Subcellular localization defines modification and production of Δ 9 -tetrahydrocannabinolic acid synthase in transiently transformed Nicotiana benthamiana

• Published in: Biotechnology letters Vol. 40; no. 6; p. 981
• Main Authors: Geissler, Marcus, Volk, Jascha, Stehle, Felix, Kayser, Oliver, Warzecha, Heribert
• Format: Journal Article
• Language: English
• Published: Netherlands 01.06.2018
• Subjects: Cannabinoids - metabolism; Cannabis - enzymology; Cannabis - genetics; Intracellular Space - chemistry; Intracellular Space - enzymology; Intracellular Space - metabolism; Intramolecular Oxidoreductases - chemistry; Intramolecular Oxidoreductases - genetics; Intramolecular Oxidoreductases - metabolism; Metabolic Engineering - methods; Nicotiana - cytology; Nicotiana - enzymology; Nicotiana - genetics; Nicotiana - metabolism; Plant Proteins - chemistry; Plant Proteins - genetics; Plant Proteins - metabolism; Plants, Genetically Modified - genetics; Plants, Genetically Modified - metabolism; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Cannabinoids; Tobacco; Cannabis sativa L; Tetrahydrocannabinolic acid synthase (THCAS); Plant metabolic engineering; Modular cloning
• ISSN: 1573-6776

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.