(284d) Engineering of Bacterial Methyl Ketone Synthesis for Biofuels: Recent Advances

Engineering of bacterial methyl ketone synthesis for biofuels: recent advances

Harry R. Beller* and Ee-Been Goh

Fuels Synthesis Division, Joint BioEnergy Institute

We have engineered Escherichia coli to overproduce saturated and monounsaturated aliphatic methyl ketones in the C₁₁ to C₁₅ (diesel) range; this group of methyl ketones includes 2-undecanone and 2-tridecanone, which are of importance to the flavor and fragrance industry and also have favorable cetane numbers (as we report here).  We describe 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 beta-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).  FadM was previously associated with oleic acid degradation, not methyl ketone synthesis, but outperformed a recently identified methyl ketone synthase (ShMKS2, a thioesterase from wild tomato) in beta-ketoacyl-CoA-overproducing strains tested.  The first generation of engineered E. coli (Goh et al. 2012) produced ~380 mg/L of methyl ketones in rich media.  We have subsequently made additional genetic modifications, including the chromosomal deletion of poxB and consolidation of all pathway genes (originally borne on two plasmids) onto a single plasmid, and have tested production in EZ-Rich medium and M9 minimal medium with 1% glucose. The poxB knockout strain accumulates less acetate (as expected) but also has a shorter lag time preceding methyl ketone biosynthesis, which increases productivity. In EZ-Rich medium and M9 minimal medium, titers of the poxB knockout strain were more than 2-fold higher than those of the poxB wild-type strain.  Our current methyl ketone titer is >1.5 g/L, which represents 40-45% of maximum theoretical yield; these are the highest titer and yield values reported to date for methyl ketones.