Catabolism of Alkylphenols in Rhodococcus via a Meta-Cleavage Pathway Associated With Genomic Islands

Abstract

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 <i>Rhodococcus rhodochrous</i> EP4 on 4-ethylphenol and applied genomic and transcriptomic approaches to elucidate alkylphenol catabolism in EP4 and <i>Rhodococcus jostii</i> RHA1. RNA-Seq and RT-qPCR revealed a pathway encoded by the <i>aphABCDEFGHIQRS</i> genes that degrades 4-ethylphenol <i>via</i> the <i>meta-</i>cleavage of 4-ethylcatechol. This process was initiated by a two-component alkylphenol hydroxylase, encoded by the <i>aphAB</i> genes, which were upregulated ~3,000-fold. Purified AphAB from EP4 had highest specific activity for 4-ethylphenol and 4-propylphenol (~2,000 U/mg) but did not detectably transform phenol. Nevertheless, a Δ<i>aphA</i> mutant in RHA1 grew on 4-ethylphenol by compensatory upregulation of phenol hydroxylase genes (<i>pheA1-3</i>). Deletion of <i>aphC</i>, encoding an extradiol dioxygenase, prevented growth on 4-alkylphenols but not phenol. Disruption of <i>pcaL</i> in the β-ketoadipate pathway prevented growth on phenol but not 4-alkylphenols. Thus, 4-alkylphenols are catabolized exclusively <i>via meta-</i>cleavage in rhodococci while phenol is subject to <i>ortho-</i>cleavage. A putative genomic island encoding <i>aph</i> genes was 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.