title: Efficient biosynthesis of rare natural product scopolamine using E. coli cells expressing a S14P/K97A mutant of hyoscyamine 6β-hydroxylase AaH6H.
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.
authors.affiliation: Laboratory of Biocatalysis and Synthetic Biotechnology, State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, PR China.
authors.forename: Yue-De
authors.initials: YD
authors.lastname: Cao
authors.affiliation: Laboratory of Biocatalysis and Bioprocessing, College of Pharmaceutical Engineering and Life Sciences, Changzhou University, Changzhou 213164, PR China.
authors.forename: Yu-Cai
authors.initials: YC
authors.lastname: He
authors.affiliation: Laboratory of Biocatalysis and Synthetic Biotechnology, State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, PR China.
authors.forename: Hao
authors.initials: H
authors.lastname: Li
authors.affiliation: Development Center of Plant Germplasm Resources, College of Life and Environment Sciences, Shanghai Normal University, Shanghai 200234, PR China.
authors.forename: Guo-Yin
authors.initials: GY
authors.lastname: Kai
authors.affiliation: 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.
authors.forename: Jian-He
authors.initials: JH
authors.lastname: Xu
authors.affiliation: 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.
authors.forename: Hui-Lei
authors.initials: HL
authors.lastname: Yu
mesh_terms: D055162:Biocatalysis; D001688:Biological Products / Q000378:metabolism*; D019149:Bioreactors; D001711:Biotransformation; D004926:Escherichia coli / Q000166:cytology*; D006900:Hydroxylation; D064692:Hyoscyamine / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000378:metabolism*; D012601:Scopolamine / Q000302:isolation & purification / Q000378:metabolism*; D019657:Solanaceae / Q000201:enzymology; D012991:Solanaceous Alkaloids / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D001688:Biological Products; D050505:Mutant Proteins; D012991:Solanaceous Alkaloids; C003922:anisodamine; D012601:Scopolamine; D006899:Mixed Function Oxygenases; C052273:hyoscyamine (6S)-dioxygenase; D064692:Hyoscyamine
keywords: Biocatalysis; Biotransformation; Hyoscyamine; Hyoscyamine 6β-hydroxylase; Scopolamine; Site-directed mutagenesis