Cofactor engineering for short and medium chain fatty acid production in E. coli

Fatty acid is an important platform chemical that is widely used in different fields, especially as a precursor of biofuels/ biodiesel and chemicals. Overexpression of an acyl-ACP thioesterase to break the fatty acid elongation cycle to produce free fatty acid has been well established. In microbial production processes, increasing precursor and cofactor availability has become an effective approach in achieving efficient production platform. An important cofactor in the production of most reduced compounds, such as fatty acids, is the availability of reducing equivalent, NAD(P)H.  There are two reduction steps in the fatty acid elongation cycle which are catalyzed by beta keto-ACP reductase (FabG) and enoyl-ACP reductase (FabI), respectively. It is reported that FabI can use either NADH or NADPH as the cofactor, while FabG only uses NADPH in E. coli. The cofactor NADPH is mainly generated through the pentose pathway, the TCA cycle, and the transhydrogenase system, such as the proton-translocating transhydrogenase PntAB and an energy dependent transhydrogenase UdhA and NAD kinase. The pntB and sthA single and double mutant strains showed dramatic drops in fatty acid production indicating that the availability of NADPH plays a very important role in fatty acid production. Increased fatty acid production was obtained in several fatty acid producer strains with enhanced NADPH availability. Experimental results demonstrating the effectiveness of cofactor engineering in enhancing the accumulation of short and medium chain length free fatty acid will be presented. The impact of various NADPH manipulations in fatty acid production will be discussed.