Cloning and Characterization of Cheilanthifoline and Stylopine Synthase Genes from Chelidonium majus

The most prominent alkaloid of Chelidonium majus is dihydrocoptisine, revealing the characteristic benzophenanthridine skeleton. To date, any informationon on the enzymes responsible for its biosynthesis and the related genes in C. majus is lacking. Based on sequence similarities to the corresponding methylenedioxy bridge-forming Cyt P450 enzymes involved in isoquinoline alkaloid biosynthesis in Eschscholzia californica, genes for a cheilanthifoline synthase and a stylopine synthase from C. majus were isolated, sequenced and heterologously expressed in yeast. The activity of the heterologously expressed Cyt P450 enzymes was determined in situ as well as on the basis of microsomal fractions. It was shown that cheilanthifoline synthase (c8931) converts scoulerine into cheilanthifoline, the latter subsequently being converted to stylopine by the action of a stylopine synthase (c1128). Based on the well-known instability of stylopine, it can be assumed that in vivo-under the acidic conditions in the vacuole-this alkaloid is converted to dihydrocoptisine, which accumulates in C. majus leaves. Both methylenedioxy bridge-forming Cyt P450 enzymes from C. majus are characterized by their high substrate specificity. Apart from their genuine substrates, i.e. scoulerine and cheilanthifoline, cheilanthifoline synthase and stylopine synthase do not accept other substrates tested; the only alternative substrate identified was scoulerine, which is converted by stylopine synthase to yield minor amounts of nandinine. Quantitative real-time PCR revealed that the expression of cheilanthifoline synthase and stylopine synthase genes is very similar in both roots and leaves from C. majus, although the alkaloid accumulation patterns in these organs are quite different.