We have engineered Escherichia coli to overproduce saturated and monounsaturated aliphatic methyl ketones in the C11 to C17 (diesel) range; this group of methyl ketones includes 2-undecanone and 2-tridecanone, which have favorable cetane numbers and are also of importance to the flavor and fragrance industry.  We have made specific improvements that resulted in more than 10,000-fold enhancement in methyl ketone titer relative to that of a fatty acid-overproducing E. coli strain, including the following: (a) overproduction of �-ketoacyl-coenzyme A (CoA) thioesters achieved by modification of the beta-oxidation pathway (specifically, overexpression of a heterologous acyl-CoA oxidase and native FadB, and chromosomal deletion of fadA) and (b) overexpression of a native thioesterase (FadM). The first generation of engineered E. coli (Goh et al. 2012) produced ~380 mg/L of methyl ketones in rich medium.  We have subsequently made additional genetic modifications, including consolidation of all pathway genes onto a single plasmid (they were originally borne on two plasmids). We have also conducted in vitro assays with purified pathway enzymes, which revealed that FadM is promiscuous and has thioesterase activity not only toward �-ketoacyl-CoAs but also toward other CoA thioester intermediates in the pathway (such as trans-2-enoyl-CoAs). These in vitro results have provided insight on how to fine-tune expression of pathway genes for further optimization of methyl ketone production. Our current methyl ketone titer is >1.5 g/L with 1% glucose (M9 minimal medium), which represents 40-45% of the maximum theoretical yield; these are the highest titer and yield values reported to date for methyl ketones.

Engineering of bacterial methyl ketone synthesis for biofuels: Recent advances

248th National Meeting (2014)

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