Innovative technologies to transform antibiotic discovery. Project 3 Rapid Access to Antibiotic Biosynthesis Machinery Using Synthetic Biology

改变抗生素发现的创新技术。

• 批准号: 10463691
• 负责人: Robert Nicol
• 金额: $ 149.65万
• 依托单位: BROAD INSTITUTE, INC.
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-07 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10463691
• 关键词: Address; Affinity; Anabolism; Antibiotic Resistance; Antibiotics; Area; Bar Codes; Biochemical Pathway; Biological; Biological Assay; Biological Models; Biological Process; Cell Survival; Cell-Free System; Cells; Centers for Disease Control and Prevention (U.S.); Chemical Engineering; Chemicals; Clinical; Complex; Cyclic Peptides; DNA; DNA Sequence; DNA sequencing; Daptomycin; Data; Defense Mechanisms; Development; Engineering; Environment; Enzymes; Experimental Designs; Foundations; Fractionation; Gene Cluster; Genomics; Goals; Gram-Negative Bacteria; Health; Human; Human Engineering; In Vitro; Individual; Instruction; Knowledge; Lectin; Libraries; Machine Learning; Macrolides; Mass Spectrum Analysis; Membrane; Methods; Microfluidics; Mining; Natural Products; Nature; Organism; Output; Pathway interactions; Peptide Antibiotics; Peptides; Periodicity; Phenotype; Polysaccharides; Process; Production; Protein Biosynthesis; Reaction; Resistance; Software Engineering; Software Tools; Structure; System; Technology; Testing; Therapeutic; Toxic effect; Translating; Ursidae Family; Variant; Work; Xenobiotics; analog; antimicrobial; antimicrobial peptide; base; beta-Lactams; chemical synthesis; combinatorial; cost; design; design-build-test; efflux pump; in vivo; innovation; innovative technologies; materials science; microbial; natural antimicrobial; next generation sequencing; novel; operation; overexpression; pathogen; protegrin PG-1; protegrins; scaffold; screening; synthetic biology; therapeutic development; tool

Project Summary/Abstract The WHO and CDC have declared Gram negative antibiotics as one of the greatest unmet needs. Indeed, the accelerating problem of antibiotic resistance threatens up to 10 million lives/year. Despite the urgent need for new antibiotics, Gram negative organisms are challenging to target because they have impermeable membranes and efflux pumps to resist xenobiotics. Complicating matters, identifying novel natural products remains, a significant challenge: i) classic approaches that use bioactivity-guided fractionation of organism extracts are slow; ii) removed from the context of their native or symbiotic environments, microbial organisms cannot always be coaxed into producing their varied metabolites in the lab; and iii) heterologous expression in different hosts remains a limitation. In work leading up to this proposal, we have developed versatile synthetic biology platform that can overcome each of these challenges. Here, we will use this platform to produce large libraries of potential antimicrobial molecules including >10,000 natural product derivatives, >100,000 lectin variants, and >1M cyclic peptides. The output from our platform will be screened against Gram negative pathogens. A key innovation of our platform is that enzymes in a biosynthetic pathway are overexpressed lysates or made in cell-free protein synthesis to construct cell-free “units” following a chemical engineering paradigm that can then be used to recreate the pathway or combinatorially diversify it. We have recently made key advancements in DNA sequencing workflows, microfluidics, cell-free systems, machine learning, and screening platforms to facilitate our goals. In Aim 1, we will develop a unit operation based antibiotic expression systems and generate libraries of novel compounds. In Aim 2, we will generate libraries of antimicrobial peptides in stable cyclic scaffolds. In Aim 3, we will extend our technology to generate libraries of lectins that target Gram negative pathogens. In Aim 4, which connects to all other aims, we will screen libraries from Aims 1, 2, and 3 for biological activity. We expect that our discovery-centered approach will be the first of its kind in offering high-throughput experimentation in a cell-free environment. It will uniquely (i) avoid inherent limitations of whole-cell viability, (ii) permit design-build-test (DBT) iterations without the need to reengineer organisms, and (iii) explore combinatorial and modular assembly of pathways through the use of well-defined experimental conditions that can use chemical and physical manipulations not possible in cells. This work will add new knowledge for the biosynthetic mechanisms responsible for the privileged class of natural product antibiotics and provide us with tools to systematically engineer them. Furthermore, it will deliver new chemical matter to serve as starting points for optimization and development of therapeutics.

项目概要/摘要 世界卫生组织和疾病预防控制中心已宣布革兰氏阴性抗生素是最大的未满足需求之一。 尽管迫切需要抗生素，但日益严重的抗生素耐药性问题每年仍然威胁着多达 1000 万人的生命。 新抗生素、革兰氏阴性生物体很难靶向，因为它们具有不渗透性 膜和外排泵来抵抗外源性物质，使问题变得复杂，识别新颖的天然产物。 仍然是一个重大挑战：i) 使用生物活性引导的生物体分级分离的经典方法 提取物缓慢；ii) 从其天然或共生环境、微生物有机体中去除 不能总是在实验室中诱导产生不同的代谢物；以及 iii) 异源表达； 在提出该提案的工作中，不同的宿主仍然是一个限制，我们已经开发出了多功能的合成材料。 可以克服这些挑战的生物平台在这里，我们将使用这个平台来生产。 潜在抗菌分子的大型库，包括超过 10,000 种天然产物衍生物， 我们平台的输出将被筛选 >100,000 个凝集素变体和 >1M 环肽。 我们平台的一个关键创新是生物合成中的酶。 途径是过表达的裂解物或在无细胞蛋白质合成中制成，以构建无细胞“单位” 一种化学范式工程，然后可用于重新创建该途径或使其组合多样化。 我们最近在 DNA 测序工作流程、微流体、无细胞系统、 机器学习和筛选平台以促进我们的目标 1，我们将开发一个单元操作。 基于抗生素表达系统并生成新化合物库 在目标 2 中，我们将生成新化合物库。 在目标 3 中，我们将扩展我们的技术来生成稳定的环状支架中的抗菌肽库。 在与所有其他目标相关的目标 4 中，我们将建立针对革兰氏阴性病原体的凝集素文库。 我们期望我们以发现为中心的方法从目标 1、2 和 3 中筛选文库。 将是同类中第一个在无细胞环境中提供高通量实验的产品。 (i) 避免全细胞活力的固有限制，(ii) 允许设计-构建-测试 (DBT) 迭代，而无需 重新设计生物体，以及（iii）通过使用以下方法探索路径的组合和模块化组装 明确的实验条件，可以使用细胞中不可能进行的化学和物理操作。 这项工作将为负责特权类别的生物合成机制增加新的知识。 天然产物抗生素并为我们提供系统地设计它们的工具此外，它将交付。 新化学物质作为优化和开发疗法的起点。