Collaborative Research: Redirecting cellular metabolism via synthetic toehold-gated dCas9 regulators

细胞代谢能够在温和的温度和压力下进行高度特异性和高效的化学合成，这远远超出了大多数合成化学途径的能力。工程特定途径可用于进一步提高可合成化合物的范围，但实现商业上可行的生产力是一个主要挑战。为了最大限度地提高生产率，对途径通量进行微调至关重要。该项目的目标是开发一种基于内源性细胞信息调节细胞代谢的新方法。一种新兴的策略是使用提供通路通量动态控制的调节器。最近发现的一种改良的基于CRISPR的工具为DNA靶向和转录调控提供了一种独特的方法。这些新一代调节剂可用于许多合成生物学和代谢工程应用的动态基因抑制和激活。除了科学进步之外，该项目还将通过整合蛋白质工程、合成生物学和细胞生理学的原理来帮助培养研究生。此外，还计划通过特拉华大学和伦斯勒理工学院现有的项目，向当地高中教师和学生开展推广活动。该项目的目标是开发一种基于内源性细胞信息调节细胞代谢的新方法。特别是，新一代的由条件sgRNA结构控制的由支点介导的链位移激活的支点门控dCas9调节剂将被创造出来，以提供细胞代谢的同步、正交和自主控制。由于基于dcas9的调节因子受结构上定义的单导RNA （sgRNA）结构控制，因此很容易设想，可以创建由内源性mrna基于支点介导的链位移激活的条件sgRNA结构。这种设计对任何内源性mRNA响应的支点门控dCas9调节因子的新框架将为实现细胞代谢动态控制的新变革方法奠定基础。将建立基于内源性细胞信息调节代谢的能力，以优化酵母中众多产品的生产。长期目标是将从这个项目中获得的知识结合起来，为任何感兴趣的代谢途径设计动态和自主的细胞控制。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Cellular metabolism is capable of highly specific and efficient chemical synthesis at mild temperatures and pressures far beyond the capability of most synthetic chemical routes. Engineering specific pathways can be used to further improve the range of compounds that can be synthesized but it is a major challenge to achieve commercially viable productivity. To maximize productivity, it is crucial to fine-tune pathway fluxes. The goal of this project is to develop a new transformative approach to modulate cell metabolism based on endogenous cellular information. An emerging strategy is the use of regulators that provide dynamic control of pathway fluxes. A recently discovered modified CRISPR based tool offers a unique approach for DNA targeting and transcriptional regulation. These new generation of regulators can be used for dynamic gene repression and activation for many synthetic-biology and metabolic engineering applications. In addition to the scientific advancements, this project will help train graduate students through the integration of principles from protein engineering, synthetic biology, and cellular physiology. Outreach activities to local high school teachers and students through existing programs available at the University of Delaware and Rensselaer Polytechnic Institute are also planned.The goal of this project is to develop a new transformative approach to modulate cell metabolism based on endogenous cellular information. In particular, a new generation of toehold-gated dCas9 regulators governed by conditional sgRNA structures that are activated by toehold-mediated strand displacement will be created to provide simultaneous, orthogonal, and autonomous control of cellular metabolism. Because dCas9-based regulators are governed by a structurally defined single guide RNA (sgRNA) structure, it is easy to envision that conditional sgRNA structures can be created that are activated by endogenous mRNAs based on toehold-mediated strand displacement. This new framework to design toehold-gated dCas9 regulators responsive to any endogenous mRNA will lay the foundation as a new transformative approach for implementing dynamic control of cellular metabolism. The ability to modulate metabolism based on endogenous cellular information in optimizing the production of numerous products in yeast will be established. The long-term goal is to combine the knowledge gained from this project toward the design of dynamic and autonomous cellular control for any metabolic pathway of interest.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.