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Title: Enabling Microbial Syringol Conversion Through Structure-Guided Protein Engineering

Abstract

Microbial conversion of aromatic compounds is an emerging and promising strategy for valorization of the plant biopolymer lignin. A critical and often rate-limiting reaction in aromatic catabolism is O-aryl-demethylation of the abundant aromatic methoxy groups in lignin to form diols, which enables subsequent oxidative aromatic ring-opening. Recently, a cytochrome P450 system, GcoAB, was discovered to demethylate guaiacol (2-methoxyphenol), which can be produced from coniferyl alcohol-derived lignin, to form catechol. However, native GcoAB has minimal ability to demethylate syringol (2,6-dimethoxyphenol), the analogous compound that can be produced from sinapyl alcohol-derived lignin. Despite the abundance of sinapyl alcohol-based lignin in plants, no pathway for syringol catabolism has been reported to date. Here we used structure-guided protein engineering to enable microbial syringol utilization with GcoAB. Specifically, a phenylalanine residue (GcoA-F169) interferes with the binding of syringol in the active site, and on mutation to smaller amino acids, efficient syringol O-demethylation is achieved. Crystallography indicates that syringol adopts a productive binding pose in the variant, which molecular dynamics simulations trace to the elimination of steric clash between the highly flexible side chain of GcoA-F169 and the additional methoxy group of syringol. Finally, we demonstrate in vivo syringol turnover in Pseudomonas putida KT2440 with themore » GcoA-F169A variant. Taken together, our findings highlight the significant potential and plasticity of cytochrome P450 aromatic O-demethylases in the biological conversion of lignin-derived aromatic compounds.« less

Authors:
 [1];  [2];  [3];  [3];  [4];  [3];  [3];  [1];  [3];  [2];  [3]; ORCiD logo [3];  [5];  [6];  [4]; ORCiD logo [3];  [2];  [1]
  1. Montana State University
  2. University of Portsmouth
  3. National Renewable Energy Laboratory (NREL), Golden, CO (United States)
  4. University of California at Los Angeles
  5. University of Georgia
  6. University of Kentucky
Publication Date:
Research Org.:
National Renewable Energy Lab. (NREL), Golden, CO (United States)
Sponsoring Org.:
USDOE Office of Energy Efficiency and Renewable Energy (EERE), Bioenergy Technologies Office (EE-3B)
OSTI Identifier:
1531144
Report Number(s):
NREL/JA-2A00-73031
DOE Contract Number:  
AC36-08GO28308
Resource Type:
Journal Article
Journal Name:
Proceedings of the National Academy of Sciences of the United States of America
Additional Journal Information:
Journal Name: Proceedings of the National Academy of Sciences of the United States of America
Country of Publication:
United States
Language:
English
Subject:
09 BIOMASS FUELS; aromatic catabolism; cytochrome P450; lignin valorization; protein engineering; Pseudomonas putida KT2440

Citation Formats

Machovina, Melodie M., Mallinson, Sam J. B., Knott, Brandon C, Meyers, Alexander, Garcia-Borras, Marc, Bu, Lintao, Gado, Japheth, Oliver, April, Schmidt, Graham, Hinchen, Daniel J., Crowley, Michael F, Johnson, Christopher W, Neidle, Ellen L., Payne, Christina M., Houk, Kendall N., Beckham, Gregg T, McGeehan, John E., and DuBois, Jennifer L/. Enabling Microbial Syringol Conversion Through Structure-Guided Protein Engineering. United States: N. p., 2019. Web. doi:10.1073/pnas.1820001116.
Machovina, Melodie M., Mallinson, Sam J. B., Knott, Brandon C, Meyers, Alexander, Garcia-Borras, Marc, Bu, Lintao, Gado, Japheth, Oliver, April, Schmidt, Graham, Hinchen, Daniel J., Crowley, Michael F, Johnson, Christopher W, Neidle, Ellen L., Payne, Christina M., Houk, Kendall N., Beckham, Gregg T, McGeehan, John E., & DuBois, Jennifer L/. Enabling Microbial Syringol Conversion Through Structure-Guided Protein Engineering. United States. doi:10.1073/pnas.1820001116.
Machovina, Melodie M., Mallinson, Sam J. B., Knott, Brandon C, Meyers, Alexander, Garcia-Borras, Marc, Bu, Lintao, Gado, Japheth, Oliver, April, Schmidt, Graham, Hinchen, Daniel J., Crowley, Michael F, Johnson, Christopher W, Neidle, Ellen L., Payne, Christina M., Houk, Kendall N., Beckham, Gregg T, McGeehan, John E., and DuBois, Jennifer L/. Mon . "Enabling Microbial Syringol Conversion Through Structure-Guided Protein Engineering". United States. doi:10.1073/pnas.1820001116.
@article{osti_1531144,
title = {Enabling Microbial Syringol Conversion Through Structure-Guided Protein Engineering},
author = {Machovina, Melodie M. and Mallinson, Sam J. B. and Knott, Brandon C and Meyers, Alexander and Garcia-Borras, Marc and Bu, Lintao and Gado, Japheth and Oliver, April and Schmidt, Graham and Hinchen, Daniel J. and Crowley, Michael F and Johnson, Christopher W and Neidle, Ellen L. and Payne, Christina M. and Houk, Kendall N. and Beckham, Gregg T and McGeehan, John E. and DuBois, Jennifer L/},
abstractNote = {Microbial conversion of aromatic compounds is an emerging and promising strategy for valorization of the plant biopolymer lignin. A critical and often rate-limiting reaction in aromatic catabolism is O-aryl-demethylation of the abundant aromatic methoxy groups in lignin to form diols, which enables subsequent oxidative aromatic ring-opening. Recently, a cytochrome P450 system, GcoAB, was discovered to demethylate guaiacol (2-methoxyphenol), which can be produced from coniferyl alcohol-derived lignin, to form catechol. However, native GcoAB has minimal ability to demethylate syringol (2,6-dimethoxyphenol), the analogous compound that can be produced from sinapyl alcohol-derived lignin. Despite the abundance of sinapyl alcohol-based lignin in plants, no pathway for syringol catabolism has been reported to date. Here we used structure-guided protein engineering to enable microbial syringol utilization with GcoAB. Specifically, a phenylalanine residue (GcoA-F169) interferes with the binding of syringol in the active site, and on mutation to smaller amino acids, efficient syringol O-demethylation is achieved. Crystallography indicates that syringol adopts a productive binding pose in the variant, which molecular dynamics simulations trace to the elimination of steric clash between the highly flexible side chain of GcoA-F169 and the additional methoxy group of syringol. Finally, we demonstrate in vivo syringol turnover in Pseudomonas putida KT2440 with the GcoA-F169A variant. Taken together, our findings highlight the significant potential and plasticity of cytochrome P450 aromatic O-demethylases in the biological conversion of lignin-derived aromatic compounds.},
doi = {10.1073/pnas.1820001116},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
number = ,
volume = ,
place = {United States},
year = {2019},
month = {6}
}