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Development of New Bio-production System for Useful Chemicals by Engineering Biosynthetic Genes

通过工程生物合成基因开发有用化学品的新型生物生产系统

基本信息

批准号：
10358015
负责人：
EBIZUKA Yutaka
金额：
$ 19.9万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A).
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10358015/
关键词：
Triterpene Synthase Saponin Oxidosqualene Biosynthetic Gene Streptomycetes Enediyne Antibiotics Self Resistance 耐性

项目摘要

(1) Molecular cloning and functional identification of triterpene synthase cDNAs. By employing homology based PCRs, more than twenty triterpene synthase cDNAs with different product specificities have been cloned from various higher plants. Identification of their enzyme functions were established by product analysis of transformants of Saccharomyces cerevisiae mutant, which carry expression plasmids with respective cDNAs. Construction of chimeras from lupeol synthase and β-amyrin synthase and some point mutants identified amino acid residues responsible for their product specificities. Antisense lupeol synthase DNA was introduced into A.thaliana by means of in planta infiltration method. were selected on plates and further grown in artificial soil. T1 progeny of transgenic plants showed marked dwarfism. Triterpene contents in T2 progeny were analysed by LC-MS revealing the complete absence of lupeol in the transgenic plants. These results, combined with those described above, transgenic technology with these triterpene synthase cDNAs would lead to the development of overproducers of pharmacologically active saponins. (2) To develop a new enediyne antitumor antibiotic, cloning of two different types of enedyne antibiotics, dynemicin and neocarzinostatin, were carried out. Out of 60 kb region cloned from Micromonospora chersina, a dynemicin producer, 24 ORFs were identified in the so far sequenced 25 kb region. Type I, type II PKS gene clones, and deoxysugar biosynthetic gene clones were also obtained from neocarzinostatin producing Streptomyces carzinostaticus. With combination of aklavinone biosynthesis genes, prodution of aklavinone-neocarzinostatin hybrid could be expected.

(1)三萜合酶cDNA的分子克隆及功能鉴定。通过采用基于同源性的PCR，已经从各种高等植物中克隆了二十多种具有不同产物特异性的三萜合酶cDNA。通过对酿酒酵母突变体的转化体进行产物分析来鉴定它们的酶功能，所述突变体携带具有各自cDNA的表达质粒。从羽扇豆醇合酶和β-香树脂合酶以及一些点突变体构建嵌合体，鉴定了负责其产物特异性的氨基酸残基。采用植物内浸润法将反义羽扇豆醇合酶DNA导入拟南芥。在平板上选择并在人造土壤中进一步生长。转基因植物的T1后代表现出明显的矮化。通过 LC-MS 分析 T2 后代中的三萜含量，表明转基因植物中完全不存在羽扇豆醇。这些结果与上述结果相结合，使用这些三萜合酶 cDNA 的转基因技术将导致药理学活性皂苷过量生产者的开发。 (2)为了开发新型烯二炔类抗肿瘤抗生素，对两种不同类型的烯二炔类抗生素——达尼霉素和新制癌菌素进行了克隆。在从动力霉素生产者小单孢菌 (Micromonospora chersina) 克隆的 60 kb 区域中，在迄今为止测序的 25 kb 区域中鉴定出了 24 个 ORF。还从产生新制癌菌素的链霉菌中获得了I型、II型PKS基因克隆和脱氧糖生物合成基因克隆。结合阿克拉维酮生物合成基因，有望生产阿克拉维酮-新制癌菌素杂合体。