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，即将代谢酶组织成顺序的多酶级联，以提高整体途径的性能。本项目采用的方法是将CRISPR(集群规则间隔短回文重复)Cas9系统重新定位为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有效性。这两个合作者在途径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概念来实现这一目标。在这个项目上接受培训的学生将在预期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的催化失活的Cas9(DCas9)蛋白，作为一种新的工具，用于体内组装酶级联，以便根据需要重新定向细胞资源。最终结果是能够提供内源过程的动态调整，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降解相结合来实现，以便在集成的合成设计中提供快速传感和执行能力，从而调节整体输出功能。研究人员将通过设计多酶级联来证明这一能力的可行性，一个使用众所周知的甲氧戊酸途径，另一个使用将甲醇转化为乳酸。该奖项由生物科学局分子和细胞生物科学部(MCB)的系统和合成生物学(SSB)计划以及工程局化学、生物工程、环境和运输系统(CBET)的生物技术和生化工程(BBE)计划共同资助。

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.