Discodermolide Biosynthesis Genes

描述(申请人提供)：我们研究的长期目标是开发一种经济的方法，通过微生物发酵生产实验性抗肿瘤药物(+)-Discodermolide。由于这种海洋聚酮供应不足，对盘状聚醚的临床前研究一直受到阻碍。目前可以通过分离或全合成的方法获得，但药物的成本非常高。为了开发基于发酵的过程，将从分离出盘状菌的海绵中克隆盘状菌生物合成基因，然后在细菌宿主中表达这些基因。第一阶段研究的近期目标是通过以下具体目标鉴定和克隆许多盘状分子生物合成基因：(1)通过对产生盘状分子的海绵细胞或细胞提取物进行PCR分析，检测模块化聚酮合成酶(PKS)和氨基甲酰磷酸转移酶(CPT)基因同源物。(2)利用从海绵或其可能的微生物共生体中提取的DNA制备Fosmid文库，并通过PCR分析鉴定包含这些PKS和CPT基因的克隆。(3)对PKS和CPT基因进行测序，以证明它们参与盘状病毒生物合成的合理性。我们的研究还将建立一个通用的方法，使许多其他目前稀缺的具有抗肿瘤活性的海洋多酮大量用于药物开发的技术基础。

DESCRIPTION (provided by applicant): The long-range goal of our research is to develop an economical method for producing the experimental anti-tumor drug, (+) -discodermolide, by microbiological fermentation. Pre-clinical investigation of discodermolide has been hampered by inadequate supply of this marine polyketide. It currently is available by isolation or total synthesis, but the cost of the drug is very high. To enable development of a fermentation based process, the discodermolide biosynthetic genes will be cloned from the marine sponge from which discodermolide is isolated, followed by expression of these genes in a bacterial host. The immediate goal of Phase I research is to identify and clone many of the discodermolide biosynthesis genes by pursuing the following specific aims: (1) Detect modular polyketide synthase (PKS) and carbamoyl phosphate transferase (CPT) gene homologs by PCR analysis of cells or cell extracts of the marine sponge that produces discodermolide. (2) Prepare a fosmid library using DNA isolated from the sponge or its putative microbial symbiont and by PCR analysis identify clones that contain these PKS and CPT genes. (3) Sequence the PKS and CPT genes to the extent required to justify their involvement in discodermolide biosynthesis. Our research will also establish the technological base for a general approach to making many other currently scarce marine polyketides with anti-tumor activity available in large amounts for drug development.