the biosynthesis of cannabinoids...16 2. the biosynthesis of cannabinoids i. setting the scene,...

C H A P T E R

13

Handbook of Cannabis and Related Pathologies. http://dx.doi.org/10.1016/B978-0-12-800756-3.00002-8Copyright © 2017 Elsevier Inc. All rights reserved.

2The Biosynthesis of Cannabinoids

F. Degenhardt, F. Stehle, O. KayserLaboratory of Technical Biochemistry, Department of Biochemical and Chemical Engineering,

TU Dortmund University, Dortmund, Germany

SUMMARY POINTS

• ThischapterfocusesonthepathwaywhichleadstothebiosynthesisofphytocannabinoidsinC. sativaL.

• CBGAisthecentralprecursorofphytocannabinoidbiosynthesisinCannabis.

• CBGAS,onlythreeenzymes—THCAS,CBDAS,andCBCAS—areinvolvedinthebiosynthesisofphytocannabinoidsinCannabisplants.

• SequencesofCBDASandTHCASareknown.• ThecarboxylgroupinCBGAseemstobe

essentialfortheenzymaticreactionscatalyzedbyCBDAS,CBCAS,andTHCAS.

• Thediversityofmorethan60cannabinoidsistheresultofnonenzymaticmodifications.

• Propylcannabinoidsoccurbytheprenylationofdivarinicacid(DA)withgeranyldiphosphate(GPP).

LIST OF ABBREVIATIONS

AAE Acyl-activatingenzymeBBE BerberinebridgeenzymeCBC CannabichromeneCBCA CannabichromenicacidCBCAS CannabichromenicacidsynthaseCBCVA CannabichrovarinicacidCBD CannabidiolCBDA CannabidiolicacidCBDAS CannabidiolicacidsynthaseCBDV CannabidivarinCBDVA CannabidivarinicacidCBG CannabigerolCBGA Cannabigerolicacid(3-geranylolivetolate)CBGAS CannabigerolicacidsynthaseCBGVA CannabigerovarinicacidCBN CannabinolCBNRA Cannabinerolicacid(cis-CBGA)CHS ChalconesynthaseCsAAE1 C. sativahexanoyl-CoAsynthetase1CsAAE3 C. sativahexanoyl-CoAsynthetase2CsHCS1 C. sativahexanoyl-CoAsynthetase1CsHCS2 C. sativahexanoyl-CoAsynthetase2DA DivarinicacidDABB Dimericα+βbarrelDMAPP Dimethylallyldiphosphate

KEY FACTS OF PHYTOCANNABINOIDS—BESIDES C. SATIVA• Phytocannabinoidsareplant-derivednaturalcompounds

thatactasligandstocannabinoidreceptors(CB1andCB2)orsharechemicalsimilaritywithcannabinoids.

• C. sativaL.isintensivelyinvestigatedforthepresenceofphytocannabinoids.Todate,onlyafewplantsarediscoveredthatcontainphytocannabinoidsotherthantheonesknownfromCannabis.

• TheNewZealandliverwortRadula marginataandJapaneseliverwortRadula perrottetiicontainperrotteti-nene,anaturallyoccurringbibenzylcannabinoid.

• Twocannabigerol-likecompoundsweredetectedintheaerialpartsofHelichrysum umbraculigerumLess.,aplantcommonintheeasternpartsofSouthAfrica.

• N-alkylamides(cannabinomimetics),foundinthemedicinalplantsEchinaceae angustifoliaandEchinaceae purpurea(purplecornflower),areknowntointeractwiththeCB2receptor.

14 2. The BiosynThesis of CannaBinoids

I. SeTTINGTHeSCeNe,BoTANICAL,GeNerALANDINTerNATIoNALASPeCTS

DOXP 1-Deoxy-d-xylulose-5-phosphateGOT Geranylpyrophosphate:olivetolate

geranyltransferaseGPP GeranyldiphosphateHTAL HexanoyltriaceticacidlactoneIPP IsopentenyldiphosphateMEP 2C-methyl-d-erythritol-4-phosphateMVA MevalonateNPP NeryldiphosphateOA olivetolicacidOAC olivetolicacidcyclaseOLS olivetolsynthasePKS PolyketidesynthaseSNP SinglenucleotidepolymorphismSTS StilbenesynthaseTHC TetrahydrocannabinolTHCA TetrahydrocannabinolicacidTHCAS TetrahydrocannabinolicacidsynthaseTHCV TetrahydrocannabivarinTHCVA Tetrahydrocannabivarinicacid

INTRODUCTION

Cannabis sativa L. (hemp) is one of the oldest do-mestic plants in the history of mankind, and has beencultivated for at least 10,000 years (Schultes, Klein,Plowman,&Lockwood,1974).TogetherwithHumulus lupulus (hop), C. sativa belongs to the small family ofCannabaceae.Cannabis isanannual,usuallydioecious,wind-pollinatedherb,withbothmaleandfemaleflow-ersgrowingonseparateplants.Theplantiswellknown

forthebiosynthesisofcannabinoids,theterpenopheno-licconstituentsthatshowpsychoactiveeffects.Butsinceotherplantsalsohavesecondarymetabolitesthatinter-actwiththehumancannabinoidreceptors,anewdefini-tionhadtobemade.Hence,phytocannabinoidsarenowdefined as any plant-derived natural compound thatcanactasaligandtohumancannabinoidreceptors(CB1andCB2)orsharechemicalsimilaritywithcannabinoids(Gertsch,Pertwee,&DiMarzo,2010). Interestingly,allpartsoftheCannabisplant,withtheexceptionofseeds,can contain cannabinoids,but theymainlyaccumulatein the glandular trichomes of female flowers (Gagneetal.,2012;vanBakeletal.,2011).

The following chapter focuses on the pathway thatleadstotheenzymaticbiosynthesisofcannabinoids.Fora long time, it was postulated that the key intermedi-ate iscannabidiol(CBD)orcannabidiolicacid(CBDA),both resulting from a condensation of a monoterpene,andolivetolorolivetolicacid(oA),respectively.In1964,Gaoni and Mechoulam postulated cannabigerol (CBG)asthekeyintermediate,thecondensationproductofge-ranyldiphosphate(GPP),andolivetoloroA.Basedonthis,theyconcludedthatthecannabinoidsCBD,tetrahy-drocannabinol(THC)andcannabinol(CBN)areallde-rivedfromCBG,andjustdifferinthewayofcyclization(Gaoni&Mechoulam,1964).Finally,incorporationstud-ieswith 13C-labeledglucosehave shown thatGPPandoAareindeedtheprecursorsforformationofcannabig-erolicacid(CBGA).Thus,thegeneralstructureofcanna-binoidsisassembledbytwoparts:(1)adiphenol(resor-cin)carryinganalkylchain(oA);and(2)amonoterpenemoiety (GPP) (Fig. 2.1). Subsequently, Fellermeier and

FIGURE 2.1 General structure of cannabinoids and their precursors, olivetolic acid, and geranyl diphosphate.Cannabinoidsarecomposedoftwoparts:acyclicmonoterpenepart(red),andadiphenol(resorcin)part,carryinganalkylchain(blue).Thedibenzopyran-numberingsystemisused.

CannaBinoid preCursor BiosynThesis 15


coworkerspostulatedCBGAasthecentralcannabinoidprecursor(Fellermeier,eisenreich,Bacher,&Zenk,2001;Fellermeier&Zenk,1998).Interestingly,freeoAhasnev-erbeendetectedinCannabisplantmaterialuntilnow.

Itisworthytonotethat,althoughmorethan60can-nabinoidsareknown,onlythreeenzymes,besidescan-nabigerolicacidsynthase(CBGAS),namelytetrahydro-cannabinolicacidsynthase(THCAS),cannabidiolicacidsynthase(CBDAS),andcannabichromenicacidsynthase(CBCAS),areinvolvedincannabinoidbiosynthesis.TheresultingacidiccannabinoidsarethemostabundantonesaccumulatinginCannabis.Theneutralandpsychoactiveforms are the results of nonenzymatic decarboxylationduringstorage,heatorsunlight;explainingtheheatingofplantmaterial(ie,smokingorbaking),duringCanna-bisconsumption(Fischedick,Hazekamp,erkelens,Choi,&Verpoorte,2010;Tauraetal.,2007a).Thus,thebroaddiversityofthedifferentcannabinoidsismainlyduetononenzymatic transformation or degradation of bothacidic and neutral cannabinoids by the effects of light(UVirradiation)andauto-oxidation(Crombie,Ponsford,Shani, Yagnitinsky, & Mechoulam, 1968; razdan,Puttick,Zitko,&Handrick,1972).Itisstillunclearifalltheseformsarepresentinlivingplantsasnaturalorar-tefacts,duetostorageandsamplepreparation(elSohly&Slade,2005).

CANNABINOID PRECURSOR BIOSYNTHESIS

Polyketide Pathway Toward Olivetolic Acid

The origin of hexanoate in trichomes has not beenelucidatedsofar.Suzuki,Kurano,esumi,Yamaguchi,andDoi (2003)showed that theside-chainmoietyofalkyl-resorcinolsisformedbyfattyacidunits,butitremainsunclearifthemoietyistheresultofbiosynthesisordeg-radationoffattyacids.Studiesregardingtheincorpora-tionof13C-labelsintocannabinoidsindicatethathexano-ateissynthesizedfromacetyl-CoAasastarterunit,andfive molecules of malonyl-CoA. These building blocksareprecursorsofthefattyacidbiosynthesis(Fellermeieretal.,2001).

Based on this, two pathways are feasibly possible,after analysis of a cDNA/eST library generated fromfemaleflowers(glands)ofC. sativa.First, thehexanoylresiduecouldbeobtainedbyanearlyterminationofthefattyacidbiosynthesis.Subsequently,thehexanoylmoi-etyoftheresultinghexanoyl-ACPwouldbecleavedbyathioesteraseortransferredtoCoAbyanACP-CoAtrans-acylase.Finally,acyl-CoAsynthetasewouldcatalyzetheconversionof theobtainedn-hexanol tohexanoyl-CoA(Marks et al., 2009). Second, n-hexanol could be pro-ducedbythebreakdownofC18unsaturatedfattyacids

viathelipoxygenasepathway(Marksetal.,2009;Stout,Boubakir,Ambrose,Purves,&Page,2012).Nevertheless,furtherstudiesarenecessarytoclarifytheoriginofthehexanolmoiety.

Hexanoyl-CoAisamedium-chainfattyacyl-CoAthatcan be detected in high amounts in Cannabis flowers(Stoutetal.,2012).Itissynthesizedbyanacyl-activatingenzyme(AAe)calledhexanoyl-CoAsynthetase(Markset al., 2009; Page & Stout, 2013). AAes can use short,medium, longaswellasverylong-chainfattyacidsascarboxylic acid substrates. Two novel enzymes wereidentified,C. sativahexanoyl-CoAsynthetase1(CsHCS1or CsAAe1) and C. sativa hexanoyl-CoA synthetase 2(CsHCS2 or CsAAe3) that are capable of producinghexanoyl-CoAusinghexanoateandCoAassubstrates.Based on transcript levels, CsHCS1 seems to be tri-chome-specific.Although CsHCS2 exhibits lower tran-scriptlevels,incomparisontoCsHCS1,itisabundantinalltissues.ThegeneofCsHCS1consistsofa2163-nucle-otideopenreadingframe,andencodesa720-aminoacidpolypeptidechain.ThegeneofCsHCS2iscomposedofa 1632-nucleotide open reading frame, and encodes a543-aminoacidpolypeptidechain.BothCsHCSsgener-allyrequiredivalentcationsforactivity.ThiswasshownbyaddingMg2+,Mn2+,andCo2+ to theenzymeassays.Thus,CsHCS1preferentiallyacceptsMg2+,andCsHCS2Co2+.Thehighestenzymeactivitywasdetectedat40°CandpH9forbothenzymes.Furthermore,bothenzymescanbeinhibitedbyhighconcentrationsofCoA(Page&Stout,2013;Stoutetal.,2012).

Taken together, the published data suggest thatCsHCS1 is theenzyme involved in thebiosynthesisofcannabinoids: (1) it is the most abundant AAe in tri-chomes;(2)itishighlyspecificforshort-chainfattyacyl-CoA,particularlyhexanoate(KMvalueinthenMrange);and(3)itislocalizedinthecytosol,assuggestedfortheolivetol synthase (see later). In contrast, CsHCS2 is lo-calizedintheperoxisomesandacceptsabroadrangeofsubstrates,while showingaKMvalue forhexanoate inthemMrange(Page&Stout,2013;Stoutetal.,2012).

The alkylresorcinol moiety of cannabinoids is de-rived from oA, the product of polyketide synthases(PKSs)thatcatalyzethealdolcondensationofhexanoyl-CoAwiththreemoleculesofmalonyl-CoA(Fellermeieretal.,2001;raharjo,Chang,Choi,Peltenburg-Looman,&Verpoorte,2004)(Fig.2.2).Thesecondprecursormal-onyl-CoA is predominantly derived from acetyl-CoAby carboxylation. TheATP-dependent reaction is cata-lyzedbyanacetyl-CoAcarboxylase(eC6.4.1.2).Theen-zymeutilizesthefirststepinthefattyacidbiosynthesis(Chen,Kim,Weng,&Browse,2011;Konishi,Shinohara,Yamada,&Sasaki,1996).Tauraetal.(2009)discoveredaplant type IIIPKS inflowersand rapidlyexpandingleavesofC. sativa.Thegeneofolivetolsynthase(oLS)encodes a 385-amino acid polypeptide chain that does



notcontainasignalpeptide(Table2.1).TheoLSproteinhasatheoreticalmolecularmassof43kDa,asconfirmedby SDS-PAGe analysis. However, size-exclusion chro-matographyexperimentsrevealedamolecularmassofabout89kDa,indicatingahomodimericenzyme(Gagneetal.,2012;Tauraetal.,2009).oLS(PKS-1)wasprelimi-narilycrystallizedbyTaguchietal.(2008)andthestruc-turewasfinallypublishedbyYangetal.(2016).ItisofinterestthattheenzymedoesnotproduceoA,butolive-tol,triketidepyrone,andtetraketidepyrone.Analysisoftheaminoacidsequencedisplayedahighsimilaritywiththose of Medicago sativa chalcone synthase (CHS), andotherplantPKSs(60–70%).Additionally,thecatalytictri-aderesiduesofCHS(Cys164-His303-Asn336)areconserved(Tauraetal.,2009).SinceCHSscatalyzeintramolecularC6→C1Claisencondensations,raharjo,andcoworkerswerethefirsttosuggestin2004(raharjoetal.,2004)thatoLScouldbeastilbenesynthase(STS).Theseenzymescatalyze C2 → C7 aldol condensations, followed by adecarboxylation step. Additionally, studies by Austin,Bowman, Ferrer, Schröder, & Noel (2004) showed thatthecyclizationreactioncanbechangedfromaClaisen-type (CHS) to an aldol-type (STS) by substitution of afewaminoacidsinCHS(=aldolswitch).

Nevertheless,sinceoLSaloneisnotcapabletoformoA, another enzyme/PKS might be involved in thebiosynthesis.ThemissingenzymeshouldcatalyzeaC2→ C7 intramolecular aldol condensation upon whichthecarboxylatemoietyispreserved.ThisisimportantsinceCBGASdoesnotacceptolivetolasaprenyldo-nor(Fellermeier&Zenk,1998).Gagneetal.(2012)iso-lated a gene encoding a 101-amino acid polypeptidechain.Thissmallprotein(12kDa)showssimilaritiestoa polyketide cyclase that belongs to the dimeric α+βbarrel (DABB)-type protein family. Furthermore, theidentifiedgeneexhibitshighexpressionlevelsinglan-dular trichomes. Together, this made the polyketidecyclaseapromisingcandidateforthemissingolivetolicacidcyclase(oAC).

Finally,usingbothoLSandoACwithhexanoyl-CoAand malonyl-CoA in one assay, the formation of oA,pentyldiaceticacid (triketidepyrone),andhexanoyltri-aceticacidlactone(HTAL;tetraketidepyrone)couldbedemonstrated (Page & Gagne, 2013) (Fig. 2.2). It is as-sumedthatoLScatalyzestheformationofaninterme-diate that is subsequently converted into oA by oAC(Gagneetal.,2012;Taguchietal.,2008).

Biosynthesis of Geranyl Diphosphate

Themonoterpenemoietyofcannabinoids(Fig.2.2)isderived from GPP. Its precursors, isopentenyl diphos-phate (IPP), and dimethylallyl diphosphate (DMAPP),are predominantly (>98%) biosynthesized via the2C-methyl-d-erythritol-4-phosphate (MeP) pathway[alsotermedasnonmevalonatepathwayor1-deoxy-d-xylulose-5-phosphate (DoXP) pathway] (Fellermeieretal.,2001).TheseresultsaresupportedbyMarksetal.(2009). They isolated rNA from the glands of a tetra-hydrocannabinolic acid (THCA)-producing CannabisstrainandgeneratedacDNAlibrary.Aftersequencing,theywereabletoidentifyallbutoneenzymeinvolvedin the MeP pathway. Additionally, Stout et al. (2012)found high expression of MeP pathway genes in Can-nabis flowers. Furthermore, in higher plants the MePpathway, mainly involved in secondary metabolism,is localized in plastids (described in detail elsewhere,for example, eisenreich, Bacher, Arigoni, & rohdich,(2004),orHunter(2007),whereasthemevalonate(MVA)pathway, predominantly contributing to primary me-tabolism,islocalizedinthecytosol.Thecompartmentalseparationbetweenthesetwopathwaysisnotabsolute.Themetabolitesofbothpathwayscanbetransportedbi-directionally across the plastid membranes (eisenreichetal.,2004).

Subsequently, the head-to-tail condensation of IPPand DMAPP to form GPP is catalyzed by geranyl di-phosphatesynthase(Fig.2.2)(Burkeetal.,1999).

TABLE 2.1 enzymes involved in Cannabinoid Biosynthesis in C. sativa L

Enzyme Accession no.a EC no. References

olivetolsynthase oLS AB164375 2.3.1.206 Tauraetal.(2009)

olivetolicacidcyclase oAC AFN42527.1 4.4.1.26 Gagneetal.(2012)

Cannabigerolicacidsynthase CBGAS US2012/0144523A1b

2.5.1.102 FellermeierandZenk(1998);PageandBoubakir(2012)

Cannabichromenicacidsynthase CBCAS 1.3.3.- Morimotoetal.(1998)

Cannabidiolicacidsynthase

CBDAS AB292682 1.21.3.8 Tauraetal.(2007a)

Tetrahydrocannabinolicacidsynthase THCAS AB057805 1.21.3.7 Sirikantaramasetal.(2004)

ThetableliststheenzymesandthecorrespondingGenBankaccessionnumbersinvolvedinbiosynthesisofC. sativaphytocannabinoids.aGenBank.bPatent number.

CannaBinoid preCursor BiosynThesis 17


Cannabigerolic Acid Biosynthesis

Cannabigerolicacidsynthase(CBGAS)orgeranylpyrophosphate:olivetolate geranyltransferase (GoT) pre-dominantly catalyzes theC-prenylationofoAbyGPPto form CBGA (Fig. 2.2). CBGA is presumed to be thecentralprecursorforcannabinoidbiosynthesis,sincedif-ferent cyclization of the prenyl moiety leads to THCAoritsisomerscannabichromenicacid(CBCA)andCBDA(Page & Boubakir, 2012; Sirikantaramas, Morimoto, &Shoyama,2007).

FellermeierandZenk(1998)detectedtheenzymeincrudehomogenatesofrapidlyexpandingyoungleavesofC. sativa.Thispartoftheplantcontainsthelateren-zymes of the THCA biosynthetic pathway (Morimoto,Komatsu,Taura,&Shoyama,1997;Tauraetal.,1995a).There are indications that CBGAS, like other prenyl-transferases, is a membrane-bound prenyltransferase(Yamamoto, Kimata, Senda, & Inoue, 1997). However,FellermeierandZenk(1998)couldnotdetectanyenzymeactivityinparticulatefractions,butinthesolublefractionofthecrudeextract.Twomajorproductswereidentified

bymassspectrometry(MS)measurementsasCBGAanditscis-isomercannabinerolicacid (CBNrA;FellermeierandZenk,1998,usedCBNAinsteadofCBNrA).Theen-zymeactivitywasfoundtobeMg2+-dependent.CBGASseemstobespecificforoAasaprenylacceptor,butalsoacceptsdifferentprenyldonorslikeGPPand,toalesserdegree,neryldiphosphate(NPP)(Fig.2.2).Theproduc-tion ratio of CBGA/CBNrA changes from 2:1 to 1:1whenNPPisusedasaprenyldonorinsteadofGPP.

However, the aromatic prenyltransferase CBGASseemstobeasolubleenzyme,butFellermeierandZenk(1998)couldnotcompletelyexcludeamembrane-boundactivity. Besides, two soluble hop prenyltransferases,involvedinthebiosynthesisofhopbitteracids,arede-scribedbyZuurbier,Fung,Scheffer,&Verpoorte(1998).Nevertheless,thesearetheonlydescriptionsofsolubleplantC-prenylatingenzymes;untilnow,itwasnotpos-sibletogetthesequenceinformationortoisolatethecor-respondinggenesorenzymes.

Contradictorily, all known sequences of plant aro-matic prenyltransferases belong to membrane-bound

FIGURE 2.2 Biosynthesis of cannabigerolic acid (CBGA).ThebiosynthesisofthecentralintermediateCBGAiscoloredindarkgreen.TheminorproductsCBNrAandCBGVAareshadedinlightgreen.Theprecursorpathwaysarehighlightedinlightblue(GPP)andblue(OA).MeP,2C-methyl-d-erythritol-4-phosphate; DoXP, 1-deoxy-d-xylulose-5-phosphate; MVA, mevalonate (Burke, Wildung, & Croteau, 1999; de Meijeretal.,2009;Fellermeier&Zenk,1998;Page&Gagne,2013;Tauraetal.,2009).



enzymes (Yamamoto et al., 1997;Yamamoto, Senda, &Inoue, 2000; Zhao, Inoue, Kouno, & Yamamoto, 2003).ThisisinaccordancewiththesecondreportdealingwiththeCBGAS(Page&Boubakir,2012).TheypublishedasequenceofCBGASthatwasmainlyexpressedinglan-dular trichomes of female flowers and young leavesofCannabisplants.Thegeneencodesa395-aminoacidpolypeptidechainshowingamembrane-boundtypeofprenyltransferases.Theywereabletoexpresstherecom-binantCBGASinSf9 insectaswellas inSaccharomyces cerevisiae cells, and verified the CBGAS activity in themicrosomal fractions. Using MS measurements, CBGA(3-geranyl olivetolate; comparison with CBGA stan-dard)wasidentifiedasthemajorproduct,and5-geranylolivetolate(identificationonlybyLC-MSanalysis)astheminorproduct.Furthermore,PageandBoubakir(2012)showedthatCBGASisspecificonlytoGPPasaprenyldonor, and approves oA, olivetol, phlorisovalerophe-none,naringenin,andresveratrolasprenylacceptor.Ad-ditionally,theenzymereactionisdependentondivalent

cations, whereas the highest enzyme activity was ob-tainedbyusingMg2+(Page&Boubakir,2012).

CANNABINOID PATHWAY

CBGA, thecentralprecursorofcannabinoidbiosyn-thesis,isconvertedbythreeenzymes(Fig.2.3):CBDAS,CBCAS, and THCAS. They predominantly use CBGAas substrate, and catalyze the stereoselective, oxida-tivecyclizationofthemonoterpenemoietyofCBGAtoCBDA,CBCA,orTHCA,respectively.TheTHCASandCBDASreactionsareoxygen-dependent,producinghy-drogenperoxideproportionaltoeitherCBDAorTHCA(Sirikantaramas et al., 2004; Taura et al., 2007b). re-markably, the CBCAS reaction is oxygen independent,and can be inhibited by hydrogen peroxide. Thus, theenzymeseemsnot tobeanoxygenaseoraperoxidase(Morimoto, Komatsu, Taura, & Shoyama, 1998). Fur-thermore, all three enzymes also convert CBNrA, the

FIGURE 2.3 Biosynthesis of cannabinoids.Theenzymaticallycatalyzedreactionsarehighlightedindarkgreen.Allnonenzyme-dependentmodificationsreactionsarecoloredinlightgreen.BiosynthesisofC3-cannabinoidsstartingfromcannabigerovarinicacid(CBGVA)iscarriedoutbythesameenzymesandforbetterclaritynotshown(Crombieetal.,1968;deMeijer,2011;Morimotoetal.,1998;Shoyama,Fujita,Yamauchi,&Nishioka,1968;Shoyama,oku,Yamauchi,&Nishioka,1972;Tauraetal.,1995a;1996).

CannaBinoid paThway 19


cis-isomerofCBGA,withalowerspecificity(Morimotoet al., 1998; Shoyama et al., 2012; Sirikantaramas,Morimoto,&Shoyama,2007;Tauraetal.,1995b;Taura,Morimoto,&Shoyama,1996).Sincenoenzymaticactiv-itywasdetectableusing theneutral cannabinoidCBG,thecarboxylgroupinCBGAseemstobeessentialfortheenzymaticreactionscatalyzedbyTHCAS,CBDAS,andCBCAS(Morimotoetal.,1997;Tauraetal.,1995a;Tauraetal.,1996).

Besides, it was postulated that THCA is biosyn-thesized and stored in the storage cavity of the glan-dular trichomes of Cannabis plants (Sirikantaramas,Morimoto,&Shoyama,2007).

Tetrahydrocannabinolic Acid Synthase

TheTHCASgeneencodesa545-aminoacidpolypep-tidechain(Table2.1).AccordingtoSirikantaramasetal.(2004),a28aminoacid longsignalpeptide iscleavedin the processed THCAS, leading to a protein of 517aminoacids.ThematureTHCAShasatheoreticalmo-lecularmassof59kDa.Anactualmassofabout75kDawas detected using SDS-PAGe (Taura et al., 1995b).Thiscouldbeexplainedbyposttranslationalmodifica-tions,sinceeightpossibleAsnglycosylationsiteswereconfirmed (Sirikantaramas et al., 2004). Furthermore,deglycosylated THCAS indeed showed a molecularmassof59kDa,andremainedfullyactive(Tauraetal.,2007b).THCASisamonomericenzymewiththehigh-est activity between pH 5.5 and pH 6.0 (Taura et al.,1995b). Sequence comparison identified similaritiesto the berberine bridge enzyme (BBe) of Eschscholzia californica (Shoyama et al., 2012). BBe belongs to thefamilyofoxidoreductasesandhasacovalentlyboundFAD(Kutchan&Dittrich,1995).TheTHCASaminoacidsequence revealedaflavinylationconsensussequence(Arg110-Ser-Gly-Gly-His114) in which His114 is probablytheFAD-bindingsite(Sirikantaramasetal.,2004).ThiscouldbeconfirmedbyX-raycrystallography(PDBID:3VTe) at a resolution of 2.75Å. The results show thattheenzymeiscomposedoftwodomainsandoneFADbinding pocket present in between. Besides His114, asecondresidue,Cys176,couldbe identifiedtobecova-lentlyboundtotheFAD(Shoyamaetal.,2012).Basedon X-ray structure data and mutational analysis ofTHCAS, a possible catalytic reaction mechanism ofTHCASwasproposedbyShoyamaetal.(2012),assign-ing a central role to Tyr484 in the catalytic mechanism(Fig.2.5).Nevertheless,sincethecrystalstructurewaspublished without substrate analoga, further studiesarenecessarytoverifythesuggestedmechanism.

Cannabis plants can be divided into two groups:“fiber-type” and “drug-type” plants (see dictionary).Alignment of THCAS coding sequences from “fiber-type” and “drug-type” plants showed 37 major amino

acidsubstitutions.Thesesubstitutionsseemtobetherea-sonfordecreasedTHCASactivityin“fiber-type”strains(Kojoma, Seki, Yoshida, & Muranaka, 2006). Minise-quencingofsamplesfrombothtypesofCannabisplantsshowedthreedifferentsinglenucleotidepolymorphism(SNP) genotypes. “Fiber-type” plants are homozygousfor the inactive THCAS form. “Drug-type” plants areeitherhomozygousorheterozygousfortheactiveformofTHCAS.Itseemsthatonlyasinglecopyofthegeneencoding the active THCAS form is necessary for thebiosynthesisofTHCA(rotherham&Harbison,2011).

Cannabidiolic Acid Synthase

ThegeneofthewildtypeCBDASencodesa544-aminoacid polypeptide (Table 2.1).According to Taura et al.(2007b), processed CBDAS consists of 517 amino ac-idsfollowingcleavageofthe28aminoacidlongsignalpeptide. The mature CBDAS has a theoretical molecu-larmassof59kDa.Anactualmassofabout74kDawasdetectedbySDS-PAGethatispossiblycausedbypost-translationalglycosylationofsevenAsnresidues(Tauraetal.,1996;Tauraetal.,2007b).CBDASisamonomericenzymewithapHoptimumof5.0 (Tauraetal.,1996).AcomparisonbetweenTHCASandCBDASrevealedasequencesimilarityof84%(Tauraetal.,2007b)(Figs.2.4and2.5).LikeTHCAS,CBDASisaflavinatedenzymeinwhichHis114andCys176aremostlikelytheFAD-bindingsites.SinceCBDASexhibitsstructuralandbiochemicalpropertiesrelatedtothoseofTHCAS,itisprobablethatthereactionmechanismofCBDAS issimilar to thatofTHCAS(Tauraetal.,2007b).

Cannabichromenic Acid Synthase (CBCAS)

ThesequenceofCBCASisstillunknown.Morimotoetal.(1998)purifiedtheenzymetoapparenthomogene-ity,butitssequenceisnotyetavailableinpublicdatabas-es.AccordingtovanBakeletal.(2011),possibleCBCAScandidatesarecurrentlyanalyzedbiochemically.

However,CBCASwasisolatedandpartiallypurifiedfrom young leaves of C. sativa (Morimoto et al., 1997;1998).IncontrasttoCBDASandTHCAS,CBCASseemsto be homodimer with a determined native molecularmassof136kDaandamaximumactivityatpH6.5.Amolecularmassof71kDawasestimatedforthemono-mersusingSDS-PAGe.Accordingtokineticdata,CBCAShasahigheraffinityforCBGAthanTHCASandCBCAS(Morimotoetal.,1998).CBCAanditsneutralformCBCarebothracemic.StudiesofMorimotoetal.(1997)sug-gested that both enantiomers of CBCA are formed bya CBCAS catalyzed reaction with a molar ratio of 5:1.Butit isstillunknownwhichofthetwoisomersisthemajor product (Gaoni & Mechoulam, 1971; Morimotoetal.,1997;Tauraetal.,2007a).



FIGURE 2.4 Alignment of amino acid sequences of THCA synthase and CBDA synthase.ThealignmentwasperformedwithCLUSTALWusingtheBLoSUM62matrix(Henikoff&Henikoff,1992;Larkinetal.,2007).Thesignalpeptidecleavagesitesareindicatedbyatriangle.Second-aryelements(α-alpha-helices;β-beta-sheets;TT-turns;η-310helix)areshownfortheTHCAS.Fullyconservedresiduesareshadedinblack.Thesequencesshowanoverallidentityof84%.ThefigurewasmadewitheSPript3.0(robert&Gouet,2014).THCAS,tetrahydrocannabinolicacidsynthase;CBDAS,cannabidiolicacidsynthase.

Mini-diCTionary 21


Cannabinoids with Propyl Side Chains

In contrast to the classic C5-phytocannabinoids,whichcontainann-pentylsidechain,cannabinoidswithann-propylsidechainarecalledC3-phytocannabinoidsorpropylcannabinoids.TheC-prenylationofdivarinicacid (DA) instead of oA by GPP yields in cannabiger-ovarinicacid(CBGVA)(Fig.2.2)(deMeijer,Hammond,&Micheler,2009).Theformationofpropylcannabinoidsdoesnotoccurbyshorteningthesidechainofpentylcan-nabinoids(Kajima&Piraux,1982).CBGVAisthecentralbranch-pointintermediateinthebiosynthesisofC3-can-nabinoidacids,likeCBGAforpentylcannabinoidacids.TheenzymesCBDAS,CBCAS,andTHCASarenotse-lectiveforthelengthofthealkylsidechain,andcanusebothasasubstrate.Theresultingcannabinoidsarecalledcannabidivarinicacid(CBDVA),cannabichrovarinicacid(CBCVA),andtetrahydrocannabivarinicacid(THCVA).The diverse amount of 2-carboxylic acids in differentCannabisstrainsiscausedbydissimilarenzymespecifici-tiesatthelevelofCBGAorCBGA-analogsformation(deMeijeretal.,2009;Shoyama,Hirano,&Nishioka,1984).relatively high amounts of tetrahydrocannabivarin

(THCV)and/orcannabidivarin(CBDV)areusuallyonlydetectableinCannabis indica(deZeeuw,1972).

MINI-DICTIONARY

Cannabinoids Cannabinoidsareagroupofterpenophenoliccompounds.Theyshowaffinitiestocannabinoidreceptors(CB1,CB2)orarestructurallyrelatedtotetrahydrocannabinol(THC).Cannabinoidscanbedifferentiatedintophytocannabinoids,syntheticcannabinoids,andendocannabinoids.CBGA Cannabigerolicacid(CBGA)isthecentralprecursorofphytocannabinoidbiosynthesis.Itisnonpsychoactive.“Drug-type” plants TheseareTHCA-richplants.TheTHCAisconvertedintopsychoactive∆9-THCbyanonenzymaticdecarboxylationthatenablestheplantstobelabelledasTHC-rich.“Fiber-type” plants “Fiber-type”plantsarealsoknownas“nondrug”plants.Theseplantshavealow(<0.2%)ornoTHCAcontent,buttheycontainahighamountofcannabidiolicacid(CBDA).Phytocannabinoids Phytocannabinoidsareauniquegroupofsecondarymetabolites(cannabinoids)occurringnaturallyinplants.othernamesincludenaturalcannabinoidsorherbalcannabinoids(seeKeyfacts).∆8-THC InCannabisplants,∆8-tetrahydrocannabinol(∆8-THC)isdetectableinlowamounts(<1%ofthepresent∆9-THC).Like

FIGURE 2.5 X-ray structure of the active center of THCAS.Thebackboneisshownascartoondiagram(PDBID:3VTe).TheFADmolecule(orange)iscovalentlyattachedtoHis114andCys176(yellow).Theactivesiteresiduesarehighlightedingreen(Shoyamaetal.,2012).Theclose-upofactivecenterwaspreparedwithPyMoL.THCAS,tetrahydrocannabinolicacidsynthase.



∆9-THC,itisalsopsychoactive.Maybe∆8-THCisanartefactofextractionand/oranalysisprocess.ThetermTHCincludesacombinationof∆8-THCand∆9-THC.∆9-THC ∆9-Tetrahydrocannabinol(∆9-THC)and∆1-THCdescribethesamecompound,differinginthenumberingsystemused(dibenzopyran-numberingandmonoterpene-numberingsystem,respectively).∆9-THCisresponsibleforthepsychoactiveeffectsofCannabisproducts.Itbindstothehumancannabinoidreceptorslocatedinthecentralandperipheralnervoussystem.Misleadingly,∆9-THCistermedasthemaincomponentofdrug-typeCannabisplants(seeTHCA).∆9-THCA ∆9-Tetrahydrocannabinolicacid(THCA),notTHC,isthemaincomponentofdrug-typeCannabisplants.Itisnonpsychoactive.THCAisconvertedintoneutralpsychoactive∆9-THCbyanonenzymaticdecarboxylationduringheatingorstorage.

AcknowledgmentWe gratefully acknowledge Parijat Kusari for critically reading thismanuscript.

ReferencesAustin,M.B.,Bowman,M.e.,Ferrer, J.L.,Schröder, J.,&Noel, J.P.

(2004).Analdolswitchdiscoveredinstilbenesynthasesmediatescyclization specificity of type III polyketide synthases. Chemistry and Biology,11,1179–1194.

Burke,C.C.,Wildung,M.r.,&Croteau,r. (1999).Geranyldiphos-phate synthase: Cloning, expression, and characterization of thisprenyltransferaseasaheterodimer.Proceedings of the National Acad-emy of Sciences of the United States of America,96,13062–13067.

Chen,H.,Kim,H.U.,Weng,H.,&Browse,J.(2011).Malonyl-CoAsyn-thetase,encodedbyACYL ACTIVATING ENZYME13,isessentialforgrowthanddevelopmentofArabidopsis.The Plant Cell,23,2247–2262.

Crombie,L.,Ponsford,r.,Shani,A.,Yagnitinsky,B.,&Mechoulam,r.(1968).Hashishcomponents.Photochemicalproductionofcanna-bicyclolfromcannabichromene.Tetrahedron Letters,9,5771–5772.

deMeijer,e.(2011).Cannabis sativaplantsrichincannabichromeneanditsacid,extracts thereofandmethodsofobtainingextracts there-from.U.S.PatentNo.2011/0098348A1

deMeijer,e.,Hammond,K.,&Micheler,M.(2009).TheinheritanceofchemicalphenotypeinCannabis sativaL.(III):variationincannabi-chromeneproportion.Euphytica,165,293–311.

deZeeuw,r.A.,Wijsbek,J.,Breimer,D.D.,Vree,T.B.,vanGinneken,C.A.,&vanrossum,J.M.(1972).CannabinoidswithapropylsidechaininCannabis:occurrenceandchromatographicbehaviour.Sci-ence,175,778–779.

eisenreich,W.,Bacher,A.,Arigoni,D.,&rohdich,F. (2004).Biosyn-thesisofisoprenoidsviathenon-mevalonatepathway.Cellular and Molecular Life Sciences,61,1401–1426.

elSohly,M.,&Slade,D.(2005).Chemicalconstituentsofmarijuana:thecomplexmixtureofnaturalcannabinoids.Life Sciences,78,539–548.

Fellermeier,M.,eisenreich,W.,Bacher,A.,&Zenk,M.H.(2001).Bio-synthesis of cannabinoids. Incorporation experiments with 13C-labeledglucoses.European Journal of Biochemistry,268,1596–1604.

Fellermeier,M.,&Zenk,M.H.(1998).Prenylationofolivetolatebyahemptransferaseyieldscannabigerolicacid,theprecursoroftetra-hydrocannabinol.FEBS Letters,427,283–285.

Fischedick,J.T.,Hazekamp,A.,erkelens,T.,Choi,Y.H.,&Verpoorte,r. (2010). Metabolic fingerprinting of Cannabis sativa L., cannabi-noidsandterpenoidsforchemotaxonomicanddrugstandardiza-tionpurposes.Phytochemistry,71,2058–2073.

Gagne,S.J.,Stout,J.M.,Liu,e.,Boubakir,Z.,Clark,S.M.,&Page,J.e.(2012).IdentificationofolivetolicacidcyclasefromCannabis sativarevealsauniquecatalyticroutetoplantpolyketides.Proceedings of

the National Academy of Sciences of the United States of America,109,12811–12816.

Gaoni, Y., & Mechoulam, r. (1964). Isolation, structure, and partialsynthesisofanactiveconstituentofhashish.Journal of the American Chemical Society,86,1646–1647.

Gaoni, Y., & Mechoulam, r. (1971). The isolation and structure of∆1-tetrahydrocannabinol and other neutral cannabinoids fromhashish.Journal of the American Chemical Society,93,217–224.

Gertsch,J.,Pertwee,r.G.,&DiMarzo,V.(2010).PhytocannabinoidsbeyondtheCannabisplant–dotheyexist?British Journal of Phar-macology,160,523–529.

Henikoff,S.,&Henikoff,J.G.(1992).Aminoacidsubstitutionmatricesfromproteinblocks.Proceedings of the National Academy of Sciences of the United States of America,89,10915–10919.

Hunter,W.N.(2007).Thenon-mevalonatepathwayofisoprenoidpre-cursorbiosynthesis.Journal of Biological Chemistry,282,21573–21577.

Kajima, M., & Piraux, M. (1982). The biogenesis of cannabinoids inCannabis sativa.Phytochemistry,21,67–69.

Kojoma,M.,Seki,H.,Yoshida,S.,&Muranaka,T.(2006).DNApoly-morphisms in the tetrahydrocannabinolic acid (THCA) synthasegene in “drug-type” and “fiber-type” Cannabis sativa L. Forensic Science International,159,132–140.

Konishi,T.,Shinohara,K.,Yamada,K.,&Sasaki,Y.(1996).Acetyl-CoAcarboxylase in higher plants: Most plants other than gramineaehaveboththeprokaryoticandtheeukaryoticformsofthisenzyme.Plant and Cell Physiology,37,117–122.

Kutchan,T.M.,&Dittrich,H.(1995).Characterizationandmechanismoftheberberinebridgeenzyme,acovalentlyflavinylatedoxidaseofbenzophenanthridinealkaloidbiosynthesis inplants. Journal of Biological Chemistry,270,24475–24481.

Larkin,M.A.,Blackshields,G.,Brown,N.P.,Chenna,r.,McGettigan,P.A.,McWilliam,H.,Valentin,F.,Wallace,I.M.,Wilm,A.,Lopez,r.,Thompson,J.D.,Gibson,T.J.,&Higgins,D.G.(2007).ClustalWandClustalXversion2.0.Bioinformatics,23,2947–2948.

Marks,M.D.,Tian,L.,Wenger,J.P.,omburo,S.N.,Soto-Fuentes,W.,He,J.,Gang,D.r.,Weiblen,G.D.,&Dixon,r.A.(2009).Identifica-tionofcandidategenesaffecting∆9-tetrahydrocannabinolbiosynthe-sisinCannabis sativa.Journal of Experimental Botany,60,3715–3726.

Morimoto,S.,Komatsu,K.,Taura,F.,&Shoyama,Y.(1997).enzymo-logicalevidenceforcannabichromenicacidbiosynthesis.Journal of Natural Products,60,854–857.

Morimoto,S.,Komatsu,K.,Taura,F.,&Shoyama,Y.(1998).Purifica-tionandcharacterizationofcannabichromenicacidsynthasefromCannabis sativa.Phytochemistry,49,1525–1529.

Page, J.e. & Boubakir, Z. (2012). Aromatic prenyltransferase fromCannabis.U.S.PatentNo.2012/0144523A1.

Page, J.e. & Gagne, S. (2013). Genes and proteins for aromaticpolyketidesynthesis.U.S.PatentNo.2013/0067619A1.

Page, J.e.&Stout, J.M. (2013).Genesandproteins foralkanoyl-CoAsynthesis.PatentNo.Wo2013/006953A1.

raharjo,T.J.,Chang,W.-T.,Choi,Y.H.,Peltenburg-Looman,A.M.G.,&Verpoorte,r.(2004).olivetolasproductofapolyketidesynthaseinCannabis sativaL.Plant Science,166,381–385.

razdan, r. K., Puttick,A. J., Zitko, B.A., & Handrick, G. r. (1972).Hashish VI1: Conversion of (-)-∆1(6)-tetrahydrocannabinol to(-)-∆1(7)-tetrahydrocannabinol.Stabilityof(-)-∆1and(-)-∆1(6)-tetrahy-drocannabinols.Experientia,28,121–122.

robert, X., & Gouet, P. (2014). Deciphering key features in proteinstructureswith theneweNDscript server.Nucleic Acids Research,42,W320–W324.

rotherham,D.,&Harbison,S.A. (2011).Differentiationofdrugandnon-drugCannabisusingasinglenucleotidepolymorphism(SNP)assay.Forensic Science International,207,193–197.

Schultes,r.,Klein,W.,Plowman,T.,&Lockwood,T.(1974).Cannabis:An example of taxonomic neglect. Botanical Museum Leaflets, Harvard University,23,337–367.

http://refhub.elsevier.com/B978-0-12-800756-3.00002-8/ref0010




































































































referenCes 23

Shoyama,Y.,Fujita,T.,Yamauchi,T.,&Nishioka, I. (1968).Cannabi-chromenicacid,agenuinesubstanceofcannabichromene.Chemical and Pharmaceutical Bulletin,16,1157–1158.

Shoyama,Y.,Hirano,H.,&Nishioka,I.(1984).Biosynthesisofpropylcannabinoidacidanditsbiosyntheticrelationshipwithpentylandmethylcannabinoidacids.Phytochemistry,23,1909–1912.

Shoyama,Y.,oku,r.,Yamauchi,T.,&Nishioka, I. (1972).Cannabis.VI. Cannabicyclolic acid. Chemical and Pharmaceutical Bulletin, 20,1927–1930.

Shoyama,Y.,Tamada,T.,Kurihara,K.,Takeuchi,A.,Taura,F.,Arai,S.,Blaber,M.,Shoyama,Y.,Morimoto,S.,&Kuroki,r.(2012).Struc-tureand functionof∆1-tetrahydrocannabinolicacid (THCA)syn-thase,theenzymecontrollingthepsychoactivityofCannabis sativa.Journal of Molecular Biology,423,96–105.

Sirikantaramas,S.,Morimoto,S.,Shoyama,Y.,Ishikawa,Y.,Wada,Y.,&Taura,F.(2004).Thegenecontrollingmarijuanapsychoactivity:molecular cloning and heterologous expression of ∆1-tetrahydro-cannabinolicacidsynthasefromCannabis sativaL.Journal of Biologi-cal Chemistry,279,39767–39774.

Sirikantaramas, S. T. S., Morimoto, S., & Shoyama, Y. (2007). recentadvances in Cannabis sativa research: biosynthetic studies and itspotential inbiotechnology.Current Pharmaceutical Biotechnology,8,237–243.

Stout, J.M.,Boubakir,Z.,Ambrose,S. J.,Purves,r.W.,&Page, J.e.(2012).Thehexanoyl-CoAprecursorforcannabinoidbiosynthesisisformedbyanacyl-activatingenzymeinCannabis sativatrichomes.The Plant Journal,71,353–365.

Suzuki, Y., Kurano, M., esumi, Y., Yamaguchi, I., & Doi, Y. (2003).Biosynthesis of 5-alkylresorcinol in rice: Incorporation of a puta-tivefattyacidunitinthe5-alkylresorcinolcarbonchain.Bioorganic Chemistry,31,437–452.

Taguchi,C.,Taura,F.,Tamada,T.,Shoyama,Y.,Tanaka,H.,Kuroki,r.,&Morimoto,S. (2008).CrystallizationandpreliminaryX-raydif-fraction studies of polyketide synthase-1 (PKS-1) from Cannabis sativa.Acta Crystallographica. Section F, Structural biology and crystal-lization communications,64,217–220.

Taura,F.,Dono,e.,Sirikantaramas,S.,Yoshimura,K.,Shoyama,Y.,&Morimoto,S.(2007).Productionof∆1-tetrahydrocannabinolicacidbythebiosyntheticenzymesecretedfromtransgenicPichia pastoris.Biochemical and Biophysical Research Communications,361,675–680.

Taura,F.,Morimoto,S.,&Shoyama,Y.(1995a).Cannabinerolicacid,acannabinoidfromCannabis sativa.Phytochemistry,39,457–458.

Taura,F.,Morimoto,S.,&Shoyama,Y. (1995b).Firstdirectevidenceforthemechanismof∆1-tetrahydrocannabinolicacidbiosynthesis.Journal of the American Chemical Society,117,9766–9767.

Taura,F.,Morimoto,S.,&Shoyama,Y. (1996).Purificationandchar-acterizationofcannabidiolicacidsynthasefromCannabis sativaL.Journal of Biological Chemistry,271,17411–17416.

Taura,F.,Sirikantaramas,S.,Shoyama,Y.,Shoyama,Y.,&Morimoto,S.(2007a).PhytocannabinoidsinCannabis sativa:recentstudiesonbiosyntheticenzymes.Chemistry and Biodiversity,4,1649–1663.

Taura,F.,Sirikantaramas,S.,Shoyama,Y.,Yoshikai,K.,Shoyama,Y.,&Morimoto,S.(2007b).Cannabidiolic-acidsynthase,thechemotype-determiningenzymeinthefiber-typeCannabis sativa.FEBS Letters,581,2929–2934.

Taura,F.,Tanaka,S.,Taguchi,C.,Fukamizu,T.,Tanaka,H.,Shoyama,Y.,&Morimoto,S.(2009).Characterizationofolivetolsynthase,apolyketide synthase putatively involved in cannabinoid biosyn-theticpathway.FEBS Letters,583,2061–2066.

vanBakel,H.,Stout,J.,Cote,A.,Tallon,C.,Sharpe,A.,Hughes,T.,&Page,J.(2011).ThedraftgenomeandtranscriptomeofCannabis sa-tiva.Genome Biology,12,r102.

Yamamoto,H.,Kimata,J.,Senda,M.,&Inoue,K.(1997).Dimethylallyldiphosphate:Kaempferol8-dimethylallyltransferaseinEpimedium diphyllumcellsuspensioncultures.Phytochemistry,44,23–28.

Yamamoto,H.,Senda,M.,&Inoue,K.(2000).Flavanone8-dimethylal-lyltransferaseinSophora flavescenscellsuspensioncultures.Phyto-chemistry,54,649–655.

Yang,X.,Matsui,T.,Kodama,T.,Mori,T.,Zhou,X.,Taura,F.,Noguchi,H.,Abe,I.,&Morita,H.(2016).StructuralbasisforolivetolicacidformationbyapolyketidecyclasefromCannabis sativa.FEBS Jour-nal,283,1088–1106.

Zhao,P.,Inoue,K.,Kouno,I.,&Yamamoto,H.(2003).CharacterizationofleachianoneG2”-dimethylallyltransferase,anovelprenylside-chainelongationenzymefortheformationofthelavandulylgroupofsophoraflavanoneGinSophora flavescensAit.cellsuspensioncul-tures.Plant Physiology,133,1306–1313.

Zuurbier,K.W.M.,Fung,S.-Y.,Scheffer,J.J.C.,&Verpoorte,r.(1998).In-vitroprenylationofaromatic intermediates in thebiosynthesisofbitteracidsinHumulus lupulus.Phytochemistry,49,2315–2322.

















































































the biosynthesis of cannabinoids...16 2. the biosynthesis of cannabinoids i. setting the scene,...

Documents