Molecular-Genetic Research of Rhodococcus rhodochrous IEGM 1362, an Active (–)-Isopulegol Biotransformer

The present study aimed to identify genes encoding enzymes involved in the biotransformation of monoterpenoid (-)-isopulegol by <i>Rhodococcus rhodochrous</i> IEGM 1362. This strain is able to transform (-)-isopulegol with formation of two novel metabolites with promising antitumor and analeptic activities. Cell fractions of rhodococci and specific inhibitor of cytochrome P450-dependent oxygenase activity were used to establish the localization and type of biotransformation enzymes. The expression of nine CYP450 genes selected by bioinformatics analysis was analyzed by quantitative real-time PCR (qRT-PCR). Selection of optimal reference genes for normalization of qRT-PCR results was performed using BestKeeper, Normfinder, geNorm, Delta CT, and RefFinder algorithms. As a result of these studies, the role of CYP450 enzyme complexes in the biotransformation of (-)-isopulegol was confirmed, and their cytoplasmic localization was established. The genes encoding DNA gyrase subunit B (<i>gyrB</i>) and protein translocase subunit A (<i>secA</i>) were selected as the most stable reference genes. The induced expression of the gene encoding CYP450 hydroxylase in the presence of (-)-isopulegol was determined. The obtained data allow us to identify the specific CYP450 enzyme involved in (-)-isopulegol biotransformation by <i>R</i>. <i>rhodochrous</i> IEGM 1362 and lay the foundation for further studies of molecular and genetic mechanisms of monoterpenoid biotransformation.