Discovery, characterization, and metabolic engineering of Rieske non-heme iron monooxygenases for guaiacol O-demethylation

Alissa Bleem, Eugene Kuatsjah, Gerald, N. Presley, Daniel James Hinchen, Michael Zahn, David, C. Garcia, William E. Michener, Gerhard Koenig, Konstantinos Tornesakis, Marco, N. Allemann, Richard, J. Giannone, John McGeehan*, Gregg T. Beckham*, Joshua, K. Michener*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

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Aryl-O-demethylation is a common rate-limiting step in the catabolism of lignin-related compounds, including guaiacol. Here we used randomly barcoded transposon insertion sequencing (RB-TnSeq) in the bacterium Novosphingobium aromaticivorans to identify a Rieske-type guaiacol O-demethylase, GdmA. Similarity searches identified GdmA homologs in other bacteria, along with candidate reductase partners, denoted GdmB. GdmAB combinations were biochemically characterized for activity with several lignin-related substrates. Structural and sequence comparisons of vanillate- and guaiacol-specific O-demethylase active sites revealed conserved hallmarks of substrate specificity. GdmAB combinations were also evaluated in Pseudomonas putida KT2440, which does not natively utilize guaiacol. GdmAB from Cupriavidus necator N-1 demonstrated the highest rate of guaiacol turnover in vitro and in engineered P. putida strains and notably higher catalytic efficiency than a cytochrome P450 system (GcoAB) and the vanillate Rieske-type O-demethylase from P. putida (VanAB). The GdmAB O-demethylases described here expand the suite of options for microbial conversion of a model lignin-derived substrate.
Original languageEnglish
Pages (from-to)1989-2011
JournalChem Catalysis
Issue number8
Early online date18 Aug 2022
Publication statusPublished - 18 Aug 2022


  • microbial lignin conversion
  • Rieske non-heme iron monooxygenase
  • biological funneling
  • Novosphingobium aromaticivorans
  • Pseudomonas putida KT2440
  • Cupriavidus necator
  • Sphingomonas wittichii
  • O-demethylation
  • biocatalysis
  • UKRI


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