Microbial syringol utilization
Inventors
BECKHAM, Gregg Tyler • DUBOIS, JENNIFER • MACHOVINA, Melodie M. • MALLINSON, Simon James Bradshaw • McGeehan, John E. • Johnson, Christopher W. • MEYERS, Alexander William
Assignees
University of Portsmouth • Montana State University Bozeman • Alliance for Sustainable Energy LLC
Publication Number
US-11208642-B2
Publication Date
2021-12-28
Expiration Date
2039-11-14
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Abstract
Disclosed herein are compositions of non-naturally occurring enzymes to enable microbial syringol utilization with GcoAB.
Core Innovation
The invention provides non-naturally occurring GcoAB enzymes that have been engineered to enable O-aryl-demethylation of syringol and guaiacol. By altering a phenylalanine residue at the active site (specifically position 169) to a smaller amino acid such as alanine, serine, histidine, valine, isoleucine, or leucine, the modified GcoAB enzymes achieve efficient catalytic conversion of syringol, which is derived from S-lignin, as well as guaiacol, derived from G-lignin.
The problem addressed is the lack of known microbial pathways for syringol O-demethylation and catabolism, despite the abundance of S-lignin in plant biomass. Native GcoAB enzymes demonstrate minimal activity towards syringol. There is a need for enzymatic solutions that can act on both major lignin subunits to facilitate the bioconversion of lignin-derived aromatic compounds into valuable chemicals for lignocellulose conversion strategies.
The engineered GcoAB enzymes with mutations at position 169 create a productive binding pocket for syringol by reducing steric hindrance, thus allowing efficient demethylation. These enzymes maintain or even improve specific activity with guaiacol while newly acquiring significant activity for syringol O-demethylation. The patent also demonstrates in vivo microbial utilization of syringol using strains expressing these engineered enzymes, thus expanding the substrate range and enhancing the value of microbial lignin conversion.
Claims Coverage
There is one independent claim in this patent covering one core inventive feature.
Non-naturally occurring GcoAB enzyme for O-aryl-demethylation of syringol and guaiacol with active site mutation
The invention comprises a non-naturally occurring GcoAB enzyme that includes the amino acid sequence of SEQ ID NO: 1, is capable of O-aryl-demethylation of both syringol and guaiacol, and has a phenylalanine residue at the active site replaced by an amino acid selected from alanine, serine, histidine, valine, isoleucine, or leucine. This replacement enables the enzyme to catalyze the O-demethylation reactions for both substrates.
The claim broadly covers engineered GcoAB enzymes with a specific active site mutation that enables efficient O-aryl-demethylation of both syringol and guaiacol.
Stated Advantages
Provides non-naturally occurring enzymes capable of efficient O-aryl-demethylation of syringol, which is not catalyzed by native enzymes.
Enables microbial utilization and conversion of syringol, expanding the substrate range for lignin valorization.
Engineered enzymes retain or improve activity toward guaiacol while acquiring new activity for syringol.
Facilitates the development of viable lignin valorization strategies for lignocellulose conversion.
Documented Applications
Degradation of lignin, including O-aryl-demethylation of both syringol and guaiacol, to assist in biomass conversion.
Microbial syringol utilization for the conversion of S-lignin derived aromatics into valuable chemicals.
Expression of engineered GcoAB enzymes in organisms to enable in vivo turnover and catabolism of syringol as demonstrated in Pseudomonas putida.
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