Disorazole Gene cluster

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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.