High glycolytic flux improves pyruvate production by a metabolically engineered Escherichia coli strain

Abstract

We report pyruvate formation in Escherichia coli strain ALS929 containing mutations in the aceEF, pfl, poxB, pps, and ldhA genes which encode, respectively, the pyruvate dehydrogenase complex, pyruvate formate lyase, pyruvate oxidase, phosphoenolpyruvate synthase, and lactate dehydrogenase. The glycolytic rate and pyruvate productivity were compared using glucose-, acetate-, nitrogen-, or phosphorus-limited chemostats at a growth rate of 0.15 h(-1). Of these four nutrient limitation conditions, growth under acetate limitation resulted in the highest glycolytic flux (1.60 g/g . h), pyruvate formation rate (1.11 g/g h), and pyruvate yield (0.70 g/g). Additional mutations in atpFH and arcA (strain ALS1059) further elevated the steady-state glycolytic flux to 2.38 g/g h in an acetate-limited chemostat, with heterologous NADH oxidase expression causing only modest additional improvement. A fed-batch process with strain ALS1059 using defined medium with 5 mM betaine as osmoprotectant and an exponential feeding rate of 0.15 h(-1) achieved 90 g/liter pyruvate, with an overall productivity of 2.1 g/liter h and yield of 0.68 g/g.

FIG. 1.

Key enzymatic reactions in the production of pyruvate by Escherichia coli strains. Enzymes: 1, PEP carboxylase, 2, PEP synthase, 3, lactate dehydrogenase, 4, pyruvate oxidase, 5, pyruvate formate lyase, 6, pyruvate dehydrogenase complex, 7, acetyl-CoA synthetase, 8, acetate kinase, 9, phosphotransacetylase.

FIG. 2.

Results of fed-batch process using E. coli strains with and without 5 mM betaine at a growth rate of 0.15 h⁻¹. OD (at 600 nm): ○, ALS929 with betaine; ▵, ALS929 without betaine; □, ALS1059 with betaine. Pyruvate: •, ALS929 with betaine; ▴, ALS929 without betaine; ▪, ALS1059 with betaine. L, liter.