Economical Modular One-Pot Multienzyme Synthesis of Human Milk Oligosaccharides

经济的模块化一锅多酶合成母乳低聚糖

• 批准号: 10575228
• 负责人: KELLEY W. MOREMEN
• 金额: $ 22.65万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-05-01 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10575228
• 关键词: Address; Applications Grants; Automobile Driving; Biological; Biological Process; Biology; Chemicals; Complex; Coupled; Couples; Coupling; Data; Development; Divalent Cations; Energy-Generating Resources; Enzyme Inhibition; Enzymes; Future; Generations; Glycoconjugates; Goals; Gut Mucosa; Human; Human Milk; Hypersensitivity; In Situ; Infant; Infant Development; Infant formula; Infection; Infection prevention; Ions; Lactose; Libraries; Lipids; Methods; Monitor; Monosaccharides; Natural regeneration; Necrotizing Enterocolitis; Nucleic Acids; Nucleoside-Diphosphate Kinase; Nucleotide Biosynthesis; Nucleotides; Oligosaccharides; Organism; Outcome; Play; Polyphosphates; Polysaccharides; Premature Infant; Production; Proteins; Reaction; Reagent; Role; Source; Specificity; Structure; Supplementation; System; Testing; Therapeutic; Therapeutic Studies; biological systems; catalyst; cost; flexibility; glycosylation; glycosyltransferase; human model; inorganic phosphate; interest; microbiota; pathogen; prevent; scale up; sialyl-Lex; sugar; sugar nucleotide; targeted treatment; therapeutic development; tool; virtual

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.

项目摘要 Glycans几乎在生物学的各个方面都起着重要作用。因此，它们有广泛的申请，包括 补充必要的人牛奶寡糖（HMO）作为婴儿配方奶粉作为预防剂 多种病原体感染，保持适当的微生物群，预防过敏并治疗坏死性小肠结肠炎 在婴儿中。但是，目前的合成方法对于更复杂的HMO在负担得起的可伸缩性中失败了 阻碍他们作为聚糖治疗学的发展。虽然当前的一锅多酶（OPME）系统用于 HMO合成简化了许多合成反应步骤，在优化和 可伸缩性。我们假设可以优化和集成用于聚糖合成的模块化OPME系统 以廉价的基于多磷酸能的能量再生来降低成本并显着提高产量 理想的聚糖。在AIM 1中，我们将建立对开始的单糖转移步骤的参考OPME反应 从简单的单糖和聚糖受体的构件作为输入，并为高级提供酶 能量糖供体合成，单糖转移和能量再生。这些研究将发展 可扩展的模块，用于优化化学输入（供体和受体构建块，催化量 核苷酸，二价阳离子，受控pH和酶催化剂）以建立跨平台兼容 反应条件。在AIM 2中，我们将建立一个可扩展的耦合的多磷酸能源再生系统 用于OPME聚糖合成。聚磷酸作为能源的优化将需要控制 多磷酸盐和二价阳离子浓度，pH变化的中和化以及酶的整合 生成ATP（RPPPK2-3）并将高能磷酸盐等效物分配到其他核苷酸形式（NDK）。 我们的目标是创建一个通用能源，以整合UDP-，GDP-，CMP-的连续合成 和大规模OPME反应中的ADP-SUGAR供体。在AIM 3中，我们将生成可扩展的概念证明 OPME生物学兴趣的模型HMO靶标的优化靶标。方法将结合在一起 糖供体合成和聚糖扩展的合成模块，并具有优化的能量再生和 确定能量耦合OPME中所有酶促步骤的跨平台兼容性条件 （Ecopme）平台。 HMO目标还提供了测试合并Ecopme反应的机会 酶竞争将产生不希望的产品的顺序添加糖。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转移步骤 再加上能量再生，从而导致概念验证模块化平台，以实现高效，灵活和 从简单的单糖构建块开始的目标治疗性HMO的可扩展合成。