The catabolism of ethylene glycol by Rhodococcus jostii RHA1 and its dependence on mycofactocin.

Ethylene glycol (EG) is a widely used industrial chemical with manifold applications and also generated in the degradation of plastics such as polyethylene terephthalate. Rhodococcus jostii RHA1 (RHA1), a potential biocatalytic chassis, grows on EG. Transcriptomic analyses revealed four clusters of genes potentially involved in EG catabolism: the mad locus, predicted to encode mycofactocin-dependent alcohol degradation, including the catabolism of EG to glycolate; two GCL clusters, predicted to encode glycolate and glyoxylate catabolism; and the mft genes, predicted to specify mycofactocin biosynthesis. Bioinformatic analyses further revealed that the mad and mft genes are widely distributed in mycolic acid-producing bacteria such as RHA1. Neither ΔmadA nor ΔmftC RHA1 mutant strains grew on EG but grew on acetate. In resting cell assays, the ΔmadA mutant depleted glycolaldehyde but not EG from culture media. These results indicate that madA encodes a mycofactocin-dependent alcohol dehydrogenase that initiates EG catabolism. In contrast to some mycobacterial strains, the mad genes did not appear to enable RHA1 to grow on methanol as sole substrate. Finally, a strain of RHA1 adapted to grow ~3× faster on EG contained an overexpressed gene, aldA2, predicted to encode an aldehyde dehydrogenase. When incubated with EG, this strain accumulated lower concentrations of glycolaldehyde than RHA1. Moreover, ecotopically expressed aldA2 increased RHA1's tolerance for EG further suggesting that glycolaldehyde accumulation limits growth of RHA1 on EG. Overall, this study provides insights into the bacterial catabolism of small alcohols and aldehydes and facilitates the engineering of Rhodococcus for the upgrading of plastic waste streams.IMPORTANCEEthylene glycol (EG), a two-carbon (C2) alcohol, is produced in high volumes for use in a wide variety of applications. There is burgeoning interest in understanding and engineering the bacterial catabolism of EG, in part to establish circular economic routes for its use. This study identifies an EG catabolic pathway in Rhodococcus, a genus of bacteria well suited for biocatalysis. This pathway is responsible for the catabolism of methanol, a C1 feedstock, in related bacteria. Finally, we describe strategies to increase the rate of degradation of EG by increasing the transformation of glycolaldehyde, a toxic metabolic intermediate. This work advances the development of biocatalytic strategies to transform C2 feedstocks.

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

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, ΔpobA, 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 ΔpcaL strain grew on neither p-MBA nor veratrate, indicating they are catabolized through the ß-ketoadipate pathway. This study expands our understanding of the bacterial catabolism of aromatic compounds and facilitates the development of biocatalysts for lignin valorization.IMPORTANCELignin, 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.

Characterization of a phylogenetically distinct extradiol dioxygenase involved in the bacterial catabolism of lignin-derived aromatic compounds.

The actinobacterium Rhodococcus jostii RHA1 grows on a remarkable variety of aromatic compounds and has been studied for applications ranging from the degradation of polychlorinated biphenyls to the valorization of lignin, an underutilized component of biomass. In RHA1, the catabolism of two classes of lignin-derived compounds, alkylphenols and alkylguaiacols, involves a phylogenetically distinct extradiol dioxygenase, AphC, previously misannotated as BphC, an enzyme involved in biphenyl catabolism. To better understand the role of AphC in RHA1 catabolism, we first showed that purified AphC had highest apparent specificity for 4-propylcatechol (kcat/KM ∼106 M-1 s-1), and its apparent specificity for 4-alkylated substrates followed the trend for alkylguaiacols: propyl > ethyl > methyl > phenyl > unsubstituted. We also show AphC only poorly cleaved 3-phenylcatechol, the preferred substrate of BphC. Moreover, AphC and BphC cleaved 3-phenylcatechol and 4-phenylcatechol with different regiospecificities, likely due to the substrates' binding mode. A crystallographic structure of the AphC·4-ethylcatechol binary complex to 1.59 Å resolution revealed that the catechol is bound to the active site iron in a bidentate manner and that the substrate's alkyl side chain is accommodated by a hydrophobic pocket. Finally, we show RHA1 grows on a mixture of 4-ethylguaiacol and guaiacol, simultaneously catabolizing these substrates through meta-cleavage and ortho-cleavage pathways, respectively, suggesting that the specificity of AphC helps to prevent the routing of catechol through the Aph pathway. Overall, this study contributes to our understanding of the bacterial catabolism of aromatic compounds derived from lignin, and the determinants of specificity in extradiol dioxygenases.

Cytochromes P450 in the biocatalytic valorization of lignin.

The valorization of lignin is critical to establishing sustainable biorefineries as we transition away from petroleum-derived feedstocks. Advances in lignin fractionation and depolymerization are yielding new opportunities for the biocatalytic upgrading of lignin-derived aromatic compounds (LDACs) using microbial cell factories. Given their roles in lignin metabolism and their catalytic versatility, cytochromes P450 are attractive enzymes in engineering such biocatalysts. Here we highlight P450s that catalyze aromatic O-demethylation, a rate-limiting step in the conversion of LDACs to valuable chemicals, including efforts to engineer the specificity of these enzymes and to use them in developing biocatalysts. We also discuss broader opportunities at the intersection of biochemistry, structure-guided enzyme engineering, and metabolic engineering for application of P450s in the emerging area of microbial lignin valorization.