Transcriptome analysis of Stephania yunnanensis and functional validation of CYP80s involved in benzylisoquinoline alkaloids biosynthesis

The medicinal plant Stephania yunnanensis is rich in aporphine alkaloids, a type of benzylisoquinoline alkaloids (BIAs), with aporphine being the representative and most abundant compound, but our understanding on the biosynthesis of BIA alkaloids in this plant have been relatively limited. Previous research has reported the genome of S. yunnanensis and preliminarily identified the upstream gene norcoclaurine synthase (NCS) in the BIA biosynthetic pathways. However, the key genes promoting the formation of the aporphine skeleton have not yet been reported. In this study, based on the differences in the content of crebanine and several other BIAs in different tissues, we conducted transcriptome sequencing of roots, stems, and leaves. We then identified candidate genes through functional annotation and sequence alignment, followed by transcriptomic and genomic analyses. Based on this analysis, we identified three CYP80 enzymes (SyCYP80Q5-1, SyCYP80Q5-3, and SyCYP80G6), which exhibited different activities towards ( S )- and ( R )-configured substrates in S. yunnanensis and demonstrated strict stereoselectivity enroute to aporphine. This study provides metabolomic and transcriptomic information on the biosynthesis of BIAs in S. yunnanensis and offers valuable insights into the elucidation of BIA biosynthesis, and lays the foundation for the complete analysis of pathways for more aporphine alkaloids.