A study on the directed engineering and multiple transformations of cannabidiolic acid synthase to enhance the expression level of the recombinant enzyme.

Lingyan Dai; Jishun Lin; Yue Kang; Jian Chi; Ning Cao; Yuke Zhang; Panjie Li

doi:10.1007/s00203-026-05097-w

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A study on the directed engineering and multiple transformations of cannabidiolic acid synthase to enhance the expression level of the recombinant enzyme.

Lingyan Dai·Jishun Lin·Yue Kang·Jian Chi·Ning Cao·Yuke Zhang·Panjie Li

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Abstract

To increase the activity of cannabidiolic acid synthase (CBDAS) and its expression levels in yeast, this study focused on the CBDASG183V-N482W mutant. Using computer-aided techniques and literature reviews, four mutation sites were further identified, resulting in the mutant CBDASH114E-S116A-C176Y-G183V-N328Q-N482W. The CBDAS gene was integrated into the Pichia pastoris genome via multiple transformation rounds, and relative enzyme activity was analyzed using high-performance liquid chromatography. The results of the molecular docking analysis revealed factors such as increased intermolecular forces, shorter bond lengths, and an increased number of amino acid-substrate interaction sites, which may have contributed to the enhanced catalytic activity of the mutant. The concentrations of CBDA and CBD produced by the CBDASH114E-S116A-C176Y-G183V-N328Q-N482W mutant were 71.543 ng/mL and 75.163 ng/mL, respectively, which were 11.87% and 11.53% greater than those produced by the CBDASG183V-N482W mutant. The recombinant CBDAS strain obtained after two consecutive transformations of the CBDASH114E-S116A-C176Y-G183V-N328Q-N482W vector presented the highest CBDAS expression levels and CBD and CBDA yields; compared with those obtained after a single transformation, the CBDA and CBD yields increased by 9.77% and 12.65%, respectively. In addition, the tolerance of the recombinant strain to induction culture conditions was analyzed, revealing that the strain could be induced to express the protein at temperatures ranging from 20 to 45 °C and at pH values ranging from 3 to 9, with optimal expression observed at 30 °C and pH 6. These findings provide theoretical and technical support for the production of enzyme preparations for the in vitro-directed biosynthesis of cannabidiol.

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Molecular Docking Simulation Mutation Recombinant Proteins Saccharomycetales Intramolecular Oxidoreductases Protein Engineering Cannabidiol Cannabidiol acid synthase Cannabis Continuous transformation Molecular docking Tolerance Journal Article

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Lingyan Dai, Jishun Lin, Yue Kang, Jian Chi, Ning Cao, Yuke Zhang, Panjie Li. 2026-08-29. A study on the directed engineering and multiple transformations of cannabidiolic acid synthase to enhance the expression level of the recombinant enzyme.. https://doi.org/10.1007/s00203-026-05097-w

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