Abstract:[Objective] In order to utilize all sugars in the raw materials and produce high value-added products, this research aims to construct a recombinant Saccharomyces cerevisiae strain that could assimilate L-arabinose and reduce xylose to xylitol. [Methods] Firstly, we used CRISPR/Cas9 gene editing technique to sequentially introduce genes in L-arabinose metabolism pathway and gene of xylose reductase into the Saccharomyces cerevisiae host strain to endow it the ability to metabolize L-arabinose and convert xylose into xylitol. Secondly, we used adaptive evolutionary engineering method to improve L-arabinose utilization efficiency of the recombinant strain. Finally, we verified the ability of the recombinant strain to metabolize L-arabinose and transfer xylose to xylitol by mixed sugar fermentation. [Results] By introducing the L-arabinose metabolic pathway of Lactobacillus plantarum, the engineered S. cerevisiae strain obtained good ability to grow on L-arabinose. After introducing the xylose reductase gene of Candida tropicalis, the strains were able to efficiently reduce xylose to xylitol using glucose as auxiliary carbon source, yet the utilization of L-arabinose decreased. After repeated batch acclimation with L-arabinose as the sole carbon source, the L-arabinose utilization ability of strains restored and improved. We isolated evolved strain KAX3-2 with good phenotype and evaluated its fermentation capacity using medium containing xylose (50 g/L) and L-arabinose (20 g/L). After 72 h fermentation, the utilization ratio of L-arabinose and xylose reached 42.1% and 65.9%, respectively, and the yield of xylitol was 64%. [Conclusion] This study successfully constructed a S. cerevisiae strain KAX3-2 which could utilize L-arabinose and convert xylose to xylitol efficiently as well as provided the base for the construction of strains with higher L-arabinose utilization efficiency and xylitol yield.
