Engineering Escherichia coli for efficient conversion of glucose to pyruvate

Abstract

Escherichia coli TC44, a derivative of W3110, was engineered for the production of pyruvate from glucose by combining mutations to minimize ATP yield, cell growth, and CO2 production (DeltaatpFH DeltaadhE DeltasucA) with mutations that eliminate acetate production [poxB::FRT (FLP recognition target) DeltaackA] and fermentation products (DeltafocA-pflB DeltafrdBC DeltaldhA DeltaadhE). In mineral salts medium containing glucose as the sole carbon source, strain TC44(DeltafocA-pflB DeltafrdBC DeltaldhA DeltaatpFH DeltaadhE DeltasucA poxB::FRT DeltaackA) converted glucose to pyruvate with a yield of 0.75 g of pyruvate per g of glucose (77.9% of theoretical yield; 1.2 g of pyruvate liters(-1).h(-1)). A maximum of 749 mM pyruvate was produced with excess glucose. Glycolytic flux was >50% faster for TC44 producing pyruvate than for the wild-type W3110 during fully aerobic metabolism. 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. In strain TC44, pyruvate yield, pyruvate titer, and the rate of pyruvate production in mineral salts medium were equivalent or better than previously reported for other biocatalysts (yeast and bacteria) requiring complex vitamin feeding strategies and complex nutrients. TC44 offers the potential to improve the economics of pyruvate production by reducing the costs of materials, product purification, and waste disposal.

Fig. 1.

Summary of central metabolism in E. coli. (A) Carbon metabolism. (B) Oxidative phosphorylation. (C) Cytoplasmic F₁ATPase subunit (active). Filled circles indicate metabolic steps that have been blocked by mutation or deletion of genes encoding respective enzymes.

Fig. 2.

Effect of oxygen level on pyruvate production by TC36. Cells were inoculated into fermentation broth at 100% air saturation and continuously sparged with air until the oxygen levels declined to 5% saturation. At this time, oxygen was blended to maintain 5% saturation during the remaining period of incubation (open symbols). Alternatively, media was sparged with a mixture of air and nitrogen to provide 5% air saturation before inoculation. Sparging was switched to air and oxygen as needed to maintain 5% air saturation (filled symbols).

Fig. 3.

Batch fermentation of glucose by mutant strains of E. coli.(A) Cell growth. (B) Glucose utilization. (C) Acetate production. (D) Pyruvate production.