Metabolic engineering of acetate metabolism in escherichia coli

Abstract

In order to investigate the physiological roles of acetate metabolism, the pta mutants of Escherichia coli defective in phosphotransacetylase, the first enzyme of the acetate pathway, was characterized. A pta mutant of E. coli W3100 excreted unusual by-products such as pyruvate,D-lactate and L-glutamate instead of acetate when grew on glucose minimal medium. The pta mutant was found to grow slowly on glucose, tryptone broth (TB), or pyruvate but it grew normally on glycerol or succinate. The defective growth and starvation survival of the pta mutant were restored by the introduction of poly-b-hydroxybutyrate (PHB) synthesis genes (phbCAB) from Alcaligenes eutrophus, indicating that the growth defect of the pta mutant was due to a perturbation of acetyl-CoA flux. By the stoichio-metric analysis of the metabolic fluxes of the central metabolism, it was found that the amount of pyruvate generated from glucose transport by the PEP-dependent phosphotransferase system (PTS) exceeded the required amount of precursor metabolites downstream of pyruvate for biomass synthesis. The perturbed acetyl-CoA flux in the pta mutant effected redirection of overall fermentation metabolism. A pta mutant of E. coli RR1 strain was found to metabolize glucose to D-lactate with a small amount of succinate by-product under anaerobic conditions. A further mutation in ppc made the mutant produce D-lactate like a homofermentative lactic acid bacterium. When this pta ppc double mutant was grown to higher biomass concentration under aerobic conditions, before shifting to the anaerobic phase of D-lactate production, more than 62.2 g/l of D-lactate was produced in 60 hours, reaching a volumetric productivity of 1.04g/L/hr. When L-lactate dehydrogenase gene from Lactobacillus casei was introduced into a pta ldhA strain, which lacked Pta and D-lactate dehydrogenase, the recombinant strain was able to metabolize glucose to L-lactate as the major fermentation product, and up to 45g/L of L-lactate w...