Economical Modular One-Pot Multienzyme Synthesis of Human Milk Oligosaccharides

项目摘要 聚糖在生物学的几乎所有方面都发挥着重要作用。因此，它们具有广泛的应用，包括 向婴儿配方食品中添加必需的人乳低聚糖（HMO）作为治疗剂， 多种病原体感染，维持适当的微生物群，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， 阻碍了它们作为聚糖治疗剂的发展。尽管目前用于制备多酶的一锅法（OPME）系统 HMO合成简化了许多合成反应步骤，但在优化和 可伸缩性我们假设一个用于聚糖合成的模块化OPME系统可以被优化和整合 利用廉价的基于多磷酸盐的能量再生，以降低成本并显著提高 所需聚糖。在目标1中，我们将建立用于开始的单个糖转移步骤的参考OPME反应。 从单糖和聚糖受体的简单结构单元作为输入，并提供酶， 能量糖供体合成、单糖转移和能量再生。这些研究将发展 用于优化化学输入的可扩展模块（供体和受体结构单元，催化量的 核苷酸、二价阳离子、受控的pH和酶催化剂）以建立跨平台相容性 反应条件在目标2中，我们将建立可扩展的耦合聚磷酸盐能量再生系统 用于OPME聚糖合成。优化聚磷酸盐作为能源将需要控制 多磷酸盐和二价阳离子浓度，pH变化的中和，以及酶的整合， 产生ATP（RpPPK 2 -3），并将高能磷酸盐当量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，它集成了UDP-，GDP-，CMP-， 和ADP-糖供体在大规模OPME反应。在目标3中，我们将生成概念验证可扩展 用于优化的生物学感兴趣的模型HMO靶标的OPME合成。该方法将联合收割机 用于糖供体合成和聚糖延伸的合成模块，具有优化的能量再生， 确定能量耦合OPME中所有酶促步骤的跨平台相容性条件 （ecOPME）平台。HMO目标还提供了测试组合ecOPME反应以及 顺序添加ecOPME糖，其中酶竞争将产生不期望的产物。这些目标是 通过选择性使用糖基转移酶受体特异性来整合多个酶促转移步骤 再加上能量再生，从而形成一个概念验证的模块化平台， 从简单的单糖结构单元开始可规模化合成目标治疗性HMO。

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.