BioChemical Engineering Program, College of Engineering, University of Georgia, Athens, GA, 30602, USA.
J Biol Eng. 2013 Jan 29;7(1):3. doi: 10.1186/1754-1611-7-3.
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.
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.
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.
与其他细菌一样，大肠杆菌必须仔细调节细胞内钠离子（Na+）的浓度。在细菌生产任何有机酸的过程中，阳离子如 Na+ 会在必须维持近中性 pH 值的过程中积累。在这项研究中，编码 Na+/H+反向转运蛋白膜蛋白的 E. coli nhaA 基因和编码 NhaA 调节蛋白的 nhaR 基因在野生型 E. coli MG1655 中过表达，以及在缺乏丙酮酸甲酸裂解酶活性并因此在厌氧条件下积累乳酸的 MG1655 pflB（ALS1317）中过表达。
在高拷贝诱导表达载体 pTrc99A 上表达 nhaA 或 nhaR 基因会导致 MG1655 的生长速率显著降低。当使用中拷贝数 pBR322 质粒过表达这两个基因时，MG1655 或 ALS1317 对 0.75-0.90 M 的 Na+浓度没有观察到生长速率的变化。在使用 NaOH 加碱控制 pH 值生产模型酸乳酸的分批补料过程中，当浓度达到 55-58 g/L 时，MG1655、MG1655/pBR322、MG1655/pBR322-nhaR 和 MG1655/pBR322-nhaA 中的乳酸积累停止。在相同的过程中，当浓度达到 70 g/L 以上时，MG1655/pBR322-nhaAR 中的乳酸积累才停止。
尽管过表达这些基因并没有在高 Na+浓度下提高生长速率，但 NhaA 和 NhaR 的共同过表达导致乳酸产量增加了 25%。因此，观察到（缺乏）这些遗传修饰对生长速率的影响是对其对酸积累影响的一个很差的指标。NhaAR 的过表达并没有导致更快的乳酸生产，但允许培养物在 10%更高的 Na+浓度下继续积累乳酸。
