Tunner J R, Robertson C R, Schippa S, Matin A
Department of Chemical Engineering, Stanford University, Stanford, California 94305-5025, USA.
Biotechnol Bioeng. 1992 Jun 20;40(2):271-9. doi: 10.1002/bit.260400211.
The use of glucose starvation to uncouple the production of recombinant beta-galactosidase from cell growth in Escherichia coli was investigated. A lacZ operon fusion to the carbon starvation-inducible cst-1 locus was used to control beta-galactosidase synthesis. beta-Galactosidase induction was observed only under aerobic starvation conditions, and its expression continued for 6 h following the onset of glucose starvation. The cessation of beta-galactosidase expression closely correlated with the exhaustion of acetate, an overflow metabolite of glucose, from the culture medium. Our results suggest the primary role of acetate in cst-1-controlled protein expression is that of an energy source. Using this information, we metered acetate to a glucose-starved culture and produced a metabolically sluggish state, where growth was limited to a low linear rate and production of recombinant beta-galactosidase occurred continuously throughout the experiment. The cst-1 controlled beta-galactosidase synthesis was also induced at low dilution rates in a glucose-limited chemostat, suggesting possible applications to high-density cell systems such as glucose-limited recycle reactors. This work demonstrates that by using an appropriate promoter system and nutrient limitation, growth can be restrained while recombinant protein production is induced and maintained.
研究了利用葡萄糖饥饿使大肠杆菌中重组β-半乳糖苷酶的产生与细胞生长解偶联的方法。使用与碳饥饿诱导型cst-1基因座融合的lacZ操纵子来控制β-半乳糖苷酶的合成。仅在有氧饥饿条件下观察到β-半乳糖苷酶的诱导，并且在葡萄糖饥饿开始后其表达持续6小时。β-半乳糖苷酶表达的停止与培养基中葡萄糖的溢流代谢产物乙酸盐的耗尽密切相关。我们的结果表明，乙酸盐在cst-1控制的蛋白质表达中的主要作用是作为能量来源。利用这一信息，我们将乙酸盐计量加入到葡萄糖饥饿的培养物中，产生了一种代谢迟缓的状态，其中生长限于低线性速率，并且在整个实验过程中持续产生重组β-半乳糖苷酶。在葡萄糖限制的恒化器中，以低稀释率也诱导了cst-1控制的β-半乳糖苷酶合成，这表明可能应用于高密度细胞系统，如葡萄糖限制的循环反应器。这项工作表明，通过使用适当的启动子系统和营养限制，可以在诱导和维持重组蛋白产生的同时抑制生长。
