权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Cloning and Analysis of Tautomycin Biosynthetic Genes

互变霉素生物合成基因的克隆与分析

基本信息

批准号：
08456062
负责人：
UBUKATA Makoto
金额：
$ 4.93万
依托单位：
Toyama Prefectural University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1998
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08456062/
关键词：
tautomycin cloning PCR polyketide gene disruption 遺伝子破壊生合成遺伝子 tautomycin 放線菌

项目摘要

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

旋转霉素(TM)是从螺旋链霉菌的发酵液中分离出来的一种蛋白磷酸酶1和2 A的抑制剂。由于TM及其类似物有望成为阐明哺乳动物细胞信号转导的有用探针，我们对聚酮化合物TM的绝对结构、构象分析、构效关系和生物合成进行了研究。在本研究中，我们对TM生物合成基因的克隆和分析进行了研究。这里我报告了1996年、1997年和1998.1年的实验结果。转化体系的建立：利用已报道的其他链霉菌原生质体形成和再生方法，实现了TM产生菌的原生质体形成和再生。在上述实验的基础上，通过对隐形质粒产生者进行热激处理，建立了转化体系。用pIJ702表达载体构建了2×10ィイD15ィエD_1转化子/μgDNA，获得了较好的转化效率，…并对转化体系的各种条件进行了优化。推测的TM生物合成基因的克隆：根据已发表的PKS Type I序列设计KS和AT引物，用于聚合酶链式反应策略以获得产物。该设计策略成功地从螺旋藻基因组DNA中扩增出一个片段，并在粘粒pWE15上扩增出一个PKS。对扩增产物的序列分析表明，我们已在TM生产菌中克隆到一个PKS基因标志。推测的TM生物合成基因的基因破坏：为了确定从PCR策略获得的PKS基因片段是否真的编码参与TM生物合成的PKS，然后将克隆的1.8kb区域重组插入到染色体上相应的区域，用于基因破坏。对得到的15株菌株进行发酵和检测TM的存在。虽然这些菌株都没有产生TM，但所有菌株都产生了另一种抗生素Xanthostatin。基因组Southern杂交分析表明，这些菌株中的TM生物合成基因从基因组DNA中缺失。较少