Repurposing the CRISPR-Cas9 system for dynamic control of cellular metabolism

该项目解决了合成生物学中的一个具有挑战性的问题，即将代谢酶组织成一个连续的多酶级联，以提高整体途径的性能。 该项目中使用的方法是将CRISPR（重复的规则间隔短回文重复序列）Cas9系统重新用作位点特异性酶组装的通用平台，并展示其在代谢工程和合成生物学应用中的实用性。这两个合作者在通路工程和蛋白质工程方面提供互补的专业知识，以追求高度创新的设计理念来实现这一目标。在该项目上接受培训的学生将在预计将显著提高合成生物学能力的研究背景下接受多学科培训。该项目的目标是开发一组正交无催化活性的Cas9（dCas9）蛋白，作为酶级联的体内组装的新工具，以便根据需要重定向细胞资源。最终结果是能够提供内源性过程的动态调节，以平衡细胞健康和途径效率的需求。这将通过将代谢物响应性dCas9表达和受控dCas9降解组合以提供快速感测和致动能力到集成合成设计中以调节整体输出功能来实现。 研究人员将通过设计多酶级联来证明这种能力的可行性，一种使用众所周知的甲羟戊酸途径，另一种使用甲醇转化为乳酸。最终目标是将所获得的知识联合收割机作为一种变革性的方法，设计任何感兴趣的代谢途径的动态酶级联。该奖项由生物科学理事会分子和细胞生物科学（MCB）部门的系统和合成生物学（SSB）计划以及化学，生物工程，生物工程和生物化学工程部门的生物技术和生物化学工程（BBE）计划共同资助。环境和运输系统（CBET）在工程局。

This project addresses a challenging problem in synthetic biology, namely the organization of metabolic enzymes into a sequential multi-enzyme cascade in order to improve the overall pathway performance. The approach used in this project is to repurpose the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) Cas9 system as a generalizable platform for site-specific enzyme assembly and to demonstrate its utility for metabolic engineering and synthetic biology applications. The two collaborators offer complementary expertise in pathway engineering and protein engineering in order to pursue a highly innovative design concept to achieve this goal. The students trained on this project will be exposed to multi-disciplinary training in the context of research that is anticipated to significantly advance synthetic biology capabilities. The goal of this project is to develop a set of orthogonal catalytically inactive Cas9 (dCas9) proteins as a new tool for in vivo assembly of enzyme cascades in order to redirect cellular resources as desired. The end result is the ability to provide dynamic tuning of endogenous processes to balance the demands of cell health and pathway efficiency. This will be achieved by combining metabolite-responsive dCas9 expression and controlled dCas9 degradation to provide both fast sensing and actuating ability into an integrated synthetic design in order to modulate the overall output function. The investigators will demonstrate the feasibility of this capability by designing multi-enzyme cascades, one using the well-known mevalonate pathway and the other using the conversion of methanol to lactate. The ultimate goal is to combine the knowledge gained as a transformative approach toward the design of dynamic enzyme cascades for any metabolic pathway of interest.This award was co-funded by the Systems and Synthetic Biology (SSB) program in the Molecular and Cellular Biosciences (MCB) Division in the Biological Sciences Directorate and the Biotechnology and Biochemical Engineering (BBE) program of the Division of Chemical, Bioengineering, Environmental and Transport Systems (CBET) in the Engineering Directorate.