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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)三萜合成酶基因的克隆和功能鉴定。通过基于同源性的PCRS，已经从不同高等植物中克隆了20多个具有不同产物特异性的三萜类合成酶cDNA。通过对酿酒酵母突变株转化子的产物分析，确定了它们的酶功能。构建了羽扇豆醇合成酶和β-香菇蛋白合成酶的嵌合体，以及一些点突变，确定了与其产物特异性有关的氨基酸残基。采用植物渗入法将反义羽扇豆醇合成酶DNA导入拟南芥。在平板上进行选择，然后在人造土壤中进一步生长。转基因植株T1代表现出明显的矮化现象。用LC-MS分析了T2代植株中的三萜烯含量，结果表明转基因植株中完全不含羽扇豆醇。这些结果与上述结果相结合，用这些三萜类合成酶cDNA进行转基因技术将导致药理活性皂苷的过度生产。(2)为开发新型烯二炔类抗肿瘤抗生素，克隆了两种不同类型的烯二炔类抗生素：动力霉素和新卡西宁。从产达尼米星的樱桃小单孢菌中克隆的60kb片段中，有24个开放阅读框存在于已测序的25kb区域。从产新卡津他汀链霉菌中还获得了I类、II类PKS基因克隆和脱氧糖生物合成基因克隆。利用阿克维酮生物合成基因的结合，可以生产出阿克维酮-新卡西宁杂交种。