Genomic approaches of discovery broad-spectrum antimicrobial agents

大多数临床使用的抗生素是细菌产生的小分子，称为天然产品，或其 衍生品。然而，天然产品并没有出现在抗生素发现计划中，因为它的价格高 再发现率和新分子发现率低。这项建议描述了一种新的基于基因组学的 利用生物信息学、微生物学 生态学、遗传学和化学，以识别隐秘的生物合成基因座并刺激它们产生 经过编码的天然产品。所有确定的分子都将被表征为具有抗微生物活性。 具体目标1.利用生物信息学鉴定生物合成基因簇并预测其产物。新的 将开发生物信息学工具来识别放线菌基因组中的生物合成基因簇 和其他微生物，并预测其小分子产物的结构元素。这些预测 将被用于抗生素的发现，并将其作为刺激生产的基础 如具体目标2和3所述的神秘天然产品： 具体目标2.通过模拟多物种环境来刺激隐蔽代谢物的产生。 大多数新天然产品的筛选都是用营养丰富的纯培养菌株进行的 成长型媒体。最近开发了一种新的筛选模式，在这种模式下菌株被培养成 在营养不良的生长介质上的微菌落。这种微菌落筛选方法将被用于 鉴定具有抗菌活性的神秘天然产品。 具体目标3.通过对生产者进行基因操纵来刺激隐蔽代谢物的产生。多数 自然产物编码基因簇被认为在标准培养条件下被抑制。 使用其生物合成基因簇已通过 具体目标1、内源性基因簇启动子将系统性地被强大的、可诱导的 启动子，能够控制合成和分离它们的小分子产物和 随后对它们的抗菌活性进行了表征。。

Most clinically-used antibiotics are bacterially-produced small molecules known as natural products, or their derivatives. However, natural products are not being featured in antibiotic discovery programs due to high rates of rediscovery and low rates of new molecule discovery. This proposal describes a new genomicsbased approach to natural product discovery that employs a combination of bioinformatics, microbial ecology, genetics, and chemistry to identify cryptic biosynthetic loci and stimulate them to produce their encoded natural products. All molecules identified will be characterized for antimicrobial activity. Specific Aim 1. Use bioinformatics to identify biosynthetic gene clusters and predict their products. New bioinformatic tools will be developed to identify biosynthetic gene clusters in the genomes of actinomycetes and other microbes, and to predict structural elements of their small molecule products. These predictions will be leveraged toward antibiotic discovery by using them as the basis for efforts to stimulate the production of cryptic natural products, as described in Specific Aims 2 and 3: Specific Aim 2. Stimulate the production of cryptic metabolites by simulating a multispecies environment. Most screens for new natural products have been performed with strains grown as pure cultures in nutrientrich growth media. A new screening format has recently been developed in which strains are grown as microcolonies on nutrient-poor growth media. This microcolony screening methodology will be used to identify cryptic natural products with antimicrobial activity. Specific Aim 3. Stimulate the production of cryptic metabolites by genetically manipulating producers. Most natural product-encoding gene clusters are thought to be repressed under standard culture conditions. Using actinomycete strains whose biosynthetic gene clusters have been identified by the efforts described in Specific Aim 1, endogenous gene cluster promoters will be systematically replaced with a strong, inducible promoter, enabling the controlled synthesis and isolation of their small molecule products and the subsequent characterization of their antimicrobial activity. .