Generating an Actinobacterial Chassis for Antimicrobial Discovery

摘要 长期以来，天然产品一直是作为治疗药物支架的主要资源，特别是针对 抗菌素耐药性的威胁。曾经处于休眠状态的天然产物发现领域正在经历 随着元基因组学领域的进步而出现的文艺复兴使人们能够研究一个巨大的、 未开发的基因簇储藏库。大型环境DNA(EDNA)片段，以前无法访问 由于寄主缺乏可培育性，现在常规构建在广泛寄主载体上并穿梭 进入异源宿主，主要是大肠杆菌和利维丹链球菌。抗菌化合物是通过 这些宿主载体中的Edna的高通量筛选，但受到非常低的‘命中率’的限制。异源的 宿主通常没有或表达不佳的转录识别元件，替代的西格玛因子， 它引导宿主RNA聚合酶激活这些生物合成基因簇(BGC)。这 提案概述了基础广泛的底盘有机体的开发，以激活BGC 放线杆菌，负责大多数抗菌剂的微生物。选择西格玛因素最多 通常在放线杆菌中发现的将在恶臭假单胞菌中组合表达，这是一种生物 它既像大肠杆菌一样具有遗传易感性，又具有高GC含量和显著耐受性 像变青链霉菌这样的外源生物。通过跨学科方法结合来自以下领域的见解 微生物学，合成生物学和分析化学，这项提议寻求提供一个系统，将 提高基因组挖掘的‘命中率’，这将是更广泛的科学研究的重要工具 社区。

Abstract Natural products have long been the primary resource as scaffolds for therapeutic drugs, particularly against the threat of antimicrobial resistance. Once dormant, the field of natural product discovery is experiencing a renaissance as advancements in the field of metagenomics has allowed the investigation of a vast, untapped reservoirs of gene clusters. Large environmental DNA (eDNA) segments, previously inaccessible due to lack of cultivability in the host, are now routinely constructed on broad-host vectors and shuttled into heterologous hosts, primarily E. coli and S. lividans. Antimicrobial compounds are discovered through high-throughput screens of eDNA in these host vectors, but are limited by very low ‘hit rates’. Heterologous hosts often do not have or poorly express the transcriptional recognition elements, alternative sigma factors, that guide the host RNA polymerase that can activate these biosynthetic gene clusters (BGCs). This proposal outlines the development of a broad-based chassis organism for activation of BGCs from actinobacteria, the organisms responsible for the majority of antimicrobials. Alternative sigma factors most commonly found in actinobacteria will be combinatorially expressed in Pseudomonas putida, an organism that is both genetically tractable like E. coli and has a high-GC content organism with significant tolerance to xenobiotics like S. lividans. Through an interdisciplinary approach combining insights from microbiology, synthetic biology and analytical chemistry, this proposal seeks to provide a system that will increase the ‘hit rate’ in genome mining, which will be an important tool to the broader scientific community.