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OentervofvDppliedvOhemistryv–nstitutevofvTechnicalvOhemistryOallinstrPv-Ev(N°qjv9annoverEvJermany
ProductionIofIsesquiterpenesIbyIbiocatalyticItransformationIofIsyntheticIfarnesylIdiphosphate
ThorevXristerEvSteffenv9artwigEvSaschavReutelEvThomasvScheper–nstitutevofvTechnicalvOhemistryEvJottfriedvWilhelmvLeibnizvUniversityv9annoverEvOallinstrPv-Ev(N°qjv9annoverEvJermany
Acknowledgements
Literature
-g
OPP
OPP
GPP
FPP
(+)-limonene
-pinene -pinene
HO
H
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humulene
HO
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HO
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O
eucalyptol
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patchoulol synthase
mevalonate
pathway
Acetyl-CoA
OPP
OPP
DMAPP
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germacrene A
gurjunene
H
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γ-
ggggggg
FPP-Synthesis Bioconversion
Introduction
Fig.I2vvvZxamplesvofvterpenesvvvvvvvvvvvvvproducedvinvnatureEvwithvXPPvandvJPPvasvavkeyvsubstrate
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Fig.I3ISynthesisvofvXPPvaccordingvtov“ellervetvalPvusingvLE,Eb-farnesolvasvstartingvmaterial
α β
Fig.I4v9PLOxchromatogramvofvavmixturevvvvvvvvvvofvfarnesylvpolyphosphatevesters
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Fig.I6IIIJOxchromatogramvofvthevbiocatalyticallyvproducedvsesquiterpenes
Retention time / min
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ty /
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2.000
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Fig.I5IIIProductionvofvsesquiterpenesvinvavtwoxphasestirredxtankvreactor
organic phase
aqueous phase
HO
H
HO
H
patchoulol
synthaseFPP
DownstreamingIandIanalytics
Xarnesol Xragrancecomponents
shortsynthesis
XPP recombinantterpene synthase downstreaming
Fig.I1vvvRioprocessvforvthevproductionvofvfragrancevcompoundsvbasedvonvrenewablevressourcesv
TerpenesvarevavlargevandvstructuallyvdiversevclassvofvsecondaryvmetabolitesEvwhichvarevmainlyvproducedvbyvplantsvasvthevmajorvcomponentsvinvresinsvandvessentialvoilsPvZspeciallyvmonoxvandvsesquiterpenesvexhibitvoftenvavstrongvsmellv andvarev thusv fequentlyvusedvasv fragrancevcompoundsv invperfumesvandvvariousv scentedvhouseholdvgoodsPvThesevdaysvthevmajorityvofvterpenevproductionvisvstillvbasedvonvthevisolationvfromvplantvderivedvrawvmaterialsPvDnvincreasingvconsumervdemandEvcombinedvwithvcropvfailuresvandvpoliticalvrisksvinvthevproducingvcountriesvisvleadingvtov higherv pricesv forv highv valuablev essentialv oilsv suchv asv patchouliv oilPv Thereforev newEv climaxindipendentv andvreliablevprocessesvforvthevproductionvofvterpenesvarevrequiredPvWevherebyvpresentvavbioprocessvbasedvonvthevlargexscalev productionvofv farnesylv diphosphatev LXPPbv asv substratev forv av recombinantlyv expressedvpatchoulolv synthasevLseevalsovpostervDvqjbv[°]P
Thev XPPv synthesisv publishedv byv “ellerv et alPv wasv foundv tov bev thev mostvconvenientv methodv forv av productionv ofv prenylv diphosphatesv inv av largervscalev[K]PvUsingvthisvsyntheticvapproachEvthevstartingvmaterialvLE,Ebxfarnesolvisv phosphorylatedv stepwisev inv av fastv andv costv effectivev reactionv atv lowvtemperaturesPv Xorv thev productv isolationEv thev crudev reactionv mixturev wasvappliedvonvavchromatographyvcolumnvL)PNNNvxv[NNvmmEvOVvVvKvlbvusingvNPjvkgvsilicavgelvqNvLK(Nx)NNvmeshbvasvstationaryvphasePvTheveluentvconsistedvinitiallyvofvavmixturevofvisoxpropanolkammoniavsolutionvLK-gvvvwkwbvwhichvwasv subsequentlyv changedv tov avhigherv ratiov ofv isoxpropanolPv Muevtov thev highv backxpressurev inv thevcolumnv av reductionv valvev wasvinstalledvtovmaintainvsafevhandlingvofvthevcoloumnvatvavconstantvflowvratev ofv °Nv mLkminPv Thisv fastvpurificationv stepv deliveredv av XPPvenrichedvsolutionvwhichvcontainedvmonoxv andv triphosphatev estersv asvwellPv Thev yieldv ofv XPPv canv bevroughlyv determinedv byv RPx9PLOvusingv av gradientv ofv N9)9OO(vL(NvmMbvsolutionvandvacetonitrileP
Thevbiocatalyticvproductionvofv sesquiterpenesvwasv performedv inv av twoxphasev stirredxtankxreactorPvDnvaqueousvMOPSOvbuffervcontainingvvthev patchoulolv synthaseEv XPPv andv MgOlKv wasvusedvforvthevenzymaticvconversionPv–nvordervtovisolatev thev veryv hydrophobicv sesquiterpenesvthev solutionv wasv coveredv withv av layerv ofvorganicv solventv [(]Pv Xorv thatv purposev severalvsolventsv havev beenv testedv inv regardv tovextractionv propertiesEv enzymev compatibilityvandv safev andv easyv handlingPv isoxoctanev andvhexamethyldisiloxanev L9MMSObv havev beenvfoundvtovbevoptimalvsolventsvinvthisvprocessP
Dfterv thev reactionEv thev organicv layerv wasvseparatedv inv av settlerPv –nv orderv tov ensurevcompletleyv productv recoveryEv thev aqueousvphasevwasv extractedvwithv organicv solventPvMuev tov thev highv proteinv contentv thevextractionvwasvsupportedvbyvcentrifugationvtov obtainv phasev separationPv SubsequentlyEvthev solventv wasv removedv carefullyv undervvacuumv resultingv inv av yellowv oilPv ThevsuccessfulvformationvofvsesquiterpenesvwasvconfirmedvbyvJOvandvJOxMSPvXorvthevexactvproductvquantificationvavmethodvbasedvonvShimadzuv JOxKN°Nv usingv av ZRxWDXplusvcolumnvwasvdevelopedPv
[°]vMeguerryvetvalPEvDrchPvRiochemPvRiophysPv)-)vLKNNqbEv°K(x°(q[K]v“ellervet alPEv:PvOhromaPvq)-vL°]](bEv°q°x°qj[(]vSieversvet alPEvMetabolicvZngineeringv°)vLKN°KbEv]°–°N(
ThisvworkvisvfundedvbyvthevZuropeanvRegionalvMevelopmentvXundvLZXRZb2v–nnovationvNetworkv“Refinmentvofvplantvresources“vLZWv[x[N°(N])NbWevwouldvlikevtovthankvProfPvRergervandvMrPv“ringsvL–nstitutevofvXoodvOhemistryEvLeibnizvUniversityv9anoverbvforvassistancevwithvterpenevanalyticsPWevwouldvlikevtovthankvSymrisevDJvforvthevprovisionvofvsubstratesPv
Wevpresentvavreliablevprocessvforvthevproductionvofvhighvvaluablevsesquiterpenesvbasedvonv thevbiocatalyticv conversionvofv thev keyxintermediatev XPPvusingv thev recombinantlyvexpressedvplantvenzymevpatchoulolvsynthasePvThisvsemixsyntheticvapproachvservesvasvavproofxofxprinciplevforvthisvplatformxproductionvstrategyPvDvscalexupvinvavmodelvreactorvbyv factorv °NNv wasv successfullyv accomplishedv alreadyPv Onev ofv thev mainv goalsv inv thevfuturevworkvisvthevsignificantvincreasevofvthevyieldvtovgetvcompetetivevvtovapproachesvbasedv onv pathwayv engineeredv microorganismsPv Xurtherv studiesv inv newv reactorvconceptsvsuchvasvbubblevcolumnsvandvenzymevimmobilizationvarevfocussedvonvpossiblevintegrationvinvindustrialvprocessesPvDlsovthevintegrationvofvnewvterpenevsynthasesvforvthevproductionvofvantibioticvterpenesvintovthevexistingvconceptvisvinvprogressPv
ConclusionIandIOutlook
phase boundary
