文献类型： 外文期刊

作者： Dai, Lingyan ¹ ; Niu, Tingli ¹ ; Luo, Ruijie ¹ ; Zhang, Liguo ² ; Zhang, Shuquan ² ; Kang, Yue ¹ ; Chi, Jian ¹ ; Feng, Xinlei ¹ ; Shi, Jiazhuo ¹ ; Tian, Yuan ³ ; Gao, Baochang ³ ; Li, Zhijiang ⁴ ;

作者机构： 1.Heilongjiang Bayi Agr Univ, Coll Life Sci & Technol, Daqing 163319, Heilongjiang, Peoples R China

2.Heilongjiang Acad Agr Sci, Inst Ind Crop, Harbin 150086, Heilongjiang, Peoples R China

3.Heilongjiang Acad Sci, Inst Phytochem, Daqing Branch, Daqing 163319, Heilongjiang, Peoples R China

4.Heilongjiang Bayi Agr Univ, Coll Food Sci, Daqing 163319, Heilongjiang, Peoples R China

关键词： Cannabidiolic acid synthase; Cannabidiol; Rational design; Pichia pastoris; Enzyme activity

期刊名称：INDUSTRIAL CROPS AND PRODUCTS （ 影响因子：5.9； 五年影响因子：6.0 ）

ISSN： 0926-6690

年卷期： 2024 年 208 卷

页码：

收录情况： SCI

摘要： Cannabidiol (CBD), a nonhallucinogenic but therapeutic compound, is widely used in fields such as medicine, food, health products, and cosmetics. Cannabidiolic acid synthase (CBDAS) can exclusively catalyze the conversion of cannabigerolic acid (CBGA), which is the common precursor of cannabinoids, to CBDA, which is then decarboxylated to form CBD. In order to determine which amino acids in the CBDAS protein undergo mutations that can significantly enhance the catalytic activity of the enzyme, molecular docking and experimental validation were performed in this study. The CBDAS gene was cloned from a high-CBD-content hemp germplasm; this 1635 base pair gene encodes 544 amino acids. Saturation and alanine scanning mutagenesis and combinatorial mutation analysis showed that a single amino acid mutation (C176W) and three dual mutations (H69G+H114L, H114L+C176W and G183V+N482W) in CBDAS reduced the binding energy of the mutant for the substrate CBGA. The molecular docking results indicated that for the mutants CBDAS(H114L+C176W) and CBDAS(G183V+N482W), the interaction force with CBGA was increased, the bond distance was shortened, and some amino acids that interacted with the substrate were also altered compared with the WT. For the mutants CBDAS(C176W) and CBDAS(H69G+H114L), compared with the WT, the interaction force with CBGA was decreased, or the interaction was eliminated. The results of enzyme catalytic activity measurement showed that compared with the WT, the catalytic activities of CBDAS(G183V+N482W) and CBDAS(H114L+C176W) were significantly increased, and that of CBDAS(H69G+H114L) was significantly decreased (P < 0.05), which was generally consistent with the expected results from molecular docking. The crude leaf extract, when used as the substrate, produced more CBDA and CBD than an equal concentration of the CBGA standard. Obviously, it is possible that the recombinant CBDAS produced with yeast mutants can be used for secondary production of CBD in the process of cannabinoid extraction with the remaining CBGA in the leaves as the substrate. The research results provide a theoretical basis for the molecular modification of other cannabinoid synthetases.