Carbohydrates, or glycans, are involved in almost every human disease and biological process. Hence, the field of glycoscience has the potential to broadly impact society in areas as diverse as medicine, energy, and materials. However, this complex and potentially transformative field has been relatively under-appreciated by the scientific community. This under-appreciation is because glycan structural analysis is tedious, synthesis is challenging, commercial availability is limited, and tools are in short supply. The field lacks a universal, easily accessible database and there is no PCR equivalent for glycans or a comprehensive glycan repository to click and “add to shopping cart.” In turn, our ability to understand and engineer glycosylation is severely restricted. In the last decade, an entirely new discipline called bacterial glycoengineering has made it possible to produce designer glycans on demand, some containing unnatural sugars, and to evolve enzymes, pathways, and host organisms that catalyze prescribed glycosylation reactions. In addition to their biotechnological potential, bacteria equipped with recombinant glycosylation pathways hold promise to improve our fundamental understanding of the glycosylation process. These developments not withstanding, cell-based production of homogeneous glycoproteins remains a significant challenge due to the complexity of this process and our inability to control glycosylation components at precise ratios. To address these challenges, this COLLABORATIVE proposal focuses on the seamless integration of (1) a cutting edge yet proven cell-free protein synthesis system, (2) new methods for bacterial glycoengineering that permit biosynthesis and conjugation of humanized glycans to target proteins of interest, and (3) metabolic introduction of sugar analogs to generate glycoproteins with complex, unnatural glycans of defined structure. Intellectual merit. By merging bottom-up design principles with innovative gene/pathway/cell engineering methodologies in a cell-free environment, our project will create a greatly simplified framework for studying and engineering glycosylation with the potential to become the first ever PCR equivalent for glycans. Moreover, by creating glycoproteins with unnatural glycan structures, our project will generate a “site-directed mutagenesis” equivalent for glycans. Such a technology platform would be instrumental in sustaining the much-anticipated revolution in glycoscience, with potential for significant breakthroughs. For example, our ability to study and control glycosylation outside the confines of a cell may 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, our cell-free platform could serve as a model for deciphering the “glycan code”—akin to the genetic code for DNA/RNA/proteins—underlying glycoprotein synthesis. From an engineering perspective, our research will enable scalable glycoconjugate biosynthesis, thereby opening the door to cheaper and more effective biomaterials, therapeutics and vaccines. Broader impacts. The proposed work aspires to catalyze a new paradigm for cell-free synthesis of glycoproteins. This is expected to enlarge the glycoengineering toolkit by enabling fundamental advances in our knowledge of glycosylation and opening the way to entirely new application
|Effective start/end date||9/1/14 → 8/31/18|
- National Science Foundation (MCB-1413563)
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