Abstract
BackgroundAcetate 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.ResultsThis 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.ConclusionResults 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.
Highlights
Acetate metabolism in Escherichia coli plays an important role in the control of the central metabolism and in bioprocess performance
We provide a further insight into the connection between the acetate/acetyl-CoA node and the central metabolism of E. coli
The activity of the central pathways of E. coli is affected by the deletion of the genes of the acetyl-CoA/acetate metabolism
Summary
Acetate metabolism in Escherichia coli plays an important role in the control of the central metabolism and in bioprocess performance. It is expressed during the stationary phase, its regulation is complex, since it involves several transcription factors, two sigma factors and two promoters [7] The gene coding this enzyme, acs, is within an operon, together with another two genes: yjcH, an inner membrane conserved protein of unknown function, and actP, which codes for an acetate permease [8,9]. This pathway excretes acetate and produces ATP, a mechanism known as "acetate overflow" [10] It is characterized by its low affinity (Km 7-10 mM), but high Vm [11], being able to dissimilate high concentrations of acetate. 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])
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