(495c) Metabolic Engineering of Saccharomyces Cerevisiae for Improved Xylose Utilization

Xylose is the major pentose and the second most abundant sugar in lignocellulosic feedstocks. Therefore, the efficient utilization of xylose is required for the cost-effective bioconversion of lignocellulose. Metabolic engineering approaches have been extensively exploited on the yeast S. cerevisiae for improved xylose utilization. However, the bioconversion of pentoses to ethanol still presents a considerable economic and technical challenge.

In this study, we started with the rational genetic engineering of a laboratory S. cerevisiae strain to express the xylose metabolic pathway, including the xylose isomerase (XI) and the xylulokinase (XK).  The non-oxidative pentose phosphate pathway (PPP) was also overexpressed to facilitate pentose assimilation. The resulting strains, H131-XYLA31, exhibited slow but significant aerobic growth (µmax=0.031±0.022 h^‑1), establishing a baseline for further advancement.

The engineered strain mentioned above was then used to initiate a three-staged evolutionary engineering, through aerobic and oxygen-limited sequential batch cultivation followed by xylose-limited anaerobic chemostat cultivation. Continuous improvement was observed during adaption of the H131-XYLA31. The finally isolated strain, H131E8-XYLA31, displayed a significantly increased anaerobic growth rate (0.120±0.004 h^-1) and xylose consumption rate (0.916 g·g^-1h^-1) compared to its parent strain.

Upon successful evolutionary engineering, the H131E8-XYLA31 was further modified by complementing the auxotrophic markers arg4 and leu2, resulting in H153E10-XYLA31 with greatly boosted aerobic growth.  Moreover, adding the anaerobic growth factors ergosterol and Tween 80 to the medium enabled a maximum anaerobic growth rate of 0.199 h-1 and a specific xylose consumption rate of 1.647g·g-1h-1 in batch fermentation, 65% and 37% higher than those of the best reported xylose-fermenting strain RWB 218, respectively.

In order to identify the genotypes responsible for quick xylose utilization in the evolutionarily engineered strain, an inverse metabolic engineering approach was applied to the evolved strain, revealing the tandem duplication of XYLA. The structure of XYLA integration, coupled with qPCR, DNA/RNA blotting, and enzyme activity assay results, suggests that the high expression level of XI is a major recombination event during the evolution and is necessary for efficient xylose assimilation.

Moreover, mutations on GRE3 that potentially abolish XR activity were discovered in the evolutionarily engineered strains, supporting the hypothesis that the elimination of XR activity was favorable for xylose utilization by reducing the production of xylitol and hence alleviating the inhibition of XI activity caused by xylitol.

In this study, we successfully applied rational and combinatorial metabolic engineering approaches for both constructing rapid xylose-fermenting strains and identifying novel genetic characteristics. The work serves as a practical milestone in lignocellulose conversions. The constructed strains can be used as hosts for further advancement, and the metabolic engineering techniques have proven to be effective tools for future strain evolution.