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Abstract - Human milk oligosaccharides (HMOs) are abundant components of human milk that hold extraordinary potential as novel formula supplements and therapeutic agents to improve gut inflammatory disorders, autoimmune and allergic diseases. Originally considered prebiotics that help shape the gut microbiome of breastfed infants, some HMOs are increasingly becoming known for their beneficial immunomodulatory and anti-inflammatory properties. HMOs prevent pathogen attachment to infant mucosal surfaces to lower the risk of infections, modulate epithelial and immune cell responses, reduce excessive mucosal leukocyte infiltration and activation, and lower the risk of necrotizing enterocolitis. However, the inability to source pure, structurally defined complex HMOs in sufficient quantities has hindered their systematic study and efforts to leverage the extraordinary biomedical potential of these commercially relevant human milk bioactives. In response to the critical need to significantly improve the production efficiency of HMOs, Zymtronix is applying its proprietary multienzyme immobilization technology to produce most HMO milk constituents via cell-free biomanufacturing, thereby removing a major barrier to their investigation in the infant and adult therapeutic space—a mission aligned with the Glycoscience NIH Common Fund. In Phase I, Zymtronix demonstrated successful production of multiple HMOs. In this Phase II project, Zymtronix proposes to significantly improve the production efficiency of human milk oligosaccharides (HMOs), initially focusing on a sialylated HMO which has been demonstrated to be highly protective against necrotizing enterocolitis (NEC) – a devastating inflammatory gastrointestinal disorder with a 10-50% mortality rate in premature infants and total annual cost burdens upwards of $1B. To validate the quality and efficacy of this compound in small and large animal models of NEC, Zymtronix is partnering with Drs. Lars Bode (UCSD) and Doug Burrin (Baylor College of Medicine), world-class experts in HMOs, pediatric nutrition, and gastroenterology. Phase II Aims are to optimize biocatalytic production (Aim 1), conduct a proof-of-concept study to replicate efficacy study in rats (Aim 2), demonstrate a scalable production and purification method (Aim 3), and for the first time, demonstrate the use of this sialylated HMO at multi-gram scale in piglets (Aim 4). The project will establish a cost-effective and industrially scalable method of production that will put 100s grams quantities into the hands of researchers and commercial partners who desire to systematically investigate its efficacy against NEC with the long-term goal of substantially improving the outcome for neonates at risk of NEC. This HMO is just one of several beneficial molecules that will be substantially more accessible as an outcome of the proposed work, creating the ability to utilize these free glycans in a wide range of applications from nutrition to therapeutics.