Natalia A. Plotnitskaya

Background: The biotransformation of the monoterpenoid (−)-isopulegol by Rhodococcus rhodochrous yields valuable diol and hydroxy acid derivatives; however, the yields are moderate, and formation of the (S)-epoxide byproduct significantly reduces the overall selectivity. Metal oxide nanoparticles (NPs) can modulate biocatalytic activity; however, their effects on monoterpenoid biotransformation are poorly understood. Methods: We investigated the effects of NiO (50 mg/L) and CuO NPs (5 mg/L) on (−)-isopulegol conversion by R. rhodochrous IEGM 1362. Products were quantified by GC–MS; cellular responses were assessed using respirometry, zeta potential measurements, AFM-CLSM, TEM, SEM, EDX, and cell viability assays. Results: NiO NPs at 50 mg/L significantly increased the total yield of target compounds (diol and hydroxy acid) from 36.1% to 48.3%. Importantly, NiO suppressed the formation of the undesired (S)-epoxide. In contrast, CuO NPs (5 mg/L) completely abolished target product formation. Both NPs reduced cell respiratory activity, shifted zeta potential toward less negative values, and induced cell aggregation and ultrastructural damage. NiO NPs were adsorbed onto the cell surface. Conclusions: NiO NPs selectively enhance the yield of valuable oxygenated metabolites of (−)-isopulegol while inhibiting the competing epoxidation pathway. Conversely, CuO NPs are detrimental and unsuitable for this bioprocess.

Non-pathogenic actinomycetes play a key role in the degradation of xenobiotics and the biotransformation of complex organic compounds. Studying gene expression in these bacteria can be challenging due to the rigid cell wall that hinders RNA extraction. Here we present the first comprehensive study of total RNA isolation from Rhodococcus and Gordonia genera, encompassing 21 strains grown under 34 conditions, including n-alkanes C₃–C₁₆, terpenoids, non-steroidal anti-inflammatory drugs, used frying oil, and mercury chloride. We compared no lysis strategy and three cell lysis approaches (freezing-thawing, bead beating, and lysozyme treatment) combined with the TRIzol or silica column-based RNA extraction kits. Bead beating, despite aggressive mechanical action, failed to reliably yield intact RNA. We show for the first time that 40 mg/mL of lysozyme with prolonged exposure (1 h) reliably lyses Rhodococcus and Gordonia cells (96–100% successful isolations), yielding pure integral RNA (118.5–485.8 ng/µL) with the RNA Solo Kit, without requiring detergents. We also developed the first protocol for RNA extraction from Rhodococcus cells grown on used frying oil, requiring pre-washing with 0.1 vol.% Tween 80 to disintegrate hard-to-harvest aggregates. These findings establish a robust strategy for RNA isolation from hydrophobic Rhodococcus and Gordonia bacteria.

Background/Objectives: Microbial biotransformation of monoterpenoids is a promising approach for obtaining bioactive compounds. Rhodococcus species are attractive biocatalysts due to their metabolic versatility and ability to transform hydrophobic substrates. In this study, we investigated the catalytic potential of Rhodococcus qingshengii IEGM 267 toward carveol isomers and explored genomic features that may underlie this activity. Methods: The strain was cultivated in mineral medium supplemented with (−)-trans-carveol. Biotransformation products were analyzed by TLC and GC–MS. The draft genome was sequenced, assembled, taxonomically assigned, and annotated using standard bioinformatics tools. Results: Rhodococcus qingshengii IEGM 267 efficiently converted (−)-trans-carveol to carvone. Genome analysis confirmed the taxonomic assignment of the strain and revealed a large repertoire of oxidoreductases, including monooxygenases, hydroxylases, and dehydrogenases. Seven genes encoding cytochrome P450-dependent oxygenases were identified as candidate enzymes potentially involved in carveol oxidation. Conclusions: R. qingshengii IEGM 267 is an efficient and stereoselective biocatalyst for (−)-trans-carveol oxidation. The results of bioinformatics analysis suggest an alternative enzymatic basis for this transformation and provide a foundation for future functional characterization.

We report the draft genome sequence of Rhodococcus rhodochrous IEGM 1360. This strain was isolated from Franz Josef Land and can transform oleanolic and glycyrrhetinic acids, pentacyclic triterpenoids commonly used for obtaining pharmacologically significant compounds. Using the sequence, putative genes encoding enzymes involved in the transformation of triterpenoids were discovered.

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.

We report the draft genome sequence of Rhodococcus pyridinivorans IEGM 66. This strain is able to transform a pentacyclic triterpenoid, betulin, commonly used as a scaffold for synthesizing bioactive compounds. Using the obtained sequence, putative genes encoding enzymes involved in the oxidative transformation of betulin were revealed.

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.

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.

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.