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Background: The production of androstenedione (AD) from phytosterols by Mycolicibacterium neoaurum is a multi-step biotransformation process, which requires degradation of sterol side chains, accompanied by the production of propionyl-CoA. However, the transient production of large amounts of propionyl-CoA can accumulate intracellularly to produce toxic effects and severely inhibit AD production. Results: In the present study, the intracellular propionyl-CoA concentration was effectively reduced and the productivity of the strain was improved by enhancing the cytosolic methyl-branched lipid synthesis pathway and increasing the expression level of nat operator gene, respectively. Subsequently, the application of a pathway combination strategy, combined and the inducible regulation strategy, further improved AD productivity with a maximum AD conversion rate of 96.88%, an increase of 13.93% over the original strain. Conclusions: Overall, we provide a new strategy for reducing propionyl-CoA stress during biotransformation for the production of AD and other steroidal drugs using phytosterols. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The low availability of hydrophobic substrates limited the bioproduction of steroid medications. As palygorskite (Pal) has excellent adsorption and dispersion capacity, Pal and modified Pal were incubated with Mycobacterium neoaurum TCCC 11979 (MNR) to investigate their effects on phytosterols (PS) biotransformation. The production of the steroid medicine intermediate androst-4-ene-3, 17-dione (AD) was remarkably improved with Pal or modified Pal as a carrier in PS biotransformation. Entropy and gas chromatography analyses confirmed that Pal samples increased the dispersion and adsorption of PS. Meanwhile, the cell permeability of MNR by nucleic acid improved. Among Pal samples, KH550 modified Pal showed superior properties, which results in the good dispersion of substrate and effectively improves the cell permeability without affecting cell growth (confirmed by TEM). Therefore, Pal, particularly KH550 modified Pal, can be used as a carrier in AD production by MNR. The data obtained will expand the application of Pal in the pharmaceutical industry and improve our understanding of the mechanism underlying the effect of Pal on the bioconversion of hydrophobic compounds. • It was the first time to apply Pal in steroids biotransformation. • The AD production was effectively improved with Pal or modified Pal as carrier in PS biotransformation of MNR. • Pal, especially KH550 modified Pal, increased the dispersion and adsorption of PS. • Pal, especially KH550 modified Pal, improved the cell permeability of MNR. [ABSTRACT FROM AUTHOR]