Enhanced Heterologous Production of Glycosyltransferase UGT76G1 by Co-Expression of Endogenous <i>prpD</i> and <i>malK</i> in <i>Escherichia coli</i> and Its Transglycosylation Application in Production of Rebaudioside

• Main Authors: Wenju Shu, Hongchen Zheng, Xiaoping Fu, Jie Zhen, Ming Tan, Jianyong Xu, Xingya Zhao, Shibin Yang, Hui Song, Yanhe Ma
• Format: Article
• Language: English
• Published: MDPI AG 2020-08-01
• Series: International Journal of Molecular Sciences
• Subjects: steviol glycosides; fusion partner; efficient <i>E. coli</i> expression system; <i>prpD</i>; <i>malK</i>; co-expression
• Online Access: https://www.mdpi.com/1422-0067/21/16/5752

Steviol glycosides (SGs) with zero calories and high-intensity sweetness are the best substitutes of sugar for the human diet. Uridine diphosphate dependent glycosyltransferase (UGT) UGT76G1, as a key enzyme for the biosynthesis of SGs with a low heterologous expression level, hinders its application. In this study, a suitable fusion partner, Smt3, was found to enhance the soluble expression of UGT76G1 by 60%. Additionally, a novel strategy to improve the expression of Smt3-UGT76G1 was performed, which co-expressed endogenous genes <i>prpD</i> and <i>malK</i> in <i>Escherichia coli</i>. Notably, this is the first report of constructing an efficient <i>E. coli</i> expression system by regulating <i>prpD</i> and <i>malK</i> expression, which remarkably improved the expression of Smt3-UGT76G1 by 200% as a consequence. Using the high-expression strain <i>E. coli</i> BL21 (DE3) M/P-3-S32U produced 1.97 g/L of Smt3-UGT76G1 with a yield rate of 61.6 mg/L/h by fed-batch fermentation in a 10 L fermenter. The final yield of rebadioside A (Reb A) and rebadioside M (Reb M) reached 4.8 g/L and 1.8 g/L, respectively, when catalyzed by Smt3-UGT76G1 in the practical UDP-glucose regeneration transformation system in vitro. This study not only carried out low-cost biotransformation of SGs but also provided a novel strategy for improving expression of heterologous proteins in <i>E. coli</i>.