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: 8593930
• 负责人: Peter Anthony Jordan
• 金额: $ 4.71万
• 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/8593930
• 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）：生物世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，包括挽救生命的抗生素青霉素和万古霉素。最近的技术和知识进步，在遗传和生物化学的理解，如何大量的植物，真菌和细菌产生这样的天然产物已经彻底改变了他们的发现。对天然产物药物开发的增长和影响至关重要的是新天然产物的不断发现及其生物合成机制的阐明。 吡咯并喹啉类生物碱是一类具有广泛生物活性的天然产物。尽管它们在许多生物体中产生，但对吡咯喹啉生物碱的生物合成知之甚少。最近的研究表明，lymphostin是一种吡咯并喹啉生物碱， 免疫抑制剂的特性，已经开始揭示一个独特的生物合成。我假设，基于对淋巴抑制素基因簇的分析和我最近的工作，描述了两种结构相关的抗癌化合物氨酰胺A和B的生物合成基因簇，这些天然产物是由核糖体合成产生的肽的化学修饰引起的。这种不寻常的机制可能被证明是普遍的吡咯喹啉生物碱，遗传和生物化学的理解可能会打开全新的方向，发现相关的天然产物药物。 本研究的目的是通过对氨基酰胺A和B生物合成的综合遗传学和生物化学表征，进一步阐明吡咯并喹啉生物碱的生物合成。由于淋巴毒素和氨基酰胺是由两种不同的细菌产生的，因此比较遗传分析将为生物合成机制的一般性提供大量信息，特别是关于核糖体肽合成在该过程中所起的假定作用。 对于拟议的工作，氨酰胺生物合成基因簇将被克隆，在异源宿主中表达，并使用定向基因缺失进行验证，这将敲除氨酰胺的生产。每个基因在生物合成中所起的功能作用将通过向敲除的基因引入化学互补物来最终恢复氨酰胺生产来询问。通过对氨酰胺生物合成早期阶段的体外生物化学研究，将获得进一步的机制细节。生物合成的知识将被用于生产新的氨基酰胺类似物的生物工程，并对其生物效力进行分析。