Oh Jeong Seok, Cho Daechul, Park Tai Hyun
School of Chemical and Biological Engineering, Seoul National University, 56-1, Shilim-Dong, Gwanak-Gu, Seoul, Korea.
Bioprocess Biosyst Eng. 2005 Nov;28(1):1-7. doi: 10.1007/s00449-005-0418-0. Epub 2005 Oct 28.
A two-stage continuous culture of Escherichia coli in combination with a bacteriophage lambda system was performed in order to overcome the intrinsic plasmid instability that is frequently observed in recombinant fermentation. A phage lambda vector with a Q(-) mutation was used to enhance the expression of the lambda system. The optimal values of the important operational variables such as the substrate concentration, the dilution rate, and the mean residence time on the expression of the cloned gene were determined in both batch and continuous cultures. For all culturing modes, the full induction of the cloned gene was observed 4 h after the temperature shift. In the two stage continuous culture, the overproduction reached their maxima at D=0.25 h(-1) with 1.5 S(0) of the medium supply. The maximum productivity of the total beta-galactosidase was 16.3x10(6) U l(-1) h(-1), which was approximately seven times higher than that in the single-copy lysogenic stage. The recombinant cells were stable in the lysogenic state for more than 260 h, while they were stable for 40 h in the lytic state. The instability that developed rapidly in the second tank is believed to be due to the accumulation of lysis proteins as a result of vector leakage during the operation.
为克服重组发酵中常见的内在质粒不稳定性，进行了大肠杆菌与噬菌体λ系统的两阶段连续培养。使用具有Q(-)突变的噬菌体λ载体来增强λ系统的表达。在分批培养和连续培养中均确定了底物浓度、稀释率和平均停留时间等重要操作变量对克隆基因表达的最佳值。对于所有培养模式，温度转换后4小时观察到克隆基因的完全诱导。在两阶段连续培养中，当培养基供应为1.5S(0)且D = 0.25 h(-1)时，过量生产达到最大值。总β-半乳糖苷酶的最大生产力为16.3×10(6) U l(-1) h(-1)，约为单拷贝溶原阶段的七倍。重组细胞在溶原状态下稳定超过260小时，而在裂解状态下稳定40小时。第二罐中迅速出现的不稳定性被认为是由于操作过程中载体泄漏导致裂解蛋白积累所致。
