Project Summary/Abstract Most infants currently rely on infant formula (IF) to meet their nutritional needs. However, existing infant formula is functionally inferior because it lacks breast milk bioactive proteins that are crucial to infant health and development. Consequently, formula-fed infants have increased risk of morbidity and mortality. Current technology limits the ability to add bioactive milk proteins to infant formula because manufacturing processes are resource intensive, costly, and difficult to scale. Thus, scalable technology to improve bioactive availability for fortification of infant formula is needed. Towards this goal, TurtleTree has developed precision fermentation technology to produce recombinant bovine lactoferrin (rbLf), a critical bioactive milk protein demonstrated to support gut health and immune function in infants. Not only will TurtleTree’s technology allow for inclusion of lactoferrin in all infant formulas, but the use of precision fermentation will also dissociate bioactive milk protein production from animal agriculture, as required for cow’s milk-derived lactoferrin (cMDLf), and the associated environmental stressors. In this proposal, we aim to answer two specific questions that will validate our ability to apply precision fermentation technology for infant health and commercialize rbLf as an ingredient for addition to infant formula: 1) Does the unique glycosolation profile of rbLf impact the protein’s immunomodulatory and gut function as compared to cMDLf? 2) Does rbLf improve gut health in an infant preclinical mouse model? Preliminary data supporting this proposal demonstrates the N-linked glycan profile is the only substantially unique structural element of rbLf, characterized by dominance of high-mannose type glycans (~99%) compared to cMDLf (~45%). Thus, we reason a proper evaluation of rbLf requires focused studies on physiological systems sensitive to glycan alterations, notably immune interactions (Aim 1) and gut development (Aim 2). Comparative results between differentially glycosylated lactoferrin treatment groups will distinguish glycan effects. In Aim 1, we will assess the role of lactoferrin N-linked glycosylation on immunomodulation mediated by the mannose receptor in order to understand glycan effect. In Aim 2, we will execute an intervention of orally-delivered rbLf to improve dysregulated gut health in infant mouse pups. With the successful completion of both aims, we expect to demonstrate rbLf promotes immune and gut interactions that maintain biological activity and efficacy as compared to cMDLf. The scope of work is designed to support regulatory approval and expedite TurtleTree’s path to market for the safe and effective application of advancing infant health.