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

项目摘要/摘要 世界卫生组织和疾控中心已宣布革兰氏阴性抗生素是最大的未得到满足的需求之一。事实上， 抗生素耐药性问题日益加剧，每年威胁着多达1000万人的生命。尽管迫切需要 新的抗生素，革兰氏阴性菌是具有挑战性的目标，因为它们具有不渗透性 膜和外排泵，以抵抗外源物质。使问题复杂化，识别新的天然产品 仍然是一个重大的挑战：i)使用生物活性引导的生物体分级的经典方法 萃取物是缓慢的；二)从其本地或共生环境、微生物生物体的背景中移除 不能总是被诱使在实验室中产生不同的代谢物；以及iii)在 不同的主机仍然是一个限制。在这项提议之前的工作中，我们开发了多功能合成材料 一个可以克服这些挑战的生物平台。在这里，我们将利用这个平台来生产 潜在抗菌分子的大型文库，包括10,000个天然产品衍生物， &gt；100,000个凝集素变异体和&gt；1M环肽。来自我们平台的输出将被筛选 对抗革兰氏阴性病原体。我们平台的一个关键创新是，生物合成中的酶 途径是过度表达的裂解产物或在无细胞蛋白质合成中构建无细胞“单位” 一种化学工程范例，然后可以用来重建这条途径或组合多样化它。 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。在目标1中，我们将开发一个单元操作 基于抗生素表达系统，并生成新化合物的文库。在目标2中，我们将生成 稳定的环状支架中的抗菌肽文库。在目标3中，我们将扩展我们的技术以产生 针对革兰氏阴性病原体的凝集素文库。在连接所有其他目标的目标4中，我们将 从AIMS 1、2和3中筛选生物活性文库。我们希望我们以发现为中心的方法 将是第一个在无细胞环境中提供高通量实验的公司。它将独一无二地 (I)避免整个细胞生存能力的固有限制；(Ii)允许设计-构建-测试(DBT)迭代而不需要 重新设计生物体，以及(Iii)通过使用 定义明确的实验条件，可以使用在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阶层的生物合成机制增加新的知识 天然产品抗生素，并为我们提供了系统化设计它们的工具。此外，它还将提供 新的化学物质作为治疗学优化和发展的起点。

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.