We report improved strain and bioprocess robustness as a result of the dynamic deregulation of central metabolism using two-stage dynamic control. Dynamic control is implemented using combinations of CRISPR interference and controlled proteolysis to reduce levels of central metabolic enzymes in the context of a standardized two-stage bioprocesses. Reducing the levels of key enzymes alters metabolite pools resulting in deregulation of the metabolic network. The deregulated network is more robust to environmental conditions improving process robustness, which in turn leads to predictable scalability from high throughput small scale screens to fully instrumented bioreactors as well as to pilot scale production. Additionally, as these two-stage bioprocesses are standardized, a need for traditional process optimization is minimized. Predictive high throughput approaches that translate to larger scales are critical for metabolic engineering programs to truly take advantage of the rapidly increasing throughput and decreasing costs of synthetic biology. In this work we demonstrate that the improved robustness of E. coli strains engineered for the improved scalability of the important industrial chemicals alanine, citramalate and xylitol, from microtiter plates to pilot reactors.

中文翻译：

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代谢的两阶段动态解除调节可改善工程大肠杆菌的工艺鲁棒性和可扩展性。

我们报告了改善的应变和生物过程的鲁棒性，这是由于使用两阶段动态控制的中央新陈代谢动态解除管制。动态控制是通过CRISPR干扰和受控蛋白水解的组合来实现的，以在标准化的两阶段生物过程中降低中央代谢酶的水平。降低关键酶的水平会改变代谢物库，从而导致代谢网络失调。解除管制的网络在环境条件下更加健壮，从而提高了过程的稳健性，进而导致了可预测的可扩展性，从高通量小型筛选器到功能齐全的生物反应器以及中试规模的生产。另外，由于这两个阶段的生物过程已标准化，因此对传统过程优化的需求降至最低。可预测的高通量方法可以转化为更大的规模，对于代谢工程计划真正利用快速增长的通量和降低合成生物学的成本至关重要。在这项工作中，我们证明了为重要工业化学品丙氨酸，柠檬醛和木糖醇从微量滴定板到中试反应器的可扩展性改进而设计的大肠杆菌菌株具有更高的鲁棒性。