State Key Laboratory of Dao-Di Herbs, National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, No.16 Neinanxiaojie, Dongzhimen, Beijing, 100700, China.
School of Pharmaceutical Sciences, Henan University of Chinese Medicine, No. 156 Jinshuidong Road, Zhengzhou, 450046, China.
Microb Cell Fact. 2023 Feb 3;22(1):23. doi: 10.1186/s12934-023-02024-2.
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.
苯并异喹啉生物碱（BIAs）是一种具有临床应用价值的次生代谢产物。（S）-阿朴啡碱是一种特殊的 BIAs，它含有亚甲二氧基桥结构。CYP719As 可以催化原小檗碱类生物碱 A 或 D 环上的亚甲二氧基桥形成，同时表现出显著的底物区域特异性。为了探索 CYP719As 的底物偏好性，我们从延胡索中克隆并鉴定了 5 种 CyCYP719A 候选物。两种 CyCYP719As（CyCYP719A39 和 CyCYP719A42）对 D 或 A 环上的亚甲二氧基桥形成具有较高的催化效率，分别进行了特征描述。通过同源建模和突变分析，鉴定出（CyCYP719A42 的 Leu294 和 CyCYP719A39 的 Asp289）残基是控制 CYP719As 区域选择性的关键，影响 A 或 D 环上的亚甲二氧基桥形成。此外，为了从头生物合成 BIAs，将 CyCYP719A39、CyCYP719A42 及其突变体引入（S）-白屈菜碱产生酵母中，产生 32mg/L 的（S）-阿朴啡碱。这些结果为理解 CYP719A 介导的亚甲二氧基桥形成的结构-功能关系奠定了基础，并为 BIAs 的生物合成提供了酵母菌株。
