Engineering a Cytochrome P450 for Demethylation of Lignin-Derived Aromatic Aldehydes
- Ellis, Emerald S. [ Montana State University: Chemistry & Biochemistry ]
- Hinchen, Daniel J.
- Bleem, Alissa
- Bu, Lintao
- Mallinson, Sam J. B.
- Allen, Mark D.
- Streit, Bennett R. [ Montana State University: Chemistry & Biochemistry ]
- Machovina, Melodie M. [ Montana State University: Chemistry & Biochemistry ]
- Doolin, Quinlan V. [ Montana State University: Chemistry & Biochemistry ]
- Michener, William E.
- Johnson, Christopher W.
- Knott, Brandon C.
- Beckham, Gregg T.
- McGeehan, John E.
- DuBois, Jennifer L. [ Montana State University: Chemistry & Biochemistry ]
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.