The Research on the Production of Usefull Compounds by the Control of Regulations in the Biosynthesis of Secondary Metabolism

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(1) The genomic gene encoding chalcone synthase which was the key enzyme of flavonoid biosynthesis was cloned from cultured Pueraria lobata cells. And also the three genomic clones of reductase which was co-acting with chalcone synthase during the formation of deoxychalcone formation were isolated from P.lobata. Since chalcone synthase and reductase are acting together, the promoter regions of chalcone synthase and reductase genes are supposed to be similar. However they have no over-all similarity, but have several similar sequences in the length of about 20 bp. The purification of 2-hydroxyisoflavone dehydratase was achieved in this period and the cloning of its cDNA is proceeding.(2) The response of cultured P.lobata cells to elicitors such as yeast extracts, jasmonic acid and CuCl_2 was investigated at compound level, enzyme level and RNA level. The results clearly showed that yeast extracts and jasmonic acid responsed very fast at any level, on the other hand CuCl_2 responsed slowly.(3) The chimeric gene of the promoter region of horse radish peroxidase gene and Sarcotoxin cDNA was introduced to Tobacco plant using the Agrobacterium and Ti-Plasmid system to produce the plant having the anti-batcerial activity.(4) The gene encoding dihydrogeodin oxygenase which catalyzed intramolecular phenol oxidative coupling reaction was cloned from Aspergillus terreus. The coned gene was transformed to Aspergillus nidulans and expressed in the enzymatically active form in large scale.