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

(1)三萜合成酶cdna的分子克隆与功能鉴定。利用同源pcr技术，从多种高等植物中克隆了20多个具有不同产物特异性的三萜合成酶cdna。通过对酿酒酵母突变体的转化体进行产物分析，确定了它们的酶功能，这些转化体携带具有各自cdna的表达质粒。从luppeol合成酶和β-amyrin合成酶和一些点突变体构建嵌合体，确定了其产物特异性的氨基酸残基。采用植物内浸润法将反义luphel合成酶DNA导入拟南芥。在培养皿上选择，然后在人工土壤中进一步生长。转基因植株的T1后代表现出明显的矮化。用LC-MS分析T2子代的三萜含量，发现转基因植株中完全不含lupeol。这些结果，结合以上所述，转基因技术与这些三萜合成酶cdna将导致过量生产的药理活性皂苷的发展。(2)为了开发一种新型的enedyne抗肿瘤抗生素，对dynemicin和neocarzinostatatin两种不同类型的enedyne抗生素进行了克隆。从产动力蛋白的chersina小单孢菌中克隆出60kb的区域，在25kb的区域中鉴定出了24个orf。从产新卡兹酸抑素链霉菌（Streptomyces carzinostaticus）中也获得了I型、II型PKS基因克隆和脱氧糖生物合成基因克隆。结合aklavinone生物合成基因，有望生产aklavinone-neocarzinostatin杂种。

(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.