ModificaUon of Methanococcus maripaludis for ProducUon of Geraniol

Modifica(onofMethanococcusmaripaludisforProduc(onofGeraniolAnnikaMcNally,TravisFetchko,andJamesBevington

UniversityofGeorgiaiGEMTeamFounda(onalAdvance−RegionalJamboreeEast−10/13/2012

METHANOCOCCUSMARIPALUDIS•  Methanogenicarchaeon•  Obligateanaerobe•  Mesophilic•  Autotrophic•  Createsisoprenoidlipids•  Doubling7meof2.3hrs

M.maripaludisbyBonnieChabon

IDEAGENERATION

•  OpportunitytoworkwithMethanococcusmaripaludis•  Wantedtomakeanaroma7ccompound•  Aisoprenoidlipidsynthesisintermediate,geranyldiphosphate,isaprecursorofgeraniol•  Geraniolisanaroma7ccompoundthatgiverosestheirdis7nctsmell

•  Thegoal:usesynthe7cbiologytechniquestomodifyMethanococcusmaripaludisfortheproduc7onofgeraniol

ABSTRACTThisstudydeterminedifaspecificallyengineeredplasmidcouldbetransformedintoMethanoccocusmaripaludisandexpressedforpersonalhygieneandenergyusage.Methanococcuswassynthe7callymodifiedviaexpressionofageneforgeraniolsynthase(GS)fromOcimumbasilicum.Theenzymecatalyzesareac7onwhichresultsingeraniol,themajoraroma7ccompoundinroses.Methanogensarearchaeathatliveinthegutofhumansandanimals,andareresponsibleforthemethanecontentofflatulence.Theyalsohavearobustisoprenoidbiosynthe7cpathway,usingisoprenoid‐basedphospholipidsasthemajorcomponentsoftheircellenvelopes.Forthatreason,theynaturallymakelargeamountsofgeranyldiphosphate,theprecursorofgeraniol.Transforma7onofMethanococcuswiththegenefprGSwasconfirmedviaPCR.Theproduc7onofsmallamountsofgeraniolinwholecellswasconfirmedwithGCandMS.Inconclusion,thisprojectconvertspersonalpollutersintoanairfreshener.Thisaccomplishmentpoten7allyhasahighimpactfactor,withthemajorityofthepopula7onaffected.

GENEDESIGN•  Iden7fiedenzymeoforigin•  Ocimumbasilicum(basil)•  shortaminoacidsequence•  insertedrestric7onsites(5’NsI1;3’BglII)

forcloning

•  Codonop7miza7onforM.maripaludis•  PlanttoArchaea

•  GenesynthesizedbyGenScript

pAW42SHUTTLEVECTOR•  Shu[levectorexpressedinE.coliandM.maripaludis•  Strongpromoter•  RBSbindingsiteupstreamoftheNsiIsite•  Ampicillinresistanceforselec7oninE.coli•  Puromycinresistanceforselec7oninM.maripaludis

Reference1.

CONSTRUCTIONOFSHUTTLEVECTOR•  PCRamplifysynthe7cGeraniolSynthase(GS)gene,

addingtheflankingrestric7onsitestotheprimers•  InsertedGSgeneintopAW42atNsiI‐Xbalsites

MEMBRANELIPIDBIOSYNTHESIS

IsoprenoidMembraneLipid

Geraniol

CLONINGINE.COLIANDVERIFICATIONOFSHUTTLEVECTOR•  Transformedshu[levectorintoE.coli•  Platedonampicillin‐containingagar•  PlanttoArchaea

•  Extracted/Purified•  Pickedresul7ngcoloniesforplasmid

miniprepara7ons•  RestrictplasmidswithEcoRVandscreened

byelectrophoresis

•  Lanes1‐4areplasmidsfromfourrepresenta7vecolonies(allcontaingeneforGS)•  Lane5wasnega7veforthegene Lanes1‐4areposi7veforGSgene

CONCLUSION

•  M.maripaludiscanmakegeranioluponthetransforma7onofapAW42shu[levectorcontainingthegeneforGS

•  Thisresearchaddstothefounda7onalknowledgeofsynthe7cbiology

•  WorkdonebythisgrouphascreatedinterestforfutureiGEMteamsatUGA

SUGGESTIONSFORFUTUREWORK

•  Increasegeraniolproduc7onbyincreasingexpressionbygrowingcellsunderdifferentgrowthcondi7ons

•  DifferentVerifica7onmethodsforexpression(enzymeassay,Microarray)

•  UseGSfromothersources,suchasCinnamomumtenuipilum

•  Inves7ga7ngtheuseofGeraniolasabiofuel•  Addi7onofGeraniolacetyltransferasegenecanconvertgeranioltogeranyl

acetate,whichisabe[erbiodieselalterna7vethanlinearesters

REFERENCES•  Whitman,WilliamB."MethanococcusmaripaludisstrainC5,strainC6andstrainC7."Methanococcus

maripaludisC6.DoeJointGenomeIns7tute,n.d.Web.8Oct.2012.<h[p://genome.jgi‐psf.org/metm6/metm6.home.html>.

•  Walters,AlisonD.,SarahSmith,andJamesChong."Shu[leVectorSystemforMethanococcusmaripaludiswithImprovedTransforma7onEfficiency."AppliedandEnvironmentalMicrobiology77.7(2011):2549‐51.Print

TRANSFORMATIONOFPLASMIDINTOM.MARIPALUDIS•  PolyethyleneGlycoltransforma7onprocedure

•  Verifica7on:•  ColonyPCR•  Lanes1‐4areselectedcolonies•  LaneCisacontrol•  Lane3isposi7veandcontainstheGSgene

GelelectrophoresisofproductsfromcolonyPCR

GERANIOL•  Hydrocarbon/alcohol•  Poten7aluseasbiofuel

•  Usedasfragrance•  Componentofrose,lemonandmanyotherscents

•  Insectrepellent•  Precursortoterpenes•  Importantcompounds•  commercialapplica7ons

ACKNOWLEDGMENTS OtherTeamMembers:JessicaCalkinsRachitJainAndrewUpchurchBriceBowerman

Advisors:Dr.WilliamWhitmanDr.YajunYan

GC/MSFacility:DennisPhillips

•  Uniqueanaerobicrespira7on

•  NovelbiochemistrywithunusualcoenzymesasC1carriers

h[p://faculty.washington.edu/leighj/mmmethanogenesis.html

PRODUCTFORMATIONANDVERIFICATION•  Geraniolwasexpectedtobenomorethan5%oftotallipidcontent•  20nmolgeraniol/5mLofculture•  0.6µg/mLofgeraniol

•  Extrac7onmethods•  Hexaneextrac7on,thenevaporatedbyN2

•  60%recoveryoncontrolexperiments•  Verifica7onofgeraniolbyGC/MS

geraniolStandard(1ng/mL)ofgeraniolinhexane

ExtractedProduct

*GC/MStracefromwild‐typeculturesshowednogeraniol

METHANOGENESIS

FromGenScript