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Molecular insigths into the biosynthesis of cannabinoids

Oliver Kayser

Cannabinoids are terpenophenolics characterized by benzochromen ring system and synthesized from olivetolic acid and geranyldiphosphate. Besides of Delta9 tetrahydro-cannabinolic acid (THCA) more than 100 other cannabinoids are biosynthesized mainly in trichomes located on leaves and with high density on flowers of C. sativa [1]. Biosynthetically, cannabinoids are prenylated polyketides derived from the polyketide and mevalonate pathway delivering olivetolic acid and geranyl diphosphate, respectively. Biosynthetic precursor of the first committed  metabolite towards a high diversiy of cannabinoids is cannabigerolic acid (CBGA) being formed by a C-C Friede-Craft alkylation of olivetolic acid (OA) in the position C10b. It is likely that olivetolic acid is biosynthesized by a candidate type III polyketide synthase (PKS) called olivetol synthase (OLS) and olivetolic acid cyclases, but detailed steps in this biosynthetic ring formation step are still unclear [2]. Towards tetrahydrocannabinolic acid (THCA) the oxidocyclase tetrahydrocannabinolic acid synthase (TCAS) is the responsible converting enzyme.

Based on laser dissection micorsopy, LC-MS and cryo-NMR the cannabinoid profile and metabolome of dissected head and stem cells was analyszed and semi-quantified [3, 4]. Here, in all cells cannabinoids and related biosynthetic precursors were identified. Additional Imaging-MS revealed that THCA is located in basal trichome cells in equal concentrations as abundant in studied head cells (Fig. 1a). Applying recent CARS microscopy (Fig. 1b), these results document and confirm distribution of cannbionids over stalked trichomes and give new insights about trafficking and localization. On the molceular biology level, transcriptome and proteome were studied. We were able to describe a network pattern, that gives first insight into biosynthetic organization of relevant enzymes like OA cyclase, CGA and THCA Synthase.

In our ongoing research, we studied metallic pattern of genuine essential oil by direct extraction (Fig. 1c). Essential oil was analyzed for abundance of proteins compared to metabolic pattern of water steam distilled extracs.



[1) Elsohly et al., 2009, Recent Pat CNS Drug Discov. 4:112-36

[2] Gagne et al., 2012, Proc Natl Acad Sci USA. 2012 Jul 31; 109(31):12811-6

[3] Happyana et al., 2013, Phytochemistry. 87:51-59.

[4] Happyana et al., 2016, Planta Med. 82:1217-1223

[5] Zirpel et al., 2015, Biotechnol Lett. 37:1869-75

Exploring the synthesis of cannabinoids in medicinal Cannabis - A qualitative and quantitative proteomic analysis of laser-microdissected trichomes in an LC-MS-based bottom up approach

Nizar Happyana, Stefan Loroch, Albert Sickmann, Oliver Kayser

Introduction: The Cannabis sativa trichomes are of high interest in biomedical research since they are the main site of cannabinoid production. More than 100 cannabinoids have been identified and structurally elucidated, like the prominent psychoactive tetrahydrocannabinolic acid (THCA) and cannabidiolic acid (CBDA). Although the distribution of the cannabinoid metabolites is well studied [1], the very complex biosynthesis pathway is only sparsely understood, not at least, because of the limited available information on the Cannabis proteome.

Aim of this work: In a shotgun proteomics approach we want to quantify the known key players of the cannabinoid synthesis pathway in Cannabis trichomes and identify new proteins via sequence similarity searches.

Methods: Extracted proteins of laser-microdissected Cannabis trichomes were digested with trypsin and separated in a 2d approach, using high-pH reversed phase and low-pH reversed phase HPLC coupled to an Orbitrap benchtop instrumetd (U3000 RSLCnano, QExactive; Thermo Scientific). Mascot database search was performed against Uniprot - Cannabaceae (~500 proteins) in a Pyrococcus furiosus backround for false discovery rate estimation [2].

Results: Using 2d-HPLC separation, we were able to identify 39 Cannabis-related proteins including the major key players of cannabinoid synthesis like the tetrahydrocannabinolic acid (THCA) synthase, the olivetolic acid cyclase and the cannabidiolic acid (CBDA) synthase. In a second experiment we could show significant qualitative and quantitative differences in the trichome proteome of four Cannabis varieties. Combining these data with metabolic profiling will lead to a deeper understanding of the cannbinoid synthesis and therefore be of increased interest in research of bioactive compounds.

[1] Happyana et al., Phytochemistry. 2013, 87: 51-59.

[2] Vaudel et al., Proteome Res. 2012, 11 (10): 5065-71


Biochemical fingerprinting of endophytes harbored in Radula marginata that confer plant fitness benefits

Parijat Kusari1, Souvik Kusari2, Michael Spiteller2, Oliver Kayser1

1Department of Biochemical and Chemical Engineering, Chair of Technical Biochemistry, Technical University Dortmund

2Institute of Environmental Research (INFU) of the Faculty of Chemistry, Technical University Dortmund


Our work focusses on the assessment and elucidation of the cost-benefit interactions of a special group of microorganisms known as endophythes which inhabit the internal tissues of the host plants without causing immediate negative effect, and remain in a mutualistic association for at least a part of their life cycle. Plants are known to produce various bioactive secondary metabolites as defensive compounds. Cannabinoids are most extensively studied secondary metabolits of Cannabis sativa L. plants. Recent work on liverworts like Radula marginata led to the identification of new cannabinoids with structural similarity to tetrahydrocannabinol, the major psychoactive compound of Cannabis plants. We have isolated a plethora of endophytes, both fungi and bacteria, from R. marginata. Since both Radula and Cannabis contain similar biosynthetic principles, we are evaluating the biocontrol potential of the endophytes against the host specific phytopathogens of C. sativa L. plants namely, Botrytis cinerea (causing gray mold disease) and Trichothecium roseum (causing pink rot disease), respectively.

We are investigating the various attack-defense-conterdefense responses of the isolated endophytes when challenged by the phytopathogens. These responses trigger the production of secondary metabolites or intermediates which are otherwise "cryptic". We are not only analyzing the varios cost-benefit tradeoffs between the endophytes and host plant but also evaluating the bioactive target and/or non-target metabolite production correlating to the endophyte-pathogen interactions. This will enable us in understanding the biochemical fingerprint of the endophytes which aid in thwarting the phytopathogens and reducing the loss of such therapeutically beneficial plants.


[1] Kusari et al. (2012) Fungal Divers. In Press (doi. 10.1007/s13225 - 012-0216-3)

[2] Kusari et al. (2013) In: Kharwar R.N. (ed.) Endophytes, (ICPMB 2012), Springer-Verlag, Heidelberg. In Press.



Monitoring metabolites production and cannabinoids analysis in medicinal Cannabis trichomes during flowering period by 1H NMR-based metabolomics

Nizar Happyana, Oliver Kayser

Department of Biochemical and Chemical Engineering, Chair of Technical Biochemistry, Technical University Dortmund


Cannabis trichomes are known as the main site for producing cannabinoids, the responsible compound for most biological activities of the plant. In this work, production of metabolites, especially cannabinoids in the triochomes of four medicinal Cannabis varieties, Bediol, Bedica, Bedrobinol, and Bedrocan during the last four weeks of flowering period was studied using 1H NMR-based metabolomics. The trichomes samples were extracted and fractionated into chloroform extracts and water extracts. Totally six cannabinoids, THCA (1), CBDA (2), CBCA (3), CBGA (4), THC (5), and CBD (6) were identified  in the chloroform extracts of all trichomes, while 20 compounds including sugar, amino acids, and other acidic compounds were detected in all water extracts. All identified cannabinoids were quantified with 1H NMR method. The concentrations of total cannabinoids in Bedrocan and Bedica trichomes increased from week five till week seven and then decreased significantly at the week eight. Meanwhile, the cannabinoids production in the Bedrobinol and Bediol trichomes incresased during the monitoring time. Different metabolites profiles within trichomes varieties were revealed by PLSDA models of metabolomics. Important differential metabolites in the discrimination were THCA (1) and CBDA (2) on the chloroform extracts, and were asparagine, choline, fructose, and glucose on the water extracts. Furthermore PLSDA models classified the trichomes of every variety based on their harvested weeks. THCA (1) was found as an important discriminant compound in the chloroform extracts of every variety. Meanwhile, threonine, asparagine and fructose were detected as differential metabolites in the water extracts for every variety. This study indicated that Cannabis triochomes during flowering period produced metabolites in different amounts depending on the time and the plant variety. Furthermore it is the first report of monitoring metabolites producition in plant trichomes using 1H NMR-based metabolomics.