Efficient biosynthesis of rare natural product scopolamine using E. coli cells expressing a S14P/K97A mutant of hyoscyamine 6β-hydroxylase AaH6H

Yue-De Cao; Yu-Cai He; Hao Li; Guo-Yin Kai; Jian-He Xu; Hui-Lei Yu

doi:10.1016/j.jbiotec.2015.07.019

Efficient biosynthesis of rare natural product scopolamine using E. coli cells expressing a S14P/K97A mutant of hyoscyamine 6β-hydroxylase AaH6H

J Biotechnol. 2015 Oct 10:211:123-9. doi: 10.1016/j.jbiotec.2015.07.019. Epub 2015 Jul 31.

Authors

Yue-De Cao¹, Yu-Cai He², Hao Li¹, Guo-Yin Kai³, Jian-He Xu⁴, Hui-Lei Yu⁵

Affiliations

¹ Laboratory of Biocatalysis and Synthetic Biotechnology, State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, PR China.
² Laboratory of Biocatalysis and Bioprocessing, College of Pharmaceutical Engineering and Life Sciences, Changzhou University, Changzhou 213164, PR China.
³ Development Center of Plant Germplasm Resources, College of Life and Environment Sciences, Shanghai Normal University, Shanghai 200234, PR China.
⁴ Laboratory of Biocatalysis and Synthetic Biotechnology, State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, PR China; Shanghai Collaborative Innovation Center for Biomanufacturing, 130 Meilong Road, Shanghai 200237, PR China.
⁵ Laboratory of Biocatalysis and Synthetic Biotechnology, State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, PR China; Shanghai Collaborative Innovation Center for Biomanufacturing, 130 Meilong Road, Shanghai 200237, PR China. Electronic address: huileiyu@ecust.edu.cn.

PMID: 26239231
DOI: 10.1016/j.jbiotec.2015.07.019

Abstract

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.

Keywords: Biocatalysis; Biotransformation; Hyoscyamine; Hyoscyamine 6β-hydroxylase; Scopolamine; Site-directed mutagenesis.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Biocatalysis
Biological Products / metabolism*
Bioreactors
Biotransformation
Escherichia coli / cytology*
Hydroxylation
Hyoscyamine / metabolism
Mixed Function Oxygenases / metabolism*
Mutagenesis, Site-Directed
Mutant Proteins / metabolism*
Scopolamine / isolation & purification
Scopolamine / metabolism*
Solanaceae / enzymology
Solanaceous Alkaloids / metabolism
Substrate Specificity

Substances

Biological Products
Mutant Proteins
Solanaceous Alkaloids
anisodamine
Scopolamine
Mixed Function Oxygenases
hyoscyamine (6S)-dioxygenase
Hyoscyamine