title: Efficient microbial production of stylopine using a Pichia pastoris expression system.
abstract: Stylopine is a protoberberine-type alkaloid that has potential biological activities. Based on the successful microbial production of (S)-reticuline, we attempted to produce stylopine from (S)-reticuline by the reaction of berberine bridge enzyme, cheilanthifoline synthase (CYP719A5), and stylopine synthase (CYP719A2). Biosynthetic enzyme expression was examined in a methanol-utilizing yeast (Pichia pastoris), and both a "consolidated" system with all genes expressed in one cell and a "co-culture" system with three cell lines that each express a single gene were examined. Although both systems efficiently converted reticuline to stylopine, the consolidated system was more rapid and efficient than the co-culture system. However, substrate-feeding experiments revealed a decrease in the conversion efficiency in the consolidated system during successive cultures, whereas the conversion efficiency in the co-culture system remained constant. Thus, the final amount of stylopine produced from reticuline after successive feedings in the co-culture system was more than 150 nmoles from 750 nmoles of (R, S)-reticuline (375 nmoles of (S)-reticuline). The advantages and drawbacks of the "consolidated" system and the "co-culture" system are discussed.
authors.affiliation: Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Kitashirakawa, Sakyo, Kyoto 606-8502, Japan.
authors.forename: Kentaro
authors.initials: K
authors.lastname: Hori
authors.affiliation: Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Kitashirakawa, Sakyo, Kyoto 606-8502, Japan.
authors.forename: Shunsuke
authors.initials: S
authors.lastname: Okano
authors.affiliation: Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Kitashirakawa, Sakyo, Kyoto 606-8502, Japan.
authors.forename: Fumihiko
authors.initials: F
authors.lastname: Sato
mesh_terms: D001600:Berberine Alkaloids / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D002853:Chromatography, Liquid; D018920:Coculture Techniques; D005285:Fermentation; D015870:Gene Expression; D013058:Mass Spectrometry; D010843:Pichia / Q000235:genetics* / Q000378:metabolism*
chemical_list: D001600:Berberine Alkaloids; C014212:stylopine