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

Abstract

Background 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. Results 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− 1 to 66.25 mg L− 1. 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− 1 7β-OH-AD in the cofactor recycling and regeneration system. Conclusions 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.

Choose Your Own Adventure: A Comprehensive Database of Reactions Catalyzed by Cytochrome P450 BM3 Variants

“…217,231,240 Screening SSM libraries at popular positions (74, 78, 82, 87, 188) within this class may yield significant benefits. These top (350, 354, 404, 409, 411, 413, 415, 417−419), 290 (164,217,334,348,393,423,424), 294 (420), 295 (217,348,423,424), 305, 420), 307 (211,212,403,406), 309 (391), 319 (211, 350, 389, 390, 399, 403, 404, 406, 409, 411−415), 324 (393, 394, 420 328 (3, 14, 211, 216, 217, 348, 393, 403, 406), 329 (211,406), 330 (3,14,21,211,216,396,403,405,406,408), 338 (391), 340 (420), 349 (348), 353 (164,211,217,334,348,393,403,423,424), 366 (217,334,348,393,394,…”

“…All positions of the P450 BM3 heme domain that have been mutated and show improvement toward drug/drug-like hydroxylation are shown (orange spheres). Mutated positions in the heme domain (residues 1−471) and in the reductase domain (472−1049) are listed, with refs: 8 (390), 11 (391, 392), 23(391,392), 24(393,394), 39 (391), 47(3, 11, 14, 164, 182, 200, 204, 205, 209−211, 213, 214, 217, 334, 348, 350, 351, 354, 389−419), 49(11,396,397,405,408,420), 51(11, 182, 204, 205, 209, 211, 395−397, 400, 403, 405, 406, 408), 52(217,348,420), 58(359,393,394,420), 64(200, 204, 205, 209, 210, 350, 354, 389, 396, 400, 401, 404, 407, 409−413, 415−420), 72(3,14,211,216,350,391,396,403,404,409,410,413,415), 74(182, 204, 205, 209, 211, 350, 393, 394, 400−404, 406, 407, 421, 422), 75(334,393,394,420), 76 (14), 77(3,14), 78(3,14,164,211,216,217,334,348,…”

Recent developments in the enzymatic modifications of steroid scaffolds

“…10). 94–96 Considering the atom economy of the HSDH cofactor system, Xu and coworkers switched the cofactor preference of 7β-HSDH from NADPH to NADH for cost-effective biotechnical applications. 97 In addition, these researchers recently characterized a novel NADH-dependent Rs 7β-HSDH from Roseococcus sp.…”

Aided‐efflux and high production of β‐amyrin realized by β‐cyclodextrin in situ synthesized on surface of Saccharomyces cerevisiae

“…Subsequently, the β‐amyrin‐β‐CD complexes (Figure 1b) diffuse in the medium for the good water solubility. Expect for β‐amyrin, β‐CD‐based transport strategy has been successfully applied to capturing carotenoids and quinones from Artemisia annua L. cell suspension cultures (Rizzello et al, 2014) and chiral 2‐alkanols in P. putida (Tieves et al, 2016), as well as to the inward delivery of steroidal substrates in sterol‑transforming mycobacteria (Xiong et al, 2020; Zhao et al, 2022). Moreover, it is reported that cyclodextrins affect the tensions, fluidities and permeabilities of membranes (Cox et al, 2021; Hammoud et al, 2019).…”