Modification of glycolysis and its effect on the production of threonine in Escherichia coli

High concentrations of acetate, the main by-product of Escherichia coli (E. coli) high cell density culture, inhibit bacterial growth and l-threonine production. Since metabolic overflux causes acetate accumulation, we attempted to reduce acetate production by redirecting glycolysis flux to the pentose phosphate pathway by deleting the genes encoding phosphofructokinase (pfk) and/or pyruvate kinase (pyk) in an l-threonine-producing strain of E. coli, THRD. pykF, pykA, pfkA, and pfkB deletion mutants produced less acetate (9.44 +/- A 0.83, 3.86 +/- A 0.88, 0.30 +/- A 0.25, and 6.99 +/- A 0.85 g/l, respectively) than wild-type THRD cultures (19.75 +/- A 0.93 g/l). THRD Delta pykF and THRD Delta pykA produced 11.05 and 5.35 % more l-threonine, and achieved a 10.91 and 5.60 % higher yield on glucose, respectively. While THRD Delta pfkA grew more slowly and produced less l-threonine than THRD, THRD Delta pfkB produced levels of l-threonine (102.28 +/- A 2.80 g/l) and a yield on glucose (0.34 g/g) similar to that of THRD. The dual deletion mutant THRD Delta pfkB Delta pykF also achieved low acetate (7.42 +/- A 0.81 g/l) and high l-threonine yields (111.37 +/- A 2.71 g/l). The level of NADPH in THRD Delta pfkA cultures was depressed, whereas all other mutants produced more NADPH than THRD did. These results demonstrated that modification of glycolysis in E. coli THRD reduced acetate production and increased accumulation of l-threonine.