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，远远超出了大多数合成化学路线的能力。工程化特定途径可用于进一步改善可合成的化合物的范围，但实现商业上可行的生产率是一个主要挑战。为了最大限度地提高生产率，关键是要微调路径通量。该项目的目标是开发一种新的变革性方法来调节细胞代谢的基础上内源性细胞信息。一种新兴的策略是使用调节剂，提供动态控制的途径通量。最近发现的基于CRISPR的工具为DNA靶向和转录调控提供了一种独特的方法。这些新一代的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，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。还计划通过特拉华州大学和伦斯勒理工学院现有的方案向当地高中教师和学生开展外展活动。该项目的目标是开发一种新的变革性方法，以内源性细胞信息为基础调节细胞代谢。特别地，将创建由通过立足点介导的链置换激活的条件性sgRNA结构控制的新一代立足点门控dCas9调节剂，以提供对细胞代谢的同时、正交和自主控制。由于基于dCas9的调节子由结构上定义的单向导RNA（sgRNA）结构控制，因此容易设想可以产生基于支点介导的链置换由内源性mRNA激活的条件性sgRNA结构。这种设计对任何内源性mRNA有响应的支点门控dCas9调节剂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在优化酵母中多种产物的生产中调节代谢的能力。长期目标是联合收割机从这个项目中获得的知识，对任何感兴趣的代谢途径的动态和自主细胞控制的设计。这个奖项反映了NSF的法定使命，并已被认为是值得通过使用基金会的智力价值和更广泛的影响审查标准进行评估的支持。

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.