DOE PAGES title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Metabolic engineering of β-oxidation to leverage thioesterases for production of 2-heptanone, 2-nonanone and 2-undecanone

Abstract

Medium-chain length methyl ketones are potential blending fuels due to their cetane numbers and low melting temperatures. Biomanufacturing offers the potential to produce these molecules from renewable resources such as lignocellulosic biomass. Here, we designed and tested metabolic pathways in Escherichia coli to specifically produce 2-heptanone, 2-nonanone and 2-undecanone. We achieved substantial production of each ketone by introducing chain-length specific acyl-ACP thioesterases, blocking the β-oxidation cycle at an advantageous reaction, and introducing active β-ketoacyl-CoA thioesterases. Using a bioprospecting approach, we identified fifteen homologs of E. coli β-ketoacyl-CoA thioesterase (FadM) and evaluated the in vivo activity of each against various chain length substrates. The FadM variant from Providencia sneebia produced the most 2-heptanone, 2-nonanone, and 2-undecanone, suggesting it has the highest activity on the corresponding β-ketoacyl-CoA substrates. We further tested enzyme variants, including acyl-CoA oxidases, thiolases, and bi-functional 3-hydroxyacyl-CoA dehydratases to maximize conversion of fatty acids to β-keto acyl-CoAs for 2-heptanone, 2-nonanone, and 2-undecanone production. In order to address the issue of product loss during fermentation, we applied a 20% (v/v) dodecane layer in the bioreactor and built an external water cooling condenser connecting to the bioreactor heat-transferring condenser coupling to the condenser. Finally, using these modifications, we were able tomore » generate up to 4.4 g/L total medium-chain length methyl ketones.« less

Authors:
ORCiD logo [1]; ORCiD logo [1]; ORCiD logo [1];  [1]; ORCiD logo [1]; ORCiD logo [1];  [1]; ORCiD logo [1]
  1. Univ. of Wisconsin, Madison, WI (United States)
Publication Date:
Research Org.:
Center for Advanced Bioenergy and Bioproducts Innovation (CABBI), Urbana, IL (United States); Univ. of Illinois at Urbana-Champaign, IL (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER); National Science Foundation (NSF)
OSTI Identifier:
1631456
Alternate Identifier(s):
OSTI ID: 1646545
Grant/Contract Number:  
SC0018420; CBET-1703504
Resource Type:
Accepted Manuscript
Journal Name:
Metabolic Engineering
Additional Journal Information:
Journal Volume: 61; Journal ID: ISSN 1096-7176
Publisher:
Elsevier
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; Thioesterase; 2-Heptanone; 2-Nonanone; 2-Undecanone; Thiolase; Metabolic engineering

Citation Formats

Yan, Qiang, Simmons, Trevor R., Cordell, William T., Hernández Lozada, Néstor J., Breckner, Christian J., Chen, Xuanqi, Jindra, Michael A., and Pfleger, Brian F. Metabolic engineering of β-oxidation to leverage thioesterases for production of 2-heptanone, 2-nonanone and 2-undecanone. United States: N. p., 2020. Web. doi:10.1016/j.ymben.2020.05.008.
Yan, Qiang, Simmons, Trevor R., Cordell, William T., Hernández Lozada, Néstor J., Breckner, Christian J., Chen, Xuanqi, Jindra, Michael A., & Pfleger, Brian F. Metabolic engineering of β-oxidation to leverage thioesterases for production of 2-heptanone, 2-nonanone and 2-undecanone. United States. https://doi.org/10.1016/j.ymben.2020.05.008
Yan, Qiang, Simmons, Trevor R., Cordell, William T., Hernández Lozada, Néstor J., Breckner, Christian J., Chen, Xuanqi, Jindra, Michael A., and Pfleger, Brian F. Fri . "Metabolic engineering of β-oxidation to leverage thioesterases for production of 2-heptanone, 2-nonanone and 2-undecanone". United States. https://doi.org/10.1016/j.ymben.2020.05.008. https://www.osti.gov/servlets/purl/1631456.
@article{osti_1631456,
title = {Metabolic engineering of β-oxidation to leverage thioesterases for production of 2-heptanone, 2-nonanone and 2-undecanone},
author = {Yan, Qiang and Simmons, Trevor R. and Cordell, William T. and Hernández Lozada, Néstor J. and Breckner, Christian J. and Chen, Xuanqi and Jindra, Michael A. and Pfleger, Brian F.},
abstractNote = {Medium-chain length methyl ketones are potential blending fuels due to their cetane numbers and low melting temperatures. Biomanufacturing offers the potential to produce these molecules from renewable resources such as lignocellulosic biomass. Here, we designed and tested metabolic pathways in Escherichia coli to specifically produce 2-heptanone, 2-nonanone and 2-undecanone. We achieved substantial production of each ketone by introducing chain-length specific acyl-ACP thioesterases, blocking the β-oxidation cycle at an advantageous reaction, and introducing active β-ketoacyl-CoA thioesterases. Using a bioprospecting approach, we identified fifteen homologs of E. coli β-ketoacyl-CoA thioesterase (FadM) and evaluated the in vivo activity of each against various chain length substrates. The FadM variant from Providencia sneebia produced the most 2-heptanone, 2-nonanone, and 2-undecanone, suggesting it has the highest activity on the corresponding β-ketoacyl-CoA substrates. We further tested enzyme variants, including acyl-CoA oxidases, thiolases, and bi-functional 3-hydroxyacyl-CoA dehydratases to maximize conversion of fatty acids to β-keto acyl-CoAs for 2-heptanone, 2-nonanone, and 2-undecanone production. In order to address the issue of product loss during fermentation, we applied a 20% (v/v) dodecane layer in the bioreactor and built an external water cooling condenser connecting to the bioreactor heat-transferring condenser coupling to the condenser. Finally, using these modifications, we were able to generate up to 4.4 g/L total medium-chain length methyl ketones.},
doi = {10.1016/j.ymben.2020.05.008},
journal = {Metabolic Engineering},
number = ,
volume = 61,
place = {United States},
year = {Fri May 29 00:00:00 EDT 2020},
month = {Fri May 29 00:00:00 EDT 2020}
}

Works referenced in this record:

Improved Prediction of Signal Peptides: SignalP 3.0
journal, July 2004

  • Dyrløv Bendtsen, Jannick; Nielsen, Henrik; von Heijne, Gunnar
  • Journal of Molecular Biology, Vol. 340, Issue 4, p. 783-795
  • DOI: 10.1016/j.jmb.2004.05.028

Crystal structure and biochemical characterization of beta-keto thiolase B from polyhydroxyalkanoate-producing bacterium Ralstonia eutropha H16
journal, February 2014

  • Kim, Eun-Jung; Son, Hyeoncheol Francis; Kim, Sangwoo
  • Biochemical and Biophysical Research Communications, Vol. 444, Issue 3
  • DOI: 10.1016/j.bbrc.2014.01.055

Review of biodiesel composition, properties, and specifications
journal, January 2012

  • Hoekman, S. Kent; Broch, Amber; Robbins, Curtis
  • Renewable and Sustainable Energy Reviews, Vol. 16, Issue 1
  • DOI: 10.1016/j.rser.2011.07.143

A Synthetic Biology Approach to Engineer a Functional Reversal of the β-Oxidation Cycle
journal, October 2012

  • Clomburg, James M.; Vick, Jacob E.; Blankschien, Matthew D.
  • ACS Synthetic Biology, Vol. 1, Issue 11
  • DOI: 10.1021/sb3000782

Engineering Escherichia coli for Microbial Production of Butanone
journal, February 2016

  • Srirangan, Kajan; Liu, Xuejia; Akawi, Lamees
  • Applied and Environmental Microbiology, Vol. 82, Issue 9
  • DOI: 10.1128/AEM.03964-15

Production of medium chain length fatty alcohols from glucose in Escherichia coli
journal, November 2013


Engineered reversal of the β-oxidation cycle for the synthesis of fuels and chemicals
journal, August 2011

  • Dellomonaco, Clementina; Clomburg, James M.; Miller, Elliot N.
  • Nature, Vol. 476, Issue 7360, p. 355-359
  • DOI: 10.1038/nature10333

Reversal of the β-Oxidation Cycle in Saccharomyces cerevisiae for Production of Fuels and Chemicals
journal, June 2014

  • Lian, Jiazhang; Zhao, Huimin
  • ACS Synthetic Biology, Vol. 4, Issue 3
  • DOI: 10.1021/sb500243c

Engineering of Ralstonia eutropha H16 for Autotrophic and Heterotrophic Production of Methyl Ketones
journal, May 2013

  • Muller, J.; MacEachran, D.; Burd, H.
  • Applied and Environmental Microbiology, Vol. 79, Issue 14
  • DOI: 10.1128/AEM.00973-13

Enzymatic Functions of Wild Tomato Methylketone Synthases 1 and 2
journal, July 2010

  • Yu, Geng; Nguyen, Thuong T. H.; Guo, Yongxia
  • Plant Physiology, Vol. 154, Issue 1
  • DOI: 10.1104/pp.110.157073

Expanding the product portfolio of fungal type I fatty acid synthases
journal, February 2017

  • Zhu, Zhiwei; Zhou, Yongjin J.; Krivoruchko, Anastasia
  • Nature Chemical Biology, Vol. 13, Issue 4
  • DOI: 10.1038/nchembio.2301

High cetane renewable diesel fuels prepared from bio-based methyl ketones and diols
journal, January 2018

  • Harrison, Kale W.; Harvey, Benjamin G.
  • Sustainable Energy & Fuels, Vol. 2, Issue 2
  • DOI: 10.1039/C7SE00415J

T OMATO , P ESTS , P ARASITOIDS , AND P REDATORS : Tritrophic Interactions Involving the Genus Lycopersicon
journal, January 2003


Growth-coupled bioconversion of levulinic acid to butanone
journal, September 2019


Revisiting metabolic engineering strategies for microbial synthesis of oleochemicals
journal, March 2020


Highly Active C 8 -Acyl-ACP Thioesterase Variant Isolated by a Synthetic Selection Strategy
journal, July 2018

  • Hernández Lozada, Néstor J.; Lai, Rung-Yi; Simmons, Trevor R.
  • ACS Synthetic Biology, Vol. 7, Issue 9
  • DOI: 10.1021/acssynbio.8b00215

Substantial improvements in methyl ketone production in E. coli and insights on the pathway from in vitro studies
journal, November 2014


Novel use of aliphatic n -methyl ketones as a fumigant and alternative to methyl bromide for insect control: Use of n -methyl ketones as a fumigant
journal, November 2017

  • Zhu, Jiwei; Dhammi, Anirudh; van Kretschmar, Jaap B.
  • Pest Management Science, Vol. 74, Issue 3
  • DOI: 10.1002/ps.4749

Lipolysis and free fatty acid catabolism in cheese: a review of current knowledge
journal, January 2003

  • Collins, Yvonne F.; McSweeney, Paul L. H.; Wilkinson, Martin G.
  • International Dairy Journal, Vol. 13, Issue 11
  • DOI: 10.1016/S0958-6946(03)00109-2

Synthesis of methyl ketones by metabolically engineered Escherichia coli
journal, August 2012

  • Park, John; Rodríguez-Moyá, María; Li, Mai
  • Journal of Industrial Microbiology & Biotechnology, Vol. 39, Issue 11, p. 1703-1712
  • DOI: 10.1007/s10295-012-1178-x

Methyl ketone production by Pseudomonas putida is enhanced by plant‐derived amino acids
journal, April 2019

  • Dong, Jie; Chen, Yan; Benites, Veronica Teixeira
  • Biotechnology and Bioengineering, Vol. 116, Issue 8
  • DOI: 10.1002/bit.26995

Improving methyl ketone production in Escherichia coli by heterologous expression of NADH-dependent FabG
journal, March 2018

  • Goh, Ee-Been; Chen, Yan; Petzold, Christopher J.
  • Biotechnology and Bioengineering, Vol. 115, Issue 5
  • DOI: 10.1002/bit.26558

Purification and Characterization of OleA from Xanthomonas campestris and Demonstration of a Non-decarboxylative Claisen Condensation Reaction
journal, January 2011

  • Frias, Janice A.; Richman, Jack E.; Erickson, Jasmine S.
  • Journal of Biological Chemistry, Vol. 286, Issue 13
  • DOI: 10.1074/jbc.M110.216127

Nitrogen-dependent regulation of medium-chain length polyhydroxyalkanoate biosynthesis genes in pseudomonads
journal, February 2005


Anaerobic production of medium-chain fatty alcohols via a β-reduction pathway
journal, July 2018


Identification and Characterization of Escherichia coli Thioesterase III That Functions in Fatty Acid β-Oxidation
journal, July 2008

  • Nie, Lina; Ren, Ying; Schulz, Horst
  • Biochemistry, Vol. 47, Issue 29
  • DOI: 10.1021/bi800595f

Engineering Escherichia coli for production of C12–C14 polyhydroxyalkanoate from glucose
journal, November 2012

  • Agnew, Daniel E.; Stevermer, Amanda K.; Youngquist, J. Tyler
  • Metabolic Engineering, Vol. 14, Issue 6
  • DOI: 10.1016/j.ymben.2012.08.003

Short-chain ketone production by engineered polyketide synthases in Streptomyces albus
journal, November 2018


The β-Oxidation Systems of Escherichia coli and Salmonella enterica Are Not Functionally Equivalent
journal, January 2006


Genetic Regulation of Octane Dissimilation Plasmid in Pseudomonas
journal, April 1973

  • Chakrabarty, A. M.; Chou, G.; Gunsalus, I. C.
  • Proceedings of the National Academy of Sciences, Vol. 70, Issue 4
  • DOI: 10.1073/pnas.70.4.1137

Metabolic engineering of 2-pentanone synthesis in Escherichia coli
journal, April 2013

  • Lan, Ethan I.; Dekishima, Yasumasa; Chuang, Derrick S.
  • AIChE Journal, Vol. 59, Issue 9
  • DOI: 10.1002/aic.14086

ThYme: a database for thioester-active enzymes
journal, November 2010

  • Cantu, D. C.; Chen, Y.; Lemons, M. L.
  • Nucleic Acids Research, Vol. 39, Issue Database
  • DOI: 10.1093/nar/gkq1072

Regulation of polyhydroxyalkanoate biosynthesis in Pseudomonas putida and Pseudomonas aeruginosa
journal, August 2004


Enzymatic assembly of DNA molecules up to several hundred kilobases
journal, April 2009

  • Gibson, Daniel G.; Young, Lei; Chuang, Ray-Yuan
  • Nature Methods, Vol. 6, Issue 5, p. 343-345
  • DOI: 10.1038/nmeth.1318

A process for microbial hydrocarbon synthesis: Overproduction of fatty acids in Escherichia coli and catalytic conversion to alkanes
journal, June 2010

  • Lennen, Rebecca M.; Braden, Drew J.; West, Ryan M.
  • Biotechnology and Bioengineering, Vol. 106, Issue 2, p. 193-202
  • DOI: 10.1002/bit.22660

Computational Redesign of Acyl-ACP Thioesterase with Improved Selectivity toward Medium-Chain-Length Fatty Acids
journal, May 2017

  • Grisewood, Matthew J.; Hernández-Lozada, Néstor J.; Thoden, James B.
  • ACS Catalysis, Vol. 7, Issue 6
  • DOI: 10.1021/acscatal.7b00408

Engineering β-oxidation in Yarrowia lipolytica for methyl ketone production
journal, July 2018


The biology of methyl ketones
journal, July 1971


Engineering of Bacterial Methyl Ketone Synthesis for Biofuels
journal, October 2011

  • Goh, Ee-Been; Baidoo, Edward E. K.; Keasling, Jay D.
  • Applied and Environmental Microbiology, Vol. 78, Issue 1
  • DOI: 10.1128/AEM.06785-11

Production of 1-octanol in Escherichia coli by a high flux thioesterase route
journal, September 2020


Spectrophotometric Studies on the Decarboxylation of β-Keto Acids
journal, May 1948


Identification of a Type III Thioesterase Reveals the Function of an Operon Crucial for Mtb Virulence
journal, May 2007