Structure-function Analysis of CYP719As Involved in Methylenedioxy Bridge-Formation in the Biosynthesis of Benzylisoquinoline Alkaloids and Its De Novo Production.
Microbial cell factories(2023)
Abstract
Benzylisoquinoline alkaloids (BIAs) are a type of secondary metabolite with clinical application value. ( S )-stylopine is a special BIA which contains methylenedioxy bridge structures. CYP719As could catalyze the methylenedioxy bridge-formation on the A or D rings of protoberberine alkaloids, while displaying significant substrate regiospecificity. To explore the substrate preference of CYP719As, we cloned and identified five CyCYP719A candidates from Corydalis yanhusuo . Two CyCYP719As (CyCYP719A39 and CyCYP719A42) with high catalytic efficiency for the methylenedioxy bridge-formation on the D or A rings were characterized, respectively. The residues (Leu 294 for CyCYP719A42 and Asp 289 for CyCYP719A39) were identified as the key to controlling the regioselectivity of CYP719As affecting the methylenedioxy bridge-formation on the A or D rings by homology modeling and mutation analysis. Furthermore, for de novo production of BIAs, CyCYP719A39, CyCYP719A42, and their mutants were introduced into the ( S )-scoulerine-producing yeast to produce 32 mg/L ( S )-stylopine. These results lay a foundation for understanding the structure-function relationship of CYP719A-mediated methylenedioxy bridge-formation and provide yeast strains for the BIAs production by synthetic biology.
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Key words
CyCYP719As,Benzylisoquinoline alkaloids (BIAs),Corydalis yanhusuo,Methylenedioxy bridge-formation,Regiospecificity,Synthetic biology
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