Rerouting carbon flux for optimized biosynthesis of mesaconate in Escherichia coli

Mesaconate, a branched unsaturated dicarboxylic acid, has drawn great interest because of its versatile applications. In this work, we optimized the fermentation efficiency of Escherichia coli to produce mesaconate from glucose. We first drove the carbon flux to 2-ketoglutarate by overexpressing genes involved in TCA precursor pathway and anaplerotic pathways. Then, to increase the pool of phosphoenolpyruvate (PEP), an upstream precursor for 2-ketoglutarate, the phosphotransferase system (PTS) of E. coli was inactivated by deleting glucose PTS permease and the import of glucose was altered by overexpressing galactose/H⁺ symporter GalP. Further, production optimization was achieved by deleting a class I fumarase (FumA) to block the hydration of mesaconate. Finally, we overexpressed PEP synthase (PpsA) to increase the availability of phosphoenolpyruvate and accelerate the production of mesaconate. These genetic modifications led to mesaconate production with a titer of 23.1 g L^-1 and a yield of 0.46 g g^-1 glucose (64% of the theoretical maximum). This work demonstrates the possibility of engineering a highly efficient bacteria strain that converts glucose into mesaconate with promising titer, rate, and yield.