Numerous metabolites from Cannabis sativa L. are interesting drug targets or lead compounds for the development of semisynthetic derivatives. To uncouple targeted cannabinoid production from a THC-producing plant, a biotechnological production system with defined products would be advantageous 1.
Here we describe the engineering of late biosynthetic genes into Nicotiana benthamiana Domin to evaluate biosynthetic capacity in a transient expression system. With THCA synthase, we were able to show that active enzyme can be obtained after ER/apoplast-targeting 2. Moreover, THCAS seems to be glycosylated in N. benthamiana, suggesting that this modification has an influence on the stability of the protein. Activity assays with cannabigerolic acid as substrate showed that the recombinant enzyme not only produces THCA (123 ± 12 fkat g- 1 FW activity towards THCA production) but also cannabichromenic acid (CBCA; 31 ± 2.6 fkat g- 1 FW activity towards CBCA production). Moreover, we achieved the specific prenylation of olivetolic acid to form cannabigerolic acid by the introduction of an orthologous microbial gene. This preliminary work shows that N. benthamiana could be a suitable host for cannabinoid production, but towards whole pathway integration careful analysis of subcellular localization is necessary.
1 Schachtsiek, J., Warzecha, H., Kayser, O., Stehle, F. (2018) Current Perspectives on Biotechnological Cannabinoid Production in Plants. Planta Med. 84, 214-220.
2 Geissler, M., Volk, J., Stehle, F., Kayser, O., Warzecha, H. (2018) Subcellular localization defines modification and production of Δ9-tetrahydrocannabinolic acid synthase in transiently transformed Nicotiana benthamiana. Biotech. Lett. (in press)