Systems Biology Engineering of the Pantothenate Pathway to Enhance 3HB Productivity in Escherichia coli

The monomer, 3-hydroxybutyrate (3HB), plays an interesting role as a precursor for antibiotics, vitamins, and bioplastics such as polyhydroxybutyrates (PHB). The biotechnological production of both compounds in Escherichia coli has seen increased interest in the last decade. The central metabolite in the 3HB production pathway is acetyl-CoA, the derivative of coenzyme A (CoA). Enriching the intracellular pool of these cofactors could improve 3HB titers. In our study, we opted to increase CoA titers by upregulating pantothenate kinase (PanK), the rate limiting step in CoA biosynthetic pathway. To this end, 4 PanKs genes of different taxonomic origins (mammalian, fungal, and bacterial) were individually expressed and evaluated in 3HB producing E. coli cells. In shake flask studies, strains expressing Aspergillus nidulans PankII and Mus musculus PanK1β achieved the highest 3HB titers. In a bioreactor fermentation, the strain harboring murine PanK1β resulted in 7.6 g/L compared to 5.4 g/L of 3HB in the control strain. Although structurally different from the bacterial PankI, our study showed that eukaryotic Panks can supplement the kinase activity in prokaryotes. Expressing the exogenous PanKs offer several advantages over the host’s enzyme; PanKII is only inhibited by acetyl-CoA, for which the 3HB-production system would provide a constant metabolic sink. Additionally, PanK1β is weakly regulated by acetyl-CoA, and its activity is stimulated by free CoA. Overexpressing eukaryotic PanKs constitutes a suitable strategy for improving 3HB titers in E. coli.