Geissler Marcus, Volk Jascha, Stehle Felix, Kayser Oliver, Warzecha Heribert
Plant Biotechnology and Metabolic Engineering, Technische Universität Darmstadt, Schnittspahnstraße 4, 64287, Darmstadt, Germany.
Laboratory of Technical Biochemistry, Department of Biochemical and Chemical Engineering, TU Dortmund University, Emil-Figge-Str. 66, 44227, Dortmund, Germany.
Biotechnol Lett. 2018 Jun;40(6):981-987. doi: 10.1007/s10529-018-2545-0. Epub 2018 Apr 4.
Through heterologous expression of the tetrahydrocannabinolic acid synthase (THCAS) coding sequence from Cannabis sativa L. in Nicotiana benthamiana, we evaluated a transient plant-based expression system for the production of enzymes involved in cannabinoid biosynthesis.
Thcas was modularized according to the GoldenBraid grammar and its expression tested upon alternative subcellular localization of the encoded catalyst with and without fusion to a fluorescent protein. THCAS was detected only when ER targeting was used; cytosolic and plastidal localization resulted in no detectable protein. Moreover, THCAS seems to be glycosylated in N. benthamiana, suggesting that this modification might have an influence on the stability of the protein. Activity assays with cannabigerolic acid as a substrate showed that the recombinant enzyme produced not only THCA (123 ± 12 fkat g activity towards THCA production) but also cannabichromenic acid (CBCA; 31 ± 2.6 fkat g activity towards CBCA production).
Nicotiana benthamiana is a suitable host for the generation of cannabinoid producing enzymes. To attain whole pathway integration, careful analysis of subcellular localization is necessary.
通过在本氏烟草中异源表达来自大麻的四氢大麻酚酸合酶（THCAS）编码序列，我们评估了一种基于植物的瞬时表达系统用于生产参与大麻素生物合成的酶。
根据GoldenBraid语法对Thcas进行模块化，并在编码催化剂的不同亚细胞定位条件下（有无与荧光蛋白融合）测试其表达。仅当使用内质网靶向时检测到THCAS；胞质和质体定位未检测到蛋白质。此外，THCAS似乎在本氏烟草中发生了糖基化，表明这种修饰可能对蛋白质的稳定性有影响。以大麻二酚酸为底物的活性测定表明，重组酶不仅产生了THCA（对THCA产生的活性为123±12 fkat g），还产生了大麻色烯酸（CBCA；对CBCA产生的活性为31±2.6 fkat g）。
本氏烟草是生产大麻素合成酶的合适宿主。为实现整个途径的整合，有必要仔细分析亚细胞定位。
