XYLOSE ISOMERASE GENES AND THEIR USE IN FERMENTATION OF PENTOSE SUGARS

Abstract

The present invention relates to eukaryotic cells which have the ability to isomerise xylose directly into xylulose. The cells have acquired this ability by transformation with nucleotide sequences coding for a xylose isomerase that has one or more specific sequence elements typical for isomerases having the ability of functional expression in yeasts, such as e.g. xylose isomerases obtainable from a bacterium of the genera Clostridium and Fusobacterium or a tunicate form the genus Ciona. The cell preferably is a yeast or a filamentous fungus, more preferably a yeast is capable of anaerobic alcoholic fermentation.

Claims

1. A yeast cell comprising a nucleic acid construct comprising a heterologous nucleic acid with a nucleotide sequence encoding a polypeptide that has xylose isomerase activity, the expression of which confers on, or increases in, the yeast cell the ability to directly isomerize xylose into xylulose, wherein the amino acid sequence of the polypeptide is at least 90% identical to SEQ ID NO:35 and comprises one or more conservative amino acid substitutions; wherein the one or more conservative amino acid substitutions is selected from the group consisting of valine to leucine or isoleucine, phenyalanine to tyrosine, lysine to arginine, and asparagine to glutamine.

2. The yeast cell according to claim 1, wherein the amino acid sequence of the polypeptide is at least 95% identical to SEQ ID NO:35.

3. The yeast cell of claim 1, wherein the amino acid sequence of the polypeptide is at least 97% identical to SEQ ID NO:35.

4. The yeast cell according to claim 1, wherein the heterologous nucleic acid is obtained from a bacterium of the genus Fusobacterium.

5. The yeast cell according to claim 1, wherein said yeast cell is a member of one of the following genera: Saccharomyces, Kluyveromyces, Candida, Pichia, Schizosaccharomyces, Hansenula, Kloeckera, Schwanniomyces, and Yarrowia.

6. The yeast cell according to claim 5, wherein the yeast cell is capable of anaerobic alcoholic fermentation.

7. The yeast cell according to claim 6, wherein the yeast cell is a member of one of the following species: S. cerevisiae, S. exiguus, S. bayanus, K. lactis, K. marxianus and Schizosaccharomyces pombe.

8. The yeast cell according to claim 1, wherein the heterologous nucleic acid is operably linked to a promoter that drives sufficient expression of the heterologous nucleic acid in the yeast cell to confer on the yeast cell the ability to isomerize xylose to xylulose.

9. The yeast cell according to claim 1, wherein said yeast cell further comprises a genetic modification that increases specific xylulose kinase activity by overexpression of a nucleic acid encoding a xylulose kinase, wherein the overexpression is by increasing the copy number of the nucleic acid or by placing the nucleic acid under the control of a heterologous promoter.

10. The yeast cell according to claim 1, wherein said yeast cell comprises a genetic modification that increases pentose phosphate pathway flux by overexpression of a nucleic acid encoding at least one enzyme selected from the group consisting of ribulose-5-phosphate isomerase, ribulose-5-phosphate 3-epimerase, transketolase and transaldolase, wherein the overexpression is by increasing the copy number of the nucleic acid or by placing the nucleic acid under the control of a heterologous promoter.

11. The yeast cell according to claim 1, wherein said yeast cell comprises a genetic modification that reduces the yeast cell's nonspecific aldose reductase activity by disruption of at least one endogenous copy of a gene encoding a nonspecific aldose reductase.

12. The yeast cell according to claim 1, wherein said yeast cell converts L-arabinose into D-xylulose 5-phosphate.

13. The yeast cell according to claim 1, wherein said yeast cell has the ability to produce at least one of the following fermentation products: ethanol, lactic acid, 3-hydroxy-propionic acid, acrylic acid, acetic acid, succinic acid, citric acid, an amino acid, 1,3-propane-diol, ethylene, glycerol, butyric acid, caproate, butanol, glyoxylate, a -lactam antibiotic or a cephalosporin.

14. A process for producing a fermentation product comprising the steps of: (a) culturing the yeast cell of claim 1 with a medium containing a source of xylose, and optionally a source of arabinose, to ferment xylose and obtain a fermentation product selected from the group consisting of ethanol, lactic acid, 3-hydroxy-propionic acid, acrylic acid, acetic acid, succinic acid, citric acid, an amino acid, 1,3-propane-diol, ethylene, glycerol, butyric acid, caproate, butanol, glyoxylate, a -lactam, an antibiotic and a cephalosporin, and, optionally, (b) recovering the fermentation product from the medium.

15. The process according to claim 14, wherein the medium also contains a source of glucose.

16. The yeast cell according to claim 1, wherein the heterologous nucleic acid is obtained from a bacterium of the family Fusobacteriaceae.

17. The yeast cell according to claim 16, wherein said yeast cell is a member of one of the following genera: Saccharomyces, Kluyveromyces, Candida, Pichia, Schizosaccharomyces, Hansenula, Kloeckera, Schwanniomyces, and Yarrowia.

18. The yeast cell according to claim 17, wherein said yeast cell is capable of anaerobic alcoholic fermentation and is a member of one the following species: S. cerevisiae, S. exiguus, S. bayanus, K. lactis, K. marxianus, and Schizosaccharomyces pombe.

19. The yeast cell according to claim 1, wherein said yeast cell (a) further comprises a genetic modification that (i) increases specific xylulose kinase activity by overexpression of a nucleic acid encoding a xylulose kinase; (ii) increases pentose phosphate pathway flux by overexpression of a nucleic acid encoding at least one enzyme selected from the group consisting of ribulose-5-phosphate isomerase, ribulose-5-phosphate 3-epimerase, transketolase and transaldolase; or (iii) reduces the cell's nonspecific aldose reductase activity by disruption of at least one endogenous copy of a gene encoding a nonspecific aldose reductase; wherein the overexpression is by increasing the copy number of the nucleic acid or by placing the nucleic acid under the control of a heterologous promoter; or (b) converts L-arabinose into D-xylulose 5-phosphate.

20. The yeast cell according to claim 16, wherein said yeast cell has the ability to produce at least one of the following fermentation products: ethanol, lactic acid, 3-hydroxy-propionic acid, acrylic acid, acetic acid, succinic acid, citric acid, an amino acid, 1,3-propane-diol, ethylene, glycerol, butyric acid, caproate, butanol, glyoxylate, a -lactam antibiotic and a cephalosporin.

21. A process for producing a fermentation product comprising the steps of: (a) culturing the yeast cell of claim 16 with a medium containing a source of xylose, and optionally a source of arabinose, to ferment xylose and obtain a fermentation product selected from the group consisting of ethanol, lactic acid, 3-hydroxy-propionic acid, acrylic acid, acetic acid, succinic acid, citric acid, an amino acid, 1,3-propane-diol, ethylene, glycerol, butyric acid, caproate, butanol, glyoxylate, a -lactam, an antibiotic and a cephalosporin, and, optionally, (b) recovering the fermentation product from the medium.

22. A eukaryotic cell comprising: a) a heterologous nucleic acid encoding the polypeptide of SEQ ID NO:35; or b) a heterologous nucleic acid encoding a polypeptide having xylose isomerase activity, wherein said polypeptide comprises all of SEQ ID NO:35 except for one amino acid substitution selected from the group consisting of valine to leucine or isoleucine, phenyalanine to tyrosine, lysine to arginine, alanine to valine, and asparagine to glutamine.

23. A process for producing a fermentation product comprising the steps of: (a) culturing the yeast cell of claim 4 with a medium containing a source of xylose, and optionally a source of arabinose, to ferment xylose and obtain a fermentation product selected from the group consisting of ethanol, lactic acid, 3-hydroxy-propionic acid, acrylic acid, acetic acid, succinic acid, citric acid, an amino acid, 1,3-propane-diol, ethylene, glycerol, butyric acid, caproate, butanol, glyoxylate, a -lactam, an antibiotic and a cephalosporin, and, optionally, (b) recovering the fermentation product from the medium.

Description

DESCRIPTION OF THE FIGURE

[0099] FIG. 1 Phylogenetic tree of xylose isomerases tested for expression in yeast.

EXAMPLES

1. Functional Expression of Xylose Isomerases of the Invention in Yeast

1.1 Host Organism

[0100] The yeast host strain was RN1000. This strain is a derivative of strain RWB 218 (Kuyper et al., FEMS Yeast Research 5, 2005, 399-409). The plasmid pAKX002 encoding the Piromyces XyIA is lost in RN1000. The genotype of the host strain is: MatA, ura3-52, leu2-112, gre3::hphMX, loxP-Ptpi::TAL1, loxP-Ptpi::RKI1, pUGPtpi-TKL1, pUGPtpi-RPE1, [p415 Padh1XKS1Tcyc1-LEU2].

1.2 Expression-Constructs With Synthetic XI Genes

[0101] Synthetic codon-optimised (for Saccharomyces cerevisiae) XI genes were cloned into a derivative of pRS306 (Sikorski R. S., Hieter P., 1989, A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae Genetics 122:19-27) comprising the Saccharomyces cerevisiae the TPI1 promoter (899bp) and the CYC1 terminator (288bp) sequences, using XbaI (at the 5-end of the synthetic genes) and BamHI (at the 3-end of the synthetic genes) restriction sites. The first three nucleotides in front of the ATG were modified into AAA in order to optimize expression. Table 1 provides a list of the XI sequence that were tested and the corresponding SEQ ID NO's depicting the synthetic sequences. Genes were synthesized by GenScript Corporation (zie www.genscript.com) and delivered cloned in pUC57:

[0102] http://www.nebi.nlm.nih.gov/entrez/viewer.fcgi?db=nuccore&id=2440162. The TPI promoter was obtained using yeast genomic DNA as template in a PCR with

TABLE-US-00001 primers: forward: AAACCGGTTTCTTCTTCAGATTCCCTC reverse: TTAGATCTCTAGATTTATGTATGTGTTTTTTGTAGT.
The CYC1 terminator was obtained using yeast genomic DNA as template in a PCR with primers:

TABLE-US-00002 forward: AAGAATTCGGATCCCCTTTTCCTTTGTCGA reverse: AACTCGAGCCTAGGAAGCCTTCGAGCGTC.

1.3 Transformation of the Host Organism and Selection of Transformants

[0103] RN1000 was transformed with plasmids using the Gietz method (Gietz et al., 1992, Nucleic Acids Res. 1992 Mar. 25; 20(6):1425.). Primary selection of transformants was done on mineral medium (YNB+2% glucose) via uracil complementation.

1.4 Enzyme Assays

[0104] Xylose isomerase activity is assayed at 37 C. in a reaction mixture containing 50 mM phosphate buffer (pH 7.0), 10 mM xylose, 10 mM MgCl.sub.2 and a suitable amount of cell-free extract. One unit of activity is defined as the amount of enzyme producing 1 nmol of xylulose per min under the assay conditions. Xylulose formed is determined by the method of Dische and Borenfreund (Dische and Borenfreund, 1951, J. Biol. Chem. 192: 583-587) or by HPLC using a Biorad HPX-87N Column operated at 80 C. and eluated at 0.6 ml/min using 0.01 M Na.sub.2HPO.sub.4 as the eluens. Xylose and xylulose are detected by a Refractive Index detector at an internal temperature of 60 C.

[0105] Specific activity is expressed as units per mg protein. Protein is determined with the Bio-Rad protein reagent (Bio-Rad Laboratories, Richmond, Calif., USA) with bovine -globulin as a standard.

1.5 Physiological Characterisation of the Transformed Cells

[0106] Transformed cells were colony-purified on minimal medium with glucose as sole carbon source several times. Subsequently colony-purified transformed cells are grown in shake flasks in the presence of oxygen on synthetic medium with 2% (w/v) xylose as carbon/energy source. The results are shown in Table 2 where + indicates that cells showed significant growth. The sign denotes that no significant growth occurred.

[0107] Strains growing at the expense of xylose were subsequently tested for their ability to grow anaerobically at the expense of xylose with a concomitant formation of ethanol. Strains transformed with the xylose isomerases of Cyllamyces aberensis Ciona intestinalis, Clostridium difficile, Bacteroides fragilis and Fusobacterium_mortiferum were able to grow anaerobically on xylose with growth rates, biomass and ethanol yields comparable to those of the prior art Piromyces enzyme, e.g. comparable to those of RWB218 as previously described by Kuyper et al. (2005, FEMS Yeast Res. 5: 925-934). However, cells transformed with the C. difficile XI showed the best performance in xylose fermentation in terms of growth rate.

[0108] Similarly the transformed cells of the invention are also capable of mixed substrate utilisation. When the transformed cells are grown in a mixture of glucose and xylose (20 g l.sup.1 each) both sugars are completely consumed but glucose was the preferred substrate. Xylose consumption commences only after approximately 80% of the glucose is consumed. The ethanol produced accounted for the consumption of the total of glucose and xylose in each instance of the strains transformed with the xylose isomerases of Cyllamyces aberensis, Ciona intestinalis, Clostridium difficile, Bacteroides fragilis and Fusobacterium_mortiferum.

TABLE-US-00003 TABLE 1 CAI nucle- Amino CAI opti- otide acid Initial mized SEQ SEQ Name length genes genes ID NO ID NO Piromyces_sp_E2 1314 0.547 0.996 18 3 Cyllamyces_aberensis 1314 0.569 0.989 19 4 Clostridium_difficile 1338 0.107 0.987 16 1 Bacteroides_fragilis 1320 0.104 0.976 20 7 Ciona_intestinalis 1371 0.119 0.993 17 2 Thermotoga_maritima 1335 0.097 0.983 23 10 Haemophilus_somnus 1320 0.145 1.000 24 11 Physcomitrella_patens 1440 0.090 0.988 26 13 Arabidopsis_thaliana 1434 0.154 1.000 25 12 Arthrobacter_aurescens 1188 0.082 0.988 22 9 Burkholderia_phytofirmans 1323 0.065 0.982 21 8 Orpinomyces_sp._Ukk1 1314 0.515 Not Not 5 tested tested Fusobacterium_mortiferum 1311 0.106 0.989 38 35

TABLE-US-00004 TABLE 2 Growth on 2% % amino acid xylose in identity with XI source organism synthetic medium Piromyces XI Piromyces + 100 Cyllamyces aberensis + 91 Physcomitrella patens 52 Arabidopsis thaliana 51 Haemophilus somnus 49 Ciona intestinalis + 47 Clostridium difficile + 54 Thermatoga maritima 51 Bacteroides fragilis + 78 Burkholderia phytofirmans 50 Arthrobacter aurescens 17 Fusobacterium_mortiferum + 51

TABLE-US-00005 TABLE 3 Pairwise amino acid identities between xylose isomerases from various organisms as indicated. 1 2 3 4 5 6 7 1 Piromyces_sp_E2 2 Bacteroides_fragilis 78, 71 3 Cyllamyces_aberensis 91, 30 77, 57 4 Clostridium_difficile 54, 00 52, 61 53, 78 5 Ciona_intestinalis 47, 83 45, 10 45, 30 52, 36 6 Fusobacterium_mortiferum 50, 92 50, 45 51, 15 71, 79 50, 46 7 Orpinomyces_sp_ukk1 94, 51 78, 03 91, 07 55, 38 47, 14 50, 23 8 Clostridium_phytofermentans 53, 55 53, 88 54, 92 62, 56 46, 80 64, 68 54, 23 9 Bacteroides_uniformis 81, 24 90, 41 80, 55 55, 03 47, 49 52, 75 80, 55 10 Thermotoga_maritima 51, 26 51, 02 51, 72 65, 99 50, 67 64, 91 50, 11 11 Haemophilus_somnus 48, 74 49, 43 49, 20 49, 89 45, 10 52, 06 48, 74 12 Arabidopsis_thaliana 51, 49 51, 71 52, 40 53, 03 51, 10 53, 90 51, 26 13 Physcomitrella_patens 51, 72 52, 39 52, 17 52, 81 51, 31 52, 75 51, 03 14 Burkholderia_ phytofirmans 49, 43 48, 74 48, 74 48, 18 44, 77 49, 09 48, 98 15 Arthrobacter_aurescens 20, 76 20, 76 20, 76 19, 75 20, 51 20, 25 20, 76 8 9 10 11 12 13 14 1 Piromyces_sp_E2 2 Bacteroides_fragilis 3 Cyllamyces_aberensis 4 Clostridium_difficile 5 Ciona_intestinalis 6 Fusobacterium_mortiferum 7 Orpinomyces_sp_ukk1 8 Clostridium_phytofermentans 9 Bacteroides_uniformis 54, 79 10 Thermotoga_maritima 59, 82 52, 28 11 Haemophilus_somnus 53, 42 50, 68 51, 03 12 Arabidopsis_thaliana 49, 77 52, 97 55, 41 48, 75 13 Physcomitrella_patens 50, 68 53, 42 55, 86 48, 29 70, 23 14 Burkholderia_ phytofirmans 50, 23 50, 23 49, 09 64, 69 46, 36 47, 05 15 Arthrobacter_aurescens 21, 52 20, 25 20, 51 19, 49 19, 24 20, 25 20, 76

TABLE-US-00006 TABLE4 CLUSTALW(1.83)multiplesequencealignment Arabidopsis_thaliana MKKVEFFMLLLOFIAASSLVSADPPTCPADLGGKOSDSDDWOGDFFPEIP Physcomitrelia_patens MKALLFSVVLLVAVLSCSGQRVADITCGVIDG-SLGSDFNWEGEFFPNIS Ciona_intestinalis ----------------------MSSFAPASGKSDLAEAGSLLTKYPLEVK Piromyces_sp_E2 ----------------------------------------MAKEYFPQIQ Orpinomyces_sp_ukk1 ----------------------------------------MTKEYFPTIG Cyllamyces_aberensis ----------------------------------------MVKEYFPAIQ Bacteroides_fragilis ---------------------------------------MATKEYFPGIG Bacteroides_uniformis ---------------------------------------MATKEYFPGIG Clostridium_difficile -----------------------------------------MNEIFKGIG Fusobacterium_mortiferum ------------------------------------------MEFFKGID Thermotoga_maritima -----------------------------------------MAEFFPEIP Clostridium_phytofermentans -----------------------------------------MKNYFPNVP Haemophilus_somnus -----------------------------------------MSNYFOKIA Burkholderia_phytofirmans ------------------------------------------MSYFEHIP Arthrobacter_aurescens -------------------------------------------------- Arabidopsis_thaliana KIKYE-GPSSKNPLAYRWYNAEEEILGKKMKDWFRFSVAFWHTFRGTGGD Physcomitrelia_patens YIKYE-GPASHNPLAYKYYNAEELIFGKKMKDWLRFSVAFWHTFRGDGGD Ciona_intestinalis KIPYKPDAKVDDVLCFRHYNESEVVMGKPMSDWLRFSVCYWHTFRGTGAD Piromyces_sp_E2 KIKFE-GKDSKNPLAFHYYDAEKEVMGKKMKDWLRFAMANWHTLCAEGAD Orpinomyces_sp_ukk1 KIPFE-GKDSKNPMAFHYYDAEKEVMGKKMKDWLRFAMAWWHTLCADGAD Cyllamyces_aberensis KIKFE-GKDSKNPMAFHYYDAEKEIMGKKMKDWLRFAMAWWMTLCAEGSD Bacteroides_fragilis KIKFE-GKDSKNPMAFRYYDAEKMINGRSMKDWZKFAMAWWHTLCAEGGD Bacteroides_uniformis KIKFE-GKESKNFMAFRYYDADKVIMCKEMSEWLKFAMANWHTLCAEGGD Clostridium_difficile QIKFE-GVKSNUELAFRYYNPEQVVGNKIMKEHLRFAMSYWHTLCGEGND Fusobacterium_mortiferum KVKYE-GVKTNNLLAFAHYNPEEVILGKEMEDHLKFAMSYWHTLTGEGTD Thermotoga_maritima KIQFE-GKESTNPLAFRFYDPNEVIDGKPLKDHLKFSVAFWHTFVNEGRD Clostridium_phytofermentans EVKYE-GPNSTNPFAFKYYDANKVVAGKTMKEHCRFALSWWHTLCAGGAD Haemophilus_somnus KVNYE-GANSTNPFAFKHYNPNEVILGKTVEEHLRLAVCYWHTFCWTGND Burkholderia_phytofirmans EIRYE-GPQSDNPLAYRHYDKSKKVLGKTLEEHLRIAVCYWHTFVWPGTD Arthrobacter_aurescens -------------------------MTPQPTPQDRFTFGLWTVG-WTGAD :::.*.** Arabidopsis_thaliana PFGAATKYWPWEDGTNSVSMAKRRMRANFEFLKKLGVDWWCFHDRDIAPD Physcomitrelia_patens PFGSPTKRWPWDDGSNSLTVAVRRMRANFEFLKKLGVEKWCFHDRDIAPE Ciona_intestinalis PFGFPTLVRPWDDGTDSIENAERRMRVAFDEMSKLGVKYWTFHERDIAPE Piromyces_sp_E2 [00001] Orpinomyces_sp_ukk1 QFGVGTKSFPWNEGTDPIAIAKQKVDAGFEIMTKLGIEHYCFHDVDLVSE Cyllamyces_aberensis QFGPGTKKTPWNEGTDPIEKAKQKVDAGFEIMTKLGIEHYCFHDVDLVDE Bacteroides_fragilis QFGGGTKQFPWNGDPDPVQAAKNEMDAGFEFMQKMGIGYYCFHDVDLVTE Bacteroides_uniformis QFGGGTKKFPWNGEADKVQAAKNEMDAGFEFMQKMGIETYCFHDVDLCEE Clostridium_difficile PFGVGTVERPWNNITDPIEIAKIKVDAGFEFMSKMGIEYFCFHDRDIAPE Fusobacterium_mortiferum PFGNATMDREWN-EYTPMEKAKARVKAGFEFMEKLGLEYFCFHDKDIAPE Thermotoga_maritima PFGDPTAERPWNRFSDPMDKAFARVDALFEFCEKLNIEYFCFHDRDIAPE Clostridium_phytofermentans PFGVTTMDRTYGNITDPMELAKAKVDAGFELMTKLGIEFFCFHDADIAPE Haemophilus_somnus MFGAGSLDRSWQKTGDLLVGAKQKAEIAFEFFQKLGVFYYSFEDVDIAPE Burkholderia_phytofirmans IFGQGTFRRPWQQAGDAMERAQQKADSAFEFFSKLGTPYYTFHDTDVSPE Arthrobacter_aurescens PFGVATR-------------PALDPVEAVHKLSELGAYGITFHDNDLVPF **:...::.****: Arabidopsis_thaliana GTTLEESNKNLDEVIELAKELQKGSKIKPLWGTAQLFLHPRYMHGGATSS Physcomitrelia_patens GSTLEESNANLDYIVSVAKKLQEGTNIRPLWGTAQLFKHPRYMHGAATSP Ciona_intestinalis GVTLSETNANLDRLAELASQLQGETGIKLLWNTCNLFAHPRYSNGAATNA Piromyces_sp_E2 [00002] Orpinomyces_sp_ukk1 GNSIEEYESNLEQVVAYLKQKQQETGIKLLWSTANVFGNPRYMNGASTNP Cyllamyces_aberensis GKNVEEYEKNLKTIVAYLKEKQKETGIKLLWSTANVFGHKRYMNGASTNP Bacteroides_fragilis ADSIEAYEANLKELVAYAKQKQAETGIKLLWGTANVFSHARYMNGAATNP Bacteroides_uniformis AETIEEYEANLKEIVAYAKQKQAETGIKLLWGTANVFGHARYMNGAATNP Clostridium_difficile GRDLEETNKILDEIVEYIKANMEKTGIKLLWGTANMFGNPRFVHGASTTC Fusobacterium_mortiferum AETLEEYHRNLDEIVDLIEEEMkRTGIKLLWGTSNMFSHPREMEGAATSC Thermotoga_maritima GKTLRETNKILDKVVERIKERMKDSNVKLLWGTANIFSHPRYMHGAATTC Clostridium_phytofermentans GDTFEESKKNLFEIVDYIKEKMDQTGIKLLWGTANNFSHPRFMHGASTSC Haemophilus_somnus SNNFKEYLHNFNTIVDILEKKQSETGVKLLWGTANCFTNPRYMSGASTNP Burkholderia_phytofirmans GSNLKEYSENFLRITDYLARKQESTGIKLLWGTANLFSHPRYAAGAATSP Arthrobacter_aurescens DATASER----DLILENFKAALAETGLKTPMVTTNLFSHPVFKDGGETSN ::::*:*::*.*. Arabidopsis_thaliana EVGVYAYAAAQVKKAMEVTHYLGGENYVFWGGREGYQTLLNTDMGRGLDH Physcomitrelia_patens DVRVYAYAAAQVKKAIEVTKLLGGENYVYWOGREGYQTLLNTDLKKELDH Ciona_intestinalis DAHVVAYAAAQVKKSLEIGKKLGAENFVFWGGREGYHTLLNTNVREELDN Piromyces_sp_E2 DFDVVARAIVQIKNAIDAGIELGAENYVFWGGREGYMSLLNTDQKREKEH Orpinomyces_sp_ukk1 DFDVVARAIVQIKNAMDAGIELGAENYVFWGGREGTMSLLNTDQKREKEH Cyllamyces_aberensis DFDVVARAIVQIKNAMDAGIELGAENYVFWGGREGTMSLLNTDQKREKEH Bacteroides_fragilis DFDVVARAAVQIKNAIDATIELGGTNYVFWGGREGYMSLLNTDQKREKEH Bacteroides_uniformis DFDVVARAAIQIKNAIDATIELGGSNYVFWGGREGYMSLLNTDQKREKEH Clostridium_difficile NADVYAYAAAQVKKAMEITKYLGGENFVFWGGREGYETLLNTNTELEMDN Fusobacterium_mortiferum NADVFAYAAAQTKKALEITKRLNGTGYVFWGGREGYETLLNTDIGLELDN Thermotoga_maritima SADVFAYAAAQVKKALEITKELGGEGYVFWGGREGYETLLNTDLGLELEN Clostridium_phytofermentans NADVFAYAAAKIKNALDATIKLGGKGYVFWGGREGYETLLUTDLGLELDN Haemophilus_somnus NPEVFAWAAAQVFTAMNATQRLGGENYVLWGGREGYEILLNTDLKREREQ Burkholderia_phytofirmans DPEVFAFAATQVRHALDATQRLGGDNYVLWGGREGYDTLLNTDLVRERDQ Arthrobacter_aurescens DRSIRRFALSKILRNIDLAAELGAETFVMWGGREGSEYDGSKDLSAALDR .:*:::*..:*:******..::. Arabidopsis_thaliana LARFFEAAVAYKKKIGFKGTLLIEPKPQEPTKAQYDWDAATAANFLRKY- Physcomitrelia_patens MATFLRSAAEYKKKIGFEGTLLLEPKPQEPTKHQYDWDAATTMGFLKNYG Ciona_intestinalis LANFFFMVVAYKKKIGFTGQFLIEPKPKEPSKHQYDYDAMTVIAFLKTY- Piromyces_sp_E2 [00003] Orpinomyces_sp_ukk1 MATMLTMARDYARSKGFKGTFLIEPKPMEPTKHQYDVDTETVIGFLRAHN Cyllamyces_aberensis MAMMLGLARDYARSKGFKGTFLIEPKPMEPTKHQYDVDTETVIGFLRAHG Bacteroides_fragilis LAQMLTIARDYGRARGFKGTFLIEPKPMEPTKHQYDVDTETVIGFLKAHG Bacteroides_uniformis LAQMLTIARDYARARGYKGTFLIEPKPMEPTKHQYDVDTETVIGFLKAHN Clostridium_difficile FARFLQMAVDYAKEIGFTGQFLIEPKPKEPTKHQYDFDTATVLGFLRKYN Fusobacterium_mortiferum LARFLQMAVDYAKKIGFEGQFFIEPKPKEPTKHQYDFDTTTVLEFLRKYN Thermotoga_maritima LARFLRMAVEYAKKIGFTGQFLIEPKPKEPTKHQYDFDVATAYAFLKNHG Clostridium_phytofermentans MARLMKMAVEYGRANGFDGDFYIEPKPKEPTKHQYDFDTATVLAFLRKYG Haemophilus_somnus IGRFMQLVVEHKYKIGFQGTLLIEPKPQEPTKHQYDYDVATVYGFLKQFG Burkholderia_phytofirmans LARFLHMVVDHAHKIGFKGSLLIEPKPQEPIKHQYDYDVATVEGFLLQHG Arthrobacter_aurescens MKEGVDTAAGYIKEKGYGLRIALEPKPNEPRGDIFLPTVGHGLAFIAQLE :..:*:::******.:.*: Arabidopsis_thaliana --GLIDEFKLNIECNHATLSGHTCHHELETARINGLLGNIDANTGDAQTG Physcomitrelia_patens LSGTNNDYKLNLECNHATLSGHSCHHELETARIYGMLGSVDANTGDAQTG Ciona_intestinalis --DLDKDFKLNIEPNETTLAGHCHEHDVVMASAYNMLGSVDSNTGSPDLG Piromyces_sp_E2 [00004] Orpinomyces_sp_ukk1 ---LDKDFKVNIEVNHATLAGHTFEHELACAVDAGMLGSIDANRGDYQNG Cyllamyces_aberensis ---LDKDFKINIEVNEATLAGHTFEHELACAVDAGMLGSIDANRGDYQNG Bacteroides_fragilis ---LNQDFKVNIEVNHATLAGHTFEHELAVAVDNGMLGSIDANRGDYQNG Bacteroides_uniformis ---LDKDFKVNIEVNEATLAGHTFEHELAVAVDNGMLGSIDANRGDYQNG Clostridium_difficile ---LDKYFKVNIEANHATLAGHTFQHELNIARINNVLGSIDANQGDLLLG Fusobacterium_mortiferum ---LDKYFKMNIEANHATLAGHTFQHELCTARINGVFGSIDANQGDMLLG Thermotoga_maritima ---LDEYFKFNIEANHATLAGHTFQHELRMARILGKLGSIDANQGDLLLG Clostridium_phytofermentans ---LEKDFKMNIEANHATLAGHTFEHELAMARVNGAFGSVDANQGDPNLG Haemophilus_somnus ---LENEIKVNIEANHATLAGHTFQHEIATATSLGIFGSIDANRGDPQLG Burkholderia_phytofirmans ---LDKEIRVNIEANHATLAGHSFHHEIATAYALGIFGSVDANPGDPQNG Arthrobacter_aurescens HG---DIVGLNPETGHEQMAGLNFTHGIAQALWAGKLFHIDLN-GQRGIK ..**.*::**:*.::***. Arabidopsis_thaliana WDTDQFLTDVGEATMVMMSVIKNGGIAPGGFNFDAKLRRES--TDVEDLF Physcomitrelia_patens WDTDQFLTDVSEATLIMLSVIKNGGLAPGGFNFDAKLRRES--VDVEDLY Ciona_intestinalis WDTDQFPMDVKNATMIMQTVLEQGGLAPGGLNFECKVRRES--TDVIDMM Piromyces_sp_E2 WDTDQFPIDQYELVQAWMEIIRGGGFVTGGTNFDAKTRRNS--TDLEDII Orpinomyces_sp_ukk1 WDTDQFPIDQYELVQAWMEIIRGGGFVTGGTNFDAKTRRNS--TDLEDII Cyllamyces_aberensis WDTDQFPIDQYELVQAWMEIIRGGGFTTGGTNFDAKTRRNS--TDLEDII Bacteroides_fragilis WDTDQFPIDNFELTQAMMQIIRNDGLGNGGTNFDAKTRRNS--TDPEDIF Bacteroides_uniformis WDTDQFPIDNFELTQAMMQIIRNGGFGNGGTNFDAKTRRNS--TDLEDIF Clostridium_difficile WDTDQFPTNIYDATLAMYEVLEQGGIAFGGENFDSKVRRAS--FEVEDLF Fusobacterium_mortiferum WDTDQFPTNVYDAVLAMYETLLAGGFKEGGLNFDAKVRRGS--FEPKDLF Thermotoga_maritima WDTDQFPTNIYDTTLAMYEVIKAGGFTKGGLNFDAKVRRAS--YKVEDLF Clostridium_phytofermentans WDTDQFPTDVHSATLAMLEVLKAGGFTNGGLNFDAKVRRGS--FEFDDIA Haemophilus_somnus WDTDQFPNSVEENTLAMYEILKAGGFTTGGFNYDAKIRRQS--TDPYDLF Burkholderia_phytofirmans WDTDQFPNSVEELTLAFYEILKHGGFTTGGMNFDSKVRRQS--VDPEDLF Arthrobacter_aurescens YDQDLVFGHGDLTSAFFTVDLLENGFPNGGPKYDGPRHFDYKPSRTDGYD :**.:.*:**::*:. Arabidopsis_thaliana IAHISGMDTMARGLRNAVKILEEGSLSELVRKRYATWDSELGKQIEEGKA Physcomitrelia_patens IAHISGMDTIARGLRNAAKLLEEGRLTKLVEDRYSSFNSPLGKTIEEGKV Ciona_intestinalis IAHVGAMDCFAKALKIAAKIREDGVLGKMKKERYASFGSGLGLKIKTGTA Piromyces_sp_E2 [00005] Orpinomyces_sp_ukk1 IAHISGMDAMARALENAAKLLQESPYCNMKKERYASFDSGIGKDFEDGKL Cyllamyces_aberensis IAHISGMDAMARALENAAKLLTESPYKKMKADRYASFDSGMGKDFEDGKL Bacteroides_fragilis IAHIAGMDAMARALESAANLLNESPYQKMKSDRYASFDAGKGKEFEEGKL Bacteroides_uniformis IAHIAGMDVMARALESAAKLLEESPYKKMKADRYASFDSGKGKEFEEGKL Clostridium_difficile LAYIAGMDTFAKGLLIAHKLLEDEVFENFTKERYASFSEGIGKDIVEGKV Fusobacterium_mortiferum YAYISGMDTFAKCLKVAAKLIEDGTFEKIKVERYSSYTTGIGKQIVNGEV Thermotoga_maritima IGHIAGMDTFALGGKIAYKLAKDGVFDKFIEEKYRSFKEGIGKEIVEGKT Clostridium_phytofermentans YGYIAGMDTFALGLIKAAEIIDDGRIAKFVDDRYASYKTGIGKAIVDGTT Haemophilus_somnus HAHIGAMDVLALSLKRAAKMIEDQTLQKVVDNRYAGWDQELGQKILNGKA Burkholderia_phytofirmans YGHIGAIDNLALAVERAAVLIENDRLDQRKRQRYSGWDAEFGRKISSGDY Arthrobacter_aurescens GVWESAKSNMSMYLLLKERALAFRADPEVQEALATSGVFELGEPTLANGE ...::.:.*. Arabidopsis_thaliana DFEYLEKKAKEF-GEPKVSSAKQELAEMIFQSAM--------- Physcomitrelia_patens GFEELEKISLEA-EEPPITSGKQELAEMIFYSYV--------- Ciona_intestinalis TLEECDSFIQEN-GEPAKLSGKQEMFEAVLNRYF--------- Piromyces_sp_E2 [00006] Orpinomyces_sp_ukk1 TLEQVYEYGKKN-GEPKVTSGKQELYEAIVAMYQ--------- Cyllamyces_aberensis TLEQVYEYGKKV-NEPKQTSGKQELYEAIVAMYM--------- Bacteroides_fragilis SLEELVAYAKAN-GEPKQTSGQQELYEALVNIYSL-------- Bacteroides_uniformis TLEDLVAYAKAN-GEPKQTSGKQELYEAIVNMYC--------- Clostridium_difficile GFKELESYALQM-PVIKNKSGRQEMLEAILNRYIYEVDTISNK Fusobacterium_mortiferum GFEELSKYALIN-GVKKNSSGRQEMLENILNRYIYE------- Thermotoga_maritima DFEKLEEYIIDK-EDIELPSGKQEYLESLLNSYIVKTIAELR- Clostridium_phytofermentans SLEELEQYVLTH-SEPVMQSGRQEVLETIVNNILFR------- Haemophilus_somnus SLEDLAKIVETQGLAPKPVSGQQEYLENLVNSYLYR------- Burkholderia_phytofirmans SLSALAEEAMARGLNPQHASGHQELMENIVNQAIYSGR----- Arthrobacter_aurescens TTADLLADASAFDTFNADQAAERSFAFVRLNQLAIEHLLGAR- :..:..