Biosynthetic Analysis of Marine Cyanobacterial Pathways

描述（由申请人提供）：海洋蓝细菌在其生物活性和结构独特的天然产物的生产方面非常丰富。许多这些次级代谢物或其衍生物是药物开发计划中的先导化合物，旨在提供治疗癌症、细菌感染、炎症反应的新疗法，以及用于作物保护以杀死有害微生物病原体和昆虫。海洋和陆地蓝藻天然产物的分离和结构分析提供了一个非常大的混合非核糖体肽合成酶/聚酮合酶（NRPS/PKS）系统的数量。相应的代谢系统由一组复杂的多功能蛋白质组成，这些蛋白质沿着相关酶通过模块化的多步骤过程产生结构复杂的分子。在过去的几年中，谢尔曼和Gerwick实验室已经开发了一个互补的计划，克隆和表征新的蓝藻次生代谢产物的生物合成途径，具有显着的生物技术应用的潜力。对蓝藻生物合成途径中的分子机制、催化活性、动力学特性和底物特异性的全面了解才刚刚开始。拟议的研究将建立在我们对curacin和jamaicamide代谢系统的研究基础上，这两个不同但相关的途径具有遗传特征，并准备进行详细的生化研究。这种详细的遗传和生物化学的理解将有助于设计新的生物合成系统，利用蓝藻次生代谢的潜力不断增长。尽管在过去几年中取得了相当大的进展，但蓝藻天然产物产生新的先导化合物作为有用的药物开发的全部承诺只有通过缩小知识和技术方面的一系列关键差距才能实现。解决这些挑战将需要开发和优化遗传和生物化学方法，使我们能够1）利用独特的次级代谢酶来创造新的小分子，2）操纵蓝藻天然产物基因簇以产生类似物结构。具体目标是：1.研究Curacin（Cur）和jamaicamide（Jam）生物合成途径的生物化学独特方面，包括环丙烷环的形成、顺式烯烃的形成和Cur的终止，以及Jam的链起始、氯乙烯的形成和终止。2.对特定目标1产生的新化合物进行生物测定，包括评价微管蛋白聚合和结合位点特异性的抑制作用，与癌症相关的生化测定，以及分别在U-M和SIO进行与抗微生物活性和神经毒性相关的内部筛选。

DESCRIPTION (provided by applicant): Marine cyanobacteria are extraordinarily rich in their production of biologically-active and structurally- unique natural products. A number of these secondary metabolites or their derivatives are lead compounds n drug development programs aimed at providing new therapies to treat cancer, bacterial infections, inflammatory responses, and in crop protection to kill harmful microbial pathogens and insects. Isolation and structural analysis of marine and terrestrial cyanobacterial natural products has provided access to an unusually large number of mixed non-ribosomal peptide synthetase/polyketide synthase (NRPS/PKS) systems. The corresponding metabolic systems are comprised of an intriguing set of complex multifunctional proteins that along with allied enzymes generate structurally complex molecules via a modular multi-step process. Over the past several years the Sherman and Gerwick laboratories have developed a complementary program to clone and characterize the biosynthetic pathways of novel cyanobacterial secondary metabolites that possess significant potential for biotechnological applications. A full understanding of the molecular mechanisms, catalytic activities, kinetic properties, and substrate specificities within cyanobacterial biosynthetic pathways is just beginning to unfold. The proposed research will build upon our studies of the curacin and jamaicamide metabolic systems, two distinct yet related pathways that are genetically characterized and poised for detailed biochemical studies. This detailed genetic and biochemical understanding will facilitate the design of new biosynthetic systems that harness the growing potential of cyanobacterial secondary metabolism. Despite considerable gains over the past few years, the full promise of cyanobacterial natural products to yield new lead compounds for development as useful Pharmaceuticals, will only be realized by closing a series of key gaps in knowledge and technology. Solving these challenges will require development and optimization of genetic and biochemical methods that allow us to 1) utilize unique secondary metabolite enzymes for creation of novel small molecules, 2) manipulate cyanobacterial natural product gene clusters to produce analog structures. The specific aims are: 1. To investigate biochemically unique aspects of the curacin (Cur), and jamaicamide (Jam) biosynthetic pathways including formation of the cyclopropane ring, cis-alkene formation, and termination in Cur, and chain initiation, vinyl chloride formation and termination in Jam. 2. Perform bioassays on new compounds resulting from Specific Aim 1 including evaluation for inhibition of tubulin polymerization and binding site specificity, biochemical assays relevant to cancer, and in house screens at U-M and SIO relevant to anti-microbial activity and neurotoxicity, respectively.