Efficient biosynthesis of rare natural product scopolamine using E. coli cells expressing a S14P/K97A mutant of hyoscyamine 6β-hydroxylase AaH6H
Hyoscyamine 6 β -hydroxylase (H6H, EC 1.14.11.11), an α-ketoglutarate dependent dioxygenase catalyzes the hydroxylation of (−)-hyoscyamine and the subsequent epoxidation of 6 β -hydroxyhyoscyamine to form scopolamine, a valuable natural alkaloid. In this study, random mutagenesis and site-directed saturation mutagenesis were used to enhance the hydroxylation activity of H6H from Anisodus acutangulus ( Aa H6H). A double mutant, Aa H6H M1 (S14P/K97A), showed a 3.4-fold improved hydroxylation activity compared with the wild-type enzyme, and the in vivo epoxidation activity was also improved by 2.3 times. After 34 h cultivation of Escherichia coli cells harboring Aa h6h m1 in a 5-L bioreactor with a working volume of 3 L, scopolamine was produced via a single-enzyme-mediated two-step transformation from 500 mg L −1 (−)-hyoscyamine in 97% conversion, and 1.068 g of the product were isolated, corresponding to a space-time yield of 251 mg L −1 d −1 . This study shows that the protein engineering of some key enzymes is a promising and effective way for improving the production of rare natural products such as scopolamine.
危险品化学品经营许可证（带存储）