Transcriptional regulation of the type II fatty acid synthase complex in Rhodococcus opacus

Rhodococcus opacus is a model oleaginous microorganism known for its ability to accumulate lipids, primarily triacylglycerols, by catabolising a variety of carbon sources, including renewable and cost-effective resources. This study systematically investigated the fatty acid biosynthetic pathways in R. opacus to enhance its application for lipid and derivative molecule production. Differential gene expression analysis revealed that the expression of the FASII gene cluster is influenced by temperature, pH, carbon to nitrogen ratio, and the addition of free fatty acids, although these effects were not always uniform across the operon. The variability observed may be due to internal transcriptional start sites within the operon, indicating a complex regulatory mechanism. Additionally, we identified and characterised three lipid-responsive regulators, MabR RO , FadR1 RO , and FadR2 RO , which bind to the fasII promoter in vitro . Electrophoretic mobility shift assays (EMSAs) showed that DNA binding of FadR1 RO and FadR2 RO is disrupted by a long-chain acyl-CoA ligand. Comparative analysis of the upstream regions of the FASII gene cluster across the Mycobacteriales taxon revealed conserved motifs, including potential -35 and -10 boxes and transcriptional regulator binding sequences. EMSAs with mutated probes identified key residues critical for binding affinity, confirming the hypothesized binding sequences. This study elucidates the intricate regulation of the FASII operon in R. opacus PD630, highlighting the complexity of its regulatory system. These findings contribute to a broader understanding of the regulatory mechanisms within Mycobacteriales, offering insights into the diversification and specialization of these mechanisms across different species, while also emphasising conserved elements.