A Novel 3-Phytosterone-9α-Hydroxylase Oxygenation Component and Its Application in Bioconversion of 4-Androstene-3,17-Dione to 9α-Hydroxy-4-Androstene-3,17-Dione Coupling with A NADH Regeneration Formate Dehydrogenase
Xian Zhang,
Manchi Zhu,
Rumeng Han,
Youxi Zhao,
Kewei Chen,
Kai Qian,
Minglong Shao,
Taowei Yang,
Meijuan Xu,
Jianzhong Xu,
Zhiming Rao
Affiliations
Xian Zhang
The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Liu Avenue, Wuxi 214122, Jiangsu, China
Manchi Zhu
The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Liu Avenue, Wuxi 214122, Jiangsu, China
Rumeng Han
The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Liu Avenue, Wuxi 214122, Jiangsu, China
Youxi Zhao
Biochemical Engineering College, Beijing Union University, Beijing 100023, China
Kewei Chen
School of Food Science and Technology, Jiangnan University, 1800 Liu Avenue, Wuxi 214122, Jiangsu, China
Kai Qian
School of Medicine, Yichun University, Yichun 336000, Jiangxi, China
Minglong Shao
The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Liu Avenue, Wuxi 214122, Jiangsu, China
Taowei Yang
The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Liu Avenue, Wuxi 214122, Jiangsu, China
Meijuan Xu
The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Liu Avenue, Wuxi 214122, Jiangsu, China
Jianzhong Xu
The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Liu Avenue, Wuxi 214122, Jiangsu, China
Zhiming Rao
The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Liu Avenue, Wuxi 214122, Jiangsu, China
9α-Hydroxy-4-androstene-3,17-dione (9-OH-AD) is one of the significant intermediates for the preparation of β-methasone, dexamethasone, and other steroids. In general, the key enzyme that enables the biotransformation of 4-androstene-3,17-dione (AD) to 9-OH-AD is 3-phytosterone-9α-hydroxylase (KSH), which consists of two components: a terminal oxygenase (KshA) and ferredoxin reductase (KshB). The reaction is carried out with the concomitant oxidation of NADH to NAD+. In this study, the more efficient 3-phytosterone-9α-hydroxylase oxygenase (KshC) from the Mycobacterium sp. strain VKM Ac-1817D was confirmed and compared with reported KshA. To evaluate the function of KshC on the bioconversion of AD to 9-OH-AD, the characterization of KshC and the compounded system of KshB, KshC, and NADH was constructed. The optimum ratio of KSH oxygenase to reductase content was 1.5:1. An NADH regeneration system was designed by introducing a formate dehydrogenase, further confirming that a more economical process for biological transformation from AD to 9-OH-AD was established. A total of 7.78 g of 9-OH-AD per liter was achieved through a fed-batch process with a 92.11% conversion rate (mol/mol). This enzyme-mediated hydroxylation method provides an environmentally friendly and economical strategy for the production of 9-OH-AD.
