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

项目总结/摘要 世卫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。持续增 抗生素耐药性问题日益严重，每年威胁着多达1000万人的生命。即使迫切需要作 新的抗生素，革兰氏阴性菌是具有挑战性的目标，因为他们有不可渗透的 膜和外排泵来抵抗异生物质。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）使用生物活性指导的生物体分级分离的经典方法 提取物是缓慢的; ii）从它们的原生或共生环境的背景中移除，微生物有机体 不能总是被诱导在实验室中产生它们不同的代谢物;和iii）在 不同的主机仍然是一个限制。在导致这一建议的工作中，我们已经开发了多功能合成 生物学平台可以克服这些挑战。在这里，我们将利用这个平台来生产 潜在抗微生物分子的大型文库，包括> 10，000种天然产物衍生物， > 100，000个凝集素变体和> 1 M环肽。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， 途径是过表达的裂解物或在无细胞蛋白质合成中制备，以构建无细胞“单元”， 这是一种化学工程范例，可以用来重建这一途径或使其多样化。 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来实现我们的目标。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产生新化合物的文库。在目标2中，我们将生成 稳定环状支架中的抗微生物肽文库。在目标3中，我们将扩展我们的技术， 靶向革兰氏阴性病原体的凝集素文库。在目标4中，它连接到所有其他目标，我们将 筛选来自目标1、2和3的文库的生物活性。我们希望我们以发现为中心的方法 这将是第一个在无细胞环境中提供高通量实验的公司。这将是独一无二的 (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 明确定义的实验条件，可以使用细胞中不可能的化学和物理操作。 这项工作将增加新的知识的生物合成机制负责特权阶级的 天然产物抗生素，并为我们提供工具来系统地设计它们。此外，它将提供 新的化学物质作为优化和开发治疗的起点。

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.