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 Rhodococcus rhodochrous 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 (gyrB) and protein translocase subunit A (secA) 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 R. rhodochrous IEGM 1362 and lay the foundation for further studies of molecular and genetic mechanisms of monoterpenoid biotransformation.