Whole-genomic and transcriptomic analyses elucidate <i>p</i> -cresol and styrene degradation metabolism in <i>Rhodococcus opacus</i> 1CP

The genus <i>Rhodococcus</i> comprises numerous strains recognized for degradation of pollutants and production of secondary metabolites, including biosurfactants, lignin breakdown, and utilization of volatile organic compounds. Often, gene redundancies and the evolution of alternative pathways are attributed to such characteristics. <i>Rhodococcus opacus</i> 1CP, initially isolated as a chlorophenol-degrading strain, was found to be a model organism comprising several such features. In this study, we analyzed the genome and transcriptome and provided evidence that the strain 1CP uses three different pathways of <i>ortho-</i>, <i>meta-</i>, and side-chain attack for the degradation of aromatic compounds. The wild-type strain and the single and double knock-out mutants of phenol hydroxylases were able to attack substituted phenols via the classical <i>ortho</i>-route. In contrast, the triple knock-out mutant expressed <i>meta</i>-pathway genes to act on <i>p</i>-cresol, indicating that this pathway serves as a reserve in strain 1CP. Growth of 1CP on phenol, <i>p</i>-cresol, and styrene induces several gene clusters that are associated with lignin metabolization. Catechol, protocatechuate, and phenylacetic acid are major key intermediates that are funneled into central metabolic pathways, which enable strain 1CP to degrade acetophenone, benzoate, phenol, 2-phenylethanol, and styrene. Strain 1CP possesses an alternative, modified <i>ortho</i>-cleavage pathway that allows it to degrade 2-chlorophenol. Interestingly, in almost all cases, redundant genes were identified; however, only in a few cases, such as phenol hydroxylases, were they found to be active and simultaneously involved in metabolic activities. The transcriptomic and kinetic data showed that the redundant styrene oxide isomerase is upregulated and involved in styrene degradation.IMPORTANCE<i>Rhodococcus opacus</i> 1CP is a model organism for various biotechnological applications due to its capabilities to metabolize a vast range of aromatic and xenobiotic compounds. Although strain 1CP has been used for decades in bioremediation, the complete metabolic pathways underlying degradation have never been elucidated. In this study, the ability of the strain to bypass phenol hydroxylase deletions and degrade substituted phenols is described using genomics, transcriptomics, and gene knock-out analyses. Despite its metabolic versatility, strain 1CP has been reported only for the <i>ortho</i>-cleavage pathway. No enzymatic or metabolic evidence has supported the presence of a <i>meta</i>-cleavage pathway to degrade aromatic compounds. Genes associated with such <i>meta</i>-pathways have been identified, but are not clustered with known degradation operons. In this study, we demonstrate that a triple knock-out mutant can utilize a <i>meta</i>-cleavage pathway for the degradation of substituted phenols.