Steroid Hydroxylation by Mutant Cytochrome P450 BM3-LG23 Using Two Expression Chassis

The unique cytochrome P450 BM3 from <i>Priestia megaterium</i> (syn. <i>Bacillus megaterium</i>) is renowned for its versatile high catalytic activity. The <i>cyp102A1-LG23</i> gene encoding its CYP102A1-LG23 mutant variant was expressed in <i>Escherichia coli</i> and <i>Mycolicibacterium smegmatis</i>. 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.