The main male hormone, testosterone is obtained from cheap and readily available phytosterol using the strains of Mycolicibacterium neoaurum VKM Ac-1815D, or Ac-1816D. During the first “oxidative” stage, phytosterol (5–10 g/L) was aerobically converted by Ac-1815D, or Ac-1816D to form 17-ketoandrostanes: androstenedione, or androstadienedione, respectively. At the same bioreactor, the 17-ketoandrostanes were further transformed to testosterone due to the presence of 17β-hydroxysteroid dehydrogenase activity in the strains (“reductive” mode). The conditions favorable for “oxidative” and “reductive” stages have been revealed to increase the final testosterone yield. Glucose supplement and microaerophilic conditions during the “reductive” mode ensured increased testosterone production by mycolicibacteria cells. Both strains effectively produced testosterone from phytosterol, but highest ever reported testosterone yield was achieved using M. neoaurum VKM Ac-1815D: 4.59 g/l testosterone was reached from 10 g/l phytosterol thus corresponding to the molar yield of over 66%. The results contribute to the knowledge on phytosterol bioconversion by mycolicibacteria, and are of significance for one-pot testosterone bioproduction from phytosterol bypassing the intermediate isolation of the 17-ketoandrostanes. Graphical Abstract

New methods for testosterone production from phytosterol were developed based on its cascade two-stage transformation by actinobacteria Mycolicibacterium neoaurum VKM Ac-1815D and Nocardioides simplex VKM Ac-2033D. Efficient oxidation of the phytosterol side chain by M. neoaurum resulted in formation of the main and side products: androst-4-en-3,20-dione (AD) and androsta-1,4-dien-3,20-dione (ADD), respectively, which were subsequently converted to testosterone by N. simplex. The latter reaction was reversible and catalyzed by the membrane-associated 17β-hydroxysteroid dehydrogenase (17β-HSD) capable of both oxidation and reduction of androstendione at C17. Addition of glucose and limited aeration were found to be the key factors providing for a shift of the 17β-HSD activity towards reduction in whole N. simplex cells. Testosterone production from phytosterol was realized using two approaches: (i) based on M. neoaurum cells inactivation after phytosterol conversion and application of the resting N. simplex biomass for androstenedione reduction and (ii) based on sequential application of the two living cultures. Under optimized conditions, the total yield of testosterone from phytosterol (10 g/L) reached 53 mol %. The results exceeded those reported so far for cascade phytosterol bioconversion to testosterone and may be used as a basis for development of new biotechnologies for production of the valuable steroid compounds, intermediates in the synthesis of modern medical preparations.

A plasmid containing the genes encoding a fungal 17β-hydroxysteroid dehydrogenase, which ca-talyzes the reduction of 17-oxo group, and a mycobacterial glucose-6-phosphate dehydrogenase, which promotes the recycling of the essential coenzyme NAD(P)H, was constructed. Its constitutive expression in well-studied Mycolicibacterium neoaurum strains made it possible to increase the yield of C-17 hydroxysteroids significantly. In particular, recombinant strains created on the basis of M. neoaurum VKM Ac-1815D and M. neoaurum NRRL B-3805 ΔkstD exhibited predominant accumulation of testosterone, while the strain based on M. neoaurum VKM Ac-1816D accumulated 1(2)-dehydrotestosterone and testosterone simultaneously.

A plasmid containing the genes of a fungal 17β-hydroxysteroid dehydrogenase, which catalyzes the reduction of the steroid core at the C17 position, and mycobacterial glucose-6-phosphate dehydrogenase, which promotes the recycling of the essential coenzyme NAD(P)H, was constructed. Its constitutive expression in well-studied Mycolicibacterium neoaurum strains made it possible to increase significantly the yield of C-17 hydroxysteroids. In particular, recombinant strains created on the basis of M. neoaurum VKM Ac-1815D and M. neoaurum NRRL B-3805 ΔkstD exhibited predominant accumulation of testosterone, while the strain based on M. neoaurum VKM Ac-1816D accumulated dehydrotestosterone and testosterone simultaneously.

In this study, for the first time, the genes encoding the mammalian steroidogenesis system—cytochrome P450scc (CYP11A1), and its native redox partners adrenodoxin and adrenodoxin reductase—were successfully expressed in the methylobacterium Methylorubrum extorquens. The advantage of using methylobacteria as an expression chassis is that they grow on inexpensive mineral media, use methanol as a carbon and energy source, and do not possess their own sterol catabolism systems. Using recombinant methylobacteria, the valuable steroid pregnenolone was obtained as a sole metabolite from cholesterol. The effect of media composition, bioconversion conditions such as methanol and N-sources content, modes of substrate addition, detergents, methyl-β-cyclodextrin, biomass, and aeration on pregnenolone accumulation was investigated. Under optimized conditions, its yield exceeded 100 mg/L. The results demonstrate a proof of concept relating to the use of bacteria lacking their own steroid degradation systems as microbial chassis for heterologous steroidogenesis systems, including mammalian cytochrome CYP11A1.