Publications by Colleges and Departments (MSU - Bozeman)
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Item Engineering a Cytochrome P450 for Demethylation of Lignin-Derived Aromatic Aldehydes(American Chemical Society, 2021-03) Ellis, Emerald S.; Hinchen, Daniel J.; Bleem, Alissa; Bu, Lintao; Mallinson, Sam J. B.; Allen, Mark D.; Streit, Bennett R.; Machovina, Melodie M.; Doolin, Quinlan V.; Michener, William E.; Johnson, Christopher W.; Knott, Brandon C.; Beckham, Gregg T.; McGeehan, John E.; DuBois, Jennifer L.Biological funneling of lignin-derived aromatic compounds is a promising approach for valorizing its catalytic depolymerization products. Industrial processes for aromatic bioconversion will require efficient enzymes for key reactions, including demethylation of O-methoxy-aryl groups, an essential and often rate-limiting step. The recently characterized GcoAB cytochrome P450 system comprises a coupled monoxygenase (GcoA) and reductase (GcoB) that catalyzes oxidative demethylation of the O-methoxy-aryl group in guaiacol. Here, we evaluate a series of engineered GcoA variants for their ability to demethylate o-and p-vanillin, which are abundant lignin depolymerization products. Two rationally designed, single amino acid substitutions, F169S and T296S, are required to convert GcoA into an efficient catalyst toward the o- and p-isomers of vanillin, respectively. Gain-of-function in each case is explained in light of an extensive series of enzyme-ligand structures, kinetic data, and molecular dynamics simulations. Using strains of Pseudomonas putida KT2440 already optimized for p-vanillin production from ferulate, we demonstrate demethylation by the T296S variant in vivo. This work expands the known aromatic O-demethylation capacity of cytochrome P450 enzymes toward important lignin-derived aromatic monomers.