Cofactor engineering of intracellular CoA/acetyl-CoA and its subsequent effect on isoamyl acetate production in Escherichia coli
Vadali, Ravishankar V.
Doctor of Philosophy
Traditional metabolic engineering focused on pathway manipulation strategies like amplification addition or deletion of pathway to manipulate fluxes. However, cofactors play an essential role in cellular metabolism and their manipulation has the potential to be used, as an additional tool to achieve desired metabolic engineering goals. Coenzyme A and its derivative acetyl-CoA are important cofactors involved in many biosynthetic pathways and precursors for many industrially useful compounds. Our study focused on increasing the intracellular level/fluxes of CoA and acetyl-CoA. This was accomplished by overexpression of key rate controlling enzyme panK in the CoA biosynthesis pathway along with simultaneous supplementation of precursor pantothenic acid. The effect of such precise alteration of CoA metabolism on extracellular metabolite formation was studied. The utility of CoA manipulation system in enhancing production of isoamyl acetate, an industrially useful compound derived from acetyl-CoA was demonstrated. This novel approach of cofactor manipulation was combined with the more traditional approach of competing pathway deletion, acetate production pathway in this case, to further enhance isoamyl acetate productivity. Overexpression of panK led to a significant increase in CoA levels. Acetyl-CoA levels also increased but not as much as CoA leaving much of it in an unacetylated form. The central carbon flux was enhanced, either by overexpression of pdh or pps to increase acetyl-CoA, under elevated CoA levels. The flux through the acetyl-CoA node increased under such conditions. This enhanced carbon flux was efficiently channeled to isoamyl acetate production pathway by inactivating the acetate production pathway. The combination of these metabolic engineering strategies led to a significant increase in isoamyl acetate production. We used production of isoamyl acetate as a model system to demonstrate the beneficial effects of CoA/acetyl-CoA manipulations in enhancing product productivity. This methodology can be easily extended to any other production systems involving the cofactors CoA/acetyl-CoA. Additional studies in our lab have shown that CoA/acetyl-CoA manipulation system is useful in improving productivities of succinate and lycopene.