A CYP80B enzyme from Stephania tetrandra enables the 3'-hydroxylation of N-methylcoclaurine and coclaurine in the biosynthesis of benzylisoquinoline alkaloids

Benzylisoquinoline alkaloids (BIAs) are a structurally diverse group of plant metabolites known for their pharmacological properties. However, the availability of sustainable sources for these compounds remains limited. Consequently, researchers are focusing on unraveling BIA biosynthetic pathways and genes to explore alternative sources using synthetic biology approaches. CYP80B, a family of CYP450 enzymes, plays a pivotal role in BIA biosynthesis. Previously reported CYP80Bs are known to catalyze the 3'-hydroxylation of (S)-N-methylcoclaurine, with the N-methyl group essential for catalytic activity. In this study, we successfully cloned a full-length CYP80B gene (StCYP80B) from Stephania tetrandra and identified its function using a yeast heterologous expression system. Both in vivo yeast feeding and in vitro enzyme analysis demonstrated that StCYP80B could catalyze N-methylcoclaurine and coclaurine into their respective 3′-hydroxylated products. Notably, StCYP80B exhibited an expanded substrate selectivity compared to previously reported wild-type CYP80Bs, as it did not require an N-methyl group for hydroxylase activity. Furthermore, StCYP80B displayed a clear preference for the (S)-configuration. Co-expression of StCYP80B with the CYP450 reductases (StCPR1 and StCPR2), also cloned from S. tetrandra, significantly enhanced the catalytic activity towards (S)-coclaurine. Site-directed mutagenesis of StCYP80B revealed that the residue H205 is crucial for coclaurine catalysis. Additionally, StCYP80B exhibited tissue-specific expression in plants. This study provides new genetic resources for the biosynthesis of BIAs and further elucidates their synthetic pathway in natural plant systems.