BIOSYNTHESIS OF ANTHRACYCLINE ANTIBIOTICS

这项建议要求支持一项分子遗传学和 链霉菌形成蒽环类抗生素的生物化学。这个 一种新的具有抗肿瘤活性的蒽环类代谢产物的生物合成 灰链霉菌对四环素C(7)的体内活性 22794将作为所有蒽环类药物形成的模型进行研究 抗生素。实验方法将首先涉及构建 一组在四环素中具有点封闭的细菌突变体 物理和生物处理灰霉菌的生物合成途径 化学诱变剂。然后，这些突变体将被用作 亲本DNA片段在鸟枪基因中的转化 使用已知功能的链霉菌克隆载体进行克隆实验 在灰斑潜蝇中。受阻人群的遗传和生化互补 突变株转化后产生7的能力将通过以下方式进行评估 恢复抗生素形成的生物测定法；化学和物理 方法将用于确认这一点以及关联的 酵素。用高拷贝数载体进行基因扩增，或使用 将探索高水平表达载体作为一种增加 四环素C酶及其中间体的生产 生物合成。将进行辅助工作以探测蜂窝位置 抗生素生产基因，以及表达的可能性 这些基因存在于其他细菌物种中。研究结果： 该计划将展示一种研究生物合成的新方法 抗生素，并将建立增加 生产有价值的抗生素。

This proposal requests support of a study of the molecular genetics and biochemistry of anthracycline antibiotic formation by streptomycetes. The biosynthesis of a new anthracycline metabolite with potent antitumor activity in vivo, tetracenomycin C (7), by Streptomyces glaucescens ETH 22794 will be studied as a model of the formation of all anthracycline antibiotics. The experimental approach will first involve construction of a set of bacterial mutants having point blocks in the tetracenomycin biosynthetic pathway by treatment of S. glaucescens with physical and chemical mutagens. These mutants will then be used as hosts for transformation with DNA segments from the parent organism in shotgun gene cloning experiments, using Streptomyces cloning vectors known to function in S. glaucescens. Genetic and biochemical complementation of the blocked mutants' ability to produce 7 after transformation will be assessed by bioassay for the restoration of antibiotic formation; chemical and physical methods will be used to affirm this and the presence of the associated enzymes. Gene amplification with high copy number vectors, or the use of high level expression vectors will be explored as a means for increasing the production of the enzymes and intermediates of tetracenomycin C biosynthesis. Subsidiary work will be done to probe the cellular location of the antibiotic production genes, and the possibility for expression of these genes in other bacterial species. The results of the research program will exemplify a new approach to research on the biosynthesis of antibiotics, and will establish general parameters necessary for increasing the production of valuable antibiotics.