Department of Technical Biochemistry, TU Dortmund University, Emil-Figge Str. 66, 44227 Dortmund, Germany.
Center for Biotechnology (CeBiTec), Bielefeld University, Universitätsstr. 27, 33615, Bielefeld, Germany.
J Biotechnol. 2018 Apr 20;272-273:40-47. doi: 10.1016/j.jbiotec.2018.03.008. Epub 2018 Mar 13.
Δ-Tetrahydrocannabinolic acid (THCA) is a secondary natural product from the plant Cannabis sativa L. with therapeutic indications like analgesics for cancer pain or reducing spasticity associated with multiple sclerosis. Here, we investigated the influence of the co-expression of 12 helper protein genes from Komagataella phaffii (formerly Pichia pastoris) on the functional expression of the Δ-tetrahydrocannabinolic acid synthase (THCAS) heterologously expressed in K. phaffii by screening 21 clones of each transformation. Our findings substantiate the necessity of a suitable screening system when interfering with the secretory network of K. phaffii. We found that co-production of the chaperones CNE1p and Kar2p, the foldase PDI1p, the UPR-activator Hac1p as well as the FAD synthetase FAD1p enhanced THCAS activity levels within the K. phaffii cells. The strongest influence showed co-expression of Hac1s - increasing the volumetric THCAS activities 4.1-fold on average. We also combined co-production of Hac1p with the other beneficial helper proteins to further enhance THCAS activity levels. An optimized strain overexpressing Hac1s, FAD1 and CNE1 was isolated that showed 20-fold increased volumetric, intracellular THCAS activity compared to the starting strain. We used this strain for a whole cell bioconversion of cannabigerolic acid (CBGA) to THCA. After 8 h of incubation at 37 °C, the cells produced 3.05 g L THCA corresponding to 12.5% g g.
Δ-四氢大麻酸 (THCA) 是一种来自大麻属植物的次生天然产物，具有治疗作用，如治疗癌症疼痛的镇痛药或减少多发性硬化症相关的痉挛。在这里，我们研究了共表达来自 Komagataella phaffii（以前是毕赤酵母）的 12 种辅助蛋白基因对在 K. phaffii 中异源表达的 Δ-四氢大麻酸合酶 (THCAS) 的功能表达的影响，通过筛选每个转化的 21 个克隆。我们的研究结果证实了在干扰 K. phaffii 的分泌网络时需要合适的筛选系统。我们发现，伴侣蛋白 CNE1p 和 Kar2p、折叠酶 PDI1p、UPR 激活剂 Hac1p 以及 FAD 合成酶 FAD1p 的共生产增加了 K. phaffii 细胞内的 THCAS 活性水平。共表达 Hac1s 的影响最强，平均使 THCAS 活性增加了 4.1 倍。我们还将 Hac1p 的共生产与其他有益的辅助蛋白结合使用，以进一步提高 THCAS 活性水平。我们分离出一株过表达 Hac1s、FAD1 和 CNE1 的优化菌株，与起始菌株相比，其比体积 THCAS 活性提高了 20 倍。我们使用该菌株进行大麻素酸 (CBGA) 到 THCA 的全细胞生物转化。在 37°C 孵育 8 小时后，细胞产生了 3.05g/L 的 THCA，相当于 12.5%g/g。
