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Modifica(on of Methanococcus maripaludis for Produc(on of Geraniol Annika McNally, Travis Fetchko, and James Bevington University of Georgia iGEM Team Founda(onal Advance − Regional Jamboree East−10/13/2012 METHANOCOCCUS MARIPALUDIS • Methanogenic archaeon • Obligate anaerobe • Mesophilic • Autotrophic • Creates isoprenoid lipids • Doubling 7me of 2.3hrs M. maripaludis by Bonnie Chabon IDEA GENERATION • Opportunity to work with Methanococcus maripaludis • Wanted to make an aroma7c compound • A isoprenoid lipid synthesis intermediate, geranyl diphosphate, is a precursor of geraniol • Geraniol is an aroma7c compound that give roses their dis7nct smell • The goal: use synthe7c biology techniques to modify Methanococcus maripaludis for the produc7on of geraniol ABSTRACT This study determined if a specifically engineered plasmid could be transformed into Methanoccocus maripaludis and expressed for personal hygiene and energy usage. Methanococcus was synthe7cally modified via expression of a gene for geraniol synthase (GS) from Ocimum basilicum. The enzyme catalyzes a reac7on which results in geraniol, the major aroma7c compound in roses. Methanogens are archaea that live in the gut of humans and animals, and are responsible for the methane content of flatulence. They also have a robust isoprenoid biosynthe7c pathway, using isoprenoid‐based phospholipids as the major components of their cell envelopes. For that reason, they naturally make large amounts of geranyl diphosphate, the precursor of geraniol. Transforma7on of Methanococcus with the gene fpr GS was confirmed via PCR. The produc7on of small amounts of geraniol in whole cells was confirmed with GC and MS. In conclusion, this project converts personal polluters into an air freshener. This accomplishment poten7ally has a high impact factor, with the majority of the popula7on affected. GENE DESIGN • Iden7fied enzyme of origin • Ocimum basilicum (basil) • short amino acid sequence • inserted restric7on sites (5’NsI1;3’BglII) for cloning • Codon op7miza7on for M. maripaludis • Plant to Archaea • Gene synthesized by GenScript pAW42 SHUTTLE VECTOR • Shu[le vector expressed in E. coli and M. maripaludis • Strong promoter • RBS binding site upstream of the NsiI site • Ampicillin resistance for selec7on in E. coli • Puromycin resistance for selec7on in M. maripaludis Reference 1. CONSTRUCTION OF SHUTTLE VECTOR • PCR amplify synthe7c Geraniol Synthase (GS) gene, adding the flanking restric7on sites to the primers • Inserted GS gene into pAW42 at NsiI‐Xbal sites MEMBRANE LIPID BIOSYNTHESIS Isoprenoid Membrane Lipid Geraniol CLONING IN E. COLI AND VERIFICATION OF SHUTTLE VECTOR • Transformed shu[le vector into E. coli • Plated on ampicillin‐containing agar • Plant to Archaea • Extracted/Purified • Picked resul7ng colonies for plasmid miniprepara7ons • Restrict plasmids with EcoRV and screened by electrophoresis • Lanes 1‐4 are plasmids from four representa7ve colonies (all contain gene for GS) • Lane 5 was nega7ve for the gene Lanes 1‐4 are posi7ve for GS gene CONCLUSION • M. maripaludis can make geraniol upon the transforma7on of a pAW42 shu[le vector containing the gene for GS • This research adds to the founda7onal knowledge of synthe7c biology • Work done by this group has created interest for future iGEM teams at UGA SUGGESTIONS FOR FUTURE WORK • Increase geraniol produc7on by increasing expression by growing cells under different growth condi7ons • Different Verifica7on methods for expression (enzyme assay, Microarray) • Use GS from other sources, such as Cinnamomum tenuipilum • Inves7ga7ng the use of Geraniol as a biofuel • Addi7on of Geraniol acetyltransferase gene can convert geraniol to geranyl acetate, which is a be[er biodiesel alterna7ve than linear esters REFERENCES • Whitman, William B. "Methanococcus maripaludis strain C5, strain C6 and strain C7." Methanococcus maripaludis C6. Doe Joint Genome Ins7tute, n.d. Web. 8 Oct. 2012. <h[p://genome.jgi‐psf.org/metm6/ metm6.home.html>. • Walters, Alison D., Sarah Smith, and James Chong. "Shu[le Vector System for Methanococcus maripaludis with Improved Transforma7on Efficiency." Applied and Environmental Microbiology 77.7 (2011): 2549‐51. Print TRANSFORMATION OF PLASMID INTO M. MARIPALUDIS • Polyethylene Glycol transforma7on procedure • Verifica7on: • Colony PCR • Lanes 1‐4 are selected colonies • Lane C is a control • Lane 3 is posi7ve and contains the GS gene Gel electrophoresis of products from colony PCR GERANIOL • Hydrocarbon/alcohol • Poten7al use as biofuel • Used as fragrance • Component of rose, lemon and many other scents • Insect repellent • Precursor to terpenes • Important compounds • commercial applica7ons ACKNOWLEDGMENTS Other Team Members: Jessica Calkins Rachit Jain Andrew Upchurch Brice Bowerman Advisors: Dr. William Whitman Dr. Yajun Yan GC/MS Facility: Dennis Phillips • Unique anaerobic respira7on • Novel biochemistry with unusual coenzymes as C1 carriers h[p://faculty.washington.edu/leighj/mmmethanogenesis.html PRODUCT FORMATION AND VERIFICATION • Geraniol was expected to be no more than 5% of total lipid content • 20 nmol geraniol/5mL of culture • 0.6 µg/mL of geraniol • Extrac7on methods • Hexane extrac7on, then evaporated by N 2 • 60% recovery on control experiments • Verifica7on of geraniol by GC/MS geraniol Standard (1ng/mL) of geraniol in hexane Extracted Product *GC/MS trace from wild‐type cultures showed no geraniol METHANOGENESIS From GenScript

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Page 1: 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

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