Collaborative Research: Synthetic CRISPR-Cas6 endonucleases for dynamic control of cellular phenotypes in yeast

该项目的总体目标是利用合成生物学的力量建立微生物工厂来生产高价值化学品。 该合作研究团队将使用创新的酶共定位/组装策略来提高酵母中选定化学物质的产量。 该项目还将通过特拉华大学和加州大学欧文分校的现有项目，促进当地高中教师和学生的外展活动。本质上，蛋白质之间的动态相互作用在定义许多细胞功能（如新陈代谢、细胞信号传导、转录调节、细胞凋亡、细胞靶向和蛋白质降解）方面发挥着至关重要的作用。 通过使用蛋白质支架控制这些超分子复合物的空间和时间组织，可以以高度动态的方式调节细胞功能以获得最佳效率。 了解这些蛋白质如何相互作用是破译它们在天然细胞功能中的作用以及为合成生物学应用创造新的细胞功能的关键。 在该项目中，研究人员提出了一种新的变革性框架，该框架结合了 RNA 杂交的可预测性和 RNA 加工的简易性，同时提供按需的可逆蛋白质组装，以创建用于蛋白酶体靶向蛋白质降解和酵母代谢途径调节的动态酶级联。 拟议的研究依赖于一种新的有效方法，该方法能够使用天然存在的 CRISPR/Cas6 系统对 RNA 转录本进行特异性处理和高亲和力结合。 虽然 Cas6 的天然功能是生成 CRISPR RNA (crRNA) 来指导 DNA 靶标的切割，但研究人员将重新利用 Cas6 家族蛋白作为位点特异性 RNA 结合/处理的通用平台，并将展示其在酵母合成生物学和代谢工程应用中组装动态酶级联的实用性。 Chen 和 Da Silva 实验室的专业知识将结合起来开发和实施代谢物。 最先进的合成生物学和遗传工具将用于实现四个总体目标：(1) 通过链置换进行动态蛋白质组装和拆卸，(2) 使用正交 Cas6 蛋白质动态蛋白质降解，(3) 动态组装代谢物以实现底物通道，以及 (4) 组合代谢物组装和蛋白质降解以增加聚酮生物合成。该奖项反映了 NSF 的法定使命，并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，被认为值得支持。

The overall goal of this project is to use the power of synthetic biology to build microbial factories for the production of high value chemicals. The collaborative research team will use an innovative enzyme colocalization/assembly strategy to boost production of selected chemicals in yeast. This project will also facilitate outreach activities to local high school teachers and students through existing programs available at the University of Delaware and University of California, Irvine.In nature, dynamic interactions between proteins play a crucial role in defining many cellular functions such as metabolism, cell signaling, transcription regulation, apoptosis, cellular targeting, and protein degradation. By controlling the spatial and temporal organization of these supramolecular complexes using a protein scaffold, cellular functions can be modulated in a highly dynamic manner for optimum efficiency. Understanding how these proteins interact holds the key to deciphering their roles in native cellular function and in creating new cellular functions for synthetic biology applications. In this project, the researchers propose a new transformative framework that combines the predictability of RNA hybridization and the ease of RNA processing, while offering reversible protein assembly on demand, to create dynamic enzyme cascades for proteasome-targeted protein degradation and for metabolic pathway regulation in yeast. The proposed research relies on a new and potent approach that enables specific processing and high-affinity binding to RNA transcripts using the naturally occurring CRISPR/Cas6 system. While the native function of Cas6 is to generate CRISPR RNAs (crRNAs) to guide the cleavage of DNA targets, the researchers will repurpose the Cas6 family proteins as a generalizable platform for site-specific RNA binding/processing and will demonstrate its utility to assemble dynamic enzyme cascades for synthetic biology and metabolic engineering applications in yeast. The expertise of the Chen and Da Silva labs will be combined to develop and implement the metabolons. State of the art synthetic biology and genetic tools will be used to accomplish four overall aims: (1) Dynamic protein assembly and disassembly by strand displacement, (2) Dynamic protein degradation using orthogonal Cas6 proteins, (3) Dynamic assembly of metabolons for substrate channeling, and (4) Combined metabolon assembly and protein degradation for increased polyketide biosynthesis.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.