U.S. patent application number 14/887018 was filed with the patent office on 2016-02-04 for polypeptides having xylanase activity and polynucleotides encoding same.
The applicant listed for this patent is Novozymes, Inc.. Invention is credited to Nikolaj Spodsberg.
Application Number | 20160032336 14/887018 |
Document ID | / |
Family ID | 55179401 |
Filed Date | 2016-02-04 |
United States Patent
Application |
20160032336 |
Kind Code |
A1 |
Spodsberg; Nikolaj |
February 4, 2016 |
Polypeptides Having Xylanase Activity and Polynucleotides Encoding
Same
Abstract
The present invention relates to isolated polypeptides having
xylanase activity and polynucleotides encoding the polypeptides.
The invention also relates to nucleic acid constructs, vectors, and
host cells comprising the polynucleotides as well as methods of
producing and using the polypeptides.
Inventors: |
Spodsberg; Nikolaj;
(Bagsvaerd, DK) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Novozymes, Inc. |
Davis |
CA |
US |
|
|
Family ID: |
55179401 |
Appl. No.: |
14/887018 |
Filed: |
October 19, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13765267 |
Feb 12, 2013 |
9169473 |
|
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14887018 |
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Current U.S.
Class: |
435/99 ; 435/106;
435/109; 435/110; 435/115; 435/116; 435/136; 435/137; 435/138;
435/139; 435/140; 435/144; 435/145; 435/146; 435/148; 435/150;
435/155; 435/158; 435/159; 435/160; 435/162; 435/166; 435/167;
435/168; 435/200; 435/252.3; 435/252.31; 435/252.33; 435/252.34;
435/252.35; 435/254.11; 435/254.2; 435/254.21; 435/254.22;
435/254.23; 435/254.3; 435/254.4; 435/254.5; 435/254.6; 435/254.7;
435/254.8; 435/320.1; 435/325; 435/348; 435/419; 800/298 |
Current CPC
Class: |
C12P 19/14 20130101;
C12Y 302/01008 20130101; C12N 9/2482 20130101; C12P 19/02 20130101;
C12P 2203/00 20130101 |
International
Class: |
C12P 19/02 20060101
C12P019/02; C12P 19/14 20060101 C12P019/14; C12N 9/24 20060101
C12N009/24 |
Goverment Interests
STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED
RESEARCH AND DEVELOPMENT
[0002] This invention was made in part with Government support
under Cooperative Agreement DE-FC36-08GO18080 awarded by the
Department of Energy. The government has certain rights in this
invention.
Claims
1. An isolated polypeptide having xylanase activity, selected from
the group consisting of: (a) a polypeptide having at least 67%,
e.g., at least 68%, at least 69%, at least 70%, at least 75%, at
least 80%, at least 85%, at least 90%, at least 91%, at least 92%,
at least 93%, at least 94%, at least 95%, at least 96%, at least
97%, at least 98%, at least 99%, or 100% sequence identity to the
mature polypeptide of SEQ ID NO: 2, a polypeptide having at least
61%, e.g., at least 62%, at least 63%, at least 65%, at least 67%,
at least 70%, at least 75%, at least 80%, at least 85%, at least
90%, at least 91%, at least 92%, at least 93%, at least 94%, at
least 95%, at least 96%, at least 97%, at least 98%, at least 99%,
or 100% sequence identity to the mature polypeptide of SEQ ID NO:
4, a polypeptide having at least 61%, e.g., at least 62%, at least
63%, at least 65%, at least 67%, at least 70%, at least 75%, at
least 80%, at least 85%, at least 90%, at least 91%, at least 92%,
at least 93%, at least 94%, at least 95%, at least 96%, at least
97%, at least 98%, at least 99%, or 100% sequence identity to the
mature polypeptide of SEQ ID NO: 6, a polypeptide having at least
90%, e.g., at least 91%, at least 92%, at least 93%, at least 94%,
at least 95%, at least 96%, at least 97%, at least 98%, at least
99%, or 100% sequence identity to the mature polypeptide of SEQ ID
NO: 8, a polypeptide having at least 79%, e.g., at least 80%, at
least 83%, at least 85%, at least 87%, at least 88%, at least 89%,
at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at least 95%, at least 96%, at least 97%, at least 98%, at
least 99%, or 100% sequence identity to the mature polypeptide of
SEQ ID NO: 10, or a polypeptide having at least 92%, e.g., at least
93%, at least 94%, at least 95%, at least 96%, at least 97%, at
least 98%, at least 99%, or 100% sequence identity to the mature
polypeptide of SEQ ID NO: 12; (b) a polypeptide encoded by a
polynucleotide that hybridizes under low stringency conditions, or
medium stringency conditions, or medium-high stringency conditions,
or high stringency conditions, or very high stringency conditions
with (i) the mature polypeptide coding sequence of SEQ ID NO: 1,
SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, or SEQ ID
NO: 11, (ii) the cDNA sequence thereof, or (iii) the full-length
complement of (i) or (ii); (c) a polypeptide encoded by a
polynucleotide having at least 60%, e.g., at least 65%, at least
70%, at least 75%, at least 80%, at least 85%, at least 85%, at
least 90%, at least 91%, at least 92%, at least 93%, at least 94%,
at least 95%, at least 96%, at least 97%, at least 98%, at least
99%, or 100% sequence identity to the mature polypeptide coding
sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7,
SEQ ID NO: 9, or SEQ ID NO: 11; or the cDNA sequence thereof; (d) a
variant of the mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4,
SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, or SEQ ID NO: 12
comprising a substitution, deletion, and/or insertion at one or
more positions; and (e) a fragment of the polypeptide of (a), (b),
(c), or (d) that has xylanase activity.
2. (canceled)
3. (canceled)
4. (canceled)
5. The polypeptide of claim 1, comprising or consisting of SEQ ID
NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, or
SEQ ID NO: 12 or the mature polypeptide of SEQ ID NO: 2, SEQ ID NO:
4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, or SEQ ID NO: 12.
6. (canceled)
7. (canceled)
8. (canceled)
9. (canceled)
10. (canceled)
11. (canceled)
12. (canceled)
13. (canceled)
14. (canceled)
15. (canceled)
16. A nucleic acid construct or expression vector comprising a
polynucleotide encoding the polypeptide of claim 1 operably linked
to one or more control sequences that direct the production of the
polypeptide in an expression host.
17. A recombinant host cell comprising the polynucleotide nucleic
acid construct or expression vector of claim 16.
18. (canceled)
19. A method of producing a polypeptide having xylanase activity,
comprising: (a) cultivating the host cell of claim 17 under
conditions conducive for production of the polypeptide; and (b)
recovering the polypeptide.
20. A transgenic plant, plant part or plant cell transformed with a
polynucleotide encoding the polypeptide of claim 1.
21. A method of producing a polypeptide having xylanase activity,
comprising: (a) cultivating the transgenic plant or plant cell of
claim 20 under conditions conducive for production of the
polypeptide; and (b) recovering the polypeptide.
22. (canceled)
23. (canceled)
24. (canceled)
25. (canceled)
26. A process for degrading a cellulosic material or
xylan-containing material, comprising: treating the cellulosic
material or xylan-containing material with an enzyme composition in
the presence of the polypeptide having xylanase activity of claim
1.
27. The process of claim 26, wherein the cellulosic material or
xylan-containing material is pretreated.
28. The process of claim 26, wherein the enzyme composition
comprises one or more enzymes selected from the group consisting of
a cellulase, a GH61 polypeptide having cellulolytic enhancing
activity, a hemicellulase, an esterase, an expansin, a laccase, a
ligninolytic enzyme, a pectinase, a peroxidase, a protease, and a
swollenin.
29. (canceled)
30. (canceled)
31. The process of claim 26, further comprising recovering the
degraded or converted cellulosic material or xylan-containing
material.
32. The process of claim 31, wherein the degraded cellulosic
material or xylan-containing material is a sugar.
33. (canceled)
34. A process for producing a fermentation product, comprising: (a)
saccharifying a cellulosic material or xylan-containing material
with an enzyme composition in the presence of the polypeptide
having xylanase activity of claim 1; (b) fermenting the
saccharified cellulosic material or xylan-containing material with
one or more fermenting microorganisms to produce the fermentation
product; and (c) recovering the fermentation product from the
fermentation.
35. The process of claim 34, wherein the cellulosic material or
xylan-containing material is pretreated.
36. The process of claim 34, wherein the enzyme composition
comprises one or more enzymes selected from the group consisting of
a cellulase, a GH61 polypeptide having cellulolytic enhancing
activity, a hemicellulase, an esterase, an expansin, a laccase, a
ligninolytic enzyme, a pectinase, a peroxidase, a protease, and a
swollenin.
37. (canceled)
38. (canceled)
39. The process of claim 34, wherein steps (a) and (b) are
performed simultaneously in a simultaneous saccharification and
fermentation.
40. (canceled)
41. A process of fermenting a cellulosic material or
xylan-containing material, comprising: fermenting the cellulosic
material or xylan-containing material with one or more fermenting
microorganisms, wherein the cellulosic material or xylan-containing
material is saccharified with an enzyme composition in the presence
of the polypeptide having xylanase activity of claim 1.
42. The process of claim 41, wherein the fermenting of the
cellulosic material or xylan-containing material produces a
fermentation product and optionally further comprising recovering
the fermentation product from the fermentation.
43. (canceled)
44. The process of claim 1, wherein the cellulosic material or
xylan-containing material is pretreated before
saccharification.
45. The process of claim 41, wherein the enzyme composition
comprises one or more enzymes selected from the group consisting of
a cellulase, a GH61 polypeptide having cellulolytic enhancing
activity, a hemicellulase, an esterase, an expansin, a laccase, a
ligninolytic enzyme, a pectinase, a peroxidase, a protease, and a
swollenin.
46-48. (canceled)
Description
CROSS-REFERENCES TO RELATED APPLICATION
[0001] This application is a divisional of U.S. patent application
Ser. No. 13/765,267, filed on Feb. 12, 2013, the contents of which
are fully incorporated herein.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] The present invention relates to polypeptides having
xylanase activity and polynucleotides encoding the polypeptides.
The invention also relates to nucleic acid constructs, vectors, and
host cells comprising the polynucleotides as well as methods of
producing and using the polypeptides.
[0005] 2. Description of the Related Art
[0006] Cellulose is a polymer of glucose linked by beta-1,4-bonds.
Many microorganisms produce enzymes that hydrolyze beta-linked
glucans. These enzymes include endoglucanases, cellobiohydrolases,
and beta-glucosidases. Endoglucanases digest the cellulose polymer
at random locations, opening it to attack by cellobiohydrolases.
Cellobiohydrolases sequentially release molecules of cellobiose
from the ends of the cellulose polymer. Cellobiose is a
water-soluble beta-1,4-linked dimer of glucose. Beta-glucosidases
hydrolyze cellobiose to glucose.
[0007] The conversion of lignocellulosic feedstocks into ethanol
has the advantages of the ready availability of large amounts of
feedstock, the desirability of avoiding burning or land filling the
materials, and the cleanliness of the ethanol fuel. Wood,
agricultural residues, herbaceous crops, and municipal solid wastes
have been considered as feedstocks for ethanol production. These
materials primarily consist of cellulose, hemicellulose, and
lignin. Once the cellulose is converted to glucose, the glucose is
easily fermented by yeast into ethanol. Xylanases degrade
beta-1,4-xylan into xylose, thus breaking down hemicellulose, one
of the major components of plant cell walls.
[0008] There is a need in the art to improve cellulolytic and
hemicellulolytic enzyme compositions through supplementation with
additional enzymes to increase efficiency and to provide
cost-effective enzyme solutions for degradation of
lignocellulose.
[0009] The present invention provides polypeptides having xylanase
activity and polynucleotides encoding the polypeptides.
SUMMARY OF THE INVENTION
[0010] The present invention relates to isolated polypeptides
having xylanase activity selected from the group consisting of:
[0011] (a) a polypeptide having at least 67% sequence identity to
the mature polypeptide of SEQ ID NO: 2, or a polypeptide having at
least 61% sequence identity to the mature polypeptide of SEQ ID NO:
4, or a polypeptide having at least 61% sequence identity to the
mature polypeptide of SEQ ID NO: 6, or a polypeptide having at
least 90% sequence identity to the mature polypeptide of SEQ ID NO:
8, or a polypeptide having at least 79% sequence identity to the
mature polypeptide of SEQ ID NO: 10, or a polypeptide having at
least 92% sequence identity to the mature polypeptide of SEQ ID NO:
12;
[0012] (b) a polypeptide encoded by a polynucleotide that
hybridizes under low, or medium, or medium-high, or high, or very
high stringency conditions with (i) the mature polypeptide coding
sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7,
SEQ ID NO: 9, or SEQ ID NO: 11, (ii) the cDNA sequence thereof, or
(iii) the full-length complement of (i) or (ii);
[0013] (c) a polypeptide encoded by a polynucleotide having at
least 60% sequence identity to the mature polypeptide coding
sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7,
SEQ ID NO: 9, or SEQ ID NO: 11; or the cDNA sequence thereof;
[0014] (d) a variant of the mature polypeptide of SEQ ID NO: 2, SEQ
ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, or SEQ ID NO:
12 comprising a substitution, deletion, and/or insertion at one or
more (e.g., several) positions; and
[0015] (e) a fragment of the polypeptide of (a), (b), (c), or (d)
that has xylanase activity.
[0016] The present invention also relates to isolated polypeptides
comprising a catalytic domain selected from the group consisting
of:
[0017] (a) a catalytic domain having at least 60% sequence identity
to the catalytic domain of SEQ ID NO: 2 (for example, amino acids
19 to 352 of SEQ ID NO: 2), a catalytic domain having at least 60%
sequence identity to the catalytic domain of SEQ ID NO: 4 (for
example, amino acids 21 to 351 of SEQ ID NO: 4), a catalytic domain
having at least 60% sequence identity to the catalytic domain of
SEQ ID NO: 6 (for example, amino acids 24 to 342 of SEQ ID NO: 6),
a catalytic domain having at least 60% sequence identity to the
catalytic domain of SEQ ID NO: 8 (for example, amino acids 82 to
395 of SEQ ID NO: 8), a catalytic domain having at least 60%
sequence identity to the catalytic domain of SEQ ID NO: 10 (for
example, amino acids 87 to 401 of SEQ ID NO: 10), or a catalytic
domain having at least 60% sequence identity to the catalytic
domain of SEQ ID NO: 12 (for example, amino acids 97 to 392 of SEQ
ID NO: 12);
[0018] (b) a catalytic domain encoded by a polynucleotide having at
least 60% sequence identity to the catalytic domain coding sequence
of SEQ ID NO: 1 (for example, nucleotides 55-1515 of SEQ ID NO: 1),
a catalytic domain encoded by a polynucleotide having at least 60%
sequence identity to the catalytic domain coding sequence of SEQ ID
NO: 3 (for example, nucleotides 61-249, 307-524, 649-665, 721-867,
930-1237, 1295-1351, and 1405-1461 of SEQ ID NO: 3), a catalytic
domain encoded by a polynucleotide having at least 60% sequence
identity to the catalytic domain coding sequence of SEQ ID NO: 5
(for example, nucleotides 79-273, 325-430, 472-598, 718-734,
789-935, 990-1297, and 1353-1400 of SEQ ID NO: 5), a catalytic
domain encoded by a polynucleotide having at least 60% sequence
identity to the catalytic domain coding sequence of SEQ ID NO: 7
(for example, nucleotides 310-453, 511-728, 786-787, 845-865,
923-1069, 1137-1441, 1503-1559, and 1618-1665 of SEQ ID NO: 7), a
catalytic domain encoded by a polynucleotide having at least 60%
sequence identity to the catalytic domain coding sequence of SEQ ID
NO: 9 (for example, nucleotides 316-459, 526-743, 799-800, 859-879,
936-1082, 1143-1447, and 1507-1614 of SEQ ID NO: 9), or a catalytic
domain encoded by a polynucleotide having at least 60% sequence
identity to the catalytic domain coding sequence of SEQ ID NO: 11
(for example, nucleotides 379-522, 586-592, 650-739, 799-1066,
1121-1229, 1286-1378, 1432-1518, and 1584-1673 of SEQ ID NO:
11);
[0019] (c) a variant of a catalytic domain comprising a
substitution, deletion, and/or insertion of one or more (several)
amino acids of the catalytic domain of SEQ ID NO: 2, SEQ ID NO: 4,
SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, or SEQ ID NO: 12;
and
[0020] (d) a fragment of a catalytic domain of (a), (b), or (c),
which has xylanase activity.
[0021] The present invention also relates to isolated
polynucleotides encoding the polypeptides of the present invention;
nucleic acid constructs; recombinant expression vectors;
recombinant host cells comprising the polynucleotides; and methods
of producing the polypeptides.
[0022] The present invention also relates to processes for
degrading a cellulosic material or xylan-containing material,
comprising: treating the cellulosic material or xylan-containing
material with an enzyme composition in the presence of a
polypeptide having xylanase activity of the present invention. In
one aspect, the processes further comprise recovering the degraded
or converted cellulosic material or xylan-containing material.
[0023] The present invention also relates to processes of producing
a fermentation product, comprising: (a) saccharifying a cellulosic
material or xylan-containing material with an enzyme composition in
the presence of a polypeptide having xylanase activity of the
present invention; (b) fermenting the saccharified cellulosic
material or xylan-containing material with one or more (e.g.,
several) fermenting microorganisms to produce the fermentation
product; and (c) recovering the fermentation product from the
fermentation.
[0024] The present invention also relates to processes of
fermenting a cellulosic material or xylan-containing material,
comprising: fermenting the cellulosic material or xylan-containing
material with one or more (e.g., several) fermenting
microorganisms, wherein the cellulosic material or xylan-containing
material is saccharified with an enzyme composition in the presence
of a polypeptide having xylanase activity of the present invention.
In one aspect, the fermenting of the cellulosic material or
xylan-containing material produces a fermentation product. In
another aspect, the processes further comprise recovering the
fermentation product from the fermentation.
[0025] The present invention also relates to a polynucleotide
encoding a signal peptide comprising or consisting of amino acids 1
to 18 of SEQ ID NO: 2, amino acids 1 to 20 of SEQ ID NO: 4, amino
acids 1 to 23 of SEQ ID NO: 6, amino acids 1 to 19 of SEQ ID NO: 8,
amino acids 1 to 19 of SEQ ID NO: 10, or amino acids 1 to 22 of SEQ
ID NO: 12, which is operably linked to a gene encoding a protein;
nucleic acid constructs, expression vectors, and recombinant host
cells comprising the polynucleotides; and methods of producing a
protein.
SEQUENCES OF THE INVENTION
[0026] Trametes versicolor Strain NN055586 Genomic Nucleotide
Sequence (SEQ ID NO: 1):
TABLE-US-00001 1 ATGCACTTCT CACTCCTCGC AGCCTTCATC GCGCTGGCTC
CGGCCGCGCT CGCGATCCCC GCGACCCCCG TGGTCGATGC 81 GAGTCTCCCC
GGCTCGACCG CGAACGTGGC GGGTCTGCAC GCCGTCGCGA AGGCGGCGGG CAAGCTCTAC
TTGGGCACTG 161 CGACGGACAA TAACGAGCTT ACCAACACGC AGTACACCGC
CATCCTCGAG GCTCCGAACA TGTTCGGCCA GATCACCGCC 241 GAGAACACCA
TGAAATGGGT CAGTTGCGCC TTGTCGCGAT GTTCCGAGCA CGTTCCGCAA GCTAACGATT
TGATGCTAGG 321 ACGCGACCGA GCCCCAGCAG AACGTGTTCA CGTTCACGCA
GGGCGACCAG ATCGCGAACC TGGCGAAGTC CCACGGGATG 401 CTGCTGCGCG
GTAACGACAG TCTTGAAGGG GCCGAAAATG TGGAAGAACT GAATGTTTTC GCAGGTCACA
ACTGCGTCTG 481 GCACCAACAG CTCCCGAGCT GGGTAACTGC CGGGAACTTC
AATGCACAGC AGCTTACGCA GATCATCCAA AACCACTGCG 561 GCACGGTCGT
CGGACACTAC AAGGGACAAG TGTGAGTGTT GGCCATATCC GCCGACGTGT ATCGTGTGCT
GACCGTGTTT 641 TATAGTTGTA CGTGACGCTT GTTGCTTGAT CGTATGAATC
CACTTAGCTG ACTCGACGCA GTGAGCTGGG ATGTAGTAAA 721 CGGTAACGGA
CATTCCTTCT CTTTGCTACA CACAACTCGG ACTCACACTC GGCTGCAGAG CCTCTCAACG
ACGACGGCTC 801 GTTCCGCCAG GACGTGTTCT TCAACACTCT CGGCTCGGGC
TACATCGCGA CGGCGCTCCG CGCTGCCAGG GCCGCAGACC 881 CTGCGGCGAA
GCTGTACATC AACGAGTTTA ACGTCGAGGG CCTAGGTTCG TCCCACCGTA TCCCCGCTCA
TTGCATCGTC 961 TGAGCCCGAA TCTTCTAGGC GCAAAGTCGA CCGCCTTGAA
GAACCTCGTC ACTTCGCTGA AGCAGCAGGG CGTCCCGATC 1041 GACGGCGTCG
GTTTCCAGTG CCACTTCATC GTCGGCCAGG TCCCCACGAC GCTCATCCAG AGCATGCAGC
AGTTCACTGC 1121 GCTCGGCCTC GAGGTGGCCA TCACGGAGCT CGACATCCGC
ATGACGCTCC CTGAAACTGC GGCGCTGCTC GAGCAGCAGA 1201 AGCAGGACTT
CCAGACTGTT ATCCACGCGT GTAAGTCCGT GGCGGGCTGT GTCGGCGTCA CTGTGTGGGA
CTTCACCGAT 1281 AAGGTATGTC GTTGTCCCGC CCCGGCGAGA TGGTTGGATT
AGCGTGCTCA CCGGTATTAT ACCAGTTCTC CTTCGTGCCG 1361 AGCACGTTCC
CGGGTCAGGG TGCTGCCACT CCTTGGGATC AGGTACGTCC CGCCGAACCT TGGGCCTTAC
AAGCTCCGGG 1441 AGAGGGCTAA CTGCGGATGC GCAGAACCTG GTGAAGAAGC
CGGCATTTGA TGGCATCGTC GCCGGATTCC AGCAGTGA
Exons/Introns (in Base Pairs) of SEQ ID NO: 1:
TABLE-US-00002 [0027] Exon 1 1-258 bp Intron 1 259-319 bp Exon 2
320-410 bp Intron 2 411-464 bp Exon 3 465-591 bp Intron 3 592-645
bp Exon 4 646-651 bp Intron 4 652-701 bp Exon 5 702-715 bp Intron 5
716-778 bp Exon 6 779-925 bp Intron 6 926-978 bp Exon 7 979-1283 bp
Intron 7 1284-1345 bp Exon 8 1346-1402 bp Intron 8 1403-1464 bp
Exon 9 1465-1518 bp
Features (in Base Pairs) of SEQ ID NO: 1:
TABLE-US-00003 [0028] Signal Peptide 1-54 bp Xylanase catalytic
site 55-1515 bp Stop codon 1516-1518 bp
Protein Sequence of Trametes versicolor Strain NN055586 Protein
(SEQ ID NO: 2):
TABLE-US-00004 1 MHFSLLAAFI ALAPAALAIP ATPVVDASLP GSTANVAGLH
AVAKAAGKLY LGTATDNNEL 61 TNTQYTAILE APNMFGQITA ENTMKWDATE
PQQNVFTFTQ GDQIANLAKS HGMLLRGHNC 121 VWHQQLPSWV TAGNFNAQQL
TQIIQNHCGT VVGHYKGQVC TELGCKPLND DGSFRQDVFF 181 NTLGSGYIAT
ALRAARAADP AAKLYINEFN VEGLGAKSTA LKNLVTSLKQ QGVPIDGVGF 241
QCHFIVGQVP TTLIQSMQQF TALGLEVAIT ELDIRMTLPE TAALLEQQKQ DFQTVIHACK
301 SVAGCVGVTV WDFTDKFSFV PSTFPGQGAA TPWDQNLVKK PAFDGIVAGF QQ
Features of SEQ ID NO: 2 (Amino Acid Positions):
TABLE-US-00005 [0029] Signal Peptide 1-18 Xylanase catalytic site
19-352
Signal Peptide Sequence of SEQ ID NO: 2:
TABLE-US-00006 [0030] MHFSLLAAFIALAPAALA
Trametes versicolor Strain NN055586 Genomic Nucleotide Sequence
(SEQ ID NO: 3):
TABLE-US-00007 1 ATGCTCTCTC TGTCAAAAAG CCTTCTTGCG CTCTCTGTCT
TGGTCCGAGG TGCGCTCGCC GTACCTGCCA GCGATGCGAG 81 TAGCGCTCTG
TTCCCATTGT CGGGGCTGAA TCTGGCCGCC AAGGGCGCGC GGAAGTTGTA CCTTGGCACG
GCAACGAACA 161 GCGAGCAGTG GAACGACACG ACGTACTTCA ACATCCTGAA
GAACAACGCC GAGTTCGGGC AGGTAACGCC CGCGAACGTC 241 ATGAAATGGG
TACGTTGTCG GCGTCCTCTT CGTACTGACG ATGTTGAGGC TAACTTTGAC GCATAGTTTG
CGACGGAGCC 321 TGAGGAGGGC GTCTTCACGT TCCAGGACGG GGATATCATC
GCGGACTTTA CCAAAAAGAC GGGGAAGCTG CTGCGCGGAC 401 ACAACTGCGT
GTGGCACAAC CAGCTCCCCG ACTGGCTAGA AACCGGCACG TTCAGTGCGC CCGAGCTCGC
ATTCATTGTC 481 TCGCGGCACT GCTTCAACCT CGTGAACCAC TACCAAGGCT
ATGTGTGAGT GCAATTCGTT ACCTGTGATC CTGCTCAACG 561 ATCTAAATCG
GTACACGGCA GGTGTACGTA CAAGCGTGTC GGTGATGTTT CATTGAGGCT GATGGCTTAT
TTTGGAAAAT 641 TCAGATAGCT GGGACGTCAT CAATGGTTCG TGCTACTTGA
CTTCCCGGAT GTGCTTGTTT CCGATCTCGA ATTTTGCTAG 721 AGGCTTTCAA
CGACGACGGA ACCTTCCGTT CGGATATCTT CTTCGACACG CTCAACACAA CCTACATCCC
GCTCGCCCTC 801 TACGCAGCAC GCGCCGCGGA CCCCAAAGCG AAGCTCTACA
TCAACGACTT CAACATCGAA GGCATAGGTA CGCCACATAA 881 CACCATCTGC
CCGCCGCAAA GCCCTGCCAC CCAACTACCC TACTCGCAGG TGCGAAGTCC GACGCGCTCA
AGAGTCTCAT 961 CAAGGAGCTC AAGAGCCAAA ACGTCCCCAT CGACGGCGTC
GGGCTGCAGT CGCACTTCGA GGTCGGCGGC GTCCCGCCCA 1041 CGCTGCAGCA
GAACATGGAG GAGTTCGTCG CGCTCGGGCT CGAGGTCGCG ATCACGGAGC TCGACATCCG
CTTCACCGCG 1121 CTCCCGCCGA CGCCTGCAGG CCTCGCGCAG CAGAAGGCGG
ACTACGAGAC CGTCGTCGCC GCGTGCAACG CGGTCCCGAA 1201 GTGCGTCGGG
GTCACGCTGT GGGACTTCAC GGACAAGGTG CGTCTGCGAG ATTGTGGTCG TGTGATGGGT
GTTGATGCCG 1281 GATGGGCGGG GTAGTACTCG TGGATCCCGG GGACCTTCCC
TGGGCAGGGA GATGCGTGTC CCTGGACGGA TGTACGTTCC 1361 TTAGTCTGTC
TCGTCCGAAG GTGTGATCTA ATGATGTACC ACAGGAATTT GTGAAGAGGC CAGCATACGA
GGGCATCATC 1441 GAGGGGTTCA AGGCCCACCA TTAG
Exons/Introns (in Base Pairs) of SEQ ID NO: 3:
TABLE-US-00008 [0031] Exon 1 1-249 bp Intron 1 250-306 bp Exon 2
307-524 bp Intron 2 525-648 bp Exon 3 649-665 bp Intron 3 666-720
bp Exon 4 721-867 bp Intron 4 868-929 bp Exon 5 930-1237 bp Intron
5 1238-1294 bp Exon 6 1295-1351 bp Intron 6 1352-1404 bp Exon 7
1405-1464 bp
Features (in Base Pairs) of SEQ ID NO: 3:
TABLE-US-00009 [0032] Signal Peptide 1-60 bp Xylanase 61-249,
307-524, 649-665, 721-867, catalytic site 930-1237, 1295-1351,
1405-1461 bp Stop codon 1462-1464 bp
Protein Sequence of Trametes versicolor Strain NN055586 Protein
(SEQ ID NO: 4):
TABLE-US-00010 1 MLSLSKSLLA LSVLVRGALA VPASDASSAL FPLSGLNLAA
KGARKLYLGT ATNSEQWNDT 61 TYFNILKNNA EFGQVTPANV MKWFATEPEE
GVFTFQDGDI IADFTKKTGK LLRGHNCVWH 121 NQLPDWLETG TFSAPELAFI
VSRHCFNLVN HYQGYVWDVI NEAFNDDGTF RSDIFFDTLN 181 TTYIPLALYA
ARAADPKAKL YINDFNIEGI GAKSDALKSL IKELKSQNVP IDGVGLQSHF 241
EVGGVPPTLQ QNMEEFVALG LEVAITELDI RFTALPPTPA GLAQQKADYE TVVAACNAVP
301 KCVGVTLWDF TDKYSWIPGT FPGQGDACPW TDEFVKRPAY EGIIEGFKAH H
Features of SEQ ID NO: 4 (Amino Acid Positions):
TABLE-US-00011 [0033] Signal Peptide 1-20 Xylanase catalytic site
21-351
Signal Peptide Sequence of SEQ ID NO: 4:
TABLE-US-00012 [0034] MLSLSKSLLALSVLVRGALA
Trametes versicolor Strain NN055586 Genomic Nucleotide Sequence
(SEQ ID NO: 5):
TABLE-US-00013 1 ATGATGACGA ACTTCCACCT AGTCTCCTCG TTGATCGCGC
TCGCGTTTCT TTCGCTGACC GGCTTAGCAT CCATTCCGTC 81 GACACGCGCT
CTGGCTGGAA ATTCCTCGAG GATCAATTCT CCTTCTGGCT TGAACTTGGC GGCTTTGGAA
GCCCGGAAGC 161 TGTACTTTGG TACTGCGACC AACAACGTCG AGCTCAACGA
CACGGCATAC TTCGACATTC TCGATGATTT CAAAATGTTC 241 GGTCAAATTA
CGCCCGCCAA AGGCATGAAA TGGGTCCGTT CTCCAACTTC TGCATCATAA ATCGCTCGCT
GATTGTTTTG 321 GAAGATGGAG ACGGAACCTG AGCGAGGCGT TTTCACCTTC
GCGCAGGCAG ACCAAATCGC GCAACTTGCC AGCGAGGGCG 401 GAAAGCTGTT
GAGAGGCTCG TACTCGAAAG GTCCCTCGCT ACCCCATACG TGCTAACATT CCGTTCTGCA
GGCCACAACT 481 GCGTATGGTA TAATGCGCTT CCCGGGTGGG TCACAAATAC
CACGTGGACG GCCTCCGAGA TGGCCGAGGT CGTACAGGAG 561 CATTGTTTCA
ACATCGTCCG TTACTGGCAA GGACAAGCGT GAGTACCGAT CTCTCTCATT AATATCGTGT
CTCTCAATTT 641 TTATTCCGAG TAGATGTGAG TATCAACGCC TTCCGGAGGA
ATCCCGCTGA ACATAGGCCG TCCTTTTACA CAGACAGCTG 721 GGACGTTATT
AACGGTGAGT TGCTCGAGAT TGAAGGCAGC TGCCCGTAGC TTACACCATT TCCCGCAGAG
CCATTCAACG 801 ATGACGGAAC GTGGCGCGAG ACCATGTGGT TTAATACTCT
CAACACGAGC TACATTCCGC TCGCGTTGCA CGCTGCGCGC 881 GCGGCCGATC
CTCATACTAA GCTGTACATC AATGAGTACA ATATCACCGG AACAGGTGCG TCGTACGCCT
CACGCTCAGA 961 CTATGCCTCC TTCATCATTC AGTATACAGG CCCGAAGGCG
ACGTCCATGA AGAACCTCAT CAAAGACTTG AAGCGCGCTG 1041 GTGTGCCCGT
TCACGGCGTT GGAGTTCAAG CGCACGAGAC CGTCGGGGAA GTTCCGACCG ACATCCGCAA
GAACCTCGGG 1121 GAGTTCGTCG CACTCGGCGT CGAGGTCGCG ATCACAGAAC
TCGACATCAA GTTCAACACG CTTCCTCCTG ATGCAGCCGG 1201 GCTCAAACAA
CAGAAGCGAG ATTACGAAGC TATTGTCTCG GCGTGCGCGG AGGTAAAAGG ATGTGTGGGC
GTGACGGTTT 1281 GGGACTTCAC GGACAAGGTG GGAAAGTATT CGACTTCACG
AAGCAATACC AAGTATTCAC CTTTGCGTAC AGTACTCATG 1361 GATCCCCGGA
ACGTTCCCTG GAACCGGCGA TGCTTGTCCT TGAgacgatg tgagcgtgat gctattgcgt
attgcttttt 1441 ctactgactg tctctcgtac ttctttgtct caggatttgc
acaagaagcc ggcgtactat ggaattttgg acgggtttgg 1521 gagatctcgc tga
Exons/Introns (in Base Pairs) of SEQ ID NO: 5:
TABLE-US-00014 [0035] Exon 1 1-273 bp Intron 1 274-324 bp Exon 2
325-430 bp Intron 2 431-471 bp Exon 3 472-598 bp Intron 3 599-717
bp Exon 4 718-734 bp Intron 4 735-788 bp Exon 5 789-935 bp Intron 5
936-989 bp Exon 6 990-1297 bp Intron 6 1298-1352 bp Exon 7
1353-1403 bp 3' UTR 1404-1533 bp
Features (in Base Pairs) of SEQ ID NO: 5:
TABLE-US-00015 [0036] Signal Peptide 1-69 bp Xylanase 79-273,
325-430, 472-598, 718-734, catalytic site 789-935, 990-1297,
1353-1400 bp Stop codon 1401-1403 bp
Protein Sequence of Trametes versicolor Strain NN055586 Protein
(SEQ ID NO: 6):
TABLE-US-00016 1 MMTNFHLVSS LIALAFLSLT GLASIPSTRA LAGNSSRINS
PSGLNLAALE ARKLYFGTAT 61 NNVELNDTAY FDILDDFKMF GQITPAKGMK
WMETEPERGV FTFAQADQIA QLASEGGKLL 121 RGSYSKGHNC VWYNALPGWV
TNTTWTASEM AEVVQEHCFN IVRYWQGQAW DVINEPFNDD 181 GTWRETMWFN
TLNTSYIPLA LHAARAADPH TKLYINEYNI TGTGPKATSM KNLIKDLKRA 241
GVPVHGVGVQ AHETVGEVPT DIRKNLGEFV ALGVEVAITE LDIKFNTLPP DAAGLKQQKR
301 DYEAIVSACA EVKGCVGVTV WDFTDKYSWI PGTFPGTGDA CP
Features of SEQ ID NO: 6 (Amino Acid Positions):
TABLE-US-00017 [0037] Signal Peptide 1-23 Xylanase catalytic site
24-342
Signal Peptide Sequence of SEQ ID NO: 6:
TABLE-US-00018 [0038] MMTNFHLVSSLIALAFLSLTGLA
Trametes versicolor Strain NN055586 Genomic Nucleotide Sequence
(SEQ ID NO: 7):
TABLE-US-00019 1 ATGAACCTCT CAGCGTCGTT CGCAGTACTT GTCGCTCTGA
TCCCGTACGC CCTCGCGCAG TCCCCGGAGT GGGGCCAATG 81 CGGCGGAACA
GGCTATACGG GCGCCACGAC TTGCGTGTCC GGAACGGTAT GCACGGTGAT CAACCCGTAC
TACTCACAAT 161 GTCTCGCAGG CACTGTAAGT ACACGACACA CGCATCTTTG
TCAGGTCACA GGGGAGCCAA ACGCTGACGG TCACCTCAAG 241 GCCACATCCG
CGCTCCTCGC TCCCAGCCCA ACTGTGAGCA CCGGCGCACC CGCCCCGAGC GTCAGCGGTC
TGCACACTCT 321 CGCCAAAGCC GCTGGGAAGC TCTACTTTGG CAGCGCGACG
GACAACCCCG AGCTGACCGA CACCGCCTAC GTCGCGAAGC 401 TCAGCGACAA
CGCCGAGTTC GGCCAGATCA CCCCCGGTAA CAGCATGAAA TGGGTGAGTA CCGCACCCTC
CATCCCCATT 481 CTCAGGTTTG TGAGAATGAA TGCGTCGTAG GACGCGACGG
AGCCGACGCG GGGGACGTTC ACGTTCACGG GCGGGGACGT 561 GGTTGCGAGC
CTGGCGGAGA AGAACGGGCA GCTGCTGCGC GGGCACAACT GCGTGTGGTA CAACCAGCTC
CCGAGCTGGG 641 TCGCGAACGG GCAGTTCACG GCTGCGGATT TGACGGACGT
GATCACGACG CACTGCGGCA CGCTCGTTGG CCACTACAAG 721 GGACAAATGT
GAGTGCCGGT CTTACTCTCG AATAATCGTG TTACAGTATG CTAATGGAGG CGCAGCTGTA
CGCATCATAG 801 GGTTGTTCGT GACTGTTGCT GGTACTGACT TGCTCGTACC
GAAGACTCTT GGGACGTCAT CAATGGTCAG TTGTCGTGAG 881 CGAGATCGTG
CATTACAGTA TGCTCAATAT TTTCGTGCCT AGAACCCTTT AACGACGACG GTACCTGGCG
CTCGGATGTG 961 TTCTTCAATA CGCTCGGTCA GTCCTACGTC TCCATCGCGC
TCAAAGCCGC ACGCGCTGCA GACCCCAACG CCAAGCTCTA 1041 CATCAACGAC
TACAACATCG AGCAGACCGG TGCGCCCCTC CTTTCCTTGA TACTTCCCTT AGCACCATCA
AACTAACCCT 1121 GCATATGATC GCACAGGCGC GAAGTCGACC GCGATGCTGA
ACCTCGTGAA GCAGCTACAA GCAGACGGCG TGCCAATCGA 1201 CGGCGTCGGC
TTCCAGAGCC ACTTCATCGT TGGCGAGGTC CCCGGCTCGT TCCAGACCGT GCTCGAGCAG
TTCACCGCGC 1281 TCGGGCTCGA GGTCGCGATC ACGGAGCTCG ACATCCGCAT
GACGCTCCCC GCGACGGACG CGCTCCTCGC GCAGCAGCAG 1361 AAGGACTACC
AGAGCGTCGT GCAGGCGTGC ATGAACGTGC AGGGCTGTGT GGGCGTCACG ATCTGGGACT
GGACGGACAA 1441 GGTGCGTGTG GTGGGGTGGA GAGAGCGAGC GAGGAGGGTG
CTGATAGGGA CTCTTGGGGC AGTACTCGTG GGTGCCGTCG 1521 ACGTTCTCGG
GACAGGGCGC GGCTCTGCCT TGGGACGAGG TGGGTGGTCC TCTCCCGCGT TCTGGGGATA
CTCAATGGAC 1601 GCATTTACGT TCGTCAGACC TTCAACAAAA AGCCCGCATA
CAGCGGCATC ACGGCGGCAC TGACGTGA
Exons/Introns (in Base Pairs) of SEQ ID NO: 7:
TABLE-US-00020 [0039] Exon 1 1-174 bp Intron 1 175-240 bp Exon 2
241-453 bp Intron 2 454-510 bp Exon 3 511-728 bp Intron 3 729-785
bp Exon 4 786-787 bp Intron 4 788-844 bp Exon 5 845-865 bp Intron 5
866-922 bp Exon 6 923-1069 bp Intron 6 1070-1136 bp Exon 7
1137-1441 bp Intron 7 1442-1502 bp Exon 8 1503-1559 bp Intron 8
1560-1617 bp Exon 9 1618-1668 bp
Features (in Base Pairs) of SEQ ID NO: 7:
TABLE-US-00021 [0040] Signal Peptide 1-57 bp Cellulose Binding
58-168 bp Module 1 (CBM 1) Linker 169-174, 241-309 bp Xylanase
310-453, 511-728, 786-787, 845-865, 923-1069, catalytic site
1137-1441, 1503-1559, 1618-1665 bp Stop codon 1666-1668 bp
Protein Sequence of Trametes versicolor Strain NN055586 Protein
(SEQ ID NO: 8):
TABLE-US-00022 1 MNLSASFAVL VALIPYALAQ SPEWGQCGGT GYTGATTCVS
GTVCTVINPY YSQCLAGTAT 61 SAPSAPSPTV STGAPAPSVS GLHTLAKAAG
KLYFGSATDN PELTDTAYVA KLSDNAEFGQ 121 ITPGNSMKWD ATEPTRGTFT
FTGGDVVASL AEKNGQLLRG HNCVWYNQLP SWVANGQFTA 181 ADLTDVITTH
CGTLVGHYKG QIYSWDVINE PFNDDGTWRS DVFFNTLGQS YVSIALKAAR 241
AADPNAKLYI NDYNIEQTGA KSTAMLNLVK QLQADGVPID GVGFQSHFIV GEVPGSFQTV
301 LEQFTALGLE VAITELDIRM TLPATDALLA QQQKDYQSVV QACMNVQGCV
GVTIWDWTDK 361 YSWVPSTFSG QGAALPWDET FNKKPAYSGI TAALT
Features of SEQ ID NO: 8 (Amino Acid Positions):
TABLE-US-00023 [0041] Signal Peptide 1-19 Cellulose Binding 20-56
Module 1 (CBM 1) Linker 57-81 Xylanase 82-395 catalytic site
Signal Peptide Sequence of SEQ ID NO: 8:
TABLE-US-00024 [0042] MNLSASFAVLVALIPYALA
Trametes versicolor Strain NN055586 Genomic Nucleotide Sequence
(SEQ ID NO: 9):
TABLE-US-00025 1 ATGCAGCTCT CGACGACCTT CACCCTCCTC GCCGCGATCA
TTCCGTTCGC CCTCGGGCAG GCCGCGGAGT GGGGCCAGTG 81 CGGTGGCATT
GGCTGGACCG GCGCGACGAC GTGCGTGGCG GGCACCACCT GCACGGTCAT GAACGCGTAC
TACTCCCAGT 161 GCCTCCCCGG TTCTGTGAGT GGCTGTGCTG TGGTAGAGAC
GTTCAACATG CTGACCGGTG AATGCTTGTA GGCTGCGCCC 241 GCGCCGACGA
CGACCCCCAC CTCGCCTTCG AGCCCGGCGA CCCCGCCGTC CGCGCCTGCG CCAACCGGCA
GCGGCCTCAA 321 CAAGCTCGCG AAGGCGGCTG GCAAGCTCTA CCTCGGCACT
GCGACGGACA ACAGCGAGCT CACCGATGCG GCGTACACCG 401 CCATCCTCGA
CGACAACTCC CAGTTCGGCC AGATCACGCC CGCCAACAGC ATGAAATGGG TGCGCATTAT
CCCTGCATCG 481 TGTACTAGAA CGCTCCTTGC TTATTGTTGT AAAATTGGAA
TGCAGGACGC GACAGAGCCG ACTCGCGGAA CGTTCACGTT 561 CTCGGGTGGT
GACCAGATCG CGAACCTGGC GAAGACGAAC GGGATGCTTC TCCGTGGACA CAACTGCGTG
TGGTACAACC 641 AGCTCCCGAG CTGGGTTGCG AACGGCCAGT TCACCGCCGC
GGACCTCACG ACCGTCATCC AGACGCACTG CAGCACCCTC 721 GTCAGCCACT
ACAAGGGTCA AGTGTACGTG ATTCCTTCTG TGTATCTACT CTCCCAATAC TGACCCCATT
TTCCGCAGTT 801 GTACGTCTAC GTTCGCATTT ATGATTCTTG TATGCATACT
GACCGACATG ACAAAAAGAC TCCTGGGACG TCGTCAACGG 881 TTAGTGGTAT
TACTCCACAA GTTCACCAGG GAAGTGTTCT GACAGTGATC TCCAGAGCCG TTCAACGACG
ATGGTACCTG 961 GCGCTCGGAC GTGTTCTACA ACACGCTCGG CACTTCGTAC
GTGCCCATCG CGCTCAAGGC TGCGCGCGCT GCGGACCCTA 1041 GCGCCAAACT
CTACATCAAC GACTACAACA TTGAGCAGAC GGGTAGGTCC CCAGCATCCA TCTCCCAGGA
GTGACGCCGC 1121 TCACGGCACA CACGCACCAC AGGCGCCAAG GCGACCGCGA
TGCTGAACCT CGTGAAGCAG CTCATCGCCG ACGGCGTTCC 1201 GATCGACGGT
GTCGGCTTCC AGTGCCACTT TATCGTTGGC GAGGTCCCCG GCTCGTTCCA GACCGTGCTC
GAGCAGTTCA 1281 CCGCGCTCGG GCTCGAGGTC GCGATCACGG AGCTCGACAT
CCGCACGACG ACGCCCGCGT CGCAGTCCGC GCTCGCACAG 1361 CAGGAGAAGG
ACTACCAGTC GGTTATCCAG GCGTGCATGA ACGTCAAGGG CTGCGTTGGT GCCACCCTCT
GGGACTTCAC 1441 CGACAAGGTT CGTAGGCAAG CTTTCTACGC GTGTAAGACG
AATTGGCTGA CGCTCTTGCG ATGCAGTACT CCTGGGTCCC 1521 CTCGACGTTC
TCCGGCCAAG GTGCGGCGTG CCCTTGGGAC CAGAACCTCG TCAAGAAGCC CGCGTACACT
GGTATCGTCA 1601 ACGCTCTCAG CGCGTGA
Exons/Introns (in Base Pairs) of SEQ ID NO: 9:
TABLE-US-00026 [0043] Exon 1 1-174 bp Intron 1 175-231 bp Exon 2
232-459 bp Intron 2 460-525 bp Exon 3 526-743 bp Intron 3 744-798
bp Exon 4 799-800 bp Intron 4 801-858 bp Exon 5 859-879 bp Intron 5
880-935 bp Exon 6 936-1082 bp Intron 6 1083-1142 bp Exon 7
1143-1447 bp Intron 7 1448-1506 bp Exon 8 1507-1617 bp
Features (in Base Pairs) of SEQ ID NO: 9:
TABLE-US-00027 [0044] Signal Peptide 1-57 bp Cellulose Binding
58-168 bp Module 1 (CBM 1) Linker 169-174, 232-315 bp Xylanase
316-459, 526-743, 799-800, 859-879, catalytic site 936-1082,
1143-1447, 1507-1614 bp Stop codon 1615-1617 bp
Protein Sequence of Trametes versicolor Strain NN055586 Protein
(SEQ ID NO: 10):
TABLE-US-00028 1 MQLSTTFTLL AAIIPFALGQ AAEWGQCGGI GWTGATTCVA
GTTCTVMNAY YSQCLPGSAA 61 PAPTTTPTSP SSPATPPSAP APTGSGLNKL
AKAAGKLYLG TATDNSELTD AAYTAILDDN 121 SQFGQITPAN SMKWDATEPT
RGTFTFSGGD QIANLAKTNG MLLRGHNCVW YNQLPSWVAN 181 GQFTAADLTT
VIQTHCSTLV SHYKGQVYSW DVVNEPFNDD GTWRSDVFYN TLGTSYVPIA 241
LKAARAADPS AKLYINDYNI EQTGAKATAM LNLVKQLIAD GVPIDGVGFQ CHFIVGEVPG
301 SFQTVLEQFT ALGLEVAITE LDIRTTTPAS QSALAQQEKD YQSVIQACMN
VKGCVGATLW 361 DFTDKYSWVP STFSGQGAAC PWDQNLVKKP AYTGIVNALS A
Features of SEQ ID NO: 10 (Amino Acid Positions):
TABLE-US-00029 [0045] Signal Peptide 1-19 Cellulose Binding 20-56
Module 1 (CBM 1) Linker 57-86 Xylanase 87-401 catalytic site
Signal Peptide Sequence of SEQ ID NO: 10:
TABLE-US-00030 [0046] MQLSTTFTLLAAIIPFALG
Trametes versicolor Strain NN055586 Genomic Nucleotide Sequence
(SEQ ID NO: 11):
TABLE-US-00031 1 ATGAAGGGCC TCGCCGCACT CGTCGCACTC GCCACCATCG
TCGCCGTCCC GGCCAACGCC GTCGCGGTCT GGGGCCAATG 81 TGAGCATCCC
TCACCCGGAC TTATACCTCT GGAATAGTAA CACTGACATG CGTTTGCAGG CGGAGTACGC
ACCTTTGCCC 161 GCTGCGCTCG TCCTGTCTAC GCTTGACACT GACCTCTCTG
TCAGGGTATC GGCTTCAGTG GATCGACCAC ATGTGATGCC 241 GGCACCACAT
GCATCGTGCT CAACTCCTAC TACTCGCAGT GCCAGCCGGG TGCGAGCGCG CCCGCGCCCA
CGACATCCGC 321 CCCCCAGCCG CCCCCGACCA CACCGGCTGG TGGCTCGCCC
GCGCCCGCGG CGACCGGACT CAACGCTGCG TTCAAGAAGC 401 ACGGCAAGAA
GTTCTGGGGC ACCGCGACGG ACTCAAACCG CTTCAGCAAC CCGACGGACT CCGCGGTCAC
CGTCCGCGAG 481 TTCGGCCAGG TCACGCCTGA GAACTCCATG AAGTGGGATG
CGGTGAGTGC CTACTGGGCG CGTCGGCGTC GAGTGAGCAT 561 GTGCTTATGA
TTATTTTCGT CGTAGACTGA GCGTGCGTAT TTAGTGAGGC TTCGGATGGT CCTCCCAGGA
AACTGACAGC 641 ATGTTGCAGC TTCCCGCAAC CAGTTCTCGT TCAGCGGCTC
TGATGCGCTG GTCAACTTCG CTACGACGAA TGGCCTGCTC 721 GTCCGCGCTC
ACACCCTCGG TAAGCATGTT CTCGTTGTCT CATCTCTGAA GTGGCGACTA ACTGTTCTTG
GGGCGCAGTC 801 TGGCATTCGC AACTGCCGTC CTGGGTCTCT GCGATCAACG
ACCGCGCGAC GCTCACGTCC GTGATCCAGA ACCACATCGC 881 GAACGTCGCA
GGCCGGTACA AGGGCAAGGT GTACTCCTGG GACGTCGTGA ACGAGATCTT CAACGAGGAC
GGCACGTTCC 961 GCTCGTCGGT GTTTTCAAAC GTCCTCGGCC AGGACTTCGT
CACGATCGCG TTCCAGGCGG CACGGGCGGC GGACCCGAAC 1041 GCGAAGCTCT
ACATCAACGA CTACAAGTGT GTCTCGCGGG TTGGCTTGGT GTGCCTTTGC TGATGCGTTT
GTGTATGCAG 1121 CCTCGACACC GTGAACCCAA AGCTCAACGG TGTTGTCAAC
CTTGTCAAGA AGATCAACGG CGGCGGCACC AAGCTGATCG 1201 ACGGTATCGG
TACTCAGGCC CACCTTTCGG TAAGTGTATC AGGACTATTT AGCAGACTGA CGTGCTGACG
CTAGAGCTCG 1281 GATAGGCTGG CGGCGCTGGC GGATTCCAGG CTGCGCTCAC
GCAGCTGGCT ACCGCCGGCA CGGAGATCGC TATCACGGAG 1361 CTCGACATTG
CGGGTGCCGT AAGTATCCGT TACAATGATT TCGCGCTGCT CCTTATTTAT GTCGCATTCA
GGCCCCCAAT 1441 GACTACTCGA CGCTGGTCAA GGCGTGTCTC GCGGTGGAGA
GCTGCGTGTC CATCACAAGC TGGGGAGTCC GCGATCCCGT 1521 AAGCAATATA
TCTTCCTTGT TGACGGTGAT GAGACGTTCT CACCATGTGC ATGCTTTTAT CAGGACTCCT
GGAGGGCGTC 1601 CACCAACCCC CTCTTGTTCG ACGCGAACTT CAACCCGAAG
CCCGCATACA CTGCGGTTAT GCAGGCCCTG GCTTGA
Exons/Introns (in Base Pairs) of SEQ ID NO: 11:
TABLE-US-00032 [0047] Exon 1 1-79 bp Intron 1 80-139 bp Exon 2
140-174 bp Intron 2 175-204 bp Exon 3 205-522 bp Intron 3 523-585
bp Exon 4 586-592 bp Intron 4 593-649 bp Exon 5 650-739 bp Intron 5
740-798 bp Exon 6 799-1066 bp Intron 6 1067-1120 bp Exon 7
1121-1229 bp Intron 7 1230-1285 bp Exon 8 1286-1378 bp Intron 8
1379-1431 bp Exon 9 1432-1518 bp Intron 9 1519-1583 bp Exon 10
1584-1676 bp
Features (in Base Pairs) of SEQ ID NO: 11:
TABLE-US-00033 [0048] Signal Peptide 1-66 bp Cellulose Binding
67-79, 140-174, 205-288 bp Module 1 (CBM 1) Linker 289-378 bp
Xylanase 379-522, 586-592, 650-739, 799-1066, 1121-1229, catalytic
site 1286-1378, 1432-1518, 1584-1673 bp Stop codon 1674-1676 bp
Protein Sequence of Trametes versicolor Strain NN055586 Protein
(SEQ ID NO: 12):
TABLE-US-00034 1 MKGLAALVAL ATIVAVPANA VAVWGQCGVR TFARCARPGI
GFSGSTTCDA GTTCIVLNSY 61 YSQCQPGASA PAPTTSAPQP PPTTPAGGSP
APAATGLNAA FKKHGKKFWG TATDSNRFSN 121 PTDSAVTVRE FGQVTPENSM
KWDATEPSRN QFSFSGSDAL VNFATTNGLL VRAHTLVWHS 181 QLPSWVSAIN
DRATLTSVIQ NHIANVAGRY KGKVYSWDVV NEIFNEDGTF RSSVFSNVLG 241
QDFVTIAFQA ARAADPNAKL YINDYNLDTV NPKLNGVVNL VKKINGGGTK LIDGIGTQAH
301 LSAGGAGGFQ AALTQLATAG TEIAITELDI AGAAPNDYST LVKACLAVES
CVSITSWGVR 361 DPDSWRASTN PLLFDANFNP KPAYTAVMQA LA
Features of SEQ ID NO: 12 (Amino Acid Positions):
TABLE-US-00035 [0049] Signal Peptide 1-22 Cellulose Binding 23-66
Module 1 (CBM 1) Linker 67-96 Xylanase 97-392 catalytic site
Signal Peptide Sequence of SEQ ID NO: 12:
TABLE-US-00036 [0050] MKGLAALVALATIVAVPANAVA
DEFINITIONS
[0051] Xylanase: The term "xylanase" means a
1,4-beta-D-xylan-xylohydrolase (E.C. 3.2.1.8) that catalyzes the
endohydrolysis of 1,4-beta-D-xylosidic linkages in xylans. For
purposes of the present invention, xylanase activity is determined
with 0.2% AZCL-arabinoxylan as substrate in 0.01% TRITON.RTM. X-100
and 200 mM sodium phosphate buffer pH 6 at 37.degree. C. One unit
of xylanase activity is defined as 1.0 .mu.mole of azurine produced
per minute at 37.degree. C., pH 6 from 0.2% AZCL-arabinoxylan as
substrate in 200 mM sodium phosphate pH 6 buffer. In one aspect,
the polypeptides of the present invention have at least 20%, e.g.,
at least 40%, at least 50%, at least 60%, at least 70%, at least
80%, at least 90%, at least 95%, or at least 100% of the xylanase
activity of the mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4,
SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, or SEQ ID NO: 12.
[0052] Acetylxylan esterase: The term "acetylxylan esterase" means
a carboxylesterase (EC 3.1.1.72) that catalyzes the hydrolysis of
acetyl groups from polymeric xylan, acetylated xylose, acetylated
glucose, alpha-napthyl acetate, and p-nitrophenyl acetate. For
purposes of the present invention, acetylxylan esterase activity is
determined using 0.5 mM p-nitrophenylacetate as substrate in 50 mM
sodium acetate pH 5.0 containing 0.01% TWEEN.TM. 20
(polyoxyethylene sorbitan monolaurate). One unit of acetylxylan
esterase is defined as the amount of enzyme capable of releasing 1
.mu.mole of p-nitrophenolate anion per minute at pH 5, 25.degree.
C.
[0053] Allelic variant: The term "allelic variant" means any of two
or more alternative forms of a gene occupying the same chromosomal
locus. Allelic variation arises naturally through mutation, and may
result in polymorphism within populations. Gene mutations can be
silent (no change in the encoded polypeptide) or may encode
polypeptides having altered amino acid sequences. An allelic
variant of a polypeptide is a polypeptide encoded by an allelic
variant of a gene.
[0054] Alpha-L-arabinofuranosidase: The term
"alpha-L-arabinofuranosidase" means an alpha-L-arabinofuranoside
arabinofuranohydrolase (EC 3.2.1.55) that catalyzes the hydrolysis
of terminal non-reducing alpha-L-arabinofuranoside residues in
alpha-L-arabinosides. The enzyme acts on
alpha-L-arabinofuranosides, alpha-L-arabinans containing (1,3)-
and/or (1,5)-linkages, arabinoxylans, and arabinogalactans.
Alpha-L-arabinofuranosidase is also known as arabinosidase,
alpha-arabinosidase, alpha-L-arabinosidase,
alpha-arabinofuranosidase, polysaccharide
alpha-L-arabinofuranosidase, alpha-L-arabinofuranoside hydrolase,
L-arabinosidase, or alpha-L-arabinanase. For purposes of the
present invention, alpha-L-arabinofuranosidase activity is
determined using 5 mg of medium viscosity wheat arabinoxylan
(Megazyme International Ireland, Ltd., Bray, Co. Wicklow, Ireland)
per ml of 100 mM sodium acetate pH 5 in a total volume of 200 .mu.l
for 30 minutes at 40.degree. C. followed by arabinose analysis by
AMINEX.RTM. HPX-87H column chromatography (Bio-Rad Laboratories,
Inc., Hercules, Calif., USA).
[0055] Alpha-glucuronidase: The term "alpha-glucuronidase" means an
alpha-D-glucosiduronate glucuronohydrolase (EC 3.2.1.139) that
catalyzes the hydrolysis of an alpha-D-glucuronoside to
D-glucuronate and an alcohol. For purposes of the present
invention, alpha-glucuronidase activity is determined according to
de Vries, 1998, J. Bacteriol. 180: 243-249. One unit of
alpha-glucuronidase equals the amount of enzyme capable of
releasing 1 .mu.mole of glucuronic or 4-O-methylglucuronic acid per
minute at pH 5, 40.degree. C.
[0056] Beta-glucosidase: The term "beta-glucosidase" means a
beta-D-glucoside glucohydrolase (E.C. 3.2.1.21) that catalyzes the
hydrolysis of terminal non-reducing beta-D-glucose residues with
the release of beta-D-glucose. For purposes of the present
invention, beta-glucosidase activity is determined using
p-nitrophenyl-beta-D-glucopyranoside as substrate according to the
procedure of Venturi et al., 2002, Extracellular beta-D-glucosidase
from Chaetomium thermophilum var. coprophilum: production,
purification and some biochemical properties, J. Basic Microbiol.
42: 55-66. One unit of beta-glucosidase is defined as 1.0 .mu.mole
of p-nitrophenolate anion produced per minute at 25.degree. C., pH
4.8 from 1 mM p-nitrophenyl-beta-D-glucopyranoside as substrate in
50 mM sodium citrate containing 0.01% TWEEN.RTM. 20.
[0057] Beta-xylosidase: The term "beta-xylosidase" means a
beta-D-xyloside xylohydrolase (E.C. 3.2.1.37) that catalyzes the
exo-hydrolysis of short beta (1.fwdarw.4)-xylooligosaccharides to
remove successive D-xylose residues from non-reducing termini. For
purposes of the present invention, one unit of beta-xylosidase is
defined as 1.0 .mu.mole of p-nitrophenolate anion produced per
minute at 40.degree. C., pH 5 from 1 mM
p-nitrophenyl-beta-D-xyloside as substrate in 100 mM sodium citrate
containing 0.01% TWEEN.RTM. 20.
[0058] cDNA: The term "cDNA" means a DNA molecule that can be
prepared by reverse transcription from a mature, spliced, mRNA
molecule obtained from a eukaryotic or prokaryotic cell. cDNA lacks
intron sequences that may be present in the corresponding genomic
DNA. The initial, primary RNA transcript is a precursor to mRNA
that is processed through a series of steps, including splicing,
before appearing as mature spliced mRNA.
[0059] Cellobiohydrolase: The term "cellobiohydrolase" means a
1,4-beta-D-glucan cellobiohydrolase (E.C. 3.2.1.91) that catalyzes
the hydrolysis of 1,4-beta-D-glucosidic linkages in cellulose,
cellooligosaccharides, or any beta-1,4-linked glucose containing
polymer, releasing cellobiose from the reducing or non-reducing
ends of the chain (Teeri, 1997, Crystalline cellulose degradation:
New insight into the function of cellobiohydrolases, Trends in
Biotechnology 15: 160-167; Teeri et al., 1998, Trichoderma reesei
cellobiohydrolases: why so efficient on crystalline cellulose?,
Biochem. Soc. Trans. 26: 173-178). Cellobiohydrolase activity is
determined according to the procedures described by Lever et al.,
1972, Anal. Biochem. 47: 273-279; van Tilbeurgh et al., 1982, FEBS
Letters, 149: 152-156; van Tilbeurgh and Claeyssens, 1985, FEBS
Letters, 187: 283-288; and Tomme et al., 1988, Eur. J. Biochem.
170: 575-581. In the present invention, the Tomme et al. method can
be used to determine cellobiohydrolase activity.
[0060] Cellulosic material: The term "cellulosic material" means
any material containing cellulose. The predominant polysaccharide
in the primary cell wall of biomass is cellulose, the second most
abundant is hemicellulose, and the third is pectin. The secondary
cell wall, produced after the cell has stopped growing, also
contains polysaccharides and is strengthened by polymeric lignin
covalently cross-linked to hemicellulose. Cellulose is a
homopolymer of anhydrocellobiose and thus a linear
beta-(1-4)-D-glucan, while hemicelluloses include a variety of
compounds, such as xylans, xyloglucans, arabinoxylans, and mannans
in complex branched structures with a spectrum of substituents.
Although generally polymorphous, cellulose is found in plant tissue
primarily as an insoluble crystalline matrix of parallel glucan
chains. Hemicelluloses usually hydrogen bond to cellulose, as well
as to other hemicelluloses, which help stabilize the cell wall
matrix.
[0061] Cellulose is generally found, for example, in the stems,
leaves, hulls, husks, and cobs of plants or leaves, branches, and
wood of trees. The cellulosic material can be, but is not limited
to, agricultural residue, herbaceous material (including energy
crops), municipal solid waste, pulp and paper mill residue, waste
paper, and wood (including forestry residue) (see, for example,
Wiselogel et al., 1995, in Handbook on Bioethanol (Charles E.
Wyman, editor), pp. 105-118, Taylor & Francis, Washington D.C.;
Wyman, 1994, Bioresource Technology 50: 3-16; Lynd, 1990, Applied
Biochemistry and Biotechnology 24/25: 695-719; Mosier et al., 1999,
Recent Progress in Bioconversion of Lignocellulosics, in Advances
in Biochemical Engineering/Biotechnology, T. Scheper, managing
editor, Volume 65, pp. 23-40, Springer-Verlag, New York). It is
understood herein that the cellulose may be in the form of
lignocellulose, a plant cell wall material containing lignin,
cellulose, and hemicellulose in a mixed matrix. In a preferred
aspect, the cellulosic material is any biomass material. In another
preferred aspect, the cellulosic material is lignocellulose, which
comprises cellulose, hemicelluloses, and lignin.
[0062] In one aspect, the cellulosic material is agricultural
residue. In another aspect, the cellulosic material is herbaceous
material (including energy crops). In another aspect, the
cellulosic material is municipal solid waste. In another aspect,
the cellulosic material is pulp and paper mill residue. In another
aspect, the cellulosic material is waste paper. In another aspect,
the cellulosic material is wood (including forestry residue).
[0063] In another aspect, the cellulosic material is arundo. In
another aspect, the cellulosic material is bagasse. In another
aspect, the cellulosic material is bamboo. In another aspect, the
cellulosic material is corn cob. In another aspect, the cellulosic
material is corn fiber. In another aspect, the cellulosic material
is corn stover. In another aspect, the cellulosic material is
miscanthus. In another aspect, the cellulosic material is orange
peel. In another aspect, the cellulosic material is rice straw. In
another aspect, the cellulosic material is switchgrass. In another
aspect, the cellulosic material is wheat straw.
[0064] In another aspect, the cellulosic material is aspen. In
another aspect, the cellulosic material is eucalyptus. In another
aspect, the cellulosic material is fir. In another aspect, the
cellulosic material is pine. In another aspect, the cellulosic
material is poplar. In another aspect, the cellulosic material is
spruce. In another aspect, the cellulosic material is willow.
[0065] In another aspect, the cellulosic material is algal
cellulose. In another aspect, the cellulosic material is bacterial
cellulose. In another aspect, the cellulosic material is cotton
linter. In another aspect, the cellulosic material is filter paper.
In another aspect, the cellulosic material is microcrystalline
cellulose. In another aspect, the cellulosic material is
phosphoric-acid treated cellulose.
[0066] In another aspect, the cellulosic material is an aquatic
biomass. As used herein the term "aquatic biomass" means biomass
produced in an aquatic environment by a photosynthesis process. The
aquatic biomass can be algae, emergent plants, floating-leaf
plants, or submerged plants.
[0067] The cellulosic material may be used as is or may be
subjected to pretreatment, using conventional methods known in the
art, as described herein. In a preferred aspect, the cellulosic
material is pretreated.
[0068] Cellulolytic enzyme or cellulase: The term "cellulolytic
enzyme" or "cellulase" means one or more (e.g., several) enzymes
that hydrolyze a cellulosic material. Such enzymes include
endoglucanase(s), cellobiohydrolase(s), beta-glucosidase(s), or
combinations thereof. The two basic approaches for measuring
cellulolytic activity include: (1) measuring the total cellulolytic
activity, and (2) measuring the individual cellulolytic activities
(endoglucanases, cellobiohydrolases, and beta-glucosidases) as
reviewed in Zhang et al., Outlook for cellulase improvement:
Screening and selection strategies, 2006, Biotechnology Advances
24: 452-481. Total cellulolytic activity is usually measured using
insoluble substrates, including Whatman No 1 filter paper,
microcrystalline cellulose, bacterial cellulose, algal cellulose,
cotton, pretreated lignocellulose, etc. The most common total
cellulolytic activity assay is the filter paper assay using Whatman
No 1 filter paper as the substrate. The assay was established by
the International Union of Pure and Applied Chemistry (IUPAC)
(Ghose, 1987, Measurement of cellulase activities, Pure Appl. Chem.
59: 257-68).
[0069] For purposes of the present invention, cellulolytic enzyme
activity is determined by measuring the increase in hydrolysis of a
cellulosic material by cellulolytic enzyme(s) under the following
conditions: 1-50 mg of cellulolytic enzyme protein/g of cellulose
in PCS (or other pretreated cellulosic material) for 3-7 days at a
suitable temperature, e.g., 50.degree. C., 55.degree. C., or
60.degree. C., compared to a control hydrolysis without addition of
cellulolytic enzyme protein. Typical conditions are 1 ml reactions,
washed or unwashed PCS, 5% insoluble solids, 50 mM sodium acetate
pH 5, 1 mM MnSO.sub.4, 50.degree. C., 55.degree. C., or 60.degree.
C., 72 hours, sugar analysis by AMINEX.RTM. HPX-87H column (Bio-Rad
Laboratories, Inc., Hercules, Calif., USA).
[0070] Coding sequence: The term "coding sequence" means a
polynucleotide, which directly specifies the amino acid sequence of
a polypeptide. The boundaries of the coding sequence are generally
determined by an open reading frame, which begins with a start
codon such as ATG, GTG, or TTG and ends with a stop codon such as
TAA, TAG, or TGA. The coding sequence may be a genomic DNA, cDNA,
synthetic DNA, or a combination thereof.
[0071] Control sequences: The term "control sequences" means
nucleic acid sequences necessary for expression of a polynucleotide
encoding a mature polypeptide of the present invention. Each
control sequence may be native (i.e., from the same gene) or
foreign (i.e., from a different gene) to the polynucleotide
encoding the polypeptide or native or foreign to each other. Such
control sequences include, but are not limited to, a leader,
polyadenylation sequence, propeptide sequence, promoter, signal
peptide sequence, and transcription terminator. At a minimum, the
control sequences include a promoter, and transcriptional and
translational stop signals. The control sequences may be provided
with linkers for the purpose of introducing specific restriction
sites facilitating ligation of the control sequences with the
coding region of the polynucleotide encoding a polypeptide.
[0072] Endoglucanase: The term "endoglucanase" means an
endo-1,4-(1,3;1,4)-beta-D-glucan 4-glucanohydrolase (E.C. 3.2.1.4)
that catalyzes endohydrolysis of 1,4-beta-D-glycosidic linkages in
cellulose, cellulose derivatives (such as carboxymethyl cellulose
and hydroxyethyl cellulose), lichenin, beta-1,4 bonds in mixed
beta-1,3 glucans such as cereal beta-D-glucans or xyloglucans, and
other plant material containing cellulosic components.
Endoglucanase activity can be determined by measuring reduction in
substrate viscosity or increase in reducing ends determined by a
reducing sugar assay (Zhang et al., 2006, Biotechnology Advances
24: 452-481). For purposes of the present invention, endoglucanase
activity is determined using carboxymethyl cellulose (CMC) as
substrate according to the procedure of Ghose, 1987, Pure and Appl.
Chem. 59: 257-268, at pH 5, 40.degree. C.
[0073] Expression: The term "expression" includes any step involved
in the production of a polypeptide including, but not limited to,
transcription, post-transcriptional modification, translation,
post-translational modification, and secretion.
[0074] Expression vector: The term "expression vector" means a
linear or circular DNA molecule that comprises a polynucleotide
encoding a polypeptide and is operably linked to control sequences
that provide for its expression.
[0075] Family 61 glycoside hydrolase: The term "Family 61 glycoside
hydrolase" or "Family GH61" or "GH61" means a polypeptide falling
into the glycoside hydrolase Family 61 according to Henrissat B.,
1991, A classification of glycosyl hydrolases based on amino-acid
sequence similarities, Biochem. J. 280: 309-316, and Henrissat B.,
and Bairoch A., 1996, Updating the sequence-based classification of
glycosyl hydrolases, Biochem. J. 316: 695-696. The enzymes in this
family were originally classified as a glycoside hydrolase family
based on measurement of very weak endo-1,4-beta-D-glucanase
activity in one family member. The structure and mode of action of
these enzymes are non-canonical and they cannot be considered as
bona fide glycosidases. However, they are kept in the CAZy
classification on the basis of their capacity to enhance the
breakdown of lignocellulose when used in conjunction with a
cellulase or a mixture of cellulases.
[0076] Feruloyl esterase: The term "feruloyl esterase" means a
4-hydroxy-3-methoxycinnamoyl-sugar hydrolase (EC 3.1.1.73) that
catalyzes the hydrolysis of 4-hydroxy-3-methoxycinnamoyl (feruloyl)
groups from esterified sugar, which is usually arabinose in
"natural" substrates, to produce ferulate
(4-hydroxy-3-methoxycinnamate). Feruloyl esterase is also known as
ferulic acid esterase, hydroxycinnamoyl esterase, FAE-III,
cinnamoyl ester hydrolase, FAEA, cinnAE, FAE-I, or FAE-II. For
purposes of the present invention, feruloyl esterase activity is
determined using 0.5 mM p-nitrophenylferulate as substrate in 50 mM
sodium acetate pH 5.0. One unit of feruloyl esterase equals the
amount of enzyme capable of releasing 1 .mu.mole of
p-nitrophenolate anion per minute at pH 5, 25.degree. C.
[0077] Fragment: The term "fragment" means a polypeptide having one
or more (e.g., several) amino acids absent from the amino and/or
carboxyl terminus of a mature polypeptide main; wherein the
fragment has xylanase activity. In one aspect, a fragment contains
at least 20 amino acid residues, e.g., at least 30 to 351 amino
acid residues or at least 50 to 340, 80 to 310, 100 to 290, 150 to
270, or 200 to 250, or any number in between, amino acid residues
of SEQ ID NO: 2. In another aspect, a fragment contains at least 20
amino acid residues, e.g., at least 30 to 350 amino acid residues
or at least 50 to 340, 80 to 320, 100 to 300, 150 to 270, or 200 to
250, or any number in between, amino acid residues of SEQ ID NO: 4.
In another aspect, a fragment contains at least 20 amino acid
residues, e.g., at least 30 to 341 amino acid residues or at least
50 to 330, 80 to 310, 100 to 290, 150 to 270, or 200 to 250, or any
number in between, amino acid residues of SEQ ID NO: 6. In another
aspect, a fragment contains at least 20 amino acid residues, e.g.,
at least 30 to 394 amino acid residues or at least 50 to 380, 80 to
360, 100 to 340, 150 to 300, or 200 to 250, or any number in
between, amino acid residues of SEQ ID NO: 8. In another aspect, a
fragment contains at least 20 amino acid residues, e.g., at least
30 to 400 amino acid residues or at least 50 to 390, 80 to 360, 100
to 340, 150 to 300, or 200 to 250, or any number in between, amino
acid residues of SEQ ID NO: 10. In another aspect, a fragment
contains at least 20 amino acid residues, e.g., at least 30 to 391
amino acid residues or at least 50 to 380, 80 to 360, 100 to 340,
150 to 300, or 200 to 250, or any number in between, amino acid
residues of SEQ ID NO: 12.
[0078] Hemicellulolytic enzyme or hemicellulase: The term
"hemicellulolytic enzyme" or "hemicellulase" means one or more
(e.g., several) enzymes that hydrolyze a hemicellulosic material.
See, for example, Shallom, D. and Shoham, Y. Microbial
hemicellulases. Current Opinion In Microbiology, 2003, 6(3):
219-228). Hemicellulases are key components in the degradation of
plant biomass. Examples of hemicellulases include, but are not
limited to, an acetylmannan esterase, an acetylxylan esterase, an
arabinanase, an arabinofuranosidase, a coumaric acid esterase, a
feruloyl esterase, a galactosidase, a glucuronidase, a glucuronoyl
esterase, a mannanase, a mannosidase, a xylanase, and a xylosidase.
The substrates of these enzymes, the hemicelluloses, are a
heterogeneous group of branched and linear polysaccharides that are
bound via hydrogen bonds to the cellulose microfibrils in the plant
cell wall, crosslinking them into a robust network. Hemicelluloses
are also covalently attached to lignin, forming together with
cellulose a highly complex structure. The variable structure and
organization of hemicelluloses require the concerted action of many
enzymes for its complete degradation. The catalytic modules of
hemicellulases are either glycoside hydrolases (GHs) that hydrolyze
glycosidic bonds, or carbohydrate esterases (CEs), which hydrolyze
ester linkages of acetate or ferulic acid side groups. These
catalytic modules, based on homology of their primary sequence, can
be assigned into GH and CE families. Some families, with an overall
similar fold, can be further grouped into clans, marked
alphabetically (e.g., GH-A). A most informative and updated
classification of these and other carbohydrate active enzymes is
available in the Carbohydrate-Active Enzymes (CAZy) database.
Hemicellulolytic enzyme activities can be measured according to
Ghose and Bisaria, 1987, Pure & Appl. Chem. 59: 1739-1752, at a
suitable temperature, e.g., 50.degree. C., 55.degree. C., or
60.degree. C., and pH, e.g., 5.0 or 5.5.
[0079] High stringency conditions: The term "high stringency
conditions" means for probes of at least 100 nucleotides in length,
prehybridization and hybridization at 42.degree. C. in
5.times.SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured
salmon sperm DNA, and 50% formamide, following standard Southern
blotting procedures for 12 to 24 hours. The carrier material is
finally washed three times each for 15 minutes using 2.times.SSC,
0.2% SDS at 65.degree. C.
[0080] Host cell: The term "host cell" means any cell type that is
susceptible to transformation, transfection, transduction, or the
like with a nucleic acid construct or expression vector comprising
a polynucleotide of the present invention. The term "host cell"
encompasses any progeny of a parent cell that is not identical to
the parent cell due to mutations that occur during replication.
[0081] Isolated: The term "isolated" means a substance in a form or
environment that does not occur in nature. Non-limiting examples of
isolated substances include (1) any non-naturally occurring
substance, (2) any substance including, but not limited to, any
enzyme, variant, nucleic acid, protein, peptide or cofactor, that
is at least partially removed from one or more or all of the
naturally occurring constituents with which it is associated in
nature; (3) any substance modified by the hand of man relative to
that substance found in nature; or (4) any substance modified by
increasing the amount of the substance relative to other components
with which it is naturally associated (e.g., multiple copies of a
gene encoding the substance; use of a stronger promoter than the
promoter naturally associated with the gene encoding the
substance). An isolated substance may be present in a fermentation
broth sample.
[0082] Low stringency conditions: The term "low stringency
conditions" means for probes of at least 100 nucleotides in length,
prehybridization and hybridization at 42.degree. C. in
5.times.SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured
salmon sperm DNA, and 25% formamide, following standard Southern
blotting procedures for 12 to 24 hours. The carrier material is
finally washed three times each for 15 minutes using 2.times.SSC,
0.2% SDS at 50.degree. C.
[0083] Mature polypeptide: The term "mature polypeptide" means a
polypeptide in its final form following translation and any
post-translational modifications, such as N-terminal processing,
C-terminal truncation, glycosylation, phosphorylation, etc. In one
aspect, the mature polypeptide is amino acids 19 to 352 of SEQ ID
NO: 2 based on the SignalP program (Nielsen et al., 1997, Protein
Engineering 10: 1-6) that predicts amino acids 1 to 18 of SEQ ID
NO: 2 are a signal peptide. In another aspect, the mature
polypeptide is amino acids 1 to 352 of SEQ ID NO: 2. In another
aspect, the mature polypeptide is amino acids 21 to 351 of SEQ ID
NO: 4 based on the SignalP program that predicts amino acids 1 to
20 of SEQ ID NO: 4 are a signal peptide. In another aspect, the
mature polypeptide is amino acids 1 to 351 of SEQ ID NO: 4. In
another aspect, the mature polypeptide is amino acids 24 to 342 of
SEQ ID NO: 6 based on the SignalP program that predicts amino acids
1 to 23 of SEQ ID NO: 6 are a signal peptide. In another aspect,
the mature polypeptide is amino acids 1 to 342 of SEQ ID NO: 6. In
another aspect, the mature polypeptide is amino acids 20 to 395 of
SEQ ID NO: 8 based on the SignalP program that predicts amino acids
1 to 19 of SEQ ID NO: 8 are a signal peptide. In another aspect,
the mature polypeptide is amino acids 1 to 395 of SEQ ID NO: 8. In
another aspect, the mature polypeptide is amino acids 20 to 401 of
SEQ ID NO: 10 based on the SignalP program that predicts amino
acids 1 to 19 of SEQ ID NO: 10 are a signal peptide. In another
aspect, the mature polypeptide is amino acids 1 to 401 of SEQ ID
NO: 10. In another aspect, the mature polypeptide is amino acids 23
to 392 of SEQ ID NO: 12 based on the SignalP program that predicts
amino acids 1 to 22 of SEQ ID NO: 12 are a signal peptide. In
another aspect, the mature polypeptide is amino acids 1 to 392 of
SEQ ID NO: 12. It is known in the art that a host cell may produce
a mixture of two of more different mature polypeptides (i.e., with
a different C-terminal and/or N-terminal amino acid) expressed by
the same polynucleotide.
[0084] Mature polypeptide coding sequence: The term "mature
polypeptide coding sequence" means a polynucleotide that encodes a
mature polypeptide having xylanase activity. In one aspect, the
mature polypeptide coding sequence is nucleotides 55 to 1515 of SEQ
ID NO: 1 or the cDNA sequence thereof based on the SignalP program
(Nielsen et al., 1997, supra) that predicts nucleotides 1 to 54 of
SEQ ID NO: 1 encode a signal peptide. In another aspect, the mature
polypeptide coding sequence is nucleotides 1 to 1515 of SEQ ID NO:
1 or the cDNA sequence thereof. In another aspect, the mature
polypeptide coding sequence is nucleotides 61 to 1461 of SEQ ID NO:
3 or the cDNA sequence thereof based on the SignalP program that
predicts nucleotides 1 to 60 of SEQ ID NO: 3 encode a signal
peptide. In another aspect, the mature polypeptide coding sequence
is nucleotides 1 to 1461 of SEQ ID NO: 3 or the cDNA sequence
thereof. In another aspect, the mature polypeptide coding sequence
is nucleotides 70 to 1400 of SEQ ID NO: 5 or the cDNA sequence
thereof based on the SignalP program that predicts nucleotides 1 to
69 of SEQ ID NO: 5 encode a signal peptide. In another aspect, the
mature polypeptide coding sequence is nucleotides 1 to 1400 of SEQ
ID NO: 5 or the cDNA sequence thereof. In another aspect, the
mature polypeptide coding sequence is nucleotides 58 to 1665 of SEQ
ID NO: 7 or the cDNA sequence thereof based on the SignalP program
that predicts nucleotides 1 to 57 of SEQ ID NO: 7 encode a signal
peptide. In another aspect, the mature polypeptide coding sequence
is nucleotides 1 to 1665 of SEQ ID NO: 7 or the cDNA sequence
thereof. In another aspect, the mature polypeptide coding sequence
is nucleotides 58 to 1614 of SEQ ID NO: 9 or the cDNA sequence
thereof based on the SignalP program that predicts nucleotides 1 to
57 of SEQ ID NO: 9 encode a signal peptide. In another aspect, the
mature polypeptide coding sequence is nucleotides 1 to 1614 of SEQ
ID NO: 9 or the cDNA sequence thereof. In another aspect, the
mature polypeptide coding sequence is nucleotides 67 to 1673 of SEQ
ID NO: 11 or the cDNA sequence thereof based on the SignalP program
that predicts nucleotides 1 to 66 of SEQ ID NO: 11 encode a signal
peptide. In another aspect, the mature polypeptide coding sequence
is nucleotides 1 to 1673 of SEQ ID NO: 11 or the cDNA sequence
thereof.
[0085] Catalytic domain: The term "catalytic domain" means the
portion of an enzyme containing the catalytic machinery of the
enzyme.
[0086] Cellulose binding domain: The term "cellulose binding
domain" means the portion of an enzyme that mediates binding of the
enzyme to amorphous regions of a cellulose substrate. The cellulose
binding domain (CBD) is found either at the N-terminal or at the
C-terminal extremity of an enzyme. A CBD is also referred to as a
cellulose binding module or CBM. In one embodiment the CBM is amino
acids 20 to 56 of SEQ ID NO: 8. In one embodiment the CBM is amino
acids 20 to 56 of SEQ ID NO: 10. In one embodiment the CBM is amino
acids 23 to 66 of SEQ ID NO: 12. The CBM is separated from the
catalytic domain by a linker sequence. The linker is in one
embodiment amino acids 57 to 81 of SEQ ID NO: 8. The linker is in
one embodiment amino acids 57 to 86 of SEQ ID NO: 10. The linker is
in one embodiment amino acids 67 to 96 of SEQ ID NO: 12.
[0087] Medium stringency conditions: The term "medium stringency
conditions" means for probes of at least 100 nucleotides in length,
prehybridization and hybridization at 42.degree. C. in
5.times.SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured
salmon sperm DNA, and 35% formamide, following standard Southern
blotting procedures for 12 to 24 hours. The carrier material is
finally washed three times each for 15 minutes using 2.times.SSC,
0.2% SDS at 55.degree. C.
[0088] Medium-high stringency conditions: The term "medium-high
stringency conditions" means for probes of at least 100 nucleotides
in length, prehybridization and hybridization at 42.degree. C. in
5.times.SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured
salmon sperm DNA, and either 35% formamide, following standard
Southern blotting procedures for 12 to 24 hours. The carrier
material is finally washed three times each for 15 minutes using
2.times.SSC, 0.2% SDS at 60.degree. C.
[0089] Nucleic acid construct: The term "nucleic acid construct"
means a nucleic acid molecule, either single- or double-stranded,
which is isolated from a naturally occurring gene or is modified to
contain segments of nucleic acids in a manner that would not
otherwise exist in nature or which is synthetic, which comprises
one or more control sequences.
[0090] Operably linked: The term "operably linked" means a
configuration in which a control sequence is placed at an
appropriate position relative to the coding sequence of a
polynucleotide such that the control sequence directs expression of
the coding sequence.
[0091] Polypeptide having cellulolytic enhancing activity: The term
"polypeptide having cellulolytic enhancing activity" means a GH61
polypeptide that catalyzes the enhancement of the hydrolysis of a
cellulosic material by enzyme having cellulolytic activity. For
purposes of the present invention, cellulolytic enhancing activity
is determined by measuring the increase in reducing sugars or the
increase of the total of cellobiose and glucose from the hydrolysis
of a cellulosic material by cellulolytic enzyme under the following
conditions: 1-50 mg of total protein/g of cellulose in PCS, wherein
total protein is comprised of 50-99.5% w/w cellulolytic enzyme
protein and 0.5-50% w/w protein of a GH61 polypeptide having
cellulolytic enhancing activity for 1-7 days at a suitable
temperature, e.g., 50.degree. C., 55.degree. C., or 60.degree. C.,
and pH, e.g., 5.0 or 5.5, compared to a control hydrolysis with
equal total protein loading without cellulolytic enhancing activity
(1-50 mg of cellulolytic protein/g of cellulose in PCS). In a
preferred aspect, a mixture of CELLUCLAST.RTM. 1.5L (Novozymes NS,
Bagsv.ae butted.rd, Denmark) in the presence of 2-3% of total
protein weight Aspergillus oryzae beta-glucosidase (recombinantly
produced in Aspergillus oryzae according to WO 02/095014) or 2-3%
of total protein weight Aspergillus fumigatus beta-glucosidase
(recombinantly produced in Aspergillus oryzae as described in WO
2002/095014) of cellulase protein loading is used as the source of
the cellulolytic activity.
[0092] The GH61 polypeptides having cellulolytic enhancing activity
enhance the hydrolysis of a cellulosic material catalyzed by enzyme
having cellulolytic activity by reducing the amount of cellulolytic
enzyme required to reach the same degree of hydrolysis preferably
at least 1.01-fold, e.g., at least 1.05-fold, at least 1.10-fold,
at least 1.25-fold, at least 1.5-fold, at least 2-fold, at least
3-fold, at least 4-fold, at least 5-fold, at least 10-fold, or at
least 20-fold.
[0093] Pretreated corn stover: The term "PCS" or "Pretreated Corn
Stover" means a cellulosic material derived from corn stover by
treatment with heat and dilute sulfuric acid, alkaline
pretreatment, or neutral pretreatment.
[0094] Sequence identity: The relatedness between two amino acid
sequences or between two nucleotide sequences is described by the
parameter "sequence identity".
[0095] For purposes of the present invention, the sequence identity
between two amino acid sequences is determined using the
Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol.
Biol. 48: 443-453) as implemented in the Needle program of the
EMBOSS package (EMBOSS: The European Molecular Biology Open
Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277),
preferably version 5.0.0 or later. The parameters used are gap open
penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62
(EMBOSS version of BLOSUM62) substitution matrix. The output of
Needle labeled "longest identity" (obtained using the -nobrief
option) is used as the percent identity and is calculated as
follows:
(Identical Residues.times.100)/(Length of Alignment-Total Number of
Gaps in Alignment)
[0096] For purposes of the present invention, the sequence identity
between two deoxyribonucleotide sequences is determined using the
Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as
implemented in the Needle program of the EMBOSS package (EMBOSS:
The European Molecular Biology Open Software Suite, Rice et al.,
2000, supra), preferably version 5.0.0 or later. The parameters
used are gap open penalty of 10, gap extension penalty of 0.5, and
the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix.
The output of Needle labeled "longest identity" (obtained using the
-nobrief option) is used as the percent identity and is calculated
as follows: (Identical Deoxyribonucleotides.times.100)/(Length of
Alignment--Total Number of Gaps in
[0097] Alignment)
[0098] Subsequence: The term "subsequence" means a polynucleotide
having one or more (e.g., several) nucleotides absent from the 5'
and/or 3' end of a mature polypeptide coding sequence; wherein the
subsequence encodes a fragment having xylanase activity. In one
aspect, a subsequence contains at least 800 nucleotides, e.g., at
least 1000 nucleotides or at least 1100 nucleotides of SEQ ID NO:
1. In another aspect, a subsequence contains at least 800
nucleotides, e.g., at least 1000 nucleotides or at least 1100
nucleotides of SEQ ID NO: 3. In another aspect, a subsequence
contains at least 800 nucleotides, e.g., at least 1000 nucleotides
or at least 1100 nucleotides of SEQ ID NO: 5. In another aspect, a
subsequence contains at least 800 nucleotides, e.g., at least 1000
nucleotides or at least 1100 nucleotides of SEQ ID NO: 7. In
another aspect, a subsequence contains at least 800 nucleotides,
e.g., at least 1000 nucleotides or at least 1100 nucleotides of SEQ
ID NO: 9. In another aspect, a subsequence contains at least 800
nucleotides, e.g., at least 1000 nucleotides or at least 1100
nucleotides of SEQ ID NO: 11.
[0099] Variant: The term "variant" means a polypeptide having
xylanase activity comprising an alteration, i.e., a substitution,
insertion, and/or deletion, at one or more (e.g., several)
positions. A substitution means replacement of the amino acid
occupying a position with a different amino acid; a deletion means
removal of the amino acid occupying a position; and an insertion
means adding an amino acid adjacent to and immediately following
the amino acid occupying a position.
[0100] Very high stringency conditions: The term "very high
stringency conditions" means for probes of at least 100 nucleotides
in length, prehybridization and hybridization at 42.degree. C. in
5.times.SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured
salmon sperm DNA, and 50% formamide, following standard Southern
blotting procedures for 12 to 24 hours. The carrier material is
finally washed three times each for 15 minutes using 2.times.SSC,
0.2% SDS at 70.degree. C.
[0101] Very low stringency conditions: The term "very low
stringency conditions" means for probes of at least 100 nucleotides
in length, prehybridization and hybridization at 42.degree. C. in
5.times.SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured
salmon sperm DNA, and 25% formamide, following standard Southern
blotting procedures for 12 to 24 hours. The carrier material is
finally washed three times each for 15 minutes using 2.times.SSC,
0.2% SDS at 45.degree. C.
[0102] Xylan-containing material: The term "xylan-containing
material" means any material comprising a plant cell wall
polysaccharide containing a backbone of beta-(1-4)-linked xylose
residues. Xylans of terrestrial plants are heteropolymers
possessing a beta-(1-4)-D-xylopyranose backbone, which is branched
by short carbohydrate chains. They comprise D-glucuronic acid or
its 4-O-methyl ether, L-arabinose, and/or various oligosaccharides,
composed of D-xylose, L-arabinose, D- or L-galactose, and
D-glucose. Xylan-type polysaccharides can be divided into
homoxylans and heteroxylans, which include glucuronoxylans,
(arabino)glucuronoxylans, (glucurono)arabinoxylans, arabinoxylans,
and complex heteroxylans. See, for example, Ebringerova et al.,
2005, Adv. Polym. Sci. 186: 1-67.
[0103] In the processes of the present invention, any material
containing xylan may be used. In a preferred aspect, the
xylan-containing material is lignocellulose.
[0104] Xylan degrading activity or xylanolytic activity: The term
"xylan degrading activity" or "xylanolytic activity" means a
biological activity that hydrolyzes xylan-containing material. The
two basic approaches for measuring xylanolytic activity include:
(1) measuring the total xylanolytic activity, and (2) measuring the
individual xylanolytic activities (e.g., endoxylanases,
beta-xylosidases, arabinofuranosidases, alpha-glucuronidases,
acetylxylan esterases, feruloyl esterases, and alpha-glucuronyl
esterases). Recent progress in assays of xylanolytic enzymes was
summarized in several publications including Biely and Puchard,
Recent progress in the assays of xylanolytic enzymes, 2006, Journal
of the Science of Food and Agriculture 86(11): 1636-1647; Spanikova
and Biely, 2006, Glucuronoyl esterase--Novel carbohydrate esterase
produced by Schizophyllum commune, FEBS Letters 580(19): 4597-4601;
Herrmann, Vrsanska, Jurickova, Hirsch, Biely, and Kubicek, 1997,
The beta-D-xylosidase of Trichoderma reesei is a multifunctional
beta-D-xylan xylohydrolase, Biochemical Journal 321: 375-381.
[0105] Total xylan degrading activity can be measured by
determining the reducing sugars formed from various types of xylan,
including, for example, oat spelt, beechwood, and larchwood xylans,
or by photometric determination of dyed xylan fragments released
from various covalently dyed xylans. The most common total
xylanolytic activity assay is based on production of reducing
sugars from polymeric 4-O-methyl glucuronoxylan as described in
Bailey, Biely, Poutanen, 1992, Interlaboratory testing of methods
for assay of xylanase activity, Journal of Biotechnology 23(3):
257-270. Xylanase activity can also be determined with 0.2%
AZCL-arabinoxylan as substrate in 0.01% TRITON.RTM. X-100
(4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol) and 200 mM
sodium phosphate buffer pH 6 at 37.degree. C. One unit of xylanase
activity is defined as 1.0 .mu.mole of azurine produced per minute
at 37.degree. C., pH 6 from 0.2% AZCL-arabinoxylan as substrate in
200 mM sodium phosphate pH 6 buffer.
[0106] For purposes of the present invention, xylan degrading
activity is determined by measuring the increase in hydrolysis of
birchwood xylan (Sigma Chemical Co., Inc., St. Louis, Mo., USA) by
xylan-degrading enzyme(s) under the following typical conditions: 1
ml reactions, 5 mg/ml substrate (total solids), 5 mg of xylanolytic
protein/g of substrate, 50 mM sodium acetate pH 5, 50.degree. C.,
24 hours, sugar analysis using p-hydroxybenzoic acid hydrazide
(PHBAH) assay as described by Lever, 1972, A new reaction for
colorimetric determination of carbohydrates, Anal. Biochem 47:
273-279.
DETAILED DESCRIPTION OF THE INVENTION
Polypeptides Having Xylanase Activity
[0107] In an embodiment, the present invention relates to isolated
polypeptides having a sequence identity to the mature polypeptide
of SEQ ID NO: 2 of at least 67%, e.g., at least 68%, at least 69%,
at least 70%, at least 75%, at least 80%, at least 85%, at least
90%, at least 91%, at least 92%, at least 93%, at least 94%, at
least 95%, at least 96%, at least 97%, at least 98%, at least 99%,
or 100%, which have xylanase activity. In an embodiment, the
present invention relates to isolated polypeptides having a
sequence identity to the mature polypeptide of SEQ ID NO: 4 of at
least 61%, e.g., at least 62%, at least 63%, at least 65%, at least
67%, at least 70%, at least 75%, at least 80%, at least 85%, at
least 90%, at least 91%, at least 92%, at least 93%, at least 94%,
at least 95%, at least 96%, at least 97%, at least 98%, at least
99%, or 100%, which have xylanase activity. In an embodiment, the
present invention relates to isolated polypeptides having a
sequence identity to the mature polypeptide of SEQ ID NO: 6 of at
least 61%, e.g., at least 62%, at least 63%, at least 65%, at least
67%, at least 70%, at least 75%, at least 80%, at least 85%, at
least 90%, at least 91%, at least 92%, at least 93%, at least 94%,
at least 95%, at least 96%, at least 97%, at least 98%, at least
99%, or 100%, which have xylanase activity. In an embodiment, the
present invention relates to isolated polypeptides having a
sequence identity to the mature polypeptide of SEQ ID NO: 8 of at
least 90%, e.g., at least 91%, at least 92%, at least 93%, at least
94%, at least 95%, at least 96%, at least 97%, at least 98%, at
least 99%, or 100%, which have xylanase activity. In an embodiment,
the present invention relates to isolated polypeptides having a
sequence identity to the mature polypeptide of SEQ ID NO: 10 of at
least 79%, e.g., at least 80%, at least 83%, at least 85%, at least
87%, at least 88%, at least 89%, at least 90%, at least 91%, at
least 92%, at least 93%, at least 94%, at least 95%, at least 96%,
at least 97%, at least 98%, at least 99%, or 100%, which have
xylanase activity. In an embodiment, the present invention relates
to isolated polypeptides having a sequence identity to the mature
polypeptide of SEQ ID NO: 12 of at least 92%, e.g., at least 93%,
at least 94%, at least 95%, at least 96%, at least 97%, at least
98%, at least 99%, or 100%, which have xylanase activity. In one
aspect, the polypeptides differ by no more than 10 amino acids,
e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9, from the mature polypeptide of
SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO:
10, or SEQ ID NO: 12.
[0108] A polypeptide of the present invention preferably comprises
or consists of the amino acid sequence of SEQ ID NO: 2, SEQ ID NO:
4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, or SEQ ID NO: 12 or
an allelic variant thereof; or is a fragment thereof having
xylanase activity. In another aspect, the polypeptide comprises or
consists of the mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4,
SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, or SEQ ID NO: 12. In
another aspect, the polypeptide comprises or consists of amino
acids 19 to 352 of SEQ ID NO: 2, amino acids 21 to 351 of SEQ ID
NO: 4, amino acids 24 to 342 of SEQ ID NO: 6, amino acids 20 to 395
of SEQ ID NO: 8, amino acids 20 to 401 of SEQ ID NO: 10, or amino
acids 23 to 392 of SEQ ID NO: 12.
[0109] In another embodiment, the present invention relates to an
isolated polypeptide having xylanase activity encoded by a
polynucleotide that hybridizes under very low stringency
conditions, or low stringency conditions, or medium stringency
conditions, or medium-high stringency conditions, or high
stringency conditions, or very high stringency conditions with (i)
the mature polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO:
3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, or SEQ ID NO: 11, (ii)
the cDNA sequence thereof, or (iii) the full-length complement of
(i) or (ii) (Sambrook et al., 1989, Molecular Cloning, A Laboratory
Manual, 2d edition, Cold Spring Harbor, N.Y.).
[0110] The polynucleotide of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO:
5, SEQ ID NO: 7, SEQ ID NO: 9, or SEQ ID NO: 11, or a subsequence
thereof, as well as the polypeptide of SEQ ID NO: 2, SEQ ID NO: 4,
SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, or SEQ ID NO: 12, or a
fragment thereof, may be used to design nucleic acid probes to
identify and clone DNA encoding polypeptides having xylanase
activity from strains of different genera or species according to
methods well known in the art. In particular, such probes can be
used for hybridization with the genomic DNA or cDNA of a cell of
interest, following standard Southern blotting procedures, in order
to identify and isolate the corresponding gene therein. Such probes
can be considerably shorter than the entire sequence, but should be
at least 15, e.g., at least 25, at least 35, or at least 70
nucleotides in length. Preferably, the nucleic acid probe is at
least 100 nucleotides in length, e.g., at least 200 nucleotides, at
least 300 nucleotides, at least 400 nucleotides, at least 500
nucleotides, at least 600 nucleotides, at least 700 nucleotides, at
least 800 nucleotides, or at least 900 nucleotides in length. Both
DNA and RNA probes can be used. The probes are typically labeled
for detecting the corresponding gene (for example, with .sup.32P,
.sup.3H, .sup.35S, biotin, or avidin). Such probes are encompassed
by the present invention.
[0111] A genomic DNA or cDNA library prepared from such other
strains may be screened for DNA that hybridizes with the probes
described above and encodes a polypeptide having xylanase activity.
Genomic or other DNA from such other strains may be separated by
agarose or polyacrylamide gel electrophoresis, or other separation
techniques. DNA from the libraries or the separated DNA may be
transferred to and immobilized on nitrocellulose or other suitable
carrier material. In order to identify a clone or DNA that
hybridizes with SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID
NO: 7, SEQ ID NO: 9, or SEQ ID NO: 11 or a subsequence thereof, the
carrier material is used in a Southern blot.
[0112] For purposes of the present invention, hybridization
indicates that the polynucleotide hybridizes to a labeled nucleic
acid probe corresponding to (i) SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID
NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, or SEQ ID NO: 11; (ii) the
mature polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3,
SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, or SEQ ID NO: 11; (iii)
the cDNA sequence thereof; (iv) the full-length complement thereof;
or (v) a subsequence thereof; under very low to very high
stringency conditions. Molecules to which the nucleic acid probe
hybridizes under these conditions can be detected using, for
example, X-ray film or any other detection means known in the
art.
[0113] In one aspect, the nucleic acid probe is a polynucleotide
that encodes the polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID
NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, or SEQ ID NO: 12; the mature
polypeptide thereof; or a fragment thereof. In another aspect, the
nucleic acid probe is SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ
ID NO: 7, SEQ ID NO: 9, or SEQ ID NO: 11; or the cDNA sequence
thereof. In another aspect, the nucleic acid probe is the
polynucleotide contained in Trametes versicolor Strain NN055586,
wherein the polynucleotide encodes a polypeptide having xylanase
activity.
[0114] In another embodiment, the present invention relates to an
isolated polypeptide having xylanase activity encoded by a
polynucleotide having a sequence identity to the mature polypeptide
coding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID
NO: 7, SEQ ID NO: 9, or SEQ ID NO: 11, or the cDNA sequence
thereof, of at least 60%, e.g., at least 65%, at least 70%, at
least 75%, at least 80%, at least 85%, at least 90%, at least 91%,
at least 92%, at least 93%, at least 94%, at least 95%, at least
96%, at least 97%, at least 98%, at least 99%, or 100%.
[0115] In another embodiment, the present invention relates to
variants of the mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4,
SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, or SEQ ID NO: 12
comprising a substitution, deletion, and/or insertion at one or
more (e.g., several) positions. In an embodiment, the number of
amino acid substitutions, deletions and/or insertions introduced
into the mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID
NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, or SEQ ID NO: 12 is not more
than 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8 or 9. The amino acid changes
may be of a minor nature, that is conservative amino acid
substitutions or insertions that do not significantly affect the
folding and/or activity of the protein; small deletions, typically
of 1-30 amino acids; small amino- or carboxyl-terminal extensions,
such as an amino-terminal methionine residue; a small linker
peptide of up to 20-25 residues; or a small extension that
facilitates purification by changing net charge or another
function, such as a poly-histidine tract, an antigenic epitope or a
binding domain.
[0116] Examples of conservative substitutions are within the groups
of basic amino acids (arginine, lysine and histidine), acidic amino
acids (glutamic acid and aspartic acid), polar amino acids
(glutamine and asparagine), hydrophobic amino acids (leucine,
isoleucine and valine), aromatic amino acids (phenylalanine,
tryptophan and tyrosine), and small amino acids (glycine, alanine,
serine, threonine and methionine). Amino acid substitutions that do
not generally alter specific activity are known in the art and are
described, for example, by H. Neurath and R. L. Hill, 1979, In, The
Proteins, Academic Press, New York. Common substitutions are
Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn,
Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile,
Leu/Val, Ala/Glu, and Asp/Gly.
[0117] Alternatively, the amino acid changes are of such a nature
that the physico-chemical properties of the polypeptides are
altered. For example, amino acid changes may improve the thermal
stability of the polypeptide, alter the substrate specificity,
change the pH optimum, and the like.
[0118] Essential amino acids in a polypeptide can be identified
according to procedures known in the art, such as site-directed
mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells,
1989, Science 244: 1081-1085). In the latter technique, single
alanine mutations are introduced at every residue in the molecule,
and the resultant mutant molecules are tested for xylanase activity
to identify amino acid residues that are critical to the activity
of the molecule. See also, Hilton et al., 1996, J. Biol. Chem. 271:
4699-4708. The active site of the enzyme or other biological
interaction can also be determined by physical analysis of
structure, as determined by such techniques as nuclear magnetic
resonance, crystallography, electron diffraction, or photoaffinity
labeling, in conjunction with mutation of putative contact site
amino acids. See, for example, de Vos et al., 1992, Science 255:
306-312; Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver
et al., 1992, FEBS Lett. 309: 59-64. The identity of essential
amino acids can also be inferred from an alignment with a related
polypeptide.
[0119] Single or multiple amino acid substitutions, deletions,
and/or insertions can be made and tested using known methods of
mutagenesis, recombination, and/or shuffling, followed by a
relevant screening procedure, such as those disclosed by
Reidhaar-Olson and Sauer, 1988, Science 241: 53-57; Bowie and
Sauer, 1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95/17413;
or WO 95/22625. Other methods that can be used include error-prone
PCR, phage display (e.g., Lowman et al., 1991, Biochemistry 30:
10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204), and
region-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145;
Ner et al., 1988, DNA 7: 127).
[0120] Mutagenesis/shuffling methods can be combined with
high-throughput, automated screening methods to detect activity of
cloned, mutagenized polypeptides expressed by host cells (Ness et
al., 1999, Nature Biotechnology 17: 893-896). Mutagenized DNA
molecules that encode active polypeptides can be recovered from the
host cells and rapidly sequenced using standard methods in the art.
These methods allow the rapid determination of the importance of
individual amino acid residues in a polypeptide.
[0121] The polypeptide may be a hybrid polypeptide in which a
region of one polypeptide is fused at the N-terminus or the
C-terminus of a region of another polypeptide.
[0122] The polypeptide may be a fusion polypeptide or cleavable
fusion polypeptide in which another polypeptide is fused at the
N-terminus or the C-terminus of the polypeptide of the present
invention. A fusion polypeptide is produced by fusing a
polynucleotide encoding another polypeptide to a polynucleotide of
the present invention. Techniques for producing fusion polypeptides
are known in the art, and include ligating the coding sequences
encoding the polypeptides so that they are in frame and that
expression of the fusion polypeptide is under control of the same
promoter(s) and terminator. Fusion polypeptides may also be
constructed using intein technology in which fusion polypeptides
are created post-translationally (Cooper et al., 1993, EMBO J. 12:
2575-2583; Dawson et al., 1994, Science 266: 776-779).
[0123] A fusion polypeptide can further comprise a cleavage site
between the two polypeptides. Upon secretion of the fusion protein,
the site is cleaved releasing the two polypeptides. Examples of
cleavage sites include, but are not limited to, the sites disclosed
in Martin et al., 2003, J. Ind. Microbiol. Biotechnol. 3: 568-576;
Svetina et al., 2000, J. Biotechnol. 76: 245-251; Rasmussen-Wilson
et al., 1997, Appl. Environ. Microbiol. 63: 3488-3493; Ward et al.,
1995, Biotechnology 13: 498-503; and Contreras et al., 1991,
Biotechnology 9: 378-381; Eaton et al., 1986, Biochemistry 25:
505-512; Collins-Racie et al., 1995, Biotechnology 13: 982-987;
Carter et al., 1989, Proteins: Structure, Function, and Genetics 6:
240-248; and Stevens, 2003, Drug Discovery World 4: 35-48.
Sources of Polypeptides Having [Enzyme] Activity
[0124] A polypeptide having xylanase activity of the present
invention may be obtained from microorganisms of any genus. For
purposes of the present invention, the term "obtained from" as used
herein in connection with a given source shall mean that the
polypeptide encoded by a polynucleotide is produced by the source
or by a strain in which the polynucleotide from the source has been
inserted. In one aspect, the polypeptide obtained from a given
source is secreted extracellularly.
[0125] The polypeptide may be a Trametes polypeptide.
[0126] In another aspect, the polypeptide is a Trametes versicolor
polypeptide, e.g., a polypeptide obtained from Trametes versicolor
Strain NN055586, or in another aspect the polypeptide is a
polypeptide from a species related to Trametes versicolor, for
example from another Trametes species.
[0127] It will be understood that for the aforementioned species,
the invention encompasses both the perfect and imperfect states,
and other taxonomic equivalents, e.g., anamorphs, regardless of the
species name by which they are known. Those skilled in the art will
readily recognize the identity of appropriate equivalents.
[0128] Strains of these species are readily accessible to the
public in a number of culture collections, such as the American
Type Culture Collection (ATCC), Deutsche Sammlung von
Mikroorganismen and Zellkulturen GmbH (DSMZ), Centraalbureau Voor
Schimmelcultures (CBS), and Agricultural Research Service Patent
Culture Collection, Northern Regional Research Center (NRRL).
[0129] The polypeptide may be identified and obtained from other
sources including microorganisms isolated from nature (e.g., soil,
composts, water, etc.) or DNA samples obtained directly from
natural materials (e.g., soil, composts, water, etc.) using the
above-mentioned probes. Techniques for isolating microorganisms and
DNA directly from natural habitats are well known in the art. A
polynucleotide encoding the polypeptide may then be obtained by
similarly screening a genomic DNA or cDNA library of another
microorganism or mixed DNA sample. Once a polynucleotide encoding a
polypeptide has been detected with the probe(s), the polynucleotide
can be isolated or cloned by utilizing techniques that are known to
those of ordinary skill in the art (see, e.g., Sambrook et al.,
1989, supra).
Catalytic Domains
[0130] The present invention also relates to isolated polypeptides
comprising a catalytic domain selected from the group consisting
of:
[0131] (a) a catalytic domain having at least 60% sequence identity
to the catalytic domain of SEQ ID NO: 2 (for example, amino acids
19 to 352 of SEQ ID NO: 2), a catalytic domain having at least 60%
sequence identity to the catalytic domain of SEQ ID NO: 4 (for
example, amino acids 21 to 351 of SEQ ID NO: 4), a catalytic domain
having at least 60% sequence identity to the catalytic domain of
SEQ ID NO: 6 (for example, amino acids 24 to 342 of SEQ ID NO: 6),
a catalytic domain having at least 60% sequence identity to the
catalytic domain of SEQ ID NO: 8 (for example, amino acids 82 to
395 of SEQ ID NO: 8), a catalytic domain having at least 60%
sequence identity to the catalytic domain of SEQ ID NO: 10 (for
example, amino acids 87 to 401 of SEQ ID NO: 10), or a catalytic
domain having at least 60% sequence identity to the catalytic
domain of SEQ ID NO: 12 (for example, amino acids 97 to 392 of SEQ
ID NO: 12);
[0132] (b) a catalytic domain encoded by a polynucleotide having at
least 60% sequence identity to the catalytic domain coding sequence
of SEQ ID NO: 1 (for example, nucleotides 55-1515 of SEQ ID NO: 1),
a catalytic domain encoded by a polynucleotide having at least 60%
sequence identity to the catalytic domain coding sequence of SEQ ID
NO: 3 (for example, nucleotides 60-249, 307-524, 649-665, 721-867,
930-1237, 1295-1351, and 1405-1461 of SEQ ID NO: 3), a catalytic
domain encoded by a polynucleotide having at least 60% sequence
identity to the catalytic domain coding sequence of SEQ ID NO: 5
(for example, nucleotides 79-273, 325-430, 472-598, 718-734,
789-935, 990-1297, and 1353-1400 of SEQ ID NO: 5), a catalytic
domain encoded by a polynucleotide having at least 60% sequence
identity to the catalytic domain coding sequence of SEQ ID NO: 7
(for example, nucleotides 310-453, 511-728, 786-787, 845-865,
923-1069, 1137-1441, 1503-1559, and 1618-1665 of SEQ ID NO: 7), a
catalytic domain encoded by a polynucleotide having at least 60%
sequence identity to the catalytic domain coding sequence of SEQ ID
NO: 9 (for example, nucleotides 316-459, 526-743, 799-800, 859-879,
936-1082, 1143-1447, and 1507-1614 of SEQ ID NO: 9), or a catalytic
domain encoded by a polynucleotide having at least 60% sequence
identity to the catalytic domain coding sequence of SEQ ID NO: 11
(for example, nucleotides 379-522, 586-592, 650-739, 799-1066,
1121-1229, 1286-1378, 1432-1518, and 1584-1673 of SEQ ID NO: 11);
(c) a variant of a catalytic domain comprising a substitution,
deletion, and/or insertion of one or more (several) amino acids of
the catalytic domain of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6,
SEQ ID NO: 8, SEQ ID NO: 10, or SEQ ID NO: 12; and
[0133] (d) a fragment of a catalytic domain of (a), (b), or (c),
which has xylanase activity.
[0134] The catalytic domain preferably has a degree of sequence
identity to the catalytic domain of SEQ ID NO: 2, SEQ ID NO: 4, SEQ
ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, or SEQ ID NO: 12 of at least
60%, e.g. at least 70%, at least 75%, at least 80%, at least 85%,
at least 90%, at least 91%, at least 92%, at least 95%, at least
96%, at least 97%, at least 98%, at least 99%, or 100%. In an
aspect, the catalytic domain comprises an amino acid sequence that
differs by ten amino acids, e.g., by five amino acids, by four
amino acids, by three amino acids, by two amino acids, and by one
amino acid from the catalytic domain of SEQ ID NO: 2, SEQ ID NO: 4,
SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, or SEQ ID NO: 12.
[0135] The catalytic domain preferably comprises or consists of the
catalytic domain of SEQ ID NO: 2 or an allelic variant thereof; or
is a fragment thereof having xylanase activity. In another
preferred aspect, the catalytic domain comprises or consists of
amino acids 19 to 352 of SEQ ID NO: 2.
[0136] The catalytic domain preferably comprises or consists of the
catalytic domain of SEQ ID NO: 4 or an allelic variant thereof; or
is a fragment thereof having xylanase activity. In another
preferred aspect, the catalytic domain comprises or consists of
amino acids 21 to 351 of SEQ ID NO: 4.
[0137] The catalytic domain preferably comprises or consists of the
catalytic domain of SEQ ID NO: 6 or an allelic variant thereof; or
is a fragment thereof having xylanase activity. In another
preferred aspect, the catalytic domain comprises or consists of
amino acids 24 to 342 of SEQ ID NO: 6.
[0138] The catalytic domain preferably comprises or consists of the
catalytic domain of SEQ ID NO: 8 or an allelic variant thereof; or
is a fragment thereof having xylanase activity. In another
preferred aspect, the catalytic domain comprises or consists of
amino acids 82 to 395 of SEQ ID NO: 8.
[0139] The catalytic domain preferably comprises or consists of the
catalytic domain of SEQ ID NO: 10 or an allelic variant thereof; or
is a fragment thereof having xylanase activity. In another
preferred aspect, the catalytic domain comprises or consists of
amino acids 87 to 401 of SEQ ID NO: 10.
[0140] The catalytic domain preferably comprises or consists of the
catalytic domain of SEQ ID NO: 12 or an allelic variant thereof; or
is a fragment thereof having xylanase activity. In another
preferred aspect, the catalytic domain comprises or consists of
amino acids 97 to 392 of SEQ ID NO: 12.
[0141] In an embodiment, the catalytic domain may be encoded by a
polynucleotide that hybridizes under very low stringency
conditions, or low stringency conditions, or medium stringency
conditions, or medium-high stringency conditions, or high
stringency conditions, or very high stringency conditions (as
defined above) with (i) the catalytic domain coding sequence of SEQ
ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9,
or SEQ ID NO: 11, (ii) the cDNA sequence contained in the catalytic
domain coding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5,
SEQ ID NO: 7, SEQ ID NO: 9, or SEQ ID NO: 11, or (iii) the
full-length complementary strand of (i) or (ii) (J. Sambrook et
al., 1989, supra).
[0142] The catalytic domain may be encoded by a polynucleotide
having a degree of sequence identity to the catalytic domain coding
sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7,
SEQ ID NO: 9, or SEQ ID NO: 11 of at least 60%, e.g. at least 70%,
at least 75%, at least 80%, at least 85%, at least 90%, at least
91%, at least 92%, at least 95%, at least 96%, at least 97%, at
least 98%, at least 99%, or 100%, which encode a polypeptide having
xylanase activity.
[0143] In one aspect, the polynucleotide encoding the catalytic
domain comprises or consists of nucleotides 55 to 1515 of SEQ ID
NO: 1 or the cDNA sequence thereof. In particular the
polynucleotide encoding the catalytic domain comprises or consists
of nucleotides 55-1515 of SEQ ID NO: 1.
[0144] In one aspect, the polynucleotide encoding the catalytic
domain comprises or consists of nucleotides 61 to 1461 of SEQ ID
NO: 3 or the cDNA sequence thereof. In particular the
polynucleotide encoding the catalytic domain comprises or consists
of nucleotides 61-249, 307-524, 649-665, 721-867, 930-1237,
1295-1351, and 1405-1461 of SEQ ID NO: 3.
[0145] In one aspect, the polynucleotide encoding the catalytic
domain comprises or consists of nucleotides 79 to 1400 of SEQ ID
NO: 5 or the cDNA sequence thereof. In particular the
polynucleotide encoding the catalytic domain comprises or consists
of nucleotides 79-273, 325-430, 472-598, 718-734, 789-935,
990-1297, and 1353-1400 of SEQ ID NO: 5.
[0146] In one aspect, the polynucleotide encoding the catalytic
domain comprises or consists of nucleotides 310 to 1665 of SEQ ID
NO: 7 or the cDNA sequence thereof. In particular the
polynucleotide encoding the catalytic domain comprises or consists
of nucleotides 310-453, 511-728, 786-787, 845-865, 923-1069,
1137-1441, 1503-1559, and 1618-1665 of SEQ ID NO:
[0147] 7.
[0148] In one aspect, the polynucleotide encoding the catalytic
domain comprises or consists of nucleotides 316 to 1614 of SEQ ID
NO: 9 or the cDNA sequence thereof. In particular the
polynucleotide encoding the catalytic domain comprises or consists
of nucleotides 316-459, 526-743, 799-800, 859-879, 936-1082,
1143-1447, and 1507-1614 of SEQ ID NO: 9.
[0149] In one aspect, the polynucleotide encoding the catalytic
domain comprises or consists of nucleotides 379 to 1673 of SEQ ID
NO: 11 or the cDNA sequence thereof. In particular the
polynucleotide encoding the catalytic domain comprises or consists
of nucleotides 379-522, 586-592, 650-739, 799-1066, 1121-1229,
1286-1378, 1432-1518, and 1584-1673 of SEQ ID NO: 11.
Polynucleotides
[0150] The present invention also relates to isolated
polynucleotides encoding a polypeptide of the present invention, as
described herein.
[0151] The techniques used to isolate or clone a polynucleotide are
known in the art and include isolation from genomic DNA or cDNA, or
a combination thereof. The cloning of the polynucleotides from
genomic DNA can be effected, e.g., by using the well known
polymerase chain reaction (PCR) or antibody screening of expression
libraries to detect cloned DNA fragments with shared structural
features. See, e.g., Innis et al., 1990, PCR: A Guide to Methods
and Application, Academic Press, New York. Other nucleic acid
amplification procedures such as ligase chain reaction (LCR),
ligation activated transcription (LAT) and polynucleotide-based
amplification (NASBA) may be used. The polynucleotides may be
cloned from a strain of Trametes, or a related organism and thus,
for example, may be an allelic or species variant of the
polypeptide encoding region of the polynucleotide.
[0152] Modification of a polynucleotide encoding a polypeptide of
the present invention may be necessary for synthesizing
polypeptides substantially similar to the polypeptide. The term
"substantially similar" to the polypeptide refers to non-naturally
occurring forms of the polypeptide. These polypeptides may differ
in some engineered way from the polypeptide isolated from its
native source, e.g., variants that differ in specific activity,
thermostability, pH optimum, or the like. The variants may be
constructed on the basis of the polynucleotide presented as the
mature polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3,
SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, or SEQ ID NO: 11, or the
cDNA sequence thereof, e.g., a subsequence thereof, and/or by
introduction of nucleotide substitutions that do not result in a
change in the amino acid sequence of the polypeptide, but which
correspond to the codon usage of the host organism intended for
production of the enzyme, or by introduction of nucleotide
substitutions that may give rise to a different amino acid
sequence. For a general description of nucleotide substitution,
see, e.g., Ford et al., 1991, Protein Expression and Purification
2: 95-107.
Nucleic Acid Constructs
[0153] The present invention also relates to nucleic acid
constructs comprising a polynucleotide of the present invention
operably linked to one or more control sequences that direct the
expression of the coding sequence in a suitable host cell under
conditions compatible with the control sequences.
[0154] A polynucleotide may be manipulated in a variety of ways to
provide for expression of the polypeptide. Manipulation of the
polynucleotide prior to its insertion into a vector may be
desirable or necessary depending on the expression vector. The
techniques for modifying polynucleotides utilizing recombinant DNA
methods are well known in the art.
[0155] The control sequence may be a promoter, a polynucleotide
that is recognized by a host cell for expression of a
polynucleotide encoding a polypeptide of the present invention. The
promoter contains transcriptional control sequences that mediate
the expression of the polypeptide. The promoter may be any
polynucleotide that shows transcriptional activity in the host cell
including mutant, truncated, and hybrid promoters, and may be
obtained from genes encoding extracellular or intracellular
polypeptides either homologous or heterologous to the host
cell.
[0156] Examples of suitable promoters for directing transcription
of the nucleic acid constructs of the present invention in a
bacterial host cell are the promoters obtained from the Bacillus
amyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformis
alpha-amylase gene (amyL), Bacillus licheniformis penicillinase
gene (penP), Bacillus stearothermophilus maltogenic amylase gene
(amyM), Bacillus subtilis levansucrase gene (sacB), Bacillus
subtilis xylA and xylB genes, Bacillus thuringiensis cryIIIA gene
(Agaisse and Lereclus, 1994, Molecular Microbiology 13: 97-107), E.
coli lac operon, E. coli trc promoter (Egon et al., 1988, Gene 69:
301-315), Streptomyces coelicolor agarase gene (dagA), and
prokaryotic beta-lactamase gene (Villa-Kamaroff et al., 1978, Proc.
Natl. Acad. Sci. USA 75: 3727-3731), as well as the tac promoter
(DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80: 21-25).
Further promoters are described in "Useful proteins from
recombinant bacteria" in Gilbert et al., 1980, Scientific American
242: 74-94; and in Sambrook et al., 1989, supra. Examples of tandem
promoters are disclosed in WO 99/43835.
[0157] Examples of suitable promoters for directing transcription
of the nucleic acid constructs of the present invention in a
filamentous fungal host cell are promoters obtained from the genes
for Aspergillus nidulans acetamidase, Aspergillus niger neutral
alpha-amylase, Aspergillus niger acid stable alpha-amylase,
Aspergillus niger or Aspergillus awamori glucoamylase (glaA),
Aspergillus oryzae TAKA amylase, Aspergillus oryzae alkaline
protease, Aspergillus oryzae triose phosphate isomerase, Fusarium
oxysporum trypsin-like protease (WO 96/00787), Fusarium venenatum
amyloglucosidase (WO 00/56900), Fusarium venenatum Dania (WO
00/56900), Fusarium venenatum Quinn (WO 00/56900), Rhizomucor
miehei lipase, Rhizomucor miehei aspartic proteinase, Trichoderma
reesei beta-glucosidase, Trichoderma reesei cellobiohydrolase I,
Trichoderma reesei cellobiohydrolase II, Trichoderma reesei
endoglucanase I, Trichoderma reesei endoglucanase II, Trichoderma
reesei endoglucanase III, Trichoderma reesei endoglucanase IV,
Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I,
Trichoderma reesei xylanase II, Trichoderma reesei beta-xylosidase,
as well as the NA2-tpi promoter (a modified promoter from an
Aspergillus neutral alpha-amylase gene in which the untranslated
leader has been replaced by an untranslated leader from an
Aspergillus triose phosphate isomerase gene; non-limiting examples
include modified promoters from an Aspergillus niger neutral
alpha-amylase gene in which the untranslated leader has been
replaced by an untranslated leader from an Aspergillus nidulans or
Aspergillus oryzae triose phosphate isomerase gene); and mutant,
truncated, and hybrid promoters thereof.
[0158] In a yeast host, useful promoters are obtained from the
genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces
cerevisiae galactokinase (GAL1), Saccharomyces cerevisiae alcohol
dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1,
ADH2/GAP), Saccharomyces cerevisiae triose phosphate isomerase
(TPI), Saccharomyces cerevisiae metallothionein (CUP1), and
Saccharomyces cerevisiae 3-phosphoglycerate kinase. Other useful
promoters for yeast host cells are described by Romanos et al.,
1992, Yeast 8: 423-488.
[0159] The control sequence may also be a transcription terminator,
which is recognized by a host cell to terminate transcription. The
terminator is operably linked to the 3'-terminus of the
polynucleotide encoding the polypeptide. Any terminator that is
functional in the host cell may be used in the present
invention.
[0160] Preferred terminators for bacterial host cells are obtained
from the genes for Bacillus clausii alkaline protease (aprH),
Bacillus licheniformis alpha-amylase (amyL), and Escherichia coli
ribosomal RNA (rrnB).
[0161] Preferred terminators for filamentous fungal host cells are
obtained from the genes for Aspergillus nidulans anthranilate
synthase, Aspergillus niger glucoamylase, Aspergillus niger
alpha-glucosidase, Aspergillus oryzae TAKA amylase, and Fusarium
oxysporum trypsin-like protease.
[0162] Preferred terminators for yeast host cells are obtained from
the genes for Saccharomyces cerevisiae enolase, Saccharomyces
cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae
glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators
for yeast host cells are described by Romanos et al., 1992,
supra.
[0163] The control sequence may also be an mRNA stabilizer region
downstream of a promoter and upstream of the coding sequence of a
gene which increases expression of the gene.
[0164] Examples of suitable mRNA stabilizer regions are obtained
from a Bacillus thuringiensis cryIIIA gene (WO 94/25612) and a
Bacillus subtilis SP82 gene (Hue et al., 1995, Journal of
Bacteriology 177: 3465-3471).
[0165] The control sequence may also be a leader, a nontranslated
region of an mRNA that is important for translation by the host
cell. The leader is operably linked to the 5'-terminus of the
polynucleotide encoding the polypeptide. Any leader that is
functional in the host cell may be used.
[0166] Preferred leaders for filamentous fungal host cells are
obtained from the genes for Aspergillus oryzae TAKA amylase and
Aspergillus nidulans triose phosphate isomerase. Suitable leaders
for yeast host cells are obtained from the genes for Saccharomyces
cerevisiae enolase (ENO-1), Saccharomyces cerevisiae
3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor,
and Saccharomyces cerevisiae alcohol
dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase
(ADH2/GAP).
[0167] The control sequence may also be a polyadenylation sequence,
a sequence operably linked to the 3'-terminus of the polynucleotide
and, when transcribed, is recognized by the host cell as a signal
to add polyadenosine residues to transcribed mRNA. Any
polyadenylation sequence that is functional in the host cell may be
used.
[0168] Preferred polyadenylation sequences for filamentous fungal
host cells are obtained from the genes for Aspergillus nidulans
anthranilate synthase, Aspergillus niger glucoamylase, Aspergillus
niger alpha-glucosidase Aspergillus oryzae TAKA amylase, and
Fusarium oxysporum trypsin-like protease.
[0169] Useful polyadenylation sequences for yeast host cells are
described by Guo and Sherman, 1995, Mol. Cellular Biol. 15:
5983-5990.
[0170] The control sequence may also be a signal peptide coding
region that encodes a signal peptide linked to the N-terminus of a
polypeptide and directs the polypeptide into the cell's secretory
pathway. The 5'-end of the coding sequence of the polynucleotide
may inherently contain a signal peptide coding sequence naturally
linked in translation reading frame with the segment of the coding
sequence that encodes the polypeptide. Alternatively, the 5'-end of
the coding sequence may contain a signal peptide coding sequence
that is foreign to the coding sequence. A foreign signal peptide
coding sequence may be required where the coding sequence does not
naturally contain a signal peptide coding sequence. Alternatively,
a foreign signal peptide coding sequence may simply replace the
natural signal peptide coding sequence in order to enhance
secretion of the polypeptide. However, any signal peptide coding
sequence that directs the expressed polypeptide into the secretory
pathway of a host cell may be used.
[0171] Effective signal peptide coding sequences for bacterial host
cells are the signal peptide coding sequences obtained from the
genes for Bacillus NCIB 11837 maltogenic amylase, Bacillus
licheniformis subtilisin, Bacillus licheniformis beta-lactamase,
Bacillus stearothermophilus alpha-amylase, Bacillus
stearothermophilus neutral proteases (nprT, nprS, nprM), and
Bacillus subtilis prsA. Further signal peptides are described by
Simonen and Palva, 1993, Microbiological Reviews 57: 109-137.
[0172] Effective signal peptide coding sequences for filamentous
fungal host cells are the signal peptide coding sequences obtained
from the genes for Aspergillus niger neutral amylase, Aspergillus
niger glucoamylase, Aspergillus oryzae TAKA amylase, Humicola
insolens cellulase, Humicola insolens endoglucanase V, Humicola
lanuginosa lipase, and Rhizomucor miehei aspartic proteinase.
[0173] Useful signal peptides for yeast host cells are obtained
from the genes for Saccharomyces cerevisiae alpha-factor and
Saccharomyces cerevisiae invertase. Other useful signal peptide
coding sequences are described by Romanos et al., 1992, supra.
[0174] The control sequence may also be a propeptide coding
sequence that encodes a propeptide positioned at the N-terminus of
a polypeptide. The resultant polypeptide is known as a proenzyme or
propolypeptide (or a zymogen in some cases). A propolypeptide is
generally inactive and can be converted to an active polypeptide by
catalytic or autocatalytic cleavage of the propeptide from the
propolypeptide. The propeptide coding sequence may be obtained from
the genes for Bacillus subtilis alkaline protease (aprE), Bacillus
subtilis neutral protease (nprT), Myceliophthora thermophila
laccase (WO 95/33836), Rhizomucor miehei aspartic proteinase, and
Saccharomyces cerevisiae alpha-factor.
[0175] Where both signal peptide and propeptide sequences are
present, the propeptide sequence is positioned next to the
N-terminus of a polypeptide and the signal peptide sequence is
positioned next to the N-terminus of the propeptide sequence.
[0176] It may also be desirable to add regulatory sequences that
regulate expression of the polypeptide relative to the growth of
the host cell. Examples of regulatory systems are those that cause
expression of the gene to be turned on or off in response to a
chemical or physical stimulus, including the presence of a
regulatory compound. Regulatory systems in prokaryotic systems
include the lac, tac, and trp operator systems. In yeast, the ADH2
system or GAL1 system may be used. In filamentous fungi, the
Aspergillus niger glucoamylase promoter, Aspergillus oryzae TAKA
alpha-amylase promoter, and Aspergillus oryzae glucoamylase
promoter may be used. Other examples of regulatory sequences are
those that allow for gene amplification. In eukaryotic systems,
these regulatory sequences include the dihydrofolate reductase gene
that is amplified in the presence of methotrexate, and the
metallothionein genes that are amplified with heavy metals. In
these cases, the polynucleotide encoding the polypeptide would be
operably linked with the regulatory sequence.
Expression Vectors
[0177] The present invention also relates to recombinant expression
vectors comprising a polynucleotide of the present invention, a
promoter, and transcriptional and translational stop signals. The
various nucleotide and control sequences may be joined together to
produce a recombinant expression vector that may include one or
more convenient restriction sites to allow for insertion or
substitution of the polynucleotide encoding the polypeptide at such
sites.
[0178] Alternatively, the polynucleotide may be expressed by
inserting the polynucleotide or a nucleic acid construct comprising
the polynucleotide into an appropriate vector for expression. In
creating the expression vector, the coding sequence is located in
the vector so that the coding sequence is operably linked with the
appropriate control sequences for expression.
[0179] The recombinant expression vector may be any vector (e.g., a
plasmid or virus) that can be conveniently subjected to recombinant
DNA procedures and can bring about expression of the
polynucleotide. The choice of the vector will typically depend on
the compatibility of the vector with the host cell into which the
vector is to be introduced. The vector may be a linear or closed
circular plasmid.
[0180] The vector may be an autonomously replicating vector, i.e.,
a vector that exists as an extrachromosomal entity, the replication
of which is independent of chromosomal replication, e.g., a
plasmid, an extrachromosomal element, a minichromosome, or an
artificial chromosome. The vector may contain any means for
assuring self-replication. Alternatively, the vector may be one
that, when introduced into the host cell, is integrated into the
genome and replicated together with the chromosome(s) into which it
has been integrated. Furthermore, a single vector or plasmid or two
or more vectors or plasmids that together contain the total DNA to
be introduced into the genome of the host cell, or a transposon,
may be used.
[0181] The vector preferably contains one or more selectable
markers that permit easy selection of transformed, transfected,
transduced, or the like cells. A selectable marker is a gene the
product of which provides for biocide or viral resistance,
resistance to heavy metals, prototrophy to auxotrophs, and the
like.
[0182] Examples of bacterial selectable markers are Bacillus
licheniformis or Bacillus subtilis dal genes, or markers that
confer antibiotic resistance such as ampicillin, chloramphenicol,
kanamycin, neomycin, spectinomycin, or tetracycline resistance.
Suitable markers for yeast host cells include, but are not limited
to, ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable
markers for use in a filamentous fungal host cell include, but are
not limited to, amdS (acetamidase), argB (ornithine
carbamoyltransferase), bar (phosphinothricin acetyltransferase),
hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG
(orotidine-5'-phosphate decarboxylase), sC (sulfate
adenyltransferase), and trpC (anthranilate synthase), as well as
equivalents thereof. Preferred for use in an Aspergillus cell are
Aspergillus nidulans or Aspergillus oryzae amdS and pyrG genes and
a Streptomyces hygroscopicus bar gene.
[0183] The vector preferably contains an element(s) that permits
integration of the vector into the host cell's genome or autonomous
replication of the vector in the cell independent of the
genome.
[0184] For integration into the host cell genome, the vector may
rely on the polynucleotide's sequence encoding the polypeptide or
any other element of the vector for integration into the genome by
homologous or non-homologous recombination. Alternatively, the
vector may contain additional polynucleotides for directing
integration by homologous recombination into the genome of the host
cell at a precise location(s) in the chromosome(s). To increase the
likelihood of integration at a precise location, the integrational
elements should contain a sufficient number of nucleic acids, such
as 100 to 10,000 base pairs, 400 to 10,000 base pairs, and 800 to
10,000 base pairs, which have a high degree of sequence identity to
the corresponding target sequence to enhance the probability of
homologous recombination. The integrational elements may be any
sequence that is homologous with the target sequence in the genome
of the host cell. Furthermore, the integrational elements may be
non-encoding or encoding polynucleotides. On the other hand, the
vector may be integrated into the genome of the host cell by
non-homologous recombination.
[0185] For autonomous replication, the vector may further comprise
an origin of replication enabling the vector to replicate
autonomously in the host cell in question. The origin of
replication may be any plasmid replicator mediating autonomous
replication that functions in a cell. The term "origin of
replication" or "plasmid replicator" means a polynucleotide that
enables a plasmid or vector to replicate in vivo.
[0186] Examples of bacterial origins of replication are the origins
of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184
permitting replication in E. coli, and pUB110, pE194, pTA1060, and
pAM111 permitting replication in Bacillus.
[0187] Examples of origins of replication for use in a yeast host
cell are the 2 micron origin of replication, ARS1, ARS4, the
combination of ARS1 and CEN3, and the combination of ARS4 and
CEN6.
[0188] Examples of origins of replication useful in a filamentous
fungal cell are AMA1 and ANSI (Gems et al., 1991, Gene 98: 61-67;
Cullen et al., 1987, Nucleic Acids Res. 15: 9163-9175; WO
00/24883). Isolation of the AMA1 gene and construction of plasmids
or vectors comprising the gene can be accomplished according to the
methods disclosed in WO 00/24883.
[0189] More than one copy of a polynucleotide of the present
invention may be inserted into a host cell to increase production
of a polypeptide. An increase in the copy number of the
polynucleotide can be obtained by integrating at least one
additional copy of the sequence into the host cell genome or by
including an amplifiable selectable marker gene with the
polynucleotide where cells containing amplified copies of the
selectable marker gene, and thereby additional copies of the
polynucleotide, can be selected for by cultivating the cells in the
presence of the appropriate selectable agent.
[0190] The procedures used to ligate the elements described above
to construct the recombinant expression vectors of the present
invention are well known to one skilled in the art (see, e.g.,
Sambrook et al., 1989, supra).
Host Cells
[0191] The present invention also relates to recombinant host
cells, comprising a polynucleotide of the present invention
operably linked to one or more control sequences that direct the
production of a polypeptide of the present invention. A construct
or vector comprising a polynucleotide is introduced into a host
cell so that the construct or vector is maintained as a chromosomal
integrant or as a self-replicating extra-chromosomal vector as
described earlier. The term "host cell" encompasses any progeny of
a parent cell that is not identical to the parent cell due to
mutations that occur during replication. The choice of a host cell
will to a large extent depend upon the gene encoding the
polypeptide and its source.
[0192] The host cell may be any cell useful in the recombinant
production of a polypeptide of the present invention, e.g., a
prokaryote or a eukaryote.
[0193] The prokaryotic host cell may be any Gram-positive or
Gram-negative bacterium. Gram-positive bacteria include, but are
not limited to, Bacillus, Clostridium, Enterococcus, Geobacillus,
Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus,
Streptococcus, and Streptomyces. Gram-negative bacteria include,
but are not limited to, Campylobacter, E. coli, Flavobacterium,
Fusobacterium, Helicobacter, Ilyobacter, Neisseria, Pseudomonas,
Salmonella, and Ureaplasma.
[0194] The bacterial host cell may be any Bacillus cell including,
but not limited to, Bacillus alkalophilus, Bacillus
amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus
clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus,
Bacillus lentus, Bacillus licheniformis, Bacillus megaterium,
Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis,
and Bacillus thuringiensis cells.
[0195] The bacterial host cell may also be any Streptococcus cell
including, but not limited to, Streptococcus equisimilis,
Streptococcus pyogenes, Streptococcus uberis, and Streptococcus
equi subsp. Zooepidemicus cells.
[0196] The bacterial host cell may also be any Streptomyces cell
including, but not limited to, Streptomyces achromogenes,
Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces
griseus, and Streptomyces lividans cells.
[0197] The introduction of DNA into a Bacillus cell may be effected
by protoplast transformation (see, e.g., Chang and Cohen, 1979,
Mol. Gen. Genet. 168: 111-115), competent cell transformation (see,
e.g., Young and Spizizen, 1961, J. Bacteriol. 81: 823-829, or
Dubnau and Davidoff-Abelson, 1971, J. Mol. Biol. 56: 209-221),
electroporation (see, e.g., Shigekawa and Dower, 1988,
Biotechniques 6: 742-751), or conjugation (see, e.g., Koehler and
Thorne, 1987, J. Bacteriol. 169: 5271-5278). The introduction of
DNA into an E. coli cell may be effected by protoplast
transformation (see, e.g., Hanahan, 1983, J. Mol. Biol. 166:
557-580) or electroporation (see, e.g., Dower et al., 1988, Nucleic
Acids Res. 16: 6127-6145). The introduction of DNA into a
Streptomyces cell may be effected by protoplast transformation,
electroporation (see, e.g., Gong et al., 2004, Folia Microbiol.
(Praha) 49: 399-405), conjugation (see, e.g., Mazodier et al.,
1989, J. Bacteriol. 171: 3583-3585), or transduction (see, e.g.,
Burke et al., 2001, Proc. Natl. Acad. Sci. USA 98: 6289-6294). The
introduction of DNA into a Pseudomonas cell may be effected by
electroporation (see, e.g., Choi et al., 2006, J. Microbiol.
Methods 64: 391-397) or conjugation (see, e.g., Pinedo and Smets,
2005, Appl. Environ. Microbiol. 71: 51-57). The introduction of DNA
into a Streptococcus cell may be effected by natural competence
(see, e.g., Perry and Kuramitsu, 1981, Infect. Immun. 32:
1295-1297), protoplast transformation (see, e.g., Catt and Jollick,
1991, Microbios 68: 189-207), electroporation (see, e.g., Buckley
et al., 1999, Appl. Environ. Microbiol. 65: 3800-3804), or
conjugation (see, e.g., Clewell, 1981, Microbiol. Rev. 45:
409-436). However, any method known in the art for introducing DNA
into a host cell can be used.
[0198] The host cell may also be a eukaryote, such as a mammalian,
insect, plant, or fungal cell.
[0199] The host cell may be a fungal cell. "Fungi" as used herein
includes the phyla Ascomycota, Basidiomycota, Chytridiomycota, and
Zygomycota as well as the Oomycota and all mitosporic fungi (as
defined by Hawksworth et al., In, Ainsworth and Bisby's Dictionary
of The Fungi, 8th edition, 1995, CAB International, University
Press, Cambridge, UK).
[0200] The fungal host cell may be a yeast cell. "Yeast" as used
herein includes ascosporogenous yeast (Endomycetales),
basidiosporogenous yeast, and yeast belonging to the Fungi
Imperfecti (Blastomycetes). Since the classification of yeast may
change in the future, for the purposes of this invention, yeast
shall be defined as described in Biology and Activities of Yeast
(Skinner, Passmore, and Davenport, editors, Soc. App. Bacteriol.
Symposium Series No. 9, 1980).
[0201] The yeast host cell may be a Candida, Hansenula,
Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or
Yarrowia cell, such as a Kluyveromyces lactis, Saccharomyces
carlsbergensis, Saccharomyces cerevisiae, Saccharomyces
diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri,
Saccharomyces norbensis, Saccharomyces oviformis, or Yarrowia
lipolytica cell.
[0202] The fungal host cell may be a filamentous fungal cell.
"Filamentous fungi" include all filamentous forms of the
subdivision Eumycota and Oomycota (as defined by Hawksworth et al.,
1995, supra). The filamentous fungi are generally characterized by
a mycelial wall composed of chitin, cellulose, glucan, chitosan,
mannan, and other complex polysaccharides. Vegetative growth is by
hyphal elongation and carbon catabolism is obligately aerobic. In
contrast, vegetative growth by yeasts such as Saccharomyces
cerevisiae is by budding of a unicellular thallus and carbon
catabolism may be fermentative.
[0203] The filamentous fungal host cell may be an Acremonium,
Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis,
Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium,
Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora,
Neocallimastix, Neurospora, Paecilomyces, Penicillium,
Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum,
Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or
Trichoderma cell.
[0204] For example, the filamentous fungal host cell may be an
Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus,
Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger,
Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsis aneirina,
Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis
pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa,
Ceriporiopsis subvermispora, Chrysosporium inops, Chrysosporium
keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium,
Chrysosporium pannicola, Chrysosporium queenslandicum,
Chrysosporium tropicum, Chrysosporium zonatum, Coprinus cinereus,
Coriolus hirsutus, Fusarium bactridioides, Fusarium cerealis,
Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum,
Fusarium graminum, Fusarium heterosporum, Fusarium negundi,
Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium
sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides,
Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides,
Fusarium venenatum, Humicola insolens, Humicola lanuginosa, Mucor
miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium
purpurogenum, Phanerochaete chrysosporium, Phlebia radiata,
Pleurotus eryngii, Thielavia terrestris, Trametes villosa, Trametes
versicolor, Trichoderma harzianum, Trichoderma koningii,
Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma
viride cell.
[0205] Fungal cells may be transformed by a process involving
protoplast formation, transformation of the protoplasts, and
regeneration of the cell wall in a manner known per se. Suitable
procedures for transformation of Aspergillus and Trichoderma host
cells are described in EP 238023, Yelton et al., 1984, Proc. Natl.
Acad. Sci. USA 81: 1470-1474, and Christensen et al., 1988,
Bio/Technology 6: 1419-1422. Suitable methods for transforming
Fusarium species are described by Malardier et al., 1989, Gene 78:
147-156, and WO 96/00787. Yeast may be transformed using the
procedures described by Becker and Guarente, In Abelson, J. N. and
Simon, M. I., editors, Guide to Yeast Genetics and Molecular
Biology, Methods in Enzymology, Volume 194, pp 182-187, Academic
Press, Inc., New York; Ito et al., 1983, J. Bacteriol. 153: 163;
and Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA 75: 1920.
Methods of Production
[0206] The present invention also relates to methods of producing a
polypeptide of the present invention, comprising (a) cultivating a
cell, which in its wild-type form produces the polypeptide, under
conditions conducive for production of the polypeptide; and (b)
recovering the polypeptide. In a preferred aspect, the cell is a
Trametes cell. In a more preferred aspect, the cell is a Trametes
versicolor cell. In a most preferred aspect, the cell is Trametes
versicolor Strain NN055586.
[0207] The present invention also relates to methods of producing a
polypeptide of the present invention, comprising (a) cultivating a
recombinant host cell of the present invention under conditions
conducive for production of the polypeptide; and (b) recovering the
polypeptide.
[0208] The host cells are cultivated in a nutrient medium suitable
for production of the polypeptide using methods known in the art.
For example, the cell may be cultivated by shake flask cultivation,
or small-scale or large-scale fermentation (including continuous,
batch, fed-batch, or solid state fermentations) in laboratory or
industrial fermentors in a suitable medium and under conditions
allowing the polypeptide to be expressed and/or isolated. The
cultivation takes place in a suitable nutrient medium comprising
carbon and nitrogen sources and inorganic salts, using procedures
known in the art. Suitable media are available from commercial
suppliers or may be prepared according to published compositions
(e.g., in catalogues of the American Type Culture Collection). If
the polypeptide is secreted into the nutrient medium, the
polypeptide can be recovered directly from the medium. If the
polypeptide is not secreted, it can be recovered from cell
lysates.
[0209] The polypeptide may be detected using methods known in the
art that are specific for the polypeptides. These detection methods
include, but are not limited to, use of specific antibodies,
formation of an enzyme product, or disappearance of an enzyme
substrate. For example, an enzyme assay may be used to determine
the activity of the polypeptide.
[0210] The polypeptide may be recovered using methods known in the
art. For example, the polypeptide may be recovered from the
nutrient medium by conventional procedures including, but not
limited to, collection, centrifugation, filtration, extraction,
spray-drying, evaporation, or precipitation.
[0211] The polypeptide may be purified by a variety of procedures
known in the art including, but not limited to, chromatography
(e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and
size exclusion), electrophoretic procedures (e.g., preparative
isoelectric focusing), differential solubility (e.g., ammonium
sulfate precipitation), SDS-PAGE, or extraction (see, e.g., Protein
Purification, Janson and Ryden, editors, VCH Publishers, New York,
1989) to obtain substantially pure polypeptides.
[0212] In an alternative aspect, the polypeptide is not recovered,
but rather a host cell of the present invention expressing the
polypeptide is used as a source of the polypeptide.
Plants
[0213] The present invention also relates to isolated plants, e.g.,
a transgenic plant, plant part, or plant cell, comprising a
polynucleotide of the present invention so as to express and
produce a polypeptide or domain in recoverable quantities. The
polypeptide or domain may be recovered from the plant or plant
part. Alternatively, the plant or plant part containing the
polypeptide or domain may be used as such for improving the quality
of a food or feed, e.g., improving nutritional value, palatability,
and rheological properties, or to destroy an antinutritive
factor.
[0214] The transgenic plant can be dicotyledonous (a dicot) or
monocotyledonous (a monocot). Examples of monocot plants are
grasses, such as meadow grass (blue grass, Poa), forage grass such
as Festuca, Lolium, temperate grass, such as Agrostis, and cereals,
e.g., wheat, oats, rye, barley, rice, sorghum, and maize
(corn).
[0215] Examples of dicot plants are tobacco, legumes, such as
lupins, potato, sugar beet, pea, bean and soybean, and cruciferous
plants (family Brassicaceae), such as cauliflower, rape seed, and
the closely related model organism Arabidopsis thaliana.
[0216] Examples of plant parts are stem, callus, leaves, root,
fruits, seeds, and tubers as well as the individual tissues
comprising these parts, e.g., epidermis, mesophyll, parenchyme,
vascular tissues, meristems. Specific plant cell compartments, such
as chloroplasts, apoplasts, mitochondria, vacuoles, peroxisomes and
cytoplasm are also considered to be a plant part. Furthermore, any
plant cell, whatever the tissue origin, is considered to be a plant
part. Likewise, plant parts such as specific tissues and cells
isolated to facilitate the utilization of the invention are also
considered plant parts, e.g., embryos, endosperms, aleurone and
seed coats.
[0217] Also included within the scope of the present invention are
the progeny of such plants, plant parts, and plant cells.
[0218] The transgenic plant or plant cell expressing the
polypeptide or domain may be constructed in accordance with methods
known in the art. In short, the plant or plant cell is constructed
by incorporating one or more expression constructs encoding the
polypeptide or domain into the plant host genome or chloroplast
genome and propagating the resulting modified plant or plant cell
into a transgenic plant or plant cell.
[0219] The expression construct is conveniently a nucleic acid
construct that comprises a polynucleotide encoding a polypeptide or
domain operably linked with appropriate regulatory sequences
required for expression of the polynucleotide in the plant or plant
part of choice. Furthermore, the expression construct may comprise
a selectable marker useful for identifying plant cells into which
the expression construct has been integrated and DNA sequences
necessary for introduction of the construct into the plant in
question (the latter depends on the DNA introduction method to be
used).
[0220] The choice of regulatory sequences, such as promoter and
terminator sequences and optionally signal or transit sequences, is
determined, for example, on the basis of when, where, and how the
polypeptide or domain is desired to be expressed. For instance, the
expression of the gene encoding a polypeptide or domain may be
constitutive or inducible, or may be developmental, stage or tissue
specific, and the gene product may be targeted to a specific tissue
or plant part such as seeds or leaves. Regulatory sequences are,
for example, described by Tague et al., 1988, Plant Physiology 86:
506.
[0221] For constitutive expression, the 35S-CaMV, the maize
ubiquitin 1, or the rice actin 1 promoter may be used (Franck et
al., 1980, Cell 21: 285-294; Christensen et al., 1992, Plant Mol.
Biol. 18: 675-689; Zhang et al., 1991, Plant Cell 3: 1155-1165).
Organ-specific promoters may be, for example, a promoter from
storage sink tissues such as seeds, potato tubers, and fruits
(Edwards and Coruzzi, 1990, Ann. Rev. Genet. 24: 275-303), or from
metabolic sink tissues such as meristems (Ito et al., 1994, Plant
Mol. Biol. 24: 863-878), a seed specific promoter such as the
glutelin, prolamin, globulin, or albumin promoter from rice (Wu et
al., 1998, Plant Cell Physiol. 39: 885-889), a Vicia faba promoter
from the legumin B4 and the unknown seed protein gene from Vicia
faba (Conrad et al., 1998, J. Plant Physiol. 152: 708-711), a
promoter from a seed oil body protein (Chen et al., 1998, Plant
Cell Physiol. 39: 935-941), the storage protein napA promoter from
Brassica napus, or any other seed specific promoter known in the
art, e.g., as described in WO 91/14772. Furthermore, the promoter
may be a leaf specific promoter such as the rbcs promoter from rice
or tomato (Kyozuka et al., 1993, Plant Physiol. 102: 991-1000), the
chlorella virus adenine methyltransferase gene promoter (Mitra and
Higgins, 1994, Plant Mol. Biol. 26: 85-93), the aldP gene promoter
from rice (Kagaya et al., 1995, Mol. Gen. Genet. 248: 668-674), or
a wound inducible promoter such as the potato pin2 promoter (Xu et
al., 1993, Plant Mol. Biol. 22: 573-588). Likewise, the promoter
may be induced by abiotic treatments such as temperature, drought,
or alterations in salinity or induced by exogenously applied
substances that activate the promoter, e.g., ethanol, oestrogens,
plant hormones such as ethylene, abscisic acid, and gibberellic
acid, and heavy metals.
[0222] A promoter enhancer element may also be used to achieve
higher expression of a polypeptide or domain in the plant. For
instance, the promoter enhancer element may be an intron that is
placed between the promoter and the polynucleotide encoding a
polypeptide or domain. For instance, Xu et al., 1993, supra,
disclose the use of the first intron of the rice actin 1 gene to
enhance expression.
[0223] The selectable marker gene and any other parts of the
expression construct may be chosen from those available in the
art.
[0224] The nucleic acid construct is incorporated into the plant
genome according to conventional techniques known in the art,
including Agrobacterium-mediated transformation, virus-mediated
transformation, microinjection, particle bombardment, biolistic
transformation, and electroporation (Gasser et al., 1990, Science
244: 1293; Potrykus, 1990, Bio/Technology 8: 535; Shimamoto et al.,
1989, Nature 338: 274).
[0225] Agrobacterium tumefaciens-mediated gene transfer is a method
for generating transgenic dicots (for a review, see Hooykas and
Schilperoort, 1992, Plant Mol. Biol. 19: 15-38) and for
transforming monocots, although other transformation methods may be
used for these plants. A method for generating transgenic monocots
is particle bombardment (microscopic gold or tungsten particles
coated with the transforming DNA) of embryonic calli or developing
embryos (Christou, 1992, Plant J. 2: 275-281; Shimamoto, 1994,
Curr. Opin. Biotechnol. 5: 158-162; Vasil et al., 1992,
Bio/Technology 10: 667-674). An alternative method for
transformation of monocots is based on protoplast transformation as
described by Omirulleh et al., 1993, Plant Mol. Biol. 21: 415-428.
Additional transformation methods include those described in U.S.
Pat. Nos. 6,395,966 and 7,151,204 (both of which are herein
incorporated by reference in their entirety).
[0226] Following transformation, the transformants having
incorporated the expression construct are selected and regenerated
into whole plants according to methods well known in the art. Often
the transformation procedure is designed for the selective
elimination of selection genes either during regeneration or in the
following generations by using, for example, co-transformation with
two separate T-DNA constructs or site specific excision of the
selection gene by a specific recombinase.
[0227] In addition to direct transformation of a particular plant
genotype with a construct of the present invention, transgenic
plants may be made by crossing a plant having the construct to a
second plant lacking the construct. For example, a construct
encoding a polypeptide or domain can be introduced into a
particular plant variety by crossing, without the need for ever
directly transforming a plant of that given variety. Therefore, the
present invention encompasses not only a plant directly regenerated
from cells which have been transformed in accordance with the
present invention, but also the progeny of such plants. As used
herein, progeny may refer to the offspring of any generation of a
parent plant prepared in accordance with the present invention.
Such progeny may include a DNA construct prepared in accordance
with the present invention. Crossing results in the introduction of
a transgene into a plant line by cross pollinating a starting line
with a donor plant line. Non-limiting examples of such steps are
described in U.S. Pat. No. 7,151,204.
[0228] Plants may be generated through a process of backcross
conversion. For example, plants include plants referred to as a
backcross converted genotype, line, inbred, or hybrid.
[0229] Genetic markers may be used to assist in the introgression
of one or more transgenes of the invention from one genetic
background into another. Marker assisted selection offers
advantages relative to conventional breeding in that it can be used
to avoid errors caused by phenotypic variations. Further, genetic
markers may provide data regarding the relative degree of elite
germplasm in the individual progeny of a particular cross. For
example, when a plant with a desired trait which otherwise has a
non-agronomically desirable genetic background is crossed to an
elite parent, genetic markers may be used to select progeny which
not only possess the trait of interest, but also have a relatively
large proportion of the desired germplasm. In this way, the number
of generations required to introgress one or more traits into a
particular genetic background is minimized.
[0230] The present invention also relates to methods of producing a
polypeptide or domain of the present invention comprising (a)
cultivating a transgenic plant or a plant cell comprising a
polynucleotide encoding the polypeptide or domain under conditions
conducive for production of the polypeptide or domain; and (b)
recovering the polypeptide or domain.
Compositions
[0231] The present invention also relates to compositions
comprising a polypeptide of the present invention. Preferably, the
compositions are enriched in such a polypeptide. The term
"enriched" indicates that the xylanase activity of the composition
has been increased, e.g., with an enrichment factor of at least
1.1.
[0232] The composition may comprise a polypeptide of the present
invention as the major enzymatic component, e.g., a mono-component
composition. Alternatively, the composition may comprise multiple
enzymatic activities, such as one or more (e.g., several) enzymes
selected from the group consisting of a cellulase, a hemicellulase,
GH61 polypeptide, an expansin, an esterase, a laccase, a
ligninolytic enzyme, a pectinase, a peroxidase, a protease, and a
swollenin.
[0233] The polypeptide compositions may be prepared in accordance
with methods known in the art and may be in the form of a liquid or
a dry composition. For instance, the polypeptide composition may be
in the form of a granulate or a microgranulate. The polypeptide to
be included in the composition may be stabilized in accordance with
methods known in the art.
[0234] Examples are given below of preferred uses of the
polypeptide compositions of the invention. The dosage of the
polypeptide composition of the invention and other conditions under
which the composition is used may be determined on the basis of
methods known in the art.
Uses
[0235] The present invention is also directed to the following
processes for using the polypeptides having xylanase activity, or
compositions thereof.
[0236] The present invention also relates to processes for
degrading a cellulosic material or xylan-containing material,
comprising: treating the cellulosic material or xylan-containing
material with an enzyme composition in the presence of a
polypeptide having xylanase activity of the present invention. In
one aspect, the processes further comprise recovering the degraded
or converted cellulosic material or xylan-containing material.
Soluble products of degradation or conversion of the cellulosic
material or xylan-containing material can be separated from
insoluble cellulosic material or xylan-containing material using a
method known in the art such as, for example, centrifugation,
filtration, or gravity settling.
[0237] The present invention also relates to processes of producing
a fermentation product, comprising: (a) saccharifying a cellulosic
material or xylan-containing material with an enzyme composition in
the presence of a polypeptide having xylanase activity of the
present invention; (b) fermenting the saccharified cellulosic
material or xylan-containing material with one or more (e.g.,
several) fermenting microorganisms to produce the fermentation
product; and (c) recovering the fermentation product from the
fermentation.
[0238] The present invention also relates to processes of
fermenting a cellulosic material or xylan-containing material,
comprising: fermenting the cellulosic material or xylan-containing
material with one or more (e.g., several) fermenting
microorganisms, wherein the cellulosic material or xylan-containing
material is saccharified with an enzyme composition in the presence
of a polypeptide having xylanase activity of the present invention.
In one aspect, the fermenting of the cellulosic material or
xylan-containing material produces a fermentation product. In
another aspect, the processes further comprise recovering the
fermentation product from the fermentation.
[0239] The processes of the present invention can be used to
saccharify the cellulosic material or xylan-containing material to
fermentable sugars and to convert the fermentable sugars to many
useful fermentation products, e.g., fuel, potable ethanol, and/or
platform chemicals (e.g., acids, alcohols, ketones, gases, and the
like). The production of a desired fermentation product from the
cellulosic material or xylan-containing material typically involves
pretreatment, enzymatic hydrolysis (saccharification), and
fermentation.
[0240] The processing of the cellulosic material or
xylan-containing material according to the present invention can be
accomplished using methods conventional in the art. Moreover, the
processes of the present invention can be implemented using any
conventional biomass processing apparatus configured to operate in
accordance with the invention.
[0241] Hydrolysis (saccharification) and fermentation, separate or
simultaneous, include, but are not limited to, separate hydrolysis
and fermentation (SHF); simultaneous saccharification and
fermentation (SSF); simultaneous saccharification and
co-fermentation (SSCF); hybrid hydrolysis and fermentation (HHF);
separate hydrolysis and co-fermentation (SHCF); hybrid hydrolysis
and co-fermentation (HHCF); and direct microbial conversion (DMC),
also sometimes called consolidated bioprocessing (CBP). SHF uses
separate process steps to first enzymatically hydrolyze the
cellulosic material or xylan-containing material to fermentable
sugars, e.g., glucose, cellobiose, and pentose monomers, and then
ferment the fermentable sugars to ethanol. In SSF, the enzymatic
hydrolysis of the cellulosic material or xylan-containing material
and the fermentation of sugars to ethanol are combined in one step
(Philippidis, G. P., 1996, Cellulose bioconversion technology, in
Handbook on Bioethanol: Production and Utilization, Wyman, C. E.,
ed., Taylor & Francis, Washington, D.C., 179-212). SSCF
involves the co-fermentation of multiple sugars (Sheehan, J., and
Himmel, M., 1999, Enzymes, energy and the environment: A strategic
perspective on the U.S. Department of Energy's research and
development activities for bioethanol, Biotechnol. Prog. 15:
817-827). HHF involves a separate hydrolysis step, and in addition
a simultaneous saccharification and hydrolysis step, which can be
carried out in the same reactor. The steps in an HHF process can be
carried out at different temperatures, i.e., high temperature
enzymatic saccharification followed by SSF at a lower temperature
that the fermentation strain can tolerate. DMC combines all three
processes (enzyme production, hydrolysis, and fermentation) in one
or more (e.g., several) steps where the same organism is used to
produce the enzymes for conversion of the cellulosic material or
xylan-containing material to fermentable sugars and to convert the
fermentable sugars into a final product (Lynd, L. R., Weimer, P.
J., van Zyl, W. H., and Pretorius, I. S., 2002, Microbial cellulose
utilization: Fundamentals and biotechnology, Microbiol. Mol. Biol.
Reviews 66: 506-577). It is understood herein that any method known
in the art comprising pretreatment, enzymatic hydrolysis
(saccharification), fermentation, or a combination thereof, can be
used in the practicing the processes of the present invention.
[0242] A conventional apparatus can include a fed-batch stirred
reactor, a batch stirred reactor, a continuous flow stirred reactor
with ultrafiltration, and/or a continuous plug-flow column reactor
(Fernanda de Castilhos Corazza, Flavio Faria de Moraes, Gisella
Maria Zanin and Ivo Neitzel, 2003, Optimal control in fed-batch
reactor for the cellobiose hydrolysis, Acta Scientiarum. Technology
25: 33-38; Gusakov, A. V., and Sinitsyn, A. P., 1985, Kinetics of
the enzymatic hydrolysis of cellulose: 1. A mathematical model for
a batch reactor process, Enz. Microb. Technol. 7: 346-352), an
attrition reactor (Ryu, S. K., and Lee, J. M., 1983, Bioconversion
of waste cellulose by using an attrition bioreactor, Biotechnol.
Bioeng. 25: 53-65), or a reactor with intensive stirring induced by
an electromagnetic field (Gusakov, A. V., Sinitsyn, A. P.,
Davydkin, I. Y., Davydkin, V. Y., Protas, 0. V., 1996, Enhancement
of enzymatic cellulose hydrolysis using a novel type of bioreactor
with intensive stirring induced by electromagnetic field, Appl.
Biochem. Biotechnol. 56: 141-153). Additional reactor types include
fluidized bed, upflow blanket, immobilized, and extruder type
reactors for hydrolysis and/or fermentation.
[0243] Pretreatment.
[0244] In practicing the processes of the present invention, any
pretreatment process known in the art can be used to disrupt plant
cell wall components of the cellulosic material or xylan-containing
material (Chandra et al., 2007, Substrate pretreatment: The key to
effective enzymatic hydrolysis of lignocellulosics?, Adv. Biochem.
Engin./Biotechnol. 108: 67-93; Galbe and Zacchi, 2007, Pretreatment
of lignocellulosic materials for efficient bioethanol production,
Adv. Biochem. Engin./Biotechnol. 108: 41-65; Hendriks and Zeeman,
2009, Pretreatments to enhance the digestibility of lignocellulosic
biomass, Bioresource Technol. 100: 10-18; Mosier et al., 2005,
Features of promising technologies for pretreatment of
lignocellulosic biomass, Bioresource Technol. 96: 673-686;
Taherzadeh and Karimi, 2008, Pretreatment of lignocellulosic wastes
to improve ethanol and biogas production: A review, Int. J. of Mol.
Sci. 9: 1621-1651; Yang and Wyman, 2008, Pretreatment: the key to
unlocking low-cost cellulosic ethanol, Biofuels Bioproducts and
Biorefining-Biofpr. 2: 26-40).
[0245] The cellulosic material or xylan-containing material can
also be subjected to particle size reduction, sieving, pre-soaking,
wetting, washing, and/or conditioning prior to pretreatment using
methods known in the art.
[0246] Conventional pretreatments include, but are not limited to,
steam pretreatment (with or without explosion), dilute acid
pretreatment, hot water pretreatment, alkaline pretreatment, lime
pretreatment, wet oxidation, wet explosion, ammonia fiber
explosion, organosolv pretreatment, and biological pretreatment.
Additional pretreatments include ammonia percolation, ultrasound,
electroporation, microwave, supercritical CO.sub.2, supercritical
H.sub.2O, ozone, ionic liquid, and gamma irradiation
pretreatments.
[0247] The cellulosic material or xylan-containing material can be
pretreated before hydrolysis and/or fermentation. Pretreatment is
preferably performed prior to the hydrolysis. Alternatively, the
pretreatment can be carried out simultaneously with enzyme
hydrolysis to release fermentable sugars, such as glucose, xylose,
and/or cellobiose. In most cases the pretreatment step itself
results in some conversion of biomass to fermentable sugars (even
in absence of enzymes).
[0248] Steam Pretreatment. In steam pretreatment, the cellulosic
material or xylan-containing material is heated to disrupt the
plant cell wall components, including lignin, hemicellulose, and
cellulose to make the cellulose and other fractions, e.g.,
hemicellulose, accessible to enzymes. The cellulosic material or
xylan-containing material is passed to or through a reaction vessel
where steam is injected to increase the temperature to the required
temperature and pressure and is retained therein for the desired
reaction time. Steam pretreatment is preferably performed at
140-250.degree. C., e.g., 160-200.degree. C. or 170-190.degree. C.,
where the optimal temperature range depends on addition of a
chemical catalyst. Residence time for the steam pretreatment is
preferably 1-60 minutes, e.g., 1-30 minutes, 1-20 minutes, 3-12
minutes, or 4-10 minutes, where the optimal residence time depends
on temperature range and addition of a chemical catalyst. Steam
pretreatment allows for relatively high solids loadings, so that
the cellulosic material or xylan-containing material is generally
only moist during the pretreatment. The steam pretreatment is often
combined with an explosive discharge of the material after the
pretreatment, which is known as steam explosion, that is, rapid
flashing to atmospheric pressure and turbulent flow of the material
to increase the accessible surface area by fragmentation (Duff and
Murray, 1996, Bioresource Technology 855: 1-33; Galbe and Zacchi,
2002, Appl. Microbiol. Biotechnol. 59: 618-628; U.S. Patent
Application No. 20020164730). During steam pretreatment,
hemicellulose acetyl groups are cleaved and the resulting acid
autocatalyzes partial hydrolysis of the hemicellulose to
monosaccharides and oligosaccharides. Lignin is removed to only a
limited extent.
[0249] Chemical Pretreatment: The term "chemical treatment" refers
to any chemical pretreatment that promotes the separation and/or
release of cellulose, hemicellulose, and/or lignin. Such a
pretreatment can convert crystalline cellulose to amorphous
cellulose. Examples of suitable chemical pretreatment processes
include, for example, dilute acid pretreatment, lime pretreatment,
wet oxidation, ammonia fiber/freeze explosion (AFEX), ammonia
percolation (APR), ionic liquid, and organosolv pretreatments.
[0250] A catalyst such as H.sub.2SO.sub.4 or SO.sub.2 (typically
0.3 to 5% w/w) is often added prior to steam pretreatment, which
decreases the time and temperature, increases the recovery, and
improves enzymatic hydrolysis (Ballesteros et al., 2006, Appl.
Biochem. Biotechnol. 129-132: 496-508; Varga et al., 2004, Appl.
Biochem. Biotechnol. 113-116: 509-523; Sassner et al., 2006, Enzyme
Microb. Technol. 39: 756-762). In dilute acid pretreatment, the
cellulosic material or xylan-containing material is mixed with
dilute acid, typically H.sub.2SO.sub.4, and water to form a slurry,
heated by steam to the desired temperature, and after a residence
time flashed to atmospheric pressure. The dilute acid pretreatment
can be performed with a number of reactor designs, e.g., plug-flow
reactors, counter-current reactors, or continuous counter-current
shrinking bed reactors (Duff and Murray, 1996, supra; Schell et
al., 2004, Bioresource Technol. 91: 179-188; Lee et al., 1999, Adv.
Biochem. Eng. Biotechnol. 65: 93-115).
[0251] Several methods of pretreatment under alkaline conditions
can also be used. These alkaline pretreatments include, but are not
limited to, sodium hydroxide, lime, wet oxidation, ammonia
percolation (APR), and ammonia fiber/freeze explosion (AFEX).
[0252] Lime pretreatment is performed with calcium oxide or calcium
hydroxide at temperatures of 85-150.degree. C. and residence times
from 1 hour to several days (Wyman et al., 2005, Bioresource
Technol. 96: 1959-1966; Mosier et al., 2005, Bioresource Technol.
96: 673-686). WO 2006/110891, WO 2006/110899, WO 2006/110900, and
WO 2006/110901 disclose pretreatment methods using ammonia.
[0253] Wet oxidation is a thermal pretreatment performed typically
at 180-200.degree. C. for 5-15 minutes with addition of an
oxidative agent such as hydrogen peroxide or over-pressure of
oxygen (Schmidt and Thomsen, 1998, Bioresource Technol. 64:
139-151; Palonen et al., 2004, Appl. Biochem. Biotechnol. 117:
1-17; Varga et al., 2004, Biotechnol. Bioeng. 88: 567-574; Martin
et al., 2006, J. Chem. Technol. Biotechnol. 81: 1669-1677). The
pretreatment is performed preferably at 1-40% dry matter, e.g.,
2-30% dry matter or 5-20% dry matter, and often the initial pH is
increased by the addition of alkali such as sodium carbonate.
[0254] A modification of the wet oxidation pretreatment method,
known as wet explosion (combination of wet oxidation and steam
explosion) can handle dry matter up to 30%. In wet explosion, the
oxidizing agent is introduced during pretreatment after a certain
residence time. The pretreatment is then ended by flashing to
atmospheric pressure (WO 2006/032282).
[0255] Ammonia fiber explosion (AFEX) involves treating the
cellulosic material or xylan-containing material with liquid or
gaseous ammonia at moderate temperatures such as 90-150.degree. C.
and high pressure such as 17-20 bar for 5-10 minutes, where the dry
matter content can be as high as 60% (Gollapalli et al., 2002,
Appl. Biochem. Biotechnol. 98: 23-35; Chundawat et al., 2007,
Biotechnol. Bioeng. 96: 219-231; Alizadeh et al., 2005, Appl.
Biochem. Biotechnol. 121: 1133-1141; Teymouri et al., 2005,
Bioresource Technol. 96: 2014-2018). During AFEX pretreatment
cellulose and hemicelluloses remain relatively intact.
Lignin-carbohydrate complexes are cleaved.
[0256] Organosolv pretreatment delignifies the cellulosic material
or xylan-containing material by extraction using aqueous ethanol
(40-60% ethanol) at 160-200.degree. C. for 30-60 minutes (Pan et
al., 2005, Biotechnol. Bioeng. 90: 473-481; Pan et al., 2006,
Biotechnol. Bioeng. 94: 851-861; Kurabi et al., 2005, Appl.
Biochem. Biotechnol. 121: 219-230). Sulphuric acid is usually added
as a catalyst. In organosolv pretreatment, the majority of
hemicellulose and lignin is removed.
[0257] Other examples of suitable pretreatment methods are
described by Schell et al., 2003, Appl. Biochem. and Biotechnol.
Vol. 105-108, p. 69-85, and Mosier et al., 2005, Bioresource
Technology 96: 673-686, and U.S. Published Application
2002/0164730.
[0258] In one aspect, the chemical pretreatment is preferably
carried out as a dilute acid treatment, and more preferably as a
continuous dilute acid treatment. The acid is typically sulfuric
acid, but other acids can also be used, such as acetic acid, citric
acid, nitric acid, phosphoric acid, tartaric acid, succinic acid,
hydrogen chloride, or mixtures thereof. Mild acid treatment is
conducted in the pH range of preferably 1-5, e.g., 1-4 or 1-2.5. In
one aspect, the acid concentration is in the range from preferably
0.01 to 10 wt % acid, e.g., 0.05 to 5 wt % acid or 0.1 to 2 wt %
acid. The acid is contacted with the cellulosic material or
xylan-containing material and held at a temperature in the range of
preferably 140-200.degree. C., e.g., 165-190.degree. C., for
periods ranging from 1 to 60 minutes.
[0259] In another aspect, pretreatment takes place in an aqueous
slurry. In preferred aspects, the cellulosic material or
xylan-containing material is present during pretreatment in amounts
preferably between 10-80 wt %, e.g., 20-70 wt % or 30-60 wt %, such
as around 40 wt %. The pretreated cellulosic material or
xylan-containing material can be unwashed or washed using any
method known in the art, e.g., washed with water.
[0260] Mechanical Pretreatment or Physical Pretreatment: The term
"mechanical pretreatment" or "physical pretreatment" refers to any
pretreatment that promotes size reduction of particles. For
example, such pretreatment can involve various types of grinding or
milling (e.g., dry milling, wet milling, or vibratory ball
milling).
[0261] The cellulosic material or xylan-containing material can be
pretreated both physically (mechanically) and chemically.
Mechanical or physical pretreatment can be coupled with
steaming/steam explosion, hydrothermolysis, dilute or mild acid
treatment, high temperature, high pressure treatment, irradiation
(e.g., microwave irradiation), or combinations thereof. In one
aspect, high pressure means pressure in the range of preferably
about 100 to about 400 psi, e.g., about 150 to about 250 psi. In
another aspect, high temperature means temperatures in the range of
about 100 to about 300.degree. C., e.g., about 140 to about
200.degree. C. In a preferred aspect, mechanical or physical
pretreatment is performed in a batch-process using a steam gun
hydrolyzer system that uses high pressure and high temperature as
defined above, e.g., a Sunds Hydrolyzer available from Sunds
Defibrator AB, Sweden. The physical and chemical pretreatments can
be carried out sequentially or simultaneously, as desired.
[0262] Accordingly, in a preferred aspect, the cellulosic material
or xylan-containing material is subjected to physical (mechanical)
or chemical pretreatment, or any combination thereof, to promote
the separation and/or release of cellulose, hemicellulose, and/or
lignin.
[0263] Biological Pretreatment: The term "biological pretreatment"
refers to any biological pretreatment that promotes the separation
and/or release of cellulose, hemicellulose, and/or lignin from the
cellulosic material or xylan-containing material. Biological
pretreatment techniques can involve applying lignin-solubilizing
microorganisms and/or enzymes (see, for example, Hsu, T.-A., 1996,
Pretreatment of biomass, in Handbook on Bioethanol: Production and
Utilization, Wyman, C. E., ed., Taylor & Francis, Washington,
D.C., 179-212; Ghosh and Singh, 1993, Physicochemical and
biological treatments for enzymatic/microbial conversion of
cellulosic biomass, Adv. Appl. Microbiol. 39: 295-333; McMillan, J.
D., 1994, Pretreating lignocellulosic biomass: a review, in
Enzymatic Conversion of Biomass for Fuels Production, Himmel, M.
E., Baker, J. O., and Overend, R. P., eds., ACS Symposium Series
566, American Chemical Society, Washington, D.C., chapter 15; Gong,
C. S., Cao, N. J., Du, J., and Tsao, G. T., 1999, Ethanol
production from renewable resources, in Advances in Biochemical
Engineering/Biotechnology, Scheper, T., ed., Springer-Verlag Berlin
Heidelberg, Germany, 65: 207-241; Olsson and Hahn-Hagerdal, 1996,
Fermentation of lignocellulosic hydrolysates for ethanol
production, Enz. Microb. Tech. 18: 312-331; and Vallander and
Eriksson, 1990, Production of ethanol from lignocellulosic
materials: State of the art, Adv. Biochem. Eng./Biotechnol. 42:
63-95).
[0264] Saccharification.
[0265] In the hydrolysis step, also known as saccharification, the
cellulosic material or xylan-containing material, e.g., pretreated,
is hydrolyzed to break down cellulose and/or hemicellulose to
fermentable sugars, such as glucose, cellobiose, xylose, xylulose,
arabinose, mannose, galactose, and/or soluble oligosaccharides. The
hydrolysis is performed enzymatically by an enzyme composition in
the presence of a polypeptide having xylanase activity of the
present invention. The enzymes of the compositions can be added
simultaneously or sequentially.
[0266] Enzymatic hydrolysis is preferably carried out in a suitable
aqueous environment under conditions that can be readily determined
by one skilled in the art. In one aspect, hydrolysis is performed
under conditions suitable for the activity of the enzyme(s), i.e.,
optimal for the enzyme(s). The hydrolysis can be carried out as a
fed batch or continuous process where the cellulosic material or
xylan-containing material is fed gradually to, for example, an
enzyme containing hydrolysis solution.
[0267] The saccharification is generally performed in stirred-tank
reactors or fermentors under controlled pH, temperature, and mixing
conditions. Suitable process time, temperature and pH conditions
can readily be determined by one skilled in the art. For example,
the saccharification can last up to 200 hours, but is typically
performed for preferably about 12 to about 120 hours, e.g., about
16 to about 72 hours or about 24 to about 48 hours. The temperature
is in the range of preferably about 25.degree. C. to about
70.degree. C., e.g., about 30.degree. C. to about 65.degree. C.,
about 40.degree. C. to about 60.degree. C., or about 50.degree. C.
to about 55.degree. C. The pH is in the range of preferably about 3
to about 8, e.g., about 3.5 to about 7, about 4 to about 6, or
about 5.0 to about 5.5. The dry solids content is in the range of
preferably about 5 to about 50 wt %, e.g., about 10 to about 40 wt
% or about 20 to about 30 wt %.
[0268] The enzyme compositions can comprise any protein useful in
degrading the cellulosic material or xylan-containing material.
[0269] In one aspect, the enzyme composition comprises or further
comprises one or more (e.g., several) proteins selected from the
group consisting of a cellulase, a GH61 polypeptide having
cellulolytic enhancing activity, a hemicellulase, an esterase, an
expansin, a laccase, a ligninolytic enzyme, a pectinase, a
peroxidase, a protease, and a swollenin. In another aspect, the
hemicellulase is preferably one or more (e.g., several) enzymes
selected from the group consisting of an acetylmannan esterase, an
acetylxylan esterase, an arabinanase, an arabinofuranosidase, a
coumaric acid esterase, a feruloyl esterase, a galactosidase, a
glucuronidase, a glucuronoyl esterase, a mannanase, a mannosidase,
a xylanase, and a xylosidase. In another aspect, the cellulase is
preferably one or more (e.g., several) enzymes selected from the
group consisting of an endoglucanase, a cellobiohydrolase, and a
beta-glucosidase.
[0270] In another aspect, the enzyme composition comprises one or
more (e.g., several) cellulolytic enzymes. In another aspect, the
enzyme composition comprises or further comprises one or more
(e.g., several) hemicellulolytic enzymes. In another aspect, the
enzyme composition comprises one or more (e.g., several)
cellulolytic enzymes and one or more (e.g., several)
hemicellulolytic enzymes. In another aspect, the enzyme composition
comprises one or more (e.g., several) enzymes selected from the
group of cellulolytic enzymes and hemicellulolytic enzymes.
[0271] In another aspect, the enzyme composition comprises an
acetylmannan esterase. In another aspect, the enzyme composition
comprises an acetylxylan esterase. In another aspect, the enzyme
composition comprises an arabinanase (e.g., alpha-L-arabinanase).
In another aspect, the enzyme composition comprises an
arabinofuranosidase (e.g., alpha-L-arabinofuranosidase). In another
aspect, the enzyme composition comprises a coumaric acid esterase.
In another aspect, the enzyme composition comprises a feruloyl
esterase. In another aspect, the enzyme composition comprises a
galactosidase (e.g., alpha-galactosidase and/or
beta-galactosidase). In another aspect, the enzyme composition
comprises a glucuronidase (e.g., alpha-D-glucuronidase). In another
aspect, the enzyme composition comprises a glucuronoyl esterase. In
another aspect, the enzyme composition comprises a mannanase. In
another aspect, the enzyme composition comprises a mannosidase
(e.g., beta-mannosidase). In another aspect, the enzyme composition
comprises a xylanase. In a preferred aspect, the xylanase is a
Family 10 xylanase. In another aspect, the enzyme composition
comprises a xylosidase (e.g., beta-xylosidase).
[0272] In another aspect, the enzyme composition comprises an
endoglucanase. In another aspect, the enzyme composition comprises
a cellobiohydrolase. In another aspect, the enzyme composition
comprises a beta-glucosidase. In another aspect, the enzyme
composition comprises a polypeptide having cellulolytic enhancing
activity. In another aspect, the enzyme composition comprises an
endoglucanase and a polypeptide having cellulolytic enhancing
activity. In another aspect, the enzyme composition comprises a
cellobiohydrolase and a polypeptide having cellulolytic enhancing
activity. In another aspect, the enzyme composition comprises a
beta-glucosidase and a polypeptide having cellulolytic enhancing
activity. In another aspect, the enzyme composition comprises an
endoglucanase and a cellobiohydrolase. In another aspect, the
enzyme composition comprises an endoglucanase and a
beta-glucosidase. In another aspect, the enzyme composition
comprises a cellobiohydrolase and a beta-glucosidase. In another
aspect, the enzyme composition comprises an endoglucanase, a
cellobiohydrolase, and a polypeptide having cellulolytic enhancing
activity. In another aspect, the enzyme composition comprises an
endoglucanase, a beta-glucosidase, and a polypeptide having
cellulolytic enhancing activity. In another aspect, the enzyme
composition comprises a cellobiohydrolase, a beta-glucosidase, and
a polypeptide having cellulolytic enhancing activity. In another
aspect, the enzyme composition comprises an endoglucanase, a
cellobiohydrolase, and a beta-glucosidase. In another aspect, the
enzyme composition comprises an endoglucanase, a cellobiohydrolase,
a beta-glucosidase, and a polypeptide having cellulolytic enhancing
activity.
[0273] In another aspect, the enzyme composition comprises an
esterase. In another aspect, the enzyme composition comprises an
expansin. In another aspect, the enzyme composition comprises a
laccase. In another aspect, the enzyme composition comprises a
ligninolytic enzyme. In a preferred aspect, the ligninolytic enzyme
is a manganese peroxidase. In another preferred aspect, the
ligninolytic enzyme is a lignin peroxidase. In another preferred
aspect, the ligninolytic enzyme is a H.sub.2O.sub.2-producing
enzyme. In another aspect, the enzyme composition comprises a
pectinase. In another aspect, the enzyme composition comprises a
peroxidase. In another aspect, the enzyme composition comprises a
protease. In another aspect, the enzyme composition comprises a
swollenin
[0274] In the processes of the present invention, the enzyme(s) can
be added prior to or during fermentation, e.g., during
saccharification or during or after propagation of the fermenting
microorganism(s).
[0275] One or more (e.g., several) components of the enzyme
composition may be wild-type proteins, recombinant proteins, or a
combination of wild-type proteins and recombinant proteins. For
example, one or more (e.g., several) components may be native
proteins of a cell, which is used as a host cell to express
recombinantly one or more (e.g., several) other components of the
enzyme composition. One or more (e.g., several) components of the
enzyme composition may be produced as monocomponents, which are
then combined to form the enzyme composition. The enzyme
composition may be a combination of multicomponent and
monocomponent protein preparations.
[0276] The enzymes used in the processes of the present invention
may be in any form suitable for use, such as, for example, a crude
fermentation broth with or without cells removed, a cell lysate
with or without cellular debris, a semi-purified or purified enzyme
preparation, or a host cell as a source of the enzymes. The enzyme
composition may be a dry powder or granulate, a non-dusting
granulate, a liquid, a stabilized liquid, or a stabilized protected
enzyme. Liquid enzyme preparations may, for instance, be stabilized
by adding stabilizers such as a sugar, a sugar alcohol or another
polyol, and/or lactic acid or another organic acid according to
established processes.
[0277] The optimum amounts of the enzymes and polypeptides having
xylanase activity depend on several factors including, but not
limited to, the mixture of component cellulolytic enzymes, the
cellulosic material or xylan-containing material, the concentration
of cellulosic material or xylan-containing material, the
pretreatment(s) of the cellulosic material or xylan-containing
material, temperature, time, pH, and inclusion of fermenting
organism (e.g., yeast for Simultaneous Saccharification and
Fermentation).
[0278] In one aspect, an effective amount of cellulolytic or
hemicellulolytic enzyme to the cellulosic material or
xylan-containing material is about 0.5 to about 50 mg, e.g., about
0.5 to about 40 mg, about 0.5 to about 25 mg, about 0.75 to about
20 mg, about 0.75 to about 15 mg, about 0.5 to about 10 mg, or
about 2.5 to about 10 mg per g of the cellulosic material or
xylan-containing material.
[0279] In another aspect, an effective amount of a polypeptide
having xylanase activity to the cellulosic material or
xylan-containing material is about 0.01 to about 50.0 mg, e.g.,
about 0.01 to about 40 mg, about 0.01 to about 30 mg, about 0.01 to
about 20 mg, about 0.01 to about 10 mg, about 0.01 to about 5 mg,
about 0.025 to about 1.5 mg, about 0.05 to about 1.25 mg, about
0.075 to about 1.25 mg, about 0.1 to about 1.25 mg, about 0.15 to
about 1.25 mg, or about 0.25 to about 1.0 mg per g of the
cellulosic material or xylan-containing material.
[0280] In another aspect, an effective amount of a polypeptide
having xylanase activity to cellulolytic or hemicellulolytic enzyme
is about 0.005 to about 1.0 g, e.g., about 0.01 to about 1.0 g,
about 0.15 to about 0.75 g, about 0.15 to about 0.5 g, about 0.1 to
about 0.5 g, about 0.1 to about 0.25 g, or about 0.05 to about 0.2
g per g of cellulolytic or hemicellulolytic enzyme.
[0281] The polypeptides having cellulolytic enzyme activity or
hemicellulolytic enzyme activity as well as other
proteins/polypeptides useful in the degradation of the cellulosic
material or xylan-containing material, e.g., GH61 polypeptides
having cellulolytic enhancing activity (collectively hereinafter
"polypeptides having enzyme activity") can be derived or obtained
from any suitable origin, including, bacterial, fungal, yeast,
plant, or mammalian origin. The term "obtained" also means herein
that the enzyme may have been produced recombinantly in a host
organism employing methods described herein, wherein the
recombinantly produced enzyme is either native or foreign to the
host organism or has a modified amino acid sequence, e.g., having
one or more (e.g., several) amino acids that are deleted, inserted
and/or substituted, i.e., a recombinantly produced enzyme that is a
mutant and/or a fragment of a native amino acid sequence or an
enzyme produced by nucleic acid shuffling processes known in the
art. Encompassed within the meaning of a native enzyme are natural
variants and within the meaning of a foreign enzyme are variants
obtained recombinantly, such as by site-directed mutagenesis or
shuffling.
[0282] A polypeptide having enzyme activity may be a bacterial
polypeptide. For example, the polypeptide may be a gram positive
bacterial polypeptide such as a Bacillus, Streptococcus,
Streptomyces, Staphylococcus, Enterococcus, Lactobacillus,
Lactococcus, Clostridium, Geobacillus, Caldicellulosiruptor,
Acidothermus, Thermobifidia, or Oceanobacillus polypeptide having
enzyme activity, or a Gram negative bacterial polypeptide such as
an E. coli, Pseudomonas, Salmonella, Campylobacter, Helicobacter,
Flavobacterium, Fusobacterium, Ilyobacter, Neisseria, or Ureaplasma
polypeptide having enzyme activity.
[0283] In one aspect, the polypeptide is a Bacillus alkalophilus,
Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans,
Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus
lautus, Bacillus lentus, Bacillus licheniformis, Bacillus
megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus
subtilis, or Bacillus thuringiensis polypeptide having enzyme
activity.
[0284] In another aspect, the polypeptide is a Streptococcus
equisimilis, Streptococcus pyogenes, Streptococcus uberis, or
Streptococcus equi subsp. Zooepidemicus polypeptide having enzyme
activity.
[0285] In another aspect, the polypeptide is a Streptomyces
achromogenes, Streptomyces avermitilis, Streptomyces coelicolor,
Streptomyces griseus, or Streptomyces lividans polypeptide having
enzyme activity.
[0286] The polypeptide having enzyme activity may also be a fungal
polypeptide, and more preferably a yeast polypeptide such as a
Candida, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces,
or Yarrowia polypeptide having enzyme activity; or more preferably
a filamentous fungal polypeptide such as an Acremonium, Agaricus,
Alternaria, Aspergillus, Aureobasidium, Botryosphaeria,
Ceriporiopsis, Chaetomidium, Chrysosporium, Claviceps,
Cochliobolus, Coprinopsis, Coptotermes, Corynascus, Cryphonectria,
Cryptococcus, Diplodia, Exidia, Filibasidium, Fusarium, Gibberella,
Holomastigotoides, Humicola, Irpex, Lentinula, Leptospaeria,
Magnaporthe, Melanocarpus, Meripilus, Mucor, Myceliophthora,
Neocallimastix, Neurospora, Paecilomyces, Penicillium,
Phanerochaete, Piromyces, Poitrasia, Pseudoplectania,
Pseudotrichonympha, Rhizomucor, Schizophyllum, Scytalidium,
Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trichoderma,
Trichophaea, Verticillium, Volvariella, or Xylaria polypeptide
having enzyme activity.
[0287] In one aspect, the polypeptide is a Saccharomyces
carlsbergensis, Saccharomyces cerevisiae, Saccharomyces
diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri,
Saccharomyces norbensis, or Saccharomyces oviformis polypeptide
having enzyme activity.
[0288] In another aspect, the polypeptide is an Acremonium
cellulolyticus, Aspergillus aculeatus, Aspergillus awamori,
Aspergillus fumigatus, Aspergillus foetidus, Aspergillus japonicus,
Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae,
Chrysosporium keratinophilum, Chrysosporium lucknowense,
Chrysosporium tropicum, Chrysosporium merdarium, Chrysosporium
inops, Chrysosporium pannicola, Chrysosporium queenslandicum,
Chrysosporium zonatum, Fusarium bactridioides, Fusarium cerealis,
Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum,
Fusarium graminum, Fusarium heterosporum, Fusarium negundi,
Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium
sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides,
Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides,
Fusarium venenatum, Humicola grisea, Humicola insolens, Humicola
lanuginosa, Irpex lacteus, Mucor miehei, Myceliophthora
thermophila, Neurospora crassa, Penicillium funiculosum,
Penicillium purpurogenum, Phanerochaete chrysosporium, Thielavia
achromatica, Thielavia albomyces, Thielavia albopilosa, Thielavia
australeinsis, Thielavia fimeti, Thielavia microspora, Thielavia
ovispora, Thielavia peruviana, Thielavia spededonium, Thielavia
setosa, Thielavia subthermophila, Thielavia terrestris, Trichoderma
harzianum, Trichoderma koningii, Trichoderma longibrachiatum,
Trichoderma reesei, Trichoderma viride, or Trichophaea saccata
polypeptide having enzyme activity.
[0289] Chemically modified or protein engineered mutants of
polypeptides having enzyme activity may also be used.
[0290] One or more (e.g., several) components of the enzyme
composition may be a recombinant component, i.e., produced by
cloning of a DNA sequence encoding the single component and
subsequent cell transformed with the DNA sequence and expressed in
a host (see, for example, WO 91/17243 and WO 91/17244). The host is
preferably a heterologous host (enzyme is foreign to host), but the
host may under certain conditions also be a homologous host (enzyme
is native to host). Monocomponent cellulolytic proteins may also be
prepared by purifying such a protein from a fermentation broth.
[0291] In one aspect, the one or more (e.g., several) cellulolytic
enzymes comprise a commercial cellulolytic enzyme preparation.
Examples of commercial cellulolytic enzyme preparations suitable
for use in the present invention include, for example, CELLIC.TM.
CTec (Novozymes NS), CELLIC.TM. CTec2 (Novozymes NS),
CELLUCLAST.TM. (Novozymes NS), NOVOZYM.TM. 188 (Novozymes NS),
CELLUZYME.TM. (Novozymes NS), CEREFLO.TM. (Novozymes NS), and
ULTRAFLO.TM. (Novozymes NS), ACCELERASE.TM. (Genencor Int.),
LAMINEX.TM. (Genencor Int.), SPEZYME.TM. CP (Genencor Int.),
FILTRASE.RTM. NL (DSM); METHAPLUS.RTM. S/L 100 (DSM). ROHAMENT.TM.
7069 W (Rohm GmbH), FIBREZYME.RTM. LDI (Dyadic International,
Inc.), FIBREZYME.RTM. LBR (Dyadic International, Inc.), or
VISCOSTAR.RTM. 150L (Dyadic International, Inc.). The cellulase
enzymes are added in amounts effective from about 0.001 to about
5.0 wt % of solids, e.g., about 0.025 to about 4.0 wt % of solids
or about 0.005 to about 2.0 wt % of solids.
[0292] Examples of bacterial endoglucanases that can be used in the
processes of the present invention, include, but are not limited
to, an Acidothermus cellulolyticus endoglucanase (WO 91/05039; WO
93/15186; U.S. Pat. No. 5,275,944; WO 96/02551; U.S. Pat. No.
5,536,655, WO 00/70031, WO 05/093050); Thermobifida fusca
endoglucanase III (WO 05/093050); and Thermobifida fusca
endoglucanase V (WO 05/093050).
[0293] Examples of fungal endoglucanases that can be used in the
present invention, include, but are not limited to, a Trichoderma
reesei endoglucanase I (Penttila et al., 1986, Gene 45: 253-263,
Trichoderma reesei Cel7B endoglucanase I (GENBANK.TM. accession no.
M15665), Trichoderma reesei endoglucanase II (Saloheimo, et al.,
1988, Gene 63:11-22), Trichoderma reesei CeI5A endoglucanase II
(GENBANK.TM. accession no. M19373), Trichoderma reesei
endoglucanase III (Okada et al., 1988, Appl. Environ. Microbiol.
64: 555-563, GENBANK.TM. accession no. AB003694), Trichoderma
reesei endoglucanase V (Saloheimo et al., 1994, Molecular
Microbiology 13: 219-228, GENBANK.TM. accession no. Z33381),
Aspergillus aculeatus endoglucanase (Ooi et al., 1990, Nucleic
Acids Research 18: 5884), Aspergillus kawachii endoglucanase
(Sakamoto et al., 1995, Current Genetics 27: 435-439), Erwinia
carotovara endoglucanase (Saarilahti et al., 1990, Gene 90: 9-14),
Fusarium oxysporum endoglucanase (GENBANK.TM. accession no.
L29381), Humicola grisea var. thermoidea endoglucanase (GENBANK.TM.
accession no. AB003107), Melanocarpus albomyces endoglucanase
(GENBANK.TM. accession no. MAL515703), Neurospora crassa
endoglucanase (GENBANK.TM. accession no. XM.sub.--324477), Humicola
insolens endoglucanase V, Myceliophthora thermophila CBS 117.65
endoglucanase, basidiomycete CBS 495.95 endoglucanase,
basidiomycete CBS 494.95 endoglucanase, Thielavia terrestris NRRL
8126 CEL6B endoglucanase, Thielavia terrestris NRRL 8126 CEL6C
endoglucanase, Thielavia terrestris NRRL 8126 CEL7C endoglucanase,
Thielavia terrestris NRRL 8126 CEL7E endoglucanase, Thielavia
terrestris NRRL 8126 CEL7F endoglucanase, Cladorrhinum
foecundissimum ATCC 62373 CEL7A endoglucanase, and Trichoderma
reesei strain No. VTT-D-80133 endoglucanase (GENBANK.TM. accession
no. M15665).
[0294] Examples of cellobiohydrolases useful in the present
invention include, but are not limited to, Aspergillus aculeatus
cellobiohydrolase II (WO 2011/059740), Chaetomium thermophilum
cellobiohydrolase I, Chaetomium thermophilum cellobiohydrolase II,
Humicola insolens cellobiohydrolase I, Myceliophthora thermophila
cellobiohydrolase II (WO 2009/042871), Thielavia hyrcanie
cellobiohydrolase II (WO 2010/141325), Thielavia terrestris
cellobiohydrolase II (CEL6A, WO 2006/074435), Trichoderma reesei
cellobiohydrolase I, Trichoderma reesei cellobiohydrolase II, and
Trichophaea saccata cellobiohydralase II (WO 2010/057086).
[0295] Examples of beta-glucosidases useful in the present
invention include, but are not limited to, beta-glucosidases from
Aspergillus aculeatus (Kawaguchi et al., 1996, Gene 173: 287-288),
Aspergillus fumigatus (WO 2005/047499), Aspergillus niger (Dan et
al., 2000, J. Biol. Chem. 275: 4973-4980), Aspergillus oryzae (WO
2002/095014), Penicillium brasilianum IBT 20888 (WO 2007/019442 and
WO 2010/088387), Thielavia terrestris (WO 2011/035029), and
Trichophaea saccata (WO 2007/019442).
[0296] The beta-glucosidase may be a fusion protein. In one aspect,
the beta-glucosidase is an Aspergillus oryzae beta-glucosidase
variant BG fusion protein (WO 2008/057637) or an Aspergillus oryzae
beta-glucosidase fusion protein (WO 2008/057637.
[0297] Other useful endoglucanases, cellobiohydrolases, and
beta-glucosidases are disclosed in numerous Glycosyl Hydrolase
families using the classification according to Henrissat B., 1991,
A classification of glycosyl hydrolases based on amino-acid
sequence similarities, Biochem. J. 280: 309-316, and Henrissat B.,
and Bairoch A., 1996, Updating the sequence-based classification of
glycosyl hydrolases, Biochem. J. 316: 695-696.
[0298] Other cellulolytic enzymes that may be used in the present
invention are described in WO 98/13465, WO 98/015619, WO 98/015633,
WO 99/06574, WO 99/10481, WO 99/025847, WO 99/031255, WO
2002/101078, WO 2003/027306, WO 2003/052054, WO 2003/052055, WO
2003/052056, WO 2003/052057, WO 2003/052118, WO 2004/016760, WO
2004/043980, WO 2004/048592, WO 2005/001065, WO 2005/028636, WO
2005/093050, WO 2005/093073, WO 2006/074005, WO 2006/117432, WO
2007/071818, WO 2007/071820, WO 2008/008070, WO 2008/008793, U.S.
Pat. No. 5,457,046, U.S. Pat. No. 5,648,263, and U.S. Pat. No.
5,686,593.
[0299] In the processes of the present invention, any GH61
polypeptide having cellulolytic enhancing activity can be used.
[0300] Examples of GH61 polypeptides having cellulolytic enhancing
activity useful in the processes of the present invention include,
but are not limited to, GH61 polypeptides from Thielavia terrestris
(WO 2005/074647, WO 2008/148131, and WO 2011/035027), Thermoascus
aurantiacus (WO 2005/074656 and WO 2010/065830), Trichoderma reesei
(WO 2007/089290), Myceliophthora thermophila (WO 2009/085935, WO
2009/085859, WO 2009/085864, WO 2009/085868), Aspergillus fumigatus
(WO 2010/138754), GH61 polypeptides from Penicillium pinophilum (WO
2011/005867), Thermoascus sp. (WO 2011/039319), Penicillium sp. (WO
2011/041397), and Thermoascus crustaceous (WO 2011/041504).
[0301] In one aspect, the GH61 polypeptide having cellulolytic
enhancing activity is used in the presence of a soluble activating
divalent metal cation according to WO 2008/151043, e.g., manganese
sulfate.
[0302] In one aspect, the GH61 polypeptide having cellulolytic
enhancing activity is used in the presence of a dioxy compound, a
bicylic compound, a heterocyclic compound, a nitrogen-containing
compound, a quinone compound, a sulfur-containing compound, or a
liquor obtained from a pretreated cellulosic material or
xylan-containing material such as pretreated corn stover (PCS).
[0303] The dioxy compound may include any suitable compound
containing two or more oxygen atoms. In some aspects, the dioxy
compounds contain a substituted aryl moiety as described herein.
The dioxy compounds may comprise one or more (e.g., several)
hydroxyl and/or hydroxyl derivatives, but also include substituted
aryl moieties lacking hydroxyl and hydroxyl derivatives.
Non-limiting examples of the dioxy compounds include pyrocatechol
or catechol; caffeic acid; 3,4-dihydroxybenzoic acid;
4-tert-butyl-5-methoxy-1,2-benzenediol; pyrogallol; gallic acid;
methyl-3,4,5-trihydroxybenzoate; 2,3,4-trihydroxybenzophenone;
2,6-dimethoxyphenol; sinapinic acid; 3,5-dihydroxybenzoic acid;
4-chloro-1,2-benzenediol; 4-nitro-1,2-benzenediol; tannic acid;
ethyl gallate; methyl glycolate; dihydroxyfumaric acid;
2-butyne-1,4-diol; (croconic acid; 1,3-propanediol; tartaric acid;
2,4-pentanediol; 3-ethyoxy-1,2-propanediol;
2,4,4'-trihydroxybenzophenone; cis-2-butene-1,4-diol;
3,4-dihydroxy-3-cyclobutene-1,2-dione; dihydroxyacetone; acrolein
acetal; methyl-4-hydroxybenzoate; 4-hydroxybenzoic acid; and
methyl-3,5-dimethoxy-4-hydroxybenzoate; or a salt or solvate
thereof.
[0304] The bicyclic compound may include any suitable substituted
fused ring system as described herein. The compounds may comprise
one or more (e.g., several) additional rings, and are not limited
to a specific number of rings unless otherwise stated. In one
aspect, the bicyclic compound is a flavonoid. In another aspect,
the bicyclic compound is an optionally subsituted isoflavonoid. In
another aspect, the bicyclic compound is an optionally substituted
flavylium ion, such as an optionally substituted anthocyanidin or
optionally substituted anthocyanin, or derivative thereof.
Non-limiting examples of thebicyclic compounds include epicatechin;
quercetin; myricetin; taxifolin; kaempferol; morin; acacetin;
naringenin; isorhamnetin; apigenin; cyanidin; cyanin; kuromanin;
keracyanin; or a salt or solvate thereof.
[0305] The heterocyclic compound may be any suitable compound, such
as an optionally substituted aromatic or non-aromatic ring
comprising a heteroatom, as described herein. In one aspect, the
heterocyclic is a compound comprising an optionally substituted
heterocycloalkyl moiety or an optionally substituted heteroaryl
moiety. In another aspect, the optionally substituted
heterocycloalkyl moiety or optionally substituted heteroaryl moiety
is an optionally substituted 5-membered heterocycloalkyl or an
optionally substituted 5-membered heteroaryl moiety. In another
aspect, the optionally substituted heterocycloalkyl or optionally
substituted heteroaryl moiety is an optionally substituted moiety
selected from pyrazolyl, furanyl, imidazolyl, isoxazolyl,
oxadiazolyl, oxazolyl, pyrrolyl, pyridyl, pyrimidyl, pyridazinyl,
thiazolyl, triazolyl, thienyl, dihydrothieno-pyrazolyl,
thianaphthenyl, carbazolyl, benzimidazolyl, benzothienyl,
benzofuranyl, indolyl, quinolinyl, benzotriazolyl, benzothiazolyl,
benzooxazolyl, benzimidazolyl, isoquinolinyl, isoindolyl,
acridinyl, benzoisazolyl, dimethylhydantoin, pyrazinyl,
tetrahydrofuranyl, pyrrolinyl, pyrrolidinyl, morpholinyl, indolyl,
diazepinyl, azepinyl, thiepinyl, piperidinyl, and oxepinyl. In
another aspect, the optionally substituted heterocycloalkyl moiety
or optionally substituted heteroaryl moiety is an optionally
substituted furanyl. Non-limiting examples of the heterocyclic
compounds include
(1,2-dihydroxyethyl)-3,4-dihydroxyfuran-2(5H)-one;
4-hydroxy-5-methyl-3-furanone; 5-hydroxy-2(5H)-furanone;
[1,2-dihydroxyethyl]furan-2,3,4(5H)-trione;
.alpha.-hydroxy-.gamma.-butyrolactone; ribonic .gamma.-lactone;
aldohexuronicaldohexuronic acid .gamma.-lactone; gluconic acid
5-lactone; 4-hydroxycoumarin; dihydrobenzofuran;
5-(hydroxymethyl)furfural; furoin; 2(5H)-furanone;
5,6-dihydro-2H-pyran-2-one; and
5,6-dihydro-4-hydroxy-6-methyl-2H-pyran-2-one; or a salt or solvate
thereof.
[0306] The nitrogen-containing compound may be any suitable
compound with one or more nitrogen atoms. In one aspect, the
nitrogen-containing compound comprises an amine, imine,
hydroxylamine, or nitroxide moiety. Non-limiting examples of
thenitrogen-containing compounds include acetone oxime; violuric
acid; pyridine-2-aldoxime; 2-aminophenol; 1,2-benzenediamine;
2,2,6,6-tetramethyl-1-piperidinyloxy; 5,6,7,8-tetrahydrobiopterin;
6,7-dimethyl-5,6,7,8-tetrahydropterine; and maleamic acid; or a
salt or solvate thereof.
[0307] The quinone compound may be any suitable compound comprising
a quinone moiety as described herein. Non-limiting examples of the
quinone compounds include 1,4-benzoquinone; 1,4-naphthoquinone;
2-hydroxy-1,4-naphthoquinone;
2,3-dimethoxy-5-methyl-1,4-benzoquinone or coenzyme Q.sub.0;
2,3,5,6-tetramethyl-1,4-benzoquinone or duroquinone;
1,4-dihydroxyanthraquinone; 3-hydroxy-1-methyl-5,6-indolinedione or
adrenochrome; 4-tert-butyl-5-methoxy-1,2-benzoquinone;
pyrroloquinoline quinone; or a salt or solvate thereof.
[0308] The sulfur-containing compound may be any suitable compound
comprising one or more sulfur atoms. In one aspect, the
sulfur-containing comprises a moiety selected from thionyl,
thioether, sulfinyl, sulfonyl, sulfamide, sulfonamide, sulfonic
acid, and sulfonic ester. Non-limiting examples of the
sulfur-containing compounds include ethanethiol; 2-propanethiol;
2-propene-1-thiol; 2-mercaptoethanesulfonic acid; benzenethiol;
benzene-1,2-dithiol; cysteine; methionine; glutathione; cystine; or
a salt or solvate thereof.
[0309] In one aspect, an effective amount of such a compound
described above to cellulosic material or xylan-containing material
as a molar ratio to glucosyl units of cellulose is about 10.sup.-6
to about 10, e.g., about 10.sup.-6 to about 7.5, about 10.sup.-6 to
about 5, about 10.sup.-6 to about 2.5, about 10.sup.-6 to about 1,
about 10.sup.-5 to about 1, about 10.sup.-5 to about 10.sup.-1,
about 10.sup.-4 to about 10.sup.-1, about 10.sup.-3 to about
10.sup.-1, or about 10.sup.-3 to about 10.sup.-2. In another
aspect, an effective amount of such a compound described above is
about 0.1 .mu.M to about 1 M, e.g., about 0.5 .mu.M to about 0.75
M, about 0.75 .mu.M to about 0.5 M, about 1 .mu.M to about 0.25 M,
about 1 .mu.M to about 0.1 M, about 5 .mu.M to about 50 mM, about
10 .mu.M to about 25 mM, about 50 .mu.M to about 25 mM, about 10
.mu.M to about 10 mM, about 5 .mu.M to about 5 mM, ord about 0.1 mM
to about 1 mM.
[0310] The term "liquor" means the solution phase, either aqueous,
organic, or a combination thereof, arising from treatment of a
lignocellulose and/or hemicellulose material in a slurry, or
monosaccharides thereof, e.g., xylose, arabinose, mannose, etc.,
under conditions as described herein, and the soluble contents
thereof. A liquor for cellulolytic enhancement of a GH61
polypeptide can be produced by treating a lignocellulose or
hemicellulose material (or feedstock) by applying heat and/or
pressure, optionally in the presence of a catalyst, e.g., acid,
optionally in the presence of an organic solvent, and optionally in
combination with physical disruption of the material, and then
separating the solution from the residual solids. Such conditions
determine the degree of cellulolytic enhancement obtainable through
the combination of liquor and a GH61 polypeptide during hydrolysis
of a cellulosic substrate by a cellulase preparation. The liquor
can be separated from the treated material using a method standard
in the art, such as filtration, sedimentation, or
centrifugation.
[0311] In one aspect, an effective amount of the liquor to
cellulose is about 10.sup.-6 to about 10 g per g of cellulose,
e.g., about 10.sup.-6 to about 7.5 g, about 10.sup.-6 to about 5,
about 10.sup.-6 to about 2.5 g, about 10.sup.-6 to about 1 g, about
10.sup.-6 to about 1 g, about 10.sup.-6 to about 10.sup.-1 g, about
10.sup.-4 to about 10.sup.-1 g, about 10.sup.-3 to about 10.sup.-1
g, or about 10.sup.-3 to about 10.sup.-2 g per g of cellulose.
[0312] In one aspect, the one or more (e.g., several)
hemicellulolytic enzymes comprise a commercial hemicellulolytic
enzyme preparation. Examples of commercial hemicellulolytic enzyme
preparations suitable for use in the present invention include, for
example, SHEARZYME.TM. (Novozymes NS), CELLIC.TM. HTec (Novozymes
NS), CELLIC.TM. HTec2 (Novozymes NS), VISCOZYME.RTM. (Novozymes
NS), ULTRAFLO.RTM. (Novozymes NS), PULPZYME.RTM. HC (Novozymes NS),
MULTIFECT.RTM. Xylanase (Genencor), ACCELLERASE.RTM. XY (Genencor),
ACCELLERASE.RTM. XC (Genencor), ECOPULP.RTM. TX-200A (AB Enzymes),
HSP 6000 Xylanase (DSM), DEPOL.TM. 333P (Biocatalysts Limit, Wales,
UK), DEPOL.TM. 740L. (Biocatalysts Limit, Wales, UK), and DEPOL.TM.
762P (Biocatalysts Limit, Wales, UK).
[0313] Examples of xylanases useful in the processes of the present
invention include, but are not limited to, xylanases from
Aspergillus aculeatus (GeneSeqP:AAR63790; WO 94/21785), Aspergillus
fumigatus (WO 2006/078256), Penicillium pinophilum (WO
2011/041405), Penicillium sp. (WO 2010/126772), Thielavia
terrestris NRRL 8126 (WO 2009/079210), and Trichophaea saccata GH10
(WO 2011/057083).
[0314] Examples of beta-xylosidases useful in the processes of the
present invention include, but are not limited to, beta-xylosidases
from Neurospora crassa (SwissProt accession number Q7SOW4),
Trichoderma reesei (UniProtKB/TrEMBL accession number Q92458), and
Talaromyces emersonii (SwissProt accession number Q8X212).
[0315] Examples of acetylxylan esterases useful in the processes of
the present invention include, but are not limited to, acetylxylan
esterases from Aspergillus aculeatus (WO 2010/108918), Chaetomium
globosum (Uniprot accession number Q2GWX4), Chaetomium gracile
(GeneSeqP accession number AAB82124), Humicola insolens DSM 1800
(WO 2009/073709), Hypocrea jecorina (WO 2005/001036), Myceliophtera
thermophila (WO 2010/014880), Neurospora crassa (UniProt accession
number q7s259), Phaeosphaeria nodorum (Uniprot accession number
QOUHJ1), and Thielavia terrestris NRRL 8126 (WO 2009/042846).
[0316] Examples of feruloyl esterases (ferulic acid esterases)
useful in the processes of the present invention include, but are
not limited to, feruloyl esterases form Humicola insolens DSM 1800
(WO 2009/076122), Neosartorya fischeri (UniProt Accession number
A1D9T4), Neurospora crassa (UniProt accession number Q9HGR3),
Penicillium aurantiogriseum (WO 2009/127729), and Thielavia
terrestris (WO 2010/053838 and WO 2010/065448).
[0317] Examples of arabinofuranosidases useful in the processes of
the present invention include, but are not limited to,
arabinofuranosidases from Aspergillus niger (GeneSeqP accession
number AAR94170), Humicola insolens DSM 1800 (WO 2006/114094 and WO
2009/073383), and M. giganteus (WO 2006/114094).
[0318] Examples of alpha-glucuronidases useful in the processes of
the present invention include, but are not limited to,
alpha-glucuronidases from Aspergillus clavatus (UniProt accession
number alcc12), Aspergillus fumigatus (SwissProt accession number
Q4WW45), Aspergillus niger (Uniprot accession number Q96WX9),
Aspergillus terreus (SwissProt accession number QOCJ P9), Humicola
insolens (WO 2010/014706), Penicillium aurantiogriseum (WO
2009/068565), Talaromyces emersonii (UniProt accession number
Q8X211), and Trichoderma reesei (Uniprot accession number
Q99024).
[0319] The polypeptides having enzyme activity used in the
processes of the present invention may be produced by fermentation
of the above-noted microbial strains on a nutrient medium
containing suitable carbon and nitrogen sources and inorganic
salts, using procedures known in the art (see, e.g., Bennett, J. W.
and LaSure, L. (eds.), More Gene Manipulations in Fungi, Academic
Press, C A, 1991). Suitable media are available from commercial
suppliers or may be prepared according to published compositions
(e.g., in catalogues of the American Type Culture Collection).
Temperature ranges and other conditions suitable for growth and
enzyme production are known in the art (see, e.g., Bailey, J. E.,
and Ollis, D. F., Biochemical Engineering Fundamentals, McGraw-Hill
Book Company, N Y, 1986).
[0320] The fermentation can be any method of cultivation of a cell
resulting in the expression or isolation of an enzyme or protein.
Fermentation may, therefore, be understood as comprising shake
flask cultivation, or small- or large-scale fermentation (including
continuous, batch, fed-batch, or solid state fermentations) in
laboratory or industrial fermentors performed in a suitable medium
and under conditions allowing the enzyme to be expressed or
isolated. The resulting enzymes produced by the methods described
above may be recovered from the fermentation medium and purified by
conventional procedures.
[0321] Fermentation.
[0322] The fermentable sugars obtained from the hydrolyzed
cellulosic material or xylan-containing material can be fermented
by one or more (e.g., several) fermenting microorganisms capable of
fermenting the sugars directly or indirectly into a desired
fermentation product. "Fermentation" or "fermentation process"
refers to any fermentation process or any process comprising a
fermentation step. Fermentation processes also include fermentation
processes used in the consumable alcohol industry (e.g., beer and
wine), dairy industry (e.g., fermented dairy products), leather
industry, and tobacco industry. The fermentation conditions depend
on the desired fermentation product and fermenting organism and can
easily be determined by one skilled in the art.
[0323] In the fermentation step, sugars, released from the
cellulosic material or xylan-containing material as a result of the
pretreatment and enzymatic hydrolysis steps, are fermented to a
product, e.g., ethanol, by a fermenting organism, such as yeast.
Hydrolysis (saccharification) and fermentation can be separate or
simultaneous, as described herein.
[0324] Any suitable hydrolyzed cellulosic material or
xylan-containing material can be used in the fermentation step in
practicing the present invention. The material is generally
selected based on the desired fermentation product, i.e., the
substance to be obtained from the fermentation, and the process
employed, as is well known in the art.
[0325] The term "fermentation medium" is understood herein to refer
to a medium before the fermenting microorganism(s) is(are) added,
such as, a medium resulting from a saccharification process, as
well as a medium used in a simultaneous saccharification and
fermentation process (SSF).
[0326] "Fermenting microorganism" refers to any microorganism,
including bacterial and fungal organisms, suitable for use in a
desired fermentation process to produce a fermentation product. The
fermenting organism can be hexose and/or pentose fermenting
organisms, or a combination thereof. Both hexose and pentose
fermenting organisms are well known in the art. Suitable fermenting
microorganisms are able to ferment, i.e., convert, sugars, such as
glucose, xylose, xylulose, arabinose, maltose, mannose, galactose,
and/or oligosaccharides, directly or indirectly into the desired
fermentation product. Examples of bacterial and fungal fermenting
organisms producing ethanol are described by Lin et al., 2006,
Appl. Microbiol. Biotechnol. 69: 627-642.
[0327] Examples of fermenting microorganisms that can ferment
hexose sugars include bacterial and fungal organisms, such as
yeast. Preferred yeast includes strains of Candida, Kluyveromyces,
and Saccharomyces, e.g., Candida sonorensis, Kluyveromyces
marxianus, and Saccharomyces cerevisiae.
[0328] Examples of fermenting organisms that can ferment pentose
sugars in their native state include bacterial and fungal
organisms, such as some yeast. Preferred xylose fermenting yeast
include strains of Candida, preferably C. sheatae or C. sonorensis;
and strains of Pichia, preferably P. stipitis, such as P. stipitis
CBS 5773. Preferred pentose fermenting yeast include strains of
Pachysolen, preferably P. tannophilus. Organisms not capable of
fermenting pentose sugars, such as xylose and arabinose, may be
genetically modified to do so by methods known in the art.
[0329] Examples of bacteria that can efficiently ferment hexose and
pentose to ethanol include, for example, Bacillus coagulans,
Clostridium acetobutylicum, Clostridium thermocellum, Clostridium
phytofermentans, Geobacillus sp., Thermoanaerobacter
saccharolyticum, and Zymomonas mobilis (Philippidis, 1996,
supra).
[0330] Other fermenting organisms include strains of Bacillus, such
as Bacillus coagulans; Candida, such as C. sonorensis, C.
methanosorbosa, C. diddensiae, C. parapsilosis, C. naedodendra, C.
blankii, C. entomophilia, C. brassicae, C. pseudotropicalis, C.
boidinii, C. utilis, and C. scehatae; Clostridium, such as C.
acetobutylicum, C. thermocellum, and C. phytofermentans; E. coli,
especially E. coli strains that have been genetically modified to
improve the yield of ethanol; Geobacillus sp.; Hansenula, such as
Hansenula anomala; Klebsiella, such as K. oxytoca; Kluyveromyces,
such as K. marxianus, K. lactis, K thermotolerans, and K. fragilis;
Schizosaccharomyces, such as S. pombe; Thermoanaerobacter, such as
Thermoanaerobacter saccharolyticum; and Zymomonas, such as
Zymomonas mobilis.
[0331] In a preferred aspect, the yeast is a Bretannomyces. In a
more preferred aspect, the yeast is Bretannomyces clausenii. In
another preferred aspect, the yeast is a Candida. In another more
preferred aspect, the yeast is Candida sonorensis. In another more
preferred aspect, the yeast is Candida boidinii. In another more
preferred aspect, the yeast is Candida blankii. In another more
preferred aspect, the yeast is Candida brassicae. In another more
preferred aspect, the yeast is Candida diddensii. In another more
preferred aspect, the yeast is Candida entomophiliia. In another
more preferred aspect, the yeast is Candida pseudotropicalis. In
another more preferred aspect, the yeast is Candida scehatae. In
another more preferred aspect, the yeast is Candida utilis. In
another preferred aspect, the yeast is a Clavispora. In another
more preferred aspect, the yeast is Clavispora lusitaniae. In
another more preferred aspect, the yeast is Clavispora opuntiae. In
another preferred aspect, the yeast is a Kluyveromyces. In another
more preferred aspect, the yeast is Kluyveromyces fragilis. In
another more preferred aspect, the yeast is Kluyveromyces
marxianus. In another more preferred aspect, the yeast is
Kluyveromyces thermotolerans. In another preferred aspect, the
yeast is a Pachysolen. In another more preferred aspect, the yeast
is Pachysolen tannophilus. In another preferred aspect, the yeast
is a Pichia. In another more preferred aspect, the yeast is a
Pichia stipitis. In another preferred aspect, the yeast is a
Saccharomyces spp. In a more preferred aspect, the yeast is
Saccharomyces cerevisiae. In another more preferred aspect, the
yeast is Saccharomyces distaticus. In another more preferred
aspect, the yeast is Saccharomyces uvarum.
[0332] In a preferred aspect, the bacterium is a Bacillus. In a
more preferred aspect, the bacterium is Bacillus coagulans. In
another preferred aspect, the bacterium is a Clostridium. In
another more preferred aspect, the bacterium is Clostridium
acetobutylicum. In another more preferred aspect, the bacterium is
Clostridium phytofermentans. In another more preferred aspect, the
bacterium is Clostridium thermocellum. In another more preferred
aspect, the bacterium is Geobacillus sp. In another more preferred
aspect, the bacterium is a Thermoanaerobacter. In another more
preferred aspect, the bacterium is Thermoanaerobacter
saccharolyticum. In another preferred aspect, the bacterium is a
Zymomonas. In another more preferred aspect, the bacterium is
Zymomonas mobilis.
[0333] Commercially available yeast suitable for ethanol production
include, e.g., BIOFERM.TM. AFT and XR (NABC--North American
Bioproducts Corporation, GA, USA), ETHANOL RED.TM. yeast
(Fermentis/Lesaffre, USA), FALI.TM. (Fleischmann's Yeast, USA),
FERMIOL.TM. (DSM Specialties), GERT STRAND.TM. (Gert Strand AB,
Sweden), and SUPERSTART.TM. and THERMOSACC.TM. fresh yeast (Ethanol
Technology, WI, USA).
[0334] In a preferred aspect, the fermenting microorganism has been
genetically modified to provide the ability to ferment pentose
sugars, such as xylose utilizing, arabinose utilizing, and xylose
and arabinose co-utilizing microorganisms.
[0335] The cloning of heterologous genes into various fermenting
microorganisms has led to the construction of organisms capable of
converting hexoses and pentoses to ethanol (co-fermentation) (Chen
and Ho, 1993, Cloning and improving the expression of Pichia
stipitis xylose reductase gene in Saccharomyces cerevisiae, Appl.
Biochem. Biotechnol. 39-40: 135-147; Ho et al., 1998, Genetically
engineered Saccharomyces yeast capable of effectively cofermenting
glucose and xylose, Appl. Environ. Microbiol. 64: 1852-1859; Kotter
and Ciriacy, 1993, Xylose fermentation by Saccharomyces cerevisiae,
Appl. Microbiol. Biotechnol. 38: 776-783; Walfridsson et al., 1995,
Xylose-metabolizing Saccharomyces cerevisiae strains overexpressing
the TKL1 and TALI genes encoding the pentose phosphate pathway
enzymes transketolase and transaldolase, Appl. Environ. Microbiol.
61: 4184-4190; Kuyper et al., 2004, Minimal metabolic engineering
of Saccharomyces cerevisiae for efficient anaerobic xylose
fermentation: a proof of principle, FEMS Yeast Research 4: 655-664;
Beall et al., 1991, Parametric studies of ethanol production from
xylose and other sugars by recombinant Escherichia coli, Biotech.
Bioeng. 38: 296-303; Ingram et al., 1998, Metabolic engineering of
bacteria for ethanol production, Biotechnol. Bioeng. 58: 204-214;
Zhang et al., 1995, Metabolic engineering of a pentose metabolism
pathway in ethanologenic Zymomonas mobilis, Science 267: 240-243;
Deanda et al., 1996, Development of an arabinose-fermenting
Zymomonas mobilis strain by metabolic pathway engineering, Appl.
Environ. Microbiol. 62: 4465-4470; WO 2003/062430, xylose
isomerase).
[0336] In a preferred aspect, the genetically modified fermenting
microorganism is Candida sonorensis. In another preferred aspect,
the genetically modified fermenting microorganism is Escherichia
coli. In another preferred aspect, the genetically modified
fermenting microorganism is Klebsiella oxytoca. In another
preferred aspect, the genetically modified fermenting microorganism
is Kluyveromyces marxianus. In another preferred aspect, the
genetically modified fermenting microorganism is Saccharomyces
cerevisiae. In another preferred aspect, the genetically modified
fermenting microorganism is Zymomonas mobilis.
[0337] It is well known in the art that the organisms described
above can also be used to produce other substances, as described
herein.
[0338] The fermenting microorganism is typically added to the
degraded cellulosic material or xylan-containing material or
hydrolysate and the fermentation is performed for about 8 to about
96 hours, e.g., about 24 to about 60 hours. The temperature is
typically between about 26.degree. C. to about 60.degree. C., e.g.,
about 32.degree. C. or 50.degree. C., and about pH 3 to about pH 8,
e.g., pH 4-5, 6, or 7.
[0339] In one aspect, the yeast and/or another microorganism are
applied to the degraded cellulosic material or xylan-containing
material and the fermentation is performed for about 12 to about 96
hours, such as typically 24-60 hours. In another aspect, the
temperature is preferably between about 20.degree. C. to about
60.degree. C., e.g., about 25.degree. C. to about 50.degree. C.,
about 32.degree. C. to about 50.degree. C., or about 32.degree. C.
to about 50.degree. C., and the pH is generally from about pH 3 to
about pH 7, e.g., about pH 4 to about pH 7. However, some
fermenting organisms, e.g., bacteria, have higher fermentation
temperature optima. Yeast or another microorganism is preferably
applied in amounts of approximately 10.sup.5 to 10.sup.12,
preferably from approximately 10.sup.7 to 10.sup.10, especially
approximately 2.times.10.sup.8 viable cell count per ml of
fermentation broth. Further guidance in respect of using yeast for
fermentation can be found in, e.g., "The Alcohol Textbook" (Editors
K. Jacques, T. P. Lyons and D. R. Kelsall, Nottingham University
Press, United Kingdom 1999), which is hereby incorporated by
reference.
[0340] For ethanol production, following the fermentation the
fermented slurry is distilled to extract the ethanol. The ethanol
obtained according to the processes of the invention can be used
as, e.g., fuel ethanol, drinking ethanol, i.e., potable neutral
spirits, or industrial ethanol.
[0341] A fermentation stimulator can be used in combination with
any of the processes described herein to further improve the
fermentation process, and in particular, the performance of the
fermenting microorganism, such as, rate enhancement and ethanol
yield. A "fermentation stimulator" refers to stimulators for growth
of the fermenting microorganisms, in particular, yeast. Preferred
fermentation stimulators for growth include vitamins and minerals.
Examples of vitamins include multivitamins, biotin, pantothenate,
nicotinic acid, meso-inositol, thiamine, pyridoxine,
para-aminobenzoic acid, folic acid, riboflavin, and Vitamins A, B,
C, D, and E. See, for example, Alfenore et al., Improving ethanol
production and viability of Saccharomyces cerevisiae by a vitamin
feeding strategy during fed-batch process, Springer-Verlag (2002),
which is hereby incorporated by reference. Examples of minerals
include minerals and mineral salts that can supply nutrients
comprising P, K, Mg, S, Ca, Fe, Zn, Mn, and Cu.
[0342] Fermentation Products:
[0343] A fermentation product can be any substance derived from the
fermentation. The fermentation product can be, without limitation,
an alcohol (e.g., arabinitol, n-butanol, isobutanol, ethanol,
glycerol, methanol, ethylene glycol, 1,3-propanediol [propylene
glycol], butanediol, glycerin, sorbitol, and xylitol); an alkane
(e.g., pentane, hexane, heptane, octane, nonane, decane, undecane,
and dodecane), a cycloalkane (e.g., cyclopentane, cyclohexane,
cycloheptane, and cyclooctane), an alkene (e.g. pentene, hexene,
heptene, and octene); an amino acid (e.g., aspartic acid, glutamic
acid, glycine, lysine, serine, and threonine); a gas (e.g.,
methane, hydrogen (H.sub.2), carbon dioxide (CO.sub.2), and carbon
monoxide (CO)); isoprene; a ketone (e.g., acetone); an organic acid
(e.g., acetic acid, acetonic acid, adipic acid, ascorbic acid,
citric acid, 2,5-diketo-D-gluconic acid, formic acid, fumaric acid,
glucaric acid, gluconic acid, glucuronic acid, glutaric acid,
3-hydroxypropionic acid, itaconic acid, lactic acid, malic acid,
malonic acid, oxalic acid, oxaloacetic acid, propionic acid,
succinic acid, and xylonic acid); and polyketide. The fermentation
product can also be protein as a high value product.
[0344] In a preferred aspect, the fermentation product is an
alcohol. It will be understood that the term "alcohol" encompasses
a substance that contains one or more hydroxyl moieties. In a more
preferred aspect, the alcohol is n-butanol. In another more
preferred aspect, the alcohol is isobutanol. In another more
preferred aspect, the alcohol is ethanol. In another more preferred
aspect, the alcohol is methanol. In another more preferred aspect,
the alcohol is arabinitol. In another more preferred aspect, the
alcohol is butanediol. In another more preferred aspect, the
alcohol is ethylene glycol. In another more preferred aspect, the
alcohol is glycerin. In another more preferred aspect, the alcohol
is glycerol. In another more preferred aspect, the alcohol is
1,3-propanediol. In another more preferred aspect, the alcohol is
sorbitol. In another more preferred aspect, the alcohol is xylitol.
See, for example, Gong, C. S., Cao, N. J., Du, J., and Tsao, G. T.,
1999, Ethanol production from renewable resources, in Advances in
Biochemical Engineering/Biotechnology, Scheper, T., ed.,
Springer-Verlag Berlin Heidelberg, Germany, 65: 207-241; Silveira,
M. M., and Jonas, R., 2002, The biotechnological production of
sorbitol, Appl. Microbiol. Biotechnol. 59: 400-408; Nigam, P., and
Singh, D., 1995, Processes for fermentative production of
xylitol--a sugar substitute, Process Biochemistry 30 (2): 117-124;
Ezeji, T. C., Qureshi, N. and Blaschek, H. P., 2003, Production of
acetone, butanol and ethanol by Clostridium beijerinckii BA101 and
in situ recovery by gas stripping, World Journal of Microbiology
and Biotechnology 19 (6): 595-603.
[0345] In another preferred aspect, the fermentation product is an
alkane. The alkane can be an unbranched or a branched alkane. In
another more preferred aspect, the alkane is pentane. In another
more preferred aspect, the alkane is hexane. In another more
preferred aspect, the alkane is heptane. In another more preferred
aspect, the alkane is octane. In another more preferred aspect, the
alkane is nonane. In another more preferred aspect, the alkane is
decane. In another more preferred aspect, the alkane is undecane.
In another more preferred aspect, the alkane is dodecane.
[0346] In another preferred aspect, the fermentation product is a
cycloalkane. In another more preferred aspect, the cycloalkane is
cyclopentane. In another more preferred aspect, the cycloalkane is
cyclohexane. In another more preferred aspect, the cycloalkane is
cycloheptane. In another more preferred aspect, the cycloalkane is
cyclooctane.
[0347] In another preferred aspect, the fermentation product is an
alkene. The alkene can be an unbranched or a branched alkene. In
another more preferred aspect, the alkene is pentene. In another
more preferred aspect, the alkene is hexene. In another more
preferred aspect, the alkene is heptene. In another more preferred
aspect, the alkene is octene.
[0348] In another preferred aspect, the fermentation product is an
amino acid. In another more preferred aspect, the organic acid is
aspartic acid. In another more preferred aspect, the amino acid is
glutamic acid. In another more preferred aspect, the amino acid is
glycine. In another more preferred aspect, the amino acid is
lysine. In another more preferred aspect, the amino acid is serine.
In another more preferred aspect, the amino acid is threonine. See,
for example, Richard, A., and Margaritis, A., 2004, Empirical
modeling of batch fermentation kinetics for poly(glutamic acid)
production and other microbial biopolymers, Biotechnology and
Bioengineering 87 (4): 501-515.
[0349] In another preferred aspect, the fermentation product is a
gas. In another more preferred aspect, the gas is methane. In
another more preferred aspect, the gas is H.sub.2. In another more
preferred aspect, the gas is CO.sub.2. In another more preferred
aspect, the gas is CO. See, for example, Kataoka, N., A. Miya, and
K. Kiriyama, 1997, Studies on hydrogen production by continuous
culture system of hydrogen-producing anaerobic bacteria, Water
Science and Technology 36 (6-7): 41-47; and Gunaseelan V. N. in
Biomass and Bioenergy, Vol. 13 (1-2), pp. 83-114, 1997, Anaerobic
digestion of biomass for methane production: A review.
[0350] In another preferred aspect, the fermentation product is
isoprene.
[0351] In another preferred aspect, the fermentation product is a
ketone. It will be understood that the term "ketone" encompasses a
substance that contains one or more ketone moieties. In another
more preferred aspect, the ketone is acetone. See, for example,
Qureshi and Blaschek, 2003, supra.
[0352] In another preferred aspect, the fermentation product is an
organic acid. In another more preferred aspect, the organic acid is
acetic acid. In another more preferred aspect, the organic acid is
acetonic acid. In another more preferred aspect, the organic acid
is adipic acid. In another more preferred aspect, the organic acid
is ascorbic acid. In another more preferred aspect, the organic
acid is citric acid. In another more preferred aspect, the organic
acid is 2,5-diketo-D-gluconic acid. In another more preferred
aspect, the organic acid is formic acid. In another more preferred
aspect, the organic acid is fumaric acid. In another more preferred
aspect, the organic acid is glucaric acid. In another more
preferred aspect, the organic acid is gluconic acid. In another
more preferred aspect, the organic acid is glucuronic acid. In
another more preferred aspect, the organic acid is glutaric acid.
In another preferred aspect, the organic acid is 3-hydroxypropionic
acid. In another more preferred aspect, the organic acid is
itaconic acid. In another more preferred aspect, the organic acid
is lactic acid. In another more preferred aspect, the organic acid
is malic acid. In another more preferred aspect, the organic acid
is malonic acid. In another more preferred aspect, the organic acid
is oxalic acid. In another more preferred aspect, the organic acid
is propionic acid. In another more preferred aspect, the organic
acid is succinic acid. In another more preferred aspect, the
organic acid is xylonic acid. See, for example, Chen, R., and Lee,
Y. Y., 1997, Membrane-mediated extractive fermentation for lactic
acid production from cellulosic biomass, Appl. Biochem. Biotechnol.
63-65: 435-448.
[0353] In another preferred aspect, the fermentation product is
polyketide.
[0354] Recovery.
[0355] The fermentation product(s) can be optionally recovered from
the fermentation medium using any method known in the art
including, but not limited to, chromatography, electrophoretic
procedures, differential solubility, distillation, or extraction.
For example, alcohol is separated from the fermented cellulosic
material or xylan-containing material and purified by conventional
methods of distillation. Ethanol with a purity of up to about 96
vol. % can be obtained, which can be used as, for example, fuel
ethanol, drinking ethanol, i.e., potable neutral spirits, or
industrial ethanol.
Signal Peptide
[0356] The present invention also relates to an isolated
polynucleotide encoding a signal peptide comprising or consisting
of amino acids 1 to 18 of SEQ ID NO: 2, amino acids 1 to 20 of SEQ
ID NO: 4, amino acids 1 to 23 of SEQ ID NO: 6, amino acids 1 to 19
of SEQ ID NO: 8, amino acids 1 to 19 of SEQ ID NO: 10, or amino
acids 1 to 22 of SEQ ID NO: 12. The polynucleotide may further
comprise a gene encoding a protein, which is operably linked to the
signal peptide. The protein is preferably foreign to the signal
peptide. In one aspect, the polynucleotide encoding the signal
peptide is nucleotides 1 to 54 of SEQ ID NO: 1. In another aspect,
the polynucleotide encoding the signal peptide is nucleotides 1 to
60 of SEQ ID NO: 3. In another aspect, the polynucleotide encoding
the signal peptide is nucleotides 1 to 69 of SEQ ID NO: 5. In
another aspect, the polynucleotide encoding the signal peptide is
nucleotides 1 to 57 of SEQ ID NO: 7. In another aspect, the
polynucleotide encoding the signal peptide is nucleotides 1 to 57
of SEQ ID NO: 9. In another aspect, the polynucleotide encoding the
signal peptide is nucleotides 1 to 66 of SEQ ID NO: 11.
[0357] The present invention also relates to nucleic acid
constructs, expression vectors and recombinant host cells
comprising such polynucleotides.
[0358] The present invention also relates to methods of producing a
protein, comprising (a) cultivating a recombinant host cell
comprising such polynucleotide; and (b) recovering the protein.
[0359] The protein may be native or heterologous to a host cell.
The term "protein" is not meant herein to refer to a specific
length of the encoded product and, therefore, encompasses peptides,
oligopeptides, and polypeptides. The term "protein" also
encompasses two or more polypeptides combined to form the encoded
product. The proteins also include hybrid polypeptides and fused
polypeptides.
[0360] Preferably, the protein is a hormone, enzyme, receptor or
portion thereof, antibody or portion thereof, or reporter. For
example, the protein may be a hydrolase, isomerase, ligase, lyase,
oxidoreductase, or transferase, e.g., an aminopeptidase, amylase,
carbohydrase, carboxypeptidase, catalase, cellobiohydrolase,
cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase,
deoxyribonuclease, endoglucanase, esterase, alpha-galactosidase,
beta-galactosidase, glucoamylase, alpha-glucosidase,
beta-glucosidase, invertase, laccase, lipase, mannosidase,
mutanase, oxidase, pectinolytic enzyme, peroxidase, phytase,
polyphenoloxidase, proteolytic enzyme, ribonuclease,
transglutaminase, xylanase, or beta-xylosidase.
[0361] The gene may be obtained from any prokaryotic, eukaryotic,
or other source.
[0362] The invention described and claimed herein is not to be
limited in scope by the specific aspects herein disclosed, since
these aspects are intended as illustrations of several aspects of
the invention. Any equivalent aspects are intended to be within the
scope of this invention. Indeed, various modifications of the
invention in addition to those shown and described herein will
become apparent to those skilled in the art from the foregoing
description. Such modifications are also intended to fall within
the scope of the appended claims. In the case of conflict, the
present disclosure including definitions will control.
Sequence CWU 1
1
1211518DNATrametes
versicolorCDS(1)..(258)sig_peptide(1)..(54)mat_peptide(55)..()Intron(259)-
..(319)CDS(320)..(410)Intron(411)..(464)CDS(465)..(591)Intron(592)..(645)C-
DS(646)..(651)Intron(652)..(701)CDS(702)..(715)Intron(716)..(778)CDS(779).-
.(925)Intron(926)..(978)CDS(979)..(1283)Intron(1284)..(1345)CDS(1346)..(14-
02)Intron(1403)..(1464)CDS(1465)..(1515) 1atg cac ttc tca ctc ctc
gca gcc ttc atc gcg ctg gct ccg gcc gcg 48Met His Phe Ser Leu Leu
Ala Ala Phe Ile Ala Leu Ala Pro Ala Ala -15 -10 -5 ctc gcg atc ccc
gcg acc ccc gtg gtc gat gcg agt ctc ccc ggc tcg 96Leu Ala Ile Pro
Ala Thr Pro Val Val Asp Ala Ser Leu Pro Gly Ser -1 1 5 10 acc gcg
aac gtg gcg ggt ctg cac gcc gtc gcg aag gcg gcg ggc aag 144Thr Ala
Asn Val Ala Gly Leu His Ala Val Ala Lys Ala Ala Gly Lys 15 20 25 30
ctc tac ttg ggc act gcg acg gac aat aac gag ctt acc aac acg cag
192Leu Tyr Leu Gly Thr Ala Thr Asp Asn Asn Glu Leu Thr Asn Thr Gln
35 40 45 tac acc gcc atc ctc gag gct ccg aac atg ttc ggc cag atc
acc gcc 240Tyr Thr Ala Ile Leu Glu Ala Pro Asn Met Phe Gly Gln Ile
Thr Ala 50 55 60 gag aac acc atg aaa tgg gtcagttgcg ccttgtcgcg
atgttccgag 288Glu Asn Thr Met Lys Trp 65 cacgttccgc aagctaacga
tttgatgcta g gac gcg acc gag ccc cag cag 340 Asp Ala Thr Glu Pro
Gln Gln 70 75 aac gtg ttc acg ttc acg cag ggc gac cag atc gcg aac
ctg gcg aag 388Asn Val Phe Thr Phe Thr Gln Gly Asp Gln Ile Ala Asn
Leu Ala Lys 80 85 90 tcc cac ggg atg ctg ctg cgc g gtaacgacag
tcttgaaggg gccgaaaatg 440Ser His Gly Met Leu Leu Arg 95 tggaagaact
gaatgttttc gcag gt cac aac tgc gtc tgg cac caa cag 490 Gly His Asn
Cys Val Trp His Gln Gln 100 105 ctc ccg agc tgg gta act gcc ggg aac
ttc aat gca cag cag ctt acg 538Leu Pro Ser Trp Val Thr Ala Gly Asn
Phe Asn Ala Gln Gln Leu Thr 110 115 120 cag atc atc caa aac cac tgc
ggc acg gtc gtc gga cac tac aag gga 586Gln Ile Ile Gln Asn His Cys
Gly Thr Val Val Gly His Tyr Lys Gly 125 130 135 caa gt gtgagtgttg
gccatatccg ccgacgtgta tcgtgtgctg accgtgtttt 641Gln Val 140 atag t
tgt ac gtgacgcttg ttgcttgatc gtatgaatcc acttagctga 691 Cys Thr
ctcgacgcag t gag ctg gga tgt a gtaaacggta acggacattc cttctctttg 745
Glu Leu Gly Cys 145 ctacacacaa ctcggactca cactcggctg cag ag cct ctc
aac gac gac ggc 798 Lys Pro Leu Asn Asp Asp Gly 150 tcg ttc cgc cag
gac gtg ttc ttc aac act ctc ggc tcg ggc tac atc 846Ser Phe Arg Gln
Asp Val Phe Phe Asn Thr Leu Gly Ser Gly Tyr Ile 155 160 165 170 gcg
acg gcg ctc cgc gct gcc agg gcc gca gac cct gcg gcg aag ctg 894Ala
Thr Ala Leu Arg Ala Ala Arg Ala Ala Asp Pro Ala Ala Lys Leu 175 180
185 tac atc aac gag ttt aac gtc gag ggc cta g gttcgtccca ccgtatcccc
945Tyr Ile Asn Glu Phe Asn Val Glu Gly Leu 190 195 gctcattgca
tcgtctgagc ccgaatcttc tag gc gca aag tcg acc gcc ttg 998 Gly Ala
Lys Ser Thr Ala Leu 200 aag aac ctc gtc act tcg ctg aag cag cag ggc
gtc ccg atc gac ggc 1046Lys Asn Leu Val Thr Ser Leu Lys Gln Gln Gly
Val Pro Ile Asp Gly 205 210 215 gtc ggt ttc cag tgc cac ttc atc gtc
ggc cag gtc ccc acg acg ctc 1094Val Gly Phe Gln Cys His Phe Ile Val
Gly Gln Val Pro Thr Thr Leu 220 225 230 235 atc cag agc atg cag cag
ttc act gcg ctc ggc ctc gag gtg gcc atc 1142Ile Gln Ser Met Gln Gln
Phe Thr Ala Leu Gly Leu Glu Val Ala Ile 240 245 250 acg gag ctc gac
atc cgc atg acg ctc cct gaa act gcg gcg ctg ctc 1190Thr Glu Leu Asp
Ile Arg Met Thr Leu Pro Glu Thr Ala Ala Leu Leu 255 260 265 gag cag
cag aag cag gac ttc cag act gtt atc cac gcg tgt aag tcc 1238Glu Gln
Gln Lys Gln Asp Phe Gln Thr Val Ile His Ala Cys Lys Ser 270 275 280
gtg gcg ggc tgt gtc ggc gtc act gtg tgg gac ttc acc gat aag 1283Val
Ala Gly Cys Val Gly Val Thr Val Trp Asp Phe Thr Asp Lys 285 290 295
gtatgtcgtt gtcccgcccc ggcgagatgg ttggattagc gtgctcaccg gtattatacc
1343ag ttc tcc ttc gtg ccg agc acg ttc ccg ggt cag ggt gct gcc act
1390 Phe Ser Phe Val Pro Ser Thr Phe Pro Gly Gln Gly Ala Ala Thr
300 305 310 cct tgg gat cag gtacgtcccg ccgaaccttg ggccttacaa
gctccgggag 1442Pro Trp Asp Gln 315 agggctaact gcggatgcgc ag aac ctg
gtg aag aag ccg gca ttt gat ggc 1494 Asn Leu Val Lys Lys Pro Ala
Phe Asp Gly 320 325 atc gtc gcc gga ttc cag cag tga 1518Ile Val Ala
Gly Phe Gln Gln 330 2352PRTTrametes versicolor 2Met His Phe Ser Leu
Leu Ala Ala Phe Ile Ala Leu Ala Pro Ala Ala -15 -10 -5 Leu Ala Ile
Pro Ala Thr Pro Val Val Asp Ala Ser Leu Pro Gly Ser -1 1 5 10 Thr
Ala Asn Val Ala Gly Leu His Ala Val Ala Lys Ala Ala Gly Lys 15 20
25 30 Leu Tyr Leu Gly Thr Ala Thr Asp Asn Asn Glu Leu Thr Asn Thr
Gln 35 40 45 Tyr Thr Ala Ile Leu Glu Ala Pro Asn Met Phe Gly Gln
Ile Thr Ala 50 55 60 Glu Asn Thr Met Lys Trp Asp Ala Thr Glu Pro
Gln Gln Asn Val Phe 65 70 75 Thr Phe Thr Gln Gly Asp Gln Ile Ala
Asn Leu Ala Lys Ser His Gly 80 85 90 Met Leu Leu Arg Gly His Asn
Cys Val Trp His Gln Gln Leu Pro Ser 95 100 105 110 Trp Val Thr Ala
Gly Asn Phe Asn Ala Gln Gln Leu Thr Gln Ile Ile 115 120 125 Gln Asn
His Cys Gly Thr Val Val Gly His Tyr Lys Gly Gln Val Cys 130 135 140
Thr Glu Leu Gly Cys Lys Pro Leu Asn Asp Asp Gly Ser Phe Arg Gln 145
150 155 Asp Val Phe Phe Asn Thr Leu Gly Ser Gly Tyr Ile Ala Thr Ala
Leu 160 165 170 Arg Ala Ala Arg Ala Ala Asp Pro Ala Ala Lys Leu Tyr
Ile Asn Glu 175 180 185 190 Phe Asn Val Glu Gly Leu Gly Ala Lys Ser
Thr Ala Leu Lys Asn Leu 195 200 205 Val Thr Ser Leu Lys Gln Gln Gly
Val Pro Ile Asp Gly Val Gly Phe 210 215 220 Gln Cys His Phe Ile Val
Gly Gln Val Pro Thr Thr Leu Ile Gln Ser 225 230 235 Met Gln Gln Phe
Thr Ala Leu Gly Leu Glu Val Ala Ile Thr Glu Leu 240 245 250 Asp Ile
Arg Met Thr Leu Pro Glu Thr Ala Ala Leu Leu Glu Gln Gln 255 260 265
270 Lys Gln Asp Phe Gln Thr Val Ile His Ala Cys Lys Ser Val Ala Gly
275 280 285 Cys Val Gly Val Thr Val Trp Asp Phe Thr Asp Lys Phe Ser
Phe Val 290 295 300 Pro Ser Thr Phe Pro Gly Gln Gly Ala Ala Thr Pro
Trp Asp Gln Asn 305 310 315 Leu Val Lys Lys Pro Ala Phe Asp Gly Ile
Val Ala Gly Phe Gln Gln 320 325 330 31464DNATrametes
versicolorCDS(1)..(249)sig_peptide(1)..(60)mat_peptide(61)..()Intron(250)-
..(306)CDS(307)..(524)Intron(525)..(648)CDS(649)..(665)Intron(666)..(720)C-
DS(721)..(867)Intron(868)..(929)CDS(930)..(1237)Intron(1238)..(1294)CDS(12-
95)..(1351)Intron(1352)..(1404)CDS(1405)..(1461) 3atg ctc tct ctg
tca aaa agc ctt ctt gcg ctc tct gtc ttg gtc cga 48Met Leu Ser Leu
Ser Lys Ser Leu Leu Ala Leu Ser Val Leu Val Arg -20 -15 -10 -5 ggt
gcg ctc gcc gta cct gcc agc gat gcg agt agc gct ctg ttc cca 96Gly
Ala Leu Ala Val Pro Ala Ser Asp Ala Ser Ser Ala Leu Phe Pro -1 1 5
10 ttg tcg ggg ctg aat ctg gcc gcc aag ggc gcg cgg aag ttg tac ctt
144Leu Ser Gly Leu Asn Leu Ala Ala Lys Gly Ala Arg Lys Leu Tyr Leu
15 20 25 ggc acg gca acg aac agc gag cag tgg aac gac acg acg tac
ttc aac 192Gly Thr Ala Thr Asn Ser Glu Gln Trp Asn Asp Thr Thr Tyr
Phe Asn 30 35 40 atc ctg aag aac aac gcc gag ttc ggg cag gta acg
ccc gcg aac gtc 240Ile Leu Lys Asn Asn Ala Glu Phe Gly Gln Val Thr
Pro Ala Asn Val 45 50 55 60 atg aaa tgg gtacgttgtc ggcgtcctct
tcgtactgac gatgttgagg 289Met Lys Trp ctaactttga cgcatag ttt gcg acg
gag cct gag gag ggc gtc ttc acg 339 Phe Ala Thr Glu Pro Glu Glu Gly
Val Phe Thr 65 70 ttc cag gac ggg gat atc atc gcg gac ttt acc aaa
aag acg ggg aag 387Phe Gln Asp Gly Asp Ile Ile Ala Asp Phe Thr Lys
Lys Thr Gly Lys 75 80 85 90 ctg ctg cgc gga cac aac tgc gtg tgg cac
aac cag ctc ccc gac tgg 435Leu Leu Arg Gly His Asn Cys Val Trp His
Asn Gln Leu Pro Asp Trp 95 100 105 cta gaa acc ggc acg ttc agt gcg
ccc gag ctc gca ttc att gtc tcg 483Leu Glu Thr Gly Thr Phe Ser Ala
Pro Glu Leu Ala Phe Ile Val Ser 110 115 120 cgg cac tgc ttc aac ctc
gtg aac cac tac caa ggc tat gt 524Arg His Cys Phe Asn Leu Val Asn
His Tyr Gln Gly Tyr Val 125 130 135 gtgagtgcaa ttcgttacct
gtgatcctgc tcaacgatct aaatcggtac acggcaggtg 584tacgtacaag
cgtgtcggtg atgtttcatt gaggctgatg gcttattttg gaaaattcag 644atag c
tgg gac gtc atc aat g gttcgtgcta cttgacttcc cggatgtgct 695 Trp Asp
Val Ile Asn 140 tgtttccgat ctcgaatttt gctag ag gct ttc aac gac gac
gga acc ttc 746 Glu Ala Phe Asn Asp Asp Gly Thr Phe 145 150 cgt tcg
gat atc ttc ttc gac acg ctc aac aca acc tac atc ccg ctc 794Arg Ser
Asp Ile Phe Phe Asp Thr Leu Asn Thr Thr Tyr Ile Pro Leu 155 160 165
gcc ctc tac gca gca cgc gcc gcg gac ccc aaa gcg aag ctc tac atc
842Ala Leu Tyr Ala Ala Arg Ala Ala Asp Pro Lys Ala Lys Leu Tyr Ile
170 175 180 aac gac ttc aac atc gaa ggc ata g gtacgccaca taacaccatc
887Asn Asp Phe Asn Ile Glu Gly Ile 185 190 tgcccgccgc aaagccctgc
cacccaacta ccctactcgc ag gt gcg aag tcc 940 Gly Ala Lys Ser gac gcg
ctc aag agt ctc atc aag gag ctc aag agc caa aac gtc ccc 988Asp Ala
Leu Lys Ser Leu Ile Lys Glu Leu Lys Ser Gln Asn Val Pro 195 200 205
210 atc gac ggc gtc ggg ctg cag tcg cac ttc gag gtc ggc ggc gtc ccg
1036Ile Asp Gly Val Gly Leu Gln Ser His Phe Glu Val Gly Gly Val Pro
215 220 225 ccc acg ctg cag cag aac atg gag gag ttc gtc gcg ctc ggg
ctc gag 1084Pro Thr Leu Gln Gln Asn Met Glu Glu Phe Val Ala Leu Gly
Leu Glu 230 235 240 gtc gcg atc acg gag ctc gac atc cgc ttc acc gcg
ctc ccg ccg acg 1132Val Ala Ile Thr Glu Leu Asp Ile Arg Phe Thr Ala
Leu Pro Pro Thr 245 250 255 cct gca ggc ctc gcg cag cag aag gcg gac
tac gag acc gtc gtc gcc 1180Pro Ala Gly Leu Ala Gln Gln Lys Ala Asp
Tyr Glu Thr Val Val Ala 260 265 270 gcg tgc aac gcg gtc ccg aag tgc
gtc ggg gtc acg ctg tgg gac ttc 1228Ala Cys Asn Ala Val Pro Lys Cys
Val Gly Val Thr Leu Trp Asp Phe 275 280 285 290 acg gac aag
gtgcgtctgc gagattgtgg tcgtgtgatg ggtgttgatg 1277Thr Asp Lys
ccggatgggc ggggtag tac tcg tgg atc ccg ggg acc ttc cct ggg cag 1327
Tyr Ser Trp Ile Pro Gly Thr Phe Pro Gly Gln 295 300 gga gat gcg tgt
ccc tgg acg gat gtacgttcct tagtctgtct cgtccgaagg 1381Gly Asp Ala
Cys Pro Trp Thr Asp 305 310 tgtgatctaa tgatgtacca cag gaa ttt gtg
aag agg cca gca tac gag ggc 1434 Glu Phe Val Lys Arg Pro Ala Tyr
Glu Gly 315 320 atc atc gag ggg ttc aag gcc cac cat tag 1464Ile Ile
Glu Gly Phe Lys Ala His His 325 330 4351PRTTrametes versicolor 4Met
Leu Ser Leu Ser Lys Ser Leu Leu Ala Leu Ser Val Leu Val Arg -20 -15
-10 -5 Gly Ala Leu Ala Val Pro Ala Ser Asp Ala Ser Ser Ala Leu Phe
Pro -1 1 5 10 Leu Ser Gly Leu Asn Leu Ala Ala Lys Gly Ala Arg Lys
Leu Tyr Leu 15 20 25 Gly Thr Ala Thr Asn Ser Glu Gln Trp Asn Asp
Thr Thr Tyr Phe Asn 30 35 40 Ile Leu Lys Asn Asn Ala Glu Phe Gly
Gln Val Thr Pro Ala Asn Val 45 50 55 60 Met Lys Trp Phe Ala Thr Glu
Pro Glu Glu Gly Val Phe Thr Phe Gln 65 70 75 Asp Gly Asp Ile Ile
Ala Asp Phe Thr Lys Lys Thr Gly Lys Leu Leu 80 85 90 Arg Gly His
Asn Cys Val Trp His Asn Gln Leu Pro Asp Trp Leu Glu 95 100 105 Thr
Gly Thr Phe Ser Ala Pro Glu Leu Ala Phe Ile Val Ser Arg His 110 115
120 Cys Phe Asn Leu Val Asn His Tyr Gln Gly Tyr Val Trp Asp Val Ile
125 130 135 140 Asn Glu Ala Phe Asn Asp Asp Gly Thr Phe Arg Ser Asp
Ile Phe Phe 145 150 155 Asp Thr Leu Asn Thr Thr Tyr Ile Pro Leu Ala
Leu Tyr Ala Ala Arg 160 165 170 Ala Ala Asp Pro Lys Ala Lys Leu Tyr
Ile Asn Asp Phe Asn Ile Glu 175 180 185 Gly Ile Gly Ala Lys Ser Asp
Ala Leu Lys Ser Leu Ile Lys Glu Leu 190 195 200 Lys Ser Gln Asn Val
Pro Ile Asp Gly Val Gly Leu Gln Ser His Phe 205 210 215 220 Glu Val
Gly Gly Val Pro Pro Thr Leu Gln Gln Asn Met Glu Glu Phe 225 230 235
Val Ala Leu Gly Leu Glu Val Ala Ile Thr Glu Leu Asp Ile Arg Phe 240
245 250 Thr Ala Leu Pro Pro Thr Pro Ala Gly Leu Ala Gln Gln Lys
Ala Asp 255 260 265 Tyr Glu Thr Val Val Ala Ala Cys Asn Ala Val Pro
Lys Cys Val Gly 270 275 280 Val Thr Leu Trp Asp Phe Thr Asp Lys Tyr
Ser Trp Ile Pro Gly Thr 285 290 295 300 Phe Pro Gly Gln Gly Asp Ala
Cys Pro Trp Thr Asp Glu Phe Val Lys 305 310 315 Arg Pro Ala Tyr Glu
Gly Ile Ile Glu Gly Phe Lys Ala His His 320 325 330 5 1533
DNATrametes
versicolorCDS(1)..(273)sig_peptide(1)..(69)mat_peptide(70)..()Intron(274)-
..(324)CDS(325)..(430)Intron(431)..(471)CDS(472)..(598)Intron(599)..(717)C-
DS(718)..(734)Intron(735)..(788)CDS(789)..(935)Intron(936)..(989)CDS(990).-
.(1297)Intron(1298)..(1352)CDS(1353)..(1400) 5atg atg acg aac ttc
cac cta gtc tcc tcg ttg atc gcg ctc gcg ttt 48Met Met Thr Asn Phe
His Leu Val Ser Ser Leu Ile Ala Leu Ala Phe -20 -15 -10 ctt tcg ctg
acc ggc tta gca tcc att ccg tcg aca cgc gct ctg gct 96Leu Ser Leu
Thr Gly Leu Ala Ser Ile Pro Ser Thr Arg Ala Leu Ala -5 -1 1 5 gga
aat tcc tcg agg atc aat tct cct tct ggc ttg aac ttg gcg gct 144Gly
Asn Ser Ser Arg Ile Asn Ser Pro Ser Gly Leu Asn Leu Ala Ala 10 15
20 25 ttg gaa gcc cgg aag ctg tac ttt ggt act gcg acc aac aac gtc
gag 192Leu Glu Ala Arg Lys Leu Tyr Phe Gly Thr Ala Thr Asn Asn Val
Glu 30 35 40 ctc aac gac acg gca tac ttc gac att ctc gat gat ttc
aaa atg ttc 240Leu Asn Asp Thr Ala Tyr Phe Asp Ile Leu Asp Asp Phe
Lys Met Phe 45 50 55 ggt caa att acg ccc gcc aaa ggc atg aaa tgg
gtccgttctc caacttctgc 293Gly Gln Ile Thr Pro Ala Lys Gly Met Lys
Trp 60 65 atcataaatc gctcgctgat tgttttggaa g atg gag acg gaa cct
gag cga 345 Met Glu Thr Glu Pro Glu Arg 70 75 ggc gtt ttc acc ttc
gcg cag gca gac caa atc gcg caa ctt gcc agc 393Gly Val Phe Thr Phe
Ala Gln Ala Asp Gln Ile Ala Gln Leu Ala Ser 80 85 90 gag ggc gga
aag ctg ttg aga ggc tcg tac tcg aaa g gtccctcgct 440Glu Gly Gly Lys
Leu Leu Arg Gly Ser Tyr Ser Lys 95 100 accccatacg tgctaacatt
ccgttctgca g gc cac aac tgc gta tgg tat 491 Gly His Asn Cys Val Trp
Tyr 105 110 aat gcg ctt ccc ggg tgg gtc aca aat acc acg tgg acg gcc
tcc gag 539Asn Ala Leu Pro Gly Trp Val Thr Asn Thr Thr Trp Thr Ala
Ser Glu 115 120 125 atg gcc gag gtc gta cag gag cat tgt ttc aac atc
gtc cgt tac tgg 587Met Ala Glu Val Val Gln Glu His Cys Phe Asn Ile
Val Arg Tyr Trp 130 135 140 caa gga caa gc gtgagtaccg atctctctca
ttaatatcgt gtctctcaat 638Gln Gly Gln Ala 145 ttttattccg agtagatgtg
agtatcaacg ccttccggag gaatcccgct gaacataggc 698cgtcctttta cacagacag
c tgg gac gtt att aac g gtgagttgct 744 Trp Asp Val Ile Asn 150
cgagattgaa ggcagctgcc cgtagcttac accatttccc gcag ag cca ttc aac 799
Glu Pro Phe Asn 155 gat gac gga acg tgg cgc gag acc atg tgg ttt aat
act ctc aac acg 847Asp Asp Gly Thr Trp Arg Glu Thr Met Trp Phe Asn
Thr Leu Asn Thr 160 165 170 agc tac att ccg ctc gcg ttg cac gct gcg
cgc gcg gcc gat cct cat 895Ser Tyr Ile Pro Leu Ala Leu His Ala Ala
Arg Ala Ala Asp Pro His 175 180 185 act aag ctg tac atc aat gag tac
aat atc acc gga aca g gtgcgtcgta 945Thr Lys Leu Tyr Ile Asn Glu Tyr
Asn Ile Thr Gly Thr 190 195 200 cgcctcacgc tcagactatg cctccttcat
cattcagtat acag gc ccg aag gcg 1000 Gly Pro Lys Ala acg tcc atg aag
aac ctc atc aaa gac ttg aag cgc gct ggt gtg ccc 1048Thr Ser Met Lys
Asn Leu Ile Lys Asp Leu Lys Arg Ala Gly Val Pro 205 210 215 220 gtt
cac ggc gtt gga gtt caa gcg cac gag acc gtc ggg gaa gtt ccg 1096Val
His Gly Val Gly Val Gln Ala His Glu Thr Val Gly Glu Val Pro 225 230
235 acc gac atc cgc aag aac ctc ggg gag ttc gtc gca ctc ggc gtc gag
1144Thr Asp Ile Arg Lys Asn Leu Gly Glu Phe Val Ala Leu Gly Val Glu
240 245 250 gtc gcg atc aca gaa ctc gac atc aag ttc aac acg ctt cct
cct gat 1192Val Ala Ile Thr Glu Leu Asp Ile Lys Phe Asn Thr Leu Pro
Pro Asp 255 260 265 gca gcc ggg ctc aaa caa cag aag cga gat tac gaa
gct att gtc tcg 1240Ala Ala Gly Leu Lys Gln Gln Lys Arg Asp Tyr Glu
Ala Ile Val Ser 270 275 280 gcg tgc gcg gag gta aaa gga tgt gtg ggc
gtg acg gtt tgg gac ttc 1288Ala Cys Ala Glu Val Lys Gly Cys Val Gly
Val Thr Val Trp Asp Phe 285 290 295 300 acg gac aag gtgggaaagt
attcgacttc acgaagcaat accaagtatt 1337Thr Asp Lys cacctttgcg tacag
tac tca tgg atc ccc gga acg ttc cct gga acc ggc 1388 Tyr Ser Trp
Ile Pro Gly Thr Phe Pro Gly Thr Gly 305 310 315 gat gct tgt cct
tgagacgatg tgagcgtgat gctattgcgt attgcttttt 1440Asp Ala Cys Pro
ctactgactg tctctcgtac ttctttgtct caggatttgc acaagaagcc ggcgtactat
1500ggaattttgg acgggtttgg gagatctcgc tga 15336342PRTTrametes
versicolor 6Met Met Thr Asn Phe His Leu Val Ser Ser Leu Ile Ala Leu
Ala Phe -20 -15 -10 Leu Ser Leu Thr Gly Leu Ala Ser Ile Pro Ser Thr
Arg Ala Leu Ala -5 -1 1 5 Gly Asn Ser Ser Arg Ile Asn Ser Pro Ser
Gly Leu Asn Leu Ala Ala 10 15 20 25 Leu Glu Ala Arg Lys Leu Tyr Phe
Gly Thr Ala Thr Asn Asn Val Glu 30 35 40 Leu Asn Asp Thr Ala Tyr
Phe Asp Ile Leu Asp Asp Phe Lys Met Phe 45 50 55 Gly Gln Ile Thr
Pro Ala Lys Gly Met Lys Trp Met Glu Thr Glu Pro 60 65 70 Glu Arg
Gly Val Phe Thr Phe Ala Gln Ala Asp Gln Ile Ala Gln Leu 75 80 85
Ala Ser Glu Gly Gly Lys Leu Leu Arg Gly Ser Tyr Ser Lys Gly His 90
95 100 105 Asn Cys Val Trp Tyr Asn Ala Leu Pro Gly Trp Val Thr Asn
Thr Thr 110 115 120 Trp Thr Ala Ser Glu Met Ala Glu Val Val Gln Glu
His Cys Phe Asn 125 130 135 Ile Val Arg Tyr Trp Gln Gly Gln Ala Trp
Asp Val Ile Asn Glu Pro 140 145 150 Phe Asn Asp Asp Gly Thr Trp Arg
Glu Thr Met Trp Phe Asn Thr Leu 155 160 165 Asn Thr Ser Tyr Ile Pro
Leu Ala Leu His Ala Ala Arg Ala Ala Asp 170 175 180 185 Pro His Thr
Lys Leu Tyr Ile Asn Glu Tyr Asn Ile Thr Gly Thr Gly 190 195 200 Pro
Lys Ala Thr Ser Met Lys Asn Leu Ile Lys Asp Leu Lys Arg Ala 205 210
215 Gly Val Pro Val His Gly Val Gly Val Gln Ala His Glu Thr Val Gly
220 225 230 Glu Val Pro Thr Asp Ile Arg Lys Asn Leu Gly Glu Phe Val
Ala Leu 235 240 245 Gly Val Glu Val Ala Ile Thr Glu Leu Asp Ile Lys
Phe Asn Thr Leu 250 255 260 265 Pro Pro Asp Ala Ala Gly Leu Lys Gln
Gln Lys Arg Asp Tyr Glu Ala 270 275 280 Ile Val Ser Ala Cys Ala Glu
Val Lys Gly Cys Val Gly Val Thr Val 285 290 295 Trp Asp Phe Thr Asp
Lys Tyr Ser Trp Ile Pro Gly Thr Phe Pro Gly 300 305 310 Thr Gly Asp
Ala Cys Pro 315 71668DNATrametes
versicolorCDS(1)..(174)sig_peptide(1)..(57)mat_peptide(58)..()Intron(175)-
..(240)CDS(241)..(453)Intron(454)..(510)CDS(511)..(728)Intron(729)..(785)C-
DS(786)..(787)Intron(788)..(844)CDS(845)..(865)Intron(866)..(922)CDS(923).-
.(1069)Intron(1070)..(1136)CDS(1137)..(1441)Intron(1442)..(1502)CDS(1503).-
.(1559)Intron(1560)..(1617)CDS(1618)..(1665) 7atg aac ctc tca gcg
tcg ttc gca gta ctt gtc gct ctg atc ccg tac 48Met Asn Leu Ser Ala
Ser Phe Ala Val Leu Val Ala Leu Ile Pro Tyr -15 -10 -5 gcc ctc gcg
cag tcc ccg gag tgg ggc caa tgc ggc gga aca ggc tat 96Ala Leu Ala
Gln Ser Pro Glu Trp Gly Gln Cys Gly Gly Thr Gly Tyr -1 1 5 10 acg
ggc gcc acg act tgc gtg tcc gga acg gta tgc acg gtg atc aac 144Thr
Gly Ala Thr Thr Cys Val Ser Gly Thr Val Cys Thr Val Ile Asn 15 20
25 ccg tac tac tca caa tgt ctc gca ggc act gtaagtacac gacacacgca
194Pro Tyr Tyr Ser Gln Cys Leu Ala Gly Thr 30 35 tctttgtcag
gtcacagggg agccaaacgc tgacggtcac ctcaag gcc aca tcc 249 Ala Thr Ser
40 gcg cct tcc gct ccc agc cca act gtg agc acc ggc gca ccc gcc ccg
297Ala Pro Ser Ala Pro Ser Pro Thr Val Ser Thr Gly Ala Pro Ala Pro
45 50 55 agc gtc agc ggt ctg cac act ctc gcc aaa gcc gct ggg aag
ctc tac 345Ser Val Ser Gly Leu His Thr Leu Ala Lys Ala Ala Gly Lys
Leu Tyr 60 65 70 ttt ggc agc gcg acg gac aac ccc gag ctg acc gac
acc gcc tac gtc 393Phe Gly Ser Ala Thr Asp Asn Pro Glu Leu Thr Asp
Thr Ala Tyr Val 75 80 85 90 gcg aag ctc agc gac aac gcc gag ttc ggc
cag atc acc ccc ggt aac 441Ala Lys Leu Ser Asp Asn Ala Glu Phe Gly
Gln Ile Thr Pro Gly Asn 95 100 105 agc atg aaa tgg gtgagtaccg
caccctccat ccccattctc aggtttgtga 493Ser Met Lys Trp 110 gaatgaatgc
gtcgtag gac gcg acg gag ccg acg cgg ggg acg ttc acg 543 Asp Ala Thr
Glu Pro Thr Arg Gly Thr Phe Thr 115 120 ttc acg ggc ggg gac gtg gtt
gcg agc ctg gcg gag aag aac ggg cag 591Phe Thr Gly Gly Asp Val Val
Ala Ser Leu Ala Glu Lys Asn Gly Gln 125 130 135 ctg ctg cgc ggg cac
aac tgc gtg tgg tac aac cag ctc ccg agc tgg 639Leu Leu Arg Gly His
Asn Cys Val Trp Tyr Asn Gln Leu Pro Ser Trp 140 145 150 gtc gcg aac
ggg cag ttc acg gct gcg gat ttg acg gac gtg atc acg 687Val Ala Asn
Gly Gln Phe Thr Ala Ala Asp Leu Thr Asp Val Ile Thr 155 160 165 acg
cac tgc ggc acg ctc gtt ggc cac tac aag gga caa at 728Thr His Cys
Gly Thr Leu Val Gly His Tyr Lys Gly Gln Ile 170 175 180 gtgagtgccg
gtcttactct cgaataatcg tgttacagta tgctaatgga ggcgcag c 786t
gtacgcatca tagggttgtt cgtgactgtt gctggtactg acttgctcgt accgaag
844ac tct tgg gac gtc atc aat g gtcagttgtc gtgagcgaga tcgtgcatta
895Tyr Ser Trp Asp Val Ile Asn 185 190 cagtatgctc aatattttcg
tgcctag aa ccc ttt aac gac gac ggt acc tgg 948 Glu Pro Phe Asn Asp
Asp Gly Thr Trp 195 cgc tcg gat gtg ttc ttc aat acg ctc ggt cag tcc
tac gtc tcc atc 996Arg Ser Asp Val Phe Phe Asn Thr Leu Gly Gln Ser
Tyr Val Ser Ile 200 205 210 215 gcg ctc aaa gcc gca cgc gct gca gac
ccc aac gcc aag ctc tac atc 1044Ala Leu Lys Ala Ala Arg Ala Ala Asp
Pro Asn Ala Lys Leu Tyr Ile 220 225 230 aac gac tac aac atc gag cag
acc g gtgcgcccct cctttccttg 1089Asn Asp Tyr Asn Ile Glu Gln Thr 235
atacttccct tagcaccatc aaactaaccc tgcatatgat cgcacag gc gcg aag 1144
Gly Ala Lys 240 tcg acc gcg atg ctg aac ctc gtg aag cag cta caa gca
gac ggc gtg 1192Ser Thr Ala Met Leu Asn Leu Val Lys Gln Leu Gln Ala
Asp Gly Val 245 250 255 cca atc gac ggc gtc ggc ttc cag agc cac ttc
atc gtt ggc gag gtc 1240Pro Ile Asp Gly Val Gly Phe Gln Ser His Phe
Ile Val Gly Glu Val 260 265 270 ccc ggc tcg ttc cag acc gtg ctc gag
cag ttc acc gcg ctc ggg ctc 1288Pro Gly Ser Phe Gln Thr Val Leu Glu
Gln Phe Thr Ala Leu Gly Leu 275 280 285 290 gag gtc gcg atc acg gag
ctc gac atc cgc atg acg ctc ccc gcg acg 1336Glu Val Ala Ile Thr Glu
Leu Asp Ile Arg Met Thr Leu Pro Ala Thr 295 300 305 gac gcg ctc ctc
gcg cag cag cag aag gac tac cag agc gtc gtg cag 1384Asp Ala Leu Leu
Ala Gln Gln Gln Lys Asp Tyr Gln Ser Val Val Gln 310 315 320 gcg tgc
atg aac gtg cag ggc tgt gtg ggc gtc acg atc tgg gac tgg 1432Ala Cys
Met Asn Val Gln Gly Cys Val Gly Val Thr Ile Trp Asp Trp 325 330 335
acg gac aag gtgcgtgtgg tggggtggag agagcgagcg aggagggtgc 1481Thr Asp
Lys 340 tgatagggac tcttggggca g tac tcg tgg gtg ccg tcg acg ttc tcg
gga 1532 Tyr Ser Trp Val Pro Ser Thr Phe Ser Gly 345 350 cag ggc
gcg gct ctg cct tgg gac gag gtgggtggtc ctctcccgcg 1579Gln Gly Ala
Ala Leu Pro Trp Asp Glu 355 360 ttctggggat actcaatgga cgcatttacg
ttcgtcag acc ttc aac aaa aag ccc 1635 Thr Phe Asn Lys Lys Pro 365
gca tac agc ggc atc acg gcg gca ctg acg tga 1668Ala Tyr Ser Gly Ile
Thr Ala Ala Leu Thr 370 375 8395PRTTrametes versicolor 8Met Asn Leu
Ser Ala Ser Phe Ala Val Leu Val Ala Leu Ile Pro Tyr -15 -10 -5 Ala
Leu Ala Gln Ser Pro Glu Trp Gly Gln Cys Gly Gly Thr Gly Tyr -1 1 5
10 Thr Gly Ala Thr Thr Cys Val Ser Gly Thr Val Cys Thr Val Ile Asn
15 20 25 Pro Tyr Tyr Ser Gln Cys Leu Ala Gly Thr Ala Thr Ser Ala
Pro Ser 30 35 40 45 Ala Pro Ser Pro Thr Val Ser Thr Gly Ala Pro Ala
Pro Ser Val Ser 50 55 60 Gly Leu His Thr Leu Ala Lys Ala Ala Gly
Lys Leu Tyr Phe Gly Ser 65 70 75 Ala Thr Asp Asn Pro Glu Leu Thr
Asp Thr Ala Tyr Val Ala Lys Leu 80 85 90 Ser Asp Asn Ala Glu Phe
Gly Gln Ile Thr Pro Gly Asn Ser Met Lys 95 100
105 Trp Asp Ala Thr Glu Pro Thr Arg Gly Thr Phe Thr Phe Thr Gly Gly
110 115 120 125 Asp Val Val Ala Ser Leu Ala Glu Lys Asn Gly Gln Leu
Leu Arg Gly 130 135 140 His Asn Cys Val Trp Tyr Asn Gln Leu Pro Ser
Trp Val Ala Asn Gly 145 150 155 Gln Phe Thr Ala Ala Asp Leu Thr Asp
Val Ile Thr Thr His Cys Gly 160 165 170 Thr Leu Val Gly His Tyr Lys
Gly Gln Ile Tyr Ser Trp Asp Val Ile 175 180 185 Asn Glu Pro Phe Asn
Asp Asp Gly Thr Trp Arg Ser Asp Val Phe Phe 190 195 200 205 Asn Thr
Leu Gly Gln Ser Tyr Val Ser Ile Ala Leu Lys Ala Ala Arg 210 215 220
Ala Ala Asp Pro Asn Ala Lys Leu Tyr Ile Asn Asp Tyr Asn Ile Glu 225
230 235 Gln Thr Gly Ala Lys Ser Thr Ala Met Leu Asn Leu Val Lys Gln
Leu 240 245 250 Gln Ala Asp Gly Val Pro Ile Asp Gly Val Gly Phe Gln
Ser His Phe 255 260 265 Ile Val Gly Glu Val Pro Gly Ser Phe Gln Thr
Val Leu Glu Gln Phe 270 275 280 285 Thr Ala Leu Gly Leu Glu Val Ala
Ile Thr Glu Leu Asp Ile Arg Met 290 295 300 Thr Leu Pro Ala Thr Asp
Ala Leu Leu Ala Gln Gln Gln Lys Asp Tyr 305 310 315 Gln Ser Val Val
Gln Ala Cys Met Asn Val Gln Gly Cys Val Gly Val 320 325 330 Thr Ile
Trp Asp Trp Thr Asp Lys Tyr Ser Trp Val Pro Ser Thr Phe 335 340 345
Ser Gly Gln Gly Ala Ala Leu Pro Trp Asp Glu Thr Phe Asn Lys Lys 350
355 360 365 Pro Ala Tyr Ser Gly Ile Thr Ala Ala Leu Thr 370 375 9
1617DNATrametes
versicolorCDS(1)..(174)sig_peptide(1)..(57)mat_peptide(58)..()Intron(175)-
..(231)CDS(232)..(459)Intron(460)..(525)CDS(526)..(743)Intron(744)..(798)C-
DS(799)..(800)Intron(801)..(858)CDS(859)..(879)Intron(880)..(935)CDS(936).-
.(1082)Intron(1083)..(1142)CDS(1143)..(1447)Intron(1448)..(1506)CDS(1507).-
.(1614) 9atg cag ctc tcg acg acc ttc acc ctc ctc gcc gcg atc att
ccg ttc 48Met Gln Leu Ser Thr Thr Phe Thr Leu Leu Ala Ala Ile Ile
Pro Phe -15 -10 -5 gcc ctc ggg cag gcc gcg gag tgg ggc cag tgc ggt
ggc att ggc tgg 96Ala Leu Gly Gln Ala Ala Glu Trp Gly Gln Cys Gly
Gly Ile Gly Trp -1 1 5 10 acc ggc gcg acg acg tgc gtg gcg ggc acc
acc tgc acg gtc atg aac 144Thr Gly Ala Thr Thr Cys Val Ala Gly Thr
Thr Cys Thr Val Met Asn 15 20 25 gcg tac tac tcc cag tgc ctc ccc
ggt tct gtgagtggct gtgctgtggt 194Ala Tyr Tyr Ser Gln Cys Leu Pro
Gly Ser 30 35 agagacgttc aacatgctga ccggtgaatg cttgtag gct gcg ccc
gcg ccg acg 249 Ala Ala Pro Ala Pro Thr 40 45 acg acc ccc acc tcg
cct tcg agc ccg gcg acc ccg ccg tcc gcg cct 297Thr Thr Pro Thr Ser
Pro Ser Ser Pro Ala Thr Pro Pro Ser Ala Pro 50 55 60 gcg cca acc
ggc agc ggc ctc aac aag ctc gcg aag gcg gct ggc aag 345Ala Pro Thr
Gly Ser Gly Leu Asn Lys Leu Ala Lys Ala Ala Gly Lys 65 70 75 ctc
tac ctc ggc act gcg acg gac aac agc gag ctc acc gat gcg gcg 393Leu
Tyr Leu Gly Thr Ala Thr Asp Asn Ser Glu Leu Thr Asp Ala Ala 80 85
90 tac acc gcc atc ctc gac gac aac tcc cag ttc ggc cag atc acg ccc
441Tyr Thr Ala Ile Leu Asp Asp Asn Ser Gln Phe Gly Gln Ile Thr Pro
95 100 105 gcc aac agc atg aaa tgg gtgcgcatta tccctgcatc gtgtactaga
489Ala Asn Ser Met Lys Trp 110 115 acgctccttg cttattgttg taaaattgga
atgcag gac gcg aca gag ccg act 543 Asp Ala Thr Glu Pro Thr 120 cgc
gga acg ttc acg ttc tcg ggt ggt gac cag atc gcg aac ctg gcg 591Arg
Gly Thr Phe Thr Phe Ser Gly Gly Asp Gln Ile Ala Asn Leu Ala 125 130
135 aag acg aac ggg atg ctt ctc cgt gga cac aac tgc gtg tgg tac aac
639Lys Thr Asn Gly Met Leu Leu Arg Gly His Asn Cys Val Trp Tyr Asn
140 145 150 cag ctc ccg agc tgg gtt gcg aac ggc cag ttc acc gcc gcg
gac ctc 687Gln Leu Pro Ser Trp Val Ala Asn Gly Gln Phe Thr Ala Ala
Asp Leu 155 160 165 acg acc gtc atc cag acg cac tgc agc acc ctc gtc
agc cac tac aag 735Thr Thr Val Ile Gln Thr His Cys Ser Thr Leu Val
Ser His Tyr Lys 170 175 180 185 ggt caa gt gtacgtgatt ccttctgtgt
atctactctc ccaatactga 783Gly Gln Val ccccattttc cgcag t t
gtacgtctac gttcgcattt atgattcttg tatgcatact 840gaccgacatg acaaaaag
ac tcc tgg gac gtc gtc aac g gttagtggta 889 Tyr Ser Trp Asp Val Val
Asn 190 195 ttactccaca agttcaccag ggaagtgttc tgacagtgat ctccag ag
ccg ttc 943 Glu Pro Phe aac gac gat ggt acc tgg cgc tcg gac gtg ttc
tac aac acg ctc ggc 991Asn Asp Asp Gly Thr Trp Arg Ser Asp Val Phe
Tyr Asn Thr Leu Gly 200 205 210 act tcg tac gtg ccc atc gcg ctc aag
gct gcg cgc gct gcg gac cct 1039Thr Ser Tyr Val Pro Ile Ala Leu Lys
Ala Ala Arg Ala Ala Asp Pro 215 220 225 230 agc gcc aaa ctc tac atc
aac gac tac aac att gag cag acg g 1082Ser Ala Lys Leu Tyr Ile Asn
Asp Tyr Asn Ile Glu Gln Thr 235 240 gtaggtcccc agcatccatc
tcccaggagt gacgccgctc acggcacaca cgcaccacag 1142gc gcc aag gcg acc
gcg atg ctg aac ctc gtg aag cag ctc atc gcc 1189Gly Ala Lys Ala Thr
Ala Met Leu Asn Leu Val Lys Gln Leu Ile Ala 245 250 255 260 gac ggc
gtt ccg atc gac ggt gtc ggc ttc cag tgc cac ttt atc gtt 1237Asp Gly
Val Pro Ile Asp Gly Val Gly Phe Gln Cys His Phe Ile Val 265 270 275
ggc gag gtc ccc ggc tcg ttc cag acc gtg ctc gag cag ttc acc gcg
1285Gly Glu Val Pro Gly Ser Phe Gln Thr Val Leu Glu Gln Phe Thr Ala
280 285 290 ctc ggg ctc gag gtc gcg atc acg gag ctc gac atc cgc acg
acg acg 1333Leu Gly Leu Glu Val Ala Ile Thr Glu Leu Asp Ile Arg Thr
Thr Thr 295 300 305 ccc gcg tcg cag tcc gcg ctc gca cag cag gag aag
gac tac cag tcg 1381Pro Ala Ser Gln Ser Ala Leu Ala Gln Gln Glu Lys
Asp Tyr Gln Ser 310 315 320 gtt atc cag gcg tgc atg aac gtc aag ggc
tgc gtt ggt gcc acc ctc 1429Val Ile Gln Ala Cys Met Asn Val Lys Gly
Cys Val Gly Ala Thr Leu 325 330 335 340 tgg gac ttc acc gac aag
gttcgtaggc aagctttcta cgcgtgtaag 1477Trp Asp Phe Thr Asp Lys 345
acgaattggc tgacgctctt gcgatgcag tac tcc tgg gtc ccc tcg acg ttc
1530 Tyr Ser Trp Val Pro Ser Thr Phe 350 tcc ggc caa ggt gcg gcg
tgc cct tgg gac cag aac ctc gtc aag aag 1578Ser Gly Gln Gly Ala Ala
Cys Pro Trp Asp Gln Asn Leu Val Lys Lys 355 360 365 370 ccc gcg tac
act ggt atc gtc aac gct ctc agc gcg tga 1617Pro Ala Tyr Thr Gly Ile
Val Asn Ala Leu Ser Ala 375 380 10401PRTTrametes versicolor 10Met
Gln Leu Ser Thr Thr Phe Thr Leu Leu Ala Ala Ile Ile Pro Phe -15 -10
-5 Ala Leu Gly Gln Ala Ala Glu Trp Gly Gln Cys Gly Gly Ile Gly Trp
-1 1 5 10 Thr Gly Ala Thr Thr Cys Val Ala Gly Thr Thr Cys Thr Val
Met Asn 15 20 25 Ala Tyr Tyr Ser Gln Cys Leu Pro Gly Ser Ala Ala
Pro Ala Pro Thr 30 35 40 45 Thr Thr Pro Thr Ser Pro Ser Ser Pro Ala
Thr Pro Pro Ser Ala Pro 50 55 60 Ala Pro Thr Gly Ser Gly Leu Asn
Lys Leu Ala Lys Ala Ala Gly Lys 65 70 75 Leu Tyr Leu Gly Thr Ala
Thr Asp Asn Ser Glu Leu Thr Asp Ala Ala 80 85 90 Tyr Thr Ala Ile
Leu Asp Asp Asn Ser Gln Phe Gly Gln Ile Thr Pro 95 100 105 Ala Asn
Ser Met Lys Trp Asp Ala Thr Glu Pro Thr Arg Gly Thr Phe 110 115 120
125 Thr Phe Ser Gly Gly Asp Gln Ile Ala Asn Leu Ala Lys Thr Asn Gly
130 135 140 Met Leu Leu Arg Gly His Asn Cys Val Trp Tyr Asn Gln Leu
Pro Ser 145 150 155 Trp Val Ala Asn Gly Gln Phe Thr Ala Ala Asp Leu
Thr Thr Val Ile 160 165 170 Gln Thr His Cys Ser Thr Leu Val Ser His
Tyr Lys Gly Gln Val Tyr 175 180 185 Ser Trp Asp Val Val Asn Glu Pro
Phe Asn Asp Asp Gly Thr Trp Arg 190 195 200 205 Ser Asp Val Phe Tyr
Asn Thr Leu Gly Thr Ser Tyr Val Pro Ile Ala 210 215 220 Leu Lys Ala
Ala Arg Ala Ala Asp Pro Ser Ala Lys Leu Tyr Ile Asn 225 230 235 Asp
Tyr Asn Ile Glu Gln Thr Gly Ala Lys Ala Thr Ala Met Leu Asn 240 245
250 Leu Val Lys Gln Leu Ile Ala Asp Gly Val Pro Ile Asp Gly Val Gly
255 260 265 Phe Gln Cys His Phe Ile Val Gly Glu Val Pro Gly Ser Phe
Gln Thr 270 275 280 285 Val Leu Glu Gln Phe Thr Ala Leu Gly Leu Glu
Val Ala Ile Thr Glu 290 295 300 Leu Asp Ile Arg Thr Thr Thr Pro Ala
Ser Gln Ser Ala Leu Ala Gln 305 310 315 Gln Glu Lys Asp Tyr Gln Ser
Val Ile Gln Ala Cys Met Asn Val Lys 320 325 330 Gly Cys Val Gly Ala
Thr Leu Trp Asp Phe Thr Asp Lys Tyr Ser Trp 335 340 345 Val Pro Ser
Thr Phe Ser Gly Gln Gly Ala Ala Cys Pro Trp Asp Gln 350 355 360 365
Asn Leu Val Lys Lys Pro Ala Tyr Thr Gly Ile Val Asn Ala Leu Ser 370
375 380 Ala 111676DNATrametes
versicolorCDS(1)..(79)sig_peptide(1)..(66)mat_peptide(67)..()Intron(80)..-
(139)CDS(140)..(174)Intron(175)..(204)CDS(205)..(522)Intron(523)..(585)CDS-
(586)..(592)Intron(593)..(649)CDS(650)..(739)Intron(740)..(798)CDS(799)..(-
1066)Intron(1067)..(1120)CDS(1121)..(1229)Intron(1230)..(1285)CDS(1286)..(-
1378)Intron(1379)..(1431)CDS(1432)..(1518)Intron(1519)..(1583)CDS(1584)..(-
1673) 11atg aag ggc ctc gcc gca ctc gtc gca ctc gcc acc atc gtc gcc
gtc 48Met Lys Gly Leu Ala Ala Leu Val Ala Leu Ala Thr Ile Val Ala
Val -20 -15 -10 ccg gcc aac gcc gtc gcg gtc tgg ggc caa t
gtgagcatcc ctcacccgga 99Pro Ala Asn Ala Val Ala Val Trp Gly Gln -5
-1 1 cttatacctc tggaatagta acactgacat gcgtttgcag gc gga gta cgc acc
153 Cys Gly Val Arg Thr 5 ttt gcc cgc tgc gct cgt cct gtctacgctt
gacactgacc tctctgtcag ggt 207Phe Ala Arg Cys Ala Arg Pro Gly 10 15
atc ggc ttc agt gga tcg acc aca tgt gat gcc ggc acc aca tgc atc
255Ile Gly Phe Ser Gly Ser Thr Thr Cys Asp Ala Gly Thr Thr Cys Ile
20 25 30 gtg ctc aac tcc tac tac tcg cag tgc cag ccg ggt gcg agc
gcg ccc 303Val Leu Asn Ser Tyr Tyr Ser Gln Cys Gln Pro Gly Ala Ser
Ala Pro 35 40 45 gcg ccc acg aca tcc gcc ccc cag ccg ccc ccg acc
aca ccg gct ggt 351Ala Pro Thr Thr Ser Ala Pro Gln Pro Pro Pro Thr
Thr Pro Ala Gly 50 55 60 65 ggc tcg ccc gcg ccc gcg gcg acc gga ctc
aac gct gcg ttc aag aag 399Gly Ser Pro Ala Pro Ala Ala Thr Gly Leu
Asn Ala Ala Phe Lys Lys 70 75 80 cac ggc aag aag ttc tgg ggc acc
gcg acg gac tca aac cgc ttc agc 447His Gly Lys Lys Phe Trp Gly Thr
Ala Thr Asp Ser Asn Arg Phe Ser 85 90 95 aac ccg acg gac tcc gcg
gtc acc gtc cgc gag ttc ggc cag gtc acg 495Asn Pro Thr Asp Ser Ala
Val Thr Val Arg Glu Phe Gly Gln Val Thr 100 105 110 cct gag aac tcc
atg aag tgg gat gcg gtgagtgcct actgggcgcg 542Pro Glu Asn Ser Met
Lys Trp Asp Ala 115 120 tcggcgtcga gtgagcatgt gcttatgatt attttcgtcg
tag act gag c 592 Thr Glu gtgcgtattt agtgaggctt cggatggtcc
tcccaggaaa ctgacagcat gttgcag 649ct tcc cgc aac cag ttc tcg ttc agc
ggc tct gat gcg ctg gtc aac 696Pro Ser Arg Asn Gln Phe Ser Phe Ser
Gly Ser Asp Ala Leu Val Asn 125 130 135 140 ttc gct acg acg aat ggc
ctg ctc gtc cgc gct cac acc ctc g 739Phe Ala Thr Thr Asn Gly Leu
Leu Val Arg Ala His Thr Leu 145 150 gtaagcatgt tctcgttgtc
tcatctctga agtggcgact aactgttctt ggggcgcag 798tc tgg cat tcg caa
ctg ccg tcc tgg gtc tct gcg atc aac gac cgc 845Val Trp His Ser Gln
Leu Pro Ser Trp Val Ser Ala Ile Asn Asp Arg 155 160 165 170 gcg acg
ctc acg tcc gtg atc cag aac cac atc gcg aac gtc gca ggc 893Ala Thr
Leu Thr Ser Val Ile Gln Asn His Ile Ala Asn Val Ala Gly 175 180 185
cgg tac aag ggc aag gtg tac tcc tgg gac gtc gtg aac gag atc ttc
941Arg Tyr Lys Gly Lys Val Tyr Ser Trp Asp Val Val Asn Glu Ile Phe
190 195 200 aac gag gac ggc acg ttc cgc tcg tcg gtg ttt tca aac gtc
ctc ggc 989Asn Glu Asp Gly Thr Phe Arg Ser Ser Val Phe Ser Asn Val
Leu Gly 205 210 215 cag gac ttc gtc acg atc gcg ttc cag gcg gca cgg
gcg gcg gac ccg 1037Gln Asp Phe Val Thr Ile Ala Phe Gln Ala Ala Arg
Ala Ala Asp Pro 220 225 230 aac gcg aag ctc tac atc aac gac tac aa
gtgtgtctcg cgggttggct 1086Asn Ala Lys Leu Tyr Ile Asn Asp Tyr Asn
235 240 tggtgtgcct ttgctgatgc gtttgtgtat gcag c ctc gac acc gtg aac
cca 1139 Leu Asp Thr Val Asn Pro 245 250 aag ctc aac ggt gtt gtc
aac ctt gtc aag aag atc aac ggc ggc ggc 1187Lys Leu Asn Gly Val Val
Asn Leu Val Lys Lys Ile Asn Gly Gly Gly 255 260 265 acc aag ctg atc
gac ggt atc ggt act cag gcc cac ctt tcg 1229Thr Lys Leu Ile Asp Gly
Ile Gly Thr Gln Ala His Leu Ser 270 275 280 gtaagtgtat caggactatt
tagcagactg acgtgctgac gctagagctc ggatag gct 1288 Ala ggc ggc gct
ggc gga ttc cag gct gcg ctc acg cag ctg gct acc gcc 1336Gly Gly Ala
Gly Gly Phe Gln Ala Ala Leu Thr Gln Leu Ala Thr Ala 285 290 295
ggc acg gag atc gct atc acg gag ctc gac att gcg ggt gcc 1378Gly Thr
Glu Ile Ala Ile Thr Glu Leu Asp Ile Ala Gly Ala 300 305 310
gtaagtatcc gttacaatga tttcgcgctg ctccttattt atgtcgcatt cag gcc 1434
Ala ccc aat gac tac tcg acg ctg gtc aag gcg tgt ctc gcg gtg gag agc
1482Pro Asn Asp Tyr Ser Thr Leu Val Lys Ala Cys Leu Ala Val Glu Ser
315 320 325 tgc gtg tcc atc aca agc tgg gga gtc cgc gat ccc
gtaagcaata 1528Cys Val Ser Ile Thr Ser Trp Gly Val Arg Asp Pro 330
335 340 tatcttcctt gttgacggtg atgagacgtt ctcaccatgt gcatgctttt
atcag gac 1586 Asp tcc tgg agg gcg tcc acc aac ccc ctc ttg ttc gac
gcg aac ttc aac 1634Ser Trp Arg Ala Ser Thr Asn Pro Leu Leu Phe Asp
Ala Asn Phe Asn 345 350 355 ccg aag ccc gca tac act gcg gtt atg cag
gcc ctg gct tga 1676Pro Lys Pro Ala Tyr Thr Ala Val Met Gln Ala Leu
Ala 360 365 370 12392PRTTrametes versicolor 12Met Lys Gly Leu Ala
Ala Leu Val Ala Leu Ala Thr Ile Val Ala Val -20 -15 -10 Pro Ala Asn
Ala Val Ala Val Trp Gly Gln Cys Gly Val Arg Thr Phe -5 -1 1 5 10
Ala Arg Cys Ala Arg Pro Gly Ile Gly Phe Ser Gly Ser Thr Thr Cys 15
20 25 Asp Ala Gly Thr Thr Cys Ile Val Leu Asn Ser Tyr Tyr Ser Gln
Cys 30 35 40 Gln Pro Gly Ala Ser Ala Pro Ala Pro Thr Thr Ser Ala
Pro Gln Pro 45 50 55 Pro Pro Thr Thr Pro Ala Gly Gly Ser Pro Ala
Pro Ala Ala Thr Gly 60 65 70 Leu Asn Ala Ala Phe Lys Lys His Gly
Lys Lys Phe Trp Gly Thr Ala 75 80 85 90 Thr Asp Ser Asn Arg Phe Ser
Asn Pro Thr Asp Ser Ala Val Thr Val 95 100 105 Arg Glu Phe Gly Gln
Val Thr Pro Glu Asn Ser Met Lys Trp Asp Ala 110 115 120 Thr Glu Pro
Ser Arg Asn Gln Phe Ser Phe Ser Gly Ser Asp Ala Leu 125 130 135 Val
Asn Phe Ala Thr Thr Asn Gly Leu Leu Val Arg Ala His Thr Leu 140 145
150 Val Trp His Ser Gln Leu Pro Ser Trp Val Ser Ala Ile Asn Asp Arg
155 160 165 170 Ala Thr Leu Thr Ser Val Ile Gln Asn His Ile Ala Asn
Val Ala Gly 175 180 185 Arg Tyr Lys Gly Lys Val Tyr Ser Trp Asp Val
Val Asn Glu Ile Phe 190 195 200 Asn Glu Asp Gly Thr Phe Arg Ser Ser
Val Phe Ser Asn Val Leu Gly 205 210 215 Gln Asp Phe Val Thr Ile Ala
Phe Gln Ala Ala Arg Ala Ala Asp Pro 220 225 230 Asn Ala Lys Leu Tyr
Ile Asn Asp Tyr Asn Leu Asp Thr Val Asn Pro 235 240 245 250 Lys Leu
Asn Gly Val Val Asn Leu Val Lys Lys Ile Asn Gly Gly Gly 255 260 265
Thr Lys Leu Ile Asp Gly Ile Gly Thr Gln Ala His Leu Ser Ala Gly 270
275 280 Gly Ala Gly Gly Phe Gln Ala Ala Leu Thr Gln Leu Ala Thr Ala
Gly 285 290 295 Thr Glu Ile Ala Ile Thr Glu Leu Asp Ile Ala Gly Ala
Ala Pro Asn 300 305 310 Asp Tyr Ser Thr Leu Val Lys Ala Cys Leu Ala
Val Glu Ser Cys Val 315 320 325 330 Ser Ile Thr Ser Trp Gly Val Arg
Asp Pro Asp Ser Trp Arg Ala Ser 335 340 345 Thr Asn Pro Leu Leu Phe
Asp Ala Asn Phe Asn Pro Lys Pro Ala Tyr 350 355 360 Thr Ala Val Met
Gln Ala Leu Ala 365 370
* * * * *