Production of Cannabidiol Acid Synthase by Chassis Pichia pastoris and of Its Catalytic Activity Analysis
2023
Tingli NIU | Yuan TIAN | Liguo ZHANG | Shuquan ZHANG | Zhijiang LI | Yue KANG | Jian CHI | Baochang GAO | Lingyan DAI
In order to obtain the higher expression of cannabinoid acid synthase (CBDAS) through the microbial chassis, the recombinant enzyme for the in vitro synthesis of cannabinoid (CBD) was studied. The CBDAS gene was firstly cloned from cannabis leaves with higher content of CBD, its basic physicochemical properties of proteins were then analyzed by bioinformatics methods. After construction of the recombinant plasmid pPIC9K-CBDAS and transformation into Pichia pastoris, conditions of the recombinant protein CBDAS were optimized and its catalytic activities were finally analyzed. The results indicated that the constructed pPIC9K-CBDAS fusion protein expression system could be successfully expressed in P. pastoris. Under conditions of 1% of methanol addition, pH6.0 of medium, 48 h of induction, the maximum expression was obtained. At 4 h reaction, the product of cannabigerol acid (CBGA) was significantly increased, in which attributed to catalysis of substrate CBGA by the recombinant CBDAS. In the following, contents of CBD were significantly increased at 8 h (P<0.05) and those of CBDA and CBD were reached to the maximum at 12 h. After reactions at 12 h in the crude extract of cannabis leaf and the standardized CBGA, CBDAS could catalyze to product 60.64 and 20.12 ng/mL of CBDA, 128.01 and 207.87 ng/mL of CBD, respectively, in which CBDAS indicated a stronger catalytic activity. In conclusion, heterologous recombinant expression of cannabis CBDAS was achieved through yeast chassis, which provided active enzymes for the efficient synthesis of CBDA and CBD.
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