Laulimalide Production Genes

劳利马内酯生产基因

• 批准号: 6584382
• 负责人: CHARLES R HUTCHINSON
• 金额: $ 16.26万
• 依托单位: KOSAN BIOSCIENCES, INC.
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-03-01 至 2004-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6584382
• 关键词: Porifera; antineoplastics; aquatic organism; artificial chromosomes; biosynthesis; biotherapeutic agent; cytochrome P450; drug design /synthesis /production; drug discovery /isolation; gene expression; genetic library; neoplasm /cancer chemotherapy; neoplasm /cancer pharmacology; nucleic acid hybridization; nucleic acid sequence; polyketide synthase; polymerase chain reaction

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.

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，从海洋资源中生产多克量的实验性抗肿瘤药物。我们的方法是基于从生产生物体中克隆生物合成基因，然后在可以在大规模发酵中生长的细菌宿主中表达这些基因。苔藓抑素，discodermolide，软海绵素，laulimalide和其他海洋细胞毒素，这是由涉及聚酮酶的途径，机制和临床前的调查已受到阻碍的海洋聚酮化合物的供应不足，因为他们是通过分离或全合成，在一个非常高的成本。第一阶段研究的目标是通过以下具体目的来鉴定含有laulimalide生物合成基因的遗传位点：（1）从可靠来源获得新鲜海绵组织，并表明其（或其共生体）含有laulimalide。(2)通过PCR分析产生laulimalide的海绵（和/或其微生物共生体）细胞或细胞提取物的DNA，检测模块聚酮合酶（PKS）、CYP 450、“羟基丙二酸（HM）”和羟甲基戊二酰辅酶A合酶（HMGS）基因同源物。(3)使用从产生laulimalide的海绵/共生体复合体或从推定的微生物共生体分离的DNA制备fosmid和BAC文库，并通过PCR和DNA杂交分析获得含有PKS、CYP 450、HM和HMGS基因的克隆。(4)对包含PKS、CYP 450、HM和HMGS基因的区域进行测序，以证明其参与laulimalide生物合成的合理性。我们的实验设计是建立在正在进行的工作中，我们试图克隆discodermolide生物合成基因。成功将导致开发基于发酵的过程，以生产大量的laulimalide或其类似物之一，用于临床前研究作为抗肿瘤药物。