Abstract: Zymtronix proposes to significantly improve production efficiency of glycans via new flow processes based on chemoenzymatic catalysis to take significant steps towards broader commercial access. The first glycans of interest are human milk oligosaccharides (HMOs), which have large commercial relevance in infant nutrition, disease prevention and therapeutics. The primary hurdle towards the broader application of complex synthetic glycans in research, preclinical and commercial applications is their affordability. The goal of the proposed research is to lower the production costs of large and complex HMOs (>5 DP) by continuous flow processes where each step of synthesis is conducted in modules. Zymtronix's technology enables (i) high enzyme activity and stability ensuring the reuse of enzyme and (ii) enables the co-immobilization of multiple enzymes to incorporate enzymatic recycling for sub-stoichiometric use of reagents to impart significant cost- reductions. For this fast-track, we will produce large human milk oligosaccharides (HMOs), the third largest component of breast milk that are particularly commercially relevant glycan models. While some simple probiotic HMOs can be effectively produced via fermentation for infant formula (2'FL, 3 DP), complex and branched HMOs are elusive and have been marginally produced at high cost via chemoenzymatic catalysis. Zymtronix's technology solution will significantly improve production of glycans, starting with HMOs, while imparting significant cost savings. Fast-track Phase I specific aims are to immobilize 6 enzymes and to combine them in a defined sequence to generate sialylated lacto-N-neopentaoses. (I) Aim 1: Immobilize 6 individual enzymes on sintered beads using ZymTrap3D technology. 4 transferases and 2 activated-sugar-producing enzymes for in-situ synthesis of GlcNAc-UDP, Gal-UDP and Neu5Ac-CMP, (II) Aim 2: Demonstrate sequential, modular synthesis of 1 mg of α- 2,3- and α-2,6-sialyl lacto-N-pentaoses on beads. (III) Aim 3: Produce 100 mg of α-2,3-sialyl lacto-N- neopentaose using a flow cell. Fast-track Phase II aims are to scale up production of three linear fucosyl- and sialyl-lacto-N-neopentaoses from Aim1. (I) Aim 1: Development of HMO fucosylation and branching modules towards the 100 mg production of 6 biantennary lacto-N-neohexaoses and heptaoses. The modularity of branching followed by three possible `functionalization' modules, and a final elongation step allows the production of 6 biantennary HMOs. (II) Aim 2: Development of flow cells with in situ reagent recycling for multi-gram scale HMO production. Production and cost metrics will be assessed. (III) Aim 3: Scaling up engineering and production of 10 g and then 100 g of fucosyl- and sialyl-lacto-N-neopentaoses. Work will include in-house enzyme production and HPLC purification of HMOs.