Economical Modular One-Pot Multienzyme Synthesis of Human Milk Oligosaccharides

项目摘要 葡聚糖在生物学的几乎所有方面都扮演着重要的角色。因此，它们有广泛的应用，包括 在婴儿配方奶粉中添加必要的人乳寡糖(HMOS)作为预防 感染多种病原体，保持适当的微生物区系，预防过敏，治疗坏死性小肠结肠炎 在婴儿身上。然而，目前用于更复杂的HMO的合成方法在负担得起的可扩展性方面失败 阻碍了它们作为葡聚糖疗法的发展。虽然目前的一锅多酶(OPME)系统 HMO合成简化了许多合成反应步骤，但在优化和 可扩展性。我们假设，用于多糖合成的模块化OPME系统可以被优化和集成 以廉价的聚磷酸盐为基础的能源再生，降低了成本，显著提高了产量 令人向往的多糖。在目标1中，我们将为开始的单糖转移步骤建立参考OPME反应 从单糖和葡聚糖受体的简单构建块作为输入，并为高... 能量糖供体合成、单糖转移和能量再生。这些研究将会发展 用于优化化学输入的可扩展模块(施主和受主构建块、催化量 核苷酸、二价阳离子、可控pH和酶催化剂)，以建立跨平台兼容性 反应条件。在目标2中，我们将建立一个可扩展的、耦合的聚磷酸盐能源再生系统 用于OPME多糖的合成。聚磷酸盐作为能源的优化将需要控制 聚磷酸盐和二价阳离子浓度，pH变化的中和，以及酶的整合 生成ATP(RpPPK2-3)并将高能磷酸等效物分配给其他核苷酸形式(NDK)。 我们的目标是创造一种通用的能源，集成UDP-，GDP-，CMP-， 以及大规模OPME反应中的ADP-糖供体。在目标3中，我们将生成可扩展的概念验证 OPME合成具有生物学意义的模型HMO靶标进行优化。该方法将结合 用于糖供体合成和糖链延伸的合成模块，具有优化的能量再生和 确定能量偶联OPME中所有酶步骤的跨平台兼容性条件 (EcOPME)平台。HMO的目标还提供了测试组合ecOPME反应以及 连续的ecOPME糖添加，其中酶竞争将产生不想要的产品。我们的目标是 通过选择性使用糖基转移酶受体特异性整合多个酶转移步骤 与能源再生相结合，形成概念验证模块化平台，实现高效、灵活和 从简单的单糖构建块开始可扩展的靶向治疗性HMOS的合成。

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.