Disorazole Gene cluster

描述（由申请人提供）：我们研究项目的长期目标是发现新的二索拉唑类似物，并将其中一种开发成有用的抗肿瘤药物。虽然二唑唑A是一种强效的细胞毒性药物，IC50低于纳摩尔范围（0.12 ~ 0.63 nM），但仍需开发具有良好治疗指标的新化合物。为了对二唑唑a进行系统的化学修饰，我们需要无限量的原料。纤维素Sorangium cellulosum菌株So ce12是二唑类化合物的天然生产者，它只能产生少量的二唑类化合物（小于100 mg/I），并且是一种生长非常缓慢的微生物（16 h加倍时间）。此外，这种细菌的遗传学尚未开发，这极大地限制了通过对二索拉唑基因簇进行遗传修饰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)采用转座子方法鉴定二唑类生物合成基因。首先，通过高效液相色谱分析培养基分离非产生突变体，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cosmid或BAC文库来鉴定基因簇，这些文库具有识别二唑基因的独特特征的探针。(2)从包含整个区域的cosmid或BAC中测序完整的基因簇。(3)通过引入整个二索拉唑基因簇，在异源宿主黄粘球菌中产生二索拉唑。这种微生物在大型发酵罐中生长得非常好，并且具有强大的遗传系统。这些目标的实现将使二索拉唑A1的大量生产成为可能，从而进一步探索其抗肿瘤特性并对其进行修饰，通过基因工程和化学合成改变化合物的化学结构来提高其药理特性。

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.