Laboratory of Biocatalysis and Synthetic Biotechnology, State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, PR China.
Laboratory of Biocatalysis and Bioprocessing, College of Pharmaceutical Engineering and Life Sciences, Changzhou University, Changzhou 213164, PR China.
J Biotechnol. 2015 Oct 10;211:123-9. doi: 10.1016/j.jbiotec.2015.07.019. Epub 2015 Jul 31.
Hyoscyamine 6β-hydroxylase (H6H, EC 1.14.11.11), an α-ketoglutarate dependent dioxygenase catalyzes the hydroxylation of (-)-hyoscyamine and the subsequent epoxidation of 6β-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 (AaH6H). A double mutant, AaH6HM1 (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 34h cultivation of Escherichia coli cells harboring Aah6hm1 in a 5-L bioreactor with a working volume of 3L, scopolamine was produced via a single-enzyme-mediated two-step transformation from 500mgL(-1) (-)-hyoscyamine in 97% conversion, and 1.068g of the product were isolated, corresponding to a space-time yield of 251mgL(-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.
莨菪碱 6β-羟化酶（H6H，EC 1.14.11.11）是一种依赖于α-酮戊二酸的双加氧酶，可催化（-）-莨菪碱的羟化以及随后的 6β-羟基莨菪碱的环氧化，生成具有重要价值的天然生物碱东莨菪碱。在本研究中，随机诱变和定点饱和诱变被用于提高来自唐古特山莨菪（AaH6H）的 H6H 的羟化活性。与野生型酶相比，双突变体 AaH6HM1（S14P/K97A）的羟化活性提高了 3.4 倍，体内环氧化活性也提高了 2.3 倍。在 5L 生物反应器中，含有 Aah6hm1 的大肠杆菌细胞在 3L 工作体积下培养 34h 后，通过两步单酶介导转化，以 97%的转化率从 500mgL（-1）（-）-莨菪碱生成 1.068g 产物，时空产率为 251mgL（-1）d（-1）。本研究表明，对一些关键酶的蛋白质工程是提高东莨菪碱等稀有天然产物产量的一种有前途且有效的方法。
