An insight into the role of phosphotransacetylase (pta) and the acetate/acetyl-CoA node in Escherichia coli

Abstract

Background: Acetate metabolism in Escherichia coli plays an important role in the control of the central metabolism and in bioprocess performance. The main problems related to the use of E. coli as cellular factory are i) the deficient utilization of carbon source due to the excretion of acetate during aerobic growth, ii) the inhibition of cellular growth and protein production by acetate and iii) the need for cofactor recycling (namely redox coenzymes and free CoASH) to sustain balanced growth and cellular homeostasis.

Results: This work analyzes the effect of mutations in the acetate excretion/assimilation pathways, acetyl-CoA synthethase (acs) and phosphotransacetylase (pta), in E. coli BW25113 grown on glucose or acetate minimal media. Biomass and metabolite production, redox (NADH/NAD+) and energy (ATP) state, enzyme activities and gene expression profiles related to the central metabolism were analyzed. The knock-out of pta led to a more altered phenotype than that of acs. Deletion of pta reduced the ability to grow on acetate as carbon source and strongly affected the expression of several genes related to central metabolic pathways.

Conclusion: Results showed that pta limits biomass yield in aerobic glucose cultures, due to acetate production (overflow metabolism) and its inefficient use during glucose starvation. Deletion of pta severely impaired growth on acetate minimal medium and under anaerobiosis due to decreased acetyl-coenzyme A synthethase, glyoxylate shunt and gluconeogenic activities, leading to lower growth rate. When acetate is used as carbon source, the joint expression of pta and acs is crucial for growth and substrate assimilation, while pta deletion severely impaired anaerobic growth. Finally, at an adaptive level, pta deficiency makes the strain more sensitive to environmental changes and de-regulates the central metabolism.

Figure 1

Simplified model for the central metabolic network of E. coli metabolism. The enzymes involved (and their codifying genes) are shown in the figure, ACEK (aceK), isocitrate dehydrogenase phosphatase/kinase; ACK (ackA), acetate kinase; ACS (acs), acetyl-CoA synthetase; ICDH (icd), isocitrate dehydrogenase; ICL (aceA), isocitrate lyase; ICLR (iclR), repressor of the glyoxylate shunt; MDH (maeB), malate dehydrogenase; ME (sfcA), malic enzyme, PFL (pfl) pyruvate:formate lyase; PTA (pta), phosphotransacetylase (Ecocyc-Metacyc [45]).

Figure 2

Growth and metabolite production of A) E. coli BW25113 (wild type.) and its B) Δacs and C) Δpta knockout derivative strains. Experiments were performed using glucose as the carbon source. Sampling times for enzyme activities and/or gene expression are indicated by arrows. Assays were carried out as indicated in the Materials and Methods section.

Figure 3

Growth and metabolite production of A) E. coli BW25113 (wild type) and its B) Δacs and C) Δpta knockout derivative strains. Experiments were performed using acetate as the carbon source. Sampling times for enzyme activities and/or gene expression are indicated by arrows. Assays were carried out as indicated in the Materials and Methods section.

Figure 4

Growth and metabolite production of A) E. coli BW25113 (wild type) and its B) Δacs and C) Δpta knockout derivative strains. Experiments were performed using glucose as the carbon source and anaerobiosis. Sampling times for enzyme activities and/or gene expression are indicated by arrows. Assays were carried out as indicated in the Materials and Methods section.

Figure 5

Simplified model for the alterations in the metabolic network of E. coli after pta deletion. Lactate is produced as a result of the knock-out of the pta gene and it is used to produce ATP by the combined action of lactate dehydrogenases, nLDH and iDLDH. Moreover, pyruvate accumulation would activate the pdh operon. The enzymes involved are shown in the figure [ACK (ackA), acetate kinase; PTA (pta), phosphotransacetylase, PDH (aceEF;lpd) pyruvate dehydrogenase (Ecocyc-Metacyc [45]).