TY - JOUR
T1 - Discovery, characterization, and metabolic engineering of Rieske non-heme iron monooxygenases for guaiacol O-demethylation
AU - Bleem, Alissa
AU - Kuatsjah, Eugene
AU - Presley, Gerald, N.
AU - Hinchen, Daniel James
AU - Zahn, Michael
AU - Garcia, David, C.
AU - Michener, William E.
AU - Koenig, Gerhard
AU - Tornesakis, Konstantinos
AU - Allemann, Marco, N.
AU - Giannone, Richard, J.
AU - McGeehan, John
AU - Beckham, Gregg T.
AU - Michener, Joshua, K.
PY - 2022/8/18
Y1 - 2022/8/18
N2 - 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.
AB - 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.
KW - microbial lignin conversion
KW - Rieske non-heme iron monooxygenase
KW - biological funneling
KW - Novosphingobium aromaticivorans
KW - Pseudomonas putida KT2440
KW - Cupriavidus necator
KW - Sphingomonas wittichii
KW - O-demethylation
KW - biocatalysis
KW - UKRI
KW - BBSRC
UR - https://www.cell.com/chem-catalysis/pdfExtended/S2667-1093(22)00228-7
UR - https://www.cell.com/chem-catalysis/fulltext/S2667-1093(22)00228-7
U2 - 10.1016/j.checat.2022.04.019
DO - 10.1016/j.checat.2022.04.019
M3 - Article
SN - 2667-1093
VL - 2
SP - 1989
EP - 2011
JO - Chem Catalysis
JF - Chem Catalysis
IS - 8
ER -