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

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.