Acetate metabolism in a pta mutant of Escherichia coli W3110: importance of maintaining acetyl coenzyme A flux for growth and survival

Abstract

In order to study the physiological role of acetate metabolism in Escherichia coli, the growth characteristics of an E. coli W3100 pta mutant defective in phosphotransacetylase, the first enzyme of the acetate pathway, were investigated. The pta mutant grown on glucose minimal medium excreted unusual by-products such as pyruvate, D-lactate, and L-glutamate instead of acetate. In an analysis of the sequential consumption of amino acids by the pta mutant growing in tryptone broth (TB), a brief lag between the consumption of amino acids normally consumed was observed, but no such lag occurred for the wild-type strain. The pta mutant was found to grow slowly on glucose, TB, or pyruvate, but it grew normally on glycerol or succinate. The defective growth and starvation survival of the pta mutant were restored by the introduction of poly-beta-hydroxybutyrate (PHB) synthesis genes (phbCAB) from Alcaligenes eutrophus, indicating that the growth defect of the pta mutant was due to a perturbation of acetyl coenzyme A (CoA) flux. By the stoichiometric analysis of the metabolic fluxes of the central metabolism, it was found that the amount of pyruvate generated from glucose transport by the phosphoenolpyruvate-dependent phosphotransferase system (PTS) exceeded the required amount of precursor metabolites downstream of pyruvate for biomass synthesis. These results suggest that E. coli excretes acetate due to the pyruvate flux from PTS and that any method which alleviates the oversupply of acetyl CoA would restore normal growth to the pta mutant.

FIG. 1

Growth and by-product excretion profiles of W3110 (a) and JP231 (b). Strains were grown in an aerated fermentor containing M9 minimal medium supplemented with 2 g of glucose liter⁻¹. Symbols: ●, dry cell weight; □, glucose; ▵, acetate; ⧫, pyruvate; ▾, d-lactate.

FIG. 2

Glutamate accumulation in a pta mutant grown on M9 glucose minimal medium. Profiles of growth (a) and glutamate accumulation (b) of W3110 (open symbols) and JP231 (solid symbols) are represented.

FIG. 3

Profiles of amino acid consumption patterns in W3110 (a) and JP231 (b) grown on TB. Symbols: ●, OD at 600 nm; ■, l-serine; □, l-aspartate; ○, l-glutamate; ▾, l-alanine; ▴, glycine; ▵, l-threonine; ▿, l-arginine.

FIG. 4

Growth and byproduct excretion profiles of recombinant strain W3110 (a) and JP231 (b) harboring phbCAB genes from A. eutrophus. Strains were grown in an aerated fermentor containing M9 minimal medium supplemented with 2 g of glucose per liter. Symbols: ●, dry cell weight; □, glucose; ▵, acetate; ⧫, pyruvate; ▾, d-lactate.

FIG. 5

Starvation survival of W3110, JP231, and their recombinant strains harboring phbCAB genes from A. eutrophus. Strains were grown in shaking flasks containing M9 minimal medium supplemented with 0.2 g of glucose liter⁻¹. After growth was arrested, incubation was continued for 9 days under the same condition. Viable cells were counted as colonies in LB plates after appropriate dilutions. One hundred percent viability corresponds to the number of viable cells counted 1 h after entering starvation. Symbols: ○, W3110; ●, W3110(pKTPHB); □, JP231; ■, JP231(pKTPHB).

FIG. 6

Stoichiometric calculation of metabolic flux distribution in E. coli. The flux distribution was analyzed by Holms’ method (16) with a slight modification in that the TCA cycle was assumed not to work in a cyclic mode but to function only for the generation of OAA, and α-ketoglutarate (2, 27, 36). Numbers in the middle of arrows indicate the fluxes through the specific metabolic step in the direction of the arrow. Italic numbers under precursor metabolites indicate the fluxes incorporated into biomass. A negative value means that the flux is in the direction opposite to the arrow. (a) Flux distribution in an ideal E. coli cell in which the central metabolism is working only for generating the exact amount of the required precursor metabolites and NADPH to make 1 g of biomass (25). The flux through a specific metabolic step was calculated by adding the amounts of precursor metabolites downstream of the metabolic step to make 1 g of biomass. In the resulting flux distribution, the amount of NADPH produced (4.319 mmol) was much smaller than that required to make 1 g of biomass (18.225 mmol). The shortage was covered by the uptake of additional (1.159 mmol) glucose and metabolizing it through the pentose phosphate pathway. (b) Flux distribution in wild-type E. coli in which the central metabolism works to supply the required amounts of precursor metabolites, NADPH, and ATP for 1 g of biomass and the production of acetate, which corresponds to 15% of input carbon. Numbers in parentheses indicate the fluxes for the pta mutant. The fluxes were calculated by adding the fluxes presented in panel a and the additional flux needed to produce acetate (wild type) or acetate, pyruvate, d-lactate, and glutamate (pta mutant). Abbreviations: AcCoA, acetyl CoA; α-KG, α-ketoglutarate; CIT, citrate; ERP, erythrose 4-phosphate; F6P, fructose 6-phosphate; G6P, glucose 6-phosphate; IsoCIT, isocitrate; 3PG, 3-phosphoglycerate; PP, pentose phosphate; PYR, pyruvate; TP, triose phosphate; SucCoA, succinyl CoA.