利用表达来自大麻的Δ⁹-四氢大麻酚酸合酶的巴斯德毕赤酵母（Komagataella）全细胞从大麻二酚酸生产Δ⁹-四氢大麻酚酸
Production of Δ9-tetrahydrocannabinolic acid from cannabigerolic acid by whole cells of Pichia (Komagataella) pastoris expressing Δ9-tetrahydrocannabinolic acid synthase from Cannabis sativa L.
作者信息
Zirpel Bastian, Stehle Felix, Kayser Oliver
机构信息
Laboratory of Technical Biochemistry, Department of Biochemical & Chemical Engineering, TU Dortmund University, Emil-Figge-Str. 66, 44227, Dortmund, Germany.
出版信息
Biotechnol Lett. 2015 Sep;37(9):1869-75. doi: 10.1007/s10529-015-1853-x. Epub 2015 May 21.
OBJECTIVE
The Δ9-tetrahydrocannabinolic acid synthase (THCAS) from Cannabis sativa was expressed intracellularly in different organisms to investigate the potential of a biotechnological production of Δ9-tetrahydrocannabinolic acid (THCA) using whole cells.
RESULTS
Functional expression of THCAS was obtained in Saccharomyces cerevisiae and Pichia (Komagataella) pastoris using a signal peptide from the vacuolar protease, proteinase A. No functional expression was achieved in Escherichia coli. The highest volumetric activities obtained were 98 pkat ml(-1) (intracellular) and 44 pkat ml(-1) (extracellular) after 192 h of cultivation at 15 °C using P. pastoris cells. Low solubility of CBGA prevents the THCAS application in aqueous cell-free systems, thus whole cells were used for a bioconversion of cannabigerolic acid (CBGA) to THCA. Finally, 1 mM (0.36 g THCA l(-1)) THCA could be produced by 10.5 gCDW l(-1) before enzyme activity was lost.
CONCLUSION
Whole cells of P. pastoris offer the capability of synthesizing pharmaceutical THCA production.
目的
将来自大麻的Δ9 - 四氢大麻酚酸合酶（THCAS）在不同生物体中进行胞内表达，以研究利用全细胞生物技术生产Δ9 - 四氢大麻酚酸（THCA）的潜力。
结果
使用来自液泡蛋白酶A的信号肽，在酿酒酵母和巴斯德毕赤酵母（Komagataella pastoris）中实现了THCAS的功能性表达。在大肠杆菌中未实现功能性表达。使用巴斯德毕赤酵母细胞在15℃培养192小时后，获得的最高体积活性分别为98 pkat ml(-1)（胞内）和44 pkat ml(-1)（胞外）。CBGA的低溶解度阻碍了THCAS在无细胞水体系中的应用，因此使用全细胞将大麻二酚酸（CBGA）生物转化为THCA。最终，在酶活性丧失前，10.5 gCDW l(-1)的细胞可产生1 mM（0.36 g THCA l(-1)）的THCA。
结论
巴斯德毕赤酵母全细胞具备合成药用THCA的能力。
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