Methyl Ketones are naturally occurring compounds originally discovered as odorants and flavor components. Along with their widespread function in biology, methyl ketones have many important industrial roles. Industrially desired methyl ketones typically fall in the medium chain length range (C7-C11). These methyl ketones are produced through the oxidation of hydrocarbons from petrochemical sources. Due to their use industrially, there has been economical as well as environmental incentives to develop an alternative route for producing methyl ketones. Attempts have been made in the past to synthesize medium chain methyl ketones form renewable feedstocks such as sugars, but to little success. This is in part to lack of known enzymes with activity and specificity towards medium chain length intermediates. Here, we aim to demonstrate the production of 2-heptanone in recombinant Escherichia coli from a glycerol feedstock through an engineered combination of the fatty acid biosynthesis and β-oxidation metabolic pathways. A significant bottleneck in methyl ketone production occurs at hydrolysis of the β-keto-acyl-CoA intermediate to the β-keto acid, which is catalyzed by the long chain acyl-CoA thioesterase of E. coli (FadM). We aim to tailor the activity and specificity of FadM towards the β-keto-acyl-CoA intermediate with a high throughput screening system that utilizes a biosensor that detects 2-heptanone. By relieving the bottleneck, we aim to improve 2-heptanone production titers.

Development of a high throughput screen to engineering a long chain acyl-CoA thioesterase for medium chain methyl ketone production in Escherichia coli

2019 Spring National Meeting

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