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

碳水化合物，或聚糖，几乎参与每一个生物过程。在体内，蛋白质被聚糖修饰，聚糖在蛋白质功能中起重要作用。 然而，理解这些聚糖如何添加到蛋白质中以及这些聚糖的位置和结构的作用背后的科学落后于生物科学的其他领域。 聚糖科学的滞后是因为聚糖结构分析繁琐，合成具有挑战性，并且工具短缺。因此，理解和工程化糖基化（将聚糖添加到蛋白质）的能力受到严重限制。为了应对这些挑战，该奖项专注于实验和计算方法的整合，以实现首个无细胞糖蛋白合成系统，该系统允许聚糖与目标蛋白的生物合成和缀合。通过在无细胞环境中将自下而上的工程设计原理与创新的分子生物学方法相结合，研究人员团队将为研究和工程化糖基化创建一个大大简化的框架。 例如，通过研究和控制细胞限制范围之外的蛋白质糖基化将有助于回答基本问题，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导致预测位点特异性蛋白质糖基化的结构后果的一般规则，反过来，设计具有有利特性的修饰蛋白质的规则。此外，无细胞平台可以作为破译“聚糖密码”的模型。“从工程的角度来看，这项研究将使可扩展的糖缀合物生物合成成为可能，为更便宜，更有效的生物制造打开大门。除了技术影响外，该奖项还将促进跨学科教育，包括为代表性不足的少数民族和妇女具体扩大STEM教育和职业机会。学生将接受培训，以整合计算生物学，系统生物学和合成生物学的原则。研究人员将开发体验式学习模块，将聚糖研究带到K-12和本科课堂，并将学生与主办机构正在进行的科学联系起来。该研究的长期目标是开发一种新的无细胞糖蛋白合成（CFGpS）系统，能够产生有用的糖蛋白。该研究小组还试图建立潜在的复杂生物过程的计算模型，可用于指导他们的实验计划。为了开发CFGpS系统，研究人员将：（i）激活产生人类样聚糖的真核糖基化途径，（ii）将真实的糖蛋白靶点引入CFGpS系统，以及（iii）设计一种用于生产CFGpS粗提物的一体化宿主菌株。同时，他们将获得对宿主菌株产生的积极和消极影响CFGpS的基因产物的综合系统水平的理解。为了实现这一目标，研究人员将开发一个数学框架，用于CFGpS的计算机评估。然后，从这些努力中获得的信息将用于指导改进的全合一CFGpS菌株的正向工程。这项工作将首次建立与无细胞蛋白质合成系统相结合的高效无细胞糖基化方法。此外，这项工作将推进糖基化的知识，并将揭示合成系统可以被工程化的程度。展望未来，研究人员认为CFGpS平台将为理解通用糖基化途径的基本原理提供一个全新的框架，剖析它们在重要生物过程中的作用，并定义控制位点特异性糖基化结构后果的规则。

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.