Engineering the metabolism of Escherichia coli for the synthesis of oxidized products: Acetate and pyruvate production

To demonstrate a novel approach that combines attributes of fermentative metabolism and oxidative metabolism into a single biocatalyst for production of oxidized chemicals, Escherichia coli was genetically engineered for acetate and pyruvate production. Acetate producing E. coli TC36 was constructed by sequentially assembling chromosomal deletions to inactivate oxidative phosphorylation (DeltaatpFH), disrupt the tricarboxylic acid cycle (DeltasucA), and eliminate fermentation pathways (DeltafocA-pflB DeltafrdBC Delta ldhA DeltaadhE). StrainTC36 produced 572 mM acetate from 6% glucose (333 mM) and a maximum of 878 mM acetate with glucose excess. Although strain TC36 produced a maximum of 4 ATP (net) per molecule of glucose as compared to 33 ATP (net) for wild type strains, it grew in glucose minimal medium (mu = 0.49, 82% of W3110) without supplements. Glycolytic flux in strain TC36 was estimated to be 0.3 mumol min-1 (mg protein)-1, twice that of the wild type parent (W3110). Pyruvate producing strain TC44 (DeltafocA-pfB Delta frdBC DeltaldhA DeltaatpFH Delta adhEDeltasucA poxB::FRT DeltaackA) was constructed from strain TC36 by inserting additional chromosomal deletions in the acetate kinase (DeltaackA) and pyruvate oxidase (DeltapoxB) genes. In mineral salts medium containing glucose as the sole carbon source, strain TC44 converted glucose to pyruvate with a yield of 0.75 g pyruvate per g glucose (77.9% of theoretical yield; 1.2 g pyruvate L-1 h-1). The tolerance of E. coli to such drastic changes in metabolic flow and energy production implies considerable elasticity in permitted pool sizes for key metabolic intermediates such as pyruvate and acetyl-CoA. The basic principles demonstrated using strains TC36 and TC44 can be applied for the production of a variety of other chemicals, irrespective of their relative oxidation states.