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: 10242005
• 负责人: Robert Nicol
• 金额: $ 146.84万
• 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/10242005
• 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; 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.

项目摘要/摘要 WHO和CDC已将革兰氏阴性抗生素宣布为最伟大的未满足需求之一。确实， 加速抗生素耐药性的问题可能会每年生命1000万。尽管迫切需要 新的抗生素，革兰氏阴性生物面临挑战，因为它们具有不可渗透 膜和外排泵可抵抗异种生物。使事情复杂化，识别新型天然产品 仍然是一个重大挑战：i）使用组织的生物活性引导分馏的经典方法 提取物很慢； ii）从其本地或共生环境的上下文中移除，微生物有机体 不能总是在实验室中生产其多样的代谢物。和iii）异源表达 不同的主机仍然是一个限制。在此提案的工作中，我们开发了多功能合成 可以克服每个挑战的生物学平台。在这里，我们将使用此平台生产 潜在抗菌分子的大型文库，包括> 10,000个天然产物衍生物， > 100,000个凝集素变体和> 1m循环宠物。我们平台的输出将被筛选 针对革兰氏阴性病原体。我们平台的关键创新是生物合成中的酶 途径过表达裂解液或无细胞蛋白质合成制造，以构建无细胞的“单位”之后 然后可以用来重新创建途径或组合多样化的化学工程范式。 最近，我们在DNA测序工作流，微流体，无细胞系统中取得了关键的进步， 机器学习和筛选平台，以促进我们的目标。在AIM 1中，我们将开发一个单位操作 基于抗生素的表达系统并生成新型化合物的库。在AIM 2中，我们将产生 稳定的循环支架中的抗菌胡椒的库。在AIM 3中，我们将扩展我们的技术以生成 针对革兰氏阴性病原体的讲座文库。在与所有其他目标相关的AIM 4中，我们将 AIMS 1、2和3的屏幕库进行生物活性。我们希望以发现为中心的方法 将是在无细胞环境中提供高通量实验的第一个同类产品。 （i）避免继承全细胞生存能力的局限 重新设计生物，以及（iii）通过使用的组合和模块化途径组合 定义明确的实验条件可以使用细胞中无法使用化学和物理操作。 这项工作将为负责特权类别的生物合成机制增加新知识 天然产品抗生素，并为我们提供系统地设计其工具。此外，它将交付 新的化学物质是优化和开发治疗的起点。