Expression of the synthetic CYP102A1-LG23 gene and functional analysis of recombinant P450 BM3-LG23 cytochrome in actinobacteria Mycolicibacterium smegmatis

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Abstract

Cytochrome CYP102A1 (P450 BM3) from Priestia megaterium (bas. Bacillus megaterium) has a number of specific features making it an ideal target for directed evolution and other synthetic applications. Previously, the CYP102A1-LG23 mutant with 14 mutations in the heme was obtained providing 7β-hydroxylation of steroid substrates of the androstane series with the formation of products possessing anti-inflammatory and neuroprotective activity. In this study, the synthetic cyp102A1-LG23 gene encoding the P450 BM3 mutant variant was expressed in Mycolicibacterium smegmatis cells as part of mono- and bicistronic operons together with the synthetic gdh or zwf2 genes encoding glucose dehydrogenase (GDH) and glucose-6-phosphate dehydrogenase (G6PD), respectively. The functional activity of the recombinant enzymes was shown in vivo by the example of hydroxylation of androst-4-ene-3,17-dione (AD) to 7β-OH-AD in growing cultures of mycolicibacteria. Biocatalytic activity was doubled by increasing the CYP102A1-LG23 protein solubility in the cell and organizing the cofactor regeneration additional system by introducing GDH and G6PD. The maximum level of 7β-OH-AD amounting 37,68 mol % was achieved by co-expression the cyp102A1-LG23 and gdh genes in M. smegmatis. The results evidence to the perspective of using synthetic genes to obtain recombinant enzymes, expand the understanding of the hydroxylation of steroid compounds by bacterial cytochromes and can be demand for the methods of microbiological production of 7β-hydroxylated steroids by genetically modified mycolicibacteria.

About the authors

V. Y Poshekhontseva

G. K. Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Federal Research Center “Pushchino Center for Biological Research of the Russian Academy of Sciences”

Email: rikahameleon@mail.ru
142290 Pushchino, Moscow Region, Russia

N. I Strizhov

G. K. Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Federal Research Center “Pushchino Center for Biological Research of the Russian Academy of Sciences”

142290 Pushchino, Moscow Region, Russia

M. V Karpov

G. K. Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Federal Research Center “Pushchino Center for Biological Research of the Russian Academy of Sciences”

142290 Pushchino, Moscow Region, Russia

V. M Nikolaeva

G. K. Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Federal Research Center “Pushchino Center for Biological Research of the Russian Academy of Sciences”

142290 Pushchino, Moscow Region, Russia

A. V Kazantsev

Faculty of Chemistry, Lomonosov Moscow State University

119991 Moscow, Russia

O. I Sazonova

G. K. Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Federal Research Center “Pushchino Center for Biological Research of the Russian Academy of Sciences”

142290 Pushchino, Moscow Region, Russia

A. A Shutov

G. K. Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Federal Research Center “Pushchino Center for Biological Research of the Russian Academy of Sciences”

142290 Pushchino, Moscow Region, Russia

M. V Donova

G. K. Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Federal Research Center “Pushchino Center for Biological Research of the Russian Academy of Sciences”

142290 Pushchino, Moscow Region, Russia

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