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

基本信息

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
项目类别：
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

项目摘要

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.

项目概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，包括在婴儿配方奶粉中补充必要的母乳低聚糖 (HMO) 作为治疗剂，以预防多种病原体感染，维持适当的菌群，预防过敏，治疗坏死性小肠结肠炎在婴儿中。然而，目前用于更复杂 HMO 的合成方法无法实现可承受的可扩展性，因此阻碍了它们作为聚糖疗法的发展。尽管目前的一锅多酶 (OPME) 系统 HMO合成简化了许多合成反应步骤，但在优化和优化方面做得很少可扩展性。我们假设用于聚糖合成的模块化 OPME 系统可以进行优化和集成使用廉价的聚磷酸盐能源再生来降低成本并显着提高产量所需的聚糖。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。这些研究将发展用于优化化学输入的可扩展模块（供体和受体构建模块、催化量核苷酸、二价阳离子、受控 pH 值和酶催化剂）以建立跨平台兼容反应条件。在目标2中，我们将建立一个可扩展的、耦合的聚磷酸盐能量再生系统用于 OPME 聚糖合成。优化聚磷酸盐作为能源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 (RpPPK2-3) 并将高能磷酸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能源，将 UDP-、GDP-、CMP-、和大规模 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 靶标以进行优化。该方法将结合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和确定能量耦合 OPME 中所有酶步骤的跨平台兼容性条件（ecOPME）平台。 HMO 目标还提供了测试组合 ecOPME 反应以及连续添加 ecOPME 糖，其中酶竞争会产生不需要的产物。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与能量再生相结合，形成一个概念验证的模块化平台，可实现高效、灵活和从简单的单糖结构单元开始可扩展地合成目标治疗性 HMO。