大肠杆菌DH5α耐乙酸突变株的选育及其代谢特性研究

Selection of Acetate-Tolerant Mutants from Escherichia coli DH5α and the Metabolic Properties of Mutant DA19

大肠杆菌DH5α是基因工程常用的宿主菌之一 ,但由于对代谢副产物乙酸十分敏感 ,影响外源基因的表达效率。为了提高E .coliDH5α乙酸耐受力 ,采用6 0 Co诱变结合连续培养 ,逐步提高稀释率和乙酸钠选择压力 ,于含乙酸钠平板进一步筛选 ,得到 5株对乙酸耐受能力显著增强的突变菌株 ,具有良好的遗传稳定性 ,其中DA1 9显示最强的耐受性能。DA1 9与DH5α相比 ,在复合培养基YPS和YPS2G中菌体浓度分别提高 1 7%和 5 % ,最大比生长速率分别提高 8%和 2 7% ,产乙酸分别减少为 6%和 5 9% ;在基本培养基中的细胞浓度提高 2 4倍 ,在含 1 0g L乙酸钠培养基中达到的细胞浓度与不加乙酸钠DH5α的细胞浓度相当。

Esherichia coli DH5α is one of the widely used host strains in genetic engineering. However, foreign gene expression level in this strain is seriously inhibited due to its great sensitivity to the accumulated metabolite, acetate. This study aimed at improving the tolerance of this strain against acetate. Cells of E. coli DH5α were irradiated with 60 Co, and subsequently continuous culture of the irradiated cells was conducted with gradual increase in the dilution rate and the selective pressure, acetate concentration in the medium. The mutants were picked up on MA plates which contained 5g/L sodium acetate. 5 strains with great improvement in acetate tolerance were obtained, among which DA19 was the best. In cultivation of DA19 in complex media YPS and YPS2G, the cell density, maximum specific growth rate and acetate produced were respectively 1.17and 1.05, 1.08 and 1.27, and 0.06 and 0.59 times of those of DH5α. In a chemically defined medium, the cell density of DA19 was 3.4-fold of that of DH5α. The cell density of DA19 in a medium containing 10g/L sodium acetate was comparable to that of DH5α in the same medium without the addition of acetate.