A Genome-Mining Strategy for identification of new antibiotics produced by short LanB enzymes

项目摘要/摘要 细菌病原菌对抗生素耐药性的发展具有遗传性 需要确定将作为新类别的新的化学实体 抗生素。蓝硫肽是一类核糖体翻译后合成的多肽 修饰多肽(RIPP)由结构多样的天然产物组成，其中许多 也就是抗生素。在这项建议中，一种用于识别黑色素瘤的基因组挖掘策略 将开发新的天然产品Ripps。最近发现了一类新的 截短的LanB酶，在这里被称为短LanB(SLanB)，促使 对他们生产的天然产品进行调查。鉴于……的丰富历史 羊毛肽，前身为抗生素，是天然的抗菌化合物。 来自sLanB酶的产品有望具有类似的治疗价值。 和结构多样性。 这项提议的第一个目标是将一种天然产品与一种 紫丁香假单胞菌中含有单一sLanB基因的模式基因簇。这个 基因簇编码的酶将通过在E. Coli.在确定了天然产物结构后，从本土生物中分离出来 将通过化学衍生法进行尝试。同源基因引起的染色体破坏 重组代表了天然产物测定的另一种方法，即 先进生物合成中间体的分离。 在我们初步研究的基础上，这项建议的第二个目标包括 桥孢霉复杂基因簇中一种天然产物的鉴定8437， 总共含有七种sLanB酶。由基因簇编码的酶 将在大肠杆菌中异源表达。7种sLAB基因在体内的共表达 标记的前体多肽将允许快速测定酶活性。这个 SLanB酶的修饰将用质谱仪进行分析。这 总体战略预计将允许快速分离来自 多种sLanB酶。从这两个基因簇中分离出的天然产物将是 评估其作为潜在治疗药物的抗菌活性。

Project Summary/Abstract Development of antibiotic resistance by bacterial pathogens poses an inherit need for the identification of new chemical entities that will serve as new classes of antibiotics. Lanthipeptides are a family of ribosomally synthesized post-translationally modified peptides (RiPPs) consisting of structurally diverse natural products many of which are antibiotics. In this proposal, a genome mining strategy for the identification of new natural product RiPPs will be explored. The recent discovery of a new class of truncated LanB enzymes, herein termed short LanBs (sLanB), has prompted investigation of the natural products they produce. Given the rich history of lanthipeptides, formerly known as lantibiotics, as antimicrobial compounds, the natural products derived from sLanB enzymes are expected to possess similar therapeutic value and structural diversity. The first aim of this proposal involves the isolation of a natural product from a model gene cluster in Pseudomonas syringae containing a single sLanB gene. The enzymes encoded by the gene cluster will be studied by heterologous expression in E. coli. After determining the natural product structure, isolation from the native organism will be attempted by chemical derivatization. Chromosomal disruption by homologous recombinations represents an alternative method for natural product determination by isolation of advanced biosynthetic intermediates. Building upon our initial studies, the second aim of this proposal involves identification of a natural product from a complex gene cluster in Desmospora sp. 8437, containing a total of seven sLanB enzymes. The enzymes encoded by the gene cluster will be heterologous expressed in E. coli. In vivo co-expression of all seven sLanBs with tagged precursor peptide will allow for rapid determination of enzyme activity. The modifications by the sLanB enzymes will be analyzed by mass spectrometry. This overall strategy is expected to allow for rapid isolation of natural products derived from multiple sLanB enzymes. Natural products isolated from these two gene clusters will be evaluated for their antimicrobial activity as potential therapeutics.