U.S. patent application number 15/835830 was filed with the patent office on 2018-05-17 for polypeptides having xylanase activity and polynucleotides encoding same.
This patent application is currently assigned to Novozymes, Inc.. The applicant listed for this patent is Novozymes Inc.. Invention is credited to Nikolaj Spodsberg.
Application Number | 20180135035 15/835830 |
Document ID | / |
Family ID | 46705039 |
Filed Date | 2018-05-17 |
United States Patent
Application |
20180135035 |
Kind Code |
A1 |
Spodsberg; Nikolaj |
May 17, 2018 |
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 |
|
|
Assignee: |
Novozymes, Inc.
Davis
CA
|
Family ID: |
46705039 |
Appl. No.: |
15/835830 |
Filed: |
December 8, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15483639 |
Apr 10, 2017 |
9885029 |
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15835830 |
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14513057 |
Oct 13, 2014 |
9637727 |
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15483639 |
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14122431 |
Nov 26, 2013 |
8859227 |
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PCT/US2012/049096 |
Aug 1, 2012 |
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14513057 |
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61531413 |
Sep 6, 2011 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 9/2482 20130101;
C12P 19/14 20130101; C12Y 302/01008 20130101; C12P 19/02 20130101;
C12N 9/248 20130101 |
International
Class: |
C12N 9/24 20060101
C12N009/24; C12P 19/02 20060101 C12P019/02; C12P 19/14 20060101
C12P019/14 |
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
1-24. (canceled)
25. A process for degrading a cellulosic material or
xylan-containing material, comprising: treating the cellulosic
material or xylan-containing material with one or more enzymes
comprising a polypeptide having xylanase activity selected from the
group consisting of (a) a polypeptide having at least 85% sequence
identity to the sequence of amino acids 18 to 364 of SEQ ID NO: 2
or amino acids 17 to 389 of SEQ ID NO: 4; and (b) a fragment of the
sequence of amino acids 18 to 364 of SEQ ID NO: 2 or amino acids 17
to 389 of SEQ ID NO: 4 that has xylanase activity.
26. The process of claim 25, wherein the one or more enzymes
further comprise a cellobiohydrolase, an endoglucanase, a
beta-glucosidase, and a GH61 polypeptide.
27. The process of claim 25, wherein the polypeptide has at least
85% sequence identity to the sequence of amino acids 18 to 364 of
SEQ ID NO: 2 or amino acids 17 to 389 of SEQ ID NO: 4.
28. The process of claim 25, wherein the polypeptide has at least
90% sequence identity to the sequence of amino acids 18 to 364 of
SEQ ID NO: 2 or amino acids 17 to 389 of SEQ ID NO: 4.
29. The process of claim 25, wherein the polypeptide has at least
95% sequence identity to the sequence of amino acids 18 to 364 of
SEQ ID NO: 2 or amino acids 17 to 389 of SEQ ID NO: 4.
30. The process of claim 25, wherein the polypeptide has at least
97% sequence identity to the sequence of amino acids 18 to 364 of
SEQ ID NO: 2 or amino acids 17 to 389 of SEQ ID NO: 4.
31. The process of claim 25, wherein the polypeptide comprises the
sequence of amino acids 18 to 364 of SEQ ID NO: 2 or amino acids 17
to 389 of SEQ ID NO: 4.
32. The process of claim 25, wherein the polypeptide is a variant
of the sequence of amino acids 18 to 364 of SEQ ID NO: 2 or amino
acids 17 to 389 of SEQ ID NO: 4 comprising a substitution, deletion
and/or insertion at one or more positions and wherein the variant
has at least 95% sequence identity to the sequence of amino acids
18 to 364 of SEQ ID NO: 2 or amino acids 17 to 389 of SEQ ID NO:
4.
33. The process of claim 25, wherein the polypeptide is a fragment
of SEQ ID NO: 2 or SEQ ID NO: 4, wherein the fragment has xylanase
activity.
34. The process of claim 25, wherein the polypeptide is encoded by
a polynucleotide that hybridizes under very high stringency
conditions with the complement of the sequence of nucleotides 52 to
1165 of SEQ ID NO: 1 or nucleotides 49 to 1705 of SEQ ID NO: 3,
wherein the very high stringency conditions are defined as
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, and washing three times each
for 15 minutes using 2.times.SSC, 0.2% SDS at 70.degree. C.
35. A process for producing a fermentation product, comprising: (a)
saccharifying a cellulosic material or xylan-containing material
with one or more enzymes comprising a polypeptide having xylanase
activity selected from the group consisting of (i) a polypeptide
having at least 85% sequence identity to the sequence of amino
acids 18 to 364 of SEQ ID NO: 2 or amino acids 17 to 389 of SEQ ID
NO: 4; and (ii) a fragment of the sequence of amino acids 18 to 364
of SEQ ID NO: 2 or amino acids 17 to 389 of SEQ ID NO: 4 that has
xylanase activity; and (b) fermenting the saccharified cellulosic
material or xylan-containing material with one or more fermenting
microorganisms to produce the fermentation product.
36. The process of claim 35, wherein the one or more enzymes
further comprise a cellobiohydrolase, and endoglucanase, a
beta-glucosidase, and a GH61 polypeptide.
37. The process of claim 35, wherein the polypeptide has at least
90% sequence identity to the sequence of amino acids 18 to 364 of
SEQ ID NO: 2 or amino acids 17 to 389 of SEQ ID NO: 4.
38. The process of claim 35, wherein the polypeptide has at least
95% sequence identity to the sequence of amino acids 18 to 364 of
SEQ ID NO: 2 or amino acids 17 to 389 of SEQ ID NO: 4.
39. The process of claim 35, wherein the polypeptide has at least
97% sequence identity to the sequence of amino acids 18 to 364 of
SEQ ID NO: 2 or amino acids 17 to 389 of SEQ ID NO: 4.
40. The process of claim 35, further comprising recovering the
fermentation product from the fermentation.
41. A process for degrading a cellulosic material or
xylan-containing material, comprising: treating the cellulosic
material or xylan-containing material with one or more enzymes
comprising a polypeptide having xylanase activity which comprises a
catalytic domain having at least 90% sequence identity to the
sequence of amino acids 18 to 364 of SEQ ID NO: 2 or amino acids 17
to 326 of SEQ ID NO: 4.
42. The process of claim 41, wherein the one or more enzymes
further comprise a cellobiohydrolase, an endoglucanase, a
beta-glucosidase, and a GH61 polypeptide.
43. The process of claim 41, wherein the polypeptide having
xylanase activity which comprises a catalytic domain has at least
95% sequence identity to the sequence of amino acids 18 to 364 of
SEQ ID NO: 2 or amino acids 17 to 326 of SEQ ID NO: 4
44. The process of claim 41, wherein the polypeptide having
xylanase activity which comprises a catalytic domain has at least
97% sequence identity to the sequence of amino acids 18 to 364 of
SEQ ID NO: 2 or amino acids 17 to 326 of SEQ ID NO: 4
45. The process of claim 41, wherein the polypeptide having
xylanase activity which comprises a catalytic domain comprises the
sequence of amino acids 18 to 364 of SEQ ID NO: 2 or amino acids 17
to 326 of SEQ ID NO: 4.
46. The process of claim 41, wherein the polypeptide further
comprises a cellulose binding domain.
47. A recombinant host cell transformed with a nucleic acid
construct or expression vector operably linked to one or more
control sequences that direct the production of the polypeptide,
wherein the polynucleotide is operably linked to one or more
control sequences that direct the production of the polypeptide in
an expression host and wherein the polypeptide is selected from the
group consisting of (a) a polypeptide having at least 85% sequence
identity to the sequence of amino acids 18 to 364 of SEQ ID NO: 2
or amino acids 17 to 389 of SEQ ID NO: 4; and (b) a fragment of the
sequence of amino acids 18 to 364 of SEQ ID NO: 2 or amino acids 17
to 389 of SEQ ID NO: 4 that has xylanase activity.
48. A method of producing a polypeptide having xylanase activity,
comprising cultivating the recombinant host cell of claim 47 under
conditions conducive for production of the polypeptide; and
49. The method of claim 48, further comprising recovering the
polypeptide.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application of U.S.
application Ser. No. 15/483,639 filed Apr. 10, 2017, which is a
divisional application of U.S. application Ser. No. 14/513,057
filed Oct. 13, 2014, now U.S. Pat. No. 9,637,727, which is a
divisional application of U.S. application Ser. No. 14/122,431
filed Aug. 1, 2012, now U.S. Pat. No. 8,859,227, which is a 35
U.S.C. .sctn. 371 national application of PCT/US2012/049096 filed
Aug. 1, 2012, which claims priority or the benefit under 35 U.S.C.
.sctn. 119 of European Application No. 11250700.9 filed Aug. 4,
2011 and U.S. Provisional Application No. 61/531,413 filed Sep. 6,
2011, the contents of which are fully incorporated herein by
reference.
REFERENCE TO A SEQUENCE LISTING
[0003] This application contains a Sequence Listing in computer
readable form, which is incorporated herein by reference.
BACKGROUND OF THE INVENTION
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.
Description of the Related Art
[0005] 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.
[0006] 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.
[0007] 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.
Xylanases are known from the prior art. A xylanase from Penicillium
canescens (Uniprot. C3VEV9) has 76.1% identity to the xylanase
disclosed as SEQ ID NO: 2. Another xylanase from Talaromyces
stipitatus (Uniprot. B8M9H8) has 76.5% identity to the xylanase
disclosed as SEQ ID NO: 4. Another xylanase from Penicillium sp
(Uniprot. AYB51189) disclosed in US2010124769-A1 has 84.0% identity
to the xylanase disclosed as SEQ ID NO: 6.
[0008] The present invention provides polypeptides having xylanase
activity and polynucleotides encoding the polypeptides.
SUMMARY OF THE INVENTION
[0009] The present invention relates to isolated polypeptides
having xylanase activity selected from the group consisting of:
[0010] (a) a polypeptide having at least 77% sequence identity to
the mature polypeptide of SEQ ID NO: 2, or a polypeptide having at
least 77% sequence identity to the mature polypeptide of SEQ ID NO:
4, or a polypeptide having at least 85% sequence identity to the
mature polypeptide of SEQ ID NO: 6;
[0011] (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, or SEQ ID NO: 5, (ii) the
cDNA sequence thereof, or (iii) the full-length complement of (i)
or (ii);
[0012] (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, or SEQ ID NO: 5; or the
cDNA sequence thereof;
[0013] (d) a variant of the mature polypeptide of SEQ ID NO: 2, SEQ
ID NO: 4, or SEQ ID NO: 6 comprising a substitution, deletion,
and/or insertion at one or more (e.g., several) positions; and
[0014] (e) a fragment of the polypeptide of (a), (b), (c), or (d)
that has xylanase activity.
[0015] The present invention also relates to isolated polypeptides
comprising a catalytic domain selected from the group consisting
of:
[0016] (a) a catalytic domain having at least 77% sequence identity
to the catalytic domain of SEQ ID NO: 2 (for example, amino acids
18 to 364 of SEQ ID NO: 2), a catalytic domain having at least 77%
sequence identity to the catalytic domain of SEQ ID NO: 4 (for
example, amino acids 17 to 326 of SEQ ID NO: 4), or a catalytic
domain having at least 85% sequence identity to the catalytic
domain of SEQ ID NO: 6 (for example, amino acids 21 to 337 of SEQ
ID NO: 6);
[0017] (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 52-240, and 314-1165 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 49-241,
302-342, 404-452, 518-639, 707-852, 912-1019, 1088-1205, 1282-1347,
and 1430-1516 of SEQ ID NO: 3), or 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 124-270, 342-474, 567-680, and 757-1313 of SEQ ID NO:
5);
[0018] (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,
or SEQ ID NO: 6; and
[0019] (d) a fragment of a catalytic domain of (a), (b), or (c),
which has xylanase activity.
[0020] 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.
[0021] 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.
[0022] 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.
[0023] 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.
[0024] The present invention also relates to a polynucleotide
encoding a signal peptide comprising or consisting of amino acids 1
to 17 of SEQ ID NO: 2, amino acids 1 to 16 of SEQ ID NO: 4, or
amino acids 1 to 20 of SEQ ID NO: 6, 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.
Definitions
[0025] 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, 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. In one aspect, the polypeptide 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. In another aspect the polypeptide 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: 4. In still another aspect the polypeptide 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: 6.
[0026] 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
pmole of p-nitrophenolate anion per minute at pH 5, 25.degree.
C.
[0027] 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.
[0028] 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).
[0029] 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 pmole of glucuronic or 4-O-methylglucuronic acid per
minute at pH 5, 40.degree. C.
[0030] 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 pmole 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.
[0031] 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 pmole 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.
[0032] 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.
[0033] 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.
[0034] 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.
[0035] 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.
[0036] 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).
[0037] 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.
[0038] 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.
[0039] 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.
[0040] 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.
[0041] 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.
[0042] 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 N21 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
N21 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).
[0043] 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).
[0044] 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. In one embodiment the
coding sequence is positions 1-240 and 314-1168 of SEQ ID NO: 1. In
another embodiment the coding sequence is positions 1-241, 302-342,
404-452, 518-639, 707-852, 912-1019, 1088-1205, 1282-1347, and
1430-1708 of SEQ ID NO: 3. In another embodiment the coding
sequence is positions 1-50, 114-270, 342-474, 567-680, 757-1520 of
SEQ ID NO: 5.
[0045] 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.
[0046] 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.
[0047] 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.
[0048] 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.
[0049] 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,
1991, A classification of glycosyl hydrolases based on amino-acid
sequence similarities, Biochem. J. 280: 309-316, and Henrissat and
Bairoch, 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.
[0050] 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.
[0051] 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 amino acid residues 18-364 of SEQ ID NO: 2. In another
aspect a fragment contains at least amino acid residues 17-326 of
SEQ ID NO: 4. In another aspect a fragment contains at least amino
acid residues 21-337 of SEQ ID NO: 6.
[0052] 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 and Shoham, 2003, Microbial
hemicellulases. Current Opinion In Microbiology 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.
[0053] 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.
[0054] 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.
[0055] 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.
[0056] 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.
[0057] 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 18 to 364 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 17 of SEQ ID
NO: 2 are a signal peptide. In another aspect, the mature
polypeptide is amino acids 17 to 389 of SEQ ID NO: 4 based on the
SignalP program that predicts amino acids 1 to 16 of SEQ ID NO: 4
are a signal peptide. In another aspect, the mature polypeptide is
amino acids 21 to 405 of SEQ ID NO: 6 based on the SignalP program
that predicts amino acids 1 to 20 of SEQ ID NO: 6 are a signal
peptide. 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.
[0058] 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 52 to 1165 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 51 of
SEQ ID NO: 1 encode a signal peptide. In another aspect, the mature
polypeptide coding sequence is nucleotides 49 to 1705 of SEQ ID NO:
3 or the cDNA sequence thereof based on the SignalP program that
predicts nucleotides 1 to 48 of SEQ ID NO: 3 encode a signal
peptide. In another aspect, the mature polypeptide coding sequence
is nucleotides 124 to 1517 of SEQ ID NO: 5 or the cDNA sequence
thereof based on the SignalP program that predicts nucleotides 1 to
123 of SEQ ID NO: 5 encode a signal peptide.
[0059] Catalytic domain: The term "catalytic domain" means the
portion of an enzyme containing the catalytic machinery of the
enzyme.
[0060] 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 354 to 389 of SEQ ID NO: 4. In one embodiment the CBM is
amino acids 370 to 405 of SEQ ID NO: 6. The CBM is separated from
the catalytic domain by a linker sequence. The linker is in one
embodiment amino acids 327 to 353 of SEQ ID NO: 4. The linker is in
one embodiment amino acids 338 to 369 of SEQ ID NO: 6.
[0061] 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.
[0062] 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.
[0063] 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.
[0064] 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.
[0065] 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 A/S,
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
02/095014) of cellulase protein loading is used as the source of
the cellulolytic activity.
[0066] 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.
[0067] 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.
[0068] Sequence identity: The relatedness between two amino acid
sequences or between two nucleotide sequences is described by the
parameter "sequence identity".
[0069] 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)
[0070] 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 Alignment)
[0071] 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 corresponds to the polynucleotide encoding
the catalytic domain.
[0072] 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.
[0073] 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.
[0074] 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.
[0075] 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.
[0076] In the processes of the present invention, any material
containing xylan may be used. In a preferred aspect, the
xylan-containing material is lignocellulose.
[0077] 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,
2006, Recent progress in the assays of xylanolytic enzymes, 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 et al., 1997, The beta-D-xylosidase of Trichoderma reesei
is a multifunctional beta-D-xylan xylohydrolase, Biochemical
Journal 321: 375-381.
[0078] 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 et al., 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.
[0079] 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
[0080] 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 77%, e.g., at least 78%, at least 79%,
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 77%, e.g., at least 78%, at
least 79%, 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 85%, e.g., at least 86%, 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 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, or SEQ ID NO:
6.
[0081] A polypeptide of the present invention preferably comprises
or consists of the amino acid sequence of SEQ ID NO: 2, SEQ ID NO:
4, or SEQ ID NO: 6 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, or SEQ ID NO: 6. In another aspect, the
polypeptide comprises or consists of amino acids 18 to 364 of SEQ
ID NO: 2, amino acids 17 to 389 of SEQ ID NO: 4, or amino acids 21
to 405 of SEQ ID NO: 6.
[0082] 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, or SEQ ID NO: 5, (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.).
[0083] The polynucleotide of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID
NO: 5, or a subsequence thereof, as well as the polypeptide of SEQ
ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6, 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.
[0084] 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, or SEQ ID NO: 5 or a
subsequence thereof, the carrier material is used in a Southern
blot.
[0085] 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, or SEQ
ID NO: 5; (ii) the mature polypeptide coding sequence of SEQ ID NO:
1, SEQ ID NO: 3, or SEQ ID NO: 5; (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.
[0086] In one aspect, the nucleic acid probe is a polynucleotide
that encodes the polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ
ID NO: 6; the mature polypeptide thereof; or a fragment thereof. In
another aspect, the nucleic acid probe is SEQ ID NO: 1, SEQ ID NO:
3, or SEQ ID NO: 5; or the cDNA sequence thereof. In another
aspect, the nucleic acid probe is the polynucleotide contained in
Talaromyces leycettanus Strain CBS 398.68, wherein the
polynucleotide encodes a polypeptide having xylanase activity.
[0087] 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, 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%. 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: 3,
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%. 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: 5, 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%.
[0088] In another embodiment, the present invention relates to
variants of the mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4,
or SEQ ID NO: 6 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, or SEQ ID NO: 6 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.
[0089] 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.
[0090] 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.
[0091] 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.
[0092] 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).
[0093] 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.
[0094] 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.
[0095] 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).
[0096] 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 Xylanase Activity
[0097] 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.
[0098] The polypeptide may be a Talaromyces polypeptide.
[0099] In another aspect, the polypeptide is a Talaromyces
leycettanus polypeptide, e.g., a polypeptide obtained from
Talaromyces leycettanus Strain CBS398.68.
[0100] 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.
[0101] 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).
[0102] 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
[0103] The present invention also relates to isolated polypeptides
comprising a catalytic domain selected from the group consisting
of:
[0104] (a) a catalytic domain having at least 77% sequence identity
to the catalytic domain of SEQ ID NO: 2 (for example, amino acids
18 to 364 of SEQ ID NO: 2), a catalytic domain having at least 77%
sequence identity to the catalytic domain of SEQ ID NO: 4 (for
example, amino acids 17 to 326 of SEQ ID NO: 4), or a catalytic
domain having at least 85% sequence identity to the catalytic
domain of SEQ ID NO: 6 (for example, amino acids 21 to 337 of SEQ
ID NO: 6);
[0105] (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 52-240, and 314-1165 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 49-241,
302-342, 404-452, 518-639, 707-852, 912-1019, 1088-1205, 1282-1347,
and 1430-1516 of SEQ ID NO: 3), or 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 124-270, 342-474, 567-680, and 757-1313 of SEQ ID NO:
5);
[0106] (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,
or SEQ ID NO: 6; and
[0107] (d) a fragment of a catalytic domain of (a), (b), or (c),
which has xylanase activity.
[0108] The catalytic domain preferably has a degree of sequence
identity to the catalytic domain of SEQ ID NO: 2, 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%. The catalytic
domain preferably has a degree of sequence identity to the
catalytic domain of SEQ ID NO: 4, 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%. The catalytic domain
preferably has a degree of sequence identity to the catalytic
domain of SEQ ID NO: 6, 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, or SEQ ID NO: 6.
[0109] 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 18 to 364 of SEQ ID NO: 2.
[0110] 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 17 to 326 of SEQ ID NO: 4.
[0111] 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 21 to 337 of SEQ ID NO: 6.
[0112] 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, or SEQ ID NO: 5, (ii) the cDNA sequence
contained in the catalytic domain coding sequence of SEQ ID NO: 1,
SEQ ID NO: 3, or SEQ ID NO: 5, or (iii) the full-length
complementary strand of (i) or (ii) (J. Sambrook et al., 1989,
supra).
[0113] 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 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.
[0114] 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: 3 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.
[0115] 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: 5 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.
[0116] In one aspect, the polynucleotide encoding the catalytic
domain comprises or consists of nucleotides 52 to 1165 of SEQ ID
NO: 1 or the cDNA sequence thereof. In particular the
polynucleotide encoding the catalytic domain comprises or consists
of nucleotides 52-240, and 314-1165 of SEQ ID NO: 1.
[0117] In one aspect, the polynucleotide encoding the catalytic
domain comprises or consists of nucleotides 49 to 1516 of SEQ ID
NO: 3 or the cDNA sequence thereof. In particular the
polynucleotide encoding the catalytic domain comprises or consists
of nucleotides 49-241, 302-342, 404-452, 518-639, 707-852,
912-1019, 1088-1205, 1282-1347, and 1430-1516 of SEQ ID NO: 3.
[0118] In one aspect, the polynucleotide encoding the catalytic
domain comprises or consists of nucleotides 124 to 1313 of SEQ ID
NO: 5 or the cDNA sequence thereof. In particular the
polynucleotide encoding the catalytic domain comprises or consists
of nucleotides 124-270, 342-474, 567-680, and 757-1313 of SEQ ID
NO: 5.
Polynucleotides
[0119] The present invention also relates to isolated
polynucleotides encoding a polypeptide of the present invention, as
described herein.
[0120] 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 Talaromyces, or a related organism and
thus, for example, may be an allelic or species variant of the
polypeptide encoding region of the polynucleotide.
[0121] 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,
or SEQ ID NO: 5, 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
substitutions, see, e.g., Ford et al., 1991, Protein Expression and
Purification 2: 95-107.
Nucleic Acid Constructs
[0122] 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.
[0123] 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.
[0124] 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.
[0125] 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.
[0126] 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 Daria (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.
[0127] 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.
[0128] 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.
[0129] 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).
[0130] 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.
[0131] 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.
[0132] 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.
[0133] 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).
[0134] 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.
[0135] Preferred leaders for filamentous fungal host cells are
obtained from the genes for Aspergillus oryzae TAKA amylase and
Aspergillus nidulans triose phosphate isomerase.
[0136] 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).
[0137] 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.
[0138] 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.
[0139] Useful polyadenylation sequences for yeast host cells are
described by Guo and Sherman, 1995, Mol. Cellular Biol. 15:
5983-5990.
[0140] 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.
[0141] 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.
[0142] 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.
[0143] 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.
[0144] 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.
[0145] 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.
[0146] 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
[0147] 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. 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.
[0148] 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.
[0149] 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.
[0150] 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.
[0151] 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.
[0152] 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.
[0153] 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.
[0154] 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.
[0155] 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
pAM.beta.1 permitting replication in Bacillus.
[0156] 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.
[0157] 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.
[0158] 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.
[0159] 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
[0160] 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.
[0161] 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.
[0162] 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.
[0163] 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.
[0164] 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.
[0165] 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.
[0166] 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.
[0167] 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.
[0168] The host cell may also be a eukaryote, such as a mammalian,
insect, plant, or fungal cell. 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).
[0169] 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).
[0170] 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.
[0171] 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.
[0172] 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, Phiebia, Piromyces, Pleurotus, Schizophyllum,
Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or
Trichoderma cell.
[0173] 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 suiphureum, Fusarium torulosum, Fusarium trichothecioides,
Fusarium venenatum, Humicola insolens, Humicola lanuginosa, Mucor
miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium
purpurogenum, Phanerochaete chrysosporium, Phiebia radiata,
Pleurotus eryngii, Thielavia terrestris, Trametes villosa, Trametes
versicolor, Trichoderma harzianum, Trichoderma koningii,
Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma
viride cell.
[0174] 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
[0175] 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
Talaromyces cell. In a more preferred aspect, the cell is a
Talaromyces leycettanus cell. In a most preferred aspect, the cell
is Talaromyces leycettanus Strain CBS398.68.
[0176] 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.
[0177] 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.
[0178] 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.
[0179] 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.
[0180] 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.
[0181] 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
[0182] 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.
The transgenic plant can be dicotyledonous (a dicot) or
monocotyledonous (a monocot).
[0183] 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).
[0184] 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.
[0185] 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.
[0186] Also included within the scope of the present invention are
the progeny of such plants, plant parts, and plant cells.
[0187] 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.
[0188] 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).
[0189] 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.
[0190] 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.
[0191] 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.
[0192] The selectable marker gene and any other parts of the
expression construct may be chosen from those available in the
art.
[0193] 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).
[0194] 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).
[0195] 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.
[0196] 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.
[0197] 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.
[0198] 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 germ plasma. In this way, the
number of generations required to introgress one or more traits
into a particular genetic background is minimized.
[0199] 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
[0200] 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.
[0201] 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.
[0202] 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.
[0203] 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
[0204] The present invention is also directed to the following
processes for using the polypeptides having xylanase activity, or
compositions thereof.
[0205] 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.
[0206] 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.
[0207] 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.
[0208] 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.
[0209] 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.
[0210] 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 et al., 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.
[0211] 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 and Sinitsyn, 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 and Lee, 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 et al., 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.
[0212] Pretreatment.
[0213] 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).
[0214] 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.
[0215] 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.
[0216] 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).
[0217] 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. 2002/0164730). 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.
[0218] 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.
[0219] 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).
[0220] 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).
[0221] 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.
[0222] 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.
[0223] 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).
[0224] 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.
[0225] 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.
[0226] Other examples of suitable pretreatment methods are
described by Schell et al., 2003, Appl. Biochem. and Biotechnol.
105-108: 69-85, and Mosier et al., 2005, Bioresource Technology 96:
673-686, and U.S. Published Application 2002/0164730.
[0227] 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.
[0228] 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.
[0229] 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).
[0230] 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.
[0231] 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.
[0232] 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).
[0233] Saccharification.
[0234] 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.
[0235] 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.
[0236] 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 %.
[0237] The enzyme compositions can comprise any protein useful in
degrading the cellulosic material or xylan-containing material.
[0238] 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.
[0239] 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.
[0240] 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).
[0241] 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.
[0242] 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
[0243] 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).
[0244] 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.
[0245] 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.
[0246] 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).
[0247] 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.
[0248] 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.
[0249] 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.
[0250] 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.
[0251] 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.
[0252] 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 A/S), CELLIC.TM. CTec2 (Novozymes A/S),
CELLUCLAST.TM. (Novozymes A/S), NOVOZYM.TM. 188 (Novozymes A/S),
CELLUZYME.TM. (Novozymes A/S), CEREFLO.TM. (Novozymes A/S), and
ULTRAFLO.TM. (Novozymes A/S), 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.
[0253] 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).
[0254] 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 Cel5A 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_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 (GENBAN K.TM. accession
no. M15665).
[0255] 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).
[0256] 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
02/095014), Penicillium brasilianum IBT 20888 (WO 2007/019442 and
WO 2010/088387), Thielavia terrestris (WO 2011/035029), and
Trichophaea saccata (WO 2007/019442).
[0257] 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.
[0258] Other useful endoglucanases, cellobiohydrolases, and
beta-glucosidases are disclosed in numerous Glycosyl Hydrolase
families using the classification according to Henrissat, 1991, A
classification of glycosyl hydrolases based on amino-acid sequence
similarities, Biochem. J. 280: 309-316, and Henrissat and Bairoch,
1996, Updating the sequence-based classification of glycosyl
hydrolases, Biochem. J. 316: 695-696.
[0259] Other cellulolytic enzymes that may be used in the present
invention are described in WO 98/13465, WO 98/15619, WO 98/15633,
WO 99/06574, WO 99/10481, WO 99/25847, WO 99/31255, WO 02/101078,
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.
[0260] In the processes of the present invention, any GH61
polypeptide having cellulolytic enhancing activity can be used.
[0261] 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).
[0262] 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.
[0263] 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).
[0264] 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.
[0265] 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 substituted 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 the bicyclic compounds include
epicatechin; quercetin; myricetin; taxifolin; kaempferol; morin;
acacetin; naringenin; isorhamnetin; apigenin; cyanidin; cyanin;
kuromanin; keracyanin; or a salt or solvate thereof.
[0266] 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
.delta.-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.
[0267] 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.
[0268] 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.
[0269] 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.
[0270] 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' 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, or about 0.1 mM to about
1 mM.
[0271] 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.
[0272] 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.-5 to about 1 g, about 10.sup.-5 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.
[0273] 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 A/S), CELLIC.TM. HTec (Novozymes
A/S), CELLIC.TM. HTec2 (Novozymes A/S), VISCOZYME.RTM. (Novozymes
A/S), ULTRAFLO.RTM. (Novozymes A/S), PULPZYME.RTM. HC (Novozymes
A/S), 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).
[0274] Examples of additional 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).
[0275] 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).
[0276] 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
Q0UHJ1), and Thielavia terrestris NRRL 8126 (WO 2009/042846).
[0277] 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).
[0278] 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).
[0279] 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 Q0CJP9), Humicola
insolens (WO 2010/014706), Penicillium aurantiogriseum (WO
2009/068565), Talaromyces emersonii (UniProt accession number
Q8X211), and Trichoderma reesei (Uniprot accession number
Q99024).
[0280] 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).
[0281] 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.
[0282] Fermentation.
[0283] 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.
[0284] 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.
[0285] 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.
[0286] 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).
[0287] "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.
[0288] 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.
[0289] 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.
[0290] 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).
[0291] 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.
[0292] 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.
[0293] 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.
[0294] 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).
[0295] 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.
[0296] 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).
[0297] 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.
[0298] It is well known in the art that the organisms described
above can also be used to produce other substances, as described
herein.
[0299] 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.
[0300] 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.
[0301] 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.
[0302] 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.
[0303] Fermentation Products:
[0304] 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.
[0305] 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 and
Singh, 1995, Processes for fermentative production of xylitol--a
sugar substitute, Process Biochemistry 30(2): 117-124; Ezeji et
al., 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.
[0306] 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.
[0307] 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.
[0308] 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.
[0309] 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 and Margaritis, 2004, Empirical modeling of
batch fermentation kinetics for poly(glutamic acid) production and
other microbial biopolymers, Biotechnology and Bioengineering
87(4): 501-515.
[0310] 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 et al., 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, 1997, Anaerobic digestion of
biomass for methane production: A review, Biomass and Bioenergy.
13(1-2): 83-114.
[0311] In another preferred aspect, the fermentation product is
isoprene.
[0312] 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.
[0313] 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 and Lee, 1997,
Membrane-mediated extractive fermentation for lactic acid
production from cellulosic biomass, Appl. Biochem. Biotechnol.
63-65: 435-448.
[0314] In another preferred aspect, the fermentation product is
polyketide.
[0315] Recovery.
[0316] 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
[0317] The present invention also relates to an isolated
polynucleotide encoding a signal peptide comprising or consisting
of amino acids 1 to 17 of SEQ ID NO: 2, amino acids 1 to 16 of SEQ
ID NO: 4, or amino acids 1 to 20 of SEQ ID NO: 6. 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 51
of SEQ ID NO: 1. In another aspect, the polynucleotide encoding the
signal peptide is nucleotides 1 to 48 of SEQ ID NO: 3. In another
aspect, the polynucleotide encoding the signal peptide is
nucleotides 1 to 50, and 114 to 123 of SEQ ID NO: 5.
[0318] The present invention also relates to nucleic acid
constructs, expression vectors and recombinant host cells
comprising such polynucleotides.
[0319] 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. 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.
[0320] 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.
[0321] The gene may be obtained from any prokaryotic, eukaryotic,
or other source.
[0322] The present invention is further described by the following
examples that should not be construed as limiting the scope of the
invention.
EXAMPLES
Strains
[0323] Talaromyces leycettanus Strain CBS398.68 was used as the
source of a polypeptide having xylanase activity. Aspergillus
oryzae MT3568 strain was used for expression of the Talaromyces
leycettanus gene encoding the polypeptide having xylanase activity.
A. oryzae MT3568 is an amdS (acetamidase) disrupted gene derivative
of Aspergillus oryzae JaL355 (WO 02/40694) in which pyrG auxotrophy
was restored by disrupting the A. oryzae acetamidase (amdS)
gene.
Media and Solutions
[0324] YP+2% glucose medium was composed of 1% yeast extract, 2%
peptone and 2% glucose.
[0325] PDA agar plates were composed of potato infusion (potato
infusion was made by boiling 300 g of sliced (washed but unpeeled)
potatoes in water for 30 minutes and then decanting or straining
the broth through cheesecloth. Distilled water was then added until
the total volume of the suspension was one liter, followed by 20 g
of dextrose and 20 g of agar powder. The medium was sterilized by
autoclaving at 15 psi for 15 minutes (Bacteriological Analytical
Manual, 8th Edition, Revision A, 1998).
[0326] LB plates were composed of 10 g of Bacto-Tryptone, 5 g of
yeast extract, 10 g of sodium chloride, 15 g of Bacto-agar, and
deionized water to 1 liter. The medium was sterilized by
autoclaving at 15 psi for 15 minutes (Bacteriological Analytical
Manual, 8th Edition, Revision A, 1998).
[0327] COVE sucrose plates were composed of 342 g Sucrose (Sigma
S-9378), 20 g Agar powder, 20 ml Cove salt solution (26 g
MgSO.sub.4.7H.sub.2O, 26 g KCL, 26 g KH.sub.2PO.sub.4, 50 ml Cove
trace metal solution) and deionized water to 1 liter), and
deionized water to 1 liter). The medium was sterilized by
autoclaving at 15 psi for 15 minutes (Bacteriological Analytical
Manual, 8th Edition, Revision A, 1998). The medium was cooled to
60.degree. C. and added 10 mM acetamide, 15 mM CsCl, Triton X-100
(50 .mu.l/500 ml)).
[0328] Cove trace metal solution was composed of 0.04 g
Na.sub.2B.sub.4O.sub.7.10H.sub.2O, 0.4 g CuSO.sub.4.5H.sub.2O, 1.2
g FeSO.sub.4.7H.sub.2O, 0.7 g MnSO.sub.4.H.sub.2O, 0.8 g
Na.sub.2MoO.sub.4.2H.sub.2O, 10 g ZnSO.sub.4.7H.sub.2O, and
deionized water to 1 liter.
[0329] Dap-4C medium was composed of 20 g Dextrose, 10 g Maltose,
11 g MgSO.sub.4.7H.sub.2O, 1 g KH.sub.2PO.sub.4, 2 g Citric Acid,
5.2 g K.sub.3PO.sub.4.H.sub.2O, 0.5 g Yeast Extract (Difco), 1 ml
Dowfax 63N10 (Dow Chemical Company), 0.5 ml KU6 trace metals
solution, 2.5 g CaCO.sub.3, and deionized water to 1 liter. The
medium was sterilized by autoclaving at 15 psi for 15 minutes
(Bacteriological Analytical Manual, 8th Edition, Revision A, 1998).
Before use, Dap-4C medium was added 3.5 ml sterile 50%
(NH.sub.4).sub.2HPO.sub.4 and 5 ml sterile 20% Lactic Acid per 150
ml medium.
[0330] KU6 trace metals solution was composed of 0.13 g NiCl.sub.2,
2.5 g CuSO.sub.4.5H.sub.2O, 13.9 g FeSO.sub.4.7H.sub.2O, 8.45 g
MnSO.sub.4.H.sub.2O, 6.8 g ZnCl.sub.2, 3 g Citric Acid, and
deionized water to 1 liter.
Example 1: Source of DNA Sequence Information for Talaromyces
leycettanus Strain CBS398.68
[0331] Genomic sequence information was generated by Illumina DNA
sequencing at the Beijing Genome Institute (BGI) in Beijing, China
from genomic DNA isolated from Talaromyces leycettanus Strain
CBS398.68. A preliminary assembly of the genome was analyzed using
the Pedant-Pro.TM. Sequence Analysis Suite (Biomax Informatics AG,
Martinsried, Germany). Gene models constructed by the software were
used as a starting point for detecting GH10 homologues in the
genome. More precise gene models were constructed manually using
multiple known GH10 protein sequences as a guide.
Example 2: Talaromyces leycettanus Strain CBS398.68 Genomic DNA
Extraction
[0332] To generate genomic DNA for PCR amplification, Talaromyces
leycettanus Strain CBS398.68 was propagated on PDA agar plates by
growing at 26.degree. C. for 7 days. Spores harvested from the PDA
plates were used to inoculate 25 ml of YP+2% glucose medium in a
baffled shake flask and incubated at 26.degree. C. for 72 hours
with agitation at 85 rpm.
[0333] Genomic DNA was isolated according to a modified DNeasy
Plant Maxi kit protocol (Qiagen Danmark, Copenhagen, Denmark). The
fungal material from the above culture was harvested by
centrifugation at 14,000.times.g for 2 minutes. The supernatant was
removed and the 0.5 g of the pellet was frozen in liquid nitrogen
with quartz sand and grinded to a fine powder in a pre-chilled
mortar. The powder was transferred to a 15 ml centrifuge tube and
added 5 ml buffer AP1 (preheated to 65.degree. C.) and 10 .mu.l
RNase A stock solution (100 mg/ml) followed by vigorous vortexing.
After incubation for 10 minutes at 65.degree. C. with regular
inverting of the tube, 1.8 ml buffer AP2 was added to the lysate by
gentle mixing followed by incubation on ice for 10 min. The lysate
was then centrifugated at 3000.times.g for 5 minutes at room
temperature and the supernatant was decanted into a QIAshredder
maxi spin column placed in a 50 ml collection tube. This was
followed by centrifugation at 3000.times.g for 5 minutes at room
temperature. The flow-through was transferred into a new 50 ml tube
and added 1.5 volumes of buffer AP3/E followed by vortexing. 15 ml
of the sample was transferred into a DNeasy Maxi spin column placed
in a 50 ml collection tube and centrifuged at 3000.times.g for 5
minutes at room temperature. The flow-through was discarded and 12
ml buffer AW was added to the DNeasy Maxi spin column placed in a
50 ml collection tube and centrifuged at 3000.times.g for 10
minutes at room temperature. After discarding the flow-through,
centrifugation was repeated to dispose of the remaining alcohol.
The DNeasy Maxi spin column was transferred to a new 50 ml tube and
0.5 ml buffer AE (preheated to 70.degree. C.) was added. After
incubation for 5 minutes at room temperature, the sample was eluded
by centrifugation at 3000.times.g for 5 minutes at room
temperature. Elution was repeated with an additional 0.5 ml buffer
AE and the eluates were combined. The concentration of the
harvested DNA was measured by a UV spectrophotometer at 260 nm.
Example 3: Construction of an Aspergillus oryzae Expression Vector
Containing Talaromyces leycettanus Strain CBS398.68 Genomic
Sequence Encoding a Family GH10 Polypeptide Having Xylanase
Activity
[0334] Two synthetic oligonucleotide primers shown below were
designed to PCR amplify the Talaromyces leycettanus Strain
CBS398.68 P24F5Z gene (SEQ ID NO: 1) from the genomic DNA prepared
in Example 2. An IN-FUSION.TM. Cloning Kit (BD Biosciences, Palo
Alto, Calif., USA) was used to clone the fragment directly into the
expression vector pDau109 (WO 2005/042735).
TABLE-US-00001 F-P24F5Z (SEQ ID NO: 7)
5'-ACACAACTGGGGATCCACCATGCGTTTCTCCTTGGCCACTG-3' R-P24F5Z (SEQ ID
NO: 8) 5'-CCCTCTAGATCTCGAGCTAGCAGACGCTGCAGGCCT-3'
Bold letters represent gene sequence. The underlined sequence is
homologous to the insertion sites of pDau109.
[0335] An MJ Research PTC-200 DNA engine was used to perform the
PCR reaction. A Phusion.RTM. High-Fidelity PCR Kit (Finnzymes Oy,
Espoo, Finland) was used for the PCR amplification. The PCR
reaction was composed of 5 .mu.l of 5.times.HF buffer (Finnzymes
Oy, Espoo, Finland), 0.5 .mu.l of dNTPs (10 mM), 0.5 .mu.l of
Phusion.RTM. DNA polymerase (0.2 units/.mu.l) (Finnzymes Oy, Espoo,
Finland), 1 .mu.l of primer F-P24F5Z (5 .mu.M), 1 .mu.l of primer
R-P24F5Z (5 .mu.M), 0.5 .mu.l of Talaromyces leycettanus genomic
DNA (100 ng/.mu.l), and 16.5 .mu.l of deionized water in a total
volume of 25 .mu.l. The PCR conditions were 1 cycle at 95.degree.
C. for 2 minutes. 35 cycles each at 98.degree. C. for 10 seconds,
60.degree. C. for 30 seconds, and 72.degree. C. for 2.5 minutes;
and 1 cycle at 72.degree. C. for 10 minutes. The sample was then
held at 12.degree. C. until removed from the PCR machine.
[0336] The reaction products were isolated by 1.0% agarose gel
electrophoresis using 40 mM Tris base, 20 mM sodium acetate, 1 mM
disodium EDTA (TAE) buffer where a 1168 bp product band was excised
from the gel and purified using an illustra GFX.RTM. PCR DNA and
Gel Band Purification Kit (GE Healthcare Life Sciences, Brondby,
Denmark) according to the manufacturer's instructions. The fragment
was then cloned into Bam HI and Xho I digested pDau109 using an
IN-FUSION.TM. Cloning Kit resulting in plasmid pP24F5Z. Cloning of
the P24F5Z gene into Bam HI-Xho I digested pDau109 resulted in the
transcription of the Talaromyces leycettanus P24F5Z gene under the
control of a NA2-tpi double promoter. NA2-tpi is a modified
promoter from the gene encoding the Aspergillus niger neutral
alpha-amylase in which the untranslated leader has been replaced by
an untranslated leader from the gene encoding the Aspergillus
nidulans triose phosphate isomerase.
[0337] The cloning protocol was performed according to the
IN-FUSION.TM. Cloning Kit instructions generating a P24F5Z GH10
construct. The treated plasmid and insert were transformed into One
Shot.RTM. TOP10F' Chemically Competent E. coli cells (Invitrogen,
Carlsbad, Calif., USA) according to the manufacturer's protocol and
plated onto LB plates supplemented with 0.1 mg of ampicillin per
ml. After incubating at 37.degree. C. overnight, colonies were seen
growing under selection on the LB ampicillin plates. Four colonies
transformed with the P24F5Z GH10 construct were cultivated in LB
medium supplemented with 0.1 mg of ampicillin per ml and plasmid
was isolated with a QIAprep Spin Miniprep Kit (QIAGEN Inc.,
Valencia, Calif., USA) according to the manufacturer's
protocol.
[0338] Isolated plasmids were sequenced with vector primers and
P24F5Z gene specific primers in order to determine a representative
plasmid expression clone that was free of PCR errors.
Example 4: Characterization of the Talaromyces Leycettanus
CBS398.68 Genomic Sequence Encoding a P24F5Z GH10 Polypeptide
Having Xylanase Activity
[0339] DNA sequencing of the Talaromyces leycettanus CBS398.68
P24F5Z GH10 genomic clone was performed with an Applied Biosystems
Model 3700 Automated DNA Sequencer using version 3.1 BIG-DYE.TM.
terminator chemistry (Applied Biosystems, Inc., Foster City,
Calif., USA) and primer walking strategy. Nucleotide sequence data
were scrutinized for quality and all sequences were compared to
each other with assistance of PHRED/PHRAP software (University of
Washington, Seattle, Wash., USA). The sequence obtained was
identical to the sequence from the JGI.
[0340] The nucleotide sequence and deduced amino acid sequence of
the Talaromyces leycettanus P24F5Z gene is shown in SEQ ID NO: 1
and SEQ ID NO: 2, respectively. The coding sequence is 1168 bp
including the stop codon and is interrupted by one intron. The
encoded predicted protein is 364 amino acids. Using the SignalP
program (Nielsen et al., 1997, Protein Engineering 10: 1-6), a
signal peptide of 17 residues was predicted. The predicted mature
protein contains 347 amino acids with a predicted molecular mass of
38.7 kDa and an isoelectric pH of 4.6.
[0341] A comparative pairwise global alignment of amino acid
sequences was determined using the Needleman and Wunsch algorithm
(Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) with gap
open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62
matrix. The alignment showed that the deduced amino acid sequence
of the Talaromyces leycettanus gene encoding the P24F5Z GH10
polypeptide having xylanase activity shares 75.6% identity
(excluding gaps) to the deduced amino acid sequence of a predicted
GH10 family protein from Penicillium canescens (accession number
UNIPROT:C3VEV9) with xylanase activity.
Example 5: Expression of the Talaromyces leycettanus GH10 Xylanase
P24F5Z
[0342] The expression plasmid pP24F5Z was transformed into
Aspergillus oryzae MT3568. Aspergillus oryzae MT3568 is an amdS
(acetamidase) disrupted derivative of JaL355 (WO 02/40694) in which
pyrG auxotrophy was restored in the process of knocking out the A,
oryzae acetamidase (amdS) gene. MT3568 protoplasts are prepared
according to the method of European Patent No. 0238023, pages
14-15, which are incorporated herein by reference.
[0343] Transformants were purified on COVE sucrose selection plates
through single conidia prior to sporulating them on PDA plates.
Production of the Talaromyces leycettanus GH10 polypeptide by the
transformants was analyzed from culture supernatants of 1 ml 96
deep well stationary cultivations at 30.degree. C. in YP+2% glucose
medium. Expression was verified on a E-Page 8% SDS-PAGE 48 well gel
(Invitrogen, Carlsbad, Calif., USA) by Coomassie staining. One
transformant was selected for further work and designated
Aspergillus oryzae 41.4.3.
[0344] For larger scale production, Aspergillus oryzae 41.4.3
spores were spread onto a PDA plate and incubated for five days at
37.degree. C. The confluent spore plate was washed twice with 5 ml
of 0.01% TWEEN.RTM. 20 to maximize the number of spores collected.
The spore suspension was then used to inoculate twenty five 500 ml
flasks containing 100 ml of Dap-4C medium. The culture was
incubated at 30.degree. C. with constant shaking at 100 rpm. At day
four post-inoculation, the culture broth was collected by
filtration through a bottle top MF75 Supor MachV 0.2 .mu.m PES
filter (Thermo Fisher Scientific, Roskilde, Denmark). Fresh culture
broth from this transformant produced a band of GH10 protein of
approximately 50 kDa with smearing indicating possible
glycosylation. The identity of the prominent band as the
Talaromyces leycettanus GH10 polypeptide was verified by peptide
sequencing.
Example 6: Alternative Method for Producing the Talaromyces
leycettanus GH10 Xylanase P24F5Z
[0345] Based on the nucleotide sequence identified as SEQ ID NO: 1,
a synthetic gene can be obtained from a number of vendors such as
Gene Art (GENEART AG BioPark, Josef-Engert-Str. 11, 93053,
Regensburg, Germany) or DNA 2.0 (DNA2.0, 1430 O'Brien Drive, Suite
E, Menlo Park, Calif. 94025, USA). The synthetic gene can be
designed to incorporate additional DNA sequences such as
restriction sites or homologous recombination regions to facilitate
cloning into an expression vector.
[0346] Using the two synthetic oligonucleotide primers F-P24F5Z and
R-P24F5Z described above, a simple PCR reaction can be used to
amplify the full-length open reading frame from the synthetic gene
of SEQ ID NO: 1. The gene can then be cloned into an expression
vector for example as described above and expressed in a host cell,
for example in Aspergillus oryzae as described above.
Example 7: Purification of the Talaromyces leycettanus GH10
Xylanase P24F5Z
[0347] 1000 ml broth of the Aspergillus oryzae expression strain
41.4.3 was adjusted to pH 7.0 and filtrated on 0.22 .mu.m PES
filter (Thermo Fisher Scientific, Roskilde, Denmark). Following,
the filtrate was added 1.8 M ammonium sulphate. The filtrate was
loaded onto a Phenyl Sepharose.TM. 6 Fast Flow column (high sub)
(GE Healthcare, Piscataway, N.J., USA) (with a column volume of 60
mL) equilibrated with 1.8 M ammonium sulphate pH 7.0, 25 mM HEPES
pH7.0. After application the column was washed with 3 column
volumes of equilibration buffer followed by 7 column volumes of 1.0
M ammonium sulphate (the protein kept binding to the column) and
the protein eluted following with 5 column volumes of 25 mM HEPES
pH 7.0 at a flow rate of 15 ml/min. Fractions of 10 mL were
collected and analyzed by SDS-page. The fractions were pooled and
applied to a Sephadex.TM. G-25 (medium) (GE Healthcare, Piscataway,
N.J., USA) column equilibrated in 25 mM HEPES pH 7.0. The fractions
were applied to a SOURCE.TM. 15Q (GE Healthcare, Piscataway, N.J.,
USA) column equilibrated in 25 mM HEPES pH 7.0 (column volumes 60
mL). After application the column was washed with 5 column volumes
equilibration buffer and bound proteins were eluted with a linear
gradient over 20 column volumes from 0-1000 mM sodium chloride.
Fractions of 10 ml were collected and analyzed by SDS-page, and
fractions with the protein were pooled. With two distinct peaks in
the chromatogram two pools, A and B, were made. The protein
concentration was determined by A280/A260 absorbance. The protein
identity was verified by MS/MS on in-gel digested sample confirming
the identity of both fractions.
Example 8: Construction of an Aspergillus oryzae Expression Vector
Containing Talaromyces leycettanus Strain CBS398.68 Genomic
Sequence Encoding a Family GH10 Polypeptide Having Xylanase
Activity
[0348] Two synthetic oligonucleotide primers shown below were
designed to PCR amplify the Talaromyces leycettanus Strain
CBS398.68 P24F61 gene (SEQ ID NO: 3) from the genomic DNA prepared
in Example 2. An IN-FUSION.TM. Cloning Kit (BD Biosciences, Palo
Alto, Calif., USA) was used to clone the fragment directly into the
expression vector pDau109 (WO 2005/042735).
TABLE-US-00002 F-P24F61 (SEQ ID NO: 9)
5'-ACACAACTGGGGATCCACCATGGTCCGTCTTTCCGCTGGA-3' R-P24F61 (SEQ ID NO:
10) 5'-CCCTCTAGATCTCGAGTTACAAGCACTGGGAGTACCACTGG-3'
Bold letters represent gene sequence. The underlined sequence is
homologous to the insertion sites of pDau109.
[0349] An MJ Research PTC-200 DNA engine was used to perform the
PCR reaction. A Phusion.RTM. High-Fidelity PCR Kit (Finnzymes Oy,
Espoo, Finland) was used for the PCR amplification. The PCR
reaction was composed of 5 .mu.l of 5.times.HF buffer (Finnzymes
Oy, Espoo, Finland), 0.5 .mu.l of dNTPs (10 mM), 0.5 .mu.l of
Phusion.RTM. DNA polymerase (0.2 units/.mu.l) (Finnzymes Oy, Espoo,
Finland), 1 .mu.l of primer F-P24F61 (5 .mu.M), 1 .mu.l of primer
R-P24F61 (5 .mu.M), 0.5 .mu.l of Talaromyces leycettanus genomic
DNA (100 ng/.mu.l), and 16.5 .mu.l of deionized water in a total
volume of 25 .mu.l. The PCR conditions were 1 cycle at 95.degree.
C. for 2 minutes. 35 cycles each at 98.degree. C. for 10 seconds,
60.degree. C. for 30 seconds, and 72.degree. C. for 2.5 minutes;
and 1 cycle at 72.degree. C. for 10 minutes. The sample was then
held at 12.degree. C. until removed from the PCR machine.
[0350] The reaction products were isolated by 1.0% agarose gel
electrophoresis using 40 mM Tris base, 20 mM sodium acetate, 1 mM
disodium EDTA (TAE) buffer where a 1708 bp product band was excised
from the gel and purified using an illustra GFX.RTM. PCR DNA and
Gel Band Purification Kit (GE Healthcare Life Sciences, Brondby,
Denmark) according to the manufacturer's instructions. The fragment
was then cloned into Bam HI and Xho I digested pDau109 using an
IN-FUSION.TM. Cloning Kit resulting in plasmid pP24F61. Cloning of
the P24F61 gene into Bam HI-Xho I digested pDau109 resulted in the
transcription of the Talaromyces leycettanus P24F61 gene under the
control of a NA2-tpi double promoter. NA2-tpi is a modified
promoter from the gene encoding the Aspergillus niger neutral
alpha-amylase in which the untranslated leader has been replaced by
an untranslated leader from the gene encoding the Aspergillus
nidulans triose phosphate isomerase.
[0351] The cloning protocol was performed according to the
IN-FUSION.TM. Cloning Kit instructions generating a P24F61 GH10
construct. The treated plasmid and insert were transformed into One
Shot.RTM. TOP10F' Chemically Competent E. coli cells (Invitrogen,
Carlsbad, Calif., USA) according to the manufacturer's protocol and
plated onto LB plates supplemented with 0.1 mg of ampicillin per
ml. After incubating at 37.degree. C. overnight, colonies were seen
growing under selection on the LB ampicillin plates. Four colonies
transformed with the P24F61 GH10 construct were cultivated in LB
medium supplemented with 0.1 mg of ampicillin per ml and plasmid
was isolated with a QIAprep Spin Miniprep Kit (QIAGEN Inc.,
Valencia, Calif., USA) according to the manufacturer's
protocol.
[0352] Isolated plasmids were sequenced with vector primers and
P24F61 gene specific primers in order to determine a representative
plasmid expression clone that was free of PCR errors.
Example 9: Characterization of the Talaromyces leycettanus
CBS398.68 Genomic Sequence Encoding a P24F61 GH10 Polypeptide
Having Xylanase Activity
[0353] DNA sequencing of the Talaromyces leycettanus CBS398.68
P24F61 GH10 genomic clone was performed with an Applied Biosystems
Model 3700 Automated DNA Sequencer using version 3.1 BIG-DYE.TM.
terminator chemistry (Applied Biosystems, Inc., Foster City,
Calif., USA) and primer walking strategy. Nucleotide sequence data
were scrutinized for quality and all sequences were compared to
each other with assistance of PHRED/PHRAP software (University of
Washington, Seattle, Wash., USA). The sequence obtained was
identical to the sequence from the JGI.
[0354] The nucleotide sequence and deduced amino acid sequence of
the Talaromyces leycettanus P24F61 gene is shown in SEQ ID NO: 3
and SEQ ID NO: 4, respectively. The coding sequence is 1708 bp
including the stop codon and is interrupted by eight introns. The
encoded predicted protein is 389 amino acids. Using the SignalP
program (Nielsen et al., 1997, Protein Engineering 10: 1-6), a
signal peptide of 16 residues was predicted. The predicted mature
protein contains 373 amino acids with a predicted molecular mass of
39.6 kDa and an isoelectric pH of 5.2.
[0355] A comparative pairwise global alignment of amino acid
sequences was determined using the Needleman and Wunsch algorithm
(Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) with gap
open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62
matrix. The alignment showed that the deduced amino acid sequence
of the Talaromyces leycettanus gene encoding the P24F61 GH10
polypeptide having xylanase activity shares 76.0% identity
(excluding gaps) to the deduced amino acid sequence of a predicted
GH10 family protein from Talaromyces stipitatus (accession number
SWISSPROT:B8M9H8) with putative xylanase activity.
Example 10: Expression of the Talaromyces leycettanus GH10 Xylanase
P24F61
[0356] The expression plasmid pP24F61 was transformed into
Aspergillus oryzae MT3568. Aspergillus oryzae MT3568 is an amdS
(acetamidase) disrupted derivative of JaL355 (WO 02/40694) in which
pyrG auxotrophy was restored in the process of knocking out the A,
oryzae acetamidase (amdS) gene. MT3568 protoplasts are prepared
according to the method of European Patent No. 0238023, pages
14-15, which are incorporated herein by reference.
[0357] Transformants were purified on COVE sucrose selection plates
through single conidia prior to sporulating them on PDA plates.
Production of the Talaromyces leycettanus GH10 polypeptide by the
transformants was analyzed from culture supernatants of 1 ml 96
deep well stationary cultivations at 30.degree. C. in YP+2% glucose
medium. Expression was verified on a E-Page 8% SDS-PAGE 48 well gel
(Invitrogen, Carlsbad, Calif., USA) by Coomassie staining. One
transformant was selected for further work and designated
Aspergillus oryzae 40.2.3.
[0358] For larger scale production, Aspergillus oryzae 40.2.3
spores were spread onto a PDA plate and incubated for five days at
37.degree. C. The confluent spore plate was washed twice with 5 ml
of 0.01% TWEEN.RTM. 20 to maximize the number of spores collected.
The spore suspension was then used to inoculate twenty five 500 ml
flasks containing 100 ml of Dap-4C medium. The culture was
incubated at 30.degree. C. with constant shaking at 100 rpm. At day
four post-inoculation, the culture broth was collected by
filtration through a bottle top MF75 Supor MachV 0.2 .mu.m PES
filter (Thermo Fisher Scientific, Roskilde, Denmark). Fresh culture
broth from this transformant produced a band of GH10 protein of
approximately 40 kDa. The identity of this band as the Talaromyces
leycettanus GH10 polypeptide was verified by peptide
sequencing.
Example 11: Alternative Method for Producing the Talaromyces
leycettanus GH10 Xylanase P24F61
[0359] Based on the nucleotide sequence identified as SEQ ID NO: 3,
a synthetic gene can be obtained from a number of vendors such as
Gene Art (GENEART AG BioPark, Josef-Engert-Str. 11, 93053,
Regensburg, Germany) or DNA 2.0 (DNA2.0, 1430 O'Brien Drive, Suite
E, Menlo Park, Calif. 94025, USA). The synthetic gene can be
designed to incorporate additional DNA sequences such as
restriction sites or homologous recombination regions to facilitate
cloning into an expression vector.
[0360] Using the two synthetic oligonucleotide primers F-P24F61 and
R-P24F61 described above, a simple PCR reaction can be used to
amplify the full-length open reading frame from the synthetic gene
of SEQ ID NO: 3. The gene can then be cloned into an expression
vector for example as described above and expressed in a host cell,
for example in Aspergillus oryzae as described above.
Example 12: Purification of the Talaromyces leycettanus GH10
Xylanase P24F61
[0361] 1000 ml broth of the Aspergillus oryzae expression strain
40.2.3 was adjusted to pH 7.0 and filtrated on 0.22 .mu.m PES
filter (Thermo Fisher Scientific, Roskilde, Denmark). Following,
the filtrate was added 1.4 M ammonium sulphate. The filtrate was
loaded onto a Phenyl Sepharose.TM. 6 Fast Flow column (high sub)
(GE Healthcare, Piscataway, N.J., USA) (with a column volume of 60
mL) equilibrated with 1.4 M ammonium sulphate pH 7.0, 25 mM HEPES
pH7.0. After application the column was washed with 3 column
volumes of equilibration buffer followed by 7 column volumes of 0.8
M ammonium sulphate (the protein kept binding to the column) and
the protein eluted following with 5 column volumes of 25 mM HEPES
pH 7.0 at a flow rate of 15 ml/min. Fractions of 10 mL were
collected and analyzed by SDS-page. The fractions were pooled and
applied to a Sephadex.TM. G-25 (medium) (GE Healthcare, Piscataway,
N.J., USA) column equilibrated in 25 mM HEPES pH 7.0. The fractions
were applied to a SOURCE.TM. 15Q (GE Healthcare, Piscataway, N.J.,
USA) column equilibrated in 25 mM HEPES pH 7.0 (column volumes 60
mL). After application the column was washed with 5 column volumes
equilibration buffer and bound proteins were eluted with a linear
gradient over 20 column volumes from 0-1000 mM sodium chloride.
Fractions of 10 ml were collected and analyzed by SDS-page. The
enzyme was in the run through and in the first fractions and pooled
accordingly. With two distinct peaks in the chromatogram two pools
were made. The enzyme was concentrated using a centrifugal
concentrator Vivaspin.RTM.20 MWCO 10,000 polyethersulfone membrane
(Sartorius Stedim Biotech GmbH, 37070 Goettingen, Germany). The
protein concentration was determined by A280/A260 absorbance. The
protein identity was verified by MS/MS on in-gel digested
sample.
Example 13: Construction of an Aspergillus oryzae Expression Vector
Containing Talaromyces leycettanus Strain CBS398.68 Genomic
Sequence Encoding a Family GH10 Polypeptide Having Xylanase
Activity
[0362] Two synthetic oligonucleotide primers shown below were
designed to PCR amplify the Talaromyces leycettanus Strain
CBS398.68 P24F62 gene (SEQ ID NO: 5) from the genomic DNA prepared
in Example 2. An IN-FUSION.TM. Cloning Kit (BD Biosciences, Palo
Alto, Calif., USA) was used to clone the fragment directly into the
expression vector pDau109 (WO 2005/042735).
TABLE-US-00003 F-P24F62 (SEQ ID NO: 11)
5'-ACACAACTGGGGATCCACCATGGTCCATCTTTCTTCCCTGGCC-3' R-P24F62 (SEQ ID
NO: 12) 5'-CCCTCTAGATCTCGAGTTACAGGCACTGGTAGTAGTAGGGATTC-3'
Bold letters represent gene sequence. The underlined sequence is
homologous to the insertion sites of pDau109.
[0363] An MJ Research PTC-200 DNA engine was used to perform the
PCR reaction. A Phusion.RTM. High-Fidelity PCR Kit (Finnzymes Oy,
Espoo, Finland) was used for the PCR amplification. The PCR
reaction was composed of 5 .mu.l of 5.times.HF buffer (Finnzymes
Oy, Espoo, Finland), 0.5 .mu.l of dNTPs (10 mM), 0.5 .mu.l of
Phusion.RTM. DNA polymerase (0.2 units/.mu.l) (Finnzymes Oy, Espoo,
Finland), 1 .mu.l of primer F-P24F62 (5 .mu.M), 1 .mu.l of primer
R-P24F62 (5 .mu.M), 0.5 .mu.l of Talaromyces leycettanus genomic
DNA (100 ng/.mu.l), and 16.5 .mu.l of deionized water in a total
volume of 25 .mu.l. The PCR conditions were 1 cycle at 95.degree.
C. for 2 minutes. 35 cycles each at 98.degree. C. for 10 seconds,
60.degree. C. for 30 seconds, and 72.degree. C. for 2.5 minutes;
and 1 cycle at 72.degree. C. for 10 minutes. The sample was then
held at 12.degree. C. until removed from the PCR machine.
[0364] The reaction products were isolated by 1.0% agarose gel
electrophoresis using 40 mM Tris base, 20 mM sodium acetate, 1 mM
disodium EDTA (TAE) buffer where a 1520 bp product band was excised
from the gel and purified using an illustra GFX.RTM. PCR DNA and
Gel Band Purification Kit (GE Healthcare Life Sciences, Brondby,
Denmark) according to the manufacturer's instructions. The fragment
was then cloned into Bam HI and Xho I digested pDau109 using an
IN-FUSION.TM. Cloning Kit resulting in plasmid pP24F62. Cloning of
the P24F62 gene into Bam HI-Xho I digested pDau109 resulted in the
transcription of the Talaromyces leycettanus P24F62 gene under the
control of a NA2-tpi double promoter. NA2-tpi is a modified
promoter from the gene encoding the Aspergillus niger neutral
alpha-amylase in which the untranslated leader has been replaced by
an untranslated leader from the gene encoding the Aspergillus
nidulans triose phosphate isomerase.
[0365] The cloning protocol was performed according to the
IN-FUSION.TM. Cloning Kit instructions generating a P24F62 GH10
construct. The treated plasmid and insert were transformed into One
Shot.RTM. TOP10F' Chemically Competent E. coli cells (Invitrogen,
Carlsbad, Calif., USA) according to the manufacturer's protocol and
plated onto LB plates supplemented with 0.1 mg of ampicillin per
ml. After incubating at 37.degree. C. overnight, colonies were seen
growing under selection on the LB ampicillin plates. Four colonies
transformed with the P24F62 GH10 construct were cultivated in LB
medium supplemented with 0.1 mg of ampicillin per ml and plasmid
was isolated with a QIAprep Spin Miniprep Kit (QIAGEN Inc.,
Valencia, Calif., USA) according to the manufacturer's
protocol.
[0366] Isolated plasmids were sequenced with vector primers and
P24F62 gene specific primers in order to determine a representative
plasmid expression clone that was free of PCR errors.
Example 14: Characterization of the Talaromyces leycettanus
CBS398.68 Genomic Sequence Encoding a P24F62 GH10 Polypeptide
Having Xylanase Activity
[0367] DNA sequencing of the Talaromyces leycettanus CBS398.68
P24F62 GH10 genomic clone was performed with an Applied Biosystems
Model 3700 Automated DNA Sequencer using version 3.1 BIG-DYE.TM.
terminator chemistry (Applied Biosystems, Inc., Foster City,
Calif., USA) and primer walking strategy. Nucleotide sequence data
were scrutinized for quality and all sequences were compared to
each other with assistance of PHRED/PHRAP software (University of
Washington, Seattle, Wash., USA). The sequence obtained was
identical to the sequence from the JGI.
[0368] The nucleotide sequence and deduced amino acid sequence of
the Talaromyces leycettanus P24F62 gene is shown in SEQ ID NO: 5
and SEQ ID NO: 6, respectively. The coding sequence is 1520 bp
including the stop codon and is interrupted by four introns. The
encoded predicted protein is 405 amino acids. Using the SignalP
program (Nielsen et al., 1997, Protein Engineering 10: 1-6), a
signal peptide of 20 residues was predicted. The predicted mature
protein contains 385 amino acids with a predicted molecular mass of
41.6 kDa and an isoelectric pH of 4.7.
[0369] A comparative pairwise global alignment of amino acid
sequences was determined using the Needleman and Wunsch algorithm
(Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) with gap
open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62
matrix. The alignment showed that the deduced amino acid sequence
of the Talaromyces leycettanus gene encoding the P24F62 GH10
polypeptide having xylanase activity shares 83.0% identity
(excluding gaps) to the deduced amino acid sequence of a predicted
GH10 family protein from Penicillium sp. (accession number
GENESEQP:AYL61291) with xylanase activity.
Example 15: Expression of the Talaromyces leycettanus GH10 Xylanase
P24F62
[0370] The expression plasmid pP24F62 was transformed into
Aspergillus oryzae MT3568. Aspergillus oryzae MT3568 is an amdS
(acetamidase) disrupted derivative of JaL355 (WO 02/40694) in which
pyrG auxotrophy was restored in the process of knocking out the A,
oryzae acetamidase (amdS) gene. MT3568 protoplasts are prepared
according to the method of European Patent No. 0238023, pages
14-15, which are incorporated herein by reference.
[0371] Transformants were purified on COVE sucrose selection plates
through single conidia prior to sporulating them on PDA plates.
Production of the Talaromyces leycettanus GH10 polypeptide by the
transformants was analyzed from culture supernatants of 1 ml 96
deep well stationary cultivations at 30.degree. C. in YP+2% glucose
medium. Expression was verified on a E-Page 8% SDS-PAGE 48 well gel
(Invitrogen, Carlsbad, Calif., USA) by Coomassie staining. One
transformant was selected for further work and designated
Aspergillus oryzae 39.3.1.
[0372] For larger scale production, Aspergillus oryzae 39.3.1
spores were spread onto a PDA plate and incubated for five days at
37.degree. C. The confluent spore plate was washed twice with 5 ml
of 0.01% TWEEN.RTM. 20 to maximize the number of spores collected.
The spore suspension was then used to inoculate twenty five 500 ml
flasks containing 100 ml of Dap-4C medium. The culture was
incubated at 30.degree. C. with constant shaking at 100 rpm. At day
four post-inoculation, the culture broth was collected by
filtration through a bottle top MF75 Supor MachV 0.2 .mu.m PES
filter (Thermo Fisher Scientific, Roskilde, Denmark). Fresh culture
broth from this transformant produced a band of GH10 protein of
approximately 50 kDa. The identity of this band as the Talaromyces
leycettanus GH10 polypeptide was verified by peptide
sequencing.
Example 16: Alternative Method for Producing the Talaromyces
leycettanus GH10 Xylanase P24F62
[0373] Based on the nucleotide sequence identified as SEQ ID NO: 5,
a synthetic gene can be obtained from a number of vendors such as
Gene Art (GENEART AG BioPark, Josef-Engert-Str. 11, 93053,
Regensburg, Germany) or DNA 2.0 (DNA2.0, 1430 O'Brien Drive, Suite
E, Menlo Park, Calif. 94025, USA). The synthetic gene can be
designed to incorporate additional DNA sequences such as
restriction sites or homologous recombination regions to facilitate
cloning into an expression vector.
[0374] Using the two synthetic oligonucleotide primers F-P24F62 and
R-P24F62 described above, a simple PCR reaction can be used to
amplify the full-length open reading frame from the synthetic gene
of SEQ ID NO: 5. The gene can then be cloned into an expression
vector for example as described above and expressed in a host cell,
for example in Aspergillus oryzae as described above.
Example 17: Purification of the Talaromyces leycettanus GH10
Xylanase P24F62
[0375] 1000 ml broth of the Aspergillus oryzae expression strain
39.3.1 was adjusted to pH 7.0 and filtrated on 0.22 .mu.m PES
filter (Thermo Fisher Scientific, Roskilde, Denmark). Following,
the filtrate was added 1.4 M ammonium sulphate. The filtrate was
loaded onto a Phenyl Sepharose.TM. 6 Fast Flow column (high sub)
(GE Healthcare, Piscataway, N.J., USA) (with a column volume of 60
mL) equilibrated with 1.4 M ammonium sulphate pH 7.0, 25 mM HEPES
pH7.0. After application the column was washed with 3 column
volumes of equilibration buffer followed by 7 column volumes of 0.8
M ammonium sulphate (the protein kept binding to the column) and
the protein eluted following with 5 column volumes of 25 mM HEPES
pH 7.0 at a flow rate of 15 ml/min. Fractions of 10 mL were
collected and analyzed by SDS-page. The fractions were pooled and
applied to a Sephadex.TM. G-25 (medium) (GE Healthcare, Piscataway,
N.J., USA) column equilibrated in 25 mM HEPES pH 7.0. The fractions
were applied to a SOURCE.TM. 15Q (GE Healthcare, Piscataway, N.J.,
USA) column equilibrated in 25 mM HEPES pH 7.0 (column volumes 60
mL). After application the column was washed with 5 column volumes
equilibration buffer and bound proteins were eluted with a linear
gradient over 20 column volumes from 0-1000 mM sodium chloride.
Fractions of 10 ml were collected and analyzed by SDS-page. The
enzyme was in the run through and in the first fractions and pooled
accordingly. With two distinct peaks in the chromatogram two pools
were made. The enzyme was concentrated using a centrifugal
concentrator Vivaspin.RTM.20 MWCO 10,000 polyethersulfone membrane
(Sartorius Stedim Biotech GmbH, 37070 Goettingen, Germany). The
protein concentration was determined by A280/A260 absorbance. The
protein identity was verified by MS/MS on in-gel digested
sample.
Example 18: Determination of Xylanase Activity of the Xylanases
According to the Invention Enzyme Activity of the Talaromyces
leycettanus GH10 Xylanase P24F5Z
[0376] Pool A and pool B of the purified xylanase were diluted in
distilled water with 0.01% Triton X-100 (100 ppm) based on a
dose-response curve to be in the linear range. The substrate was
AZCL-arabinoxylan (Megazyme Wicklow, Ireland) in 0.2% (w/v) in pH
6.0 in (50 mM phosphoric acid, 50 mM acetic acid, 50 mM boric
acid), 50 mM KCl, 1 mM CaCl.sub.2, 0.01% Triton X-100; pH adjusted
with NaOH. The substrate was equilibrated to 37.degree. C. 1000
.mu.l 0.2% (w/v) AZCL-arabinoxylan was mixed with 20 .mu.l diluted
enzyme. The tube was incubated at 37.degree. C. for 15 minutes on
an Eppendorf Comfort thermomixer (Eppendorf AG, Hamburg, Germany)
at 1.400 rpm. The reaction was stopped by placing the tube on ice
for 5 minutes. The eppendorf tube was centrifuged 5 minutes with
10,000 rpm at 4.degree. C. 200 microliter supernatant is
transferred to a flat bottom MicroWell plate (NUNC, Roskilde,
Denmark) and the absorbance was read at 595 nm in a
spectrophotometer. Relative to Shearzyme (Novozymes, Bagsvaerd,
Denmark), the specific activity on AZCL-arabinoxylan was found to
be 2% for pool A and 4% for pool B.
Enzyme Activity of the Talaromyces leycettanus GH10 Xylanase
P24F61
[0377] The purified xylanase was diluted in distilled water with
0.01% Triton X-100 (100 ppm) based on a dose-response curve to be
in the linear range. The substrate was AZCL-arabinoxylan (Megazyme
Wicklow, Ireland) in 0.2% (w/v) in pH 6.0 in (50 mM phosphoric
acid, 50 mM acetic acid, 50 mM boric acid), 50 mM KCl, 1 mM
CaCl.sub.2, 0.01% Triton X-100; pH adjusted with NaOH. The
substrate was equilibrated to 37.degree. C. 1000 .mu.l 0.2% (w/v)
AZCL-arabinoxylan was mixed with 20 .mu.l diluted enzyme. The tube
was incubated at 37.degree. C. for 15 minutes on an Eppendorf
Comfort thermomixer (Eppendorf AG, Hamburg, Germany) at 1.400 rpm.
The reaction was stopped by placing the tube on ice for 5 minutes.
The eppendorf tube was centrifuged 5 minutes with 10,000 rpm at
4.degree. C. 200 microliter supernatant is transferred to a flat
bottom MicroWell plate (NUNC, Roskilde, Denmark) and the absorbance
was read at 595 nm in a spectrophotometer. Relative to Shearzyme
(Novozymes, Bagsvaerd, Denmark), the specific activity on
AZCL-arabinoxylan was found to be 830% for the purified
xylanase.
Enzyme Activity of the Talaromyces leycettanus GH10 Xylanase
P24F62
[0378] The purified xylanase was diluted in distilled water with
0.01% Triton X-100 (100 ppm) based on a dose-response curve to be
in the linear range. The substrate was AZCL-arabinoxylan (Megazyme
Wicklow, Ireland) in 0.2% (w/v) in pH 6.0 in (50 mM phosphoric
acid, 50 mM acetic acid, 50 mM boric acid), 50 mM KCl, 1 mM
CaCl.sub.2, 0.01% Triton X-100; pH adjusted with NaOH. The
substrate was equilibrated to 37.degree. C. 1000 .mu.l 0.2% (w/v)
AZCL-arabinoxylan was mixed with 20 .mu.l diluted enzyme. The tube
was incubated at 37.degree. C. for 15 minutes on an Eppendorf
Comfort thermomixer (Eppendorf AG, Hamburg, Germany) at 1.400 rpm.
The reaction was stopped by placing the tube on ice for 5 minutes.
The eppendorf tube was centrifuged 5 minutes with 10,000 rpm at
4.degree. C. 200 microliter supernatant is transferred to a flat
bottom MicroWell plate (NUNC, Roskilde, Denmark) and the absorbance
was read at 595 nm in a spectrophotometer. Relative to Shearzyme
(Novozymes, Bagsvaerd, Denmark), the specific activity on
AZCL-arabinoxylan was found to be 950% for the purified
xylanase.
[0379] 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.
[0380] The present invention is further described by the following
numbered paragraphs:
[1] An isolated polypeptide having xylanase activity, selected from
the group consisting of:
[0381] (a) a polypeptide having at least 77%, e.g., at least 78%,
at least 79%, 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, or a
polypeptide having at least 77%, e.g., at least 78%, at least 79%,
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, or a polypeptide having at
least 85%, e.g., at least 86%, 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: 6;
[0382] (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, or SEQ ID NO: 5, (ii) the cDNA sequence thereof, or (iii) the
full-length complement of (i) or (ii);
[0383] (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, or SEQ ID NO: 5; or the cDNA sequence thereof;
[0384] (d) a variant of the mature polypeptide of SEQ ID NO: 2, SEQ
ID NO: 4, or SEQ ID NO: 6 comprising a substitution, deletion,
and/or insertion at one or more positions; and
[0385] (e) a fragment of the polypeptide of (a), (b), (c), or (d)
that has xylanase activity.
[2] The polypeptide of paragraph 1, having at least 77%, at least
78%, at least 79%, 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; having
at least 77%, at least 78%, at least 79%, 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; or having at least 85%, at least 86%, 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: 6. [3] The polypeptide of
paragraph 1 or 2, which is encoded by a polynucleotide that
hybridizes under low stringency conditions, or low-medium
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, or SEQ ID NO: 5,
(ii) the cDNA sequence thereof, or (iii) the full-length complement
of (i) or (ii). [4] The polypeptide of any of paragraphs 1-3, which
is encoded by a polynucleotide having at least 60%, 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% sequence identity to the mature polypeptide coding sequence
of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5 or the cDNA sequence
thereof. [5] The polypeptide of any of paragraphs 1-4, comprising
or consisting of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6 or the
mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6.
[6] The polypeptide of paragraph 5, wherein the mature polypeptide
is amino acids 18 to 364 of SEQ ID NO: 2, amino acids 17 to 389 of
SEQ ID NO: 4, or amino acids 21 to 405 of SEQ ID NO: 6. [7] The
polypeptide of any of paragraphs 1-4, which is a variant of the
mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6
comprising a substitution, deletion, and/or insertion at one or
more positions. [8] The polypeptide of paragraph 1, which is a
fragment of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6, wherein
the fragment has xylanase activity. [9] An isolated polypeptide
comprising a catalytic domain selected from the group consisting
of:
[0386] (a) a catalytic domain having at least 77% sequence identity
to the catalytic domain of SEQ ID NO: 2 or SEQ ID NO: 4, or a
catalytic domain having at least 85% sequence identity to the
catalytic domain of SEQ ID NO: 6;
[0387] (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, SEQ ID NO: 3, or SEQ ID NO: 5;
[0388] (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,
or SEQ ID NO: 6; and
[0389] (d) a fragment of a catalytic domain of (a), (b), or (c),
which has xylanase activity.
[10] The polypeptide of paragraph 9, comprising or consisting of
the catalytic domain of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO:
6. [11] The polypeptide of paragraph 10, wherein the catalytic
domain is amino acids 18 to 364 of SEQ ID NO: 2, amino acids 17 to
326 of SEQ ID NO: 4, or amino acids 21 to 337 of SEQ ID NO: 6. [12]
The polypeptide of any of paragraphs 9-11, further comprising a
cellulose binding domain. [13] The polypeptide of any of paragraphs
1-12, which is encoded by the polynucleotide contained in
Talaromyces leycettanus Strain CBS398.68. [14] A composition
comprising the polypeptide of any of paragraphs 1-13. [15] An
isolated polynucleotide encoding the polypeptide of any of
paragraphs 1-13. [16] A nucleic acid construct or expression vector
comprising the polynucleotide of paragraph 15 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 of paragraph 15 operably linked to
one or more control sequences that direct the production of the
polypeptide. [18] A method of producing the polypeptide of any of
paragraphs 1-13, comprising:
[0390] (a) cultivating a cell, which in its wild-type form produces
the polypeptide, under conditions conducive for production of the
polypeptide; and
[0391] (b) recovering the polypeptide.
[19] A method of producing a polypeptide having xylanase activity,
comprising:
[0392] (a) cultivating the host cell of paragraph 17 under
conditions conducive for production of the polypeptide; and
[0393] (b) recovering the polypeptide.
[20] A transgenic plant, plant part or plant cell comprising a
polynucleotide encoding the polypeptide of any of paragraphs 1-13.
[21] A method of producing a polypeptide having xylanase activity,
comprising:
[0394] (a) cultivating the transgenic plant or plant cell of
paragraph 20 under conditions conducive for production of the
polypeptide; and
[0395] (b) recovering the polypeptide.
[22] An isolated polynucleotide encoding a signal peptide
comprising or consisting of amino acids 1 to 17 of SEQ ID NO: 2,
amino acids 1 to 16 of SEQ ID NO: 4, or amino acids 1 to 20 of SEQ
ID NO: 6. [23] A nucleic acid construct or expression vector
comprising a gene encoding a protein operably linked to the
polynucleotide of paragraph 22, wherein the gene is foreign to the
polynucleotide encoding the signal peptide. [24] A recombinant host
cell comprising a gene encoding a protein operably linked to the
polynucleotide of paragraph 22, wherein the gene is foreign to the
polynucleotide encoding the signal peptide. [25] A method of
producing a protein, comprising:
[0396] (a) cultivating a recombinant host cell comprising a gene
encoding a protein operably linked to the polynucleotide of
paragraph 22, wherein the gene is foreign to the polynucleotide
encoding the signal peptide, under conditions conducive for
production of the protein; and
[0397] (b) recovering the protein.
[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 any of
paragraphs 1-13. [27] The process of paragraph 26, wherein the
cellulosic material or xylan-containing material is pretreated.
[28] The process of paragraph 26 or 27, 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] The process of paragraph 28,
wherein the hemicellulase is one or more enzymes selected from the
group consisting of a xylanase, an acetylxylan esterase, a feruloyl
esterase, an arabinofuranosidase, a xylosidase, and a
glucuronidase. [30] The process of paragraph 28, wherein the
cellulase is one or more enzymes selected from the group consisting
of an endoglucanase, a cellobiohydrolase, and a beta-glucosidase.
[31] The process of any of paragraphs 26-30, further comprising
recovering the degraded cellulosic material or xylan-containing
material. [32] The process of paragraph 31, wherein the degraded
cellulosic material or xylan-containing material is a sugar. [33]
The process of paragraph 32, wherein the sugar is selected from the
group consisting of glucose, xylose, mannose, galactose, and
arabinose. [34] A process for producing a fermentation product,
comprising:
[0398] (a) saccharifying a cellulosic material or xylan-containing
material with an enzyme composition in the presence of the
polypeptide having xylanase activity of any of paragraphs 1-13;
[0399] (b) fermenting the saccharified cellulosic material or
xylan-containing material with one or more fermenting
microorganisms to produce the fermentation product; and
[0400] (c) recovering the fermentation product from the
fermentation.
[35] The process of paragraph 34, wherein the cellulosic material
or xylan-containing material is pretreated. [36] The process of
paragraph 34 or 35, 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] The process of paragraph 36, wherein the hemicellulase is one
or more enzymes selected from the group consisting of a xylanase,
an acetylxylan esterase, a feruloyl esterase, an
arabinofuranosidase, a xylosidase, and a glucuronidase. [38] The
process of paragraph 36, wherein the cellulase is one or more
enzymes selected from the group consisting of an endoglucanase, a
cellobiohydrolase, and a beta-glucosidase. [39] The process of any
of paragraphs 34-38, wherein steps (a) and (b) are performed
simultaneously in a simultaneous saccharification and fermentation.
[40] The process of any of paragraphs 34-39, wherein the
fermentation product is an alcohol, an alkane, a cycloalkane, an
alkene, an amino acid, a gas, isoprene, a ketone, an organic acid,
or polyketide. [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 any of paragraphs
1-13. [42] The process of paragraph 41, wherein the fermenting of
the cellulosic material or xylan-containing material produces a
fermentation product. [43] The process of paragraph 42, further
comprising recovering the fermentation product from the
fermentation. [44] The process of any of paragraphs 41-43, wherein
the cellulosic material or xylan-containing material is pretreated
before saccharification. [45] The process of any of paragraphs
41-44, 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] The process of paragraph 45, wherein the hemicellulase is one
or more enzymes selected from the group consisting of a xylanase,
an acetylxylan esterase, a feruloyl esterase, an
arabinofuranosidase, a xylosidase, and a glucuronidase. [47] The
process of paragraph 45, wherein the cellulase is one or more
enzymes selected from the group consisting of an endoglucanase, a
cellobiohydrolase, and a beta-glucosidase. [48] The process of any
of paragraphs 41-47, wherein the fermentation product is an
alcohol, an alkane, a cycloalkane, an alkene, an amino acid, a gas,
isoprene, a ketone, an organic acid, or polyketide.
Sequence CWU 1
1
1211168DNATalaromyces leycettanus 1atgcgtttct ccttggccac tgcagctctt
ctcgctggcc ctgccctggc agcgccacca 60gctcctcgtc acgatgacaa ggatgttggg
ctcaacgccc tggcccagag agcaggcaag 120ctctggttcg gcactgctgc
tgatatcccc ggcaccgacg agacgaccga tgctgcgtac 180ctaaaaatct
tggaaaatcc cgccaacttc ggcgagatca cccctgccaa cgccatgaag
240gtacggagct gcttgacaag tcggaggatc atgcctacga gcgaacaaac
ttccgatgct 300gacgcattga cagttcatgt acaccgagcc agagcagaac
gtgttcaact acaccggcgg 360tgactacgtc ctgaacctcg ccgagcgcca
cggccagcgt gtccgctgcc acaacctcgt 420ctgggccagc cagctgtccg
acttcgtcaa caacggcaac tggaccaagg agtccctcac 480ggccgtgatg
cggaaccaca tcttccacgt cgtccagcac ttcggccggc gctgctactc
540gtgggatgtc gtcaacgagg ccctcaacgg cgacggcacc ttctcctcca
gcatctggta 600cgacaccatc ggcgaggact acttctacct cgccttccag
tacgcccagg aggccctcgc 660ggagatccac gccaacgacg tcaagctcta
ctacaacgac tacggcatcg agaaccccgg 720caccaaggcc gatgccgtgc
acaacctcgt caaggagctg cgcaagcgcg acatccgcat 780cgacggcatc
ggtctcgagt cccacttcga ggtcggtttc accccctccc tacaggacca
840gctcagcacc aagcagggct acatcgcgct cggtctcgac gtcgccatca
ccgagctgga 900cgtgcgcttc acccaggccc cttactacga tgccgcgggc
gagaagcagc aggcccagga 960ctactatacc agcgtttcta gctgcatcga
ggccggcccc aagtgcatcg gtatcaccgt 1020ctgggacttc gatgacaagt
actcgtgggt tccttacact ttcgccggcc agggtggtgc 1080agatatctac
aatgctacct tgcaggccaa gcctgcctac tatgccattg ccgatgctct
1140tcagggcaag gcctgcagcg tctgctag 11682364PRTTalaromyces
leycettanus 2Met Arg Phe Ser Leu Ala Thr Ala Ala Leu Leu Ala Gly
Pro Ala Leu 1 5 10 15 Ala Ala Pro Pro Ala Pro Arg His Asp Asp Lys
Asp Val Gly Leu Asn 20 25 30 Ala Leu Ala Gln Arg Ala Gly Lys Leu
Trp Phe Gly Thr Ala Ala Asp 35 40 45 Ile Pro Gly Thr Asp Glu Thr
Thr Asp Ala Ala Tyr Leu Lys Ile Leu 50 55 60 Glu Asn Pro Ala Asn
Phe Gly Glu Ile Thr Pro Ala Asn Ala Met Lys 65 70 75 80 Phe Met Tyr
Thr Glu Pro Glu Gln Asn Val Phe Asn Tyr Thr Gly Gly 85 90 95 Asp
Tyr Val Leu Asn Leu Ala Glu Arg His Gly Gln Arg Val Arg Cys 100 105
110 His Asn Leu Val Trp Ala Ser Gln Leu Ser Asp Phe Val Asn Asn Gly
115 120 125 Asn Trp Thr Lys Glu Ser Leu Thr Ala Val Met Arg Asn His
Ile Phe 130 135 140 His Val Val Gln His Phe Gly Arg Arg Cys Tyr Ser
Trp Asp Val Val 145 150 155 160 Asn Glu Ala Leu Asn Gly Asp Gly Thr
Phe Ser Ser Ser Ile Trp Tyr 165 170 175 Asp Thr Ile Gly Glu Asp Tyr
Phe Tyr Leu Ala Phe Gln Tyr Ala Gln 180 185 190 Glu Ala Leu Ala Glu
Ile His Ala Asn Asp Val Lys Leu Tyr Tyr Asn 195 200 205 Asp Tyr Gly
Ile Glu Asn Pro Gly Thr Lys Ala Asp Ala Val His Asn 210 215 220 Leu
Val Lys Glu Leu Arg Lys Arg Asp Ile Arg Ile Asp Gly Ile Gly 225 230
235 240 Leu Glu Ser His Phe Glu Val Gly Phe Thr Pro Ser Leu Gln Asp
Gln 245 250 255 Leu Ser Thr Lys Gln Gly Tyr Ile Ala Leu Gly Leu Asp
Val Ala Ile 260 265 270 Thr Glu Leu Asp Val Arg Phe Thr Gln Ala Pro
Tyr Tyr Asp Ala Ala 275 280 285 Gly Glu Lys Gln Gln Ala Gln Asp Tyr
Tyr Thr Ser Val Ser Ser Cys 290 295 300 Ile Glu Ala Gly Pro Lys Cys
Ile Gly Ile Thr Val Trp Asp Phe Asp 305 310 315 320 Asp Lys Tyr Ser
Trp Val Pro Tyr Thr Phe Ala Gly Gln Gly Gly Ala 325 330 335 Asp Ile
Tyr Asn Ala Thr Leu Gln Ala Lys Pro Ala Tyr Tyr Ala Ile 340 345 350
Ala Asp Ala Leu Gln Gly Lys Ala Cys Ser Val Cys 355 360
31708DNATalaromyces leycettanus 3atggtccgtc tttccgctgg acttatcgtc
ctccccctcg tgtccgccgc ggccgtcgat 60ctccagagcc gccaggcggc acagagcatc
aacaccctca tccaggccaa gggcaagaag 120tactggggca cttgcgccga
tgagggccga ttgaccgaga actcgcaaaa cccggccatc 180gccaaggcgg
actttggcca ggtgacgcca gagaacagca tgaagtggga tgctactgag
240cgttagtcag gatgtccatg tgcaatatat agatggatgg ctaactgctg
ttgatgtgta 300gcaagccagg gccagttcaa ctttgctcag gctgattggt
tggtaagtga agctggcatg 360ttgttcagat cctagactgt acggctggct
gactgttctc aaggtcaact gggcgcagca 420aaatggcaag ctgatccgag
gccacaacct gggtgagtcc tgttcatcat cacaatcgtc 480atagtagcta
cgaacacgct aacttatgtg tcaacagtgt ggcactccca gctcccatcc
540tgggtgtgcg gtatcaccga caagacggca ttgaccaatg ccatgaccaa
ccacatcacc 600accctggtga gccgctataa ggggaagatc tatgcctggg
taagtgtttt tcttttctct 660actgatatca gttccagaga aacggaggcc
actgacagtt gtacaggacg tcgtcaacga 720acccttcaac gaagatggaa
gtcttcgtca gacctgcttc tacaacgtca tcggacctga 780ctacatcaag
attgccttcc aaacagctcg tgcggccgat ccgaacgcga agctctacgt
840caatgattac aagtaagact gatcaagtcc cagccttagc tgcgttaatc
cgctgacatt 900cataacctca gccttgactc cgcttcctac gccaagacca
ccggcgtggc gaaccaggtc 960aagcagtgga ttgcacaggg tgtcccgatt
gacggtattg gttctgagtc tcaccttagg 1020tatagctgac tcccatgtct
caagtcaatg ctagttgtga acgtgtactt acgactttca 1080aaaacagcgc
cggtgcagga gcgggtgtgc cagctgccct gcaagtgctc gccaattccg
1140gagtctccga ggtcgcgatc accgaactcg atatcgccca agcctcatcc
actgactatg 1200acaacgtaag aagaactctt ctgatatcct ctttaaacca
taaagtgtcg tgtgactaac 1260actctttttt tttcaacata ggtcgcccaa
gcctgcctga acgtcgcgaa gtgtgttggt 1320atcacttcct ggggtatctc
tgataaggtc cgtttcatgc ctgcatttcc cttccctgaa 1380agacaagatg
tgtctttcca ttgattgctg acgcctaaat caataacagg actcctggcg
1440ctccagcgag aaccctgatc tcttcgacag caactatcag cccaaggctg
cctacaatgc 1500tcttgtgacc ttgctcggtg gaagctccgg ctccggctct
ggctctggct ctggctctgg 1560ctctggctca ggctcaggct caggctcagg
ctccggccag gctcaacact ggggtcagtg 1620tggtggcgaa ggctggaccg
gaccaacgtc ctgtgtctct ccatacactt gccagtacca 1680gaaccagtgg
tactcccagt gcttgtaa 17084389PRTTalaromyces leycettanus 4Met Val Arg
Leu Ser Ala Gly Leu Ile Val Leu Pro Leu Val Ser Ala 1 5 10 15 Ala
Ala Val Asp Leu Gln Ser Arg Gln Ala Ala Gln Ser Ile Asn Thr 20 25
30 Leu Ile Gln Ala Lys Gly Lys Lys Tyr Trp Gly Thr Cys Ala Asp Glu
35 40 45 Gly Arg Leu Thr Glu Asn Ser Gln Asn Pro Ala Ile Ala Lys
Ala Asp 50 55 60 Phe Gly Gln Val Thr Pro Glu Asn Ser Met Lys Trp
Asp Ala Thr Glu 65 70 75 80 Pro Ser Gln Gly Gln Phe Asn Phe Ala Gln
Ala Asp Trp Leu Val Asn 85 90 95 Trp Ala Gln Gln Asn Gly Lys Leu
Ile Arg Gly His Asn Leu Val Trp 100 105 110 His Ser Gln Leu Pro Ser
Trp Val Cys Gly Ile Thr Asp Lys Thr Ala 115 120 125 Leu Thr Asn Ala
Met Thr Asn His Ile Thr Thr Leu Val Ser Arg Tyr 130 135 140 Lys Gly
Lys Ile Tyr Ala Trp Asp Val Val Asn Glu Pro Phe Asn Glu 145 150 155
160 Asp Gly Ser Leu Arg Gln Thr Cys Phe Tyr Asn Val Ile Gly Pro Asp
165 170 175 Tyr Ile Lys Ile Ala Phe Gln Thr Ala Arg Ala Ala Asp Pro
Asn Ala 180 185 190 Lys Leu Tyr Val Asn Asp Tyr Asn Leu Asp Ser Ala
Ser Tyr Ala Lys 195 200 205 Thr Thr Gly Val Ala Asn Gln Val Lys Gln
Trp Ile Ala Gln Gly Val 210 215 220 Pro Ile Asp Gly Ile Gly Ser Glu
Ser His Leu Ser Ala Gly Ala Gly 225 230 235 240 Ala Gly Val Pro Ala
Ala Leu Gln Val Leu Ala Asn Ser Gly Val Ser 245 250 255 Glu Val Ala
Ile Thr Glu Leu Asp Ile Ala Gln Ala Ser Ser Thr Asp 260 265 270 Tyr
Asp Asn Val Ala Gln Ala Cys Leu Asn Val Ala Lys Cys Val Gly 275 280
285 Ile Thr Ser Trp Gly Ile Ser Asp Lys Asp Ser Trp Arg Ser Ser Glu
290 295 300 Asn Pro Asp Leu Phe Asp Ser Asn Tyr Gln Pro Lys Ala Ala
Tyr Asn 305 310 315 320 Ala Leu Val Thr Leu Leu Gly Gly Ser Ser Gly
Ser Gly Ser Gly Ser 325 330 335 Gly Ser Gly Ser Gly Ser Gly Ser Gly
Ser Gly Ser Gly Ser Gly Ser 340 345 350 Gly Gln Ala Gln His Trp Gly
Gln Cys Gly Gly Glu Gly Trp Thr Gly 355 360 365 Pro Thr Ser Cys Val
Ser Pro Tyr Thr Cys Gln Tyr Gln Asn Gln Trp 370 375 380 Tyr Ser Gln
Cys Leu 385 51520DNATalaromyces leycettanus 5atggtccatc tttcttccct
ggccctggct ttggccgccg gctcgcagct gtatgtgatc 60catgccatga ctcgagaagt
gctcccaaaa ctgactccaa gtctcaatct tagtgcccaa 120gctgcaggtc
ttaacactgc tgccaaagcg attggaaagc tctatttcgg taccgcaacc
180gacaacccgg agctgtccga cagcacatac atgcaggaga cggataacac
cgatgatttc 240ggccaactca ccccagctaa ctccatgaag gttcgctgac
atcttagttc cccccccctt 300ttgggaatct gcgcggagat atgctgagcc
ttcaaaacta gtgggatgcc accgagccct 360ctcagaacac cttcaccttc
accaacggtg atcagatcgc aaaccttgct aagagcaacg 420gtcagatgct
gagatgccac aacctggtgt ggtacaacca gttgcccagc tggggtaagc
480aaccggttct gttaatatca tcagcgtgac cgcatcgatc gtattgcgcg
gagattggaa 540agatttgcaa gctaatgtca ctacagtcac cagcggatct
tggaccaatg ccacgcttct 600tgcggccatg aagaaccaca tcaccaacgt
tgtgacccac tacaagggac agtgctacgc 660ttgggatgtt gtcaacgaag
gtacgtttcg attcggcttc cctcggaccg tatctgcagg 720caaaaaggtc
aatcaattga caatcgtgat ccccagctct caacgatgat ggcacctacc
780gatccaatgt cttctatcag tacatcggcg aggcatacat tcccattgcc
tttgcgaccg 840ctgccgccgc cgatccaaac gcgaagctct actacaacga
ctacaacatt gagtaccccg 900gcgccaaggc caccgccgcc cagaacatcg
tcaagatggt caaggcttac ggcgcgaaaa 960tcgacggtgt cggtctgcaa
tctcacttca tcgttggcag cacccctagc cagagctccc 1020agcagagcaa
catggctgct ttcaccgcgc tcggcgtcga ggtcgccatc accgaactgg
1080atatccgcat gacgttgcct tccaccagtg ctctcttggc ccagcaatcc
accgattacc 1140agagcactgt gtcggcttgc gtgaacactc cgaagtgcat
tggtatcacc ctctgggact 1200ggaccgacaa gtactcctgg gttcccaaca
ccttctccgg ccaaggtgac gcctgcccct 1260gggattctaa ctaccagaag
aagcctgcct actacggtat cttgactgcg ctcggaggca 1320gcgcttccac
ctccaccacc accactctgg tgacctccac caggacttcg actacgacca
1380gcacttcggc cacctccacg tctactggcg ttgctcagca ctggggccag
tgcggtggta 1440tcggctggac agggccgact acctgcgcta gcccctacac
ctgccaggaa ctgaatccct 1500actactacca gtgcctgtaa
15206405PRTTalaromyces leycettanus 6Met Val His Leu Ser Ser Leu Ala
Leu Ala Leu Ala Ala Gly Ser Gln 1 5 10 15 Leu Ala Gln Ala Ala Gly
Leu Asn Thr Ala Ala Lys Ala Ile Gly Lys 20 25 30 Leu Tyr Phe Gly
Thr Ala Thr Asp Asn Pro Glu Leu Ser Asp Ser Thr 35 40 45 Tyr Met
Gln Glu Thr Asp Asn Thr Asp Asp Phe Gly Gln Leu Thr Pro 50 55 60
Ala Asn Ser Met Lys Trp Asp Ala Thr Glu Pro Ser Gln Asn Thr Phe 65
70 75 80 Thr Phe Thr Asn Gly Asp Gln Ile Ala Asn Leu Ala Lys Ser
Asn Gly 85 90 95 Gln Met Leu Arg Cys His Asn Leu Val Trp Tyr Asn
Gln Leu Pro Ser 100 105 110 Trp Val Thr Ser Gly Ser Trp Thr Asn Ala
Thr Leu Leu Ala Ala Met 115 120 125 Lys Asn His Ile Thr Asn Val Val
Thr His Tyr Lys Gly Gln Cys Tyr 130 135 140 Ala Trp Asp Val Val Asn
Glu Ala Leu Asn Asp Asp Gly Thr Tyr Arg 145 150 155 160 Ser Asn Val
Phe Tyr Gln Tyr Ile Gly Glu Ala Tyr Ile Pro Ile Ala 165 170 175 Phe
Ala Thr Ala Ala Ala Ala Asp Pro Asn Ala Lys Leu Tyr Tyr Asn 180 185
190 Asp Tyr Asn Ile Glu Tyr Pro Gly Ala Lys Ala Thr Ala Ala Gln Asn
195 200 205 Ile Val Lys Met Val Lys Ala Tyr Gly Ala Lys Ile Asp Gly
Val Gly 210 215 220 Leu Gln Ser His Phe Ile Val Gly Ser Thr Pro Ser
Gln Ser Ser Gln 225 230 235 240 Gln Ser Asn Met Ala Ala Phe Thr Ala
Leu Gly Val Glu Val Ala Ile 245 250 255 Thr Glu Leu Asp Ile Arg Met
Thr Leu Pro Ser Thr Ser Ala Leu Leu 260 265 270 Ala Gln Gln Ser Thr
Asp Tyr Gln Ser Thr Val Ser Ala Cys Val Asn 275 280 285 Thr Pro Lys
Cys Ile Gly Ile Thr Leu Trp Asp Trp Thr Asp Lys Tyr 290 295 300 Ser
Trp Val Pro Asn Thr Phe Ser Gly Gln Gly Asp Ala Cys Pro Trp 305 310
315 320 Asp Ser Asn Tyr Gln Lys Lys Pro Ala Tyr Tyr Gly Ile Leu Thr
Ala 325 330 335 Leu Gly Gly Ser Ala Ser Thr Ser Thr Thr Thr Thr Leu
Val Thr Ser 340 345 350 Thr Arg Thr Ser Thr Thr Thr Ser Thr Ser Ala
Thr Ser Thr Ser Thr 355 360 365 Gly Val Ala Gln His Trp Gly Gln Cys
Gly Gly Ile Gly Trp Thr Gly 370 375 380 Pro Thr Thr Cys Ala Ser Pro
Tyr Thr Cys Gln Glu Leu Asn Pro Tyr 385 390 395 400 Tyr Tyr Gln Cys
Leu 405 741DNAArtificialPCR primer 7acacaactgg ggatccacca
tgcgtttctc cttggccact g 41836DNAArtificialPCR primer 8ccctctagat
ctcgagctag cagacgctgc aggcct 36940DNAArtificialPCR primer
9acacaactgg ggatccacca tggtccgtct ttccgctgga 401041DNAArtificialPCR
primer 10ccctctagat ctcgagttac aagcactggg agtaccactg g
411143DNAArtificialPCR primer 11acacaactgg ggatccacca tggtccatct
ttcttccctg gcc 431243DNAArtificialPCR primer 12acacaactgg
ggatccacca tggtccatct ttcttccctg gcc 43
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