Effect of overexpressing nhaA and nhaR on sodium tolerance and lactate production in Escherichia coli

Abstract

Background: Like other bacteria, Escherichia coli must carefully regulate the intracellular concentration of sodium ion (Na+). During the bacterial production of any organic acid, cations like Na+ invariably accumulate during a process which must maintain a near neutral pH. In this study, the E. coli nhaA gene encoding the Na+/H+ antiporter membrane protein and the nhaR gene encoding the NhaA regulatory protein were overexpressed in wild-type E. coli MG1655 and in MG1655 pflB (ALS1317) which lacks pyruvate formate lyase activity and thus accumulates lactate under anaerobic conditions.

Results: Expression of either the nhaA or nhaR gene on the high copy inducible expression vector pTrc99A caused a significant reduction in the growth rate of MG1655. No change in growth rate was observed for MG1655 or ALS1317 for Na+ concentrations of 0.75-0.90 M when the medium copy pBR322 plasmid was used to overexpress the two genes. In a fed-batch process to produce the model acid lactate with NaOH addition for pH control, lactate accumulation ceased in MG1655, MG1655/pBR322, MG1655/pBR322-nhaR and MG1655/pBR322-nhaA when the concentration reached 55-58 g/L. In an identical process lactate accumulation in MG1655/pBR322-nhaAR did not terminate until the concentration reached over 70 g/L.

Conclusions: Although overexpression the genes did not improve growth rate at high Na+ concentrations, the overexpression of nhaA and nhaR together led to a 25% increase in lactate production. Thus, the observed (absence of) impact that these genetic modifications had on growth rate is a poor indicator of their effect on acid accumulation. The overexpression of nhaAR did not cause faster lactate production, but permitted the culture to continue accumulating lactate at 10% greater Na+ concentration.

Figure 1

Maximum specific growth rate (μ_MAX) of MG1655/pTrc99A-nhaA (○, ●) and MG1655/pTrc99A-nhaR (◊, ♦) at low (<0.10 M) Na⁺ concentration (hollow symbols) and at 0.75 M Na⁺ concentration (solid symbols). The specific growth rate of MG1655 was about 0.69 h^-1 with <0.10 M Na⁺ and 0.17 h^-1 at 0.75 M Na⁺ concentration.

Figure 2

Maximum specific growth rate (μ_MAX) of MG1655 (○), MG1655/pBR322 (□), MG1655/pBR322-nhaA (Δ), MG1655/pBR322-nhaR (◊), and MG1655/pBR322-nhaAR (∇) with increasing total Na⁺ concentration.