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

DOI: 10.1016/j.checat.2022.04.019 DOI Help

Authors: Alissa Bleem (National Renewable Energy Laboratory; Oak Ridge National Laboratory) , Eugene Kuatsjah (National Renewable Energy Laboratory; Oak Ridge National Laboratory) , Gerald N. Presley (Oak Ridge National Laboratory; Oregon State University) , Daniel J. Hinchen (University of Portsmouth) , Michael Zahn (University of Portsmouth) , David C. Garcia (University of Tennessee) , William E. Michener (National Renewable Energy Laboratory) , Gerhard K├Ânig (University of Portsmouth) , Konstantinos Tornesakis (University of Portsmouth) , Marco N. Allemann (Oak Ridge National Laboratory) , Richard J. Giannone (Oak Ridge National Laboratory) , John E. Mcgeehan (University of Portsmouth) , Gregg T. Beckham (National Renewable Energy Laboratory; Oak Ridge National Laboratory) , Joshua K. Michener (Oak Ridge National Laboratory)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Chem Catalysis , VOL 55

State: Published (Approved)
Published: May 2022

Abstract: 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.

Journal Keywords: microbial lignin conversion; Rieske non-heme iron monooxygenase; biological funneling; Novosphingobium aromaticivorans; Pseudomonas putida KT2440; Cupriavidus necator; Sphingomonas wittichii; O-demethylationbio; catalysis

Diamond Keywords: Biofuel; Enzymes; Bacteria

Subject Areas: Biology and Bio-materials, Chemistry, Energy


Instruments: I03-Macromolecular Crystallography

Added On: 25/05/2022 10:47

Discipline Tags:

Bioenergy Earth Sciences & Environment Sustainable Energy Systems Energy Climate Change Biochemistry Catalysis Chemistry Structural biology Organic Chemistry Life Sciences & Biotech

Technical Tags:

Diffraction Macromolecular Crystallography (MX)