Enabling Microbial Syringol Conversion Through Structure-Guided Protein Engineering

Gregg Beckham, Brandon Knott, Lintao Bu, Japheth Gado, Michael Crowley, Christopher Johnson, Melodie Machovina, Sam Mallinson, Marc Garcia-Borras, April Oliver, Daniel Hinchen, Ellen Neidle, Christina Payne, Kendall Houk, John McGeehan, Jennifer DuBois, Alexander Meyers, Graham Schmidt

Research output: Contribution to journalArticlepeer-review

41 Scopus Citations


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.

Original languageAmerican English
Pages (from-to)13970-13976
Number of pages7
JournalProceedings of the National Academy of Sciences of the United States of America
Issue number28
StatePublished - 2019

Bibliographical note

Publisher Copyright:
© 2019 National Academy of Sciences. All rights reserved.

NREL Publication Number

  • NREL/JA-2A00-73031


  • Biorefinery
  • Demethylase
  • Lignin
  • P450


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