Supporting Information

Transkript

1 Biosynthesis of Novel Statins by Combining Heterologous Genes from Xylaria and Aspergillus Hiroya Itoh, Makoto Matsui, Yuki Miyamura, Itaru Takeda, Jun Ishii, Toshitaka Kumagai, Masayuki Machida, Takashi Shibata, Masanori Arita Supporting Information Contents 1. Sequence Alignment of FrlB (X. grammica), LovB (A. terreus), and MlcA (C. pilosus) proteins: 6 pages with designation of six PKS domains 2. Figures S1-S6: MS, MS/MS analysis of FR91512, lovastatin, and the two new statins 3. Table S1: Identified gene clusters in four filamentous fungi by the MIPS-CG software program 4. Table S2: Result of Quantitative PCR 5. Cloning, sequencing, and qpcr primer sequences

2 Supporting Information Sequence Alignment of FrlB (X. grammica), LovB (A. terreus), and MlcA (P. Citrinumu) proteins. FrlB 1 MAPSI-PNEPIAVIGSGCRFPGGINTPSKLWEQLKVPTDLQRPIPQSRFNIESFYNPDGS LovB 1 MAQSMYPNEPIVVVGSGCRFPGDANTPSKLWELLQHPRDVQSRIPKERFDVDTFYHPDGK mlca 1 MDQANYPNEPIVVVGSGCRFPGGVNTPSKLWELLKEPRDVQTKIPKERFDVDTFYSPDGT consensus 1 *...*****.*.********..********.*. *.*.*..**..**...** ***. KS domain FrlB 6 HPGRTNAKHAYTLSEDTHAFDAPFFNIQPHEAEAIDPQQRLLLETVYEGIASAGLKVEDL LovB 61 HHGRTNAPYAYVLQDDLGAFDAAFFNIQAGEAESMDPQHRLLLETVYEAVTNAGMRIQDL mlca 61 HPGRTNAPFAYLLQEDLRGFDASFFNIQAGEAETIDPQQRLLLETVYEAVSNAGLRIQGL consensus 61 *.*****..**.*..*...*** *****..***..***.*********...**...* FrlB 12 AGSPTAVYVGVMTHDFETNITRDLSSVPTHTATGISLCIASNRLSYFFDWHGPSMTLDTA LovB 121 QGTSTAVYVGVMTHDYETVSTRDLESIPTYSATGVAVSVASNRISYFFDWHGPSMTIDTA mlca 121 QGSSTAVYVGMMTHDYETIVTRELDSIPTYSATGVAVSVASNRVSYFFDWHGPSMTIDTA consensus 121.*..******.****.**..**.*.*.**..***...****.************.*** FrlB 18 CSSSLVAVHLAVQHLRSGNGTLAVAAGANLILDPMPYVLESKLNMLSPTGRSRMWDAAAD LovB 181 CSSSLVAVHLAVQQLRTGQSSMAIAAGANLILGPMTFVLESKLSMLSPSGRSRMWDAGAD mlca 181 CSSSLAAVHLAVQQLRTGESTMAVAAGANLILGPMTFVMESKLNMLSPNGRSRMWDAAAD consensus 181 *****.*******.**.*...*.********.**..*.****.****.********.** FrlB 24 GYARGEGVACVILKTLSRALADGDQIECIIRETGVNQDGRTPGITMPNNEAQAALIRDTY LovB 241 GYARGEAVCSVVLKTLSQALRDGDTIECVIRETGVNQDGRTTGITMPNHSAQEALIKATY mlca 241 GYARGEGVCSIVLKTLSQALRDGDSIECVIRETGINQDGRTTGITMPNHSAQEALIRATY consensus 241 ******.*...*****.**.***.***.*****.******.******..**.***..** FrlB 3 SRAGLDLAKTSDRCQFFEAHGTGTPAGDPKEAEAISTAFLSHGQ----RGSSESPLYVGS LovB 31 AQAGLDITKAEDRCQFFEAHGTGTPAGDPQEAEAIATAFFGHEQVARSDGNERAPLFVGS mlca 31 AKAGLDITNPQERCQFFEAHGTGTPAGDPQEAEAIATAFFGHKDGTIDSDGEKDELFVGS consensus 31..****....*****************.*****.***..*.....*.*** FrlB 356 IKTVIGHTEGTAGIAGLMKASLAVQHGIIPPNLLFEELSPRVAAFYDGLRIVKEAQRWPE LovB 361 AKTVVGHTEGTAGLAGLMKASFAVRHGVIPPNLLFDKISPRVAPFYKNLRIPTEATQWPA mlca 361 IKTVLGHTEGTAGIAGLMKASFAVRNGVIPPNLLFEKISPRVAPFYTHLKIATEATEWPI consensus 361.***.********.*******.**..*.*******...*****.** *.*.**. ** FrlB 416 LAPGQPRRASVNSFGFGGTNAHAIIENYIPSGNGKLSLPAGQHQQDIPKPVSICTLPLVI LovB 421 LPPGQPRRASVNSFGFGGTNAHAIIEEYMEPEQNQLRV---SNNEDCPPMTGVLSLPLVL mlca 421 VAPGQPRRVSVNSFGFGGTNAHAIIEEYMAPPHKPTAV-VTEVTSD----ADACSLPLVL consensus 421..******.*****************.* *....****. FrlB 476 SAKSKRSMKSVMKDLVSFLKTNPGIQMQDVLWTHLKKRSTFNVRRAIAGARTQDEACAAL LovB 478 SAKSQRSLKIMMEEMLQFLDSHPEIHLHDLTWSLLRKRSVLPFRRAIVG-HSHETIRRAL mlca 476 SSKSQRSMKATLENMLQFLETHDDVDMHDIAYTLLEKRSILPFRRAIAA-HNKEVARAAL consensus 481 *.**.**.*...**.....*....*.***...**** ** FrlB 536 DREIASFDAKEDQWVSTTDLSSKPGILGIFTGQGAQWPSMGQKLLATSALARETLAELDE LovB 537 EDAIEDGIVSSD---FTTEVRGQPSVLGIFTGQGAQWPGMLKNLIEASPYVRNIVRELDD mlca 535 EAAIADGEVVTD---FRTDANDNPRVLGVFTGQGAQWPGMLKKLMVGMPFVRGILEELDN consensus 541..*....*..*...*.**.*********.*...*....*.. ***. AT domain

3 FrlB 596 SLQTLPPEYRPGWKLYDEIMRPKDESNVMNASFSQPLCTAVQIMLVRMLEAVGINFTKVI LovB 594 SLQSLPEKYRPSWTLLDQFMLEGEASNVQYATFSQPLCCAVQIVLVRLLEAARIRFTAVV mlca 592 SLQTLPEKYRPTWTLYDQLMLEGDASNVRLASFSQPLCCAVQIVLVRLLAAAGIEFSAIV consensus 61 ***.**..***.*.*.*..*...*** *.******.****.***.*.*..* *... FrlB 656 GHSSGEIACAYAANFITAVQAIRIAYLRGAVVAKNSASPTRVKGAMLAAGASYEDALDLC LovB 654 GHSSGEIACAFAAGLISASLAIRIAYLRGVVSAGGA---RGTPGAMLAAGMSFEEAQEIC mlca 652 GHSSGEIACAFAAGFISATQAIRIAHLRGVVSAEHASSPSGQTGAMLAAGMSYDDAKELC consensus 661 **********.**..*.*..*****.***.*.*.... *******.*...*..* FrlB 716 GLEAFQGRVCVAAHNSPDSVTLSGDEDGILEIKEVLEDESKFARLLNVDMAYHSHHMLPC LovB 711 ELDAFEGRICVAASNSPDSVTFSGDANAIDHLKGMLEDESTFARLLKVDTAYHSHHMLPC mlca 712 ELEAFEGRVCVAASNSPDSVTFSGDMDAIQHVEGVLEDESTFARILRVDKAYHSHHMHPC consensus 721.*.**.**.****.*******.***..*...*****.***.*.** *******.** FrlB 776 APHYTHSMEVCGYSIND-GNLVESSSPTWYSSVH-EGKVMTPADTDQNYWKDNLVSPVLF LovB 771 ADPYMQALEECGCAVADAGS--PAGSVPWYSSVDAENRQMAARDVTAKYWKDNLVSPVLF mlca 772 AAPYVKALLECDCAVAD-GQ--GNDSVAWFSAVHETSKQMTVQDVMPAYWKDNLVSPVLF consensus 781 *.*...*...* *. *. *.*.*....*. *. ************ FrlB 834 SKALTCALGDDSKQIDIAIEVGPHPALKGPALSTMK-AMTGNELPYTGCMQRWGDDADAF LovB 829 SHAVQRAV-VTHKALDIGIEVGCHPALKSPCVATIKDVLSGVDLAYTGCLERGKNDLDSF mlca 829 SQAVQKAV-ITHRLIDVAIEIGAHPALKGPCLATIKDALAGVELPYTGCLARNVDDVDAF consensus 841 *.*...*.....*..**.* *****.*...*.*...*..*.****. *.*.*.* FrlB 893 SGVLGFLWERFGSGCLDV PRPVDGESLAKALPLYPWDHSRSYWATSRRAHA LovB 888 SRALAYLWERFGASSFDADEFMRAVAPDRPC--MSVSKLLPAYPWDRSRRYWVESRATRH mlca 888 AGGLGYIWERFGVRSIDAEGFVQQVRPDRAV--QNLSKSLPTYSWDHTRQYWAESRSTRQ consensus 91...*...*****..* *.....* ** *.**..* **..**.. FrlB 944 FLQGGRPHLLLGKLSADSTTSTLVWDNSIRPHDIEWLDGHGLQGQTVFPGAGYVIMAMEA LovB 946 HLRGPKPHLLLGKLSEYSTPLSFQWLNFVRPRDIEWLDGHALQGQTVFPAAGYIVMAMEA mlca 946 HLRGGAPHLLLGKLSSYSTASTFQWTNFIRPRDLEWLDGHALQGQTVFPAAGYIIMAMEA consensus 961.*.*..*********.**...* *..**.*.******.********.***..***** DH domain FrlB 4 ALH--ATSGRQVQLLEVMGLSIDKAITFEDEQSSVEISLKAQIIKPESSGTNTVVLSFGI LovB 6 ALMIAGTHAKQVKLLEILDMSIDKAVIFDDEDSLVELNLTADVSR-NAGEAGSMTISFKI mlca 6 AMKVAGERAAQVQLLEILDMSINKAIVFEDENTSVELNLTAEVTS-DNDADGQVTVKFVI consensus 121 *...**.***...**.**..*.**...**..*.* * * FrlB 162 DSCLSKESNMSSSASGQIVVTLG---PTVSSG---THLPPAREELPHMNKVNLDTFYKEL LovB 165 DSCLSKEGNLSLSAKGQLALTIEDVNPRTTSASDQHHLPPPEEEHPHMNRVNINAFYHEL mlca 165 DSCLAKESELSTSAKGQIVITLGEASPSS------QLLPPPEEEYPQMNNVNIDFFYREL consensus 181 ****.**...*.**.**...*... *....***..** *.**.**.. **.** FrlB 1116 SGIGYQYTKDFREILSLKRTDGKTTGSISF-PQCPDGDG-ALILHPATLDLTFQTIIAAY LovB 1125 GLMGYNYSKDFRRLHNMQRADLRASGTLDFIPLMDEGNGCPLLLHPASLDVAFQTVIGAY mlca 1119 DLLGYDYSKDFRRLQTMRRADSKASGTLAFLPLKDELRNEPLLLHPAPLDIAFQTVIGAY consensus **.*.****.....*.*...*.. *.*......*.****.**..***.*.**

4 FrlB 1174 SSPGDKRLRSLHVPTNIDRIAIVPSLCATLYEQGIDKIHFNSSTRVPTSNAIRGDIEAFT LovB 1185 SSPGDRRLRCLYVPTHVDRITLVPSLCLATAESGCEKVAFNTINTYDKGDYLSGDIVVFD mlca 1179 SSPGDRRLRSLYVPTHVDRVTLIPSLCISAGNSGETELAFDTINTHDKGDFLSGDITVYD consensus 121 *****.***.*.***..**...****.....*...*......***... FrlB 1234 TTDGSMPGGERTTLFQVEGITFKPFTPPSAADDHRMFSKWAWGPLAPRSLLDNEAYWATA LovB 1245 A EQTTLFQVENITFKPFSPPDASTDHAMFARWSWGPLTPDSLLDNPEYWATA mlca 1239 S TKTTLFQVDNIVFKPFSPPTASTDHRIFAKWVWGPLTPEKLLEDPATLIIA consensus ******..*.****.**.*..**..*..* ****.*..**...* FrlB 1294 EDKTVMPIMERAVYFYIRDFLEQLTKEDRQHVSPHHEQQIKWFEYISVEAKEGRLHLWYD LovB 1297 QDKEAIPIIERIVYFYIRSFLSQLTLEERQQAAFHLQKQIEWLEQVLASAKEGR-HLWYD mlca 1291 RDKEDILTIERIVYFYIKSFLAQITPDDRQNADLHSQKYIEWCDQVQADARAGH-HQWYQ consensus 1321 **....**.*****..** *.*..**.. *...*.*.....*..*. *.**. FrlB 1354 VSWEADTHVTITQMCERYSYHPHVRLIRRIGENLTDIITYGTRNPFELMDHDGLLTDFYA LovB 1356 PGWENDTEAQIEHLCTANSYHPHVRLVQRVGQHLLPTVR-SNGNPFDLLDHDGLLTEFYT mlca 135 ESWEEDTSVHIEQMCESNSSHPHVRLIQRVGKELISIVR-GNGDPLDIMNRDGLFTEYYT consensus 1381.** **. *...*..*.******..*.* * *...***.*..*. cmt domain FrlB 1414 DPHSSGPSLSYFEDLVTDITHRYQNMDILEIGAGTGGATKHVLNSKHHTINSYTFTDLSS LovB 1415 NTLSFGPALHYARELVAQIAHRYQSMDILEIGAGTGGATKYVLATPQLGFNSYTYTDIST mlca 149 NKLAFGSAIHVVQDLVSQIAHRYQSIDILEIGLGTGIATKRVLASPQLGFNSYTCTDISA consensus *....**..*.****..******.***.***.**...****.**.*. FrlB 1474 SLLDKARDSFAENETRIEFRRLDICQKPEEQGFTSQGYDMVIASNVLHATPRLEETMANV LovB 1475 GFFEQAREQFAPFEDRMVFEPLDIRRSPAEQGFEPHAYDLIIASNVLHATPDLEKTMAHA mlca 1469 DVIGKAREQLSEFDGLMQFEALDINRSPAEQGFKPHSYDLIIASDVLHASSNFEEKLAHI consensus **..... *. ***..*.****...**..***.****...*...*.. FrlB 1534 RSLLKPGGRLVIVELTHRKHSRLGFIFGLFPEWWAGQGEGRTWEPFVSMDEWNGILKRTG LovB 1535 RSLLKPGGQMVILEITHKEHTRLGFIFGLFADWWAGVDDGRCTEPFVSFDRWDAILKRVG mlca 1529 RSLLKPGGHLVTFGVTHREPARLAFISGLFADRWTGEDETRALSASGSVDQWEHTLKRVG consensus 1561 ********..*...**...**.**.***...*.*...*...*.* *...***.* FrlB 1594 FSGIDSRTSDRDAALFPNSCFSTHAVNSRVMQLDNPL--SVPAKDENSPAVVVIGGATSR LovB 1595 FSGVDSRTTDRDANLFPTSVFSTHAIDATVEYLDAPLASSGTVKD-SYPPLVVVGGQTPQ mlca 1589 FSGVDSRTLDREDDLIP-SVFSTHAVDATVERLYDPL--SAPLKD-SYPPLVVIGGESTK consensus 1621 ***.****.**.. *.* *.*****...*. *. ** *...**..*..**.**... FrlB 1652 TKPMIERLQEILPRRRFVHIQYLRDIHETD-LEPNSSFLVLSELDDALFLDLDPDKFEAI LovB 1654 SQRLLNDIKAIMPPRPLQTYKRLVDLLDAEELPMKSTFVMLTELDEELFAGLTEETFEAT mlca 1645 TERILNDMKAALPHRHIHSVKRLESVLDDPALQPKSTFVILSELDDEVFCNLEEDKFEAV consensus *.*.....*.... *..*.*..*.***...* *...***. FrlB 1711 KVILHYASNLLFVTENAWVQHPQQAMVIGLLRSLRLENPGVSMQVLDADCLENVSIETLA LovB 1714 KLLLTYASNTVWLTENAWVQHPHQASTIGMLRSIRREHPDLGVHVLDVDAVETFDATFLV mlca 175 KSLLFYAGRMMWLTENAWIDHPHQASTIGMLRTIKLENPDLGTHVFDVDTVENLDTKFFV consensus 1741 *..* **...*****..**.**..**.**...*.*...*.*.*.*......

5 FrlB 1771 EQLLRLEYGAGWQESGLVWTQEPEIYLSNGRLMIPRLRHDAERNARLNATRRPILADVNL LovB 1774 EQVLRLEEHTDELASSTTWTQEPEVSWCKGRPWIPRLKRDLARNNRMNSSRRPIYEMIDS mlca 1765 EQLLRFEESDDQLLESITWTHEPEVYWCKGRAWVPRLKQDIARNDRMNSSRRPIFGNFNS consensus 181 **.**.*......**.***...**..***..*..** *.*..****..... FrlB 1831 NDSPVILRQTPHG----VHLECSSVKGEPSAVE------IRVRFALAKSIYVTKLGFLYL LovB 1834 SRAPVALQTARDSSS--YFLESAETWFVPESVQQMETKTIYVHFSCPHALRVGQLGFFYL mlca 1825 SKTAIALKEARGASSSMYYLESTETCDSLEDARHAGKATVRVRYALPQAIRVGHLGYFHV consensus * ** *...*..**... FrlB 1881 TQG--VDTSSGKPVIALSQENGSTICVPASHVFDLNSTN----NDQCVLLHLAAELTAQN LovB 1892 VQGHVQEGNREVPVVALAERNASIVHVRPDYIYTEADNNLSEGGGSLMVTVLAAAVLAET mlca 1885 VQGSILENTCEVPVVALAEKNGSILHVPRNYMHSLPDNM-AEGEDSSFLLSTAAALLAET consensus 1921.**......**.**...*.*...* **...*.. FrlB 1935 IMSHVSS---GAAVLILEPPEFCVEALVRAAKSKAVTVKLATMQPARLDTVRGRETQWIL LovB 1952 VISTAKCLGVTDSILVLNPPSICGQMLLHAGEEIGLQVHLATTSGNRSSVSAGDAKSWLT mlca 1944 ILSSAQSFGSDASILIMEPPIFCVKAILESAKTYGVQVHLATT----LSDVKTIPAPWIR consensus * *...**.* *.*** *. FrlB 1992 LHEKETHSVIESKLSASNVSHFYDFSADQGPFSLGRRLATCLPPTCLVHGTERLFQDLAT LovB 212 LHARDTDWHLRRVL-PRGVQALVDLSADQSCEGLTQRMMKVLMPGCAHYRAADLFTDTVS mlca 2 LHAKETDARLKHSL-PTNMMAFFDLSTDRTAAGITNRLAKLLPPSCFMYSGDYLIRSTAS consensus 241 **...*.... *......*.*.*.....*...*.*.*.... *.... FrlB 252 QTIYD---EDTSAQRGLVDYSLATVTND---PAISPS--IIPADQVV GSEGP LovB 271 TELHSGSRHQASLPAAYWEHVVSLARQGL--PSVSEGWEVMPCTQFAA------HADKTR mlca 259 TYKVS---HVEDIP--ILEHSVAMAKNTVSASTVDDTEKVITATQILLPGQLSVNHNDQR consensus *..... FrlB 296 WDISTVIDW---HSDQTLTARIRPVDATDLFTSTKTYILVGLAGDLGRSLARWMIKHGAR LovB 2123 PDLSTVISWPRESDEATLPTRVRSIDAETLFAADKTYLLVGLTGDLGRSLGRWMVQHGAC mlca 2114 FNLATVIDW----KENEVSARICPIDSGNLFSNKKTYLLVGLTGDLGRSLCRWMILHGAR consensus ***.*...*...*. **. ***.****.*******.***. ***. KR domain FrlB 2153 HIVLSSRNPQVDDRWLEETRKTGGNVLVLPMDVSDKPSVDAGLAKISASGMPVIGGVAFG LovB 2183 HIVLTSRNPQVNPKWLAHVEELGGRVTVLSMDVTSQNSVEAGLAKLKDLHLPPVGGIAFG mlca 217 HVVLTSRNPRLDPKWIANMEALGGDITVLSMDVANEDSVDAGLGKLVDMKLPPVAGIAFG consensus 2221 *.**.****...*.....**..**.***. **.***.*....*...*.*** FrlB 2213 PLVLQDILFKNMDLEMMEMVLAPKVTGATILNEALA----HTALDFFVMFSSVVAVLGNP LovB 2243 PLVLQDVMLNNMELPMMEMVLNPKVEGVRILHEKFSDPTSSNPLDFFVMFSSIVAVMGNP mlca 223 PLVLQDVMLKNMDHQMMDMVLKPKVQGARILHERFSEQTGSKALDFFIMFSSIVAVIGNP consensus 2281 ******...**.. **.*** *** *..**.*......****.****.***.*** FrlB 2269 GQTAYSAANSYMHGLAQQRRAQGLAGSTIDIGAVYGVGYVTRAGREEEFDAVRFLFDTVS LovB 233 GQANYSAANCYLQALAQQRVASGLAASTIDIGAVYGVGFVTRAELEEDFNAIRFMFDSVE mlca 229 GQSNYGAANAYLQALAQQRCARGLAGSTIDIGAVYGVGFVTRAEMEEDFDAIRFMFDSVE consensus 2341 **..*.*** *...***** * ***.************.****..**.*.*.**.**.*.

6 FrlB 2329 EDELHALFAEAVIAGQPG TPL-TQDVEVITGVPFINPVYRERIPFFDDP LovB 2363 EHELHTLFAEAVVAGRRAVHQQEQQRKFATVLDMADLELTTGIPPLDPALKDRITFFDDP mlca 235 EHELHTLFAEAVVSDQRA--RQQPQRK--TVIDMADLELTTGIPDLDPALQDRIIYFNDP consensus 241 *.***.****** *...*.*..**.*..*...**.*.** FrlB 2377 RFGYYLLKSEES-QGEVSGAASNGTIKEQLAKAESMDKVREIVANGLSLKLRSTLQTAAE LovB 2423 RIGNLKIPEYRGAKAGEGAAGSKGSVKEQLLQATNLDQVRQIVIDGLSAKLQVTLQIPDG mlca 246 RFGNFKIPGQRG-DGGDNGSGSKGSIADQLKQATTLDQVRQIVIDGLSEKLRVTLQVSDG consensus 2461 *.* *.*...**.*...*.**.**..*** **..***... FrlB 2436 DSIDVTSSLIDQGVDSLGAVTIGTWFSKQLNLDLPLLKVLGGASITDLATEAATRLPPSA LovB 2483 ESVHPTIPLIDQGVDSLGAVTVGTWFSKQLYLDLPLLKVLGGASITDLANEAAARLPPSS mlca 2465 ESVDPTIPLIDQGVDSLGAVTVGSWFSKQLYLDLPLLRVLGGASVADLADDAATRLPATS consensus 2521.*...*..*************.*.******.******.******..***.**.***... FrlB 2496 IPLVLSTSSADSEDTSSAGGHVPSSDETTTDSLGGSKSSAVSSV------TESDDENDEA LovB 2543 IPLVAAT DGGAE--STDNTSENEVSGREDTDLSAAATITEPSSADEDDTEP mlca 2525 IPLLLQI--GDSTGTSDSGAS--PTPTDSHDEASSATSTDASSA----EEDEEQEDDNEQ consensus 2581 *** * * * FrlB 255 KELMLVRREK-LSLTQEYSWAIQKQVQDPSILNSTIGMFMEGPIDFNLLAKAVNAVLRRH LovB 2592 GDEDVPRSHHPLSLGQEYSWRIQQGAEDPTVFNNTIGMFMKGSIDLKRLYKALRAVLRRH mlca 2577 GGRKILRRER-LSLGQEYSWRQQQMVKDHTIFNNTIGMFMKGTIDLDRLRRALKASLRRH consensus *... ***.*****..*.. *...*.******.*.**..*.*..*.**** ACP domain FrlB 269 DIFRTRF------HESANGPTQDVMSLPSPLVQLQNIPVANRAAALEGFTQLEQQKYAHG LovB 2652 EIFRTGFA-----NVDENGMAQLVFGQTKNKVQT--IQVSDRAGAEEGYRQLVQTRYNPA mlca 2636 EIFRTCFVTGDDYSSDLNGPVQVVLKNPENRVHF--VQVNNAAEAEEEYRKLEKTNYSIS consensus 271.**** *. **. *.*.....*...*..*.*.*...*...*. FrlB 2663 EGDTTRFIIYHWSSEHHLLVLGYSRFVGDGQTTENIFNEIGQYYAGASVPSPAQQYADFT LovB 275 AGDTLRLVDFFWGQDDHLLVVAYHRLVGDGSTTENIFVEAGQLYDGTSLSPHVPQFADLA mlca 2694 TGDTLRLVDFYWGTDDHLLVIGYHRLVGDGSTTENLFNEIGQIYSGVKMQRPSTQFSDLA consensus 2761 ***.*...*...****..*.*.****.****.*.*.**.* *... *..*.. FrlB 2723 VRQLRDYESGRMDEDIDFWVSLHKS-----P--VPVLPMLALPQAQPRGAAADGRV---- LovB 2765 ARQRAMLEDGRMEEDLAYWKKMHYR-----PSSIPVLPLMRPLVGNSSRSDTPNFQHCGP mlca 2754 VQQRENLENGRMGDDIAFWKSMHSKVSSSAPTVLPIMNLINDPAANSEQQQIQPFT---- consensus *..* ***..*...*...*. *..* FrlB 2772 WGQHTASVRLNPMIAIRIKERSRKHKAAPMHFYLTAFHVLLARLTSTADVAIGFADTNRH LovB 282 WQQHEAVARLDPMVAFRIKERSRKHKATPMQFYLAAYQVLLARLTDSTDLTVGLADTNRA mlca 281 WQQYEAIARLDPMVAFRIKERSRKHKATPMQFYLAAYHVLLARLTGSKDITIGLAETNRS consensus 2881 *.*..*..**.**.*.***********.**.***.*..*******. *...*.*.*** FrlB 2832 NLQDMTTMGYFANLLPVRM-AYAPGDTFGDELVATKDFLRGALPHSRVPYGVVLERLGLA LovB 288 TVDEMAAMGFFANLLPLRFRDFRPHITFGEHLIATRDLVREALQHARVPYGVLLDQLGLE mlca 287 TMEEISAMGFFANVLPLRFDEFVGSKTFGEHLVATKDSVREAMQHARVPYGVILDCLGLN consensus **.***.**.*.... ***..*.**.*.*.*..*.******.*. ***

7 FrlB 2891 SP-PAQEEPAAAAAAAAPLFQAVFDYRQGQAESGSIGAAKITEVVASRERTPHDVVLEIS LovB 294 VPVPTSNQP APLFQAVFDYKQGQAESGTIGGAKITEVIATRERTPYDVVLEMS mlca 293 LP-TSGEEP--KTQTHAPLFQAVFDYKQGQAESGSIGNAKMTSVLASRERTPYDIVLEMW consensus 31.*....* **********.*******.**.**.*.*.*.*****.*.***.. FrlB 295 DDPTKTPLITFKLKDAMYGPGDVQYIANAYLAVLSVFSRNPALKVNEGRLDQTAKGVAG LovB 2993 DDPTKDPLLTAKLQSSRYEAHHPQAFLESYMSLLSMFSMNPALKL A mlca 2987 DDPTKDPLIHVKLQSSLYGPEHAQAFVDHFSSILTMFSMNPALKL A consensus 361 *****.**.. **...*... *......*..**.*****.. The key domains [ketosynthase (KS), acyltransferase (AT), dehydratase (DH), carbon methyltransferase (cmt), ketoreductase (KR), and acyl carrier protein (ACP)] are displayed with an underline. Three of the consensus motifs in methyltransferases domain are indicated by the red boxes.

8 A) B) _FR91512_STD _FR91512_STD e-1 2.5e e-1 2.e e-1 3: Diode Array Range: 9.67e _FR91512_STD _FR91512_STD 3.47 TIC 2.7e6 AU 1.5e e-1 1.e-1 7.5e-2 5.e-2 2.5e C) D) _FR91512_STD _FR91512_STD Da 9.64e _FR91512_STD_MSMS _FR91512_STD_MSMS 397 (3.465) : TOF MSMS ES- 5.18e m/z Figure S1. Analysis of FR91512 standard. A) UPLC chromatogram ( nm), B) Total ion current chromatogram, C) MS spectrum (m/z: ), D) MS/MS result detected by negative ion mode with collision energy of 2 ev.

9 A) B) _FRFull_ _FRFull_ : Diode Array Range: _FRFull_ _FRFull_ TIC 1.67e AU 1. 8.e e-1 4.e-1 2.e C) D) _FRFull_ _FRFull_ Da 2.27e _nega_21_FRFull_ECM_14_1_MSMS _nega_21_FRFull_ECM_14_1_MSMS2 396 (3.456) : TOF MSMS ES m/z Figure S2. Analysis of FR91512 produced by yeast. A) UPLC chromatogram ( nm), B) Total ion current chromatogram, C) MS spectrum (m/z: ), D) MS/MS result detected by negative ion mode with collision energy of 2 ev.

10 A) B) _nega_1214_ecm_FRdF_LovD_addLovF_ _nega_1214_ecm_FRdF_LovD_addLovF_ : Diode Array Range: _nega_1214_ecm_FRdF_LovD_addLovF_ _nega_1214_ecm_FRdF_LovD_addLovF_1.55 TIC 1.8e AU e e-1 4.e-1 2.e _nega_FRdF_ecm22_lovDaddafoG_1_MSMS _nega_FRdF_ecm22_lovDaddafoG_1_MSMS (5.171) : TOF MSMS ES- 2.8e C) D) _nega_1214_ecm_FRdF_LovD_addLovF_ _nega_1214_ecm_FRdF_LovD_addLovF_ Da 2.49e m/z Figure S3. Analysis of Methylbutyryl-DA-FR91512 produced by yeast. A) UPLC chromatogram ( nm), B) Total ion current Methylbutyryl_DA_FR chromatogram, C) MS spectrum (m/z: ), D) MS/MS (2 ev) shares fragments of MW212, 254, and 316 with the standard (Fig S1).

11 A) B) _lovastatin_STD _lovastatin_STD.46 3: Diode Array Range: _lovastatin_STD _lovastatin_STD 5.22 TIC 4.4e6 3.5e-1 3.e-1 2.5e AU 2.e-1 1.5e e e _lovastatin_STD _lovastatin_STD Da 1.99e _lovastatin_STD_MSMS 21738_lovastatin_STD_MSMS 596 (5.196) : TOF MSMS ES- 1.11e C) D) m/z Figure S4. Analysis of Lovastatin standard. A) UPLC chromatogram ( nm), B) Total ion current chromatogram, C) MS spectrum Lovastatin STD (m/z: ), D) MS/MS result detected by negative ion mode with collision energy of 2 ev.

12 A) B) _lov _lov9.64 3: Diode Array Range: _lov _lov9.54 TIC 1.28e AU e e e-1 2.e C) D) _lov _lov Da 1.53e _lov9_MSMS _lov9_MSMS (5.248) : TOF MSMS ES m/z Figure S5. Analysis of Lovastatin produced by yeast. A) UPLC chromatogram ( nm), B) Total ion current chromatogram, C) MS Lovastatin Sample spectrum (m/z: ), D) MS/MS result detected by negative ion mode with collision energy of 2 ev.

13 A) B) 21738_lovdDF_frlD _lovdDF_frlD : Diode Array Range: _lovdDF_frlD _lovdDF_frlD1.55 TIC 1.7e AU 1. 8.e e-1 4.e-1 2.e C) D) 21738_lovdDF_frlD _lovdDF_frlD Da 7.62e _lovdDF_frlD1_MSMS _lovdDF_frlD11_MSMS5 48 (3.566) : TOF MSMS ES m/z Figure S6. Analysis of O-acetylmonacolin J produced by yeast. A) UPLC chromatogram ( nm), B) Total ion current chromatogram, O-acetyl_monacolin J Sample C) MS spectrum (m/z: ), D) MS/MS (2 ev) shares fragments of MW32 as the base peak (MW85 is a noise).

14 Table S1. Identified gene clusters in the four filamentous fungi Metarhizium majus ARSEF 297 Metarhizium brunneum ARSEF 3297 Pestalotiopsis fici W16-1 Aspergillus clavatus NRRL 1 Query locus ID Max score Total score Query cover E value Ident Accession FrlA MAJ_ E KID FrlB FrlC MAJ_9235 MAJ_ E+ 9.E KID KID MAJ_9237 MAJ_ E-25 9.E KID KID FrlG MAJ_ E KID FrlA MBR_ E XP_ FrlB MBR_ E+ 39 XP_ FrlC MBR_ E XP_ FrlG MBR_ E XP_ FrlA PFICI_ E XP_ FrlB PFICI_ E+ 41 XP_ FrlC PFICI_ E XP_ FrlG PFICI_ E XP_ FrlA PFICI_ E XP_ FrlB PFICI_ E+ 51 XP_ PFICI_ E+ 31 XP_ FrlC PFICI_ E XP_ FrlG PFICI_ E XP_ FrlA FrlB ACLA_5574 ACLA_ E-14.E XP_ XP_ ACLA_5578 ACLA_ E-55 2.E XP_ XP_ FrlC ACLA_ E XP_ FrlG ACLA_ E XP_ Ident: indicates the amino acid sequence identity with each FR91512 biosynthesis gene.

15 Table S2. Results of qpcr analysis Promoter HIS3 Gene FR91512 lovastatin Empty FR91512 FR91512 FR91512 FR91512 FR91512 lovastatin Vector FR91512 FR91512 FR91512 FR91512 FR91512 FR91512 lovastatin lovastatin lovastatin ΔfrlF ΔfrlF ΔfrlF ΔfrlF ΔfrlF ΔlovF all ΔfrlF ΔfrlDF ΔfrlADF Ex.F Ex.DF all ΔlovDF ΔlovADF Ex.A Ex.B Ex.C Ex.D Ex.G Ex.D 2.3±.6 1.8± ± ± ± ± ± ± ±.3 8.2±.2 6.2± ± ±3. 1.4±1. 8.8± ±2.2 patp423 P ADH1 P TDH3 P PGK3 frla 21.9± ± ± ± ± ±.5 2.4± ± ±2.8 lova 15.3± ± ± ±5.2 frlc 43.7± ± ± ±1. 8.9± ± ± ± ± ±11. lovc 37.± ± ± ± ±8.8 frld 19.6±2. 3.3± ± ± ± ± ±7.6 lovd 13.4± ± ±3.4 Gene Expression LEU2 3.2± ±3. 24.± ± ±.2 1.1±.6 3.±1. 2.4± ± ±.9 8.9± ± ± ±.2 8.9± ±1.9 P ADH1 npga 11.4± ± ± ±2. 7.9± ± ± ± ±.6 8.7± ± ± ±.3 8.2± ±7. Relative to HTA1 expression patp425 P TDH3 frlb 28.8± ± ± ± ±.5 4.3± ± ±2. 19.± ±2.4 lovb 5.± ± ± ± ±7.4 frlg 1.7±.6.9±.2 2.3±.1 1.8±.1.9±. 2.1±.1 2.±.3 2.4±.1 1.2±.1 5.9±.5 P PGK3 lovg 4.5±.8 6.7±.9 6.8±.3 5.2±.9 1.2±.1 URA3 1.8±.5 4.2±.3 7.4± ±.6 5.±.6 3.4±. 7.9±.3 7.5± ±.5 4.9±.4 2.8±.5 3.9±.2 3.8±.3 2.9±.1 2.9±.4 4.3±.5 patp426 P ADH1 P TDH3 frlh 2.3±.1 1.6± ± ±.6 3.±.2 1.1±.6 8.2± ±.5 6.9± ±.3 4.5±. ivra 7.8± ± ± ±8.8 frlf 48.5± ±4.5 lovf 14.4± ± ±.5 P PGK3 lovi 8.3±1.5 The expression level of each gene is shown as a relative value with HTA1 expression. The values are Mean value ± Standard deviation of three replicate experiments.

16 Supporting Information: Cloning primers 1/2 Lower case letters indicate overlapping sequences for in fusion cloning. name sequence template organism frla_pmei-f atacacctagggtttaaacatggaggccttcaacactct Xylaria grammica frla_pmei-r cgcggccggccgtttaaacctagacgtggaattcctcct Xylaria grammica frlb_noti-f gacacgcgtgcggccgcatggctccttcaatacccaa Xylaria grammica frlb_noti-r taaattcacgcggccgcttaacccgctacccccttgg Xylaria grammica frlb_mid1-r cttgtcgatgctcaatcccatg Xylaria grammica frlb_mid2-f ttgagcatcgacaaggccatca Xylaria grammica frlb_mid2-r actcagcttggattcgataacg Xylaria grammica frlb_mid3-f gaatccaagctgagtgcttcga Xylaria grammica frlc_noti-f gacacgcgtgcggccgcatggggtcgtcggcgccaga Xylaria grammica frlc_noti-r taaattcacgcggccgcctaggccacccgaaccacaa Xylaria grammica frld_xmai-f aatataaaaccccgggatgcaagatattgaaagagc Xylaria grammica frld_xmai-r tcggcgcgcccccgggtcaatcgttcctcagctgct Xylaria grammica frlf_noti-f gacacgcgtgcggccgcatgtctgtcgaacccattgc Xylaria grammica frlf_noti-r taaattcacgcggccgctcaagagatcttggcattac Xylaria grammica frlf_mid1-r agtcgatgttccacgattgatg Xylaria grammica frlf_mid2-f cgtggaacatcgactggaagcc Xylaria grammica frlg_xmai-f aatataaaaccccgggatgaccgcacaacaggtgga Xylaria grammica frlg_xmai-r tcggcgcgcccccgggtcattcatttggtataactc Xylaria grammica frlh_pmei-f atacacctagggtttaaacatgtttggccgcggcatcgttga Xylaria grammica frlh_pmei-r cgcggccggccgtttaaactcaggccccagccggcggag Xylaria grammica lova_pmei-f atacacctagggtttaaacatgactgtcgacgcgctcac Aspergillus terreus ATCC2542 lova_pmei-r cgcggccggccgtttaaacctatagtgaaccaggaaggc Aspergillus terreus ATCC2542 lovb_noti-f gacacgcgtgcggccgcatggctcaatctatgtatcc Aspergillus terreus ATCC2542 lovb_noti-r taaattcacgcggccgctcatgccagcttcagggcgg Aspergillus terreus ATCC2542 lovb_mid1-r atgcatgtccatcaagccattc Aspergillus terreus ATCC2542 lovb_mid2-f ttgatggacatgcattgcaagg Aspergillus terreus ATCC2542 lovb_mid2-r gcatgcaatgttagccaggact Aspergillus terreus ATCC2542 lovb_mid3-f gctaacattgcatgctcgcgac Aspergillus terreus ATCC2542 lovc_noti-f gacacgcgtgcggccgcatgggcgaccagccattcat Aspergillus terreus ATCC2542 lovc_noti-r taaattcacgcggccgcttacggcccctcgagccgaa Aspergillus terreus ATCC2542 lovd_xmai-f aatataaaaccccgggatgggatccatcattgatgc Aspergillus terreus ATCC2542 lovd_xmai-r tcggcgcgcccccgggttaaccctgctggtactgcg Aspergillus terreus ATCC2542

17 Supporting Information: Cloning primers 2/2 Lower case letters indicate overlapping sequences for in fusion cloning. name sequence template organism lovf_noti-f gacacgcgtgcggccgcatgacaccattagatgcgcc Aspergillus terreus ATCC2542 lovf_noti-r taaattcacgcggccgcctaagctttgtatctctgca Aspergillus terreus ATCC2542 lovf_mid1-r gatgtctggagcccaatgccag Aspergillus terreus ATCC2542 lovf_mid2-f tgggctccagacatcagcttag Aspergillus terreus ATCC2542 lovg_xmai-f aatataaaaccccgggatgcgttaccaagcatctcc Aspergillus terreus ATCC2542 lovg_xmai-r tcggcgcgcccccgggctactccaatgtctgggccg Aspergillus terreus ATCC2542 ivra_pmei-f atacacctagggtttaaacatggatccggtggttagaaa Aspergillus terreus ATCC2542 ivra_pmei-r cgcggccggccgtttaaacttacctacctaagttacatg Aspergillus terreus ATCC2542 lovi_xmai-f aatataaaaccccgggatgacatcccaccacggtga Aspergillus terreus ATCC2542 lovi_xmai-r tcggcgcgcccccgggtcattcgctccgtcctttctc Aspergillus terreus ATCC2542 npga_pmei-f atacacctagggtttaaacatggtgcaagacacatcaag Aspergillus nidulans FGSC A4 npga_pmei-r cgcggccggccgtttaaacttaggataggcaattacaca Aspergillus nidulans FGSC A4 afog_noti_f gacacgcgtgcggccgcatgggcagcacatcttccgag Aspergillus nidulans FGSC A4 afog_noti_r taaattcacgcggccgctcaagcaacaacgacagttccc Aspergillus nidulans FGSC A4 afog_mid1_r ccacgactgtcacgttcgcttc Aspergillus nidulans FGSC A4 afog_mid2_f acgtgacagtcgtggacgaggc Aspergillus nidulans FGSC A4 A_ACLA_5574_PmeI-F atacacctagggtttaaacatgttccgcgagaatcttgg Aspergillus clavatus NRRL 1 A_ACLA_5574_PmeI-R cgcggccggccgtttaaacttatgaagaaatctctttcctccg C Aspergillus clavatus NRRL 1 B_ACLA_5568_NotI-F gacacgcgtgcggccgcatgacccaatctgaagcccg Aspergillus clavatus NRRL 1 B_ACLA_5568_mid1-R cgatggagaggttcagaacttcc Aspergillus clavatus NRRL 1 B_ACLA_5568_mid2-F tgaacctctccatcgacaaggc Aspergillus clavatus NRRL 1 B_ACLA_5568_mid2-R gcctcaaccagacactgagacgc Aspergillus clavatus NRRL 1 B_ACLA_5568_mid3-F gtgtctggttgaggctgtccgg Aspergillus clavatus NRRL 1 B_ACLA_5568_NotI-R taaattcacgcggccgctcaagccctcgatttggccc Aspergillus clavatus NRRL 1 C_ACLA_5566_NotI-F gacacgcgtgcggccgcatgggatcggttgatactgc Aspergillus clavatus NRRL 1 C_ACLA_5566_NotI-R taaattcacgcggccgcctagctgactagacgaaccacac Aspergillus clavatus NRRL 1 G_ACLA_5567_XmaI-F aatataaaaccccgggatgtctagggtaccgtccaaagc Aspergillus clavatus NRRL 1 G_ACLA_5567_XmaI-R tcggcgcgcccccgggtcaatttggcgcgtcaattg Aspergillus clavatus NRRL 1

18 Supporting Information: Sequencing primers 1/4 name sequence ADH1_seqF CTCGTCATTGTTCTCGTTCC ADH1_seqR CCTACAGGAAAGAGTTACTC TDH3_seqF CGGTAGGTATTGATTGTAATTCTG TDH3_seqR CAATGCAATAGCGCATCAAG PGK1_seqF CTTGCATAAATTGGTCAATGC PGK1_seqR CATAAAGGCATTAAAAGAGGAGCG frla_seq CGCGCATCTAGTCAACAATG frlb_seq1 TCGGCTGTCGTATTTCTTCG frlb_seq2 TGAAAACATTAAGTCGGGCAC frlb_seq3 AACCATCTAACTTCTTCACTGC frlb_seq4 ATCTCAAGAAACGCTCAACC frlb_seq5 GCGTTTCAAGGTCGCGTCTG frlb_seq6 AGGCTCTCCCACTGTATCCC frlb_seq7 TGGAAATCTCTCTCAAGGCG frlb_seq8 ACAAGATCCACTTCAATTCCTC frlb_seq9 ACAGATGTGTGAGAGGTGCG frlb_seq1 ACCTTTACTGATCTCTCTTC frlb_seq11 GAGCCCAACTCTTCCTTCCTTG frlb_seq12 CTGGGATTTCTGTACCTTAC frlb_seq13 GCAACATGTCTTCCACCAACG frlb_seq14 CGAGATGATGGAAATGGTCC frlb_seq15 AAGAGCGAAGAGAGCCAAGG frlb_seq16 GAGGCGGCAACAAGACTACC frlb_seq17 TATTGGTCCTCGGCTACAGC frlc_seq GAGAATGTGCTGGTTTATGGC frld_seq CGAACCAACCCCTTTCCGTC frlf_seq1 GTTCATAGAGAAGAGAAAGTGC frlf_seq2 TTGTCCTTAAGCGGATGTCG frlf_seq3 CTGTCATTAAGAGCATTCTC frlf_seq4 AAGAACTCACTTCAACTGGC frlf_seq5 GTTTGTATGTTTGGTGACAC frlf_seq6 ACACGAAATCTCTAGGCGAG frlf_seq7 AATATGGCGGAACTGGATTC

19 Supporting Information: Sequencing primers 2/4 name sequence frlf_seq8 TCGGGCTTGTATTTGGCAAG frlf_seq9 AGCAAGACTTTCGGGACCG frlf_seq1 GACAAGCTATCCAACATCCGC frlf_seq11 AAGGTTATCGACGGGGTGGTC frlf_seq12 ATATTGGAGACTTGGGAAAG frlf_seq13 CAACAGAATGACATGGGACG frlf_seq14 TCTTCAAAAATCCGAGCTGC frlh_seq1 AACAGATGCAAAGAGCCAGG frlh_seq2 TGTCTTGTCCCATGCGCTGG frlh_seq3 AGATACGATCCTAAACGCCG frlh_seq4 GCCATCTCCGTCGTGTTCTAC frlh_seq5 TTGACGGACGAAGTTATTTC lova_seq1 ATGCACATCTCATCACCAAG lova_seq2 ATCCAGTGGTTCGAGGATACTG lovb-seq1 TGCGTCCAACCGCATCTCG lovb_seq2 ACAGGTGCCAATTCTTCGAG lovb_seq3 CATGGAGCCAGAGCAAAACC lovb_seq4 TCCAACGTCCAATATGCTAC lovb_seq5 CTAGAAGAGTGTGGTTGTGC lovb_seq6 CTGTATGAGTGTGTCGAAGC lovb_seq7 TAGCGACCAGCACCATCTTC lovb_seq8 TGACATTGTGGTGTTTGACG lovb_seq9 GATATTCTGGAGATTGGAGC lovb_seq1 TAGTACCCATGCAATTGACG lovb_seq11 ATAACCGAATGAACTCCTCG lovb_seq12 CTCGTTCTGAATCCCCCCAG lovb_seq13 AACATATCTCCTGGTCGGAC lovb_seq14 TGCAATTCGGTTCATGTTCG lovb_seq15 GAGAATGAAGTTTCGGGACG lovb_seq16 TCTGCTGGTTGTGGCTTACC lovc_seq CTGGATTGGCGATGAAGTTG lovd_seq TCGTACGTCTTCCTCCATCC lovf_seq1 AGTACCGACTCATACTTGAAG

20 Supporting Information: Sequencing primers 3/4 name sequence lovf_seq2 TCCGATGGGATTTCCTACTC lovf_seq3 TCATGGATACCAACCGATGG lovf_seq4 AAGCAAGTCAGCCGACCAAG lovf_seq5 ACAGGTTCCATTACAGAGTG lovf_seq6 CGTGTGACATCCCTTATCTG lovf_seq7 CGACATCAGTTACATCCTAC lovf_seq8 GTAGCAGTTTTTGATGAGGC lovf_seq9 GAAATGATCTGTCGCCTGGG lovf_seq1 TCACTAAGCCCTACGATGTG lovf_seq11 CAAGGAGCGACAATGAAGTC lovf_seq12 GACCACAAGCCGGACCAACG lovf_seq13 TGATATTCTCTACTGGCAGC lovf_seq14 GTTCAGGGAAGTTGGAATCTG ivra_seq1 CTCTTGTCACCCTCGATCTAC ivra_seq2 GTGCTGTTCTCTGCGTGTCG ivra_seq3 ATATGGCTAATGGTACTCTGC ivra_seq4 AGTCATTGCTCCCTTATCGC ivra_seq5 AAGAACCTGGTCGGGAGTGC lovi_seq1 GCTCACCAATGGCGCAATCACC lovi_seq2 CTCCCAGGAACTGATACATGAC afog_seq1 CAAGCGTGACTGTAGACACC afog_seq2 GGATTGGCTCTGTCAAGACG afog_seq3 AACGCAGCACCGCACTCGCTTC afog_seq4 CCATGGCAGCAGACAAGACG afog_seq5 TTCACTGATATGGTGCTTGG afog_seq6 TGCAGGATTCGTGTGTCTCG afog_seq7 GACGACGCTCGACTCAATCG afog_seq8 TGGCAAATCTCGCATTCCAC afog_seq9 AGCTGGCACTTTACTGACATC afog_seq1 TTCGAGCCCTGAGAAGGTGG afog_seq11 GAGTTCGCTCTGCGAGACG afog_seq12 ACGACCACATCTTCAACTCTC afog_seq13 AGCTGGATGGTCGAACACG

21 Supporting Information: Sequencing primers 4/4 name afog_seq14 afog_seq15 sequence AATCAAAGATGTCGGCTACGTC TAGTATCCTGCAGAGTGTG

22 Supporting Information: Quantitative PCR primers 1/2 name Sequence HTA1_qF GCTGGTTTGACATTCCCAGT HTA1_qR CCAATTGCAAATGTCTTGGA HIS3_qF CCTCCACGTTGATTGTCTGCG HIS3_qR GGAACATCGTTGGTACCATTGG LEU2_qF CCAGCAACATGTTTGGTGATATC LEU2_qR CGTGGCATGGTTCGTACAAAC URA3_qF CAGTATAGAACCGTGGATGATGTGG URA3_qR CTGGCCGCATCTTCTCAAATATG frla_qf CTAACACGAGCGTGGATCAGTATG frla_qr GAAAGCACGATAGACCGGCTTG frlb_qf CAAGACGCCGCTCATCACGTTC frlb_qr GCTGTCTGGTCCAGACGTCCTTC frlc_qf CAGTGACGAGATGAGGCAGTTC frlc_qr CGAACCACAATCTTCTCTCCGG frld_qf GTCGGCAAGGTTCGATGAGC frld_qr CTCTCAAATGCGCAACCAAGTTC frlf_qf CTCGAGGCGTTGCTAGAGAAGC frlf_qr GGAGACGTCGGAGCCAATCTC frlg_qf CGAGAAACCGCTCATCAACGTC frlg_qr GTCTCAACGTCGTGTCGCATG frlh_qf CATCATCGCAGCTGCAGTCATG frlh_qr CTAGAACCATGGCTCGCAGGTG lova_qf CCGGTTCTTTGCCTCTAAGGAG lova_qr CATGAGCTTCGTGGTGGGATG lovb_qf GGGTGCCAAGATAACCGAGGTG lovb_qr GTGAGCCTCGTAGCGGGAACTC lovc_qf GCAGTTCGGCGAGGATCTGTG lovc_qr CGACAGCTCTCCCTTCCGGAC lovd_qf CGCCGAAAAGGTTCCTTGACC lovd_qr GCTCGAATGTGCGTGTCAGATC lovf_qf GTGATCATGGAGGCGATGAGC lovf_qr CTGTGATCCAGTTCCGGAGCTC lovg_qf GGAACTGGTCATTCGGATACCG

23 Supporting Information: Quantitative PCR primers 2/2 name lovg_qr ivra_qr ivra_qf lovi_qf lovi_qr npga_qf npga_qr Sequence GTCGATTGGGAAACTGATGCTGC GCACGAAATGGATGTCTCCCG GGTCACTTGGTCAATGCCCATATG CAATATGCGCCAACCCTCAATG CAGGCGAAGATGAAGGTAGCTCC CGGTGATTACCTATTTGCAACGG CACAGGGCTGGATATCTCGCTC