Ideas Lab: USPRD: Enabling cell-free synthesis of longer human milk oliogosaccharides

Enabling Cell-free Synthesis of Longer Human Milk Oliogosaccharides This project will support bio-based production of essential nutrients in human breast milk that support infant health and development. These nutrients include complex sugars that are known as human milk oligosaccharides (HMOs). Current infant formulas only contain a fraction of the simplest known HMOs, which are already difficult to produce in large quantities. This effort seeks to enable the cost-effective synthesis of longer, more complex HMOs at-scale. This project also emphasizes educational outreach and workforce development for the next generation of American biotechnology researchers. These approaches will strengthen the U.S. bioeconomy through new, globally competitive bioproducts. This collaborative effort seeks to overcome the challenges of producing complex HMOs by developing advanced enzymatic and computational strategies. Key objectives include engineering glycosyltransferases and transglucosidases to synthesize longer, more complex HMOs, leveraging recent advances in cell-free protein synthesis, protein engineering, and machine learning. The project will employ tools such as AlphaFold, ESM2, ProteinMPNN, LigandMPNN, and RFDiffusion to analyze enzyme structure-function relationships and design libraries for activity and selectivity. Cell-free protein synthesis will facilitate rapid screening and iterative refinement of enzyme variants, while use of industrially relevant processes will validate the economic feasibility of production. Ultimately, the best performing enzymes will be evaluated in cell-free process at lab-scale to demonstrate process feasibility for producing commercially relevant HMOs. Beyond addressing infant nutrition, this work will contribute significantly to the protein design community by developing reusable models, establishing new design algorithms, and sharing datasets for glycosylation enzyme development. These innovations will not only accelerate HMO research but also pave the way for broader applications in synthetic biology, enabling a new generation of AI-driven breakthroughs in enzyme and protein design, and supporting the bioeconomy. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.