Cytochrome CYP102A1 (P450 BM3) of Priestia megaterium (bas. Bacillus megaterium) has several unique functional features and thus provides an ideal object for directed evolution and other synthetic applications. Previously, the CYP102A1-LG23 mutant with 14 mutations in the heme part was obtained that hydroxylates several androstanes at C7β with the formation of products with the anti-inflammatory and neuroprotective activities. In this study, synthetic cyp102A1-LG23 gene encoding the P450 BM3 mutant was expressed as a component of either monocistronic operon or bicistronic operon containing the gdh (glucose dehydrogenase, GDH) or zwf2 (glucose 6-phosphate dehydrogenase, G6PD) gene in Mycolicibacterium smegmatis BD cells. The recombinant bacteria were able hydroxylate androst-4-ene-3,17-dione (AD) into 7β-OH-AD. Their biocatalytic activity was increased twice by increasing the solubility of CYP102A1-LG23 protein in the cells and supplementing the cells with the additional cofactor regeneration system by introducing GDH and G6PD. The maximum 7β-OH-AD yield (37.68 mol%) was achieved by co-expression of cyp102A1-LG23 and gdh genes in M. smegmatis. These results demonstrate the possibility of using synthetic genes to obtain recombinant enzymes and expand our understanding of the processes involved in steroid hydroxylation by bacterial cytochromes. The data obtained can be used to develop new approaches for microbiological production of 7β-hydroxylated steroids in genetically modified Mycolicibacterium species.

The unique cytochrome P450 BM3 from Priestia megaterium (syn. Bacillus megaterium) is renowned for its versatile high catalytic activity. The cyp102A1-LG23 gene encoding its CYP102A1-LG23 mutant variant was expressed in Escherichia coli and Mycolicibacterium smegmatis. The in vivo activity of the heterologous enzyme was assessed with respect to androstenedione (AD), androstadienedione (ADD), testosterone (TS) and dehydroepiandrosterone (DHEA). Alongside 7β-hydroxylation, the heterologous enzyme catalyzed the mono- and dihydroxylation of C19 steroids. For the first time, the formation of 7β-, 6β- and 11α-hydroxylated derivatives of ADD using a bacterial enzyme, as well as the hydroxylation of DHEA at the C7α and C7β positions, and its dihydroxylation with the formation of the 7α,15α-dihydroxylated derivative using the mutant cytochrome P450 BM3 were demonstrated. The steroid structures were confirmed using mass spectrometry and ¹H NMR spectroscopy. The advantages of using mycolicibacteria as a bacterial chassis for gene expression were also shown. The results demonstrate the unusual properties of the mutant cytochrome P450 BM3-LG23 and open up prospects for its application in the biotechnological production of valuable hydroxysteroids.