Terpenoids and steroids are secondary plant and animal metabolites and are widely used to produce highly effective pharmacologically significant compounds. One of the promising approaches to the transformation of these compounds to form bioactive metabolites is their transformation using microorganisms. Rhodococcus spp. are one of the most developed objects in biotechnology due to their exceptional metabolic capabilities and resistance to extreme environmental conditions. In this review, information on the processes of biotransformation of terpenoid and steroid compounds by actinomycetes of the genus Rhodococcus and their molecular genetic bases are most fully collected and analyzed for the first time. Examples of the use of both native whole-cell catalysts and mutant strains and purified enzyme systems for the production of derivatives of terpenoids and steroids are given.

The ability of actinobacteria of the genus Rhodococcus to biotransform the monoterpenoid (–)-isopulegol has been established for the first time. R. rhodochrous strain IEGM 1362 was selected as a bacterium capable of metabolizing (–)-isopulegol to form new, previously unknown, 10-hydroxy (2) and 10-carboxy (3) derivatives, which may presumably have antitumor activity and act as respiratory stimulants and cancer prevention agents. In the experiments, optimal conditions were selected to provide the maximum target catalytic activity of rhodococci. Using up-to-date (TEM, AFM-CLSM, and EDX) and traditional (cell size, roughness, and zeta potential measurements) biophysical and microbiological methods, it was shown that (–)-isopulegol and halloysite nanotubes did not negatively affect the bacterial cells. The data obtained expand our knowledge of the biocatalytic potential of rhodococci and their possible involvement in the synthesis of pharmacologically active compounds from plant derivatives.

Background: This study explored the biotransformation of (–)-isopulegol using immobilized cells of Rhodococcus rhodochrous IEGM 1362 to optimize the production of new bioactive compounds. Methods: An efficient biocatalyst based on R. rhodochrous IEGM 1362 cells immobilized in a macroporous polyvinyl alcohol (PVA) cryogel matrix was developed for the production of bioactive derivatives of (–)-isopulegol. The biological characteristics of the immobilized cells were investigated using scanning and transmission electron microscopy and energy-dispersive X-ray spectroscopy methods. Results: The use of the biocatalyst increased the overall yield of target products from 54% with free cells to 87% with immobilized cells in a single cycle. Major derivatives identified included (1R,2S,5R)-5-(hydroxymethyl)-2-(prop-1-en-2-yl)cyclohexanol and (1R,3R,4S)-3-hydroxy-4-(prop-1-en-2-yl)cyclohexanecarboxylic acid, both exhibiting potential pharmacological activity. The biocatalyst retained functional activity toward monoterpenoid over 13 exploitation cycles, meeting industrial biotechnology requirements. Immobilized cells were characterized by the absence of endogenous reserve inclusions (in particular lipids) and a high intracellular iron content. Conclusions: The developed immobilized biocatalyst is promising for scaling up the production of biologically active compounds.

A genome of Rhodococcus rhodochrous IEGM 1362 was sequenced and annotated. This strain can transform monoterpene alcohol (–)-isopulegol with the formation of two novel pharmacologically promising metabolites. Nine genes encoding cytochrome P450, presumably involved in (–)-isopulegol transformation, were found in the genome of R. rhodochrous IEGM 1362. Primers and PCR conditions for their detection were selected. The obtained data can be used for the further investigation of genes encoding enzymes involved in monoterpene biotransformation.

Background/Objectives: Rhodococcus rhodochrous IEGM 107 cells exhibit pronounced catalytic activity toward mono- and diterpenoids. However, the genetics and enzymatic foundations underlying this activity remain poorly understood. Methods: Using new-generation sequencing, the R. rhodochrous IEGM 107 whole genome was sequenced. Bioinformatic analysis and PCR were employed to identify and characterize genes, with a focus on cytochromes P450 (CYP450s). Results: The catalytic potential of R rhodochrous IEGM 107 was revealed. Its CYP450 genes were detected and analyzed, providing information on the enzymatic base of the strain related to the biotransformation of terpenoids. Conclusions: These findings enhance the understanding of the molecular and genetic basis for terpenoid transformations in R. rhodochrous actinomycetes. The results provide a foundation for future studies on gene expression and enzyme characterization aimed at developing efficient and selective biocatalysts for mono- and diterpenoid transformations.

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.