Engineering a Cytochrome P450 O-Demethylase for the Bioconversion of Hardwood Lignin

Lignin is a sustainable 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 bioconversion of the depolymerized lignin by microbial cell factories. A rate-limiting step in this bioconversion 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. The former are O-demethylated by AgcA, a cytochrome P450, and AgcB, the cognate reductase, but there are no known enzymes that convert the latter. To develop a biocatalyst that can efficiently transform these monomers, we studied and rationally engineered AgcAB. A 1.82 Å 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 homologue, AgcARHA1 Y166A, catalyzed the O-demethylation 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. Engineering RHA1 to express the variant yielded a strain that transformed 4-propylsyringol and 4-propylguaiacol simultaneously. Moreover, the engineered strain converted some of the 4-propylsyringol to pentanoyl-CoA, consistent with catabolism via the meta-cleavage pathway that catabolizes 4-alkylguaiacols. Exometabolomics validated the conversion of 4-propylsyringol via this pathway and identified O-demethylation and extradiol ring cleavage as bottlenecks for its transformation. These studies improve our understanding of a critical lignin-degrading enzyme system and significantly advance the development of a biocatalyst to convert these monomers.