Cannabis sativa L. is an important herbaceous species originating from Central Asia, which has been used in folk medicine and as a source of textile fiber since the dawn of times. This fast-growing plant has recently seen a resurgence of interest because of its multi-purpose applications: it is indeed a treasure trove of phytochemicals and a rich source of both cellulosic and woody fibers. Equally highly interested in this plant are the pharmaceutical and construction sectors, since its metabolites show potent bioactivities on human health and its outer and inner stem tissues can be used to make bioplastics and concrete-like material, respectively. In this review, the rich spectrum of hemp phytochemicals is discussed by putting a special emphasis on molecules of industrial interest, including cannabinoids, terpenes and phenolic compounds, and their biosynthetic routes. Cannabinoids represent the most studied group of compounds, mainly due to their wide range of pharmaceutical effects in humans, including psychotropic activities. The therapeutic and commercial interests of some terpenes and phenolic compounds, and in particular stilbenoids and lignans, are also highlighted in view of the most recent literature data. Biotechnological avenues to enhance the production and bioactivity of hemp secondary metabolites are proposed by discussing the power of plant genetic engineering and tissue culture. In particular two systems are reviewed, i.e., cell suspension and hairy root cultures. Additionally, an entire section is devoted to hemp trichomes, in the light of their importance as phytochemical factories. Ultimately, prospects on the benefits linked to the use of the -omics technologies, such as metabolomics and transcriptomics to speed up the identification and the large-scale production of lead agents from bioengineered Cannabis cell culture, are presented.

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Cannabis sativa: The Plant of the Thousand and One Molecules

https://pubs.rsc.org/en/Content/ArticlePDF/2016/NP/C6NP00074F

Phytocannabinoids: a unified critical inventory.

Cannabis sativa L. has been cultivated and used around the globe for its medicinal properties for millennia1. Some cannabinoids, the hallmark constituents of Cannabis, and their analogues have been investigated extensively for their potential medical applications2. Certain cannabinoid formulations have been approved as prescription drugs in several countries for the treatment of a range of human ailments3. However, the study and medicinal use of cannabinoids has been hampered by the legal scheduling of Cannabis, the low in planta abundances of nearly all of the dozens of known cannabinoids4, and their structural complexity, which limits bulk chemical synthesis. Here we report the complete biosynthesis of the major cannabinoids cannabigerolic acid, Δ9-tetrahydrocannabinolic acid, cannabidiolic acid, Δ9-tetrahydrocannabivarinic acid and cannabidivarinic acid in Saccharomyces cerevisiae, from the simple sugar galactose. To accomplish this, we engineered the native mevalonate pathway to provide a high flux of geranyl pyrophosphate and introduced a heterologous, multi-organism-derived hexanoyl-CoA biosynthetic pathway5. We also introduced the Cannabis genes that encode the enzymes involved in the biosynthesis of olivetolic acid6, as well as the gene for a previously undiscovered enzyme with geranylpyrophosphate:olivetolate geranyltransferase activity and the genes for corresponding cannabinoid synthases7,8. Furthermore, we established a biosynthetic approach that harnessed the promiscuity of several pathway genes to produce cannabinoid analogues. Feeding different fatty acids to our engineered strains yielded cannabinoid analogues with modifications in the part of the molecule that is known to alter receptor binding affinity and potency9. We also demonstrated that our biological system could be complemented by simple synthetic chemistry to further expand the accessible chemical space. Our work presents a platform for the production of natural and unnatural cannabinoids that will allow for more rigorous study of these compounds and could be used in the development of treatments for a variety of human health problems.

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Complete biosynthesis of cannabinoids and their unnatural analogues in yeast

Cannabis sativa has been employed for thousands of years, primarily as a source of a stem fiber (both the plant and the fiber termed “hemp”) and a resinous intoxicant (the plant and its drug preparations commonly termed “marijuana”). Studies of relationships among various groups of domesticated forms of the species and wild-growing plants have led to conflicting evolutionary interpretations and different classifications, including splitting C. sativa into several alleged species. This review examines the evolving ways Cannabis has been used from ancient times to the present, and how human selection has altered the morphology, chemistry, distribution and ecology of domesticated forms by comparison with related wild plants. Special attention is given to classification, since this has been extremely contentious, and is a key to understanding, exploiting and controlling the plant. Differences that have been used to recognize cultivated groups within Cannabis are the results of disruptive selection for characteristics selected by humans. Wild-growing plants, insofar as has been determined, are either escapes from domesticated forms or the results of thousands of years of widespread genetic exchange with domesticated plants, making it impossible to determine if unaltered primeval or ancestral populations still exist. The conflicting approaches to classifying and naming plants with such interacting domesticated and wild forms are examined. It is recommended that Cannabis sativa be recognized as a single species, within which there is a narcotic subspecies with both domesticated and ruderal varieties, and similarly a non-narcotic subspecies with both domesticated and ruderal varieties. An alternative approach consistent with the international code of nomenclature for cultivated plants is proposed, recognizing six groups: two composed of essentially non-narcotic fiber and oilseed cultivars as well as an additional group composed of their hybrids; and two composed of narcotic strains as well as an additional group composed of their hybrids.

Evolution and Classification of Cannabis sativa (Marijuana, Hemp) in Relation to Human Utilization

https://uknowledge.uky.edu/cgi/viewcontent.cgi?article=1077&context=pss_facpub

Challenges towards Revitalizing Hemp: A Multifaceted Crop

Δ9-Tetrahydrocannabinol (THC) and other cannabinoids are responsible for the psychoactive and medicinal properties of Cannabis sativa L. (marijuana). The first intermediate in the cannabinoid biosynthetic pathway is proposed to be olivetolic acid (OA), an alkylresorcinolic acid that forms the polyketide nucleus of the cannabinoids. OA has been postulated to be synthesized by a type III polyketide synthase (PKS) enzyme, but so far type III PKSs from cannabis have been shown to produce catalytic byproducts instead of OA. We analyzed the transcriptome of glandular trichomes from female cannabis flowers, which are the primary site of cannabinoid biosynthesis, and searched for polyketide cyclase-like enzymes that could assist in OA cyclization. Here, we show that a type III PKS (tetraketide synthase) from cannabis trichomes requires the presence of a polyketide cyclase enzyme, olivetolic acid cyclase (OAC), which catalyzes a C2–C7 intramolecular aldol condensation with carboxylate retention to form OA. OAC is a dimeric α+β barrel (DABB) protein that is structurally similar to polyketide cyclases from Streptomyces species. OAC transcript is present at high levels in glandular trichomes, an expression profile that parallels other cannabinoid pathway enzymes. Our identification of OAC both clarifies the cannabinoid pathway and demonstrates unexpected evolutionary parallels between polyketide biosynthesis in plants and bacteria. In addition, the widespread occurrence of DABB proteins in plants suggests that polyketide cyclases may play an overlooked role in generating plant chemical diversity.

https://www.pnas.org/content/pnas/109/31/12811.full.pdf

Identification of olivetolic acid cyclase from Cannabis sativa reveals a unique catalytic route to plant polyketides.

The FAD2-like desaturases comprise a group of membrane-bound oxygenases involved in the modification of fatty acyl groups in plants and fungi. This group includes typical oleate desaturases which introduce a Δ12 cis double bond and more unusual enzymes such as Crep1, an acetylenase from the plant Crepis alpina, which introduces a triple bond in linoleate at the Δ12 position. In this study, the structure−function relationship between FAD2-like acetylenases and desaturases was examined through site-directed mutagenesis and heterologous expression. Eleven amino acid positions were identified that show complete evolutionary conservation within acetylenases or desaturases but have different amino acids in the other class of enzyme. Point mutants in Crep1 were constructed and expressed in yeast to test the role in fatty acid modification of the amino acids at the 11 positions. Results indicate the importance of five amino acid positions within Crep1 with regard to desaturase and acetylenase chemoselectivity, st...

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Steve J. Gagne

Papers

Identification of olivetolic acid cyclase from Cannabis sativa reveals a unique catalytic route to plant polyketides.

Structural control of chemoselectivity, stereoselectivity, and substrate specificity in membrane-bound fatty acid acetylenases and desaturases.