The catabolism of lignin-derived p -methoxylated aromatic compounds by Rhodococcus jostii RHA1

ABSTRACT Emergent strategies to valorize lignin, an abundant but underutilized aromatic biopolymer, include tandem processes that integrate chemical depolymerization and biological catalysis. To date, aromatic monomers from C–O bond cleavage of lignin have been converted to bioproducts, but the presence of recalcitrant C–C bonds in lignin limits the product yield. A promising chemocatalytic strategy that overcomes this limitation involves phenol methyl protection and autoxidation. Incorporating this into a tandem process requires microbial cell factories able to transform the p -methoxylated products in the resulting methylated lignin stream. In this study, we assessed the ability of Rhodococcus jostii RHA1 to catabolize the major aromatic products in a methylated lignin stream and elucidated the pathways responsible for this catabolism. RHA1 grew on a methylated pine lignin stream, catabolizing the major aromatic monomers: p -methoxybenzoate ( p -MBA), veratrate, and veratraldehyde. Bioinformatic analyses suggested that a cytochrome P450, PbdA, and its cognate reductase, PbdB, are involved in p -MBA catabolism. Gene deletion studies established that both pbdA and pbdB are essential for growth on p -MBA and several derivatives. Furthermore, a deletion mutant of a candidate p- hydroxybenzoate ( p -HBA) hydroxylase, Δ pobA , did not grow on p -HBA. Veratraldehyde and veratrate catabolism required both vanillin dehydrogenase (Vdh) and vanillate O -demethylase (VanAB), revealing previously unknown roles of these enzymes. Finally, a Δ pcaL strain grew on neither p -MBA nor veratrate, indicating they are catabolized through the β-ketoadipate pathway. This study expands our understanding of the bacterial catabolism of aromatic compounds and facilitates the development of biocatalysts for lignin valorization. IMPORTANCE Lignin, an abundant aromatic polymer found in plant biomass, is a promising renewable replacement for fossil fuels as a feedstock for the chemical industry. Strategies for upgrading lignin include processes that couple the catalytic fractionation of biomass and biocatalytic transformation of the resulting aromatic compounds with a microbial cell factory. Engineering microbial cell factories for this biocatalysis requires characterization of bacterial pathways involved in catabolizing lignin-derived aromatic compounds. This study identifies new pathways for lignin-derived aromatic degradation in Rhodococcus , a genus of bacteria well suited for biocatalysis. Additionally, we describe previously unknown activities of characterized enzymes on lignin-derived compounds, expanding their utility. This work advances the development of strategies to replace fossil fuel-based feedstocks with sustainable alternatives.