Subcellular localization defines modification and production of Δ9-tetrahydrocannabinolic acid synthase in transiently transformed Nicotiana benthamiana

Geissler, Marcus; Volk, Jascha; Stehle, Felix; Kayser, Oliver; Warzecha, Heribert

doi:10.1007/s10529-018-2545-0

Cited by 17 publications

(17 citation statements)

References 14 publications

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“…The geranyl diphosphate (GPP) used for prenylation of olivetolic acid (OA) originates from the methylerythritol 4-phosphate (MEP) pathway, which typically operates in plastidial organelles in eukaryotic cells (normally chloroplasts) [96]. Oxidative cyclization and storage of the final products take place outside the gland cells in the resin cavity [97,98]. It remains to be resolved how the pathway intermediates are transported between the different compartments.…”

Section: Cannabinoid Biosynthesis In Cannabis Sativamentioning

confidence: 99%

Phytocannabinoids: Origins and Biosynthesis

Gülck

Møller

2020

Trends in Plant Science

239

240

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Phytocannabinoids are bioactive natural products found in some flowering plants, liverworts, and fungi that can be beneficial for the treatment of human ailments such as pain, anxiety, and cachexia. Targeted biosynthesis of cannabinoids with desirable properties requires identification of the underlying genes and their expression in a suitable heterologous host. We provide an overview of the structural classification of phytocannabinoids based on their decorated resorcinol core and the bioactivities of naturally occurring cannabinoids, and we review current knowledge of phytocannabinoid biosynthesis in Cannabis, Rhododendron, and Radula species. We also highlight the potential in planta roles of phytocannabinoids and the opportunity for synthetic biology approaches based on combinatorial biochemistry and protein engineering to produce cannabinoid derivatives with improved properties. Phytocannabinoids The term phytocannabinoid (see Glossary, also cannabinoid) defines meroterpenoids with a resorcinyl core typically decorated with a para-positioned isoprenyl, alkyl, or aralkyl side chain [1]. The alkyl side chain typically contains an odd number of carbon atoms, where orcinoids contain one carbon, varinoids three, and olivetoids five. Cannabinoids with an even number of carbon atoms in the side chain are known but rare. The term cannabinoid generally refers to molecules with a characteristic chemical structure; however, the term may also refer to pharmacological ligands of human endocannabinoid receptors [2]. In this review we use the chemical definition of cannabinoids. Phytocannabinoids occur in flowering plants, liverworts, and fungi (Figure 1, Key Figure). They were first isolated from Cannabis sativa L. (Cannabaceae), a plant with a long and controversial history of use and abuse [3]. The mammalian brain has receptors that respond to compounds found in C. sativa. Accordingly, these receptors were named cannabinoid receptors (CB x) and are the basis of the endocannabinoid system. Studies in human and animals demonstrated that the endocannabinoid system regulates a broad range of biological functions, including memory, mood, brain reward systems, and drug addiction, as well as metabolic processes such as lipolysis, glucose metabolism, and energy balance [2]. More than 113 different cannabinoids have been isolated from C. sativa and these are classified into distinct types: cannabigerols (CBGs), cannabichromenes (CBCs), cannabidiols (CBDs), (−)-Δ 9-trans-tetrahydrocannabinols (Δ 9-THCs), (−)-Δ 8-trans-tetrahydrocannabinols (Δ 8-THCs), cannabicyclols (CBLs), cannabielsoins (CBEs), cannabinols (CBNs), cannabinodiols (CBNDs), cannabitriols (CBTs), and the miscellaneous cannabinoids (Figure 2) [4]. C. sativa predominantly produces alkyl type cannabinoids that carry a monoterpene isoprenyl moiety (C10) and a pentyl side chain (C5) [1]. The most abundant constituents are trans-Δ 9-THC, CBD, CBC, and CBG, together with their respective acid forms (Δ 9-THCA, CBDA, CBCA, and CBGA) [5]. Cannabinoid biosynthetic pathways ty...

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Section: Cannabinoid Biosynthesis In Cannabis Sativamentioning

confidence: 99%

Phytocannabinoids: Origins and Biosynthesis

Gülck

Møller

2020

Trends in Plant Science

239

240

View full text Add to dashboard Cite

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“…These enzymes do not accept CBG as a substrate, since the interaction of carboxylate group with a histidine and an adjacent tyrosine residue is important for substrate binding as demonstrated by site directed mutagenesis and reported crystal structures [31]. Albeit forming the respective product as their name suggests, all three enzymes were shown to form small amounts of other cannabinoids when tested in vitro and in vivo, which was also influenced by pH [36,72,74]. The major by-product of THCAS is CBCA (ca.…”

Section: Fad-depended Cyclases Form Various Cannabinoidsmentioning

confidence: 99%

“…This is supported by the fact that none of the BBE-like enzymes have been expressed as an active enzyme in E. coli, a prokaryote with an N-glycosylation process fundamentally different from that of eukaryotes. Expression of the recombinant proteins in tobacco was also only possible when they were tagged for secretion or endoplasmic reticulum (ER) localization, whereas the deletion of the signal peptides or replacement of a chloroplast targeting signal peptide did not yield detectable proteins [74]. However, the N-glycans are not needed for enzyme activity after protein maturation, since deglycosylated THCAS still showed comparable activity in vitro [39].…”

Section: Fad-depended Cyclases Form Various Cannabinoidsmentioning

confidence: 99%

“…Such localization is perhaps evolutionarily preferential, as the hydrophobic environment in the glandular trichome storage cavity allows optimal enzymatic activity and accumulation of the hydrophobic products to concentrations that are cytotoxic to plant cells [76]. When expressed in tobacco leaves, the recombinant proteins were also detected in the chloroplast in addition to the apoplast [74]. Additionally, the signal peptide is cleaved in the mature enzyme.…”

Section: Fad-depended Cyclases Form Various Cannabinoidsmentioning

confidence: 99%

“…Expression of these cyclases has been demonstrated using insect Spodoptera frugiperda (Sf9) cell culture [50], yeast (K. phaffii [71], S. cerevisiae [37,40]), plant (N. benthamiana [38,50,74], and also claimed in a number of patents and patent applications. In all above systems, active enzymes can be produced without complications.…”

Section: Fad-depended Cyclases Form Various Cannabinoidsmentioning

confidence: 99%

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The Biochemistry of Phytocannabinoids and Metabolic Engineering of Their Production in Heterologous Systems

Blatt-Janmaat

2021

IJMS

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The medicinal properties of cannabis and the its legal status in several countries and jurisdictions has spurred the massive growth of the cannabis economy around the globe. The value of cannabis stems from its euphoric activity offered by the unique phytocannabinoid tetrahydrocannabinol (THC). However, this is rapidly expanding beyond THC owing to other non-psychoactive phytocannabinoids with new bioactivities that will contribute to their development into clinically useful drugs. The discovery of the biosynthesis of major phytocannabinoids has allowed the exploration of their heterologous production by synthetic biology, which may lead to the industrial production of rare phytocannabinoids or novel synthetic cannabinoid pharmaceuticals that are not easily offered by cannabis plants. This review summarizes the biosynthesis of major phytocannabinoids in detail, the most recent development of their metabolic engineering in various systems, and the engineering approaches and strategies used to increase the yield.

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