A minimally invasive synthetic bio-driven approach for natural products discovery

 描述（由申请人提供）：本提案中概述的工作旨在建立一个新的交钥匙天然产物发现平台——从模型菌株的开发和用于捕获和生物信息学鉴定新型生物合成多样性的简化方法，到天然产物本身的表达和表征。我们的努力最终将不仅开发新的天然产物发现管道，而且开发一系列工具/资源（优化的菌株、改进的簇克隆和注释方法、改进的天然产物生产门菌株的基因/基因组编辑方法等），旨在消除基因组DNA转化为天然产物的现有障碍。我们的方法致力于开发工具，最大限度地减少 DNA 序列生产分子所需的操作，从而最大限度地发挥真正高通量小分子发现管道的潜力。在我们的首要目标中，我们将通过凭经验选择细菌，开发传统上与天然产物发现相关的门（放线菌、β/γ-变形菌）的改进模型宿主 显示出天然产物生产的最大先天倾向的菌株。然后，利用现代合成生物学/基因组编辑工具和高通量经验簇表达筛选，我们将进一步优化这些菌株，以提高它们转录诱导和支持从不同的生物合成基因簇集合中生产天然产物的能力。第二个目标侧重于建立在模型天然产物生产属中使用现代基因组/基因簇编辑工具（CRISPR、MAGE）的协议。在我们的第三个目标中，我们建议开发一个生物信息学管道来识别基因簇家族，通过 基因内容和/或基因组织，与任何已知的基因簇家族（即神秘基因簇家族，CCF）都不相似。第三个目标还建立了改进的簇克隆和注释方法，允许以单一格式快速归档微生物生物合成多样性（无论是来自培养的微生物还是未培养的微生物），并且可以使用最少的测序工作轻松注释和挖掘 CCF。在我们的第四个目标中，我们建议利用目标 1-3 中开发的新工具来建立更有效地从 DNA 序列获取天然产物的方法。该目标包括建立改进的功能（元）基因组学平台以及两种可扩展的基于 CRISPR 的、与基因簇无关的激活策略，这些策略利用 CRISPR 系统的体内或体外能力，同时靶向簇内的多个启动子区域进行转录激活。该目标还包括开发基于 MS/NMR 的半自动化结构评估方法，对小规模发酵的肉汤提取物中存在的异源生产的天然产物进行结构评估。我们的发现策略提供了更积极的簇诱导方法，我们相信，作为一个整体，代表了一种从基因组序列获取天然产物的稳健、全面的方法。

 DESCRIPTION (provided by applicant): The work outlined in this proposal is designed to establish a new turnkey natural product discovery platform -- from the development of model strains and streamlined methods for the capture and bioinformatics identification of novel biosynthetic diversity, through to the expression and characterization of natural products themselves. Our efforts will culminate in the development of not only a new natural product discovery pipeline but also a series of tools/resources (optimized strains, improved cluster cloning and annotation methods, improved gene/genome editing methods for strains from natural product producing phyla, etc.) that are designed to remove existing roadblocks in the conversion of genomic DNA into natural products. Our approach strives to develop tools that will minimize the manipulations required for molecule production from DNA sequence and, thereby, maximize the potential for truly high throughput small molecule discovery pipelines. In our First Aim we will develop improved model hosts from Phyla that are traditionally associated with natural product discovery (actinomycetes, ß/γ-proteobacteria), by empirically selecting bacterial strains that show the greatest innate tendency for natural product production. Then, using modern synthetic biology/genome editing tools and high throughput empirical cluster expression screening, we will further optimize these strains to improve their ability to transcriptionally indce and support the production of natural products from diverse collections of biosynthetic gene clusters. The Second Aim focuses on establishing protocols for using modern genome/gene cluster editing tools (CRISPR, MAGE) in model natural product producing genera. In our Third Aim, we propose to develop a bioinformatics pipeline for identifying gene cluster families that, by gene content and/or gene organization, do not resemble any known gene cluster family (i.e. cryptic gene cluster families, CCF). The Third Aim also establishes improved cluster cloning and annotation methods that will allow for the rapid archiving of microbial biosynthetic diversity (whether from cultured or uncultured microbes) in a single format that can be easily annotated and mined for CCFs using minimal sequencing effort. In our Fourth Aim we propose to leverage the new tools developed in Aims 1-3 to establish methods for more efficiently accessing natural products from DNA sequence. This Aim includes the establishment of an improved functional (meta)genomics platform as well as two scalable CRISPR-based, gene cluster agnostic, activation strategies that take advantage of either the in vivo or in vitro ability of the CRISPR system to simultaneously target multiple promoter regions within a cluster for transcriptional activation. This aim also includes the development of methods for semi-automated, MS/NMR-based structural evaluation of heterologously produced natural products present in broth extracts from small-scale fermentations. Our discovery strategy provides successively more aggressive cluster induction methods and we believe, as a whole, represents a robust, comprehensive approach for accessing natural products from genomic sequence.