Journal
METABOLIC ENGINEERING
Volume 61, Issue -, Pages 335-343Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ymben.2020.05.008
Keywords
Thioesterase; 2-Heptanone; 2-Nonanone; 2-Undecanone; Thiolase; Metabolic engineering
Categories
Funding
- National Science Foundation [CBET-1703504]
- DOE Center for Advanced Bioenergy and Bioproducts Innovation (U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research) [DE-SC0018420]
- NIH Chemistry-Biology Interface Training Program fellowship [T32 GM008505]
- UW-Madison College of Engineering
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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. In this work, 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 beta-oxidation cycle at an advantageous reaction, and introducing active beta-ketoacyl-CoA thioesterases. Using a bioprospecting approach, we identified fifteen homologs of E. coli beta-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 2heptanone, 2-nonanone, and 2-undecanone, suggesting it has the highest activity on the corresponding beta-ketoacyl-CoA substrates. We tested enzyme variants, including acyl-CoA oxidases, thiolases, and bi-functional 3hydroxyacyl-CoA dehydratases to maximize conversion of fatty acids to beta-keto acyl-CoAs for 2-heptanone, 2nonanone, 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. Using these modifications, we were able to generate up to 4.4 g/L total medium-chain length methyl ketones.
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