Evaluation of Biosynthetic Pathway and Engineered Biosynthesis of Alkaloids

19 Pages·2016·3.07 MB·English

molecules
Review
Evaluation of Biosynthetic Pathway and Engineered
Biosynthesis of Alkaloids
ShinjiKishimoto,MichioSato,YutaTsunematsuandKenjiWatanabe*
DepartmentofPharmaceuticalSciences,UniversityofShizuoka,Shizuoka422-8526,Japan;
[email protected](S.K.);[email protected](M.S.);
[email protected](Y.T.)
* Correspondence:[email protected];Tel.:[phone];Fax:[phone]
AcademicEditor:MichaelWink
Received:20July2016;Accepted:15August2016;Published:18August2016
Abstract: Varietiesofalkaloidsareknowntobeproducedbyvariousorganisms,includingbacteria,
fungiandplants,assecondarymetabolitesthatexhibitusefulbioactivities. However,understanding
ofhowthosemetabolitesarebiosynthesizedstillremainslimited,becausemostofthesecompounds
are isolated from plants and at a trace level of production. In this review, we focus on recent
effortsinidentifyingthegenesresponsibleforthebiosynthesisofthosenitrogen-containingnatural
products and elucidating the mechanisms involved in the biosynthetic processes. The alkaloids
discussed in this review are ditryptophenaline (dimeric diketopiperazine alkaloid), saframycin
(tetrahydroisoquinolinealkaloid), strictosidine(monoterpeneindolealkaloid), ergotamine(ergot
alkaloid) and opiates (benzylisoquinoline and morphinan alkaloid). This review also discusses
theengineeredbiosynthesisofthesecompounds,primarilythroughheterologousreconstitutionof
targetbiosyntheticpathwaysinsuitablehosts,suchasEscherichiacoli,Saccharomycescerevisiaeand
Aspergillusnidulans. Thoseheterologousbiosyntheticsystemscanbeusedtoconfirmthefunctionsof
theisolatedgenes,economicallyscaleuptheproductionofthealkaloidsforcommercialdistributions
andengineerthebiosyntheticpathwaystoproducevaluableanalogsofthealkaloids. Inparticular,
extensive involvement of oxidation reactions catalyzed by oxidoreductases, such as cytochrome
P450s,duringthesecondarymetabolitebiosynthesisisdiscussedindetails.
Keywords: alkaloids;biosynthesis;enzymes;engineeredbiosynthesis;aromaticaminoacids
1. Introduction
Alkaloids were originally defined as organic compounds of plant origin that possess strong
bioactivities and exhibit basicity that is attributed to the presence of nitrogen [1]. However,
harmful nitrogen-containing compounds, such as tetrodotoxin and saxitoxin (Figure 1), that are
isolated from animal or microorganism sources are frequently categorized as alkaloids recently.
Similarly, compounds that do not show basicity due to conversion of an amine into an amide in
themoleculeasseenincolchicine(Figure1)arealsogenerallyidentifiedasalkaloid-typecompounds.
Even synthetic compounds, such as procaine (Figure 1), that have similar chemical structures or
biological properties to plant alkaloids can be referred to as alkaloids. As such, it has become
commonplace nowadays to define alkaloids as nitrogen-containing organic compounds that can
producepronouncedphysiologicalresponses,withnotableexceptionsbeingaminoacids,nucleicacids
andcertainnaturalnitrogen-containingcompounds,suchastetracycline(polyketide)andkanamycin
(aminoglycoside). Thealkaloidclassofnaturalproductshasbeenveryimportantforhumanityas
a valuable source of pharmaceutical products, because they show a variety of biological activities
ataverylowdosage. Therefore,alkaloidshavebeenfrequentlyemployedasleadcompoundsfor
drugdiscovery. Ontheotherhand,duringthepastdecade,developmentofnewDNAsequencing
Molecules2016,21,1078;doi:10.3390/molecules21081078 www.mdpi.com/journal/molecules
Molecules2016,21,1078 2of19
technologies,inparticularnext-generationsequencing,hasenabledustoeasilyexplore“themaster
planofaMlkolaecluoleisd 20p16r,o 21d, u10c7t8i on”;thatis,thegenesresponsibleforthebiosynthesisofalkaloids.2B oyf 1s9t udying
the master plan, we started to gain deep insight into “the implementation of the master plan”,
has enabled us to easily explore “the master plan of alkaloid production”; that is, the genes responsible
meaning the mechanism of the biosynthesis of alkaloids. Furthermore, we are using the gained
for the biosynthesis of alkaloids. By studying the master plan, we started to gain deep insight into “the
knowledimgpeletmoelnotoatkiofno rofn tehwe malaksatelro ipdlasna”n, dmeaatntinhge tshaem meetcihmaneismm oodf ifthyet hbieos“ymntahsetseisr opfl aanlk”altooides.n gineer
productFiuornthoerfmmoroed, wifiee adre nusaitnugr tahle gpariondedu kcntso.wlIendgteh tios loaorkti cfolre n,ewwe alfkoaclouidsso anndfi avt tehes usacmcee stismfue lmeoxdaifmy ples of
reconstitthuetM i“oomlencauolsetsfe 2ra0 1pl6kl, aa2n1l,”o 1 0itd7o8 ebnigoisnyenert hperotdicupctaiotnh wof amyosdiinfiehde nteartuorlaolg poruodsumctsic. rIno bthiaisl ahrtoicsltes, .wTeh foocsues2fi oovfn 1e 9f ipvea thways
successful examples of reconstitution of alkaloid biosynthetic pathways in heterologous microbial hosts.
areresponsiblefortheformationofditryptophenaline(dimericdiketopiperazinealkaloid),saframycin
Thohsaes efinvaeb lpeadt huws taoy esa sairley erexspploornes “ibthlee fmoar sttheer pfolarnm oaft aiolkna loofi dd iptrryodputocptihoenn”;a tlhinaet i(sd, itmhee griecn desik reetsoppoinpseibralez ine
(tetrahydroisoquinoline alkaloid), strictosidine (monoterpene indole alkaloid), ergotamine (ergot
alkfaolro tihde) ,b sioafsryanmthyecsiisn o (ft eatlrkaahloyiddsr.o Bisyo sqtuudinyoinlign eth ael mkaalsotiedr )p, lsatnri, cwtoes sitdairnteed ( mto ognaoint edrepeepn ien sinigdhot lien taol k“athloei d),
alkaloide)rgaiomntdpamleomipneiean (tteaertsigoo(nbt eaonlfk zathyloeli idsmo) aaqsnutedir n opoplliaiannt”ee,s am(bneedannzmiynlgois rotphqheu iimnnoaelcnihnaaenl akisnamdlo moidfo )rt.hpeDh ibinsiaocnsuy asnlstkihaoelnsoiisdo )of.f tD haielskcauulsosseidioosn.f omf icrobes
as cataltyhseFt suusrfteoh oerfr mtmhoiercer,ob wbioees ca aoresn ucvasietnarglsy tishotesn gfoaoirnf tehaded k bnviooawcnolcenedvdgeer satiool klnoa oolkfo faioddrv naiennwtc eeadrlkm aalleokidadliosa iadtne disn atcte tarhmne esbdameiatefe otsim ucanen md boeed lfisofyeu wndh ere [2].
Similarleyl,srtehewea hd“emerraess t[ae2rr] .ep Slraienmf”ei ltraorr eelynd,g tirnoeeaoedrt ehprreso rdarrueecv triioeenfwe orfra emrdto itdcoilf eioestdhf noerar turtehrvaeli erpweroc deaurntctitcsel.e fIfsno ftrohtriss ttahoretwi crlaeecr, edwneet nfeogfcfuionsr etosen rt foiinvwega rpdl antsfor
improveedngspuinrcoeceedsrsuifnucglt iepoxlnaamnotpsfl efaosl rko fia mrleopciordonssvteaitdun tpdiorntoh doeuf iacrltkiaoannloa iodlfo bagilosksa[y3lno,ti4hd]es.t iaWcn pdea twthhweilialry aas lnisnao lhodegtiessr [co3ul,o4sg]so. uWths eme wriceirlcol ebainlastlo hp dorsiostscg.u rsess sinthe
Those five pathways are responsible for the formation of ditryptophenaline (dimeric diketopiperazine
the recent progress in the development of methodology for generating various compounds through
developmaelnktalooifdm), seatfhraomdyoclion g(tyetfroahrygderoniesorqautiinnoglivnea railkoaulosidc)o, mstrpicotousniddinset (hmroonuogtehrpheentee irnodlooleg oalukasloriedc)o, nstitution
heterologous reconstitution and engineering of biosynthesis pathways, especially those involved in
andengineeregroitnamgionfe b(eirogsoyt anltkhaleosidis) apnadt hopwiaateyss (,beesnpzyelcisioaqlluyintohlionsee anind vmoolrvpehdinainn atlhkealobiido)p. Droisdcuuscsitoion nofo fopioids.
the bioproduction of opioids.
the use of microbes as catalysts for the bioconversion of advanced alkaloid intermediates can be found
elsewhere [2]. Similarly, readers are referred to other review articles for the recent efforts toward
engineering plants for improved production of alkaloids and their analogs [3,4]. We will also discuss
the recent progress in the development of methodology for generating various compounds through
heterologous reconstitution and engineering of biosynthesis pathways, especially those involved in
the bioproduction of opioids.

Figure 1. Chemical structures of alkaloids in a broad sense: natural alkaloids of non-plant origin,
Figure 1. Chemical structures of alkaloids in a broad sense: natural alkaloids of non-plant origin,
tetrodotoxin and saxitoxin; a natural but non-basic alkaloid, colchicine; and a synthetic alkaloid, procaine.
tetrodotoxinandsaxitoxin;anaturalbutnon-basicalkaloid,colchicine;andasyntheticalkaloid,procaine.
2. Ditryptophenaline
2. Ditryptophenaline
Ditryptophenaline (1) is a dimeric diketopiperazine alkaloid [5] and has a complicated structure
Figure 1. Chemical structures of alkaloids in a broad sense: natural alkaloids of non-plant origin,
Dit(rFyigputroep 2h). eDniamleinrieza(t1io)ni sisa foduinmd erreliactidvieklye ftroepquipenetrlayz aimneonagl knaatluoriadl p[5ro]dauncdts,h aas sita isc coomnspidleicreadt etdo bset ructure
tetrodotoxin and saxitoxin; a natural but non-basic alkaloid, colchicine; and a synthetic alkaloid, procaine.
an efficient way of enhancing the chemical complexity and diversity of metabolites being biosynthesized.
(Figure2). Dimerizationisfoundrelativelyfrequentlyamongnaturalproducts,asitisconsidered
It c2a.n D ailtsroy pintocprehaesnea tlhine ev alency of a compound, affording the molecule a higher affinity for its target or
to be an efficient way of enhancing the chemical complexity and diversity of metabolites being
higher stability upon forming a complex with its target [6]. Compound 1 [7] and its related compound
biosynthesizeDdi.tryIpttocpahnenaallsinoe i(1n)c irse aa dseimtehriec dvikaelteonpcipyeroazfinae calokmalopido [u5n] adn,da hfafso rad cionmgpltichaetedm sotrluecctuurlee a higher
WIN 64821 (Figure 2) [8] originally isolated from Aspergillus versicolor exhibit useful biological activities,
(Figure 2). Dimerization is found relatively frequently among natural products, as it is considered to be
affinitysfuocrhi tass taanr ignehtiboitriohni gohf seurbssttaanbcieli Pty reucpepotnorf foorrm pointegntaiacl oanmalpgleesixc wanidth anittis-itnaflragmemt[a6to].ryC aocmtivpitoieus.n d1[7]
an efficient way of enhancing the chemical complexity and diversity of metabolites being biosynthesized.
and itsDreelsaptiteed thceoimr pcoomupnledx WchIeNmic6a4l 8s2t1ru(cFtuirgeus,r eth2ei)r [f8o]rmoraitgioinn asleleymiesdo lraetleatdivefrlyo msimApslpe,e rignivlolulvsinvge rsicolor
It can also increase the valency of a compound, affording the molecule a higher affinity for its target or
dimerization of diketopiperazine monomers formed through condensation of two amino acids at
exhibitusehfiguhlebr isotalboiglitiyc aulpaocnt fiovrimtiiensg, as cuocmhpalesx awnithin iths itbaritgieot n[6]o. fCsoumbpsotuanndc 1e [P7] raencde iptst roerlafteodr cpoomtpeonutniadl analgesic
the initial stage. Judging from the structure of the diketopiperazine monomer of 1, it appeared that
andanti-inWflINam 64m82a1 t(oFirgyuraec 2t)i [v8i] toireisg.inDalleys ipsoiltaetetdh feroirmc Aosmpeprglielluxs cvheresimcoliocra elxhsitbriut ucsteufruel bs,iotlhogeiicralf aocrtimvitaietiso, nseemed
L-trsyupchto apsh aann i nahnidb itLi-opnh oefn syulablsatannincee Pc oreucledp tboer cfoorn pdoetnesnetdia lt aongaeltgheesric t aon fdo ramnt it-ihnef ladmikmetaotopriyp earcatizviintiee sc. ore
relativelysimple,involvingdimerizationofdiketopiperazinemonomersformedthroughcondensation
struDcetsupriet eo ft htheeir mcoonmopmleexr . cOhnemcei ctahle sptrhuecntyulraelsa, ntihneei-rd efroirvmedat iaomn idsee enmiterdo greenl aotfiv tehley dsiikmeptolep,i pienrvaozlivnien gc ore
oftwoaism Ndi-nimmoeertahiczyaildtaitosenda ,ot aft hrdiienkgei ntcolioptsiipuaerlresa wtzaiingthee imn. Jtouhnedo mmgieonrnsgo fmofrremor maendd tth hare souusbtgrshue qccuotneundrtee nmosoaftnitoohmne eodrf– itmkweoont oaompmiienpro ec rraoacsizdsilsni naekt imn go nomer
of 1, it athprptohueeag ihnrei todiaxlit dshtaaatgitoe.Ln J -urtdreyagcipntitgoo nfprso hmina vnthoealv nsitndrugc Lttu-hpree h torefyn tphyteol apdlhiakanentio nipneidpceorolaeuz rilnidneg bm ecoancnoo mlneeadrd eo nft o1s ,e tihdt ea tpfoopgremeartaehtdie otrnha toot f fao rm the
diketophipomLe-rtoardyzpiimtnoeeprh coaofn r1 ae. nsdt Lr-upchteunryelaloafnitnhe ecomulodn boe mcoenrd.enOsendc etogthetehepr htoe nfoyrmla ltahne idnikee-tdoepripivereadzinaem coidree nitrogen
structure of the monomer. Once the phenylalanine-derived amide nitrogen of the diketopiperazine core
ofthediketopiperazinecoreisN-methylated,aringclosurewithinthemonomerandasubsequent
is N-methylated, a ring closure within the monomer and a subsequent monomer–monomer crosslinking
monomer–monomercrosslinkingthroughoxidationreactionsinvolvingthetryptophanindolering
through oxidation reactions involving the tryptophan indole ring can lead to the formation of a
canleadtohotmheodfiomremr oaft i1o. nofahomodimerof1.

Figure 2. Chemical structures of dimeric diketopiperazine alkaloids, (–)-ditryptophenaline 1 [7] and
(+)-WIN 64821 [8].

Figure2.CFihguerme i2c. aClhsetmruiccalt ustrreusctuorfeds oimf deimriecridc idkieketotoppiippeerraaziznien aelkaallkoiadlso, i(d–)s-d,i(t–ry)-pdtoiptrhyenpatloinpeh 1e [n7]a alinnde 1[7]and
(+)-WIN 64821 [8].
(+)-WIN64821[8].
Molecules2016,21,1078 3of19
Morerecently,1wasfoundtobeproducedbyanotherfungus,Aspergillusflavus[9]. Toelucidate
Molecules 2016, 21, 1078 3 of 19
thebiosyntheticpathwayof1,experimentsofdeficiencyofthebiosyntheticgenewereperformedto
identifydMitroyrpe troecpehnetlnya, l1i nweabs ifoosuynndt htoe btiec pgreondeucceluds btye ra.nBoathseedr founngtuhse, Ahospmerogliollugsy floafvuasm [9in].o Toa ceilducsiedqautee nces
dedutcheedM boifleorcosuylmesn 2t0ht1he6t,e i2c1A ,p 1.0a7flt8ha wvuasy gofe n1,o emxpeersiemqeunetns coef d[1ef0i]citeonckyn oofw thne ebniozsyynmtheest,icth greenee gweenree sp,edrftopr3Am o,f e1dd9t ptoB and
dtpC,idcleunstitfeyr edditrtyopgteotphheernaolninteh beioAsy.nfltahveutisc ggeenneo mcluestwere. rBeapserde donic ttheed htoomcooldogeyf oorf aamnionno raicbido ssoeqmuaelnpceesp tide
More recently, 1 was found to be produced by another fungus, Aspergillus flavus [9]. To elucidate
deduced from the A. flavus genome sequence [10] to known enzymes, three genes, dtpA, dtpB and dtpC,
synthetathsee b(iNosRynPtSh)e,tiacn paNth-mwaeyt hoyf l1t,r eaxnpsefreimraesnetsa onfd deaficcyietnoccyh orfo tmhee bPio4s5y0nt(hPe4ti5c0 g)e,nree swpeerec tpiverefloyrm(Feidg tuor e3)[9].
clustered together on the A. flavus genome were predicted to code for a nonribosomal peptide synthetase
Knockoiudetnotiffya ndiytryopnteopohfenthaleinteh brieoesygnethneetsic ignenteh celuAst.eflr.a Bvaussed∆ opny trhGe /ho∆mkoul7o0gys otrfa aimninAo1 a4c2id1 s[e1q1u]enrceessu ltedin
(NRPS), an N-methyltransferase and a cytochrome P450 (P450), respectively (Figure 3) [9]. Knockout
thelossdoedfupcreodd fruocmti othne Ao.f f1la,viuns dgeicnaotmine gsetqhuaentcteh [e1s0e] ttoh krneoewgne ennezsymweesr, ethirneev goelnveesd, ditnpAt,h detpbB iaonsdy dnttphCe, sisof1.
of any one of the three genes in the A. flavus ∆pyrG/∆ku70 strain A1421 [11] resulted in the loss of
clustered together on the A. flavus genome were predicted to code for a nonribosomal peptide synthetase
BasedontheinvitroassaysofDtpBusingasubstrate2isolatedfromthedtpBknockoutmutant,DtpB
production of 1, indicating that these three genes were involved in the biosynthesis of 1. Based on the
(NRPS), an N-methyltransferase and a cytochrome P450 (P450), respectively (Figure 3) [9]. Knockout
wassihno vowift rnaon tyao soscnaaeyt sao lfoy ftz hDeet atphnBr eNues -gimnegne eatsh siyunlb atshttiero aAnte. o f2lfa iv2suotslo a∆tbepdiyor fGsryo/∆nmktu ht7he0es isdzttrepaBitnh k eAni1on4ct2ke1or u[m1t1 em]d ruieatsatuneltt3e, d.D Itinnpt Bteh rwee aslosti ssnsh goolfwy ,n3 was
directtloy pccarootandlvuyzceteri toeannd o fNt o1-,m 1inebdtyhicyaDltaitntpigoC nth (aoPtf4 t2h5 e0ts)oe, wtbhiorhesieyc hgnetwhneeasssi wzpee rrteeh piena virnoetldevremads eiand itmahteei c b3rio.o ssIynontmethraeelsstfiisrn aogcfl ty1i.,o B3na swoefdaSs o andc ictrhheeac trloym yces
cerevicsoianienv everixtterpdor aetosss s1ai ynbsyg o Dfd tDtpptCpC B(P rue4sc5ino0g)m, awb shiunibcahsnt rwtaltayes. 2p Tirsehopleaatraeednd a farlsoy mas im sthioec rfdottsphoBme kaninlo fcrvkaioctutrioto mne uoxtfpa Sneartc,i cDmhatepronBm twsyacsesus s gchegorewevsnits eiade that
DtpCexcpatotrae cslaystizanelgys zdetth paCen rCNe2c-om–mNetb1hi0ynlaranitintolgyn. coTlfho 2es uatonr eablwiyossiiytshn otihfn ethsthiez eein mt hveiot rniono teemxrmpeererdaiminateden t3ths. esInudtgeigrmeessettierndigz ltayht,a i3ot nDwtoapsfC td hciraeetcattlelyytzr easc yclic
thec oCn2v–eNrt1e0d rtion 1g b cylo DsutprCe w(Pi4t5h0i)n, wthhei cmh ownaosm pererp aanrded t hase ad mimicerroiszoamtioaln f roafc tthioen t oeft rSaaccychcalirco mmyocneso cmereevrsis,i abeo th
monomers, both of which likely proceed through a radical pathway. In the proposed mechanism
of wexhpircehs sliinkge ldyt ppCr orecceoemd btihnraonutlgyh. Tah rea adniaclayl spisa othf wthaey in. Ivni ttrhoe e xppreorpimoseendts m suegcgheasnteisdm th (aFti DgutpreC 4c)a,t aal yrazedsi cal
(Figure4),aradicalformedatN10throughhydrogenatomabstractionbythehemeironofP450can
fortmhee dC 2a–t NN1100 r tihngro culogshu hrey wdriothgienn t haeto mmo naobmstrear catniodn t hbey dthime ehreizmatei oirno onf othf eP t4e5t0ra ccayncl imc imgroantoem toe rCs,3 b wotihth a
migratetoC3withaconcomitantC2–N10ringclosureandundergoaradical-mediatedcouplingwith
conocfo wmhiitcahn tl iCke2l–yN p1ro0c reiendg tchlroosuugrhe aan rdad uicnadl epragtoh wa arya.d Iinc atlh-em perdoipaotesded c omuepchlianngi swmi t(hF iagnuoreth 4e)r, aC 3ra rdaidcaicl al-
anothbeeraforCirnm3ger mdad aoitnc Noaml1-0be treh atroor iufnoggrhm mh ytohdnero obgmreinde gartitonomg f baoobrnsmtdra tichnti eothnbe rb diydi tmghieen rhg leimbkeoe n1ir.d oni noft hP4e5d0 icmane mrligikraete1 .to C3 with a
concomitant C2–N10 ring closure and undergo a radical-mediated coupling with another C3 radical-
bearing monomer to form the bridging bond in the dimer like 1.

Figure 3. Aspergillus flavus ditryptophenaline biosynthetic gene cluster and predicted func tions of the
Figure3.Aspergillusflavusditryptophenalinebiosyntheticgeneclusterandpredictedfunctionsofthe
genFeigs uinre t 3h.e A cslpuesrtgeirll.u s flavus ditryptophenaline biosynthetic gene cluster and predicted functions of the
genesinthecluster.
genes in the cluster.

FigurFeig4Fui.greuP rr4eo. 4pP.o rPosrpeoodpsoemsde edmc mhecaehncahinsamnisimsmo fo ofD f DtDpttpCpCC--c-ccaaatttaaalllyyyzzzeeeddd dddiimmimeerreiizrzaiaztitaoiotnin oo nof fi oninftetirenmrtmeederimdaitaeestde 3isa a3ten asdn 34d at4on tdfoo r4fmotr om fo rm
(−)-d(i−t)r-(yd−)pi-tdrtyoitpprythopeptonhpeahnleiannliaenlie1n ,e1 ,(1 −(,− ())−--)dd-dibiibbrerrevevviaiianananammaimiddeie d FFe 55 F aann5dd a((−n−))d--ttrry(y−pprr)oo-ptprhhyeenpnaraloilnipneh e6 e 6vn ivaai laai nara erdai6dcaivcl airalo uraoteur t[a9ed] [. 9iDc].at pDlAtrp,o Au,t e[9].
ditryptophenaline nonribosomal peptide synthetase (NRPS); DtpB, N-methyltransferase; DtpC,
ditryptophenaline nonribosomal peptide synthetase (NRPS); DtpB, N-methyltransferase; DtpC,
DtpA,dciyttroycphtroomphe ePn4a50li;n FetmnAo,n fruimboitsroemmoarlgpine NptRidPSe. synthetase(NRPS);DtpB,N-methyltransferase;DtpC,
cytochrome P450; FtmA, fumitremorgin NRPS.
cyt ochromeP450;FtmA,fumitremorginNRPS.
Molecules2016,21,1078 4of19
ToexaminethesubstratetoleranceofDtpCandtobiosynthesizenewdimericproductsusing
DtpCinthisstudy,brevianamideF(4)wasprovidedtothisenzymeasasubstrate[9]. Whenadding
Molecules 2016, 21, 1078 4 of 19
only4,compound5wasproduced. Furthermore,addingboththenaturalsubstrate2andthesubstrate
analog4Tpor oevxaidmeidnec othmep souubnstdraste6 taoslewraenllcea so5f .DUtpsCin ganNd MtoR baionsdynXth-reasyizcer nyeswta ldloimgrearpich picroddautcatso nus5inagn d6,
thecDhtepmCi cina lthstisr usctutudrye, sborefvtihaensaemciodme Fp o(4u)n wdass, ipnrcolvuiddiendg toth tehiirs aebnszoylmutee acso an sfiugbusrtraattieo n[9s],. wWehreend aedtedrimngi ned.
Theohnolmy 4o, dcoimmeproiucnpdr o5d wuacst p5rowdauscenda.m Fuerdth(e−rm)-doribe,r aedvdiainnga mboitdhe thFe, annatdurtahle suhbestetrraotde i2m anerdi cthper soudbustcrta6te was
namaendal(o−g )4-t pryropvriodpehde cnoamlipnoeuansdsn e6w asa wlkealll oasid 5s. U(Fsiignugr NeM5)R. Aansdin X1-r,atyh cersytsetarelloocghraepmhiics tdryatao focnh 5i raanldc a6r, bon
atomthsei nch5emanicdal6 swtruacstudreetse romf tihneesde ctoombpeoaullnidns,t hineclSudcoinngfi tghueirra atibosnolbuyte tchoenFfilgaucrkatpioanras,m weetreer dveatleurmeoinbetda.i ned
The homodimeric product 5 was named (−)-dibrevianamide F, and the heterodimeric product 6 was
fromthecrystallographicdata. Thus,thebiosyntheticmechanismfortheformationof5and6islikely
named (−)-tryprophenaline as new alkaloids (Figure 5). As in 1, the stereochemistry of chiral carbon
thesameasthatdescribedabovefortheformationof1(Figure4). Furthermore, asanexampleof
atoms in 5 and 6 was determined to be all in the S configuration by the Flack parameter value obtained
engineeredbiosynthesisofnon-nativedimericproducts,theheterologoushostAspergillusnigerA1179
from the crystallographic data. Thus, the biosynthetic mechanism for the formation of 5 and 6 is likely
modified[12]withtheintroductionofftmA(fumitremorginNRPS[13])geneswasprovidedwiththe
the same as that described above for the formation of 1 (Figure 4). Furthermore, as an example of
dtpC gene [14]. This engineered strain was able to isolate additional dimeric compounds 7 and 8
engineered biosynthesis of non-native dimeric products, the heterologous host Aspergillus niger
(Figure6). Thesetwocompoundswereanalogsofthehomodimericproduct5previouslyproduced
A1179 modified [12] with the introduction of ftmA (fumitremorgin NRPS [13]) genes was provided
(Figuwrieth4 t)h.eE dlutpcCid gaetnioe n[1o4f]. tThheisc henegminiceaelresdtr sutcratuinr ewsa7s aabnlde t8o sihsoolwatee dad7dtiotiobneala dnimewericco cmompopuonudndasn 7d a8ndto be
nase8s e(Faizgiunree B6)i.s Tohlaetseed twreoc econmtlypofruonmds awmeraer iannea-ldogws eolfl itnhge shtorempotdoimmyecriect eprboadcutectr i5u pmre[v1i5o]u.sDlyi mpreordizuacteido nin
7an(dFi8gudried 4)n. oEtlufcoirdmatiothne oCf t3h–eC ch3’emlinickaal gsterufcotuurneds 7in an1d, 58 sahnodw6e.d I7n to7 ,bde iam neerwiz caotmiopnooucncdu arnredd 8 btoe tbwe een
C3annadseNse1a’,zwinhe eBr eisaoslaptoesdi trieocnesnCtly3 farnodmC a6 mwaerirneei-ndvwoellvliendg isntrtehpetodmimyceertize abtaioctnertihuamt f[o1r5m]. eDdim8.eTrihzaotsieond iimn eric
com7p oanudn d8s daidre nsopte fcourmlat tehde aCls3o–Ca3s’p lirnokdaugcet sfooufnadr iand 1ic, a5l -amnde d6i. aItne d7, odximideartiizvaetidoinm oecrciuzrarteido nbeotfwtreyepn tCo3p han
and N1’, whereas positions C3 and C6 were involved in the dimerization that formed 8. Those dimeric
derivatives,wherethelinkagesareformedatvariousdifferentpositions. DtpCisthoughttoextract
compounds are speculated also as products of a radical-mediated oxidative dimerization of tryptophan
thehydrogenfromthediketopiperazinecoreamidenitrogenN10of2toyieldaradical. Then,the
derivatives, where the linkages are formed at various different positions. DtpC is thought to extract the
radicalcanmigratetoC3throughtheC2–N10ringclosure,andthesubsequentcouplingcanproceed
hydrogen from the diketopiperazine core amide nitrogen N10 of 2 to yield a radical. Then, the radical
toformtheC3–C3’linkage,givingthedimericproduct1. However,whenDtpCwasprovidedwith
can migrate to C3 through the C2–N10 ring closure, and the subsequent coupling can proceed to form
theunnaturalsubstrate4,whichlacksthebulkyphenylsidechaingroup,thepositioningof4within
the C3–C3’ linkage, giving the dimeric product 1. However, when DtpC was provided with the
theactivesiteofDtpCmaybeshiftedfromwhere2wouldbebound. Thismaycauseanoccasional
unnatural substrate 4, which lacks the bulky phenyl side chain group, the positioning of 4 within the
extraaccttiivoen soiftet hoef hDytpdCro gmeany frboem shtihfteedin fdroomle nwithreorgee 2n wtooyuiledl dbea braodunicda.l TahtiNs 1m,awyh cicahuscea nans toacrctamsioignraal ting
fromextthraecitniodno olef tnheit hroygderongteon Cfr6oman thdeb inadckolteo nNitr1og(eFnig tuo ryeie6ld, rai grahdticcaoll uatm Nn1), .wChoicuhp clainn gstaorft tmhiegrraatdinicga lat
C3ifnroomn ethme oinndoomlee nritfroorgmene dtov Cia6 athned Nba1c0k etox tNra1c (tFioignurpea 6th, rwigahyt c(olalubmelned). CasouCp3liinng Foifg tuhere ra6d,ilceaflt acto Clu3 mn)
withint hoener amdoincaolmaetr Nfo1rminedth veiao tthhee Nr1m0o enxotrmacetironfo prmathewdavyi a(lathbeeleNd1 ase xCt3r ainc tFioignurpea 6t,h lwefat yco(lluambenl)e wditahs N1
in Ftihgeu rraed6ic,arl iagth Nt1c ionl uthme ont)hcera nmopnroomduerc feortmheedd vimia ethriec Nc1o emxtpraocutinodn p7a.thSwimayi l(alarblye,lecdo ausp Nli1n gin oFfigtuhree 6s,a me
C3rraigdhict acloilunmonn)e cmano pnroomduecre wthiet hditmheerCic 6cormadpiocuanldin 7.t hSiemoiltahrelyr, mcoounpolimnge orf( tlhabe esalemdea Cs3C ra6dNi1cailn inF iognuer e6,
monomer with the C6 radical in the other monomer (labeled as C6N1 in Figure 6, right column)
rightcolumn)canproduce8. Thetiterofthesecompoundswassignificantlylowerthanthatofthe
can produce 8. The titer of these compounds was significantly lower than that of the native product
nativeproduct1(approximately350µg/Lofculturefor7and8ascomparedto2.3mg/Lofculture
1 (approximately 350 µg/L of culture for 7 and 8 as compared to 2.3 mg/L of culture for 1) [9,14],
for1)[9,14],presumablybecauseofthelowfrequencyoftheN1hydrogenextractionhappeninginside
presumably because of the low frequency of the N1 hydrogen extraction happening inside the active
theactivesiteofDtpC.Thereareotherknownnaturalproductshavingsimilardimerizationpatterns.
site of DtpC. There are other known natural products having similar dimerization patterns. For
Forexample,(–)-asperazineA[16]and(+)-pestalazineB[17,18]havethesameC3–N1linkageas7,
example, (–)-asperazine A [16] and (+)-pestalazine B [17,18] have the same C3–N1 linkage as 7, whereas
whereas(+)-asperazine[19]and(+)-pestalazineA[17,20]haveaC3–C7linkagethatwasnotobserved
(+)-asperazine [19] and (+)-pestalazine A [17,20] have a C3–C7 linkage that was not observed in our
in osutrudsytu (dFiygu(rFei g6u, broex6e,d)b. oPxreesdu)m. aPbrlye,s uthme abbiolsyy,ntthheesbisi oosf ythnothsee sdiismoerfict hdoikseetodpiimpeerraizcindei kcoetmoppoipuenrdasz ine
comips oaulsnod csatiaslyazlseod cbayt aPl4y5z0e denbzyymPe4s5 0weitnhz ryemlaxeesdw siutbhsrtrealatex sepdescuifibcsittyra, tmeuscphe cliikfiec Ditytp,Cm. uchlikeDtpC.

FiguFriegu5r.e 5C. hCehmemicaiclals tsrtuructcuturreess ooff tthhee nneewwllyy ffoorrmmeedd ccoommppouonudnsd, s(,–)(-–d)i-bdriebvrieavniaamniadme iFd e5 Fan5d a nd
(–)-t(r–y)p-trroypphroepnhaelinnaelin6e[ 96] [.9].
Molecules2016,21,1078 5of19
Molecules 2016, 21, 1078 5 of 19

FFiigguurree 6.6 P.ropoPserodp moescehdanmismec hofa DnitspmC-coatfalyDztepdC f-ocramtaaltyiozne dof dfoimrmeraiztiaotinon opfrodduimctes r(i−z)a-dtiiobnrevpiarnoadmuicdtes
(F− 5) -[d9i]b, rae vnieawna cmomidpeoFun5d[ 97] ,[1a4]n eawndc noamsepsoeuanzidne7 B[1 84 ][1a5n]d. Rnaadsiecsaela-mziendeiaBte8d [f1o5r]m. aRtiaodni coafl -7m aenddia 8te ids
fthoormugahtito tno boef i7niatinadted8 bisy tDhtopuCg fhotrmtoinbge ai nraitdiaictaeld atb Ny1D itnpsCteafdo romf Nin1g0 aasr padroicpaolseadt Nfo1r tihnes tfeoarmdaotfioNn 1o0f
a5.s Tphreo praodsiecdal fsopretchiees faorrem laabtieolendo bfy5 t.heT hpeosriatidoinc aolf sthpee criaedsicaarle. Tlaob delieffderbenytitahtee bpeotswiteioenn roafdtihcael rsapdeicciaesl.
Tdoerdivifefder fernotmia theybdertowgeeenn erxatdraiccatilosnp aetc iNes10d e(lreivfte dcoflruommnh) yvds.r oNg1e n(riegxhtrta ccotilounmant)N, t1h0os(ele ffotrcmoleudm vnia) tvhse. NN11
(hryigdhrotgcoenlu emxntr)a,ctthioonse afroer mgievdenv iaa ltahbeeNl w1ihthy dar osguepneresxctrriapctt iNon1. aCrehegmiviecnala sltarbuecltuwreitsh oaf srueplaetresdc rkipntoNw1n.
Cnahteumrailc aplrsotdruuccttus raerseo sfhroewlante idn kthneo wbonxn. aTthuer amlopnroodmuecrt-sbarridegsihnogw bnonindtsh aereb osxh.oTwhne imn orendo.m er-bridging
bondsareshowninred.
3. Saframycin
3. Saframycin
Saframycin A (9) isolated from Streptomyces lavendulae is a representative member of the
tetrahSyafdrraomisyocqiuninAoli(n9e) aislkoalaloteidds f(rFoigmurSe t7r)e p[2to1m]. yTchesis lcalvaesnsd oufl naeatiusraal preropdreuscetns thaatisv beeemne imsoblaetredo ffrothme
tveatrriaohuysd orrogisaonqisuminso, liinnceluadlkinaglo sidoisl (bFaicgtuerriea 7a)n[d2 1m].arTinheis inclvaesrsteobfrnataetsu, rsaulcphr oasd uspctosnhgaess abneden asiscoidlaiatends
f[r2o2m]. Tvhaerisoeu csoomrgpaonuinsdmss ,exinhcibluitd ivnagrisoouisl baanctitbeiroiatica nadctimviatireins ebiuntv aelrstoeb praotsesse,sss uac hpoatsenspt oanngtietsumanodr
aascctiivdiitayn,s w[2h2i]c.hT ihse tsheocuogmhpt otou nbde seeffxehcitbeidt vtahrrioouugsha nctoivbiaoletincta mctiovditiifeicsabtuiotna losof DpoNsAse sbsya ipmoitneinutman itoitnusm voiar
acactribviintyo,lawmhiinceh misothieotyu gahntdt oitsb eeqeuffievcatleednttsh r[2o3u]g. hAcmoovnagle nthtem soafdriafimcaytciionn aonfaDloNgsA, ebcyteiimnainsciuidmini o7n43s v(1ia0
cina rFbiignuorlea m7)i [n2e4]m shooiewtyeda nad reimtsaerqkuaibvlyal penottsen[t2 3a]n.tiAtummoonrg actthievistya fargamainyscti nnoatn oanlolyg sc,oemctmeionna stucimdionr 7ce4l3l
(li1n0eisn bFuitg aulrseo 7r)es[i2s4t]ansht ocewlle ldinaesr e[m25a].r Rkaebcelyntplyo,t e1n0t waanst iatupmproorvaecdt iavsi tayna agnatiincasntcneort dornulgy icno mthme Eounrtoupmeaonr
cUenlliolinn efosrb puattiaelnsots rwesitisht asonftt cteisllsulien seasr[c2o5m].a R[2e6c]e.n Btleyc,a1u0sew oafs thaep plirmoviteedd sauspapnlya nfrtoicman ncaetrudrarul sgoiunrctehse,
E10u riso pcuerarnenUtlnyi opnrofdourcpeda tbieyn ats sewmitihsysnothftettiics spureocseasrsc o[2m7]a. H[2o6w].evBeerc,a tuhsise soyfntthheesilsim reitqeudirseus pmpolyre ftrhoamn
n25a tcuhreamlsicoaulr tcreasn,s1f0orimscautirorne nsttelypsp, rmodaukicnegd ibt ya sausbeompitsimynatlh meteicthpordo cfoers sa [c2o7m].mHeorwciaelv perr,otdhuisctsioynn tohfe 1s0is.
rEenqguiinreesermedo rbeiothsaynnt2h5ecshise fmoirc galentrearnastfionrgm tahtiiso inmspteoprsta,mnta akliknagloitida asus baonp atilmteranlamtievteh osdtrafotergayc iosm stmroenrcgilayl
pdreosdiruedct,i eosnpoecfi1a0ll.yE fnrogmin etheere pdebrsiposeyctnivthee osifs sfuosrtaginenaebrilaittiyn agntdh iesnivmirpoonrmtaennttaallk baulordidena sofa nthael tseyrnntahteivtiec
smtraanteugfaycitsusrtirnogn gplryodceesssir. eTdo, epsupte tchiaisll byiforpormodthuectpioerns papecptrivoeacohf sinutsota pinraabctiilcitey, adnedtaeilnevdi ruonndmeernsttaanldbuinrgd eonf
the biosynthetic mechanism of this class of natural products is essential. Therefore, there has been a
considerable study on the subject involving gene isolation [28–30], substrate feeding experiments
Molecules2016,21,1078 6of19
ofthesyntheticmanufacturingprocess. Toputthisbioproductionapproachintopractice,detailed
understandingofthebiosyntheticmechanismofthisclassofnaturalproductsisessential. Therefore,
Molecules 2016, 21, 1078 6 of 19
therehasbeenaconsiderablestudyonthesubjectinvolvinggeneisolation[28–30],substratefeeding
[e3x1p,3e2r]i,m beioncthse[m31ic,3a2l ]c,hbairoacchteermiziactaiolnchs a[3r0a,c3t3e]r aiznadti boinosin[f3o0r,m33a]tiacn adnabliyosiensf o[3r0m].a Itnic thains arleyvsieesw[,3 t0h]e. cIunrrtehnist
urenvdieewrs,tathnedicnugr roefn tthuen mdeercshtaannidsimng oof fbtihoesymntehcehsains iosmf 9 oisf bdieosscyrnibtehdes. isof9isdescribed.

FFiigguurree 77.. CChheemmiiccaall ssttrruuccttuurreess ooff tteettrraahhyyddrrooiissooqquuiinnoolliinnee aallkkaallooiiddss,, ssaaffrraammyycciinn AA 99 [[2211]] aanndd eecctteeiinnaasscciiddiinn
774433 1100 [[2244]]..
Investigation of the biosynthesis of 9 and related compounds has been ongoing for quite some
Investigationofthebiosynthesisof9andrelatedcompoundshasbeenongoingforquitesome
time after the initial discovery of 9 in 1977 [21]. By 1985, it was determined that the main framework of
timeaftertheinitialdiscoveryof9in1977[21]. By1985,itwasdeterminedthatthemainframeworkof
9 was comprised of one molecule each of L-alanine and glycine and two molecules of L-tyrosine [31].
9wascomprisedofonemoleculeeachofL-alanineandglycineandtwomoleculesofL-tyrosine[31].
The alanyl-glycyl terminal moiety was found to be modifiable by feeding of particular amino acids,
Thealanyl-glycylterminalmoietywasfoundtobemodifiablebyfeedingofparticularaminoacids,
such as 2-amino-n-butylic acid [32]. The initial discovery of a saframycin biosynthetic gene cluster
suchas2-amino-n-butylicacid[32]. Theinitialdiscoveryofasaframycinbiosyntheticgenecluster
was made in Myxococcus xanthus, where two NRPS-encoding genes and an O-methyltransferase gene
wasmadeinMyxococcusxanthus,wheretwoNRPS-encodinggenesandanO-methyltransferasegene
responsible for the formation of the core structure were identified [28]. The findings from this study
responsiblefortheformationofthecorestructurewereidentified[28]. Thefindingsfromthisstudy
confirmed that the main framework of 9 was biosynthesized by NRPS. The subsequent study on the
confirmedthatthemainframeworkof9wasbiosynthesizedbyNRPS.Thesubsequentstudyonthe
gene cluster for the biosynthesis of safracin, a compound closely related to 9, revealed additional
gene cluster for the biosynthesis of safracin, a compound closely related to 9, revealed additional
genes proposed to be involved in the modification of building block amino acids and post-NRPS
genes proposed to be involved in the modification of building block amino acids and post-NRPS
intermediates [29]. The most recent study provided the most complete information on the saframycin
intermediates[29]. Themostrecentstudyprovidedthemostcompleteinformationonthesaframycin
biosynthetic gene cluster with a total of 30 genes that are proposed to be involved in the biosynthetic
biosyntheticgeneclusterwithatotalof30genesthatareproposedtobeinvolvedinthebiosynthetic
process [30].
process[30].
Based on the bioinformatically predicted functions of the genes that were identified as described
Basedonthebioinformaticallypredictedfunctionsofthegenesthatwereidentifiedasdescribed
above, various biosynthetic schemes were hypothesized [29,31,34]. However, no actual experimental
above,variousbiosyntheticschemeswerehypothesized[29,31,34]. However,noactualexperimental
data were available to verify the proposed mechanisms. Thus, to establish the mechanism of how the
datawereavailabletoverifytheproposedmechanisms. Thus,toestablishthemechanismofhowthe
complex core structure of 9 was biosynthesized, in vitro assays on the recombinantly prepared SfmA
complexcorestructureof9wasbiosynthesized,invitroassaysontherecombinantlypreparedSfmA
and SfmB were carried out [30]. As predicted from the adenylation domain amino acid sequence [30],
andSfmBwerecarriedout[30]. Aspredictedfromtheadenylationdomainaminoacidsequence[30],
two NRPSs, SfmA and SfmB, were shown to specifically activate L-alanine and glycine, respectively,
twoNRPSs,SfmAandSfmB,wereshowntospecificallyactivateL-alanineandglycine,respectively,
indicating that those two enzymes were responsible for the formation of the non-tyrosine dipeptide
indicatingthatthosetwoenzymeswereresponsiblefortheformationofthenon-tyrosinedipeptide
segment of 9 (Figure 8). However, amino acid sequence analysis of SfmA also revealed the presence of
segmentof9(Figure8). However,aminoacidsequenceanalysisofSfmAalsorevealedthepresenceof
an N-terminal acyl-CoA ligase (AL)-acyl carrier protein (ACP) didomain [30,35], which is commonly
anN-terminalacyl-CoAligase(AL)-acylcarrierprotein(ACP)didomain[30,35],whichiscommonly
found in NRPSs that form lipopeptides having a long fatty acyl chain extension at the N-terminus of
foundinNRPSsthatformlipopeptideshavingalongfattyacylchainextensionattheN-terminusofthe
the core peptide [36,37]. This finding prompted analysis for the requirement of an N-terminal acyl
corepeptide[36,37]. ThisfindingpromptedanalysisfortherequirementofanN-terminalacylchain
chain in the L-alanyl-glycyl intermediate. In vitro assays with synthetically prepared L-alanyl-glycyl-
intheL-alanyl-glycylintermediate. InvitroassayswithsyntheticallypreparedL-alanyl-glycyl-S-CoA
S-CoA substrates having an N-acyl chain of various length and SfmC that was recombinantly prepared
substrates having an N-acyl chain of various length and SfmC that was recombinantly prepared
in Escherichia coli clearly indicated that the N-myristoylated dipeptide was the actual substrate of SfmC
in Escherichia coli clearly indicated that the N-myristoylated dipeptide was the actual substrate of
[35]. No reaction took place when substrates lacking a long acyl extension were provided to SfmC.
SfmC[35]. Noreactiontookplacewhensubstrateslackingalongacylextensionwereprovidedto
Because the final product 9 does not carry any acyl chain, the requirement of SfmC for a cryptic acyl
SfmC.Becausethefinalproduct9doesnotcarryanyacylchain,therequirementofSfmCforacryptic
chain in its substrate is a completely unexpected finding.
acylchaininitssubstrateisacompletelyunexpectedfinding.
Once the actual substrate for SfmC was identified, the mechanism of the formation of the
Once the actual substrate for SfmC was identified, the mechanism of the formation of the
pentacyclic tetrahydroisoquinoline core was investigated. Further detailed examinations, which
pentacyclic tetrahydroisoquinoline core was investigated. Further detailed examinations, which
involved the use of additional synthetic substrates and SfmC mutants [35], demonstrated that SfmC
was solely responsible for the formation of the pentacyclic scaffold from two molecules of a L-tyrosine
derivative, 3-hydroxy-5-methyl-O-methyltyrosine (Figure 8) [30]. Based on the results from those
experiments, the reaction mechanism was suggested with the Re (reduction) domain of SfmC
catalyzing three reduction reactions and the C (condensation) domain catalyzing two cycles of the
Molecules2016,21,1078 7of19
involvedtheuseofadditionalsyntheticsubstratesandSfmCmutants[35],demonstratedthatSfmC
wassolelyresponsiblefortheformationofthepentacyclicscaffoldfromtwomoleculesofaL-tyrosine
derivative, 3-hydroxy-5-methyl-O-methyltyrosine (Figure 8) [30]. Based on the results from those
experiments,thereactionmechanismwassuggestedwiththeRe(reduction)domainofSfmCcatalyzing
Molecules 2016, 21, 1078 7 of 19
threereductionreactionsandtheC(condensation)domaincatalyzingtwocyclesofthePictet-Spengler
Preiactcettio-Snpteonfgolremr rethaecttieotnr atoh yfodrrmoi stohqe uteintroalhinyedcrooirseooqfu9in[o35li]n.eT choerela osft 9c y[3cl5i]z.a Ttihoen laresta ccyticolnizaafttieornt rheeacthtiiornd
areftdeur ctthioe nthsitredp rmedauycatiloson bsetecpa tmalayyz eadlsob ybeS fcmatCa,lyazltehdo buyg hSftmheCr,e aalctthiooungcho uthlde preraoccteieodn scpoounldta pnreoocueseldy.
Wsphoinletafnuerothuesrlyin. vWeshtiiglea tifounrsthienrt oitnhveebstiiogsaytniothness iisnotof 9tahned brieolsaytendthceosmisp oouf n9d saanrde rreeqlautierded ,ceolmucpioduatniodns
aorfet hriesqcuoimrepdle, xerlueaccidtiaotniomn eochf atnhiissm cwomouplldexp rroevaicdteiouns wmietchhaannoisvmel cwhoeumldoe npzryomviadteic uasp pwroitahc hat onwoavredl
cshynemthoeesinzzinygmsautifcfi acipepnrtoaamcho utonwtoarfd1 0syanntdheitssizainnagl osgusf.ficient amount of 10 and its analogs.

FFiigguurree 88.. PPrrooppoosseedd oovveerraallll sscchheemmeef foorrt thheep peenntataccyycclilcicc ocoreres ksekleelteotnona sassesmemblbylyd udruinrigntgh tehbei obsioysnythnethsiessoisf
osaf fsraafmraymcyinciAn A9 [93 [53]5.]S. fSmfmAA,S, fSmfmBBa annddS SfmfmCCa arreet thhrreeees saaffrraammyycciinn AA nnoonnrriibboossoommaall ppeeppttiiddee ssyynntthheettaasseess..
CC,, ccoonnddeennssaattiioonn ddoommaaiinn; ;AA, ,aaddeennyylalatitoionn ddoommaainin; ;TT, ,ththioiolalatitoionn ddomomaiani;n R;Re,e r,erdeudcutcatsaes deodmomaiani.n .
44.. SStrtircictotossididininee
TTeerrppeennee iinnddoollee aallkkaallooiiddss ((TTIIAAss)) ccoonnssttiittuuttee oonnee ooff tthhee llaarrggeesstt aanndd mmoosstt ddiivveerrssee ffaammiilliieess ooff
ccoommpplleexx nniittrrooggeenn--ccoonnttaaiinniinngg mmeettaabboolliitteess ffoouunndd iinn ppllaannttss [[3388,,3399]].. MMaannyy TTIIAAss,, eessppeecciiaallllyy mmoonnootteerrppeennee
iinnddoollee aallkkaallooiiddss ((MMIIAAss)) aanndd tthheeiirr aannaallooggss,, hhaavvee mmeeddiicciinnaall pprrooppeerrttiieess:: ccaammppttootthheecciinn aanndd vviinnbbllaassttiinnee
aarree aapppprroovveedd aass aannttiiccaanncceerr tthheerraappeeuuttiiccss,, wwhheerreeaass rreesseerrppiinnee iiss uusseedd aass aann aannttiihhyyppeerrtteennssiivvee [[4400]]..
PPhhyyssoossttiiggmmiinnee iiss aann aannttiiddoottee ooff aannttiicchhoolliinneerrggiicc ttooxxiicciittyy aanndd iiss uusseedd pprriimmaarriillyy ffoorr ttrreeaattiinngg ggllaauuccoommaa [[4411]]..
HHoowweevveerr,, tthheessee ssttrruuccttuurraalllyly ccoommpplelexx cocmompopuonudnsd scacna nbeb deifdfiicffiucltu tlot tsoynstyhnetshizeesi czheecmheicmalilcya [ll4y2,[4432],.4 T3o].
sTuopspulyp pthlyeseth ceosmepcooumnpdos uthnrdosugthhr boiuogshynbthioestiycn rtohuetteics, rthoeu tbeiso,sythnethbetiiocs pyantthhwetaicy fpoart ah wtaragyetf ocormaptoaurgnedt
mcoumstp obue nedlumciduasttebde. eOlunccied aaltle do.f Othnec egeanlleos finthveolgveende sini ntvhoel vpeadthiwnatyh eaprea tihdwenatyifiaerde, iad ehnotsifit eodrg,aanhisomst
tohragta nisi smmotrhea ctoisnvmeonrieencto ntov ecnuiletinvtattoe ctuhlatniv aat epltahnatn caanp labne tucsaendb feour sheedtefroorlhogetoeurso lporgooduuscptiroond uocf titohne
dofesthireedd ecsoirmedpocuonmdp. oFuunrdth.eFrumrothree,r melourceid,ealtuiocnid oaft iothneo bfitohseybnitohseytinct hpeattihcwpaayth awnady easntadbelissthabmliesnhtm oef nat
hoeftaerhoeltoegrooulosg soyusstesmy swteomulwd oaulllodwa ellnogwineenegriinnge eorfi nthgeo pfatthhewpaayt hfowra eyfffiocireneftfi acnieanlotga nparolodgucptrioodn.u Fcrtioomn.
tFhreosme tvhieewsepvoiienwtsp, otihnet sm,tohset mimosptoirmtapnot rctaonmtpcooumnpdo uinn dthiins tchliasscs laosfs noaftnuaratul rparlopdruocdtus cttos teonegningeinere ear
haehteetreorloolgooguosu psrpordoudcutcitoino nsyssytsetmem wwoouuldld bbee stsrtircictotossididininee (1(111),) ,bbeeccaauussee iitt iiss tthhee uunniivveerrssaall iinntteerrmmeeddiiaattee
ffoorr tthhee bbiioossyynntthheessiiss ooff aallll MMIIAAss.. BBeellooww,, tthhee bbiioossyynntthheettiicc ppaatthhwwaayy lleeaaddiinngg ttoo tthhee ffoorrmmaattiioonn ooff 1111 aanndd
rreecceenntt eeffffoorrttss iinn tthhee eennggiinneeeerreedd bbiioossyynntthheessiiss ooff 1111 uussiinngg aa yyeeaasstt hhoosstt ssyysstteemm aarree ddiissccuusssseedd..
TThhee ggeenneerraall mmeecchhaanniissmm ooff tthhee bbiioossyynntthheessiiss ooff 1111 hhaass bbeeeenn rreevviieewweedd eellsseewwhheerree [[3388]].. BBrriieeffllyy,, 1111 iiss
ffoorrmmeedd wwiitthh ttwwoo ssttaarrttiinngg ssuubbssttrraatteess,,g geerraannioiolla annddL L-t-rtyrypptotopphhanan. T. Thheefi frisrtsst tsatratrintigngsu sbusbtrsatrtaetgee grearnaionlioisl
is initially converted into a dihydroxy intermediate, 8-hydroxygeraniol, by geraniol 8-hydroxylase
(G8H). Then, both hydroxyl groups are oxidized to form 8-oxogeranial. Subsequent cyclization and
oxidation of 8-oxogeranial yields loganic acid, and methylation of the carboxylic acid moiety of
loganic acid results in the formation of loganin (12) (Figure 9). The following transformation of 12
into secologanin (13) is an interesting reaction that is catalyzed by secologanin synthase (SLS, a
cytochrome P450). This step is thought to be the rate-limiting step in the biosynthetic pathway leading
to the formation of 11 [44]. SLS is proposed to catalyze the opening of the 5-membered ring of 12 by
Molecules2016,21,1078 8of19
initiallyconvertedintoadihydroxyintermediate,8-hydroxygeraniol,bygeraniol8-hydroxylase(G8H).
Then,bothhydroxylgroupsareoxidizedtoform8-oxogeranial. Subsequentcyclizationandoxidation
of8-oxogeranialyieldsloganicacid,andmethylationofthecarboxylicacidmoietyofloganicacid
resultsintheformationofloganin(12)(Figure9). Thefollowingtransformationof12intosecologanin
(13) is an interesting reaction that is catalyzed by secologanin synthase (SLS, a cytochrome P450).
Thisstepisthoughttobetherate-limitingstepinthebiosyntheticpathwayleadingtotheformationof
Molecules 2016, 21, 1078 8 of 19
11[44]. SLSisproposedtocatalyzetheopeningofthe5-memberedringof12byaradical-mediated
ah yrdadroicxayll-amtieodni,atfeodll ohwydedrobxyylaatdioenh, yfdorllaotwioendt obyy iae lddeh13yd(Fraigtiuorne t9o) y[4ie5l]d. 1T3h e(Foigthuerre s9t)a r[4ti5n]g. Tshueb sottrhaeter
sLt-atrrytipntgo psuhbasntriastme Lo-dtrifiypedtotphhraonu gihs madodecifairebdo xthyrloatuigohn sat edpectoarabfofoxrydlattrioynp tsatmepin teo, wafhfoicrhd itsrycopmtabminineed,
wwihtihch1 3ist ocogmivbein11edb ywtihthe 1a3c ttioo ngiovfes t1r1i cbtoys tihdein aectsiyonnt hoaf ssetr(iScTtoRs,idainnae msyinnethlaysaes e()S,TaRk, eayn eanmzyinme elyinasMe)I, Aa
kbeioys eynnzthyemsies .inT MhisIAtr baniossfyonrmthaetsiiosn. Twhoisu tlrdanbsefothrmematoiosnt cwhoemulidc ablely thinet meroesstti cnhgesmteipcailnlyt hinetesrtreiscttionsgi dstienpe
ibnio tshyen sthtreitcitcospiadtihnwe abyio;siytnisthaentiac npoatthhewraeyx;a mit pisle aonf athneotPhiecrt eetx-Sapmepnlgel eorf rtehaec tPioicnteat-pSppleiendglteorw raeradctitohne
abpiopslyiendth teoswisaorfda ltkhael obiidoss,yanstihtewsiass oafl saolkmaelonitdiosn, aeds idt uwriansg atlhseo dmisecnutsisoinoendo fdtuhreinsagf rthame dycisincubsisoisoynn tohfe tshise,
(sFaifgraumrey9c)in. Absiotsoytnhtehefsoirsm, (aFtiigounroe f91)1. ,Athse tore athctei ofnorimspatrioopno osef d11to, tohcec urreabcettiwone eins pthreopaoldseedh ytdoe oocfcu13r
banetdwteheen atmhei naeldoefhtyrdype toafm 1i3n eantod cthoeu palmeitnhee otwf torysputbasmtriantee stot hcroouupgleh tthhee tfworom sautbiosntraotfesa tβh-rcoaurgbho ltinhee
fmoromieatytio[4n6 o].f a β-carboline moiety [46].

Figure 9. Proposed biosynthetic pathway of strictosidine 11, the universal intermediate in the monoterpene
Figure 9. Proposed biosynthetic pathway of strictosidine 11, the universal intermediate in the
indole alkaloid biosynthesis [38]. G8H, geraniol 8-hydroxylase; SLS, secologanin synthase; STR,
monoterpeneindolealkaloidbiosynthesis[38].G8H,geraniol8-hydroxylase;SLS,secologaninsynthase;
strictosidine synthase.
STR,strictosidinesynthase.
To develop a yeast strain that can biosynthesize 11 de novo, 21 gene additions and three gene
Todevelopayeaststrainthatcanbiosynthesize11denovo,21geneadditionsandthreegene
deletions were introduced into the yeast genome [47]. Fourteen of the added genes were known genes
deletions were introduced into the yeast genome [47]. Fourteen of the added genes were known
from a MIA producer plant Catharanthus roseus [48]. Of the 14 genes, 11 constituted the actual strictosidine
genesfromaMIAproducerplantCatharanthusroseus[48]. Ofthe14genes,11constitutedtheactual
biosynthesis pathway and the other three were involved in enhancing the activity of P450s. Among
strictosidine biosynthesis pathway and the other three were involved in enhancing the activity of
the remaining seven added genes, five were existing yeast genes that were included as an extra copy
P450s. Amongtheremainingsevenaddedgenes,fivewereexistingyeastgenesthatwereincluded
of the genes in the genome. Incorporation of multiple copies of genes is frequently performed in
asanextracopyofthegenesinthegenome. Incorporationofmultiplecopiesofgenesisfrequently
building a heterologous production system to increase the expression level of specific genes for
performedinbuildingaheterologousproductionsystemtoincreasetheexpressionlevelofspecific
improved productivity of the biosynthetic pathway of interest [49]. This strategy was also applied to
genesforimprovedproductivityofthebiosyntheticpathwayofinterest[49]. Thisstrategywasalso
the strictosidine biosynthetic gene G8H, where four copies of the codon-optimized G8H gene were
appliedtothestrictosidinebiosyntheticgeneG8H,wherefourcopiesofthecodon-optimizedG8H
incorporated into the yeast genome to allow production of 11 without exogenous feeding of geraniol.
genewereincorporatedintotheyeastgenometoallowproductionof11withoutexogenousfeeding
Lastly, two more genes, AgGPPS2 and mFPS144, were taken from exogenous sources and incorporated
of geraniol. Lastly, two more genes, AgGPPS2 and mFPS144, were taken from exogenous sources
into the yeast genome to increase the cellular pool of geranyl pyrophosphate (GPP). AgGPPS2 is a
andincorporatedintotheyeastgenometoincreasethecellularpoolofgeranylpyrophosphate(GPP).
GPP synthase from grand fir (Abies grandis) that is known to produce only GPP and does not convert
AgGPPS2isaGPPsynthasefromgrandfir(Abiesgrandis)thatisknowntoproduceonlyGPPanddoes
GPP further into farnesyl pyrophosphate (FPP) [50], and mFPS144 is an N114W mutant of avian FPP
notconvertGPPfurtherintofarnesylpyrophosphate(FPP)[50],andmFPS144isanN114Wmutantof
synthase from the red junglefowl (Gallus gallus) that produces GPP nearly exclusively [51]. Those two
genes were used to replace the yeast FPP synthase gene ERG20, which was one of the three yeast genes
deleted from the genome for the biosynthesis of 11. The other two genes were ATF1 and OYE2, both of
which are involved in metabolizing geraniol into other products. After those extensive modifications,
the engineered yeast successfully produced 11 at a yield of approximately 0.5 mg/L of culture medium
comprised of only simple carbon and nitrogen sources [47]. This yeast strain provides an important first
step toward building a microbial system for the production of expensive, complex molecules that
only plants produce in small amounts.
Molecules2016,21,1078 9of19
avianFPPsynthasefromtheredjunglefowl(Gallusgallus)thatproducesGPPnearlyexclusively[51].
ThosetwogeneswereusedtoreplacetheyeastFPPsynthasegeneERG20,whichwasoneofthethree
yeastgenesdeletedfromthegenomeforthebiosynthesisof11. TheothertwogeneswereATF1and
OYE2,bothofwhichareinvolvedinmetabolizinggeraniolintootherproducts. Afterthoseextensive
modifications,theengineeredyeastsuccessfullyproduced11atayieldofapproximately0.5mg/Lof
culturemediumcomprisedofonlysimplecarbonandnitrogensources[47]. Thisyeaststrainprovides
animportantfirststeptowardbuildingamicrobialsystemfortheproductionofexpensive,complex
Mmoolelceucleus l2e0s16t,h 2a1t, 1o0n7l8y plantsproduceinsmallamounts. 9 of 19
55.. EErgrgootatammininee
EErrggoott aallkkaallooiiddss ffoorrmm aannootthheerr ffaammiillyy ooff aallkkaallooiiddss uunnddeerr tthhee ssttrruuccttuurraallllyy ddiivveerrssee ggrroouupp ooff TTIIAAss..
UUnnlliikkee MMIAIAss ddesecsrcirbiebde dini tnheth perepvrieovuiso suesctsieocnt itohnatt uhsaet GuPseP GanPdP La-tnrdypLto-tpryhpanto apsh tahneira sbuthilediirngb ubillodciknsg,
ebrlgoockt sa,lekraglooitdasl kaarleo iddesraivreedd efrroivmed dfirmomethdyimlaleltyhl yplaylrloyplhpoysropphhatoes p(DhaMteA(PDPM) AanPdP )La-tnrdypLt-otrpyhpatno p[5h2a]n. T[5h2e] .
eTrhgeote trygpoet otfy npaetuorfaln partoudraulctpsr eoxdeurtcst sbieoxloegrticsabl iaocltoivgiitcieasl bayc taivctiitniegs abs yeitahcetirn agn aasgoenitihste orr aann aangtoangiosntiostr
taonwaanrtdag voanriisotutso wreacredptvoarrsi ofours are ncuepmtobresr foofr naenuuromtrbaenrsomfnitetuerrso,t sruanchsm asit tdeorps,asmucinhea,s sderooptaomniinn,e a,dserreontaolninine,
aanddre nnaolrianderaenndalninoera [d5r3e]n. aClienreta[i5n3 ]m. Cemerbtaeirns mofe merbgeorts aolfkearlgooidtsa lhkaalvoei daslshoa vbeeeanls oshboewenns htoo wexnhtiobietx ahnibtii-t
taunmti-otru amctoivriaticetsiv [i5t4ie].s T[5h4e] p.oTtheentp boitoeancttibviiotaiecst iovfi teiregsoot faelkrgaolotiadlsk haalovied lsehda pveeolpelde tpoe uospel ethteomu sien tvhaermiouins
mvaerdioicuasl mapepdliiccaaltiaopnps lsicinatcieo neasrsliyn icne tehaer lhyuimntahne hhiustmorayn, whiisttho rthy,ew oiltdhestht ereoclodreds tdraetcinogrd bdacakti ntog abraocukntdo
1a0ro00u nBd.C1.0E0. 0[5B5.]C. .TEh.e[5 e5r]g.oTth aelkearglooitdasl karaelo pidrosdaurecepdr obdyu fcuendgbi yanfudn pgliaanntsd, bpulatn tthse, bCultavthiceipCiltaavceicaiep iftaamceialye
ofaf mfuinlygio ifs fmunogsti wisemll oksntowwenll fkonr oewrgnotf oprroedrguocttiponro. dAumctoionng .thAem ColanvgicthipeitCalcaevaiec fipamitailcye,a Celfaavmiceilpys, pCulrapvuicreepas,
apnu repnudreoap,ahnyteinc dpoaprhasyittiec pthaarta siist eatlshoa tkinsoawlson kanso twhen aesrgtohte feurgnogtufsu nofg uryseo,f arnyde, aNnedotNypehootydpiuhmod iluomlii,l oalini,
eanndeonpdhoypthicy tsiycmsybmiobnito,n atr,ea rteheth tewtow momosot-sstt-ustduideide deregrogto-tp-rpordoduucicningg fufunnggi i[5[522].] .AAnnooththeerr ffuunngguuss kknnoowwnn
ffoorr pprroodduuccttiioonn ooff tthhee eerrggoott ffaammiillyy ooff nnaattuurraall pprroodduuccttss,, pprriimmaarriillyy ccllaavviinnee--ttyyppee aallkkaallooiiddss,, iiss AAssppeerrggiilllluuss
ffuummiiggaattuuss,, aann ooppppoorrttuunniissttiicc ppaatthhooggeenn ooff mmaammmmaallss [[5566]].. BBeellooww,, wwee wwiillll ffooccuuss oonn tthhee bbiioossyynntthheettiicc
ppaatthhwwaayy ooff eerrggoott aallkkaallooiiddss,, aanndd iinn ppaarrttiiccuullaarr ddiissccuussss tthhee mmeecchhaanniissmm iinnvvoollvveedd iinn tthhee ffoorrmmaattiioonn ooff tthhee
iinntteerreessttiinngg bbaacckkbboonnee ccoorree ooff 66,,88--ddiimmeetthhyylleerrggoolliinnee tteettrraaccyycclliicc rriinngg ssttrruuccttuurree ((1144)) tthhaatt iiss sshhaarreedd bbyy aallll
eerrggoott aallkkaallooiiddss ((FFiigguurree 1100))..

FFiigguurree 1100.. PProrpoposoesde dbbioiosysnynthtehteitci cppatahthwwaya yoof fDD-l-ylysesergrgici caaccidid 2200 [[5533]],, hhaavviinngg tthhee pprriinncciippaall
66,,88--ddiimmeetthhyylleerrggoolliinnee tteettrraaccyycclliicc rriinngg ssttrruuccttuurree 1144 ooff eerrggoott aallkkaallooiiddss..
Biosynthesis of ergot alkaloids was initially investigated through feeding experiments that used
Biosynthesisofergotalkaloidswasinitiallyinvestigatedthroughfeedingexperimentsthatused
isotopically labeled substrates in the cultures of C. purpurea [53]. Based on those experiments, the
isotopically labeled substrates in the cultures of C. purpurea [53]. Based on those experiments, the
ergot biosynthetic pathway was determined to be initiated by the prenylation of L-tryptophan with
ergotbiosyntheticpathwaywasdeterminedtobeinitiatedbytheprenylationofL-tryptophanwith
DMAPP that forms 4-(γ,γ-dimethylallyl) tryptophan (15) (Figure 10) [57,58]. The next step involves
DMAPPthatforms4-(γ,γ-dimethylallyl)tryptophan(15)(Figure10)[57,58]. Thenextstepinvolves
the N-methylation of 15 to yield 4-dimethyl-L-abrine (16) [59]. Subsequently, a series of successive
theN-methylationof15toyield4-dimethyl-L-abrine(16)[59]. Subsequently,aseriesofsuccessive
oxidation steps is proposed to take place, resulting in the intramolecular cyclization of the prenyl and
oxidation steps is proposed to take place, resulting in the intramolecular cyclization of the prenyl
indole moieties to form the tricyclic chanoclavine-I (17) along with ring C of the tetracyclic core of
and indole moieties to form the tricyclic chanoclavine-I (17) along with ring C of the tetracyclic
ergot alkaloids [60]. Compound 17 is then oxidized to form chanoclavine-I-aldehyde (18) from which
ergot alkaloid biosynthesis can diverge. In one pathway, 18 can undergo intramolecular cyclization to
form ring D to complete the tetracyclic ergoline core structure, yielding agroclavine (19). Subsequently,
19 is converted to D-lysergic acid (20) via an oxidation of a methyl group as illustrated in Figure 10. In
another pathway, 18 undergoes a similar cyclization to form a closely related tetracyclic intermediate,
festuclavine, which becomes the core molecule for the clavine-type fumigaclavines and related
compounds produced by A. fumigatus. In this section, we will focus on the biosynthesis of ergotamine
(21) from 20, which takes place in C. purpurea and N. lolii.
Further steps in the biosynthesis of ergotamine (21) have been elucidated predominantly by
Keller et al. [61–64]. Those steps mainly concern the formation and the modification of the peptide-
derived segment of 21 (Figure 11). The peptide extension of 20 is initiated by LPS2 (lysergic peptide
synthetase 2, NRPS). The adenylation domain of LPS2 accepts 20 as the starter unit, and the condensation
Molecules2016,21,1078 10of19
core of ergot alkaloids [60]. Compound 17 is then oxidized to form chanoclavine-I-aldehyde (18)
fromwhichergotalkaloidbiosynthesiscandiverge. Inonepathway,18canundergointramolecular
cyclizationtoformringDtocompletethetetracyclicergolinecorestructure,yieldingagroclavine(19).
Subsequently,19isconvertedtoD-lysergicacid(20)viaanoxidationofamethylgroupasillustratedin
Figure10. Inanotherpathway,18undergoesasimilarcyclizationtoformacloselyrelatedtetracyclic
intermediate,festuclavine,whichbecomesthecoremoleculefortheclavine-typefumigaclavinesand
relatedcompoundsproducedbyA.fumigatus. Inthissection,wewillfocusonthebiosynthesisof
ergotamine(21)from20,whichtakesplaceinC.purpureaandN.lolii.
Further steps in the biosynthesis of ergotamine (21) have been elucidated predominantly
by Keller et al. [61–64]. Those steps mainly concern the formation and the modification of the
peptide-derivedsegmentof21(Figure11). Thepeptideextensionof20isinitiatedbyLPS2(lysergic
Molecules 2016, 21, 1078 10 of 19
peptidesynthetase2,NRPS).TheadenylationdomainofLPS2accepts20asthestarterunit,andthe
cdoonmdaeinns caotiuopnleds oimt waiinthc Lo-uaplalnesiniet, wwihthichL- aisl aancitnivea,twedh bicyh tihsea fcitrisvt aatdeednbyylattihoen fidrosmtaadine noyf lLatPiSo1n (dlyosmeragiinc
opfepLtPidSe1 s(ylynstheergtaiscep 1e, pNtiRdPeSs),y tnhtrhoeutgahs ean1 ,aNmRidPeS l)i,ntkhargoeu fgohrmaantiaomn.i Tdheel irneksualgtaenfto Drm-lyasteirogny.l-Tmhoenroepseuplttaidnet
iDn-tleyrsmeregdyila-mteo ins oepxteepntiddeedi nfuterrthmeerd wiaitteh iLs-epxhteennydleadlafnuirnthe earnwd iLth-pLr-oplhineen bylya lLaPnSin1e toa nydieLld-p trhoeli Dn-elybsyeLrgPySl1-
ttroipyeiepltdidteh, ewDh-ilcyhs erergmyali-ntrsi pteetphteidreed, wtoh tihceh prheomsapihnosptaenthteetrheedintyol tahremp ohfo tshpeh tohpiradn tthetihoelaintiyoln adrommoafint hoef
tLhPirSd1. thTihoel atteiormnidnoaml cayincliozfatLioPnS 1o.r Tlahcetatmerimzaintiaolnc ydcolmizaaitnio nis otrholaucgthamt tioz actaiotanlydzoem tahien riesletahsoeu gohf tthtoe
cDa-tlyalsyezrgeythl-etrripeleepatsiedeo ffrthomeD L-lPySs1e ragsy lN-t-r(iDp-elpystiedregyfrlo-Lm-ALlaP)S-L1-aPshNe--L(-DP-rloy-slearcgtayml-L (-LA,Lla-e)-rLg-oPthame-La-mP)r.o F-liancatlalmy,
(LL,L,L-e-ergrgootatammaamm i)s. cFoinnavlelryt,eLd, Li-netrog oetragmotaammiinsec o(2n1v)e brtye dthien thoyedrrgooxtyalmatiinoen (a2c1t)ivbiytyt hoef hthyed rnooxny-lhaetimone
adciotixvyitgyeonfasthe eEnaosHn- h(Feem2+e/2d-kioextoyggleuntaasreatEea-dsHep(eFned2+en/t2 -dkieotxoyggluentaarsaet)e [-6d4e]p. endentdioxygenase)[64].

FFiigguurree 1111.. NNoonnrriibboossoommaall aasssseemmbbllyy ooff tthhee ppeeppttiiddee ppoorrttiioonn ooff eerrggoottaammiinnee 2211,, iinniittiiaatteedd wwiitthh DD--llyysseerrggiicc
aacciidd 2200 asa as parimpreirm uenrit [u6n4i]t. L[P64S], .D-lyLsPeSrg,yDl -plyepsetirdgey slypnethpettiadsee; sCy,n ctohnedtaesnes;atiCo,n cdoonmdaeinns; aAt,i oandednyolmataioinn;
Ado,madaeinny; Tla,t tiohniodlaotmionai nd;oTm,athinio; lCatyico,n cydcolmolaizinat;iCony cd,ocmycaloinli.z ationdomain.
Very recently, heterologous partial reconstitution of the ergot alkaloid biosynthetic pathway was
Very recently, heterologous partial reconstitution of the ergot alkaloid biosynthetic pathway
accomplished in Aspergillus nidulans by introducing a segment of the A. fumigatus genome covering
was accomplished in Aspergillus nidulans by introducing a segment of the A. fumigatus genome
a portion of the ergot alkaloid biosynthetic gene cluster into the A. nidulans chromosome [65].
coveringaportionoftheergotalkaloidbiosyntheticgeneclusterintotheA.nidulanschromosome[65].
The integrated A. fumigatus genomic DNA segment contained four genes, dimethylallyltryptophan
TheintegratedA.fumigatusgenomicDNAsegmentcontainedfourgenes,dimethylallyltryptophan
synthase-coding dmaW, dimethylallyltryptophan N-methyltransferase-coding easF, oxidoreductase-
synthase-codingdmaW,dimethylallyltryptophanN-methyltransferase-codingeasF,oxidoreductase-like
like protein-coding easE and catalase-like protein-coding easC. The transformed A. nidulans was able
protein-coding easE and catalase-like protein-coding easC. The transformed A. nidulans was able
to produce 16 and 17, early intermediates in the D-lysergic acid biosynthetic pathway (Figure 10).
to produce 16 and 17, early intermediates in the D-lysergic acid biosynthetic pathway (Figure 10).
Using this heterologous production system, gene deletion experiments were performed to determine
Usingthisheterologousproductionsystem,genedeletionexperimentswereperformedtodetermine
for the first time that the four genes were sufficient for the biosynthesis of 17 [65]. This successful
forthefirsttimethatthefourgenesweresufficientforthebiosynthesisof17[65]. Thissuccessful
production of ergot alkaloid biosynthetic intermediates in A. nidulans would facilitate the effort
productionofergotalkaloidbiosyntheticintermediatesinA.nidulanswouldfacilitatetheefforttoward
toward achieving engineered biosynthesis of this class of natural products. Such a heterologous
achievingengineeredbiosynthesisofthisclassofnaturalproducts. Suchaheterologousbiosynthetic
biosynthetic platform would promote the study on the complex mechanism of ergot alkaloid biosynthesis
and advance the drug discovery efforts that evolve around ergot-type natural products.
6. Opiates
The last section will look at opiates, which is a group of alkaloids that has been known to humans
for about 6000 years and played a significant role as analgesic therapeutics and as abused recreational
substances [66]. Opiates are naturally occurring members of opioids that can be isolated from the
latex collected from opium poppy Papaver somniferum. The representative examples of opiates include
reticuline, thebaine, codeine and morphine (Figures 12–15). Opioids also include semi-synthetic and
synthetic compounds, for example oxycodone and fentanyl, that interact with opioid receptors to effect
physiological responses, such as analgesia, euphoria and respiratory depression [67]. Currently, the
pharmaceutical industry relies heavily on plants as the predominant source of opiates and opiate-derived
compounds. However, plant cultivation is labor intensive, difficult to scale up and hard to maintain
yield consistency. While metabolic engineering of plants has been attempted to improve growth
parameters and opiate productivity, the efforts have generally been met with difficulties thus far [68].