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Diarrheal disease is one of the top ten leading causes of death worldwide. The rise of antibiotic resistance and decline of new antibiotics entering the clinical setting have created a need for a safe and effective strategy to prevent life-threatening bacterial diarrheal diseases. Glycoconjugate vaccines, which are composed of a pathogen-specific sugar antigen attached to a protein that stimulates the immune system, are an effective method to prevent bacterial infection. Currently, vaccine production relies of living cells, requiring centralized facilities and distribution under refrigerated conditions. The Jewett Lab has developed a freeze-dried, cell-free system to produce glycoconjugate vaccines at the point-of-care. Within this cell-free glycoprotein synthesis (CFGpS) platform, a specialized protein, known as PglB, transfers sugars to proteins forming the glycoconjugate vaccine. However, two main challenges prevent the adoption of this technology. First, the PglB enzyme is specific to certain sugars and cannot transfer all vaccine-relevant sugar antigens, such as those from diarrheal disease-causing bacteria Shigella flexneri and Shigella dysenteriae. Second, the current CFGpS reaction formulation has limited thermostability and a high cost per dose, limiting its relevance for low-resource settings where diarrheal diseases are often most prevalent. To address the first challenge, we hypothesized mutating PglB would expand its transfer capabilities for non-native sugars. Through western blotting, we demonstrated that mutating PglB increased transfer efficiency of sugar antigens from Shigella flexneri 5-fold and Shigella dysenteriae 2-fold compared to wild-type PglB. To address the second challenge of affordability and thermostability, we developed a low-cost, thermostable CFGpS reaction formulation by screening low-cost alternative energy sources that could also increase thermostability. Using this formulation, we produced glycoconjugate vaccines against enterotoxigenic E. coli 078 for ~$0.50 per dose after storage for up to four weeks at 37⁰C. Overall, we expanded the capabilities for cell-free glycoconjugate vaccine production in resource-limited regions by increasing the number of potential disease targets and developing a low-cost, thermostable formulation for accessible distribution.