Enediyne Antitumor Antibiotic Synthesis - Streptomycetes

描述（由申请人提供）：烯二炔抗生素是最有效的， 高活性的抗肿瘤药物。新制癌素（NCS）和 其缀合物在抗癌化疗中显示出巨大的临床前景， 并且几种卡奇霉素-抗体和C-1027-抗体缀合物具有 在最近的临床试验或模型研究中取得了出色的结果。 因此，关键的研究目标是开发方法以使 烯二炔及其类似物的研究。本申请建议 链霉菌NCS产生基因的克隆和鉴定 carzinostaticus，补充我们正在进行的努力，为C-1027在链霉菌 globisporus，试图通过操纵合成新的抗肿瘤药物， 控制烯二炔抗生素生物合成的基因。该提案基于 NCS发色团来源于三种生物合成的假设， 结构单元烯二炔核、脱氧氨基糖和聚酮化合物 萘甲酸部分。这一假设的三个推论是：（1） 放线菌中脱氧糖和聚酮生物合成的广泛知识 这将极大地帮助克隆脱氧氨基糖的基因， 从S.（2）结构性 NCS和C-1027的烯二炔核之间的相似性提供了独特的 有机会破译烯二炔核心的遗传和生化基础 （3）遗传工具和技术的发展， 链霉菌属的抗生素生物合成应直接适用 涉及操纵NCS和C-1027生物合成基因以提高产量， 类似物或新的烯二炔生产。五年奖的具体目标 阶段是（1）克隆和测序整个NCS生物合成基因簇 来自酿脓链球菌（2）建立了一套适合于该菌的遗传系统。 （3）为了证实在体内的NCS生物合成， 克隆S.编码NCS生物合成的carzinostaticus DNA，并定位 通过基因破坏和替换的NCS基因簇的边界 实验，以及（4）鉴定烯二炔核心所必需的基因 通过比较NCS和C-1027生物合成基因簇， 通过表达以下来演示烯二炔核心结构的合成： 异源宿主中的相应基因。这些研究的结果将 揭示了对烯二炔家族生物合成的新见解， 抗生素，阐明合理工程的烯二炔生物合成 在这些以及其他产生烯二炔的生物体中， 实际上导致了一种全新的，临床上有用的， 抗肿瘤剂

DESCRIPTION (provided by applicant): Enediyne antibiotics are the most potent, highly active antitumor agents in existence today. Neocarzinostatin (NCS) and its conjugates have shown great clinical promise in anticancer chemotherapy, and several calicheamicin-antibody and C-1027-antibody conjugates have produced outstanding results in recent clinical trials or model studies. Therefore, it is a critical research goal to develop ways to make the enediynes and their analogs for further studies. This application proposes to clone and characterize the genes for NCS production in Streptomyces carzinostaticus, complementing our on going effort for C-1027 in Streptomyces globisporus, in an attempt to synthesize novel antitumor drugs by manipulating genes governing enediyne antibiotic biosynthesis. This proposal is based on the hypothesis that the NCS chromophore is derived from three biosynthetic building blocks an enediyne core, a deoxy aminosugar, and a polyketide naphthoic acid moiety. Three corollaries of this hypothesis are (1) that the extensive knowledge of deoxysugar and polyketide biosynthesis in actinomycetes should greatly aid the effort to clone the genes for deoxy aminosugar and naphthoic acid biosynthesis from S. carzinostaticus, (2) that the structural similarity between the enediyne cores of NCS and C-1027 provides an unique opportunity to decipher the genetic and biochemical basis of enediyne core assembly, and (3) that the genetic tools and technology developed in Streptomyces species for antibiotic biosynthesis should be directly applicable to manipulating NCS and C-1027 biosynthesis genes for yield improvement and analog or novel enediyne production. The specific aims for the five-year award period are (1) to clone and sequence the entire NCS biosynthetic gene cluster from S. carzinostaticus, (2) to develop a genetic system for S. carzinostaticus to manipulate NCS biosynthesis in viva, (3) to confirm the cloned S. carzinostaticus DNA encoding NCS biosynthesis and to localize the boundaries of the NCS gene cluster by gene disruption and replacement experiments, and (4) to identify genes essential for the enediyne core biosynthesis by comparing the NCS and C-1027 biosynthetic gene clusters and to demonstrate the synthesis of an enediyne core structure by expressing the corresponding genes in a heterologous host. The outcome of these studies will reveal new insights into the biosynthesis of the enediyne family of antibiotics, shedding light on rational engineering of enediyne biosynthesis in these as well as other enediyne-producing organisms, and could realistically lead to the making of fundamentally new, clinically useful antitumor agents