Collaborative Research: Glycoengineering Without Borders: Bacterial Cell-Free Glycoprotein Synthesis

合作研究：无国界糖工程：细菌无细胞糖蛋白合成

• 批准号: 1413563
• 负责人: Michael Jewett
• 金额: $ 30万
• 依托单位: Northwestern University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-01 至 2018-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1413563&HistoricalAwards=false
• 关键词: Collaborative; Research; Glycoengineering; Without; Borders

Carbohydrates, or glycans, are involved in almost every biological process. In vivo, proteins are decorated with glycans that play an important role in protein function. However, the science behind understanding how these glycans are added to proteins and the role of position and structure of these glycans has lagged behind other fields of biological sciences. This lag in glycan science is because glycan structural analysis is tedious, synthesis is challenging, and tools are in short supply. Thus, the ability to understand and engineer glycosylation (the addition of glycans to proteins) is severely restricted. To address these challenges, this award focuses on the integration of experimental and computational approaches to enable a first-of-its-kind cell-free glycoprotein synthesis system that permits biosynthesis and conjugation of glycans to target proteins of interest. By merging bottom-up engineering design principles with innovative molecular biology methodologies in a cell-free environment, the team of investigators will create a greatly simplified framework for studying and engineering glycosylation. For example, by studying and controlling protein glycosylation outside the restrictive confines of a cell will help answer fundamental questions such as how glycan attachment affects protein folding and stability. Answers to these questions could lead to general rules for predicting the structural consequences of site-specific protein glycosylation and, in turn, rules for designing modified proteins with advantageous properties. Further, the cell-free platform could serve as a model for deciphering the "glycan code." From an engineering perspective, the research in this grant will enable scalable glycoconjugate biosynthesis, opening the door to cheaper and more effective biomanufacturing. Beyond technological impact, this award will also promote interdisciplinary education, including the specific expansion of STEM education and career opportunities for underrepresented minorities and women. Students will be trained to integrate principles from computational biology, systems biology, and synthetic biology. The investigators will develop experiential learning modules that bring glycan research to K-12 and undergraduate classrooms and connect students to the science being done at the host institutions. The long-term goal of the proposed research is to develop a novel cell-free glycoprotein synthesis (CFGpS) system capable of producing useful glycoproteins. The research team also seeks to build computational models of the underlying complex biological processes that can be used to guide their experimental program. To develop the CFGpS system, the researchers will: (i) activate a eukaryotic glycosylation pathway that produces human-like glycans, (ii) introduce authentic glycoprotein targets to CFGpS system, and (iii) engineer an all-in-one host strain for producing CFGpS crude extracts. In parallel, they will gain an integrated systems-level understanding of gene products made by the host strain that positively and negatively influence CFGpS. To accomplish this goal, the investigators will develop a mathematical framework for in silico assessment of CFGpS. Then, information gained from these efforts will be used to guide forward engineering of improved all-in-one CFGpS strains. The work will establish for the first time efficient cell-free glycosylation methods integrated with a cell free protein synthesis system. Further, this work will advance the knowledge of glycosylation and will reveal the extent to which synthetic systems can be engineered. Looking forward, the investigators believe that the CFGpS platform will provide an entirely new framework for understanding the fundamentals of universal glycosylation pathways, dissecting their role in important biological processes, and defining the rules governing structural consequences of site-specific glycosylation.

碳水化合物或聚糖几乎参与了几乎每个生物学过程。在体内，蛋白质饰有聚糖，在蛋白质功能中起重要作用。 但是，了解如何将这些聚糖添加到蛋白质中的科学以及这些聚糖的位置和结构的作用落后于其他生物科学领域。 聚糖科学中的这种滞后是因为聚糖结构分析很乏味，合成具有挑战性，工具供不应求。因此，严格限制了理解和设计糖基化（向蛋白质添加聚糖）的能力。为了应对这些挑战，该奖项的重点是实验和计算方法的整合，以使初始的无细胞糖蛋白合成系统允许生物合成和聚糖偶联以靶向感兴趣的蛋白质。通过将自下而上的工程设计原理与无细胞环境中创新的分子生物学方法合并，研究人员将创建一个非常简化的框架，用于研究和工程糖基化。 例如，通过研究和控制细胞限制性范围之外的蛋白质糖基化，将有助于回答基本问题，例如聚糖附着如何影响蛋白质折叠和稳定性。这些问题的答案可能会导致一般规则，以预测位点特异性蛋白质糖基化的结构后果，进而是设计具有优势性能的修饰蛋白质的规则。此外，无单元的平台可以用作解密“聚糖代码”的模型。从工程的角度来看，这项赠款的研究将使可扩展的糖缀合物生物合成，打开便宜，更有效的生物制造之门。除了技术影响之外，该奖项还将促进跨学科教育，包括STEM教育的特定扩展以及代表性不足的少数族裔和妇女的职业机会。将对学生进行培训，以整合计算生物学，系统生物学和合成生物学的原则。调查人员将开发经验式学习模块，这些模块将聚糖研究带到K-12和本科教室，并将学生联系到主持机构的科学。拟议的研究的长期目标是开发一种能够产生有用的糖蛋白的新型无细胞糖蛋白合成（CFGPS）系统。研究团队还试图建立可用于指导其实验计划的基础复杂生物学过程的计算模型。为了开发CFGPS系统，研究人员将：（i）激活一种产生类似人类的聚糖的真核糖基化途径，（ii）将正宗的糖蛋白靶标引入CFGPS系统，以及（III）工程师，一种用于生产CFGP的宿主菌株来生产CFGPS粗糙的提取物。同时，它们将获得对宿主菌株产生的基因产物的综合系统级别的理解，从而对CFGP产生积极和负面影响。为了实现这一目标，研究人员将开发用于CFGP的硅评估中的数学框架。然后，从这些工作中获得的信息将用于指导改进的多合一CFGP菌株的前进工程。这项工作将首次建立与无细胞蛋白质合成系统集成的无细胞糖基化方法。此外，这项工作将提高糖基化的知识，并将揭示可以设计合成系统的程度。展望未来，调查人员认为，CFGPS平台将为了解通用糖基化途径的基本原理，解剖其在重要的生物学过程中的作用，并定义有关位置特定糖基化的结构后果的规则的全新框架。