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Title: Muconic acid production from methane using rationally-engineered methanotrophic biocatalysts

Abstract

Here, we demonstrate bioconversion of methane to muconic acid, a dicarboxylic acid that can be upgraded to an array of platform chemicals, by three gammaproteobacterial methanotrophs. All engineered methanotrophs expressing a heterologous dihydroxyshikimate dehydratase, protocatechuate decarboxylase, and catechol dioxygenase produced muconic acid from methane, with the highest titer (12.4 mg MA per L), yield (2.8 mg MA per g CH 4), and specific productivity (1.2 mg MA per g dcw, 48 hr) synthesized by Methylotuvimicrobium buryatense, Methylococcus capsulatus, and Methylotuvimicrobium alcaliphilium, respectively. Methylotuvimicrobium alcaliphilum genome-scale model-guided strain engineering predicted that disruption of the pyruvate dehydrogenase or shikimate dehydrogenase would significantly enhance flux to the heterologous muconic acid pathway in this organism. However, knock-out of these targets caused a growth defect, and coupled with similar muconic acid titers (~1 mg L –1), resulted in minimal flux enhancement to muconic acid in these genetically-modified strains. The shikimate dehydrogenase mutant's ability to grow without aromatic amino acid supplementation revealed that M. alcaliphilum likely encodes an unidentified enzyme or pathway with shikimate biosynthetic capacity, which prevents maximal flux through the synthetic muconic acid pathway. This study expands the suite of products that can be generated from methane using methanotrophic biocatalysts, lays the foundation formore » green production of muconic acid-derived polymers from methane, and highlights the need for further analysis of methanotroph biosynthetic potential to guide refinement of metabolic models and strain engineering.« less

Authors:
ORCiD logo [1]; ORCiD logo [2]; ORCiD logo [3]; ORCiD logo [4]
  1. National Renewable Energy Lab. (NREL), Golden, CO (United States)
  2. San Diego State Univ., CA (United States); Russian Academy of Sciences (RAS), Novosibirsk (Russia). Federal Research Center Inst. of Cytology and Genetics of the Siberian Branch
  3. San Diego State Univ., CA (United States)
  4. National Bioenergy Center; National Renewable Energy Laboratory; Golden; USA
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:
1575818
Alternate Identifier(s):
OSTI ID: 1579635
Report Number(s):
NREL/JA-5100-75388
Journal ID: ISSN 1463-9262; GRCHFJ
Grant/Contract Number:  
AC36-08GO28308; FOA-0001085
Resource Type:
Published Article
Journal Name:
Green Chemistry
Additional Journal Information:
Journal Volume: 21; Journal Issue: 24; Journal ID: ISSN 1463-9262
Publisher:
Royal Society of Chemistry
Country of Publication:
United States
Language:
English
Subject:
09 BIOMASS FUELS; methanotroph; methane; muconic acid; biocatalysis

Citation Formats

Henard, Calvin A., Akberdin, Ilya R., Kalyuzhnaya, Marina G., and Guarnieri, Michael T. Muconic acid production from methane using rationally-engineered methanotrophic biocatalysts. United States: N. p., 2019. Web. doi:10.1039/C9GC03722E.
Henard, Calvin A., Akberdin, Ilya R., Kalyuzhnaya, Marina G., & Guarnieri, Michael T. Muconic acid production from methane using rationally-engineered methanotrophic biocatalysts. United States. doi:10.1039/C9GC03722E.
Henard, Calvin A., Akberdin, Ilya R., Kalyuzhnaya, Marina G., and Guarnieri, Michael T. Mon . "Muconic acid production from methane using rationally-engineered methanotrophic biocatalysts". United States. doi:10.1039/C9GC03722E.
@article{osti_1575818,
title = {Muconic acid production from methane using rationally-engineered methanotrophic biocatalysts},
author = {Henard, Calvin A. and Akberdin, Ilya R. and Kalyuzhnaya, Marina G. and Guarnieri, Michael T.},
abstractNote = {Here, we demonstrate bioconversion of methane to muconic acid, a dicarboxylic acid that can be upgraded to an array of platform chemicals, by three gammaproteobacterial methanotrophs. All engineered methanotrophs expressing a heterologous dihydroxyshikimate dehydratase, protocatechuate decarboxylase, and catechol dioxygenase produced muconic acid from methane, with the highest titer (12.4 mg MA per L), yield (2.8 mg MA per g CH4), and specific productivity (1.2 mg MA per g dcw, 48 hr) synthesized by Methylotuvimicrobium buryatense, Methylococcus capsulatus, and Methylotuvimicrobium alcaliphilium, respectively. Methylotuvimicrobium alcaliphilum genome-scale model-guided strain engineering predicted that disruption of the pyruvate dehydrogenase or shikimate dehydrogenase would significantly enhance flux to the heterologous muconic acid pathway in this organism. However, knock-out of these targets caused a growth defect, and coupled with similar muconic acid titers (~1 mg L–1), resulted in minimal flux enhancement to muconic acid in these genetically-modified strains. The shikimate dehydrogenase mutant's ability to grow without aromatic amino acid supplementation revealed that M. alcaliphilum likely encodes an unidentified enzyme or pathway with shikimate biosynthetic capacity, which prevents maximal flux through the synthetic muconic acid pathway. This study expands the suite of products that can be generated from methane using methanotrophic biocatalysts, lays the foundation for green production of muconic acid-derived polymers from methane, and highlights the need for further analysis of methanotroph biosynthetic potential to guide refinement of metabolic models and strain engineering.},
doi = {10.1039/C9GC03722E},
journal = {Green Chemistry},
number = 24,
volume = 21,
place = {United States},
year = {2019},
month = {11}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record
DOI: 10.1039/C9GC03722E

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