Overexpression of cytochrome p450 125 in Mycobacterium: a rational strategy in the promotion of phytosterol biotransformation

Abstract

Androst-4-ene-3, 17-dione (AD) and androst-1, 4-diene-3, 17-dione (ADD) are generally produced by the biotransformation of phytosterols in Mycobacterium. The AD (D) production increases when the strain has high NAD/NADH ratio. To enhance the AD (D) production in Mycobacterium neoaurum TCCC 11978 (MNR M3), a rational strategy was developed through overexpression of a gene involved in the phytosterol degradation pathway; NAD was generated as well. Proteomic analysis of MNR cultured with and without phytosterols showed that the steroid C27-monooxygenase (Cyp125-3), which performs sequential oxidations of the sterol side chain at the C27 position and has the oxidative cofactor of NAD generated, played an important role in the phytosterol biotransformation process of MNR M3. To improve the productivity of AD (D), the cyp125-3 gene was overexpressed in MNR M3. The specific activity of Cyp125-3 in the recombinant strain MNR M3C3 was improved by 22% than that in MNR M3. The NAD/NADH ratio in MNR M3C3 was 131% higher than that in the parent strain. During phytosterol biotransformation, the conversion of sterols increased from 84 to 96%, and the yield of AD (D) by MNR M3C3 was increased by approximately 18% for 96 h fermentation. This rational strain modification strategy may also be applied to develop strains with important application values for efficient production of cofactor-dependent metabolites.

Biotransformation of Phytosterols into Androstenedione—A Technological Prospecting Study

“…2–4 M) was able to produce 9-OH-AD as a major product (50% molar yield), in addition to AD (22% molar yield). Su et al [ 81 ], for example, overexpressed the cyp125-3 gene in Mycolicibacterium neoaurum (basonym: Mycobacterium neoaurum ) TCCC 11978 to increase AD productivity, since this gene is directly involved in phytosterol degradation and the generation of NAD+ (which is needed for AD production).…”

Rational development of mycobacteria cell factory for advancing the steroid biomanufacturing

“…Deletion of the gene greatly affected the growth of mycobacteria utilizing cholesterol as carbon source (Capyk et al 2009 ). Overexpression of CYP 125–3 in M. neoaurum M3 increased the specific activity by 22%, and the NAD + /NADH ratio was increased by 31%, the yield of AD was improved approximately 18% in the biotransformation of phytosterol (Su et al 2018 ). In addition, three isoenzymes (SMO1, SMO2, SMO3) of steroid C27 monooxygenase were identified in M. neoaurum JC-12.…”

Structures and molecular mechanisms of action of the cholesterol C17 side-chain-degrading enzymes

“…Overexpression of a ChOx, ChoM2, in M. neoaurum NwIB-R10 resulted in a 40% increase in ADD production [77]. In M. neoaurum TCCC 11978 (MNR M3), by overexpressing the gene encoding CYP125-3, the AD(D) yield was increased by approximately 18% [78]. By overexpressing hsd4A, the yield of ADD was increased by 50% and the C22-steroid by-products halved during the steroid biotransformation of M. neoaurum strain III [79].…”