Disorazole Gene cluster

描述(由申请人提供)：我们研究计划的长期目标是发现新的双唑类化合物，并将其中一种开发为有用的抗肿瘤药物。虽然二唑A是一种有效的细胞毒剂，但其IC_(50)‘S低于纳米分子范围(0.12~0.63 nm)，但仍需开发具有良好治疗指数的新化合物。为了对二唑甲进行系统的化学修饰，我们需要无限量的起始原料。天然产生二唑类化合物的纤维索兰菌菌株SOCE12只产生少量的这类化合物(不到100 mg/l)，而且是一种生长非常缓慢的微生物(16小时倍增时间)。此外，该细菌的遗传学尚未开发，这极大地限制了通过对异丙唑基因簇进行遗传修饰来产生类似物的可能性。为了突破这些限制，我们将追求以下具体目标。(1)通过基于转座子的方法鉴定双唑类生物合成基因。首先，通过对生长介质的高效液相分析，分离出不产生突变的菌株，然后克隆和测序整合转座子两侧的基因组序列。或者，我们将通过筛选粘粒或BAC文库来识别基因簇，该文库带有识别地索拉唑基因的独特特征的探针。(2)从包含整个区域的粘粒或BAC中测序完整的基因簇。(3)在异源寄主黄色粘球菌中通过导入完整的异源双唑基因簇来生产异源双唑。这种微生物在大型发酵罐中生长得非常好，并拥有强大的遗传系统。这些目标的实现将使二唑醇A1的大量生产成为可能，以进一步探索其抗肿瘤性质并对其进行修饰，通过基因工程和化学合成改变化合物的化学结构来改善其药理性质。

DESCRIPTION (provided by applicant): The long-range goal of our research program is to discover novel disorazole analogs and to develop one of them into a useful antitumor drug. Although disorazole A is a potent cytotoxic agent, with IC50's below the nanomolar range (0.12 to 0.63 nM), novel compounds with good therapeutic index should be developed. In order to initiate a systematic program of chemical modifications on disorazole A, we will need unlimited amounts of the starting material. Sorangium cellulosum strain So ce12, the natural producer of disorazoles, only makes small amounts of this family of compounds (less than 100 mg/I) and it is a very slow growing microorganism (16 h doubling time). Moreover, the genetics of this bacterium has not yet been developed, which restricts enormously the possibility of generating analogs through genetic modification of the disorazole gene cluster. In order to break through these limitations we will pursuit the following Specific Aims. (1) Identify the disorazole biosynthetic genes by following a transposon-based approach. First, isolating non-producing mutants by HPLC analysis of the growth medium, then cloning and sequencing the genome sequences flanking the integrated transposon. Alternatively, we will identify the gene cluster by screening a cosmid or a BAC library with probes that recognize distinctive features of the disorazole genes. (2) Sequencing the complete gene cluster from a cosmid or a BAC containing the entire region. (3) Production of disorazole in the heterologous host Myxococcus xanthus by introducing the entire disorazole gene cluster. This microorganism grows very well in large fermenters and has a robust genetic system. Achievement of these objectives will enable the production of large amounts of disorazole A1 for further exploration of its antitumor properties and modification of them to improve the pharmacological properties by changing the chemical structure of the compound through genetic engineering and chemical synthesis.