Engineering Research Center of Ministry of Education on Food Synthetic Biotechnology, School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China.
Science Center for Future Foods, Jiangnan University, 1800 Lihu Rd, Wuxi, Jiangsu 214122, China.
J Agric Food Chem. 2024 Apr 10;72(14):8140-8148. doi: 10.1021/acs.jafc.4c01483. Epub 2024 Apr 2.
Rebaudioside (Reb) M is an important sweetener with high sweetness, but its low content in 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 , several key issues must be resolved including knocking out endogenous hydrolases, enhancing glycosylation, and extending the enzyme catalytic process. Herein, endogenous glycosyl hydrolase was knocked out in . The glycosylation process was enhanced by screening glycosyltransferases, and UGT91D2 from was identified as the optimum glycosyltransferase. The UDP-glucose supply was enhanced by overexpressing , 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 () which can prolong the growth cycle of 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.
瑞鲍迪苷 M（Reb）是一种高甜度的重要甜味剂，但由于其在 的含量低，以及异源微生物中糖基转移酶的催化能力低，限制了其生产。为了提高 通过转化甜菊苷生产瑞鲍迪苷 M 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、增强糖基化和延长酶的催化过程。在此， 通过敲除内源性糖苷水解酶 。通过筛选糖基转移酶增强了糖基化过程，鉴定出 来自 的 UGT91D2 为最佳糖基转移酶。通过过表达 增强 UDP-葡萄糖的供应，共表达 UGT91D2 和 UGT76G1 可实现甜菊苷向瑞鲍迪苷 M 的高效转化。为了延长催化过程，引入了沉默信息调节因子 2（ ），它可以延长 的生长周期。最终，通过这些修饰，在 5 L 生物反应器中以 10.0 g/L 甜菊苷生产出 12.5 g/L 的瑞鲍迪苷 M，得率达到 77.9%，这是迄今为止从甜菊糖苷到瑞鲍迪苷 M 的最高产量。该工程菌株可以促进瑞鲍迪苷 M 的工业化生产，该策略为甜菊糖苷的生产提供了参考。
