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

用于鉴定由短 LanB 酶产生的新抗生素的基因组挖掘策略

• 批准号: 9767527
• 负责人: Chi Pan Ting
• 金额: $ 4.72万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-16 至 2020-06-02
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9767527
• 关键词: Amino Acids; Anabolism; Antibiotics; Antifungal Agents; Bacterial Antibiotic Resistance; Bioinformatics; Biological; C-terminal; Carbon; Chemicals; Complex; Cyclization; Cysteine; Dehydration; Development; Enzymes; Escherichia coli; Excision; Exhibits; Family; Food Preservatives; Gene Cluster; Genes; Genetic; Genome; Gram-Negative Bacteria; Health; Human; Hydro-Lyases; Infection; Inherited; Investigation; Lead; Light; Logic; Mass Spectrum Analysis; Methods; Mining; Modeling; Modification; Natural Products; Nisin; Organism; Pathogenicity; Penicillins; Peptides; Production; Pseudomonas syringae; Reaction; Recording of previous events; Resistance; Ribosomes; Roentgen Rays; Role; Serine; Societies; Source; Structure; System; Testing; Therapeutic; Threonine; Time; Transfer RNA; antimicrobial; bacterial resistance; base; combat; dehydroalanine; dehydrobutyrine; enzyme activity; enzyme biosynthesis; glutamic acid-tRNA; homologous recombination; in vivo; novel; pathogenic bacteria; scaffold

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.

项目总结/摘要 细菌病原体对抗生素耐药性的发展构成了一种遗传 需要确定新的化学实体， 抗生素羊毛硫肽是一类翻译后合成的核糖体肽 修饰肽（RIPPs）由结构多样的天然产物组成， 抗生素在这项提议中，一种用于识别 将探索新的天然产物RIPPs。最近发现了一类新的 截短的LanB酶，在本文中称为短LanB（sLanB）， 研究他们生产的天然产品。鉴于丰富的历史， 羊毛硫肽，以前称为羊毛硫抗生素，作为抗微生物化合物，天然的 预期从sLanB酶衍生的产物具有类似的治疗价值 结构多样性。 该提案的第一个目的涉及从天然产物中分离天然产物。 包含单个sLanB基因的假单胞菌中的模式基因簇。的 将通过在大肠杆菌中异源表达来研究由基因簇编码的酶。 杆菌确定天然产物结构后，从天然生物体中分离 将尝试通过化学衍生化。同源染色体断裂 重组是天然产物测定的另一种方法， 高级生物合成中间体的分离。 在初步研究的基础上，这项建议的第二个目的是 桥孢菌8437中复杂基因簇天然产物鉴定， 含有总共七种sLanB酶。基因簇编码的酶 将在E.杆菌所有七种sLanB与 标记的前体肽将允许酶活性的快速测定。的 通过质谱分析sLanB酶的修饰。这 总体战略预计将允许快速分离天然产物， 多种sLanB酶。从这两个基因簇中分离的天然产物将是 评价它们作为潜在治疗剂的抗微生物活性。