Abstract：
Rebaudioside (Reb) M is an important sweetener with high sweetness, but its low content in Stevia rebaudiana and low catalytic capacity of the glycosyltransferases in heterologous microorganisms limit its production. In order to improve the catalytic efficiency of the conversion of stevioside to Reb M by Saccharomyces cerevisiae, several key issues must be resolved including knocking out endogenous hydrolases, enhancing glycosylation, and extending the enzyme catalytic process. Herein, endogenous glycosyl hydrolase SCW2 was knocked out in S. cerevisiae. The glycosylation process was enhanced by screening glycosyltransferases, and UGT91D2 from S. rebaudiana was identified as the optimum glycosyltransferase. The UDP-glucose supply was enhanced by overexpressing UGP1, and co-expressing UGT91D2 and UGT76G1 achieved efficient conversion of stevioside to Reb M. In order to extend the catalytic process, the silencing information regulator 2 (SIR2) which can prolong the growth cycle of S. cerevisiae was introduced. Finally, combining these modifications produced 12.5 g/L Reb M and the yield reached 77.9% in a 5 L bioreactor with 10.0 g/L stevioside, the highest titer from steviol glycosides to Reb M reported to date. The engineered strain could facilitate the industrial production of Reb M, and the strategies provide references for the production of steviol glycosides.
摘要：
莱鲍迪甙（Reb）M是一种重要的甜味剂，具有高甜度，但是甜叶菊中的低含量和异源微生物中糖基转移酶的低催化能力限制了其生产。为了提高酿酒酵母对甜菊苷转化为RebM的催化效率,必须解决几个关键问题，包括敲除内源性水解酶，增强糖基化，延伸酶催化过程。在这里，内源性糖基水解酶SCW2在酿酒酵母中被敲除。通过筛选糖基转移酶增强了糖基化过程，来自S.rebaudiana的UGT91D2被鉴定为最佳糖基转移酶。通过过表达UGP1增强了UDP-葡萄糖供应，共表达UGT91D2和UGT76G1实现了甜菊苷向RebM的有效转化。为了延长催化过程，介绍了能延长酿酒酵母生长周期的沉默信息调节因子2(SIR2)。最后,结合这些修饰产生12.5g/LRebM，在5L生物反应器中，用10.0g/L甜菊糖苷的产率达到77.9％，迄今为止报道的从甜菊醇糖苷到RebM的最高滴度。工程菌株可以促进RebM的工业化生产，为甜菊糖苷的生产提供参考。
