One-pot biosynthesis of 7β-hydroxyandrost-4-ene-3,17-dione from phytosterols by cofactor regeneration system in engineered mycolicibacterium neoaurum

• Published in: Microbial cell factories Vol. 21; no. 1; p. 59
• Main Authors: Zhao, Yun-Qiu, Liu, Yong-Jun, Ji, Wei-Ting, Liu, Kun, Gao, Bei, Tao, Xin-Yi, Zhao, Ming, Wang, Feng-Qing, Wei, Dong-Zhi
• Format: Journal Article
• Language: English
• Published: England BioMed Central 09-04-2022; BMC
• Subjects: 7β-hydroxyandrost-4-ene-3,17-dione; Adenine; Biosynthesis; Biotransformation; Chromatography; Conversion; Dehydrogenases; Enzymatic activity; Enzyme activity; Enzymes; Glucose 6 phosphate dehydrogenase; Glucosephosphate dehydrogenase; Hydroxylation; Inflammation; Kinases; mP450-BM3; Mutagenesis; Mutation; Mycobacteriaceae; Mycolicibacterium; NAD kinase; NADPH-diaphorase; Neuroprotection; Nicotinamide; Nicotinamide adenine dinucleotide; Nicotinamide adenine dinucleotide phosphate regeneration; Phytosterols; Phytosterols - metabolism; Precursors; Protein engineering; Raw materials; Regeneration; Steroid hormones; Steroids; 7β-hydroxyandrost-4-ene-3,17-dione; Nicotinamide adenine dinucleotide phosphate regeneration; Phytosterols; mP450-BM3; Mycolicibacterium
• ISSN: 1475-2859; 1475-2859
• DOI: 10.1186/s12934-022-01786-5

7β-hydroxylated steroids (7β-OHSt) possess significant activities in anti-inflammatory and neuroprotection, and some of them have been widely used in clinics. However, the production of 7β-OHSt is still a challenge due to the lack of cheap 7β-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 7β-hydroxy precursor, 7β-hydroxyandrost-4-ene-3,17-dione (7β-OH-AD) in Mycolicibacterium, for 7β-OHSt synthesis.
A mutant of P450-BM3, mP450-BM3, was mutated and engineered into an AD producing strain for the efficient production of 7β-OH-AD. The enzyme activity of mP450-BM3 was then increased by 1.38 times through protein engineering and the yield of 7β-OH-AD was increased from 34.24 mg L
to 66.25 mg L
. To further enhance the performance of 7β-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
7β-OH-AD in the cofactor recycling and regeneration system.
This was the first report on the one-pot biosynthesis of 7β-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.