Substantial improvements in methyl ketone production in E. coli and insights on the pathway from in vitro studies |
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| Affiliation: | 1. Joint BioEnergy Institute, 5885 Hollis Avenue, Emeryville, CA 94608, United States;2. Earth Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, United States;3. Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, United States;4. Departments of Chemical & Biomolecular Engineering and of Bioengineering, University of California, Berkeley, CA 94720, United States;1. Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences, Nanjing, Jiangsu, China;2. Department of Plant Pathology, Nanjing Agricultural University, Nanjing, Jiangsu, China;3. College of Plant Protection, Anhui Agricultural University, Hefei, Anhui, China;1. Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA;2. Synthetic Biology Engineering Research Center (SynBERC), Massachusetts Institute of Technology, Cambridge, MA 02139, USA;1. INRA, AgroParisTech, UMR1319 Micalis, F-78350 Jouy-en-Josas, France;2. Department of Biotechnology and Food Microbiology, Wroclaw University of Environmental and Life Sciences, Chełmońskiego 37/41, 51-630 Wroclaw, Poland;1. Biotechnology Core Laboratory, National Institute of Diabetes and Digestive and Kidney Diseases, National Institute of Health, Bethesda, MD, 20892, United States;2. Laboratorio de Ecología Molecular Microbiana, Centro de Investigaciones en Ciencias Microbiológicas, Instituto de Ciencias, Benemérita Universidad Autónoma de Puebla (BUAP), Puebla 72000, Mexico;3. Mass Spectrometry Facility, National Institute of Diabetes and Digestive and Kidney Diseases, National Institute of Health, Bethesda, MD, 20892, United States |
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| Abstract: | We previously reported development of a metabolic pathway in Escherichia coli for overproduction of medium-chain methyl ketones (MK), which are relevant to the biofuel and flavor-and-fragrance industries. This MK pathway was a re-engineered version of β-oxidation designed to overproduce β-ketoacyl-CoAs and involved overexpression of the fadM thioesterase gene. Here, we document metabolic engineering modifications that have led to a MK titer of 3.4 g/L after ~45 h of fed-batch glucose fermentation and attainment of 40% of the maximum theoretical yield (the best values reported to date for MK). Modifications included balancing overexpression of fadR and fadD to increase fatty acid flux into the pathway, consolidation of the pathway from two plasmids into one, codon optimization, and knocking out key acetate production pathways. In vitro studies confirmed that a decarboxylase is not required to convert β-keto acids into MK and that FadM is promiscuous and can hydrolyze several CoA-thioester pathway intermediates. |
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| Keywords: | Methyl ketones Fatty acid Biofuels FadM Beta-oxidation Acetate |
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