Benzylisoquinoline alkaloids(BIAs)are a structurally diverse group of plant metabolites renowned for their pharmacological properties.However,sustainable sources for these compounds remain limited.Consequently,researc...Benzylisoquinoline alkaloids(BIAs)are a structurally diverse group of plant metabolites renowned for their pharmacological properties.However,sustainable sources for these compounds remain limited.Consequently,researchers are focusing on elucidating BIA biosynthetic pathways and genes to explore alternative sources using synthetic biology approaches.CYP80B,a family of cytochrome P450(CYP450)enzymes,plays a crucial role in BIA biosynthesis.Previously reported CYP80Bs are known to catalyze the 3′-hydroxylation of(S)-Nmethylcoclaurine,with the N-methyl group essential for catalytic activity.In this study,we successfully cloned a full-length CYP80B gene(St CYP80B)from Stephania tetrandra(S.tetrandra)and identified its function using a yeast heterologous expression system.Both in vivo yeast feeding and in vitro enzyme analysis demonstrated that St CYP80B could catalyze Nmethylcoclaurine and coclaurine into their respective 3'-hydroxylated products.Notably,St CYP80B exhibited an expanded substrate selectivity compared to previously reported wildtype CYP80Bs,as it did not require an N-methyl group for hydroxylase activity.Furthermore,St CYP80B displayed a clear preference for the(S)-configuration.Co-expression of St CYP80B with the CYP450 reductases(CPRs,StCPR1,and StCPR2),also cloned from S.tetrandra,significantly enhanced the catalytic activity towards(S)-coclaurine.Site-directed mutagenesis of St CYP80B revealed that the residue H205 is crucial for coclaurine catalysis.Additionally,St CYP80B 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.展开更多
文摘Benzylisoquinoline alkaloids(BIAs)are a structurally diverse group of plant metabolites renowned for their pharmacological properties.However,sustainable sources for these compounds remain limited.Consequently,researchers are focusing on elucidating BIA biosynthetic pathways and genes to explore alternative sources using synthetic biology approaches.CYP80B,a family of cytochrome P450(CYP450)enzymes,plays a crucial role in BIA biosynthesis.Previously reported CYP80Bs are known to catalyze the 3′-hydroxylation of(S)-Nmethylcoclaurine,with the N-methyl group essential for catalytic activity.In this study,we successfully cloned a full-length CYP80B gene(St CYP80B)from Stephania tetrandra(S.tetrandra)and identified its function using a yeast heterologous expression system.Both in vivo yeast feeding and in vitro enzyme analysis demonstrated that St CYP80B could catalyze Nmethylcoclaurine and coclaurine into their respective 3'-hydroxylated products.Notably,St CYP80B exhibited an expanded substrate selectivity compared to previously reported wildtype CYP80Bs,as it did not require an N-methyl group for hydroxylase activity.Furthermore,St CYP80B displayed a clear preference for the(S)-configuration.Co-expression of St CYP80B with the CYP450 reductases(CPRs,StCPR1,and StCPR2),also cloned from S.tetrandra,significantly enhanced the catalytic activity towards(S)-coclaurine.Site-directed mutagenesis of St CYP80B revealed that the residue H205 is crucial for coclaurine catalysis.Additionally,St CYP80B 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.
