FigS5 A E coli coexpressionassaysofPxaTPS8proteinvariantsFig4comparedwiththewildtype enzymeandtheemptyvectorcontrolpET28b PeakISinternalstandard1eicosenepeak1pseudolaratrienepeak10 ID: 839822
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1 Fig.S5. ( A )Totalionchromatogramsofprod
Fig.S5. ( A )Totalionchromatogramsofproductsresultingfrom E . coli coexpressionassaysofPxaTPS8proteinvariants(Fig.4)comparedwiththewild-type enzymeandtheemptyvectorcontrolpET28b( + ).PeakIS,internalstandard1-eicosene;peak1,pseudolaratriene;peak10,dephosphorylatedGGPP;peaks11 14, unidentifiedputativediterpeneolefins.( B )Massspectracorrespondingtopeaks11 14in A . Mafuetal. www.pnas.org/cgi/content/short/1612901114 4of8 Fig.S4. Massspectraofcompoundsidentifiedin P . amabilis .Shownaremassspectraofselectcompoundsidentifiedin P . amabilis root,bark,andneedle tissuethatmayrepresentendogenousditerpenesasbasedonsignaturemassions. Mafuetal. www.pnas.org/cgi/content/short/1612901114 3of8 Fig.S3. ProductsofinvitroassaysofPxaTPS5andPxaTPS8.( A and B )GC-MSanalysisofreactionproductsobtainedfrominvitroenzymeassaysof recombinantPxaTPS5withFPPasasubstrate( A )andwithPxaTPS8withGGPPasasubstrate( B ).( C )ActivityassaysofPxaTPS8withGPPorFPPassubstrate resultedonlyinthecorrespondingdephosphorylatedsubstrates,demonstratin
2 gnoconversionbyPxaTPS8.( D ) E . coli co
gnoconversionbyPxaTPS8.( D ) E . coli coexpressionassaysofPxaTPS8witha ( + )-CPPsynthasefrom Marrubiumvulgare oran ent -CPPsynthasefrom Zeamays .NoadditionalproductswereobservedcomparedwiththeindividualCPP synthasereactionproducts.GC/MStracesareillustratedasTICs. Mafuetal. www.pnas.org/cgi/content/short/1612901114 2of8 SupportingInformation Mafuetal.10.1073/pnas.1612901114 Fig.S1. Naturalditerpenescaffoldsingymnosperms. Fig.S2. TPSgeneannotationinthe P . amabilis roottranscriptome.TPScandidategeneswereidentifiedwithan E -valuethresholdof1E-50.Relativetranscript abundanceisbasedonfragmentsperkilobaseofexonpermillionfragmentsmapped(FPKM)valuesobtainedviamappingofIlluminareadsagainstassemb led TPSreads.F,full-lengthtranscript;P,partialtranscript. Mafuetal. www.pnas.org/cgi/content/short/1612901114 1of8 DatasetS1.NMRandquantumchemicalanalysisofpseudolaratriene DatasetS1 Mafuetal. www.pnas.org/cgi/content/short/1612901114 8of8 TableS2.Oligonucleotidesusedinthisstudy DesignationGeneSequence(5 3 ) pET2
3 8b( + )PxaTPS5-forward TATCATATGGCTGGCGT
8b( + )PxaTPS5-forward TATCATATGGCTGGCGTTTCTGTTG PxaTPS5-reverse TATGTCGACTTAAAGTGGGAGCGGATCA PxaTPS8-forward TATCATATGATACGGTCAAATACTTCG PxaTPS8-reverse TGAGAGCTCTCATAAAGACACAGCTTG pET28b( + )PxaTPS8variantsR558A-sense CCACGTTTCTATGTGCAGTGAACCTC R558A-antisense GAGGTTCACTGCACATAGAAACGTGG S696I-sense ATGTAAACCAATCGTGACCTTGCTGTTC S696I-antisense GAACAGCAAGGTCACGATTGGTTTACAT S696V-sense CTATATGTAAACCAACCGTGACCTTGCTGT S696V-antisense ACAGCAAGGTCACGGTTGGTTTACATATAG G697S-sense ATCGCTATATGTAAACTACTCGTGACCTTGCTG G697S-antisense CAGCAAGGTCACGAGTAGTTTACATATAGCGAT Y564T-sense CCATAGTGTAAGTTTCCACGTTTC Y564T-antisense GAAACGTGGAAACTTACACTATGG Y564V-sense CCATAGTGTAAACTTCCACGTTTC Y564V-antisense GAAACGTGGAAGTTTACACTATGG Y564H-sense ATAGAAACGTGGAACATTACACTATGGC Y564H-antisense GCCATAGTGTAATGTTCCACGTTTCTAT Y564F-sense CCATAGTGTAAAATTCCACGTTTC Y564F-antisense GAAACGTGGAATTTTACACTATGG Y564W-sense TAGAAACGTGGAATGGTACACTATGGCAT Y564W-antisense ATGCCATAGTGTACCATTCCACGTTTCTA A591T-sense AATTGTGTGGAATCACTACTTGTATCGATG A591T-ant
4 isense CATCGATACAAGTAGTGATTCCACACAATT A5
isense CATCGATACAAGTAGTGATTCCACACAATT A591Q-sense ATTGTGTGGAATCCCAACTTGTATCGAT A591Q-antisense ATCGATACAAGTTGGGATTCCACACAAT H670A-sense TATAATTGATGCGGCTCTGGTAGAAGC H670A-antisense GCTTCTACCAGAGCCGCATCAATTATA H670Y-sense TATAATTGATGCGTATCTGGTAGAAGCG H670Y-antisense CGCTTCTACCAGATACGCATCAATTATA H670W-sense TATAATTGATGCGTGGCTGGTAGAAG H670W-antisense CTTCTACCAGCCACGCATCAATTATA A701C-sense GTGAGGATGGGCAATATACATATATGTAAACCAC A701C-antisense GTGGTTTACATATATGTATATTGCCCATCCTCAC A701L-sense GATGGGCAATATCAGTATATGTAAACCACTCG A701L-antisense CGAGTGGTTTACATATACTGATATTGCCCATC G735S-sense GCCTCCACCATTAGTCGACTAAGTGGT G735S-antisense ACCACTTAGTCGACTAATGGTGGAGGC G735W-sense ACTTGGCCTCCACCATTTGGCGACTAAGT G735W-antisense ACTTAGTCGCCAAATGGTGGAGGCCAAGT G739N-sense TTGGTCGACTAAGTAATGACATGAACG G739N-antisense CGTTCATGTCATTACTTAGTCGACCAA G739D-sense GGTCGACTAAGTGATGACATGAACGCC G739D-antisense GGCGTTCATGTCATCACTTAGTCGACC A743T-sense GGTGACATGAACACCTACAAGATTGACC A743T-antisense GGTCAATCTTGTAGGTGTTCATGTCACC PxaTPS8:helixHchimera5
5 -forward CAAATGGGTCGCGGATCCATGTCAAATACTT
-forward CAAATGGGTCGCGGATCCATGTCAAATACTTCGAAATATG PxaTPS8:helixHchimera3 -reverse GGTGGTGGTGCTCGAGTTATAAAGACACAGCTTGAACG PxaTPS8:helixHchimerainternal-forward GGTCTTTGTGTCGTCTGCCAGGCGGGAAATGAGGCTGTTAAGCTCTGTGAGAACA- CGCCTAGACAGAGTATCAATTTCCTGCA PxaTPS8:helixHchimerainternal-reverse GCCTCATTTCCCGCCTGGCAGACGACACAAAGACCTACAAAGCTGAGAAGGCTCGT- GGAGAAGAATCTTCATGCATATCGTGTTATATG pLIFE33PxaTPS5-forward GGCTTAAUAATGGCTGGCGTTTCTGTTG PxaTPS5-reverse GGTTTAAUTTAAAGTGGGAGCGGATCAATTAGAC PxaTPS8-forward GGCTTAAUAATGTCAAGATTTACATCTGCTGC PxaTPS8-reverse GGTTTAAUTTATAAAGACACAGCTTGAACGAG pESC-HisPxaTPS8-forward GAATTCAACCCTCACTAAAGGGCGGCCGCATGACGAAAAGGAGTGAAGCAGATG PxaTPS8-reverse CTTGTAATCCATCGATACTAGTGCGGCCGCTTATAAAGACACAGCTTGAACGAG- AGTTC qRT-PCRPxaTPS5-forward CTTGATTCGAAGAACGGCCAA PxaTPS5-reverse TCGTTGATTCTCCATCTCCTGC PxaTPS8-forward TGCTAGAAACGCTTGGGAGG PxaTPS8-reverse ATGGTGGAGGCCAAGTGATG EF-1 -forward CCTGGCCATCGTGACTTCAT EF-1 -reverse AAGTGAACGCCAATAGGGCA Mafuetal. www.pnas.org/cgi/content/short/16129
6 01114 7of8 TableS1.AbbreviationsandGenBa
01114 7of8 TableS1.AbbreviationsandGenBank/EuropeanBioinformaticsInstituteDataBankaccession numbersofditerpenesynthasesusedforphylogeneticanalyses AbbreviationProteinAccessionno. AbCAS Cis -abienolsynthase, Abiesbalsamea JN254808 AbISOIsopimaradienesynthase, Abiesbalsamea JN254806 AbLASLevopimaradiene/abietadienesynthase, Abiesbalsamea JN254805 AgASAbietadienesynthase, Abiesgrandis AAK83563 AgBIS E - -Bisabolenesynthase, Abiesgrandis AF006195 AgCAMCamphenesynthase, Abiesgrandis AAB70707 AgHUM -Humulenesynthase, Abiesgrandis AAC05728 AgLIM4 S -Limonenesynthase, Abiesgrandis AAB70907 AgSEL -Selinenesynthase, Abiesgrandis AAC05727 AgTOLTerpinolenesynthase, Abiesgrandis AAF61454 GbLSLevopimaradienesynthase, Ginkgobiloba AAS89668 PaBIS E - -bisabolenesynthase, Piceaabies AY473619 PaFAR -Farnesenesynthase, Piceaabies AAS47697 PaISOIsopimaradienesynthase, Piceaabies AY473620 PaLASLevopimaradiene/abietadienesynthase, Piceaabies AAS47691 PaLIMLimonenesynthase, Piceaabies AAS47694 PaLIN(-)-Li
7 naloolsynthase, Piceaabies AAS47693 PaLO
naloolsynthase, Piceaabies AAS47693 PaLONLongifolenesynthase, Piceaabies AAS47695 PaMYRMyrcenesynthase, Piceaabies AAS47696 PaPIN / -Pinenesynthase, Piceaabies AAS47692 PbaISOIsopimaradienesynthase, Pinusbanksiana M4HXW5 PbaLASLevopimaradiene/abietadienesynthase, Pinusbanksiana M4HXU6 PbaPIMPimaradienesynthase, Pinusbanksiana M4HY08 PcISOIsopimaradienesynthase, Pinuscontorta JQ240314 PcLASLevopimaradiene/abietadienesynthase, Pinuscontorta JQ240310 PcPIMPimaradienesynthase, Pinuscontorta JQ240315 PgCAR3-Carenesynthase, Piceaglauca ADZ45510 PgCIN1,8-Cineolesynthase, Piceaglauca ADZ45498 PgLIN(-)-Linaloolsynthase, Piceaglauca ADZ45500 PmBIS E - -Bisabolenesynthase, Pseudotsugamenziesii AAX07266 PmFAR -Farnesenesynthase, Pseudotsugamenziesii AAX07265 PmTOLTerpinolenesynthase, Pseudotsugamenziesii AAX07264 PpCPSKS Ent -kaurene/kaurenolsynthase, Physcomitrellapatens BAF61135 PsiCAR3-Carenesynthase, Piceasitchensis ADU85924 PsiISOIsopimaradienesynthase, Piceasitchensis ADZ45512 PsiLASLevopimara
8 diene/abietadienesynthase, Piceasitchens
diene/abietadienesynthase, Piceasitchensis ADZ45517 PsiLONLongipinenesynthase, Piceasitchensis ADZ45516 PsiPHE -Phellandrenesynthase, Piceasitchensis ADZ45506 PsiSABSabinenesynthase, Piceasitchensis ADU85929 PsyCARY -Caryophyllenesynthase, Pinussylvestris B4XAK4 PtFAR -Farnesenesynthase, Pinustaeda AAO61226 PtTOL -Terpineolsynthase, Pinustaeda Q84KL4 PxaTPS4Levopimaradiene/abietadienesynthase, Pseudolarixamabilis AGN70885 TbaTXSTaxadienesynthase, Taxusbaccata AAR02861 TbrTXSTaxadienesynthase, Taxusbrevifolia U48796 TcTXSTaxadienesynthase, Taxuscuspidata DQ305407 TxmTXSTaxadienesynthase, Taxusxmedia AAS18603 Mafuetal. www.pnas.org/cgi/content/short/1612901114 6of8 Fig.S6. Pseudolaratrieneformationinengineeredyeast.ShownareGC/MSTICsofthePxaTPS8productionaftercoexpressionwiththeyeastGGPPsynthase (BTS1)intheengineeredyeast( S . cerevisiae )strainAM94.Pseudolaratrienewaspurifiedonsilicamatrix.GGPP-ol,geranylgeraniol(dephosphorylatedGGPP). Mafuetal. www.pnas.org/cgi/content/short/1612901114 5o