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Enabling microbial syringol conversion through structure-guided protein engineering

Research output: Contribution to journalArticlepeer-review

  • Melodie M. Machovina
  • Sam Mallinson
  • Brandon C. Knott
  • Alexander W. Meyers
  • Marc Garcia-Borràs
  • Lintao Bu
  • Japheth E. Gado
  • April Oliver
  • Graham P. Schmidt
  • Daniel James Hinchen
  • Michael F. Crowley
  • Christopher W. Johnson
  • Ellen L. Neidle
  • Christina M. Payne
  • Kendall N. Houk
  • Gregg T. Beckham
  • Professor John McGeehan
  • Jennifer L. DuBois
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 languageEnglish
Article number0
Pages (from-to)13970-13976
Number of pages7
JournalProceedings of the National Academy of Sciences
Issue number28
Early online date24 Jun 2019
Publication statusPublished - 9 Jul 2019


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