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

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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.