Laulimalide Production Genes

描述(由申请人提供)： 我们研究的长期目标是开发经济的方法，从海洋来源生产多克数量的实验性抗肿瘤药物。我们的方法是从生产有机体中克隆生物合成基因，然后在细菌宿主中表达这些基因，这些宿主可以在大规模发酵中生长。苔藓素类、盘状菌素、灯盏花素、劳利马利德和其他海洋细胞毒素的机制和临床前研究一直受到海洋聚酮类化合物供应不足的阻碍，因为它们只能以非常高的成本通过分离或全合成获得。第一阶段研究的目标是通过以下具体目标确定包含劳利马利生物合成基因的遗传位点：(1)从可靠的来源获得新鲜的海绵组织，并证明其(或其共生体)含有劳利马利。(2)通过对产生劳利马利的海绵(和/或它们的微生物共生体)的细胞或细胞提取液中的DNA进行PCR分析，检测模块聚酮合成酶(PKS)、细胞色素P450(CyP450)、羟基丙二酸(HM)和羟甲基戊二酰辅酶A合成酶(HMG)基因的同源性。(3)利用从产生月桂胺的海绵/共生体或可能的微生物共生体中分离的DNA制备Fosid和BAC文库，通过聚合酶链式反应和DNA杂交分析获得含有PKS、CYP450、HM和HMGs基因的克隆。(4)对含有PKS、CYP450、HM和HMGS基因的区域进行测序，以证明它们参与了劳利马利的生物合成。我们的实验设计是建立在正在进行的工作的基础上的，在这项工作中，我们试图克隆盘状病毒的生物合成基因。成功将导致基于发酵的过程的开发，以制造大量的劳利马利德或其类似物，用于临床前研究作为抗肿瘤药物。

DESCRIPTION (provided by applicant): The long-range goal of our research is to develop economical methods for producing multigram quantities of experimental antitumor drugs from marine sources. Our approach is based on cloning the biosynthetic genes from the producing organism followed by expression of these genes in a bacterial host that can be grown in large-scale fermentations. Mechanistic and preclinical investigations of bryostatins, discodermolide, halichondrins, laulimalide and other marine cytotoxins, which are made by pathways involving polyketide synthases, have been hampered by inadequate supplies of the marine polyketides because they are available only by isolation or total synthesis at a very high cost. The goal of Phase I research is to identify the genetic locus that contains the biosynthesis genes for laulimalide by the following specific aims: (1) Obtain fresh sponge tissue from a reliable source, and show that it (or its symbionts) contains laulimalide. (2) Detect modular polyketide synthase (PKS), CYP450, "hydroxymalonate (HM)" and hydroxymethylglutaryI-Coenzyme A synthase (HMGS) gene homologs by PCR analysis of DNA from cells or cell extracts of the marine sponge (and/or their microbial symbionts) that produce laulimalide. (3) Prepare fosmid and BAC libraries using DNA isolated from the sponge/symbiont complex that produces laulimalide or from a putative microbial symbiont, and by PCR and DNA hybridization analyses obtain clones that contain the PKS, CYP450, HM and HMGS genes. (4) Sequence the region that contains the PKS, CYP450, HM and HMGS genes to the extent required to justify their involvement in laulimalide biosynthesis. Our experimental design is built on work in progress in which we are attempting to clone the discodermolide biosynthesis genes. Success will lead to development of a fermentation-based process to make large amounts of laulimalide or one of its analogs for preclinical studies as an antitumor drug.