title: Substantial improvements in methyl ketone production in E. coli and insights on the pathway from in vitro studies.
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.
authors.affiliation: Joint BioEnergy Institute, 5885 Hollis Avenue, Emeryville, CA 94608, United States; Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, United States.
authors.forename: Ee-Been
authors.initials: EB
authors.lastname: Goh
authors.affiliation: Joint BioEnergy Institute, 5885 Hollis Avenue, Emeryville, CA 94608, United States; Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, United States.
authors.forename: Edward E K
authors.initials: EEK
authors.lastname: Baidoo
authors.affiliation: Joint BioEnergy Institute, 5885 Hollis Avenue, Emeryville, CA 94608, United States; Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, United States.
authors.forename: Helcio
authors.initials: H
authors.lastname: Burd
authors.affiliation: Joint BioEnergy Institute, 5885 Hollis Avenue, Emeryville, CA 94608, United States; Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, United States.
authors.forename: Taek Soon
authors.initials: TS
authors.lastname: Lee
authors.affiliation: Joint BioEnergy Institute, 5885 Hollis Avenue, Emeryville, CA 94608, United States; Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, United States; Departments of Chemical & Biomolecular Engineering and of Bioengineering, University of California, Berkeley, CA 94720, United States.
authors.forename: Jay D
authors.initials: JD
authors.lastname: Keasling
authors.affiliation: Joint BioEnergy Institute, 5885 Hollis Avenue, Emeryville, CA 94608, United States; Earth Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, United States. Electronic address: HRBeller@lbl.gov.
authors.forename: Harry R
authors.initials: HR
authors.lastname: Beller
mesh_terms: D004926:Escherichia coli / Q000502:physiology*; D029968:Escherichia coli Proteins / Q000235:genetics*; D024861:Genetic Enhancement / Q000379:methods*; D006588:Hexanones / Q000378:metabolism*; D060847:Metabolic Engineering / Q000379:methods*; D015398:Signal Transduction / Q000502:physiology
chemical_list: D029968:Escherichia coli Proteins; D006588:Hexanones
keywords: Acetate; Beta-oxidation; Biofuels; FadM; Fatty acid; Methyl ketones