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

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. 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.sup.- 1 to 66.25 mg L.sup.- 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.sup.- 1 7[beta]-OH-AD in the cofactor recycling and regeneration system. 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.