Dynamic Control of Glycan Biosynthesis with Synthetic RNA Circuitry

建议编号：CBET-1402843首席研究员：朱利叶斯·卢克斯研究所：康奈尔大学标题：利用合成核糖核酸电路动态控制糖的生物合成存在于我们体内的许多蛋白质，大多数通过一种称为糖基化的过程被称为糖链的复杂糖装饰。糖基化通常是这些蛋白质正常运作所必需的。对于人类的治疗用途，糖蛋白必须具有与人类相似的糖链。因此，生产往往限于哺乳动物细胞培养，这是耗时、昂贵的，而且容易受到病毒污染。一种能够产生与人类相似的糖蛋白的大肠杆菌菌株可以克服其中的一些障碍。该项目寻求通过使用RNA工程中最先进的技术来动态优化糖基化途径的输出，将细菌生产的糖基化蛋白提高到一个新的水平。在这个项目中，研究人员将使用RNA工程的原理来创建遗传网络，在需要时动态调整糖基化酶的表达，以提高产品产量和纯度。拟议的研究和研究培训活动预计将对社会产生广泛影响，从糖生物学科学和RNA基因调控工程科学到人类糖疗法的发展。该项目还将培养下一代训练有素的研究生，他们将被介绍给生物技术研究的广泛、跨学科性质。此外，该计划将积极和积极地扩大对科学和工程的参与。这将通过为本科生提供跨学科研究机会，为本科生和高中生开发体验式糖科学学习模块，以及创建生物分子工程和合成生物学的定量研究生水平课程来实现。最后，将通过与当地生物技术公司的合作，将细菌糖基化和RNA工程用于生物技术应用的开发带到更大的研究社区。这项研究项目的长期目标是对细菌细胞进行基因工程和优化，以便常规生产真正的人类N-连接糖蛋白。到目前为止，研究人员已经在大肠杆菌中重现了这一复杂过程的最早步骤。这一特殊应用的目的是通过设计基于合成RNA的遗传电路来最大限度地提高这一途径的生产率，该电路将使用两种不同的策略动态控制糖基化酶的表达。第一种方法是通过创建不同阶段的糖链构建，然后进行糖链靶向，从而消除竞争性副反应，我们预计这将极大地提高糖蛋白的纯度。同时，途径生产率将通过按照需要的顺序及时表达途径酶来优化，就像在细胞使用的几个基本代谢途径中所做的那样。由于糖基化包括在细胞不同部分发生的一系列酶步骤，控制酶表达的动态使其在需要时最活跃，有望显著促进途径的产生。这些研究的成功完成将导致一种新型细菌糖蛋白表达平台的开发，该平台有可能克服现有真核平台的许多局限性。此外，拟议的研究和研究培训活动将影响：(1)用于研究、工业和治疗应用的新型糖结合物和潜在免疫刺激剂的生物技术合成；(2)开发可用于优化一系列代谢过程的新的、广泛适用的战略；(3)开发新的工具和设计原则，以控制具有深远潜力的基因电路的细胞行为。由于该项目的跨学科性质，该奖项由CBET部门的生物技术、生化和生物质工程项目共同资助，由分子和细胞生物学部门的系统和合成生物学项目共同资助。

Proposal Number: CBET - 1402843 Principal Investigator: Julius Lucks Institution: Cornell University Title: Dynamic Control of Glycan Biosynthesis with Synthetic RNA Circuitry Of the many proteins that exist inside our bodies, most are decorated with complex sugars called glycans through a process called glycosylation. Glycosylation is often necessary for these proteins to function correctly. For therapeutic use in humans, glycoproteins must have human-like glycans. Thus production is often limited to mammalian cell culture, which is time-consuming, expensive, and susceptible to viral contamination. A strain of Escherichia coli capable of producing human-like glycoproteins could overcome some of these hurdles. This project seeks to take bacterial production of glycosylated proteins to the next level by using state-of-the art techniques in RNA engineering to dynamically optimize the output of the glycosylation pathway. In this project, the investigators will use the principles of RNA engineering to create genetic networks that dynamically tune the expression of glycosylation enzymes as they are needed in order to increase product output and purity. The proposed studies and research training activities are expected to have a broad impact on society, ranging from the science of glycobiology and the engineering science of RNA gene regulation, to the development of human glycotherapeutics. This project will also cultivate the next generation of highly trained graduate students who will be introduced to the broad, interdisciplinary nature of biotechnology research. Moreover, this program will actively and aggressively broaden participation in science and engineering. This will be accomplished by providing interdisciplinary research opportunities for undergraduate students, developing experiential glycoscience learning modules for undergraduate and high school students, and creating a quantitative graduate-level course for biomolecular engineering and synthetic biology. Finally, the development of bacterial glycosylation and RNA engineering for biotechnological applications will be brought to a larger research community through partnership with local biotechnology companies. The long-term goal of this research project is to genetically engineer and optimize bacterial cells for the routine production of authentic human N-linked glycoproteins. To date, the investigators have recreated the earliest steps of this complicated process in Escherichia coli. The objective of this particular application is to maximize the productivity of this pathway by engineering synthetic RNA-based genetic circuitry that will dynamically control the expression of glycosylation enzymes using two distinct strategies. The first will eliminate competitive side reactions by creating distinct stages of glycan construction followed by glycan targeting, which we anticipate will dramatically increase the purity of the glycoproteins produced. In parallel, pathway productivity will be optimized by expressing pathway enzymes 'just-in-time' in the order they are needed as is done in several essential metabolic pathways used by cells. Since glycosylation consists of sequential enzyme steps that take place in different parts of the cell, controlling the dynamics of enzyme expression so that they are most active when needed is expected to significantly boost pathway production. Successful completion of these studies will lead to the development of a novel bacterial glycoprotein expression platform with the potential to overcome many of the limitations of existing eukaryotic platforms. Moreover, the proposed studies and research training activities will impact: (i) biotechnological synthesis of novel glycoconjugates and potential immunostimulating agents for research, industrial and therapeutic applications; (ii) the development of new, broadly applicable strategies that can be used to optimize a wide array of metabolic processes; and (iii) the development of new tools and design principles for engineering genetic circuitry to control cellular behavior with far-reaching potential. Due to the interdisciplinary nature of the project, this award by the Biotechnology, Biochemical, and Biomass Engineering Program of the CBET Division is co-funded by the Systems and Synthetic Biology Program of the Division of Molecular and Cellular Biology.