Engineering a cytochrome P450 O-demethylase for the bioconversion of hardwood lignin

Lignin is a promising alternative to petroleum as a feedstock for the chemical industry. Emergent strategies for lignin valorization involve tandem processes in which biomass is chemo-catalytically fractionated followed by biotransformation of the depolymerized lignin by microbial cell factories. A rate-limiting step in this biotransformation is O-demethylation of the lignin-derived monomers. The reductive catalytic fractionation of hardwood biomass generates high yields of two classes of monomers: 4-alkylguaiacols and 4-alkylsyringols. To better understand the biotransformation of these monomers, we studied AgcA, a cytochrome P450, and AgcB, the cognate reductase, that together catalyze the O-demethylation of 4-alkylguaiacols. A 1.82 [A] resolution crystal structure of AgcAEP4 from Rhodococcus rhodochrous EP4 in complex with 4-ethylguaiacol identified residues Leu78, Ala293 and Phe166 as potential specificity determinants. Substitution of Ala293 and Leu78 decreased the specificity of AgcAEP4 for alkylguaiacols. Substitution of Phe166 yielded a variant that bound 4-propylsyringol but did not transform it. In contrast, the corresponding variant in the Rhodococcus aromaticivorans RHA1 homolog, AgcARHA1 Y166A, catalyzed the O-demethylated of both methoxy groups of 4-propylsyringol with a kcat/Km of 8500 M-1 s-1 for the first O-demethylation, nearly 7-fold higher than WT AgcARHA1. A strain of RHA1 harboring the variant did not grow on 4-propylsyringol but consumed it at approximately the same rate as 4-propylguaiacol and transformed some of it to pentanoyl-CoA, consistent with metabolism via the meta-cleavage pathway that catabolizes 4-alkylguaiacols. These studies improve our understanding of a critical lignin-degrading enzyme system and facilitate its efficient implementation into biocatalysts. SignificanceLignin is a highly abundant source of aromatic carbon and a promising alternative to petroleum to generate materials. Fulfilling this promise depends on technological advances in areas such as catalytic fractionation and biocatalysis. Catalytic fractionation of hardwood biomass generates mixtures of aromatics enriched in 4-propylguaiacol and 4-propylsyringol. Here, we biochemically and structurally characterized a cytochrome P450 that initiates 4-propylguaiacol catabolism. Informed by the structure, we engineered the enzyme to have dual activity on both 4-propylguaiacol and 4-propylsyringol, and implemented this enzyme into a bacterial biocatalyst. Metabolomic analysis of this strain provided insights into the catabolism of both aromatics. Overall, these findings greatly facilitate the engineering of P450s and bacteria to biocatalytically upgrade lignin.