Project Summary Glycans play essential roles in virtually all aspects of biology. Thus, they have broad applications, including supplementation of necessary human milk oligosaccharides (HMOs) to infant formula as therapeutics to prevent infection by multiple pathogens, maintain proper microbiota, prevent allergies, and treat necrotizing enterocolitis in infants. However, present synthesis methods for the more complex HMOs fail in affordable scalability that hinders their development as glycan therapeutics. Although current one-pot multienzyme (OPME) systems for HMO synthesis have streamlined numerous synthetic reaction steps, little has been done on optimization and scalability. We hypothesize that a modular OPME system for glycan synthesis can be optimized and integrated with inexpensive polyphosphate-based energy regeneration to decrease cost and significantly increase yield of desirable glycans. In Aim 1, we will establish reference OPME reactions for single sugar transfer steps that start from simple building blocks of monosaccharides and glycan acceptors as input, and provide enzymes for high- energy sugar donor synthesis, monosaccharide transfer and energy regeneration. These studies will develop scalable modules for optimization of chemical input (donor and acceptor building blocks, catalytic quantities of nucleotides, divalent cations, controlled pH, and enzyme catalysts) to establish cross-platform compatible reaction conditions. In Aim 2, we will establish a scalable, coupled polyphosphate energy regeneration system for OPME glycan synthesis. Optimization of polyphosphate as an energy source will require control of polyphosphate and divalent cation concentrations, neutralization of pH changes, and integration of enzymes that generate ATP (RpPPK2-3) and distribute high-energy phosphate equivalents to other nucleotide forms (NDK). Our goals are to create a universal energy source that integrates the continual synthesis of UDP-, GDP-, CMP-, and ADP-sugar donors in large-scale OPME reactions. In Aim 3, we will generate proof-of-concept scalable OPME synthesis of model HMO targets of biological interest for optimization. The approach will combine synthetic modules for sugar donor synthesis and glycan extension with optimized energy regeneration and determine conditions for cross-platform compatibility for all enzymatic steps in the energy-coupled OPME (ecOPME) platform. The HMO targets also provide opportunities to test combined ecOPME reactions as well as sequential ecOPME sugar additions where enzyme competition would yield undesired products. The goals are to integrate multiple enzymatic transfer steps through the selective use of glycosyltransferase acceptor specificity coupled with energy regeneration to result in a proof-of-concept modular platform for efficient, flexible, and scalable synthesis of target therapeutic HMOs starting from simple monosaccharide building blocks.