Applied Microbiology and Biotechnology Electronic supplementary material A family of AA9 lytic polysaccharide monooxygenases in Aspergillus nidulans is differentially regulated by multiple substrates and at least one is active on cellulose and xyloglucan Guru Jagadeeswaran 1, Lawrie Gainey 1, Rolf Prade 2 and Andrew J. Mort 1* Author Affiliations 1 Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater, OK, 74078, USA 2 Department of Microbiology and Molecular Genetics, Oklahoma State University, Stillwater, OK, 74078, USA *Corresponding author; email: andrew.mort@okstate.edu
Supplementary material Table S1 Sequence for primers used in RT-PCR expression analysis Table S2 List of LPMOs in Aspergillus species and in N. crassa Table S3 Pairwise identity (percent) of LPMOs in A. nidulans Table S4 Pairwise identity (percent) of the LPMOs in four Aspergilli species Table S5 Putative cis elements in promoter sequences (1 KB upstream) of LPMOs in A. nidulans Fig S1 Expression of AN3046 and protein identity confirmation a. SDS-PAGE analysis of purified AN3046 enzyme from A. nidulans after maltose induction. Protein band corresponding to AN3046 indicated by an arrow (M - protein marker) b. Protein identity confirmation using peptide mass fingerprinting. The MALDI-TOF mass spectrum from a trypsin digest of protein band cut from SDS gel c. Histogram showing the results from a protein database search with the MS spectrum peak list data using Mascot search. The top score matches AN3046 of A. nidulans FGSCA4 d. Matched peptides observed in the spectrum shown in red Fig S2. Multiple sequence alignment of LPMOs in A. nidulans showing the conserved HXGP and Q/EXYXXC motifs. Copper coordinating residues shown with an asterisk mark Fig S3 Domain features of AA9 LPMO proteins in A. nidulans Fig S4 CBM1 domain in AN1602 and AN9524
Table S1 Sequence for primers used in expression analysis primer AN1041 For AN1041 Rev AN1602 For AN1602 Rev AN2388 For AN2388 Rev AN3046 For AN3046 Rev AN3860 For AN3860 Rev AN3511 For AN3511 Rev AN6428 For AN6428 Rev AN7891 For AN7891 Rev AN9524 For AN9524 Rev CDH1 For CDH1 Rev CDH2 For CDH2 Rev ACTIN For ACTIN Rev sequence TCA TCT GCC ACA AGG ACG CT CCA TTC GAG GGT GGT CTT GT CTG GCT CCA TCC AAA TCT ACC AG CGT CTG GAC TCA GGA CCT CAA T ATG CGA TGG CGT GTG GTT ATG TTG TGG GAT CCG TCG ATG G GTC GGA GCA TAT CGG CGT C CCA GAC GCT GAA GTT GAA CGA G CGA ATC TCA TAA GGG CCC CAT C AAC CCA GGT GTT GGT GGA A CCA GAT GAT GAA AGC GTC GCT TCA ACG CCC CAG TCC TCA A CGT ACA TGG CCA AAG CAC C GGT AGT CGC CAT CAG GTA GAG TGG CCC TCA TCC ACT GCT AC GAG AGC TGC AGG ATG CCA ACA ACG ACA TCC ATT GCC ACC CAC TGA GCG TAC AAG GGG TTA G CGA CTA CTA TGC CGC CTA CG AGT TGT CGG GTT ACG TTG TCG GTA TTG ACA GCA GTC CGT ATA CGC TCA TGC ATC CTG GCT GTT AGT C GTC CGT AAG GAT CTG TAC GGC CAC ATC TGT TGG AAG GTG GAC A
Table S2 List of LPMOs in Aspergillus species and in N. crassa ID Species GenBank Uniprot An04g08550 A. niger CAK38942.1 A2QJX0 An08g05230 A. niger CAK45495.1 A2QR94 An12g02540 A. niger CAK41095.1 A2QYU6 An12g04610 A. niger CAK97151.1 A2QZE1 An14g02670 A. niger CAK46515.1 A2R313 An15g04570 A. niger CAK97324.1 A2R5J9 AO090001000221 A. oryzae BAE56764.1 Q2UNV1 AO090005000531 A. oryzae BAE55582.1 Q2US83 AO090012000090 A. oryzae BAE60320.1 Q2UDP5 AO090023000056 A. oryzae BAE58643.1 Q2UIH2 AO090023000159 A. oryzae BAE58735.1 Q2UI80 AO090023000787 A. oryzae BAE59290.1 Q2UGM5 AO090103000087 A. oryzae BAE65561.1 Q2TYW2 AO090138000004 A. oryzae BAE64395.1 Q2U220 Afu1g12560 A. fumigatus Afu2g14540 A. fumigatus Afu3g03870 A. fumigatus CAF31975.1 Q6MYM8 Afu6g09540 A. fumigatus Afu8g06830 A. fumigatus AN1041.2 A. nidulans EAA65609.1 Q5BEI9 An1602.2 A. nidulans EAA64722.1 Q5BCX8 An2388.2 A. nidulans EAA64499.1 Q5BAP2 AN3046.2 A. nidulans EAA63617.1 Q5B8T4 AN3511.2 A. nidulans EAA59072.1 Q5B7G9 AN3860.2 A. nidulans EAA59125.1 Q5B6H0 AN6428.2 A. nidulans EAA58450.1 Q5AZ52 AN7891.2 A. nidulans EAA59545.1 Q5AUY9 AN9524.2 A. nidulans EAA66740.1 Q5AQA6 LPMO in N. crassa NCU01050 N. crassa CAD21296.1 Q1K8B6 NCU03328 N. crassa CAD70347.1 Q1K4Q1 NCU01867 N. crassa CAE81966.1 Q7SHD9 NCU02344 N. crassa CAF05857.1 Q7S411 NCU07898 N. crassa EAA33178.1 Q7SA19 NCU05969 N. crassa EAA29347.1 Q7S1V2 NCU02916 N. crassa EAA36362.1 Q7SHI8 NCU07760 N. crassa EAA29018.1 Q7S111 NCU07520 N. crassa EAA29132.1 Q7S1A0 NCU02240 N. crassa EAA30263.1 Q7S439 NCU00836 N. crassa EAA34466.1 Q7SCJ5 NCU08760 N. crassa EAA26873.1 Q7RWN7 NCU07974 N. crassa EAA33408.1 Q7SAR4 NCU03000 N. crassa EAA36150.1 Q7RV41
Table S3 Pairwise identity (percent) of LPMOs in A. nidulans AN3860 AN7891 AN1041 AN3511 AN9524 AN1602 AN3046 AN6428 AN2388 AN3860 100.0 58.0 53.1 51.0 50.4 35.5 25.3 28.2 24.6 AN7891 100.0 48.2 42.2 41.8 27.3 22.5 25.4 19.8 AN1041 100.0 39.4 42.6 33.3 21.0 31.6 17.3 AN3511 100.0 42.7 25.3 20.3 29.3 13.4 AN9524 100.0 32.2 18.3 29.2 19.1 AN1602 100.0 26.1 32.4 23.0 AN3046 100.0 44.9 21.9 AN6428 100.0 26.7 AN2388 100.0 Table S4. Pairwise identity (percent) of the LPMOs in four Aspergilli species #1 #2 #3 #4 #5 #6 #7 #8 #9 # 10 # 11 # 12 # 13 # 14 # 15 # 16 # 17 # 18 # 19 # 2 0 # 2 1 # 2 2 # 2 3 # 2 4 # 2 5 # 2 6 # 2 7 # 2 8 # 2 9 AO090023000056 (#1) 100.0 42.8 18.4 26.1 24.9 20.1 26.4 32.9 25.7 23.9 23.9 24.3 27.8 28.0 25.2 23.6 17.7 20.9 24.6 20.6 19.5 20.3 19.5 29.3 23.9 21.3 21.1 20.6 27.6 AN2388 (#2) 100.0 21.5 24.0 25.0 20.2 26.9 29.3 26.5 26.9 25.2 26.4 26.4 26.6 22.8 25.0 16.5 20.6 23.4 22.4 18.9 20.2 20.5 27.5 20.9 20.6 21.7 19.7 28.1 An15g04570 (#3) 100.0 43.5 41.8 39.6 43.5 39.7 42.5 41.4 33.1 32.3 31.9 32.9 20.4 24.2 20.6 27.7 23.1 25.7 18.4 19.9 21.0 23.0 20.8 21.2 22.2 18.9 23.6 Afu2g14540 (#4) 100.0 73.2 71.4 74.3 41.6 43.1 43.1 33.5 32.2 32.9 33.3 23.5 22.9 23.6 24.2 22.7 23.6 23.6 22.9 22.5 27.6 22.5 24.1 23.9 23.0 26.1 AO090023000159 (#5) 100.0 72.1 76.8 43.4 41.8 41.7 32.4 32.7 36.8 35.6 24.0 22.6 22.4 23.4 21.9 21.5 22.3 23.8 24.2 27.2 24.2 25.5 23.4 22.3 25.2 An12g02540 (#6) 100.0 79.1 43.4 38.3 41.0 25.5 27.4 28.9 32.0 20.8 20.0 19.2 17.3 21.2 19.2 18.1 19.7 17.2 28.9 20.5 20.3 19.6 20.7 27.8 AN3046 (#7) 100.0 42.5 40.1 43.1 32.7 33.0 36.0 35.9 23.9 21.9 21.4 22.3 21.0 21.9 22.7 23.1 23.8 28.9 24.0 24.2 24.2 22.2 28.3 AN6428 (#8) 100.0 45.7 43.9 40.9 41.3 40.6 40.4 33.2 33.2 33.6 35.3 32.3 31.0 32.1 31.7 38.4 33.9 36.2 33.6 33.9 29.0 33.3 AO090103000087 (#9) 100.0 67.4 36.3 33.1 34.1 33.6 25.0 26.2 24.7 30.6 25.1 27.9 25.3 25.3 25.8 29.8 25.0 26.8 24.4 23.4 29.8 Afu6g09540 (#10) 100.0 34.3 32.1 31.7 31.8 23.7 26.3 22.7 28.5 24.2 25.9 26.1 25.9 25.0 28.1 23.5 25.0 23.9 23.5 27.6 An15g04900 (#11) 100.0 62.6 59.9 65.2 30.7 34.5 24.6 31.7 32.8 32.5 27.0 28.6 28.3 36.4 29.8 28.9 31.9 24.9 36.0 AN1602 (#12) 100.0 63.0 62.3 32.9 34.7 28.2 38.5 33.3 34.6 29.2 29.6 34.3 42.1 33.8 31.4 31.6 29.1 40.3 AO090005000531 (#13) 100.0 68.6 32.5 34.2 27.9 33.5 32.3 32.2 26.6 27.5 32.4 38.1 31.0 30.1 30.3 27.1 38.6 Afu8g06830 (#14) 100.0 30.3 35.3 29.2 33.8 31.7 34.3 29.9 28.7 32.7 37.4 32.2 31.0 31.1 27.8 36.0 AO090012000090 (#15) 100.0 58.3 47.9 54.6 60.5 57.5 39.2 41.1 46.9 51.4 46.6 44.8 45.3 41.8 49.8 An14g02670 (#16) 100.0 45.0 54.5 64.3 64.8 42.2 41.8 47.0 52.2 43.7 45.0 48.5 41.8 49.4 AN3511 (#17) 100.0 41.5 63.0 55.9 31.0 32.3 37.0 51.0 38.3 35.4 36.3 36.7 50.2 AN9524 (#18) 100.0 64.0 57.6 36.3 39.4 43.3 52.1 42.4 43.3 38.6 41.4 48.8 An12g04610 (#19) 100.0 81.2 48.4 49.5 53.5 48.6 51.3 50.0 49.5 46.9 48.6 Afu3g03870 (#20) 100.0 37.9 39.1 48.2 49.0 42.3 42.1 41.6 43.4 49.8 An04g08550 (#21) 100.0 72.1 37.8 60.3 42.6 43.8 44.3 51.2 57.6 AO090138000004 (#22) 100.0 40.9 61.1 46.2 44.9 47.2 53.4 57.6 AN1041 (#23) 100.0 61.9 53.4 51.6 49.5 43.6 52.2 Ao090023000787 (#24) 100.0 66.0 64.3 66.8 56.3 57.5 Afu1g12560 (#25) 100.0 64.1 54.4 49.6 55.9 Ao090001000221 (#26) 100.0 58.9 49.2 54.5 An08g05230 (#27) 100.0 48.2 52.7 AN7891 (#28) 100.0 58.0 AN3860 (#29) 100.0
Table S5 Putative cis elements in promoter sequences (1 KB upstream) of A. nidulans LPMOs Motif Strand Location Gene XlnR GGCTAA - -391 to -396 AN2388 GGCTAG - -569 to -574 AN3046 GGCTAA + -249 to -244 AN3860 GGCTAG + -327 to -322 AN3860 GGCTAA + -823 to -818 AN7891 GGCTAA - -427 to -432 AN7891 CreA CCGGAG + -789 to -784 AN1041 GTGGAG + -767 to -762 AN1041 GCGGAG - -538 to -543 AN1041 CCGGAG - -786 to -791 AN1041 GTGGAG - -700 to -705 AN1041 GCGGAG + -201 to -196 AN1602 GCGGAG - -388 to -393 AN3046 GCGGAG + -289 to -284 AN3046 GCGGAG - -927 to -932 AN9524 GTGGAG + -541 to -536 AN7891 GTGGAG - -849 to -854 AN9524 GTGGAG + -288 to -283 AN9524 CTGGAG - -869 to -874 AN1041 CTGGAG - -539 to -544 AN1602 GCGGGG - -299 to -304 AN3860 GCGGGG - -229 to -234 AN3860 GCGGGG - -987 to -992 AN6428 CCGGGG none GTGGGG + -574 to -569 AN3860 CTGGGG + -59 to -54 AN1602 CeRE CCTTAAAAGG + -799 to -790 AN3046
Fig S1 MS/MS analysis of trypsin digested AN3046 M a) b) c) d) Protein sequence: Matched peptides observed in the spectrum shown in red HGYLTIPFSR TRLGAEAGLD TCPECSILEP VTAWPNVTEA KVGRSGPCGY NARVSIDYNQ PATNWGNSPV VTYTAGDTVD VQWCVDHNGD HGGMFSYRIC QDQELVNKFL TPGYLPTEAE KQAAEDCFEK GTLPCTDVNG QSCDFSPDCQ QGQACWRNDW FTCNAFQADS RRGCQGVDNA ALGSCFTTIA GGYTVTKKIK IPNYISGHTL LSFRWNSFQT AQVYLSCADI AIVGDSASTT KVSATATTLV TSSKTASASC TPAATVAVTF NHLASTSYGE SIKIVGSISQ LGSWSASSGV ALSASQYTTS NPLWTATVSL PAGTKFEYKF VKVSSEGSAV TWESDPNRSY TVPQSCAESV AVESSWK
Fig S2 Multiple sequence alignment of LPMOs in A. nidulans showing the conserved HXGP and Q/EXYXXC motifs. Copper coordinating residues shown with an asterisk mark AN3046 HYFFDTLVIDGQETTPNQYVRSNTRPEKYNPTKWVNTRDDMTPD------MPDFRCNKGSFTFAGQTDTAEVKAGSKLAMKLGVGA---------TMQHPGP 87 AN6428 HYVFPALVQDGAATGDWKYVRDWTGSYGNGPVEDVTS--------------LDIRCNKDASTNGNATETLPVKAGEEIGFTVRT-----------NIGHPGP 77 AN2388 HTLMTTLYVDGENQGDGVCIRMNRNAEKATFPISPLAN-------------DAMACGYDGEI---AAARTCAVSQSSTLTFEFRAYPDGSQPGSIDGSHKGP 86 AN1602 HATVFAVWINDEDQ-GLGNTADGYIRTPPNN--SPVTDVTST----------DLTCNVNGDQ---AAAKTLEVAAGDKITFEWHHNSRDSSDDIIADSHKGP 80 AN3860 HGYVTKMTIDGEEYGGWLADSYYYMDSPPDNYGWSTTVTDNGFVSPDAFGTDDITCHRGATP---GALSAPVTAGSKIDITWNT----------WPESHKGP 89 AN1041 HGYVTGIVADGTYYGGYLVNQYPYSNDPPAVVGWAEDATDLGFVDGSGYTSGDIICHKDATN---AQASATVAAGGTVELQWTE----------WPESHHGP 89 AN3511 HGYVSNIVINGVSYRGWLPSQDPYSPSPPIGVGWETPNLSNGFVTPEEASTDAIICHKEATP---ARGHATVAAGDKIYIQWQPIP--------WPDSHHGP 91 AN7891 HGYVTKIDVDGTTYGGYLVDTYSYEPDPPKLIAWSTTATDTGYVSPSAYGTSDIVCHRGAEP---GALSAETLPGGSVTLYWNT----------WPTDHHGP 89 AN9524 HGYVQNIVVNGVYYSGWEINTYPYMTDPPVVAAWQIPNSNGPVDVSNGYTTEDIICNLNATN---AAGYVEVAAGDKINLQWSA----------WPDTHHGP 89 * * AN3046 GLVYMSKAPGAANQYEGDG-DWFKIHEEGICDTSKDIKTDAWCTWDKDR--IEFTIPADLPDGEYLIRSEHIGVHGA--HDGQAEFYYECAQVKVTGGGNGN 184 AN6428 LLAYMAKAPGDASDFDGDGQVWFKIYEDGPTVTDDGLT---WPSDGATN--VNFTIPSSLPDGDYLLRVEHIALHGAG-TEGGAQFYLSCGQVSVTGGGNGD 173 AN2388 CAVYMKPVANATSDNNAAGDGWFKIYELDYDSSTS---QWCTEKLIANNGFLSVQIPEGLRGGDYLVRTELLALHAAQDSPPDPQFYVGCAQVFLEGSESGD 185 AN1602 VLVYMAPTEAGSAGKN-----WVKIYEDGYN-----DGTWAVDTLIANKGKHSVTVP-DVPAGNYLFRPEIIALHEGN-REGGAQLYMECVQFKVTSDGTTQ 170 AN3860 IINYLAKCNGDCSSADKTSLEFVKIQAEAIVDASTN--TWVTDELIENSFTTSVTIPASIAPGNYVLRHEIIALHSAG-QQNGAQAYPQCLNLVVSGSGT-D 187 AN1041 VIDYIASCNGDCTTVDKTTLEWVKISESGLVDGSSAPGTWASDNLISNNNSWTVTIPSSLAAGGYVLRHEIIALHSAG-NENGAQNYPQCVNLEVTGGGS-A 189 AN3511 VLDYLAPCNGDCQTVDKNSLEFFKISGVGLIDGSSPPGYWADDELIENGNGWLVQIPADIKPGNYVLRHEIIALHGAG-SQNGAQLYPQCFNLKITGSGT-A 191 AN7891 VITYLANCNGDCASVDKSTLKFFKIDAGGLVDNSAVPGTWATDELIAADFSRTVTIPSDIASGNYVLRHEIIALHSAG-NKDGAQNYPQCINLKITGSGT-A 189 AN9524 VISYLADCGDDCTTVDKTTLEFFKIDAVGLVDDSTVPGTWGDDELIENNNSWMVEIPTSIAPGNYVLRHEIIALHSAG-TEGGAQNYPQCFNLKVTGSGT-D 189 * Fig S3 Domain features of AA9 LPMO proteins in A. nidulans AN1041 An1602 An2388 An3046 An3511 AN3860 AN6428 AN7891 AN9524 20 19 AA9 AA9 238 223 17 AA9 233 18 AA9 231 23 AA9 242 22 AA9 241 19 AA9 222 20 AA9 238 17 AA9 234 CBM1 CBM1 Fig S4 CBM1 domain in AN1602 and AN9524 CBM1 domain in AN1602(aa 327 to 354) MKFSSVLALAASAKLVASHATVFAVWINDEDQGLGNTADGYIRTPPNNSPVTDVTSTDLTCNVNGDQAAAKTLEVAAGDKITFEWHHNSRDSSDDIIADSH KGPVLVYMAPTEAGSAGKNWVKIYEDGYNDGTWAVDTLIANKGKHSVTVPDVPAGNYLFRPEIIALHEGNREGGAQLYMECVQFKVTSDGTTQLPEGVSLP GAYTATDEGILFDIYSSFDSYPIPGPAVWDGASSGSGSSGSGSSSSAAATSSAEKTATSTTAAATTTAVATSTSSATQVQPTSVATFTTSVRPTTSAAPTT SAPTSSAAPTGGTGTGSIQIYQQCGGMNYKGATGCASGLTCKQWNPYYHQCVQA CBM1 domain in AN9524(aa 372 to 399) MSRLVSFASLLAAVNAHGYVQNIVVNGVYYSGWEINTYPYMTDPPVVAAWQIPNSNGPVDVSNGYTTEDIICNLNATNAAGYVEVAAGDKINLQWSAWPDT HHGPVISYLADCGDDCTTVDKTTLEFFKIDAVGLVDDSTVPGTWGDDELIENNNSWMVEIPTSIAPGNYVLRHEIIALHSAGTEGGAQNYPQCFNLKVTGS GTDSPAGTLGTELYNLDDPGILVNIYASLSTYVIPGPTLYSGATSIAQATSAITATGSATSGAGGAAATGSSAATTTAAAASTTATPTTAAAQTAKSASAP SSAATGSVPAAPTTATVSTTTSIATSVGTTLTRTTLATTTTAAAAEPSASAPAPSGNSASGSNPLYAQCGGLNFKGASGCVAGATCKKMNPYYSQCVSA General pattern of CBM1 domain CXXXXXXXXXXCXXXXXCXXXXXXXXXC