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

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(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.