Enabling microbial syringol conversion through structure-guided protein engineering
[5];
[4];
[7];
[8];
[9];
[2];
- Department of Chemistry and Biochemistry, Montana State University, Bozeman, MT 59717,
- Centre for Enzyme Innovation, School of Biological Sciences, Institute of Biological and Biomedical Sciences, University of Portsmouth, Portsmouth PO1 2UP, United Kingdom,
- Biosciences Center, National Renewable Energy Laboratory, Golden, CO 80401,
- National Bioenergy Center, National Renewable Energy Laboratory, Golden, CO 80401,
- Department of Chemistry and Biochemistry, University of California, Los Angeles, CA 90095,
- National Bioenergy Center, National Renewable Energy Laboratory, Golden, CO 80401,, Department of Chemical Engineering, University of Kentucky, Lexington, KY 40506,
- Department of Microbiology, University of Georgia, Athens, GA 30602,
- Department of Chemical Engineering, University of Kentucky, Lexington, KY 40506,
- National Bioenergy Center, National Renewable Energy Laboratory, Golden, CO 80401,, Center for Bioenergy Innovation, Oak Ridge National Laboratory, Oak Ridge, TN 37830
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.
- Research Organization:
- National Renewable Energy Laboratory (NREL), Golden, CO (United States)
- Sponsoring Organization:
- USDOE Office of Energy Efficiency and Renewable Energy (EERE), Sustainable Transportation Office. Bioenergy Technologies Office (BETO); USDOE Office of Science (SC), Workforce Development for Teachers and Scientists (WDTS); USDOE Office of Science (SC), Biological and Environmental Research (BER)
- Grant/Contract Number:
- AC36-08GO28308; SC0014664
- OSTI ID:
- 1529168
- Alternate ID(s):
- OSTI ID: 1531144
- Report Number(s):
- NREL/JA-2A00-73031
- Journal Information:
- Proceedings of the National Academy of Sciences of the United States of America, Journal Name: Proceedings of the National Academy of Sciences of the United States of America Vol. 116 Journal Issue: 28; ISSN 0027-8424
- Publisher:
- Proceedings of the National Academy of SciencesCopyright Statement
- Country of Publication:
- United States
- Language:
- English
Web of Science
Similar Records
A promiscuous cytochrome P450 aromatic O-demethylase for lignin bioconversion
Accelerating pathway evolution by increasing the gene dosage of chromosomal segments