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 »
- Authors:
-
- 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}
}
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