Expression of Synthetic cyp102A1-LG23 Gene and Functional Analysis of Recombinant Cytochrome P450 BM3-LG23 in the Actinobacterium Mycolicibacterium smegmatis

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Veronika Y. Poshekhontseva^1,a*, Nikolai I. Strizhov^1,b, Mikhail V. Karpov^1,c, Vera M. Nikolaeva^1,d, Alexey V. Kazantsev^2,e, Olesya I. Sazonova^1,f, Andrey A. Shutov^1,g, and Marina V. Donova^1,h

¹Skryabin Institute of Biochemistry and Physiology of Microorganisms, Pushchino Center for Biological Research, Russian Academy of Sciences, 142290 Pushchino, Moscow Region, Russia

²Faculty of Chemistry, Lomonosov Moscow State University, 119991 Moscow, Russia

^* To whom correspondence should be addressed.

Received May 16, 2023; Revised June 15, 2023; Accepted June 16, 2023

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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.

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KEY WORDS: cytochrome CYP102A1 (P450 BM3), heterologous expression, hydroxylation, 7β-hydroxyandrost-4-ene-3,17-dione, Mycolicibacterium smegmatis, bioconversion, steroids

DOI: 10.1134/S0006297923090146

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