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
Aporphine alkaloids have diverse pharmacological activities;however,our understanding of their biosynthesis is relatively limited.Previous studies have classified aporphine alkaloids into two categories based on the c...Aporphine alkaloids have diverse pharmacological activities;however,our understanding of their biosynthesis is relatively limited.Previous studies have classified aporphine alkaloids into two categories based on the configuration and number of substituents of the D-ring and have proposed preliminary biosynthetic pathways for each category.In this study,we identified two specific cytochrome P450 enzymes(CYP80G6 and CYP80Q5)with distinct activities toward(S)-configured and(R)-configured substrates from the herbaceous perennial vine Stephania tetrandra,shedding light on the biosynthetic mechanisms and stereochemical features of these two aporphine alkaloid categories.Additionally,we characterized two CYP719C enzymes(CYP719C3 and CYP719C4)that catalyzed the formation of the methylenedioxy bridge,an essential pharmacophoric group,on the A-and D-rings,respectively,of aporphine alkaloids.Leveraging the functional characterization of these crucial cytochrome P450 enzymes,we reconstructed the biosynthetic pathways for the two types of aporphine alkaloids in budding yeast(Saccharomyces cerevisiae)for the de novo production of compounds such as(R)-glaziovine,(S)-glaziovine,and magnoflorine.This study provides key insight into the biosynthesis of aporphine alkaloids and lays a foundation for producing these valuable compounds through synthetic biology.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.
基金supported by the National Key R&D Program of China(2020YFA0908000)the National Natural Science Foundation of China(82011530137,31961133007)+2 种基金Scientific and technological innovation project of CACMS(CI2023D002,CI2023E002)Key project at central government level:The ability to establish sustainable use of valuable Chinese medicine resources(2060302)Vetenskapsradet(2018-06003),Stiftelsen for internationalisering av hogre utbildning och forskning。
文摘Aporphine alkaloids have diverse pharmacological activities;however,our understanding of their biosynthesis is relatively limited.Previous studies have classified aporphine alkaloids into two categories based on the configuration and number of substituents of the D-ring and have proposed preliminary biosynthetic pathways for each category.In this study,we identified two specific cytochrome P450 enzymes(CYP80G6 and CYP80Q5)with distinct activities toward(S)-configured and(R)-configured substrates from the herbaceous perennial vine Stephania tetrandra,shedding light on the biosynthetic mechanisms and stereochemical features of these two aporphine alkaloid categories.Additionally,we characterized two CYP719C enzymes(CYP719C3 and CYP719C4)that catalyzed the formation of the methylenedioxy bridge,an essential pharmacophoric group,on the A-and D-rings,respectively,of aporphine alkaloids.Leveraging the functional characterization of these crucial cytochrome P450 enzymes,we reconstructed the biosynthetic pathways for the two types of aporphine alkaloids in budding yeast(Saccharomyces cerevisiae)for the de novo production of compounds such as(R)-glaziovine,(S)-glaziovine,and magnoflorine.This study provides key insight into the biosynthesis of aporphine alkaloids and lays a foundation for producing these valuable compounds through synthetic biology.
