Biosynthesis and bioengineering of the marine anti-tumor compounds, ammosamides A

海洋抗肿瘤化合物氨酰胺A的生物合成和生物工程

• 批准号: 8715322
• 负责人: Peter Anthony Jordan
• 金额: $ 5.15万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-01 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8715322
• 关键词: Alkaloids; Anabolism; Antineoplastic Agents; Bacteria; Biochemical; Bioinformatics; Biological; Biological Assay; Biological Factors; Biomedical Engineering; Chemicals; Cloning; Complement; Culture Media; Data; Development; Engineering; Enzymatic Biochemistry; Enzymes; Exhibits; Family; Family member; Future; Gene Cluster; Gene Deletion; Gene Expression; Genes; Genetic; Genetic Recombination; Genome; Genomics; Goals; Growth; Health; Human; Hybrids; Immunosuppressive Agents; In Vitro; Investigation; Knock-out; Knowledge; Libraries; Life; Light; Link; Marines; Mining; Modification; Natural Product Drug; Open Reading Frames; Organism; Oxidoreductase; Pathway interactions; Penicillins; Peptide Hydrolases; Peptide Synthesis; Peptides; Phase; Play; Post-Translational Protein Processing; Process; Production; Property; Protein Biosynthesis; Reporting; Role; Scheme; Series; Source; Staging; Streptomyces; Tryptophan; Vancomycin; Variant; Work; analog; base; cancer cell; cancer therapy; comparative; cytotoxic; cytotoxicity test; drug development; experience; genetic analysis; genome sequencing; insight; knockout gene; member; mutant; novel; pathogen; peptide synthase; plant fungi; polyketide synthase; public health relevance; pyrroloquinoline; tumor

DESCRIPTION (provided by applicant): The biological world is the source of numerous naturally produced chemicals of significance to human health, including the life saving antibiotics penicillin and vancomycin. Recent technological and intellectual advances in the genetic and biochemical understanding of how the multitude of plants, fungi and bacteria produce such natural products has revolutionized their discovery. Critical to the growth and impact of natural product drug development is the continued discovery of new natural products and the elucidation of the mechanisms of their biosynthesis. The pyrroloquinoline alkaloids are a diverse class of natural products which exhibit broad biological activity. Despite their production in a number of organisms, little is known about the biosynthesis of the pyrroloquinoline alkaloids. Recently studies of lymphostin, a pyrroloquinoline alkaloid with potent immunosuppressant properties, have begun to shed light on a unique biosynthesis. I hypothesize, based upon an analysis of the lymphostin gene cluster and my recent work characterizing the gene cluster for the biosynthesis of two structurally related anticancer compounds, ammosamide A and B, that these natural products arise from the chemical modification of a peptide produced by ribosomal synthesis. This unusual mechanism may prove to be general for the pyrroloquinoline alkaloids, and a genetic and biochemical understanding may open entirely new directions for the discovery of related natural product drugs. The goal of this proposal is to further elucidate the biosynthesis of pyrroloquinoline alkaloids by the comprehensive genetic and biochemical characterization of the biosynthesis of ammosamides A and B. Because lymphostin and the ammosamides are produced by two different bacteria, a comparative genetic analysis will be highly informative of the generality of the biosynthetic mechanism, especially with regard to the putative role that ribosomal peptide synthesis plays in the process. For the proposed work, the ammosamide biosynthetic gene cluster will be cloned, expressed in a heterologous host and validated using directed gene deletions, which will knockout the production of the ammosamides. The functional role that each gene plays in the biosynthesis will be interrogated by introducing chemical complements to the knocked out genes to ultimately restore ammosamide production. Further mechanistic detail will be achieved through in vitro biochemical studies of the early stages of ammosamide biosynthesis. The knowledge of the biosynthesis will then be used to bioengineer the production of new analogues of the ammosamides, which will be assayed for their biological potency.

描述（由申请人提供）：生物界是许多对人类健康具有重要意义的天然化学物质的来源，包括救生抗生素青霉素和万古霉素。最近在对众多植物、真菌和细菌如何产生此类天然产物的遗传和生化理解方面的技术和知识进步彻底改变了他们的发现。天然产物药物开发的增长和影响的关键是不断发现新的天然产物并阐明其生物合成机制。 吡咯喹啉生物碱是一类具有广泛生物活性的天然产物。尽管吡咯喹啉生物碱在许多生物体中产生，但人们对吡咯喹啉生物碱的生物合成知之甚少。最近对淋巴素（lymphostin）的研究，一种吡咯并喹啉生物碱，具有强效 免疫抑制特性，已开始揭示其独特的生物合成。根据对淋巴毒素基因簇的分析以及我最近对生物合成两种结构相关的抗癌化合物氨酰胺 A 和 B 的基因簇进行表征的工作，我推测这些天然产物来自核糖体合成产生的肽的化学修饰。这种不寻常的机制可能被证明对于吡咯并喹啉生物碱来说是普遍的，并且遗传和生化的理解可能为相关天然产物药物的发现开辟全新的方向。 该提案的目标是通过氨酰胺 A 和 B 生物合成的全面遗传和生化表征，进一步阐明吡咯喹啉生物碱的生物合成。由于淋巴抑素和氨酰胺是由两种不同的细菌产生，因此比较遗传分析将为生物合成机制的普遍性提供大量信息，特别是在 核糖体肽合成在此过程中发挥的假定作用。 对于拟议的工作，将克隆氨酰胺生物合成基因簇，在异源宿主中表达并使用定向基因删除进行验证，这将敲除氨酰胺的产生。通过向被敲除的基因引入化学互补物以最终恢复氨酰胺的生产，可以探究每个基因在生物合成中所发挥的功能作用。进一步的机制细节将通过氨酰胺生物合成早期阶段的体外生化研究来实现。然后，生物合成知识将用于生物工程生产新的氨酰胺类似物，并对其生物效力进行测定。