大肠杆菌BL21(DE3)磷酸转乙酰基酶缺陷变株的发酵研究

研究了E.coliBL21(DE3)及其磷酸转乙酰基酶(PTA)缺陷变株FR55发酵过程中菌体生长和有机酸产生情况，并以肿瘤坏死因子(TNF)为外源蛋白表达的模型考察了pta基因缺陷对外源蛋白表达的影响。在摇瓶培养条件下，pta变株TNF的表达水平比亲株提高了23%。在5L发酵罐中进行了补料分批培养试验，在不限制比生长速率的条件下pta变株能够以较长时间和较高比生长速率保持对数生长，最终达到32.5g(DCW)/L的菌密度，TNF的总表达量达2.8g/L；而在相同条件下，以BL21(DE3)为受体菌的对照组最高菌密度为19.5g(DCW)/L,TNF总表达量只有0.84g/L。表明pta变株对于提高工程菌外源蛋白的表达和实现高密度培养具有一定应用价值。分析了补料分批培养过程中发酵液有机酸组成和含量的动态变化情况，发现pta变株乙酸累积水平明显降低(为亲株乙酸累积水平的42%)的同时，其他几种有机酸(丙酮酸、乳酸、琥珀酸)的累积有显著增加的趋势，使发酵液中总有机酸浓度增加了123%，其中乳酸的累积是影响菌体进一步生长的主要因素。

High Cell Density Culture of Phosphotransacetylase Mutants of Escherichia coli BL21 (DE3)

Cell culture, organic acid production and foreign protein (TNF) expression of E. coli BL21(DE3) and its pta mutant were investigated. Under shaking conditions, TNF expression in pta mutant increased by 23%. During the fed-batch culture without limitation of specific growth rates, the mutant reached a cell density as high as 32.5 g(DCW)/L and total TNF expression at 2.8 g/L, while the parental strain only obtained 19.5 g(DCW)/L and 0.84 g/L. The results indicate that utility of pta mutant as a host is advantageous in foreign protein expression and high cell density culture. Meanwhile, the analysis data of organic acids accumulated during fed-batch culture showed that as the decrease of acetate production(42% of the parental strain), the accumulation of other organic acids(mainly pyruvate, lactate and succinate) obviously increased. As a result, the amount of total organic acids increased by 123% over its parent. The lactate production may be the main obstacle in further growth of the cells.