Cloning and Analysis of Tautomycin Biosynthetic Genes

Tautomycin (TM) isolated from a culture broth of Streptomyces spiroverticillatus is an inhibitor ofprotein phosphatases 1 and 2a .自TM and its analogues are expected to be有用的probes forelucidating the signal trasduction of mammalian cells, we studied on the absolute structureconformational analysis, structure-activity relationshipand biosynthesis of the polyketide compound， TM. In the present study，我们已经研究了TM biosynthetic genes的cloning and analysis . Here I report the results ofexperiments done in 1996, 1997和1998.1. Development of transformation system：Protoplast formation and regeneration of the TM producer were achieved by using the reportedprocedure for其他Streptomyces sp. In addition to above experiments，转换系统was established by curing the producer of the cryptic plasmid by a heatshock treatment. The satisfactory efficiency of 2x10 . D15 . D1 transformants/μgDNA was obtained使用pIJ702 plasmid， with the result th…我们optimized various conditions of the transformation system.2. Cloning of the More at we optimized various conditions of the transformation system.2putative TM biosynthetic gene：The KS and AT primers, which were designed from The published sequences in PKS Type I，在PCR产品中使用PCR策略用于obtain products.原始设计策略是成功的identifying a fragment from S. spiroverticillatus genomic DNA and a PKS on cosmid pWE15. Sequenceanalysis of the PCR product strongly suggests that we have cloned a PKS gene flagment in the TM3. Gene disruption of the putative TM biosynthetic Gene：To determine whether the PKS gene fragment obtained from PCR strategy indeed encodes PKS involvingin TM biosynthesis，the fragments were then used for gene disruption by recombinational insertion of the cloned 1.8 kbregion into the corresponding region on the chromosome. the resulting 15 strains were fermented andassayed for the presence of TM. Although none of the strains produced TM，an another antibiotic xanthostatin was produced by all strains. Genomic Southern hybridizationanalyses showed that TM biosynthetic genes in these strains deleted from the genomic DNA. Less

Tautomycin (TM) isolated from a culture broth of Streptomyces spiroverticillatus is an inhibitor of protein phosphatases 1 and 2A. Since TM and its analogues are expected to be useful probes for elucidating the signal trasduction of mammalian cells, we studied on the absolute structure, conformational analysis, structure-activity relationship, and biosynthesis of the polyketide compound, TM. In the present study, we have studied on the cloning and analysis of TM biosynthetic genes. Here I report the results of experiments done in 1996, 1997 and 1998.1. Development of transformation system: Protoplast formation and regeneration of the TM producer were achieved by using the reported procedure for other Streptomyces sp. In addition to above experiments, the transformation system was established by curing the producer of the cryptic plasmid by a heat shock treatment. The satisfactory efficiency of 2 x 10ィイD15ィエD1 transformants/μgDNA was obtained using the pIJ702 plasmid, with the result th … More at we optimized various conditions of the transformation system.2. Cloning of the putative TM biosynthetic gene: The KS and AT primers, which were designed from the published sequences in PKS Type I, were used in PCR strategy to obtain products. The primer design strategy was successful in identifying a fragment from S. spiroverticillatus genomic DNA and a PKS on cosmid pWE15. Sequence analysis of the PCR product strongly suggests that we have cloned a PKS gene flagment in the TM producer.3. Gene disruption of the putative TM biosynthetic gene: To determine whether the PKS gene fragment obtained from PCR strategy indeed encodes PKS involving in TM biosynthesis, the fragments were then used for gene disruption by recombinational insertion of the cloned 1.8 kb region into the corresponding region on the chromosome. The resulting 15 strains were fermented and assayed for the presence of TM. Although none of the strains produced TM, an another antibiotic xanthostatin was produced by all strains. Genomic Southern hybridization analyses showed that TM biosynthetic genes in these strains were deleted from the genomic DNA. Less