大麻素前体物2,4-二羟基-6-正庚基苯甲酸的生物合成及产量优化

Biosynthesis and yield improvement of cannabinoid precursor sphaerophorolcarboxylic acid

摘要:

背景 大麻素是植物的天然活性产物，是一种重要的临床药物。目前药学相关大麻素的生产仍然依赖植物，但植物生产效率低、周期长并且受安全等因素限制。利用生物方法合成大麻素及其类似物具有重要意义。 目的 在酿酒酵母(Saccharomyces cerevisiae) BJ5464-npgA中重构大麻素前体物2,4-二羟基-6-正庚基苯甲酸(sphaerophorolcarboxylic acid, SA)的生物合成途径，优化发酵培养基组分，利用生物合成途径高效生产SA。 方法 从罗伯茨绿僵菌(Matarhizium robertsii) ARSEF 23基因组扩增Mr_OvaA、Mr_OvaB、Mr_OvaC基因，分别构建到不同酵母-大肠杆菌穿梭质粒中，共同转化酿酒酵母BJ5464-npgA表达，获得酵母工程菌株CLB2。通过单因素试验及Plackett-Burman、最陡爬坡试验和响应面法设计优化发酵培养基，使用Design Expert 8.0对试验数据进行分析。 结果 酵母工程菌株CLB2可以合成63.75 mg/L SA。培养基各成分中影响SA产量的3个主要因素为：蔗糖、KH₂PO₄和维生素溶液，其最佳浓度分别为蔗糖7.26 g/L、KH₂PO₄·7H₂O 6.08 g/L、维生素溶液5.67 mL/L，预测产量最大值为93.15 mg/L，实际产量为93.75 mg/L，较优化前提高了47%。 结论 大麻素前体物SA可以在酿酒酵母中高效合成。试验获得的高产发酵培养基配方为后续SA及大麻素的研究提供了可靠的支持。

关键词:2,4-二羟基-6-正庚基苯甲酸;生物合成;Plackett-Burman;响应面法

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.

Key words:sphaerophorolcarboxylic acid (SA);biosynthesis;Plackett-Burman;response surface method