utilization pathway. The aerobic xylose growth rate of recombinant S. cerevisiae was distinctly improved when utilizing the TDH3 promoter for XR expression instead of the ADH1 promoter. Traditionally, xylose-utilizing S. cerevisiae with the xylose reductase (XR) and xylitol dehydrogenase (XDH) from Pichia stipitis produce low ethanol yields due to the formation of xylitol by-product, which has been attributed to the different cofactor preferences of the NAD(P)H-dependent XR and the NAD+-dependent XDH. With the purpose of engineering the enzyme towards NADH-preference, the cofactor binding site of P. stipitis XR was altered by site-directed mutagenesis. Native P. stipitis XR and several mutants were kinetically characterized, and the effects of the mutations were investigated in vivo. The K270R mutation enabled increased ethanol yields, decreased xylitol yields and higher xylose consumption rates in anaerobic xylose fermentations. Additionally, traits important for ethanolic xylose fermentation were investigated at the transcriptional level. Four recombinant S. cerevisiae strains with enhanced xylose utilization capabilities were compared with two control strains in a genome-wide transcription analysis. Thirteen genes, with altered expression levels in all improved strains, were singled out for further analysis. It was found that the overexpression of SOL3 and TAL1, and the deletion of YLR042C, MNI1 and RPA49, enhanced the aerobic xylose growth by recombinant S. cerevisiae