Chemoenzymatic construction of a programmable synthetic endoplasmic reticulum

可编程合成内质网的化学酶构建

基本信息

批准号：
2124105
负责人：
Neal Devaraj
金额：
$ 150万
依托单位：
University of California-San Diego
依托单位国家：
美国
项目类别：
Standard Grant
财政年份：
2021
资助国家：
美国
起止时间：
2021-08-01 至 2026-07-31
项目状态：
未结题

来源：
https://www.nsf.gov/awardsearch/showAward?AWD_ID=2124105&HistoricalAwards=false
关键词：
Chemoenzymatic construction programmable synthetic endoplasmic

项目摘要

A fundamental goal in synthetic biology is to build artificial cells that have many of the basic functions of natural cells, as well as specific functions that are useful in medicine and biotechnology. This project focuses on building a synthetic subcellular compartment or organelle known as the endoplasmic reticulum, since this organelle would provide artificial cells with essential functions such as the ability to synthesize proteins, process proteins and synthesize lipids. Broader outcomes of this proposal include graduate student training and educating the public about designer cell organelles and their importance through outreach lectures to high school students and the general public. Bioethical consequences related to generating a synthetic organelle will also be considered. To generate a synthetic ER de novo, the chemoenzymatic synthesis of non-canonical phospholipids will be combined with recombinant proteins that influence membrane curvature and fusion. The proposed program to generate and study a synthetic ER will follow these three closely coupled objectives: (1) chemical and enzymatic steps will be developed to generate a library of non-canonical phospholipid membranes similar to the lipid membranes of living cells. (2) the minimal lipid composition requirements necessary for generating an ER-like reticulated membrane architecture will be explored. The interactions of these lipids with membrane-bound proteins that play a critical role in membrane curvature stabilization and fusion respectively in yeast, will be monitored. Furthermore, different combinations of non-canonical lipids will be tested to explore how biomimetic phospholipids affect membrane properties; and (3) the differences in properties between natural and synthetic spherical, tubular, and reticular membrane geometries will be investigated. In particular, the kinetics of internalized reactions, reversibility of the steady-state morphology, mixing of lipids in reticular membranes, diffusion through the lumen of the synthetic ER, and network connectivity will be characterized. To equip the synthetic ER with functionality, a simple post-translational network by localizing two fluorescently labeled glycosyltransferases will be developed within a synthetic reticular network for post-translational modification of a protein substrate. The proposed studies will establish the groundwork for developing artificial reticulated organelles that mimic the ER and facilitate future programmable functions such as post-translational modification, biomolecular trafficking, sensing, and protein turnover.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.

合成生物学的一个基本目标是建立具有自然细胞的许多基本功能的人造细胞，以及在医学和生物技术中有用的特定功能。该项目的重点是建造一个合成的亚细胞室或细胞器，称为内质网，因为该细胞器将为人造细胞提供必不可少的功能，例如合成蛋白质，过程蛋白质和合成脂质的能力。该建议的更广泛的结果包括研究生培训和对公众进行有关设计师细胞器的教育及其通过向高中生和公众的宣传讲座的重要性进行教育。还将考虑与生成合成细胞器有关的生物伦理后果。为了产生从头开始的合成ER，非经典磷脂的化学酶合成将与影响膜曲率和融合的重组蛋白结合使用。提出的生成和研究合成ER的程序将遵循这三个紧密耦合的目标：（1）将开发化学和酶促步骤，以生成类似于活细胞脂质膜的非典型磷脂膜的库。（2）将探索产生类似ER的网状膜结构所需的最小脂质组成要求。这些脂质与膜结合的蛋白的相互作用将在酵母中分别在膜曲率稳定和融合中起关键作用。此外，将测试非经典脂质的不同组合，以探讨仿生磷脂如何影响膜特性。（3）将研究天然球形，管状和网状膜几何形状之间的性质差异。特别是，将表征内部反应的动力学，稳态形态的可逆性，网状膜中脂质的混合，通过合成ER的腔内扩散以及网络连接性。为了使合成ER配备功能，通过定位两个荧光标记的糖基转移酶的简单后翻译网络将在合成网络网络中开发，用于蛋白质底物的翻译后修饰。拟议的研究将建立基础，以开发模仿ER并促进可编程功能的人工网状细胞器，例如翻译后修改，生物分子贩运，感应和蛋白质转移。该奖项反映了NSF的法定任务，并通过评估智力的Merit和Broadia and Intfactial和Broadia and Impactia and Spractial and Impactia和Broadia and crowia and crowia and crowia and crowia and crowia and crowia and crowia and rockia and crowisa奖。