Discovery, Characterization, and Metabolic Engineering of Rieske Non-Heme Iron Monooxygenases for Guaiacol O-Demethylation

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

Research output: Contribution to journalArticlepeer-review

9 Scopus Citations


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 languageAmerican English
Pages (from-to)1989-2011
Number of pages23
JournalChem Catalysis
Issue number8
StatePublished - 18 Aug 2022

Bibliographical note

Publisher Copyright:
© 2022 Elsevier Inc.

NREL Publication Number

  • NREL/JA-2800-81928


  • biocatalysis
  • biological funneling
  • Cupriavidus necator
  • microbial lignin conversion
  • Novosphingobium aromaticivorans
  • O-demethylation
  • Pseudomonas putida KT2440
  • Rieske non-heme iron monooxygenase
  • SDG 7: Affordable and clean energy
  • SDG 9: Industry, innovation, and infrastructure
  • SDG13: Climate action
  • Sphingomonas wittichii


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