Metabolic flux reorientation in escherichia coli pta mutant

Abstract

It was set out to manipulate the central metabolism of Escherichia coli in order to redirect acetate flow to the formation of either L- or D-lactic acid, or PHB. The metabolic flux of pta (phosphotransacetylase) mutant of E. coli RRl and its parental strain in anaerobic condition was analyzed. The production of acetate in E. coli was coordinately regulated with ethanol production. The pta mutant of E. coli RRl under anaerobic condition fermented glucose more like a homo-fermentative bacteria with D-lactate as major product. More than 60 g/l of D-lactate was accumulated under anaerobic cultivation of this mutant. When the L-lactate deydrogenase of Lactobacillus casei introduced in this mutant, metabolic redirection to L-lactate did not occur. Therefore, a pta ldh double mutant of E. coli RRl was constructed, and L-lactate dehydrogenase gene of L. casei introduced. In the anaerobic cultivation of this recombinant, glucose was mainly catabolized to L-lactate, in which more than 25g/l of L-lactate was accumulated. To know whether the perturbed intracellular redox potential was the real cause for this redirection of metabolic flow in pta mutant or in pta ldh mutant containing L-lactate gene, PHB accumulation in both strain, E. coli RRl and its pta mutant were compared. As expected, pta mutant accumulated PHB more efficiently than E. coli RRl. In both aerobic and anaerobic cultivation of pta mutant, in which introduced PHB synthetic genes of Alcailgenes eutrophus, PHB was accumulated over than 55\% of dry cell weight. In contrast, its parental strain RRl accumulated PHB less than 30\% of dry cell weight. PHB accumulation, a possible competitive metabolic pathway to D-lactate formation, did not affect significantly the production of D-lactate under oxygen-limited condition.