文献类型：期刊文献

英文题名：One-pot biosynthesis of 7β-hydroxyan drost-4-ene-3,17-dione from phytosterols by cofactor regeneration system in engineered mycolicibacterium neoaurum

作者：Zhao, Yun-Qiu[1];Liu, Yong-Jun[1];Ji, Wei-Ting[1];Liu, Kun[1];Gao, Bei[1];Tao, Xin-Yi[1];Zhao, Ming[1];Wang, Feng-Qing[1];Wei, Dong-Zhi[1]

机构：[1]East China Univ Sci & Technol, Newworld Inst Biotechnol, State Key Lab Bioreactor Engn, Shanghai 200237, Peoples R China

年份：2022

卷号：21

期号：1

外文期刊名：MICROBIAL CELL FACTORIES

收录：;WOS:【SCI-EXPANDED(收录号:WOS:000781361900002)】；

基金：This work was financially supported by the National Natural Science Foundation of China (No. 21776075), the Natural Science Foundation of Shanghai (No. 20ZR1415100), and the National Key Research and Development Program of China (No. SQ2020YFC210061).

语种：英文

外文关键词：7 beta-hydroxyandrost-4-ene-3,17-dione; Phytosterols; mP450-BM3; Nicotinamide adenine dinucleotide phosphate regeneration; Mycolicibacterium

摘要：Background: 7 beta-hydroxylated steroids (7 beta-OHSt) possess significant activities in anti-inflammatory and neuroprotection, and some of them have been widely used in clinics. However, the production of 7 beta-OHSt is still a challenge due to the lack of cheap 7 beta-hydroxy precursor and the difficulty in regio- and stereo-selectively hydroxylation at the inert C7 site of steroids in industry. The conversion of phytosterols by Mycolicibacterium species to the commercial precursor, androst-4-ene-3,17-dione (AD), is one of the basic ways to produce different steroids. This study presents a way to produce a basic 7 beta-hydroxy precursor, 7 beta-hydroxyandrost-4-ene-3,17-dione (7 beta-OH-AD) in Mycolicibacterium, for 7 beta-OHSt synthesis. Results: A mutant of P450-BM3, mP450-BM3, was mutated and engineered into an AD producing strain for the efficient production of 7 beta-OH-AD. The enzyme activity of mP450-BM3 was then increased by 1.38 times through protein engineering and the yield of 7 beta-OH-AD was increased from 34.24 mg L-1 to 66.25 mg L-1. To further enhance the performance of 7 beta-OH-AD producing strain, the regeneration of nicotinamide adenine dinucleotide phosphate (NADPH) for the activity of mP450-BM3-0 was optimized by introducing an NAD kinase (NADK) and a glucose-6-phosphate dehydrogenase (G6PDH). Finally, the engineered strain could produce 164.52 mg L-1 7 beta-OH-AD in the cofactor recycling and regeneration system. Conclusions: This was the first report on the one-pot biosynthesis of 7 beta-OH-AD from the conversion of cheap phytosterols by an engineered microorganism, and the yield was significantly increased through the mutation of mP450-BM3 combined with overexpression of NADK and G6PDH. The present strategy may be developed as a basic industrial pathway for the commercial production of high value products from cheap raw materials.