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Enabling microbial syringol conversion through structure-guided protein engineering.

  • Author(s): Machovina, Melodie M;
  • Mallinson, Sam JB;
  • Knott, Brandon C;
  • Meyers, Alexander W;
  • Garcia-Borràs, Marc;
  • Bu, Lintao;
  • Gado, Japheth E;
  • Oliver, April;
  • Schmidt, Graham P;
  • Hinchen, Daniel J;
  • Crowley, Michael F;
  • Johnson, Christopher W;
  • Neidle, Ellen L;
  • Payne, Christina M;
  • Houk, Kendall N;
  • Beckham, Gregg T;
  • McGeehan, John E;
  • DuBois, Jennifer L
  • et al.
Abstract

Microbial conversion of aromatic compounds is an emerging and promising strategy for valorization of the plant biopolymer lignin. A critical and often rate-limiting reaction in aromatic catabolism is O-aryl-demethylation of the abundant aromatic methoxy groups in lignin to form diols, which enables subsequent oxidative aromatic ring-opening. Recently, a cytochrome P450 system, GcoAB, was discovered to demethylate guaiacol (2-methoxyphenol), which can be produced from coniferyl alcohol-derived lignin, to form catechol. However, native GcoAB has minimal ability to demethylate syringol (2,6-dimethoxyphenol), the analogous compound that can be produced from sinapyl alcohol-derived lignin. Despite the abundance of sinapyl alcohol-based lignin in plants, no pathway for syringol catabolism has been reported to date. Here we used structure-guided protein engineering to enable microbial syringol utilization with GcoAB. Specifically, a phenylalanine residue (GcoA-F169) interferes with the binding of syringol in the active site, and on mutation to smaller amino acids, efficient syringol O-demethylation is achieved. Crystallography indicates that syringol adopts a productive binding pose in the variant, which molecular dynamics simulations trace to the elimination of steric clash between the highly flexible side chain of GcoA-F169 and the additional methoxy group of syringol. Finally, we demonstrate in vivo syringol turnover in Pseudomonas putida KT2440 with the GcoA-F169A variant. Taken together, our findings highlight the significant potential and plasticity of cytochrome P450 aromatic O-demethylases in the biological conversion of lignin-derived aromatic compounds.

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