Catabolism of alkylphenols in Rhodococcus via a meta-cleavage pathway associated with genomic islands

The bacterial catabolism of aromatic compounds has considerable promise to convert lignin depolymerization products to commercial chemicals. Alkylphenols are a key class of depolymerization products whose catabolism is not well elucidated. We isolated Rhodococcus rhodochrous EP4 on 4-ethylphenol and applied genomic and transcriptomic approaches to elucidate alkylphenol catabolism in EP4 and Rhodococcus jostii RHA1. RNA-Seq and RT-qPCR revealed a pathway encoded by the aphABCDEFGHIQRS genes that degrades 4-ethylphenol via the meta-cleavage of 4-ethylcatechol. This process was initiated by a two-component alkylphenol hydroxylase, encoded by the aphAB genes, which were up-regulated ~3,000-fold. Purified AphAB from EP4 had highest specific activity for 4-ethylphenol and 4-propylphenol (~2000 U/mg) but did not detectably transform phenol. Nevertheless, a {Delta}aphA mutant in RHA1 grew on 4-ethylphenol by compensatory up-regulation of phenol hydroxylase genes (pheA1-3). Deletion of aphC, encoding an extradiol dioxygenase, prevented growth on 4-alkylphenols but not phenol. Disruption of pcaL in the {beta}-ketoadipate pathway prevented growth on phenol but not 4-alkylphenols. Thus, 4-ethylphenol and 4-propylphenol are catabolized exclusively via meta-cleavage in rhodococci while phenol is subject to ortho-cleavage. Putative genomic islands encoding aph genes were identified in EP4 and several other rhodococci. Overall, this study identifies a 4-alkylphenol pathway in rhodococci, demonstrates key enzymes involved, and presents evidence that the pathway is encoded in a genomic island. These advances are of particular importance for wide-ranging industrial applications of rhodococci, including upgrading of lignocellulose biomass.\n\nImportanceElucidation of bacterial alkylphenol catabolism is important for the development of biotechnologies to upgrade the lignin component of plant biomass. We isolated a new strain, Rhodococcus rhodochrous EP4, on 4-ethylphenol, an alkylphenol that occurs in lignin-derived streams, including reductive catalytic fractionation products of corn stover. We further demonstrated its degradation via a meta-cleavage pathway (Aph) with transcriptomics. A new class of Actinobacterial hydroxylase, AphAB, acts specifically on alkylphenols. Phylogenomic analysis indicated that the aph genes occur on putative genomic islands in several rhodococcal strains. These genes were identified in the genetically-tractable strain Rhodococcus jostii RHA1. Strains missing this element cannot metabolise 4-ethylphenol and 4-propylphenol. Overall, we advanced the understanding of how aromatic compounds are degraded by environmental bacteria and identified enzymes that can be employed in the transition away from petro-chemicals towards renewable alternatives.