可同化阿拉伯糖的木糖还原酿酒酵母菌株构建

Construction of a xylose-reducing and L-arabinose-assimilating Saccharomyces cerevisiae strain by genetic and evolutionary engineering

【目的】以秸秆等木质纤维素类生物质为原料生产液体生物燃料乙醇,目前生产成本高,大规模工业化生产尚有较大难度。构建能同化阿拉伯糖进行木糖还原生产木糖醇的重组酿酒酵母菌株,以实现原料中全糖利用、生产高附加值产品,实现产品多元化。【方法】首先,利用CRISPR/Cas9基因编辑技术依次向出发菌株中导入阿拉伯糖代谢途径和木糖还原酶基因,使菌株获得代谢阿拉伯糖和将木糖转化为木糖醇的能力;其次,通过适应性驯化的进化工程手段,提高重组菌株对阿拉伯糖的利用效率;最后,通过混合糖发酵验证重组菌株利用阿拉伯糖和还原木糖产木糖醇的能力。【结果】通过导入植物乳杆菌的阿拉伯糖代谢途径,酿酒酵母菌株获得了较好的利用阿拉伯糖生长繁殖的能力;进一步导入假丝酵母的木糖还原酶基因后,重组菌株在葡萄糖作为辅助碳源条件下可高效还原木糖产木糖醇,但阿拉伯糖的利用能力下降。利用以阿拉伯糖为唯一碳源的培养基进行反复批次驯化,阿拉伯糖的利用能力得以恢复和提升,得到表型较好的重组菌株KAX3-2。该菌株在木糖(50 g/L)和阿拉伯糖(20 g/L)混合糖发酵条件下发酵72 h时,对阿拉伯糖和木糖利用率分别达到42.1%和65.9%,木糖醇的收率为64%。【结论】本研究成功构建了一株能有效利用阿拉伯糖并能将木糖转化为木糖醇的重组酿酒酵母菌株KAX3-2,为后续构建、获得阿拉伯糖代谢能力更强、木糖醇积累效率更高菌株的工作奠定了基础。

[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.