EAGER: NSF/ABF Dynamic Control of Metabolism Using Redesigned Proteins

微生物可以从生物质中产生燃料和化学品。生物燃料有助于促进美国的能源安全。目前使用微生物进行生产的一个限制是，微生物也使用相同的生物质来生长。快速增长减少了燃料和化学品的生产。然而，控制微生物新陈代谢的新策略可能会将生长与产品形成分开。该项目的目标是开发一种可应用于多种不同生物体的模块化控制系统。该项目的成功将提高燃料和化学品的微生物产量，从而促进生物经济。本项目的目标是评估一种新的动态代谢控制策略。我们将使用先进的计算蛋白质设计和工程来改造T7RNA聚合酶(T7RNAP)，T7RNAP将DNA转录成mRNA，因此它需要用户定义的小分子才能发挥作用。这种改造后的聚合酶将被构件性表达。这种诱导分子激活聚合酶，导致T7特异性基因的表达。我们将(I)确定对各种非模式寄主生物无毒和不可代谢的廉价化学品；(Ii)在恶臭假单胞菌中部署动态控制电路以生产变酸，以显示滴度/产量/生产率的改进指标；以及(Iii)展示T7 RNAP电路对其他与生物制造相关的组织的效用。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

Microbes can create fuels and chemicals from biomass. Bio-based fuels help contribute to energy security in the US. One current limitation to using microbes for production is that the microbes also use the same biomass to grow. Rapid growth reduces fuel and chemical production. However, new strategies for controlling the microbial metabolism could separate growth from product formation. The objective of this project is to develop a modular control system to be applied across many different organisms. Success of this project will enhance microbial production of fuels and chemicals, thus advance the bioeconomy. The objective of this project is to evaluate a novel strategy for dynamic metabolic control. We will use advanced computational protein design and engineering to remodel T7 RNA polymerase (T7RNAP), which transcribes DNA into mRNA, such that it requires a user-defined small molecule to function. This remodeled polymerase would be constitutively expressed. This induction molecule activates the polymerase, resulting in expression of T7-specific genes. We will (i) identify inexpensive chemicals that are non-toxic and non-metabolizable for diverse non-model host organisms; (ii) deploy dynamically controlled circuits in Pseudomonas putida for production of muconate to show improved metrics of titer/yield/productivity; and (iii) demonstrate the utility of T7 RNAP circuits for other biomanufacturing-relevant organisms.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.