title: Metabolic engineering of cannabinoid biosynthesis in tobacco
Creators: name: Geißler, Marcus Frank
Publisher: name: UNSPECIFIED
Year: 2021

description: For millennia, Cannabis has been used in various cultural groups because of its versatile applications. Among other things, the plant served as a source of textile fibers, but was also used as a medicine to treat inflammation, cramps or epilepsy. With the discovery of the endocannabinoid system, the plant, which had fallen into disrepute in the mid-20th century because of its excessive use as a recreational drug and is therefore still subject to heavy restrictions in most countries, gained new prestige. Thus, it was found that certain secondary metabolites, known as cannabinoids, could modulate a variety of physiological processes, potentially offering new therapeutic applications. To date, more than 100 of these cannabinoids have been isolated, the best known of which are the psychoactive-acting Δ9 tetrahydrocannabinol (THC) and the non-psychotropic cannabidiol (CBD). In this context, THC- and CBD-containing products such as Sativex® and Epidyolex®, which are already approved as pharmaceuticals, are finding application in medicine. However, THC is considered a major limitation for clinical use due to its psychoactive character, so non-psychotropic cannabinoids, as well as synthetic non-naturally-occurring cannabinoids that have similar medicinal properties to THC, but do not have the undesirable side effect, will be of key importance in the future. Given that chemical production of synthetic cannabinoids is very costly, heterologous production in host organisms could provide a remedy, potentially leading to industrial-scale production of rare phytocannabinoids or novel synthetic cannabinoid pharmaceuticals not readily offered by cannabis plants. In this regard, the aim was to establish tobacco as an alternative host organism for the biosynthetic production of cannabinoids. It was shown that it was possible to produce all enzymes involved in cannabinoid biosynthesis in transiently transformed Nicotiana benthamiana plants and to detect their activity in vitro. Moreover, transient expression of aae1, ols, and oac and supplementation of hexanoic acid in vivo resulted in the formation of both, the cannabinoid precursor olivetolic acid (OA) and the new-to-nature C-4 OA glucoside. However, beyond the synthesis of OA, it was not yet possible to reconstruct the biosynthetic pathway in vivo, probably due to the lack of sufficient geranyl diphosphate (GPP) supply within tobacco plants. To enable efficient production of cannabinoids in the future, it is therefore essential to eliminate this bottleneck in the biosynthetic pathway. In addition to the transient approach, stably transformed liquid cell cultures were generated, which expressed the necessary genes for the production of OA or its glucoside. This should, with a view to use in industrial production, enable large-scale cultivation in bioreactors under GMP conditions. In this context, it was possible to integrate the individual genes into Nicotiana tabacum by stable transformation. However, in contrast to transient expression, neither the synthesis of OA nor its glucoside could be detected, most likely due to a mutation in the OLS gene that was used. The second part of the work dealt with the characterization of the cannabinoid-forming synthases Δ9 tetrahydrocannabinolic acid synthase (THCAS), cannabichromenic acid synthase (CBCAS) and cannabidiolic acid synthase (CBDAS). It was found that N-glycosylation and therefore localization of the proteins via the secretory pathway, either into the apoplast or vacuole, is required for the production of the enzymes in planta. In addition, in vitro experiments with THCAS and CBDAS showed that when organic solvents such as acetonitrile or acetone were added, the product specificity of the enzymes changed from Δ9 tetrahydrocannabinolic acid (THCA) and cannabidiolic acid (CBDA), respectively, to the synthesis of cannabichromenic acid (CBCA). This in turn indicates that, among other things, also the hydrophobic environment in the glandular trichomes of Cannabis could be responsible for the cannabinoid diversity in different Cannabis strains. Since CBCAS has a 93 % amino acid identity to THCAS but does not produce THCA, it stands to reason that small differences in amino acid sequence outside the catalytic center affect cyclization specificities. Therefore, final mutagenesis studies were performed with CBCAS and the goal of producing THCA to gain further insight into the catalytic mechanisms of the synthases. However, none of the so far introduced mutations resulted in the production of the desired cannabinoid, thus further site-directed mutagenesis have to be performed in the future, including the initially neglected amino acids.