Biosynthesis and yield improvement of cannabinoid precursor sphaerophorolcarboxylic acid

Abstract:

Background Cannabinoids are natural active products of plants that serve as key clinical drugs. At present, the production of pharmaceutical cannabinoids still relies on plants, which has low yield, long cycles, and safety problems. Biosynthesis is of great significance to the production of cannabinoids and their analogues. Objective To reconstruct the biosynthetic pathway of the cannabinoid precursor sphaerophorolcarboxylic acid (SA) in Saccharomyces cerevisiae BJ5464-npgA and optimize the fermentation medium to increase the yield of SA. Methods Mr_OvaA, Mr_OvaB, and Mr_OvaC were amplified by PCR with the genomic DNA of Matarhizium robertsii ARSEF 23 as the template and respectively integrated into S. cerevisiae-Escherichia coli shuttle plasmids. All the three recombinant plasmids were transformed into S. cerevisiae BJ5464-npgA and the engineered strain CLB2 was obtained. Single factor experiments, Plackett-Burman design, method of steepest ascent, and response surface method were employed to optimize the fermentation medium. The experimental data were analyzed in Design Expert 8.0. Results The S. cerevisiae engineered strain CLB2 yielded 63.75 mg/L SA. The three main factors affecting SA yield were sucrose, KH₂PO₄, and vitamin solution. The optimized medium was composed of 7.26 g/L sucrose, 6.08 g/L KH₂PO₄·7H₂O, 5.67 mL/L vitamin solution, in which the predicted SA yield was 93.15 mg/L. The actual yield of SA in the optimized medium was 93.75 mg/L, which was 47% higher than that before optimization. Conclusion Cannabinoid precursor SA can be synthesized efficiently in S. cerevisiae. The fermentation medium formula for high production of SA in this study provides reliable support for the subsequent research on SA and cannabinoid.