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Mycolicibacterium smegmatis mc2155 has been genetically modified to be used as a platform for the expression of heterologous cytochrome P450 monooxygenases by introducing deletions in the kshB and kstD genes that encode key stages of the steroid nucleus enzymatic degradation. Three sets of genetic constructs have been created for heterologous expression of the genes encoding P450 cytochromes CYP106A1 from Bacillus megaterium DSM319 and CYP106A2 from B. megaterium ATCC13368 in M. smegmatis mc2155 (ΔkshBΔkstD) cells. The recombinant plasmids contained monocistronic expression cassettes of cytochrome genes (pNS31 and pNS32), tricistronic cassettes of cytochrome genes together with cDNA copies of adrenodoxin and adrenodoxin reductase genes of the bovine adrenal cortex (pNS33 and pNS34), or monocistronic gene cassettes of chimeric cytochromes fused with the DNA sequence encoding the CYP116B2 reductase domain from the soil bacterium Rhodococcus sp. NCIMB 9784 (pNS35 and pNS36). The recombinant strains of mycolicibacteria were shown to selectively monohydroxylate androstenedione (AD) under growth conditions. The product was identified as 15β-hydroxyandrostenedione (15β-OH-AD) by mass spectrometry and 1H and 13C NMR spectroscopy. The maximum level of 15β-OH-AD production (17.3 ± 1.5 mg/L) was observed when using the recombinant M. smegmatis mc2155 (ΔkshBΔkstD) (pNS32) strain, which expresses a single cyp106A2 gene from B. megaterium ATCC13368. Host proteins of M. smegmatis mc2155 were shown to be capable of supplying electrons to heterologous cytochromes to support their hydroxylation activity. The results we obtained are of a priority character, expand the understanding of the hydroxylation of steroid compounds by bacterial cytochromes CYP106A1/A2, and are important for the creation of microbial strains for producing valuable hydroxysteroids. [ABSTRACT FROM AUTHOR]