Improving E. coli Methyl Ketone Production during Fed-Batch Fermentation By Co-Factor Balancing

We have engineered Escherichia coli to overproduce aliphatic methyl ketones (MK) in the C₁₁ to C₁₅ (diesel) range; this group of MK includes 2-undecanone and 2-tridecanone, which have favorable cetane numbers and are also of importance to the flavor and fragrance industry. Overall, we have made specific improvements that resulted in 50,000-fold enhancement in MK titer relative to that of a fatty acid-overproducing E. coli strain. The first generation of engineered E. coli (Goh et al. 2012) had modifications that included: (a) overproduction of β-ketoacyl-coenzyme A (CoA) thioesters achieved by modification of the β-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). Subsequent genetic 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 (resulting in strain EGS1895). This strain produced an MK titer of 1.4 g/L with 1% glucose in shake-flask experiments, representing 40% of the maximum theoretical yield. ¹³C-based metabolic flux analysis with EGS1895 showed increased carbon flux through the pentose phosphate pathway (PPP), which we hypothesized is to generate more NADPH - an important co-factor for FabG (β-ketoacyl-ACP reductase) activity during fatty acid biosynthesis. In an attempt to divert flux away from PPP, we overexpressed alternate FabG variants that we have demonstrated preferentially use NADH instead of NADPH. Specifically, overexpression of the NADH-dependent Acholeplasma laidlawii FabG increased MK production by 20-50% in shake-flask experiments and had more sustained MK production in fed-batch fermentation compared to EGS1895. We have achieved MK titers of ~6.0 g/L in fed-batch fermentation, which is the best MK titer observed to date.