AbstractStylopine 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.