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Steroid-26-monooxygenases belong to the cytochrome P450 superfamily and function as part of three-component systems together with ferredoxins and ferredoxin reductases providing electron transport. The P450-dependent redox partners of the actinobacterial strain Mycolicibacterium smegmatis mc2155 were investigated. The genes encoding mycolibacterial ferredoxins (FdxD and FdxE) and ferredoxin reductases (FdrA and FprA) were overexpressed in E. coli cells. A scheme for isolation and purification of synthesized recombinant proteins using affinity chromatography was developed, resulting in electrophoretically homogeneous preparations. Spectral analysis of ferredoxin reductase showed absorption peaks characteristic of FAD-containing proteins. The reaction of cytochrome c reduction using recombinant proteins was carried out, demonstrating that FdxD, FdxE, FdrA, and FprA can act as components of electron transport from the reducing equivalents of NAD(P)H. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]