A novel genetic switch with an optimal ON/OFF ratio to preserve growth performance prior to Escherichia coli autolysis for enhanced plasmid release

当生物技术的主力生物体--大肠杆菌(E.coli)被用来繁殖具有商业价值的质粒时，从大肠杆菌细胞中高效地回收质粒是关键。然而，回收所需的质粒产物可能被证明是具有挑战性的，因为需要大规模的细胞破坏和从复杂的流程流中分离出质粒。本研究的目的是应用一种新的遗传开关，具有最佳的开/关比来控制大肠杆菌的自溶，从而在保持正常生长性能的同时允许更简单、剪切诱导较少的分离。已经报道了几种技术，在这些技术中，触发大肠杆菌自溶以提高重组蛋白或质粒DNA产物的释放。然而，到目前为止，这些大肠杆菌菌株一直表现出生长受阻，因此产量和表现都受到影响，这是由于控制自溶事件启动的遗传触发机制的“泄漏”。如果这项技术成功，将为减少DNA制造中的步骤提供关键方法，使这一过程更具成本效益和可持续性。

When the biotechnology workhorse organism, Escherichia coli (E. coli) is used to propagate commercially valuable plasmids, such as those demanded by the globally expanding cell and gene therapy sector, efficient recovery of plasmids from the E. coli cell is key. However, this recovery of the desired plasmid product can prove challenging, with requirements for cell disruption and plasmid isolation from a complex process stream at scale.The aim of this study is to apply a novel genetic switch, with an optimal ON/OFF ratio to control E.coli autolysis such that normal growth performance is maintained whilst allowing simpler less shear induced separation. Several technologies have been reported in which E. coli autolysis is triggered to improve liberation of recombinant protein or plasmid DNA products. However, to date these E. coli strains consistently exhibit compromised growth, and therefore yield, performance due to 'leakiness' of the genetic trigger mechanism that controls initiation of autolytic events. This technology if successful will provide a key approach in reducing the number of steps in DNA manufacture, making the process more cost effective and sustainable.