Characterizafion of gene network in isoquinoline alkaloid biosynthesis and its application

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1. Isolation and characterization of biosynthetic enzyme genes(1) Identification and characterization of norcoclaurine synthase and tetrahydroberberine oxidase in berberine biosynthesis from Coptis japonica(2) Identification and characterization of corytuberine synthase (CYP80G2) from Coptis japonica in magnoflorine biosynthesis(3) Identification and characterization of stylopine synthase (CYP719A2/A3) from California poppy in sanguinarine biosynthesis2. Metabolic engineering of isoquinoline alkaloid biosynthesis using biosynthetic genes isolated(1) Increase of the isoquinoline alkaloid production by the overexpression of rate-limiting enzyme gene; e.g., overexpression of norcoclaurine 6-O-methyltransferase isolated from Coptis japonica in California poppy cells(2) Accumulation of intermediate by the metabolic block using gene silencing with RNAi technology; e.g., reticuline accumulation in transgenic California poppy cells transformed with RNAi vector for berberine bridge enzyme(3) Pr … More oduction of novel metabolites by the introduction of new branch pathway ; e.g., introduction of scoulerine 9-O- methyltransferase isolated from Coptis japonica in California poppy cells. Modification of metabolite profiles in transgenic cells has been characterized with LC-MS, LC-NMR.(4) Increase of isoquinolie alkaloid accumulation by the overexpression of biosynthetic gene (3'-hydroxy N-methylcoclaurine 4' O-methyltransferase) was determined in transgenic Coptis japonica plants.3. Characterization of transcriptional regulation system in isoquinoline alkaloid biosynthesis(1) Development of novel screening system to isolate transcriptional factor genes using transient RNAi in Coptis japonica protoplasts.(2) Identification of WRKY1 and bHLH1 as the comprehensive gene regulator specific for isoquinoline alkaloid biosynthesis in Coptis japonica cells(3) Isolation of promoter sequences for some biosynthetic genes in isoquinoline alkaloid biosynthesis in Coptis japonica and characterization of promoter activities. Minimum promoter sequences for the expression in Coptis japonica protoplasts were identified.4. Reconstruction of isoquinoline alkaloid biosynthesis in microbe(1) Using isolated biosynthetic genes, isoquinoline alkaloid biosynthetic pathway from dopamine to reticuline was reconstructed in Escherichia coli.(2) Using budding yeast with CYP80G2 gene and above transgenic E. coli, magnoflorine was produced from dopamine Less