Poster: Secondary Metabolism

Abs # 282: Molecular characterization of isoquinoline alkaloid biosynthesis in Coptis japonica cells

Presenter:	Sato, Fumihiko , fumihiko@kais.kyoto-u.ac.jp
Authors	Sato, Fumihiko  (A)   Kato, Nobuhiko  (A)   Dubouzet, Emilyn  (A)   Morishige, Takashi  (A)
Affiliations:	(A): Grad. Sch. Biostudies, Kyoto University

The biosynthetic pathway to berberine, anti-microbial isoquinoline alkaloid, has been clarified. Of 13 enzyme steps from two molecules of L-tyrosine to one molecule of berberine, we have isolated 7 cDNAs such as norcoclaurine 6-O-methyltransferase (6OMT); 3'-hydroxy-N-methylcoclaurine 4'-O-methyltransferase (4'OMT); coclaurine N-methyltransferase (CNMT), etc from high berberine producing cultured Coptis japonica cells (e.g., ref. 1). We report here the molecular basis of benzylisoquinoline alkaloid biosynthesis in C. japonica. First, we characterized the expression of biosynthetic genes in several lines of C. japonica with different alkaloid productivity. A high berberine producing cell line 156-S showed quite high expression of all biosynthetic genes examined. On the other hand, CjY, non-selected cell line with low berberine production showed low expression of all genes examined. Interestingly, Cj8, a sister line of high producing line which have reduced productivity, showed relatively high expression of examined biosynthetic genes. These data suggested that whole transcriptional activation in berberine biosynthesis occurred in 156-S, and not in CjY. Whereas the situation in Cj8 was little complicated, we speculated that some early biosynthetic step(s) might block the biosynthesis. Thus, we have characterized the EST profile in high berberine producing cells. Sequence analysis of ESTs isolated clearly showed that the ESTs contained high portion of secondary metabolite related genes. Further characterization revealed the cell-specific expression profiles with different alkaloid productivity. The regulatory network in berberine biosynthesis will also be discussed. 1) Choi, K.-B., et al., J. Biol. Chem., 277: 830-835 (2002)