Metabolic engineering is the cornerstone of microbial syntheses of value-added, heterologous metabolites, and its toolbox has been extensively employed for maximizing the biosynthetic yields of heterologous molecules. However, there are fewer examples of applying metabolic engineering for improving the productivity of the strains. Productivities of fermentations have hitherto been largely improved through bioprocess engineering. We posited that re-tooling the expression machinery of the host so that it abruptly transitions from biomass accumulation to product generation at a defined time could improve productivity. We verified this hypothesis using a simple mathematical model and subsequently re-engineered the expression machinery in E. coli to switch between these regimes in response to an external stimulus. Specifically, we modified the T7 RNA polymerase that drives expression of the desired metabolic pathway by interrupting its sequence with a temperature-sensitive mutant of the vacuolar membrane ATPase (VMA) intein of S. cerevisiae. This modification temporarily inactivates the T7 RNA polymerase and turns off product formation in favour of biomass accumulation. The polymerase is only activated within the cell when the temperature of the culture is lowered from 37 °C to 18 °C at a defined time, which then coaxes the cells to transition exclusively to product formation. When we tested this molecular control scheme in a strain of E. coli that also expresses the lycopene biosynthetic pathway, we observed that the cells exhibited improved resource allocation, greater stringency of control over expression of the production pathway, and a 15% improvement in productivity. Our results establish a robust and generalizable model for applying metabolic engineering to improve productivity; and, most importantly, our approach seamlessly interfaces metabolic and macroscopic process control.

中文翻译：

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一种分子开关，可提高异源代谢物生产的生物过程的生产率

代谢工程是增值异源代谢产物微生物合成的基础，其工具箱已被广泛用于最大化异源分子的生物合成产率。然而，很少有应用代谢工程来提高菌株生产力的例子。迄今为止，发酵的生产率已经通过生物工艺工程得到了很大的提高。我们假设重新改造宿主的表达机制，使其在定义的时间从生物质积累突然转变为产物产生，可以提高生产率。我们使用简单的数学模型验证了这一假设，然后重新设计了大肠杆菌中的表达机制在外部刺激下在这些制度之间进行切换。具体而言，我们修饰了T7 RNA聚合酶，该酶通过用酿酒酵母液泡膜ATPase（VMA）内含肽的温度敏感突变体中断其序列来驱动所需代谢途径的表达。这种修饰暂时使T7 RNA聚合酶失活，并关闭了有利于生物质积累的产物形成。仅当在规定的时间将培养物温度从37°C降至18°C时，聚合酶才会在细胞内被激活，然后诱使细胞仅转移至产物形成。当我们在大肠杆菌菌株中测试这种分子控制方案时它也表达番茄红素的生物合成途径，我们观察到细胞表现出改善的资源分配，对生产途径表达的控制更加严格，生产率提高了15％。我们的结果建立了一个健壮且可推广的模型，用于应用代谢工程来提高生产率。而且，最重要的是，我们的方法无缝地衔接了代谢和宏观过程控制。