U.S. patent application number 15/885244 was filed with the patent office on 2018-06-28 for polypeptides having endoglucanase activity and polynucleotides encoding same.
This patent application is currently assigned to Novozymes, Inc.. The applicant listed for this patent is Novozymes A/S, Novozymes, Inc.. Invention is credited to Pierre Cassland, Brett McBrayer, Nikolaj Spodsberg.
Application Number | 20180179505 15/885244 |
Document ID | / |
Family ID | 46172886 |
Filed Date | 2018-06-28 |
United States Patent
Application |
20180179505 |
Kind Code |
A1 |
Spodsberg; Nikolaj ; et
al. |
June 28, 2018 |
Polypeptides Having Endoglucanase Activity And Polynucleotides
Encoding Same
Abstract
The present invention relates to isolated polypeptides having
endoglucanase activity, catalytic domains, cellulose binding
domains and polynucleotides encoding the polypeptides, catalytic
domains or cellulose binding domains. 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, catalytic domains or cellulose binding domains.
Inventors: |
Spodsberg; Nikolaj;
(Bagsvaerd, DK) ; McBrayer; Brett; (Sacramento,
CA) ; Cassland; Pierre; (Vellinge, SE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Novozymes, Inc.
Novozymes A/S |
Davis
Bagsvaerd |
CA |
US
DK |
|
|
Assignee: |
Novozymes, Inc.
Davis
CA
Novozymes A/S
Bagsvaerd
|
Family ID: |
46172886 |
Appl. No.: |
15/885244 |
Filed: |
January 31, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14112022 |
Jan 17, 2014 |
9926547 |
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PCT/US2012/035257 |
Apr 26, 2012 |
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15885244 |
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61480245 |
Apr 28, 2011 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 9/2437 20130101;
C12Y 302/01004 20130101 |
International
Class: |
C12N 9/42 20060101
C12N009/42 |
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
1-20. (canceled)
21. A method for degrading a cellulosic material, comprising:
treating the cellulosic material with an enzyme composition
comprising a polypeptide having endoglucanase activity, wherein the
polypeptide having endoglucanase activity is selected from the
group consisting of: (a) a polypeptide having endoglucanase
activity having at least 95% sequence identity to amino acids 18 to
419 of SEQ ID NO: 2; and (b) a polypeptide having endoglucanase
activity encoded by a polynucleotide having at least 95% sequence
identity to nucleotides 51 to 1257 of SEQ ID NO: 1; (c) a
polypeptide comprising a catalytic domain having endoglucanase
activity having at least 95% sequence identity to amino acids 93 to
419 of SEQ ID NO: 2; and (d) a polypeptide comprising a catalytic
domain having endoglucanase activity encoded by a polynucleotide
having at least 95% sequence identity to nucleotides 277 to 1317 of
SEQ ID NO: 1.
22. The method of claim 21, wherein the polypeptide having
endoglucanase activity has at least 96% sequence identity to amino
acids 18 to 419 of SEQ ID NO: 2.
23. The method of claim 21, wherein the polypeptide having
endoglucanase activity has at least 97% sequence identity to amino
acids 18 to 419 of SEQ ID NO: 2.
24. The method of claim 21, wherein the polypeptide having
endoglucanase activity has at least 98% sequence identity to amino
acids 18 to 419 of SEQ ID NO: 2.
25. The method of claim 21, wherein the polypeptide having
endoglucanase activity has at least 99% sequence identity to amino
acids 18 to 419 of SEQ ID NO: 2.
26. The method of claim 21, wherein the polypeptide having
endoglucanase activity comprises amino acids 18 to 419 of SEQ ID
NO: 2.
27. The method of claim 21, wherein the catalytic domain has at
least 96% sequence identity to amino acids 93 to 419 of SEQ ID NO:
2.
28. The method of claim 21, wherein the catalytic domain has at
least 97% sequence identity to amino acids 93 to 419 of SEQ ID NO:
2.
29. The method of claim 21, wherein the catalytic domain has at
least 98% sequence identity to amino acids 93 to 419 of SEQ ID NO:
2.
30. The method of claim 21, wherein the catalytic domain has at
least 99% sequence identity to amino acids 93 to 419 of SEQ ID NO:
2.
31. The method of claim 21, wherein the catalytic domain comprises
amino acids 93 to 419 of SEQ ID NO: 2.
32. The method of claim 21, wherein the cellulosic material is
pretreated.
33. The method of claim 21, wherein the enzyme composition further
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.
34. The method of claim 33, wherein the cellulase is one or more
enzymes selected from the group consisting of an endoglucanase, a
cellobiohydrolase, and a beta-glucosidase.
35. The method of claim 33, 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.
36. The method of claim 21, further comprising recovering the
degraded cellulosic material.
37. The method of claim 36, wherein the degraded cellulosic
material is a sugar.
38. The method of claim 37, wherein the sugar is selected from the
group consisting of glucose, xylose, mannose, galactose, and
arabinose.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application of U.S.
application Ser. No. 14/112,022 filed on Jan. 17, 2014, which is a
35 U.S.C. .sctn. 371 national application of PCT/US2012/035257
filed on Apr. 26, 2012, which claims priority or the benefit under
35 U.S.C. .sctn. 119 of U.S. Provisional Application No. 61/480,245
filed on Apr. 28, 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
endoglucanase activity, catalytic domains, and cellulose binding
domains, and polynucleotides encoding the polypeptides, catalytic
domains, and cellulose binding domains. 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, catalytic domains, and cellulose binding domains.
Description of the Related Art
[0005] Cellulose is a polymer of the simple sugar glucose
covalently 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 lignocellulose is converted to fermentable sugars,
e.g., glucose, the fermentable sugars are easily fermented by yeast
into ethanol.
[0007] The present invention provides polypeptides having
endoglucanase activity and polynucleotides encoding the
polypeptides.
SUMMARY OF THE INVENTION
[0008] The present invention relates to isolated polypeptides
having endoglucanase activity selected from the group consisting
of:
[0009] (a) a polypeptide having at least 80% sequence identity to
the mature polypeptide of SEQ ID NO: 2;
[0010] (b) a polypeptide encoded by a polynucleotide that
hybridizes under at least high stringency conditions with (i) the
mature polypeptide coding sequence of SEQ ID NO: 1, (ii) the cDNA
sequence thereof, or (iii) the full-length complement of (i) or
(ii);
[0011] (c) a polypeptide encoded by a polynucleotide having at
least 80% sequence identity to the mature polypeptide coding
sequence of SEQ ID NO: 1 or the cDNA sequence thereof;
[0012] (d) a variant of the mature polypeptide of SEQ ID NO: 2
comprising a substitution, deletion, and/or insertion at one or
more (e.g., several) positions; and
[0013] (e) a fragment of the polypeptide of (a), (b), (c), or (d)
that has endoglucanase activity.
[0014] The present invention also relates to isolated polypeptides
comprising a catalytic domain selected from the group consisting
of:
[0015] (a) a catalytic domain having at least 85% sequence identity
to amino acids 93 to 419 of SEQ ID NO: 2;
[0016] (b) a catalytic domain encoded by a polynucleotide that
hybridizes under at least high stringency conditions with (i)
nucleotides 277 to 1317 of SEQ ID NO: 1, (ii) the cDNA sequence
thereof, or (iii) the full-length complement of (i) or (ii);
[0017] (c) a catalytic domain encoded by a polynucleotide having at
least 85% sequence identity to nucleotides 277 to 1317 of SEQ ID
NO: 1 or the cDNA sequence thereof;
[0018] (d) a variant of amino acids 93 to 419 of SEQ ID NO: 2
comprising a substitution, deletion, and/or insertion at one or
more (e.g., several) positions; and
[0019] (e) a fragment of the catalytic domain of (a), (b), (c), or
(d) that has endoglucanase activity.
[0020] The present invention also relates to isolated polypeptides
comprising a cellulose binding domain selected from the group
consisting of:
[0021] (a) a cellulose binding domain having at least 80% sequence
identity to amino acids 23 to 58 of SEQ ID NO: 2;
[0022] (b) a cellulose binding domain encoded by a polynucleotide
that hybridizes under at least high stringency conditions with
nucleotides 67 to 174 of SEQ ID NO: 1 or the full-length complement
thereof;
[0023] (c) a cellulose binding domain encoded by a polynucleotide
having at least 80% sequence identity to nucleotides 67 to 174 of
SEQ ID NO: 1 or the cDNA sequence thereof;
[0024] (d) a variant of amino acids 23 to 58 of SEQ ID NO: 2
comprising a substitution, deletion, and/or insertion at one or
more (e.g., several) positions; and
[0025] (e) a fragment of the cellulose binding domain of (a), (b),
(c), or (d) that has cellulose binding activity.
[0026] 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.
[0027] The present invention also relates to methods for degrading
or converting a cellulosic material, comprising: treating the
cellulosic material with an enzyme composition in the presence of a
polypeptide having endoglucanase activity of the present invention.
In one aspect, the method further comprises recovering the degraded
or converted cellulosic material.
[0028] The present invention also relates to methods of producing a
fermentation product, comprising: (a) saccharifying a cellulosic
material with an enzyme composition in the presence of a
polypeptide having endoglucanase activity of the present invention;
(b) fermenting the saccharified cellulosic material with one or
more (e.g., several) fermenting microorganisms to produce the
fermentation product; and (c) recovering the fermentation product
from the fermentation.
[0029] The present invention also relates to methods of fermenting
a cellulosic material, comprising: fermenting the cellulosic
material with one or more (e.g., several) fermenting
microorganisms, wherein the cellulosic material is saccharified
with an enzyme composition in the presence of a polypeptide having
endoglucanase activity of the present invention. In one aspect, the
fermenting of the cellulosic material produces a fermentation
product. In another aspect, the method further comprises recovering
the fermentation product from the fermentation.
[0030] The present invention also relates to processes for
manufacturing a paper material, which processes comprise treating a
paper-making pulp and/or process water with a polypeptide having
endoglucanase activity of the present invention.
[0031] 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, which is operably linked to a gene encoding
a protein; nucleic acid constructs, expression vectors, and
recombinant host cells comprising the polynucleotide; and methods
of producing a protein.
BRIEF DESCRIPTION OF THE FIGURES
[0032] FIG. 1 shows a restriction map of pDAU222-PE03860005710.
[0033] FIG. 2 shows the effect of Chaetomium virescens GH5
endoglucanase on a high-temperature enzyme composition in the
hydrolysis of milled unwashed PCS at 50-65.degree. C.
[0034] FIG. 3 shows the effect of C. virescens GH5 endoglucanase on
handsheet tensile index of unbleached eucalyptus Kraft pulp.
[0035] FIG. 4 shows the effect of C. virescens GH5 endoglucanase on
handsheet tear index of unbleached eucalyptus Kraft pulp.
[0036] FIG. 5 shows a comparison of the effect of Chaetomium
virescens GH5 endoglucanase and Trichoderma reesei GH5
endoglucanase II in the hydrolysis of milled unwashed PCS by a
cellulase enzyme composition.
DEFINITIONS
[0037] Acetylxylan esterase: The term "acetylxylan esterase" means
a carboxylesterase (EC 3.1.1.72) that catalyzes the hydrolysis of
acetyl groups from polymeric xylan, acetylated xylose, acetylated
glucose, alpha-napthyl acetate, and p-nitrophenyl acetate. For
purposes of the present invention, acetylxylan esterase activity is
determined using 0.5 mM p-nitrophenylacetate as substrate in 50 mM
sodium acetate pH 5.0 containing 0.01% TWEEN.TM. 20
(polyoxyethylene sorbitan monolaurate). One unit of acetylxylan
esterase is defined as the amount of enzyme capable of releasing 1
.mu.mole of p-nitrophenolate anion per minute at pH 5, 25.degree.
C.
[0038] 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.
[0039] 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).
[0040] Alpha-glucuronidase: The term "alpha-glucuronidase" means an
alpha-D-glucosiduronate glucuronohydrolase (EC 3.2.1.139) that
catalyzes the hydrolysis of an alpha-D-glucuronoside to
D-glucuronate and an alcohol. For purposes of the present
invention, alpha-glucuronidase activity is determined according to
de Vries, 1998, J. Bacteriol. 180: 243-249. One unit of
alpha-glucuronidase equals the amount of enzyme capable of
releasing 1 .mu.mole of glucuronic or 4-O-methylglucuronic acid per
minute at pH 5, 40.degree. C.
[0041] Beta-glucosidase: The term "beta-glucosidase" means a
beta-D-glucoside glucohydrolase (E.C. 3.2.1.21) that catalyzes the
hydrolysis of terminal non-reducing beta-D-glucose residues with
the release of beta-D-glucose. For purposes of the present
invention, beta-glucosidase activity is determined using
p-nitrophenyl-beta-D-glucopyranoside as substrate according to the
procedure of Venturi et al., 2002, Extracellular beta-D-glucosidase
from Chaetomium thermophilum var. coprophilum: production,
purification and some biochemical properties, J. Basic Microbiol.
42: 55-66. One unit of beta-glucosidase is defined as 1.0 .mu.mole
of p-nitrophenolate anion produced per minute at 25.degree. C., pH
4.8 from 1 mM p-nitrophenyl-beta-D-glucopyranoside as substrate in
50 mM sodium citrate containing 0.01% TWEEN.RTM. 20.
[0042] Beta-xylosidase: The term "beta-xylosidase" means a
beta-D-xyloside xylohydrolase (E.C. 3.2.1.37) that catalyzes the
exo-hydrolysis of short beta (1.fwdarw.4)-xylooligosaccharides to
remove successive D-xylose residues from non-reducing termini. For
purposes of the present invention, one unit of beta-xylosidase is
defined as 1.0 .mu.mole of p-nitrophenolate anion produced per
minute at 40.degree. C., pH 5 from 1 mM
p-nitrophenyl-beta-D-xyloside as substrate in 100 mM sodium citrate
containing 0.01% TWEEN.RTM. 20.
[0043] Catalytic domain: The term "catalytic domain" means the
region of an enzyme containing the catalytic machinery of the
enzyme.
[0044] 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.
[0045] Cellobiohydrolase: The term "cellobiohydrolase" means a
1,4-beta-D-glucan cellobiohydrolase (E.C. 3.2.1.91 and E.C.
3.2.1.176) 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.
[0046] Cellulose binding domain: The term "cellulose binding
domain" means the region of an enzyme that mediates binding of the
enzyme to amorphous regions of a cellulose substrate. The cellulose
binding domain (CBD) is typically found either at the N-terminal or
at the C-terminal extremity of an endoglucanase. The term
"cellulose binding domain" is also known in the art as a
"carbohydrate binding module".
[0047] Cellulolytic enzyme or cellulase: The term "cellulolytic
enzyme" or "cellulase" means one or more (e.g., several) enzymes
that hydrolyze a cellulosic material. Such enzymes include
endoglucanase(s), cellobiohydrolase(s), beta-glucosidase(s), or
combinations thereof. The two basic approaches for measuring
cellulolytic activity include: (1) measuring the total cellulolytic
activity, and (2) measuring the individual cellulolytic activities
(endoglucanases, cellobiohydrolases, and beta-glucosidases) as
reviewed in Zhang et al., Outlook for cellulase improvement:
Screening and selection strategies, 2006, Biotechnology Advances
24: 452-481. Total cellulolytic activity is usually measured using
insoluble substrates, including Whatman No 1 filter paper,
microcrystalline cellulose, bacterial cellulose, algal cellulose,
cotton, pretreated lignocellulose, etc. The most common total
cellulolytic activity assay is the filter paper assay using Whatman
No 1 filter paper as the substrate. The assay was established by
the International Union of Pure and Applied Chemistry (IUPAC)
(Ghose, 1987, Measurement of cellulase activities, Pure Appl. Chem.
59: 257-68).
[0048] 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).
[0049] 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.
[0050] 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.
[0051] 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).
[0052] 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.
[0053] 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.
[0054] 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.
[0055] 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.
[0056] 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.
[0057] 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.
[0058] 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.
[0059] 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.
[0060] In one aspect, the polypeptides of the present invention
have at least 20%, e.g., at least 40%, at least 50%, at least 60%,
at least 70%, at least 80%, at least 90%, at least 95%, or at least
100% of the endoglucanase activity of the mature polypeptide of SEQ
ID NO: 2.
[0061] 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.
[0062] 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.
[0063] Family 61 glycoside hydrolase: The term "Family 61 glycoside
hydrolase" or "Family GH61" or "GH61" means a polypeptide falling
into the glycoside hydrolase Family 61 according to Henrissat B.,
1991, A classification of glycosyl hydrolases based on amino-acid
sequence similarities, Biochem. J. 280: 309-316, and Henrissat B.,
and Bairoch A., 1996, Updating the sequence-based classification of
glycosyl hydrolases, Biochem. J. 316: 695-696. The enzymes in this
family were originally classified as a glycoside hydrolase family
based on measurement of very weak endo-1,4-beta-D-glucanase
activity in one family member. The structure and mode of action of
these enzymes are non-canonical and they cannot be considered as
bona fide glycosidases. However, they are kept in the CAZy
classification on the basis of their capacity to enhance the
breakdown of lignocellulose when used in conjunction with a
cellulase or a mixture of cellulases.
[0064] 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
biomass 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.
[0065] Fragment: The term "fragment" means a polypeptide or a
catalytic or cellulose binding domain having one or more (e.g.,
several) amino acids absent from the amino and/or carboxyl terminus
of a mature polypeptide or domain; wherein the fragment has
endoglucanase. In one aspect, a fragment having endoglucanase
activity contains at least 340 amino acid residues, e.g., at least
360 amino acid residues or at least 380 amino acid residues. In
another aspect, a fragment of a catalytic domain having
endoglucanase activity contains at least 280 amino acid residues,
e.g., at least 295 amino acid residues or at least 310 amino acid
residues. In another aspect, a fragment of a cellulose binding
domain contains at least 30 amino acids, e.g., at least 32 amino
acid residues or at least 34 amino acid residues.
[0066] Hemicellulolytic enzyme or hemicellulase: The term
"hemicellulolytic enzyme" or "hemicellulase" means one or more
(e.g., several) enzymes that hydrolyze a hemicellulosic material.
See, for example, Shallom, D. and Shoham, Y. Microbial
hemicellulases. Current Opinion In Microbiology, 2003, 6(3):
219-228). Hemicellulases are key components in the degradation of
plant biomass. Examples of hemicellulases include, but are not
limited to, an acetylmannan esterase, an acetylxylan esterase, an
arabinanase, an arabinofuranosidase, a coumaric acid esterase, a
feruloyl esterase, a galactosidase, a glucuronidase, a glucuronoyl
esterase, a mannanase, a mannosidase, a xylanase, and a xylosidase.
The substrates of these enzymes, the hemicelluloses, are a
heterogeneous group of branched and linear polysaccharides that are
bound via hydrogen bonds to the cellulose microfibrils in the plant
cell wall, crosslinking them into a robust network. Hemicelluloses
are also covalently attached to lignin, forming together with
cellulose a highly complex structure. The variable structure and
organization of hemicelluloses require the concerted action of many
enzymes for its complete degradation. The catalytic modules of
hemicellulases are either glycoside hydrolases (GHs) that hydrolyze
glycosidic bonds, or carbohydrate esterases (CEs), which hydrolyze
ester linkages of acetate or ferulic acid side groups. These
catalytic modules, based on homology of their primary sequence, can
be assigned into GH and CE families. Some families, with an overall
similar fold, can be further grouped into clans, marked
alphabetically (e.g., GH-A). A most informative and updated
classification of these and other carbohydrate active enzymes is
available in the Carbohydrate-Active Enzymes (CAZy) database.
Hemicellulolytic enzyme activities can be measured according to
Ghose and Bisaria, 1987, Pure & Appl. Chem. 59: 1739-1752, at a
suitable temperature, e.g., 50.degree. C., 55.degree. C., or
60.degree. C., and pH, e.g., 5.0 or 5.5.
[0067] 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.
[0068] 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.
[0069] 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., recombinant production
in a host cell; multiple copies of a gene encoding the substance;
and use of a stronger promoter than the promoter naturally
associated with the gene encoding the substance).
[0070] 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.
[0071] 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 419 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. 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.
[0072] Mature polypeptide coding sequence: The term "mature
polypeptide coding sequence" means a polynucleotide that encodes a
mature polypeptide having endoglucanase activity. In one aspect,
the mature polypeptide coding sequence is nucleotides 52 to 1317 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.
[0073] 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.
[0074] 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 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.
[0075] 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.
[0076] 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.
[0077] 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,
Bagsvaerd, Denmark) in the presence of 2-3% of total protein weight
Aspergillus oryzae beta-glucosidase (recombinantly produced in
Aspergillus oryzae according to WO 02/095014) or 2-3% of total
protein weight Aspergillus fumigatus beta-glucosidase
(recombinantly produced in Aspergillus oryzae as described in WO
2002/095014) of cellulase protein loading is used as the source of
the cellulolytic activity.
[0078] 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.
[0079] 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.
[0080] Sequence identity: The relatedness between two amino acid
sequences or between two nucleotide sequences is described by the
parameter "sequence identity".
[0081] 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)
[0082] 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)
[0083] 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 endoglucanase activity. In
one aspect, a subsequence contains at least 1020 nucleotides, e.g.,
at least 1080 nucleotides or at least 1140 nucleotides. In another
aspect, a subsequence of a coding sequence of a catalytic domain
having endoglucanase activity contains at least 840 nucleotides,
e.g., at least 885 nucleotides or at least 930 nucleotides. In
another aspect, a subsequence of a coding sequence of a cellulose
binding domain contains at least 90 nucleotides, e.g., at least 96
nucleotides or at least 102 nucleotides.
[0084] Variant: The term "variant" means a polypeptide having
endoglucanase 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.
[0085] 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.
[0086] 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.
[0087] 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.
[0088] In the methods of the present invention, any material
containing xylan may be used. In a preferred aspect, the
xylan-containing material is lignocellulose.
[0089] Xylan degrading activity or xylanolytic activity: The term
"xylan degrading activity" or "xylanolytic activity" means a
biological activity that hydrolyzes xylan-containing material. The
two basic approaches for measuring xylanolytic activity include:
(1) measuring the total xylanolytic activity, and (2) measuring the
individual xylanolytic activities (e.g., endoxylanases,
beta-xylosidases, arabinofuranosidases, alpha-glucuronidases,
acetylxylan esterases, feruloyl esterases, and alpha-glucuronyl
esterases). Recent progress in assays of xylanolytic enzymes was
summarized in several publications including Biely and Puchard,
Recent progress in the assays of xylanolytic enzymes, 2006, Journal
of the Science of Food and Agriculture 86(11): 1636-1647; Spanikova
and Biely, 2006, Glucuronoyl esterase--Novel carbohydrate esterase
produced by Schizophyllum commune, FEBS Letters 580(19): 4597-4601;
Herrmann, Vrsanska, Jurickova, Hirsch, Biely, and Kubicek, 1997,
The beta-D-xylosidase of Trichoderma reesei is a multifunctional
beta-D-xylan xylohydrolase, Biochemical Journal 321: 375-381.
[0090] Total xylan degrading activity can be measured by
determining the reducing sugars formed from various types of xylan,
including, for example, oat spelt, beechwood, and larchwood xylans,
or by photometric determination of dyed xylan fragments released
from various covalently dyed xylans. The most common total
xylanolytic activity assay is based on production of reducing
sugars from polymeric 4-O-methyl glucuronoxylan as described in
Bailey, Biely, Poutanen, 1992, Interlaboratory testing of methods
for assay of xylanase activity, Journal of Biotechnology 23(3):
257-270. Xylanase activity can also be determined with 0.2%
AZCL-arabinoxylan as substrate in 0.01% TRITON.RTM. X-100
(4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol) and 200 mM
sodium phosphate buffer pH 6 at 37.degree. C. One unit of xylanase
activity is defined as 1.0 .mu.mole of azurine produced per minute
at 37.degree. C., pH 6 from 0.2% AZCL-arabinoxylan as substrate in
200 mM sodium phosphate pH 6 buffer.
[0091] 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.
[0092] Xylanase: The term "xylanase" means a
1,4-beta-D-xylan-xylohydrolase (E.C. 3.2.1.8) that catalyzes the
endohydrolysis of 1,4-beta-D-xylosidic linkages in xylans. For
purposes of the present invention, xylanase activity is determined
with 0.2% AZCL-arabinoxylan as substrate in 0.01% TRITON.RTM. X-100
and 200 mM sodium phosphate buffer pH 6 at 37.degree. C. One unit
of xylanase activity is defined as 1.0 .mu.mole of azurine produced
per minute at 37.degree. C., pH 6 from 0.2% AZCL-arabinoxylan as
substrate in 200 mM sodium phosphate pH 6 buffer.
DETAILED DESCRIPTION OF THE INVENTION
Polypeptides Having Endoglucanase Activity
[0093] 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 80%, e.g., at least 81%, at least 82%,
at least 83%, at least 84%, 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%, which have
endoglucanase activity. In one aspect, the polypeptides differ by
up to 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, from
the mature polypeptide of SEQ ID NO: 2.
[0094] A polypeptide of the present invention preferably comprises
or consists of the amino acid sequence of SEQ ID NO: 2 or an
allelic variant thereof; or is a fragment thereof having
endoglucanase activity. In another aspect, the polypeptide
comprises or consists of the mature polypeptide of SEQ ID NO: 2. In
another aspect, the polypeptide comprises or consists of amino
acids 18 to 419 of SEQ ID NO: 2.
[0095] In another embodiment, the present invention relates to
isolated polypeptides having endoglucanase activity encoded by
polynucleotides that hybridize under very low stringency
conditions, low stringency conditions, medium stringency
conditions, medium-high stringency conditions, high stringency
conditions, or very high stringency conditions with (i) the mature
polypeptide coding sequence of SEQ ID NO: 1, (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.).
[0096] The polynucleotide of SEQ ID NO: 1 or a subsequence thereof,
as well as the polypeptide of SEQ ID NO: 2 or a fragment thereof,
may be used to design nucleic acid probes to identify and clone DNA
encoding polypeptides having endoglucanase 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.
[0097] 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 endoglucanase
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 or a
subsequence thereof, the carrier material is used in a Southern
blot.
[0098] 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; (ii) the mature
polypeptide coding sequence of SEQ ID NO: 1; (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.
[0099] In one aspect, the nucleic acid probe is nucleotides 52 to
1317 of SEQ ID NO: 1. In another aspect, the nucleic acid probe is
a polynucleotide that encodes the polypeptide of SEQ ID NO: 2; the
mature polypeptide thereof; or a fragment thereof. In another
aspect, the nucleic acid probe is SEQ ID NO: 1 or the cDNA sequence
thereof.
[0100] In another embodiment, the present invention relates to
isolated polypeptides having endoglucanase activity encoded by
polynucleotides having a sequence identity to the mature
polypeptide coding sequence of SEQ ID NO: 1, or the cDNA sequence
thereof, of at least 80%, at least 81%, at least 82%, at least 83%,
at least 84%, 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%.
[0101] In another embodiment, the present invention relates to
variants of the mature polypeptide of SEQ ID NO: 2 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 is up to 10, e.g., 1, 2, 3, 4,
5, 6, 7, 8, 9, or 10. 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.
[0102] 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.
[0103] 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.
[0104] 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 endoglucanase
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.
[0105] 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).
[0106] 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.
[0107] 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.
[0108] 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).
[0109] 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 Endoglucanase Activity
[0110] A polypeptide having endoglucanase 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.
[0111] The polypeptide may be a bacterial polypeptide. For example,
the polypeptide may be a Gram-positive bacterial polypeptide such
as a Bacillus, Clostridium, Enterococcus, Geobacillus,
Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus,
Streptococcus, or Streptomyces polypeptide having endoglucanase
activity, or a Gram-negative bacterial polypeptide such as a
Campylobacter, E. coli, Flavobacterium, Fusobacterium,
Helicobacter, Ilyobacter, Neisseria, Pseudomonas, Salmonella, or
Ureaplasma polypeptide.
[0112] In one aspect, the polypeptide is a Bacillus alkalophilus,
Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans,
Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus
lautus, Bacillus lentus, Bacillus licheniformis, Bacillus
megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus
subtilis, or Bacillus thuringiensis polypeptide.
[0113] In another aspect, the polypeptide is a Streptococcus
equisimilis, Streptococcus pyogenes, Streptococcus uberis, or
Streptococcus equi subsp. Zooepidemicus polypeptide.
[0114] In another aspect, the polypeptide is a Streptomyces
achromogenes, Streptomyces avermitilis, Streptomyces coelicolor,
Streptomyces griseus, or Streptomyces lividans polypeptide.
[0115] The polypeptide may also be a fungal polypeptide. For
example, the polypeptide may be a yeast polypeptide such as a
Candida, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces,
or Yarrowia polypeptide; or a filamentous fungal polypeptide such
as an Acremonium, Agaricus, Alternaria, Aspergillus, Aureobasidium,
Botryospaeria, Ceriporiopsis, Chaetomidium, Chrysosporium,
Claviceps, Cochliobolus, Coprinopsis, Coptotermes, Corynascus,
Cryphonectria, Cryptococcus, Dipodia, Exidia, Filibasidium,
Fusarium, Gibberella, Holomastigotoides, Humicola, lrpex,
Lentinula, Leptospaeria, Magnaporthe, Melanocarpus, Meripilus,
Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces,
Penicillium, Phanerochaete, Piromyces, Poitrasia, Pseudoplectania,
Pseudotrichonympha, Rhizomucor, Schizophyllum, Scytalidium,
Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trichoderma,
Trichophaea, Verticillium, Volvariella, or Xylaria polypeptide.
[0116] In another aspect, the polypeptide is a Saccharomyces
carisbergensis, Saccharomyces cerevisiae, Saccharomyces
diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri,
Saccharomyces norbensis, or Saccharomyces oviformis
polypeptide.
[0117] In another aspect, the polypeptide is an Acremonium
cellulolyticus, Aspergillus aculeatus, Aspergillus awamori,
Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus,
Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae,
Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium
lucknowense, Chrysosporium merdarium, Chrysosporium pannicola,
Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium
zonatum, Fusarium bactridioides, Fusarium cerealis, Fusarium
crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium
graminum, Fusarium heterosporum, Fusarium negundi, Fusarium
oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium
sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides,
Fusarium suiphureum, Fusarium torulosum, Fusarium trichothecioides,
Fusarium venenatum, Humicola grisea, Humicola insolens, Humicola
lanuginosa, Irpex lacteus, Mucor miehei, Myceliophthora
thermophila, Neurospora crassa, Penicillium funiculosum,
Penicillium purpurogenum, Phanerochaete chrysosporium, Thielavia
achromatica, Thielavia albomyces, Thielavia albopilosa, Thielavia
australeinsis, Thielavia fimeti, Thielavia microspora, Thielavia
ovispora, Thielavia peruviana, Thielavia setosa, Thielavia
spededonium, Thielavia subthermophila, Thielavia terrestris,
Trichoderma harzianum, Trichoderma koningii, Trichoderma
longibrachiatum, Trichoderma reesei, or Trichoderma viride
polypeptide.
[0118] In another aspect, the polypeptide is a Chaetomium virescens
polypeptide, e.g., a polypeptide obtained from Chaetomium virescens
ATCC 32319.
[0119] 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.
[0120] 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).
[0121] 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
[0122] In one embodiment, the present invention also relates to
catalytic domains having a sequence identity to amino acids 18 to
419 of SEQ ID NO: 2 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%. In one aspect,
the catalytic domains comprise amino acid sequences that differ by
up to 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, from
amino acids 93 to 419 of SEQ ID NO: 2.
[0123] The catalytic domain preferably comprises or consists of
amino acids 93 to 419 of SEQ ID NO: 2 or an allelic variant
thereof; or is a fragment thereof having endoglucanase
activity.
[0124] In another embodiment, the present invention also relates to
catalytic domains encoded by polynucleotides that hybridize under
very low stringency conditions, low stringency conditions, medium
stringency conditions, medium-high stringency conditions, high
stringency conditions, or very high stringency conditions (as
defined above) with (i) the nucleotides 277 to 1317 of SEQ ID NO:
1, (ii) the cDNA sequence thereof, or (iii) the full-length
complement of (i) or (ii) (Sambrook et al., 1989, supra).
[0125] In another embodiment, the present invention also relates to
catalytic domains encoded by polynucleotides having a sequence
identity to nucleotides 277 to 1317 of SEQ ID NO: 1 or the cDNA
sequence thereof, 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%.
[0126] The polynucleotide encoding the catalytic domain preferably
comprises or consists of nucleotides 277 to 1317 of SEQ ID NO:
1.
[0127] In another embodiment, the present invention also relates to
catalytic domain variants of amino acids 93 to 419 of SEQ ID NO: 2
comprising a substitution, deletion, and/or insertion at one or
more (e.g., several) positions. In one aspect, the number of amino
acid substitutions, deletions and/or insertions introduced into the
sequence of amino acids 93 to 419 of SEQ ID NO: 2 is up to 10,
e.g., 1, 2, 3, 4, 5, 6, 8, 9, or 10.
Cellulose Binding Domains
[0128] In one embodiment, the present invention also relates to
cellulose binding domains having a sequence identity to amino acids
23 to 58 of SEQ ID NO: 2 of at least 80%, e.g., at least 81%, at
least 82%, at least 83%, at least 84%, 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%. In
one aspect, the cellulose binding domains comprise amino acid
sequences that differ by up to 10 amino acids, e.g., 1, 2, 3, 4, 5,
6, 7, 8, 9, or 10, from amino acids 23 to 58 of SEQ ID NO: 2.
[0129] The cellulose binding domain preferably comprises or
consists of amino acids 23 to 58 of SEQ ID NO: 2 or an allelic
variant thereof; or is a fragment thereof having cellulose binding
activity.
[0130] In another embodiment, the present invention also relates to
cellulose binding domains encoded by polynucleotides that hybridize
under very low stringency conditions, low stringency conditions,
medium stringency conditions, medium-high stringency conditions,
high stringency conditions, or very high stringency conditions (as
defined above) with nucleotides 67 to 174 of SEQ ID NO: 1 or the
full-length complement thereof (Sambrook et al., 1989, supra).
[0131] In another embodiment, the present invention also relates to
cellulose binding domains encoded by polynucleotides having a
sequence identity to nucleotides 67 to 174 of SEQ ID NO: 1 of at
least 80%, e.g., at least 81%, at least 82%, at least 83%, at least
84%, 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%.
[0132] The polynucleotide encoding the cellulose binding domain
preferably comprises or consists of nucleotides 67 to 174 of SEQ ID
NO: 1.
[0133] In another embodiment, the present invention also relates to
cellulose binding domain variants of amino acids 23 to 58 of SEQ ID
NO: 2 comprising a substitution, deletion, and/or insertion at one
or more (e.g., several) positions. In one aspect, the number of
amino acid substitutions, deletions and/or insertions introduced
into the sequence of amino acids 23 to 58 of SEQ ID NO: 2 is up to
10, e.g., 1, 2, 3, 4, 5, 6, 8, 9, or 10.
[0134] A catalytic domain operably linked to the cellulose binding
domain may be from 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. The polynucleotide
encoding the catalytic domain may be obtained from any prokaryotic,
eukaryotic, or other source.
Polynucleotides
[0135] The present invention also relates to isolated
polynucleotides encoding a polypeptide, a catalytic domain, or
cellulose binding domain of the present invention, as described
herein.
[0136] The techniques used to isolate or clone a polynucleotide
encoding a polypeptide 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 Chaetomium, or a
related organism and thus, for example, may be an allelic or
species variant of the polypeptide encoding region of the
polynucleotide.
[0137] 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 or the cDNA
sequence thereof, e.g., a subsequence thereof, and/or by
introduction of nucleotide substitutions that do not result in a
change in the amino acid sequence of the polypeptide, but which
correspond to the codon usage of the host organism intended for
production of the enzyme, or by introduction of nucleotide
substitutions that may give rise to a different amino acid
sequence. For a general description of nucleotide substitution,
see, e.g., Ford et al., 1991, Protein Expression and Purification
2: 95-107.
Nucleic Acid Constructs
[0138] 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.
[0139] The 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.
[0140] 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.
[0141] 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.
[0142] 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 V,
Trichoderma reesei xylanase I, Trichoderma reesei xylanase II,
Trichoderma reesei xylanase III, Trichoderma reesei
beta-xylosidase, and Trichoderma reesei translation elongation
factor, 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. Other promoters are
described in U.S. Pat. No. 6,011,147.
[0143] 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.
[0144] 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.
[0145] 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).
[0146] Preferred terminators for filamentous fungal host cells are
obtained from the genes for Aspergillus nidulans acetamidase,
Aspergillus nidulans anthranilate synthase, Aspergillus niger
glucoamylase, Aspergillus niger alpha-glucosidase, Aspergillus
oryzae TAKA amylase, Fusarium oxysporum trypsin-like protease,
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 V, Trichoderma reesei xylanase I, Trichoderma
reesei xylanase II, Trichoderma reesei xylanase III, Trichoderma
reesei beta-xylosidase, and Trichoderma reesei translation
elongation factor.
[0147] 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.
[0148] 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.
[0149] 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).
[0150] 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.
[0151] Preferred leaders for filamentous fungal host cells are
obtained from the genes for Aspergillus oryzae TAKA amylase and
Aspergillus nidulans triose phosphate isomerase.
[0152] 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).
[0153] 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.
[0154] 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.
[0155] Useful polyadenylation sequences for yeast host cells are
described by Guo and Sherman, 1995, Mol. Cellular Biol. 15:
5983-5990.
[0156] 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.
[0157] 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.
[0158] 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.
[0159] 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.
[0160] 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.
[0161] 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.
[0162] 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 sequences 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 sequences 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, Trichoderma reesei cellobiohydrolase I promoter, and
Trichoderma reesei cellobiohydrolase II 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 to the regulatory
sequence.
Expression Vectors
[0163] 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.
[0164] 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.
[0165] 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.
[0166] 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.
[0167] 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, adeA
(phosphoribosylaminoimidazole-succinocarboxamide synthase), adeB
(phosphoribosyl-aminoimidazole synthase), 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. Preferred for use
in a Trichoderma cell are adeA, adeB, amdS, hph, and pyrG
genes.
[0168] The selectable marker may be a dual selectable marker system
as described in WO 2010/039889. In one aspect, the dual selectable
marker is a hph-tk dual selectable marker system.
[0169] 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.
[0170] 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.
[0171] 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.
[0172] 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
pAN/AMB 1 permitting replication in Bacillus.
[0173] 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.
[0174] Examples of origins of replication useful in a filamentous
fungal cell are AMA1 and ANS1 (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.
[0175] 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.
[0176] 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
[0177] The present invention also relates to recombinant host
cells, comprising a polynucleotide of the present invention
operably linked to one or more control sequences that direct the
production of a polypeptide of the present invention. A construct
or vector comprising a polynucleotide is introduced into a host
cell so that the construct or vector is maintained as a chromosomal
integrant or as a self-replicating extra-chromosomal vector as
described earlier. The term "host cell" encompasses any progeny of
a parent cell that is not identical to the parent cell due to
mutations that occur during replication. The choice of a host cell
will to a large extent depend upon the gene encoding the
polypeptide and its source.
[0178] 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.
[0179] 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.
[0180] 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.
[0181] 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.
[0182] 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.
[0183] 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 etal., 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.
[0184] The host cell may also be a eukaryote, such as a mammalian,
insect, plant, or fungal cell.
[0185] 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).
[0186] 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).
[0187] The yeast host cell may be a Candida, Hansenula,
Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or
Yarrowia cell such as a Kluyveromyces lactis, Saccharomyces
carisbergensis, Saccharomyces cerevisiae, Saccharomyces
diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri,
Saccharomyces norbensis, Saccharomyces oviformis, or Yarrowia
lipolytica cell.
[0188] 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.
[0189] 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.
[0190] 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, Phlebia radiata,
Pleurotus eryngii, Thielavia terrestris, Trametes villosa, Trametes
versicolor, Trichoderma harzianum, Trichoderma koningii,
Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma
viride cell.
[0191] 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
[0192] 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
optionally (b) recovering the polypeptide. In one aspect, the cell
is a Chaetomiun cell. In another aspect, the cell is a Chaetomiun
virescens cell. In another aspect, the cell is Chaetomiun virescens
ATCC 32319.
[0193] 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 optionally (b)
recovering the polypeptide.
[0194] The host cells are cultivated in a nutrient medium suitable
for production of the polypeptide using methods known in the art.
For example, the cells 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.
[0195] 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.
[0196] 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. In one aspect, a
fermentation broth comprising the polypeptide is recovered.
[0197] 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.
[0198] 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
[0199] 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.
[0200] The transgenic plant can be dicotyledonous (a dicot) or
monocotyledonous (a monocot). Examples of monocot plants are
grasses, such as meadow grass (blue grass, Poa), forage grass such
as Festuca, Lolium, temperate grass, such as Agrostis, and cereals,
e.g., wheat, oats, rye, barley, rice, sorghum, and maize
(corn).
[0201] 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.
[0202] 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.
[0203] Also included within the scope of the present invention are
the progeny of such plants, plant parts, and plant cells.
[0204] 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.
[0205] 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).
[0206] 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.
[0207] 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.
[0208] 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.
[0209] The selectable marker gene and any other parts of the
expression construct may be chosen from those available in the
art.
[0210] 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).
[0211] 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).
[0212] 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.
[0213] 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.
[0214] 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.
[0215] Genetic markers may be used to assist in the introgression
of one or more transgenes of the invention from one genetic
background into another. Marker assisted selection offers
advantages relative to conventional breeding in that it can be used
to avoid errors caused by phenotypic variations. Further, genetic
markers may provide data regarding the relative degree of elite
germplasm in the individual progeny of a particular cross. For
example, when a plant with a desired trait which otherwise has a
non-agronomically desirable genetic background is crossed to an
elite parent, genetic markers may be used to select progeny which
not only possess the trait of interest, but also have a relatively
large proportion of the desired germplasm. In this way, the number
of generations required to introgress one or more traits into a
particular genetic background is minimized.
[0216] 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.
Removal or Reduction of Endoglucanase Activity
[0217] The present invention also relates to methods of producing a
mutant of a parent cell, which comprises disrupting or deleting a
polynucleotide, or a portion thereof, encoding a polypeptide of the
present invention, which results in the mutant cell producing less
of the polypeptide than the parent cell when cultivated under the
same conditions.
[0218] The mutant cell may be constructed by reducing or
eliminating expression of the polynucleotide using methods well
known in the art, for example, insertions, disruptions,
replacements, or deletions. In a preferred aspect, the
polynucleotide is inactivated. The polynucleotide to be modified or
inactivated may be, for example, the coding region or a part
thereof essential for activity, or a regulatory element required
for expression of the coding region. An example of such a
regulatory or control sequence may be a promoter sequence or a
functional part thereof, i.e., a part that is sufficient for
affecting expression of the polynucleotide. Other control sequences
for possible modification include, but are not limited to, a
leader, polyadenylation sequence, propeptide sequence, signal
peptide sequence, transcription terminator, and transcriptional
activator.
[0219] Modification or inactivation of the polynucleotide may be
performed by subjecting the parent cell to mutagenesis and
selecting for mutant cells in which expression of the
polynucleotide has been reduced or eliminated. The mutagenesis,
which may be specific or random, may be performed, for example, by
use of a suitable physical or chemical mutagenizing agent, by use
of a suitable oligonucleotide, or by subjecting the DNA sequence to
PCR generated mutagenesis. Furthermore, the mutagenesis may be
performed by use of any combination of these mutagenizing
agents.
[0220] Examples of a physical or chemical mutagenizing agent
suitable for the present purpose include ultraviolet (UV)
irradiation, hydroxylamine, N-methyl-N'-nitro-N-nitrosoguanidine
(MNNG), O-methyl hydroxylamine, nitrous acid, ethyl methane
sulphonate (EMS), sodium bisulphite, formic acid, and nucleotide
analogues.
[0221] When such agents are used, the mutagenesis is typically
performed by incubating the parent cell to be mutagenized in the
presence of the mutagenizing agent of choice under suitable
conditions, and screening and/or selecting for mutant cells
exhibiting reduced or no expression of the gene.
[0222] Modification or inactivation of the polynucleotide may be
accomplished by insertion, substitution, or deletion of one or more
nucleotides in the gene or a regulatory element required for
transcription or translation thereof. For example, nucleotides may
be inserted or removed so as to result in the introduction of a
stop codon, the removal of the start codon, or a change in the open
reading frame. Such modification or inactivation may be
accomplished by site-directed mutagenesis or PCR generated
mutagenesis in accordance with methods known in the art. Although,
in principle, the modification may be performed in vivo, i.e.,
directly on the cell expressing the polynucleotide to be modified,
it is preferred that the modification be performed in vitro as
exemplified below.
[0223] An example of a convenient way to eliminate or reduce
expression of a polynucleotide is based on techniques of gene
replacement, gene deletion, or gene disruption. For example, in the
gene disruption method, a nucleic acid sequence corresponding to
the endogenous polynucleotide is mutagenized in vitro to produce a
defective nucleic acid sequence that is then transformed into the
parent cell to produce a defective gene. By homologous
recombination, the defective nucleic acid sequence replaces the
endogenous polynucleotide. It may be desirable that the defective
polynucleotide also encodes a marker that may be used for selection
of transformants in which the polynucleotide has been modified or
destroyed. In an aspect, the polynucleotide is disrupted with a
selectable marker such as those described herein.
[0224] The present invention also relates to methods of inhibiting
the expression of a polypeptide having endoglucanase activity in a
cell, comprising administering to the cell or expressing in the
cell a double-stranded RNA (dsRNA) molecule, wherein the dsRNA
comprises a subsequence of a polynucleotide of the present
invention. In a preferred aspect, the dsRNA is about 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, 25 or more duplex nucleotides in
length.
[0225] The dsRNA is preferably a small interfering RNA (siRNA) or a
micro RNA (miRNA). In a preferred aspect, the dsRNA is small
interfering RNA for inhibiting transcription. In another preferred
aspect, the dsRNA is micro RNA for inhibiting translation.
[0226] The present invention also relates to such double-stranded
RNA (dsRNA) molecules, comprising a portion of the mature
polypeptide coding sequence of SEQ ID NO: 1 for inhibiting
expression of the polypeptide in a cell. While the present
invention is not limited by any particular mechanism of action, the
dsRNA can enter a cell and cause the degradation of a
single-stranded RNA (ssRNA) of similar or identical sequences,
including endogenous mRNAs. When a cell is exposed to dsRNA, mRNA
from the homologous gene is selectively degraded by a process
called RNA interference (RNAi).
[0227] The dsRNAs of the present invention can be used in
gene-silencing. In one aspect, the invention provides methods to
selectively degrade RNA using a dsRNAi of the present invention.
The process may be practiced in vitro, ex vivo or in vivo. In one
aspect, the dsRNA molecules can be used to generate a
loss-of-function mutation in a cell, an organ or an animal. Methods
for making and using dsRNA molecules to selectively degrade RNA are
well known in the art; see, for example, U.S. Pat. Nos. 6,489,127;
6,506,559; 6,511,824; and 6,515,109.
[0228] The present invention further relates to a mutant cell of a
parent cell that comprises a disruption or deletion of a
polynucleotide encoding the polypeptide or a control sequence
thereof or a silenced gene encoding the polypeptide, which results
in the mutant cell producing less of the polypeptide or no
polypeptide compared to the parent cell.
[0229] The polypeptide-deficient mutant cells are particularly
useful as host cells for expression of native and heterologous
polypeptides. Therefore, the present invention further relates to
methods of producing a native or heterologous polypeptide,
comprising: (a) cultivating the mutant cell under conditions
conducive for production of the polypeptide; and (b) recovering the
polypeptide. The term "heterologous polypeptides" means
polypeptides that are not native to the host cell, e.g., a variant
of a native protein. The host cell may comprise more than one copy
of a polynucleotide encoding the native or heterologous
polypeptide.
[0230] The methods used for cultivation and purification of the
product of interest may be performed by methods known in the
art.
[0231] The methods of the present invention for producing an
essentially endoglucanase-free product are of particular interest
in the production of eukaryotic polypeptides, in particular fungal
proteins such as enzymes. The endoglucanase-deficient cells may
also be used to express heterologous proteins of pharmaceutical
interest such as hormones, growth factors, receptors, and the like.
The term "eukaryotic polypeptides" includes not only native
polypeptides, but also those polypeptides, e.g., enzymes, which
have been modified by amino acid substitutions, deletions or
additions, or other such modifications to enhance activity,
thermostability, pH tolerance and the like.
[0232] In a further aspect, the present invention relates to a
protein product essentially free from endoglucanase activity that
is produced by a method of the present invention.
Fermentation Broth Formulations or Cell Compositions
[0233] The present invention also relates to a fermentation broth
formulation or a cell composition comprising a polypeptide of the
present invention. The fermentation broth product further comprises
additional ingredients used in the fermentation process, such as,
for example, cells (including, the host cells containing the gene
encoding the polypeptide of the present invention which are used to
produce the polypeptide of interest), cell debris, biomass,
fermentation media and/or fermentation products. In some
embodiments, the composition is a cell-killed whole broth
containing organic acid(s), killed cells and/or cell debris, and
culture medium.
[0234] The term "fermentation broth" as used herein refers to a
preparation produced by cellular fermentation that undergoes no or
minimal recovery and/or purification. For example, fermentation
broths are produced when microbial cultures are grown to
saturation, incubated under carbon-limiting conditions to allow
protein synthesis (e.g., expression of enzymes by host cells) and
secretion into cell culture medium. The fermentation broth can
contain unfractionated or fractionated contents of the fermentation
materials derived at the end of the fermentation. Typically, the
fermentation broth is unfractionated and comprises the spent
culture medium and cell debris present after the microbial cells
(e.g., filamentous fungal cells) are removed, e.g., by
centrifugation. In some embodiments, the fermentation broth
contains spent cell culture medium, extracellular enzymes, and
viable and/or nonviable microbial cells.
[0235] In an embodiment, the fermentation broth formulation and
cell compositions comprise a first organic acid component
comprising at least one 1-5 carbon organic acid and/or a salt
thereof and a second organic acid component comprising at least one
6 or more carbon organic acid and/or a salt thereof. In a specific
embodiment, the first organic acid component is acetic acid, formic
acid, propionic acid, a salt thereof, or a mixture of two or more
of the foregoing and the second organic acid component is benzoic
acid, cyclohexanecarboxylic acid, 4-methylvaleric acid,
phenylacetic acid, a salt thereof, or a mixture of two or more of
the foregoing.
[0236] In one aspect, the composition contains an organic acid(s),
and optionally further contains killed cells and/or cell debris. In
one embodiment, the killed cells and/or cell debris are removed
from a cell-killed whole broth to provide a composition that is
free of these components.
[0237] The fermentation broth formulations or cell compositions may
further comprise a preservative and/or anti-microbial (e.g.,
bacteriostatic) agent, including, but not limited to, sorbitol,
sodium chloride, potassium sorbate, and others known in the
art.
[0238] The cell-killed whole broth or composition may contain the
unfractionated contents of the fermentation materials derived at
the end of the fermentation. Typically, the cell-killed whole broth
or composition contains the spent culture medium and cell debris
present after the microbial cells (e.g., filamentous fungal cells)
are grown to saturation, incubated under carbon-limiting conditions
to allow protein synthesis. In some embodiments, the cell-killed
whole broth or composition contains the spent cell culture medium,
extracellular enzymes, and killed filamentous fungal cells. In some
embodiments, the microbial cells present in the cell-killed whole
broth or composition can be permeabilized and/or lysed using
methods known in the art.
[0239] A whole broth or cell composition as described herein is
typically a liquid, but may contain insoluble components, such as
killed cells, cell debris, culture media components, and/or
insoluble enzyme(s). In some embodiments, insoluble components may
be removed to provide a clarified liquid composition.
[0240] The whole broth formulations and cell compositions of the
present invention may be produced by a method described in WO
90/15861 or WO 2010/096673.
Enzyme Compositions
[0241] 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 endoglucanase activity of the
composition has been increased, e.g., with an enrichment factor of
at least 1.1.
[0242] The compositions may comprise a polypeptide of the present
invention as the major enzymatic component, e.g., a mono-component
composition. Alternatively, the compositions may comprise multiple
enzymatic activities, such as one or more (e.g., several) enzymes
selected from the group consisting of hydrolase, isomerase, ligase,
lyase, oxidoreductase, or transferase, e.g., an
alpha-galactosidase, alpha-glucosidase, aminopeptidase, amylase,
beta-galactosidase, beta-glucosidase, beta-xylosidase,
carbohydrase, carboxypeptidase, catalase, cellobiohydrolase,
cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase,
deoxyribonuclease, endoglucanase, esterase, glucoamylase,
invertase, laccase, lipase, mannosidase, mutanase, oxidase,
pectinolytic enzyme, peroxidase, phytase, polyphenoloxidase,
proteolytic enzyme, ribonuclease, transglutaminase, or
xylanase.
[0243] The 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. The compositions may be stabilized in accordance with
methods known in the art.
[0244] Examples are given below of preferred uses of the
compositions of the present invention. The dosage of the
composition and other conditions under which the composition is
used may be determined on the basis of methods known in the
art.
Uses
[0245] The present invention is also directed to the following
methods for using the polypeptides having endoglucanase activity,
or compositions thereof.
Processing of Cellulosic Material
[0246] The present invention also relates to methods for degrading
or converting a cellulosic material, comprising: treating the
cellulosic material with an enzyme composition in the presence of a
polypeptide having endoglucanase activity of the present invention.
In one aspect, the methods further comprise recovering the degraded
or converted cellulosic material. Soluble products of degradation
or conversion of the cellulosic material can be separated from
insoluble cellulosic material using a method known in the art such
as, for example, centrifugation, filtration, or gravity
settling.
[0247] The present invention also relates to methods of producing a
fermentation product, comprising: (a) saccharifying a cellulosic
material with an enzyme composition in the presence of a
polypeptide having endoglucanase activity of the present invention;
(b) fermenting the saccharified cellulosic material with one or
more (e.g., several) fermenting microorganisms to produce the
fermentation product; and (c) recovering the fermentation product
from the fermentation.
[0248] The present invention also relates to methods of fermenting
a cellulosic material, comprising: fermenting the cellulosic
material with one or more (e.g., several) fermenting
microorganisms, wherein the cellulosic material is saccharified
with an enzyme composition in the presence of a polypeptide having
endoglucanase activity of the present invention. In one aspect, the
fermenting of the cellulosic material produces a fermentation
product. In another aspect, the methods further comprise recovering
the fermentation product from the fermentation.
[0249] The methods of the present invention can be used to
saccharify the cellulosic 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 typically involves pretreatment, enzymatic hydrolysis
(saccharification), and fermentation.
[0250] The processing of the cellulosic material according to the
present invention can be accomplished using processes conventional
in the art. Moreover, the methods of the present invention can be
implemented using any conventional biomass processing apparatus
configured to operate in accordance with the invention.
[0251] 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 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 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 to fermentable sugars and to
convert the fermentable sugars into a final product (Lynd, L. R.,
Weimer, P. J., van Zyl, W. H., and Pretorius, I. S., 2002,
Microbial cellulose utilization: Fundamentals and biotechnology,
Microbiol. Mol. Biol. Reviews 66: 506-577). It is understood herein
that any method known in the art comprising pretreatment, enzymatic
hydrolysis (saccharification), fermentation, or a combination
thereof, can be used in the practicing the methods of the present
invention.
[0252] A conventional apparatus can include a fed-batch stirred
reactor, a batch stirred reactor, a continuous flow stirred reactor
with ultrafiltration, and/or a continuous plug-flow column reactor
(Fernanda de Castilhos Corazza, Flavio Faria de Moraes, Gisella
Maria Zanin and Ivo Neitzel, 2003, Optimal control in fed-batch
reactor for the cellobiose hydrolysis, Acta Scientiarum. Technology
25: 33-38; Gusakov, A. V., and Sinitsyn, A. P., 1985, Kinetics of
the enzymatic hydrolysis of cellulose: 1. A mathematical model for
a batch reactor process, Enz. Microb. Technol. 7: 346-352), an
attrition reactor (Ryu, S. K., and Lee, J. M., 1983, Bioconversion
of waste cellulose by using an attrition bioreactor, Biotechnol.
Bioeng. 25: 53-65), or a reactor with intensive stirring induced by
an electromagnetic field (Gusakov, A. V., Sinitsyn, A. P.,
Davydkin, I. Y., Davydkin, V. Y., Protas, O. V., 1996, Enhancement
of enzymatic cellulose hydrolysis using a novel type of bioreactor
with intensive stirring induced by electromagnetic field, Appl.
Biochem. Biotechnol. 56: 141-153). Additional reactor types
include: fluidized bed, upflow blanket, immobilized, and extruder
type reactors for hydrolysis and/or fermentation.
[0253] Pretreatment. In practicing the methods of the present
invention, any pretreatment process known in the art can be used to
disrupt plant cell wall components of the cellulosic 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).
[0254] The cellulosic 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.
[0255] 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.
[0256] The cellulosic 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).
[0257] Steam Pretreatment. In steam pretreatment, the cellulosic
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 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 is generally only moist during the
pretreatment. The steam pretreatment is often combined with an
explosive discharge of the material after the pretreatment, which
is known as steam explosion, that is, rapid flashing to atmospheric
pressure and turbulent flow of the material to increase the
accessible surface area by fragmentation (Duff and Murray, 1996,
Bioresource Technology 855: 1-33; Galbe and Zacchi, 2002, Appl.
Microbiol. Biotechnol. 59: 618-628; U.S. Patent Application No.
20020164730). During steam pretreatment, hemicellulose acetyl
groups are cleaved and the resulting acid autocatalyzes partial
hydrolysis of the hemicellulose to monosaccharides and
oligosaccharides. Lignin is removed to only a limited extent.
[0258] 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.
[0259] 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 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).
[0260] 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).
[0261] 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.
[0262] 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.
[0263] 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).
[0264] Ammonia fiber explosion (AFEX) involves treating the
cellulosic 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.
[0265] Organosolv pretreatment delignifies the cellulosic 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.
[0266] Other examples of suitable pretreatment methods are
described by Schell et al., 2003, Appl. Biochem. and Biotechnol.
Vol. 105-108, p. 69-85, and Mosier et al., 2005, Bioresource
Technology 96: 673-686, and U.S. Published Application
2002/0164730.
[0267] 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 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.
[0268] In another aspect, pretreatment takes place in an aqueous
slurry. In preferred aspects, the cellulosic 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 can be unwashed or washed using any method
known in the art, e.g., washed with water.
[0269] 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).
[0270] The cellulosic 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.
[0271] Accordingly, in a preferred aspect, the cellulosic 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.
[0272] 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. 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).
[0273] Saccharification. In the hydrolysis step, also known as
saccharification, the cellulosic 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 endoglucanase activity of the
present invention. The enzymes of the compositions can be added
simultaneously or sequentially.
[0274] 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 is
fed gradually to, for example, an enzyme containing hydrolysis
solution.
[0275] 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 %.
[0276] The enzyme compositions can comprise any protein useful in
degrading or converting the cellulosic material.
[0277] 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
cellulase is preferably one or more (e.g., several) enzymes
selected from the group consisting of an endoglucanase, a
cellobiohydrolase, and a beta-glucosidase. 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.
[0278] 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. 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.
[0279] 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).
[0280] 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.
[0281] In the methods of the present invention, the enzyme(s) can
be added prior to or during saccharification, saccharification and
fermentation, or fermentation.
[0282] 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.
[0283] The enzymes used in the methods of the present invention may
be in any form suitable for use, such as, for example, a
fermentation broth formulation or a cell composition, 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.
[0284] The optimum amounts of the enzymes and polypeptides having
endoglucanase activity depend on several factors including, but not
limited to, the mixture of component cellulolytic enzymes and/or
hemicellulolytic enzymes, the cellulosic material, the
concentration of cellulosic material, the pretreatment(s) of the
cellulosic material, temperature, time, pH, and inclusion of
fermenting organism (e.g., yeast for Simultaneous Saccharification
and Fermentation).
[0285] In one aspect, an effective amount of cellulolytic or
hemicellulolytic enzyme to the cellulosic 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.
[0286] In another aspect, an effective amount of a polypeptide
having endoglucanase activity to the cellulosic 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.
[0287] In another aspect, an effective amount of a polypeptide
having endoglucanase 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.
[0288] The polypeptides having cellulolytic enzyme activity or
hemicellulolytic enzyme activity as well as other
proteins/polypeptides useful in the degradation of the cellulosic
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.
[0289] A polypeptide having enzyme activity may be a bacterial
polypeptide. For example, the polypeptide may be a Gram positive
bacterial polypeptide such as a Bacillus, Streptococcus,
Streptomyces, Staphylococcus, Enterococcus, Lactobacillus,
Lactococcus, Clostridium, Geobacillus, Caldicellulosiruptor,
Acidothermus, Thermobifidia, or Oceanobacillus polypeptide having
enzyme activity, or a Gram negative bacterial polypeptide such as
an E. coli, Pseudomonas, Salmonella, Campylobacter, Helicobacter,
Flavobacterium, Fusobacterium, Ilyobacter, Neisseria, or Ureaplasma
polypeptide having enzyme activity.
[0290] In one aspect, the polypeptide is a Bacillus alkalophilus,
Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans,
Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus
lautus, Bacillus lentus, Bacillus licheniformis, Bacillus
megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus
subtilis, or Bacillus thuringiensis polypeptide having enzyme
activity.
[0291] In another aspect, the polypeptide is a Streptococcus
equisimilis, Streptococcus pyogenes, Streptococcus uberis, or
Streptococcus equi subsp. Zooepidemicus polypeptide having enzyme
activity.
[0292] In another aspect, the polypeptide is a Streptomyces
achromogenes, Streptomyces avermitilis, Streptomyces coelicolor,
Streptomyces griseus, or Streptomyces lividans polypeptide having
enzyme activity.
[0293] The polypeptide having enzyme activity may also be a fungal
polypeptide, and more preferably a yeast polypeptide such as a
Candida, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces,
or Yarrowia polypeptide having enzyme activity; or more preferably
a filamentous fungal polypeptide such as an Acremonium, Agaricus,
Alternaria, Aspergillus, Aureobasidium, Botryospaeria,
Ceriporiopsis, Chaetomidium, Chrysosporium, Claviceps,
Cochliobolus, Coprinopsis, Coptotermes, Corynascus, Cryphonectria,
Cryptococcus, Diplodia, Exidia, Filibasidium, Fusarium, Gibberella,
Holomastigotoides, Humicola, Irpex, Lentinula, Leptospaeria,
Magnaporthe, Melanocarpus, Meripilus, Mucor, Myceliophthora,
Neocallimastix, Neurospora, Paecilomyces, Penicillium,
Phanerochaete, Piromyces, Poitrasia, Pseudoplectania,
Pseudotrichonympha, Rhizomucor, Schizophyllum, Scytalidium,
Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trichoderma,
Trichophaea, Verticillium, Volvariella, or Xylaria polypeptide
having enzyme activity.
[0294] In one aspect, the polypeptide is a Saccharomyces
carlsbergensis, Saccharomyces cerevisiae, Saccharomyces
diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri,
Saccharomyces norbensis, or Saccharomyces oviformis polypeptide
having enzyme activity.
[0295] In another aspect, the polypeptide is an Acremonium
cellulolyticus, Aspergillus aculeatus, Aspergillus awamori,
Aspergillus fumigatus, Aspergillus foetidus, Aspergillus japonicus,
Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae,
Chrysosporium keratinophilum, Chrysosporium lucknowense,
Chrysosporium tropicum, Chrysosporium merdarium, Chrysosporium
inops, Chrysosporium pannicola, Chrysosporium queenslandicum,
Chrysosporium zonatum, Fusarium bactridioides, Fusarium cerealis,
Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum,
Fusarium graminum, Fusarium heterosporum, Fusarium negundi,
Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium
sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides,
Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides,
Fusarium venenatum, Humicola grisea, Humicola insolens, Humicola
lanuginosa, Irpex lacteus, Mucor miehei, Myceliophthora
thermophila, Neurospora crassa, Penicillium funiculosum,
Penicillium purpurogenum, Phanerochaete chrysosporium, Thielavia
achromatica, Thielavia albomyces, Thielavia albopilosa, Thielavia
australeinsis, Thielavia fimeti, Thielavia microspora, Thielavia
ovispora, Thielavia peruviana, Thielavia spededonium, Thielavia
setosa, Thielavia subthermophila, Thielavia terrestris, Trichoderma
harzianum, Trichoderma koningii, Trichoderma longibrachiatum,
Trichoderma reesei, Trichoderma viride, or Trichophaea saccata
polypeptide having enzyme activity.
[0296] Chemically modified or protein engineered mutants of
polypeptides having enzyme activity may also be used.
[0297] 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.
[0298] 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.RTM.
CTec (Novozymes A/S), CELLIC.RTM. CTec2 (Novozymes A/S),
CELLIC.RTM. CTec3 (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.
[0299] Examples of bacterial endoglucanases that can be used in the
methods 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).
[0300] 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 (GENBANK.TM. accession
no. M15665).
[0301] 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 cellobiohydrolase II (WO 2010/057086).
[0302] Examples of beta-glucosidases useful in the present
invention include, but are not limited to, beta-glucosidases from
Aspergillus aculeatus (Kawaguchi et al., 1996, Gene 173: 287-288),
Aspergillus fumigatus (WO 2005/047499), Aspergillus niger (Dan et
al., 2000, J. Biol. Chem. 275: 4973-4980), Aspergillus oryzae (WO
2002/095014), Penicillium brasilianum IBT 20888 (WO 2007/019442 and
WO 2010/088387), Thielavia terrestris (WO 2011/035029), and
Trichophaea saccata (WO 2007/019442).
[0303] 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).
[0304] Other useful endoglucanases, cellobiohydrolases, and
beta-glucosidases are disclosed in numerous Glycosyl Hydrolase
families using the classification according to Henrissat B., 1991,
A classification of glycosyl hydrolases based on amino-acid
sequence similarities, Biochem. J. 280: 309-316, and Henrissat B.,
and Bairoch A., 1996, Updating the sequence-based classification of
glycosyl hydrolases, Biochem. J. 316: 695-696.
[0305] Other cellulolytic enzymes that may be used in the present
invention are described in WO 98/13465, WO 98/015619, WO 98/015633,
WO 99/06574, WO 99/10481, WO 99/025847, WO 99/031255, WO
2002/101078, WO 2003/027306, WO 2003/052054, WO 2003/052055, WO
2003/052056, WO 2003/052057, WO 2003/052118, WO 2004/016760, WO
2004/043980, WO 2004/048592, WO 2005/001065, WO 2005/028636, WO
2005/093050, WO 2005/093073, WO 2006/074005, WO 2006/117432, WO
2007/071818, WO 2007/071820, WO 2008/008070, WO 2008/008793, U.S.
Pat. No. 5,457,046, U.S. Pat. No. 5,648,263, and U.S. Pat. No.
5,686,593.
[0306] In the methods of the present invention, any GH61
polypeptide having cellulolytic enhancing activity can be used.
[0307] In a first aspect, the GH61 polypeptide having cellulolytic
enhancing activity comprises the following motifs:
TABLE-US-00001 (SEQ ID NO: 21 or SEQ ID NO: 22)
[ILMV]-P-X(4,5)-G-X-Y-[ILMV]-X-R-X-[EQ]-X(4)-[HNQ] and
[FW]-[TF]-K-[AIV],
wherein X is any amino acid, X(4,5) is any amino acid at 4 or 5
contiguous positions, and X(4) is any amino acid at 4 contiguous
positions.
[0308] The isolated polypeptide comprising the above-noted motifs
may further comprise:
TABLE-US-00002 (SEQ ID NO: 23 or SEQ ID NO: 24)
H-X(1,2)-G-P-X(3)-[YW]-[AILMV], (SEQ ID NO: 25)
[EQ]-X-Y-X(2)-C-X-[EHQN]-[FILV]-X-[ILV], or (SEQ ID NO: 26 or SEQ
ID NO: 27) H-X(1,2)-G-P-X(3)-[YW]-[AILMV] and (SEQ ID NO: 28)
[EQ]-X-Y-X(2)-C-X-[EHQN]-[FILV]-X-[ILV],
[0309] wherein X is any amino acid, X(1,2) is any amino acid at 1
position or 2 contiguous positions, X(3) is any amino acid at 3
contiguous positions, and X(2) is any amino acid at 2 contiguous
positions. In the above motifs, the accepted IUPAC single letter
amino acid abbreviation is employed.
[0310] In a preferred aspect, the isolated GH61 polypeptide having
cellulolytic enhancing activity further comprises
H-X(1,2)-G-P-X(3)-[YW]-[AILMV] (SEQ ID NO: 23 or SEQ ID NO: 24). In
another preferred embodiment, the isolated GH61 polypeptide having
cellulolytic enhancing activity further comprises
[EQ]-X-Y-X(2)-C-X-[EHQN]-[FILV]-X-[ILV] (SEQ ID NO: 25). In another
preferred embodiment, the isolated GH61 polypeptide having
cellulolytic enhancing activity further comprises
H-X(1,2)-G-P-X(3)-[YW]-[AILMV] (SEQ ID NO: 26 or SEQ ID NO: 27) and
[EQ]-X-Y-X(2)-C-X-[EHQN]-[FILV]-X-[ILV] (SEQ ID NO: 28).
[0311] In a second aspect, the GH61 polypeptide having cellulolytic
enhancing activity comprises the following motif:
TABLE-US-00003 (SEQ ID NO: 29 or SEQ ID NO: 30)
[ILMV]-P-X(4,5)-G-X-Y-[ILMV]-X-R-X-[EQ]-X(3)-A- [HNQ],
wherein x is any amino acid, x(4,5) is any amino acid at 4 or 5
contiguous positions, and x(3) is any amino acid at 3 contiguous
positions. In the above motif, the accepted IUPAC single letter
amino acid abbreviation is employed.
[0312] 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).
[0313] 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.
[0314] In another 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 such as
pretreated corn stover (PCS).
[0315] 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.
[0316] 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.
[0317] 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.
[0318] 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 the
nitrogen-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.
[0319] 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.
[0320] 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.
[0321] In one aspect, an effective amount of such a compound
described above to cellulosic material as a molar ratio to glucosyl
units of cellulose is about 10.sup.-6 to about 10, e.g., about
10.sup.-6 to about 7.5, about 10.sup.-6 to about 5, about 10.sup.-6
to about 2.5, about 10.sup.-6 to about 1, about 10.sup.-5 to about
1, about 10.sup.-5 to about 10.sup.-1, about 10.sup.-4 to about
10.sup.-1, about 10.sup.-3 to about 10.sup.-1, or about 10.sup.-3
to about 10.sup.-2. In another aspect, an effective amount of such
a compound described above is about 0.1 .mu.M to about 1 M, e.g.,
about 0.5 .mu.M to about 0.75 M, about 0.75 .mu.M to about 0.5 M,
about 1 .mu.M to about 0.25 M, about 1 .mu.M to about 0.1 M, about
5 .mu.M to about 50 mM, about 10 .mu.M to about 25 mM, about 50
.mu.M to about 25 mM, about 10 .mu.M to about 10 mM, about 5 .mu.M
to about 5 mM, or about 0.1 mM to about 1 mM.
[0322] 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.
[0323] 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 g,
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.-1g, 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.
[0324] 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.RTM. HTec (Novozymes
A/S), CELLIC.RTM. HTec2 (Novozymes A/S), CELLIC.RTM. HTec3
(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).
[0325] Examples of xylanases useful in the methods 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).
[0326] Examples of beta-xylosidases useful in the methods 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).
[0327] Examples of acetylxylan esterases useful in the methods 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).
[0328] Examples of feruloyl esterases (ferulic acid esterases)
useful in the methods 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).
[0329] Examples of arabinofuranosidases useful in the methods 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).
[0330] Examples of alpha-glucuronidases useful in the methods 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).
[0331] The polypeptides having enzyme activity used in the methods
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, CA,
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, NY, 1986).
[0332] 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.
[0333] Fermentation. The fermentable sugars obtained from the
hydrolyzed cellulosic 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.
[0334] In the fermentation step, sugars, released from the
cellulosic 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.
[0335] Any suitable hydrolyzed cellulosic 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.
[0336] 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).
[0337] "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.
[0338] Examples of bacterial and fungal fermenting organisms
producing ethanol are described by Lin et al., 2006, Appl.
Microbiol. Biotechnol. 69: 627-642.
[0339] 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.
[0340] 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.
[0341] 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).
[0342] 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.
[0343] 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 another 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.
[0344] 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 Geobacilus 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.
[0345] 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).
[0346] 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.
[0347] 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 TAL1 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).
[0348] 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.
[0349] It is well known in the art that the organisms described
above can also be used to produce other substances, as described
herein.
[0350] The fermenting microorganism is typically added to the
degraded cellulosic 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.
[0351] In one aspect, the yeast and/or another microorganism are
applied to the degraded cellulosic 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.
[0352] 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.
[0353] 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.
[0354] Fermentation products: 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.
[0355] In a preferred aspect, the fermentation product is an
alcohol. It will be understood that the term "alcohol" encompasses
a substance that contains one or more hydroxyl moieties. In a more
preferred aspect, the alcohol is n-butanol. In another more
preferred aspect, the alcohol is isobutanol. In another more
preferred aspect, the alcohol is ethanol. In another more preferred
aspect, the alcohol is methanol. In another more preferred aspect,
the alcohol is arabinitol. In another more preferred aspect, the
alcohol is butanediol. In another more preferred aspect, the
alcohol is ethylene glycol. In another more preferred aspect, the
alcohol is glycerin. In another more preferred aspect, the alcohol
is glycerol. In another more preferred aspect, the alcohol is
1,3-propanediol. In another more preferred aspect, the alcohol is
sorbitol. In another more preferred aspect, the alcohol is xylitol.
See, for example, Gong, C. S., Cao, N. J., Du, J., and Tsao, G. T.,
1999, Ethanol production from renewable resources, in Advances in
Biochemical Engineering/Biotechnology, Scheper, T., ed.,
Springer-Verlag Berlin Heidelberg, Germany, 65: 207-241; Silveira,
M. M., and Jonas, R., 2002, The biotechnological production of
sorbitol, Appl. Microbiol. Biotechnol. 59: 400-408; Nigam, P., and
Singh, D., 1995, Processes for fermentative production of
xylitol--a sugar substitute, Process Biochemistry 30 (2): 117-124;
Ezeji, T. C., Qureshi, N. and Blaschek, H. P., 2003, Production of
acetone, butanol and ethanol by Clostridium beijerinckii BA101 and
in situ recovery by gas stripping, World Journal of Microbiology
and Biotechnology 19 (6): 595-603.
[0356] 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.
[0357] 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.
[0358] 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.
[0359] In another preferred aspect, the fermentation product is an
amino acid. In another more preferred aspect, the organic acid is
aspartic acid. In another more preferred aspect, the amino acid is
glutamic acid. In another more preferred aspect, the amino acid is
glycine. In another more preferred aspect, the amino acid is
lysine. In another more preferred aspect, the amino acid is serine.
In another more preferred aspect, the amino acid is threonine. See,
for example, Richard, A., and Margaritis, A., 2004, Empirical
modeling of batch fermentation kinetics for poly(glutamic acid)
production and other microbial biopolymers, Biotechnology and
Bioengineering 87 (4): 501-515.
[0360] In another preferred aspect, the fermentation product is a
gas. In another more preferred aspect, the gas is methane. In
another more preferred aspect, the gas is H.sub.2. In another more
preferred aspect, the gas is CO.sub.2. In another more preferred
aspect, the gas is CO. See, for example, Kataoka, N., A. Miya, and
K. Kiriyama, 1997, Studies on hydrogen production by continuous
culture system of hydrogen-producing anaerobic bacteria, Water
Science and Technology 36 (6-7): 41-47; and Gunaseelan V. N. in
Biomass and Bioenergy, Vol. 13 (1-2), pp. 83-114, 1997, Anaerobic
digestion of biomass for methane production: A review.
[0361] In another preferred aspect, the fermentation product is
isoprene.
[0362] 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.
[0363] In another preferred aspect, the fermentation product is an
organic acid. In another more preferred aspect, the organic acid is
acetic acid. In another more preferred aspect, the organic acid is
acetonic acid. In another more preferred aspect, the organic acid
is adipic acid. In another more preferred aspect, the organic acid
is ascorbic acid. In another more preferred aspect, the organic
acid is citric acid. In another more preferred aspect, the organic
acid is 2,5-diketo-D-gluconic acid. In another more preferred
aspect, the organic acid is formic acid. In another more preferred
aspect, the organic acid is fumaric acid. In another more preferred
aspect, the organic acid is glucaric acid. In another more
preferred aspect, the organic acid is gluconic acid. In another
more preferred aspect, the organic acid is glucuronic acid. In
another more preferred aspect, the organic acid is glutaric acid.
In another preferred aspect, the organic acid is 3-hydroxypropionic
acid. In another more preferred aspect, the organic acid is
itaconic acid. In another more preferred aspect, the organic acid
is lactic acid. In another more preferred aspect, the organic acid
is malic acid. In another more preferred aspect, the organic acid
is malonic acid. In another more preferred aspect, the organic acid
is oxalic acid. In another more preferred aspect, the organic acid
is propionic acid. In another more preferred aspect, the organic
acid is succinic acid. In another more preferred aspect, the
organic acid is xylonic acid. See, for example, Chen, R., and Lee,
Y. Y., 1997, Membrane-mediated extractive fermentation for lactic
acid production from cellulosic biomass, Appl. Biochem. Biotechnol.
63-65: 435-448.
[0364] In another preferred aspect, the fermentation product is
polyketide.
[0365] Recovery. 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 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.
Paper-Making
[0366] The present invention also relates to processes for
manufacturing a paper material, which processes comprise treating a
paper-making pulp and/or process water with a polypeptide having
endoglucanase activity of the present invention.
[0367] The term "a paper-making process" means a process, wherein
the pulp is suspended in water, mixed with various additives, and
then passed to equipment in which paper, cardboard, tissue, towel
etc. is formed, pressed and dried.
[0368] The term "paper material" means products, which can be made
out of pulp, such as paper, linerboard, corrugated paperboard,
tissue, towels, corrugated containers, or boxes.
[0369] The term "a paper-making pulp" or "pulp" means any pulp
which can be used for the production of a paper material. For
example, the pulp can be supplied as a virgin pulp, or can be
derived from a recycled source. The paper-making pulp may be a wood
pulp, a non-wood pulp or a pulp made from waste paper. A wood pulp
may be made from softwood such as pine, redwood, fir, spruce,
cedar, and hemlock or from hardwood such as maple, alder, birch,
hickory, beech, aspen, acacia, and eucalyptus. A non-wood pulp may
be made, e.g., from bagasse, bamboo, cotton, or kenaf. A waste
paper pulp may be made by re-pulping waste paper such as newspaper,
mixed office waste, computer print-out, white ledger, magazines,
milk cartons, paper cups, etc.
[0370] In a particular embodiment, the paper-making pulp to be
treated comprises both hardwood pulp and softwood pulp.
[0371] The wood pulp to be treated may be mechanical pulp (such as
ground wood pulp, GP), chemical pulp (such as Kraft pulp or sulfite
pulp), semichemical pulp (SCP), thermomechanical pulp (TMP),
chemithermomechanical pulp (CTMP), or bleached
chemithermomechanical pulp (BCTMP).
[0372] Mechanical pulp is manufactured by grinding and refining
methods, wherein the raw material is subjected to periodical
pressure impulses. The following are examples of mechanical pulps:
TMP is thermomechanical pulp, GW is groundwood pulp, PGW is
pressurized groundwood pulp, RMP is refiner mechanical pulp, PRMP
is pressurized refiner mechanical pulp, and CTMP is
chemithermomechanical pulp.
[0373] Chemical pulp is manufactured by alkaline cooking whereby
most of the lignin and hemicellulose components are removed. In
Kraft pulping or sulphate cooking sodium sulphide or sodium
hydroxide are used as principal cooking chemicals.
[0374] The Kraft pulp to be treated may be a bleached Kraft pulp,
which may consist of softwood bleached Kraft (SWBK, also called
NBKP, Nadel Holz Bleached Kraft Pulp), hardwood bleached Kraft
(HWBK, also called LBKP, Laub Holz Bleached Kraft Pulp), or a
mixture of these.
[0375] The pulp to be used in the processes of the present
invention is a suspension of mechanical pulp, chemical pulp, or a
combination thereof. For example, the pulp to be used in the
processes of the present invention may comprise 0%, 10-20%, 20-30%,
30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100% of
chemical pulp. In a particular embodiment, a chemical pulp forms
part of the pulp being used for manufacturing the paper material.
The expression "forms part of" means that the percentage of
chemical pulp lays within the range of 1-99%. In particular
embodiments, the percentage of chemical pulp lies within the range
of 2-98%, 3-97%, 4-96%, 5-95%, 6-94%, 7-93%, 8-92%, 9-91%, 10-90%,
15-85%, 20-80%, 25-75%, 30-70%, 40-60%, or 45-55%.
[0376] In a particular embodiment, the chemical pulp is a Kraft
pulp, a sulfite pulp, a semichemical pulp (SCP), a thermomechanical
pulp (TMP), a chemithermomechanical pulp (CTMP), or a bleached
chemithermomechanical pulp (BCTMP). In particular embodiments, the
Kraft pulp is bleached Kraft pulp, for example, softwood bleached
Kraft (SWBK, also called NBKP, Nadel Holz Bleached Kraft Pulp),
hardwood bleached Kraft (HWBK, also called LBKP, Laub Holz Bleached
Kraft Pulp), or a mixture thereof.
[0377] In the case of pulp and paper processing, the processes of
the present invention can be carried out at any pulp production
stage. The enzyme can be added to any holding tank, e.g., to a pulp
storing container (storage chest), storage tower, mixing chest, or
metering chest. The enzyme treatment can be performed before the
bleaching of pulp, in connection with the pulp bleaching process,
or after the bleaching. When carried out in connection with pulp
bleaching the enzyme preparation may be added together with
bleaching chemicals such as chlorine or chlorine dioxide.
[0378] Oxygen gas, hydrogen peroxide, ozone, or combinations
thereof may also be applied in the bleaching of pulp. The enzyme
preparation may also be added together with these substances.
[0379] Preferably the enzyme preparation is added prior to
bleaching. The enzyme can also be added to the circulated process
water (white water) originating from bleaching and process water
(brown water) originating from the mechanical or chemimechanical
pulping process. In a particular embodiment of a Kraft pulping
process, the enzyme is added during the brown-stock washing.
[0380] The term "process water" comprises (1) water added as a raw
material to the paper manufacturing process; (2) intermediate water
products resulting from any step of the process for manufacturing
the paper material; and/or (3) waste water as an output or
by-product of the process. In a particular embodiment, the process
water is, has been, is being, or is intended for being circulated
(re-circulated), i.e., re-used in another step of the process. The
term "water" means any aqueous medium, solution, suspension, e.g.,
ordinary tap water, and tap water in admixture with various
additives and adjuvants commonly used in paper manufacturing
processes. In a particular embodiment, the process water has a low
content of solid (dry) matter, e,g., below 20%, 18%, 16%, 14%, 12%,
10%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% dry matter.
[0381] The processes of the present invention may be carried out
under conventional conditions in the pulp and paper processing. The
process conditions will be a function of the enzyme(s) applied, the
reaction time, and the conditions used.
[0382] The drainability of paper-making pulps may also be improved
by treatment of the pulp with a polypeptide of the present
invention. Use of a polypeptide of the present invention may be
more effective, e.g., result in a higher degree of loosening
bundles of strongly hydrated microfibrils in the fines fraction
that limits the rate of drainage by blocking hollow spaces between
the fibers and in the wire mesh of the paper machine.
[0383] A polypeptide having endoglucanase activity of the present
invention should be added in an effective amount. The term
"effective amount" means the amount sufficient to achieve the
desired and expected effect, such as (a) an increase in handsheet
tensile index, and/or (b) an increase in handsheet tear index,
relative to no treatment with the polypeptide having endoglucanase
activity.
[0384] In a particular embodiment, the dosage of a polypeptide of
the present invention and additional enzymes, if any, is from about
0.1 mg enzyme protein to about 100,000 mg enzyme protein (of each
enzyme) per ton of paper pulp.
[0385] In further particular embodiments, the amount of the
polypeptide having endoglucanase activity and additional enzymes,
if any, is in the range of 0,00001-20 mg of enzyme (calculated as
pure enzyme protein) per gram (dry weight) of lignocellulosic
material, e.g., 0.0001-20, 0.0001-10, 0.0001-1, 0.001-1, 0.001-0.1,
or 0.01-0.1 mg of enzyme per gram of lignocellulosic material.
These amounts refer to the amount of each enzyme.
[0386] The enzymatic treatment can be performed at conventional
consistency, e.g., 0.5-10% dry substance. In particular
embodiments, the consistency is within the range of 0.5-45, 0.5-40,
0.5-35, 0.5-30, 0.5-25, 0.5-20, 0.5-15, 0.5-10, 0.5-8, 0.5-6, or
0.5-5% dry substance.
[0387] The enzymatic treatment may be carried out at a temperature
of from about 10 to about 100.degree. C. Further examples of
temperature ranges (all "from about" and "to about") are the
following: 20-100, 30-100, 35-100, 37-100, 40-100, 50-100, 60-100,
70-100, 10-90, 10-80, 10-70, 10-60, or 30-66.degree. C., as well as
any combination of the upper and lower values indicated herein. A
typical temperature is from about 20 to 90.degree. C., or 20 to
95.degree. C., preferably from about 40 to 70.degree. C., or 40 to
75.degree. C.
[0388] The enzymatic treatment may be carried out at a pH from
about 2 to about 12. Further examples of pH ranges (all "from
about" and "to about") are the following: 3-12, 4-12, 5-12, 6-12,
7-12, 8-12, 9-12, 2-11, 2-10, 2-9, 2-8, 4-10, or 5-8 as well as any
combination of the upper and lower values indicated herein. A
typical pH range is from about 2 to 11, preferably within the range
from about 4 to 9.5, or 6 to 9.
[0389] A suitable duration of the enzymatic treatment may be in the
range from a few seconds to several hours, e.g., about 30 seconds
to about 48 hours, about 1 minute to about 24 hours, about 1 minute
to about 18 hours, about 1 minute to about 12 hours, about 1 minute
to 5 hours, about 1 minute to about 2 hours, about 1 minute to
about 1 hour, or about 1 minute to about 30 minutes. A typical
reaction time is from about 10 minutes to about 3 hours, about 10
minutes to about 10 hours, preferably about 15 minutes to about 1
hour, or about 15 minutes to about 2 hours.
[0390] Molecular oxygen from the atmosphere will usually be present
in sufficient quantity, if required. Therefore, the reaction may be
conveniently performed in an open reactor, i.e., at atmospheric
pressure.
[0391] Various additives over and above a polypeptide having
endoglucanase activity of the present invention and additional
enzymes, if any, can be used in the processes of the present
invention. Surfactants and/or dispersants are often present in,
and/or added to, a paper-making pulp. Thus the processes of the
present invention may be carried out in the presence of an anionic,
non-ionic, cationic, and/or zwitterionic surfactant and/or
dispersant conventionally used in a paper-making pulp. Examples of
anionic surfactants are carboxylates, sulphates, sulphonates, or
phosphates of alkyl, substituted alkyl, or aryl. Fatty acids are
examples of alkyl-carboxylates. Examples of non-ionic surfactants
are polyoxyethylene compounds, such as alcohol ethoxylates,
propoxylates, or mixed ethoxy/propoxylates, poly-glycerols and
other polyols, as well as certain block-copolymers. Examples of
cationic surfactants are water-soluble cationic polymers, such as
quaternary ammonium sulphates and certain amines, e.g.,
epichlorohydrin/dimethylamine polymers (EPI-DMA) and cross-linked
solutions thereof, polydiallyl dimethyl ammonium chloride (DADMAC),
DADMAC/acrylamide co-polymers, and ionene polymers, such as those
disclosed in U.S. Pat. Nos. 5,681,862 and 5,575,993. Examples of
zwitterionic or amphoteric surfactants are betaines, glycinates,
amino propionates, imino propionates, and various
imidazoline-derivatives. Also the polymers disclosed in U.S. Pat.
No. 5,256,252 may be used.
[0392] Also according to the present invention, surfactants such as
the above, including any combination thereof, may be used in a
paper making process together with a polypeptide having
endoglucanase activity of the present invention, and included in a
composition together with such enzyme. The amount of each
surfactant in such composition may amount to about 8 to about 40%
(w/w) of the composition. In particular embodiments, the amount of
each surfactant is from about 10 to about 38, about 12 to about 36,
about 14 to about 34, about 16 to about 34, about 18 to about 34,
about 20 to about 34, about 22 to about 34, about 24 to about 34,
about 26 to about 34, or about 28 to about 32% (w/w).
[0393] In another particular embodiment, each of the above ranges
refers to the total amount of surfactants.
[0394] In the processes of the present invention, a polypeptide of
the present invention may be applied alone or together with an
additional enzyme. The term "an additional enzyme" means at least
one additional enzyme, e.g., one, two, three, four, five, six,
seven, eight, nine, ten or even more additional enzymes.
[0395] The term "applied together with" (or "used together with")
means that the additional enzyme may be applied in the same step or
in another step of the process. The other process step may be
upstream or downstream in the paper manufacturing process, as
compared to the step in which the paper making pulp or process
water is treated with a polypeptide of the present invention.
[0396] In particular embodiments, the additional enzyme is an
enzyme which has protease, lipase, xylanase, cutinase,
oxidoreductase, cellulase, endoglucanase, amylase, mannanase,
steryl esterase, fatty acid oxidizing enzyme, and/or cholesterol
esterase activity. Examples of oxidoreductase enzymes are enzymes
with laccase and/or peroxidase activity. In a preferred embodiment,
the additional enzyme is lipase.
[0397] The term "a step" of a process means at least one step, and
it could be one, two, three, four, five or even more process steps.
In other words, a polypeptide of the present invention may be
applied in at least one process step, and the additional enzyme(s)
may also be applied in at least one process step, which may be the
same or a different process step compared to the step where the
polypeptide of the present invention is used.
[0398] The term "enzyme preparation" means a product containing at
least one polypeptide of the present invention. The enzyme
preparation may also comprise enzymes having other enzyme
activities, preferably lipolytic enzymes or enzymes having
oxidoreductase activity, most preferably lipolytic enzymes. In
addition to the enzymatic activity such a preparation preferably
contains at least one adjuvant. Examples of adjuvants used in
enzyme preparations for the pulp and paper industry are buffers,
polymers, surfactants, and stabilizing agents.
[0399] Any enzyme having protease, lipase, xylanase, cutinase,
oxidoreductase, cellulase, endoglucanase, amylase, mannanase,
steryl esterase, fatty acid oxidizing enzyme, and/or cholesterol
esterase can be used as additional enzymes in the processes of the
present invention. Non-limiting examples are listed below of such
additional enzymes. The activity of any of the additional enzymes
can be analyzed using any method known in the art for the enzyme in
question, including the methods mentioned in the references
cited.
[0400] Examples of cutinases are those derived from Humicola
insolens (U.S. Pat. No. 5,827,719); or a strain of Fusarium, e.g.,
F. roseum culmorum, or particularly F. solani pili (WO 90/09446; WO
94/14964, WO 94/03578). The cutinase may also be derived from a
strain of Rhizoctonia, e.g., R. solani, or a strain of Alternaria,
e.g., A. brassicicola (WO 94/03578), or variants thereof such as
those described in WO 00/34450, or WO 01/92502.
[0401] Examples of proteases are the ALCALASE.TM., ESPERASE.TM.,
SAVINASE.TM., NEUTRASE.TM., and DURAZYM.TM. proteases (Novozymes
A/S, Bagsvaerd, Denmark). Other proteases are derived from
Nocardiapsis, Aspergillus, Rhizopus, Bacillus alcalophilus, B.
cereus, B., naffo, B. vulgatus, B. mycoide, especially proteases
from the species Nocardiopsis sp. and Nocardiopsis dassonvillei
such as those disclosed in WO 88/03947, and mutants thereof, e.g.,
those disclosed in WO 91/00345 and EP 415296.
[0402] Examples of amylases are the BAN.TM., AQUAZYM.TM.,
TERMAMYL.TM., and AQUAZYM.TM. Ultra amylases (Novozymes A/S,
Bagsvaerd, Denmark). An example of a lipase is RESINASE.TM. A2X
lipase (Novozymes A/S, Bagsvaerd, Denmark). An example of a
xylanase is the PULPZYME.TM. HC hemicellulase (Novozymes Bagsvaerd,
Denmark). Examples of other endoglucanases are the NOVOZYM 613,
342, and 476 enzyme products (Novozymes A/S, Bagsvaerd,
Denmark).
[0403] Examples of mannanases are the Trichoderma reesei
endo-beta-mannanases described by Stahlband et al., 1993, J.
Biotechnol. 29: 229-242.
[0404] Examples of steryl esterases, peroxidases, laccases, and
cholesterol esterases are disclosed in WO 00/53843; U.S. Pat. No.
6,066,486; JP 2000080581; Karlsson et al, 2001, Appl. Microbiol.
Biotechnol. 55: 317-320; and Zhang, 2000; Pulp & Paper Canada
101: 59-62. Further examples of oxidoreductases are the peroxidases
and laccases disclosed in EP 730641; WO 01/98469; EP 719337; EP
765394; EP 767836; EP 763115; and EP 788547. Whenever an
oxidoreductase enzyme is mentioned that requires or benefits from
the presence of acceptors (e.g., oxygen or hydrogen peroxide),
enhancers, mediators and/or activators, such compounds should be
considered to be included. Examples of enhancers and mediators are
disclosed in EP 705327; WO 98/56899; EP 677102; EP 781328; and EP
707637. If desired a distinction could be made by defining an
oxidoreductase enzyme system (e.g., a laccase, or a peroxidase
enzyme system) as the combination of the enzyme in question and its
acceptor, and optionally also an enhancer and/or mediator for the
enzyme in question.
[0405] Examples of fatty acid oxidizing enzymes are disclosed in WO
2003/035972.
Signal Peptide
[0406] 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. 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.
[0407] The present invention also relates to nucleic acid
constructs, expression vectors, and recombinant host cells
comprising such a polynucleotide.
[0408] The present invention also relates to methods of producing a
protein, comprising: (a) cultivating a recombinant host cell
comprising such a polynucleotide; and optionally (b) recovering the
protein.
[0409] 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.
[0410] Preferably, the protein is a hormone or variant thereof,
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.
[0411] The gene may be obtained from any prokaryotic, eukaryotic,
or other source.
[0412] The present invention is further described by the following
examples that should not be construed as limiting the scope of the
invention.
EXAMPLES
Materials
[0413] Chemicals used as buffers and substrates were commercial
products of at least reagent grade.
Strains
[0414] Chaetomium virescens ATCC 32319 was used as the source of a
polypeptide having endoglucanase activity. Aspergillus oryzae
MT3568 strain was used for expression of the Chaetomium virescens
gene encoding the polypeptide having endoglucanase activity. A.
oryzae MT3568 is an amdS (acetamidase) disrupted gene derivative of
Aspergillus oryzae JaL355 (WO 2002/40694) in which pyrG auxotrophy
was restored by disrupting the A. oryzae acetamidase (amdS)
gene.
Media and Solutions
[0415] YP+2% glucose medium was composed of 1% yeast extract, 2%
peptone, and 2% glucose.
[0416] PDA plates were composed of potato infusion 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).
[0417] LB medium was composed of 10 g of Bacto-Tryptone, 5 g of
yeast extract, 10 g of sodium chloride, and deionized water to 1
liter.
[0418] 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).
[0419] COVE sucrose plates were composed of 342 g of sucrose, 20 g
of agar powder, 20 ml of COVE salt solution, 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 10 mM
acetamide, 15 mM CsCl, and TRITON.RTM. X-100 (50 .mu.l/500 ml) were
added.
[0420] COVE salt solution was composed of 26 g of
MgSO.sub.4.7H.sub.2O, 26 g of KCl, 26 g of KH.sub.2PO.sub.4, 50 ml
of COVE trace metals solution, and deionized water to 1 liter.
[0421] COVE trace metals solution was composed of 0.04 g of
Na.sub.2B.sub.4O.sub.7.10H.sub.2O, 0.4 g of CuSO.sub.4.5H.sub.2O,
1.2 g of FeSO.sub.4.7H.sub.2O, 0.7 g of MnSO.sub.4.H.sub.2O, 0.8 g
of Na.sub.2MoO.sub.4.2H.sub.2O, 10 g of ZnSO.sub.4.7H.sub.2O, and
deionized water to 1 liter.
[0422] Dap-4C medium was composed of 20 g of dextrose, 10 g of
maltose, 11 g of MgSO.sub.4.7H.sub.2O, 1 g of KH.sub.2PO.sub.4, 2 g
of citric acid, 5.2 g of K.sub.3PO.sub.4.H.sub.2O, 0.5 g of yeast
extract (Difco), 1 ml of Dowfax 63N10 (Dow Chemical Company), 0.5
ml of KU6 trace metals solution, 2.5 g of 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, 3.5 ml of sterile 50%
(NH.sub.4).sub.2HPO.sub.4 and 5 ml of sterile 20% lactic acid were
added per 150 ml of Dap-4C medium.
[0423] KU6 trace metals solution was composed of 0.13 g of
NiCl.sub.2, 2.5 g of CuSO.sub.4.5H.sub.2O, 13.9 g of
FeSO.sub.4.7H.sub.2O, 8.45 g of MnSO.sub.4.H.sub.2O, 6.8 g of
ZnCl.sub.2, 3 g of citric acid, and deionized water to 1 liter.
[0424] MDU2BP medium was composed of 45 g of maltose, 1 g of
MgSO.sub.4.7H.sub.2O, 1 g of NaCl, 2 g of K.sub.2SO.sub.4, 12 g of
KH.sub.2PO.sub.4, 7 g of yeast extract, 2 g of urea, 0.5 ml of AMG
trace metals solution, and deionized water to 1 liter; pH 5.0.
[0425] AMG trace metals solution was composed of 14.3 g of
ZnSO.sub.4.7H.sub.2O, 2.5 g of CuSO.sub.4.5H.sub.2O, 0.5 g of
NiCl.sub.2.6H.sub.2O, 13.8 g of FeSO.sub.4.7H.sub.2O, 8.5 g of
MnSO.sub.4.7H.sub.2O, 3 g of citric acid, and deionized water to 1
liter.
[0426] 2XYT plates were composed of 16 g of tryptone, 10 g of yeast
extract, 5 g of NaCl, 15 g of Noble agar, and deionized water to 1
liter.
Example 1
Preparation of Chaetomium Virescens ATCC 32319 Cel5
Endoglucanase
[0427] The Chaetomium virescens ATCC 32319 GH5 endoglucanase gene
(SEQ ID NO: 1 [DNA sequence] and SEQ ID NO: 2 [deduced amino acid
sequence]) was cloned into an Aspergillus oryzae expression vector
as described below.
[0428] Two synthetic oligonucleotide primers, shown below, were
designed to PCR amplify the endoglucanase gene from C. virescens
ATCC 32319 genomic DNA. Genomic DNA was isolated using a
FASTDNA.RTM. Spin for Soil Kit (MP Biomedicals, OH, USA).
TABLE-US-00004 Primer F-Cv1: (SEQ ID NO: 3)
5'-ACACAACTGGGGATCCACCATGCCTTCAGTCGTCCTTTCTC-3' Primer R-Cv1: (SEQ
ID NO: 4) 5'-CCCTCTAGATCTCGAGCAACAATGCCGACACACTCCA-3'
[0429] Bold letters represent gene sequence. The underlined
sequence is homologous to the insertion sites of plasmid pDau109
(WO 2005/042735).
[0430] The amplification reaction was composed of 1 .mu.l of C.
virescens ATCC 32319 genomic DNA, 5 .mu.l of 5.times. PHUSION.TM.
HF Buffer (Finnzymes, Espoo, Finland), 0.5 .mu.l (2 Units/.mu.l) of
PHUSION.TM. High-Fidelity DNA Polymerase (Finnzymes, Espoo,
Finland), 1 .mu.l of 5 .mu.M primer F-Cv1, 1 .mu.l of 5 .mu.M
primer R-Cv1, 0.5 .mu.l of 10 mM dNTP, and 16 .mu.l of H.sub.2O.
The amplification reaction was incubated in a PTC-200 DNA
ENGINE.TM. Thermal Cycler (MJ Research Inc., Waltham, Mass., USA)
programmed for 1 cycle at 95.degree. C. for 2 minutes; and 35
cycles each at 98.degree. C. for 10 seconds, 60.degree. C. for 30
seconds, and 72.degree. C. for 2.5 minutes.
[0431] A 1.34 kbp PCR reaction product was isolated by 1% agarose
gel electrophoresis using 40 mM Tris base-20 mM sodium acetate-1 mM
disodium EDTA (TAE) buffer and staining with SYBR.RTM. Safe DNA Gel
Stain (Invitrogen Corp., Carlsbad, Calif., USA). The DNA band was
visualized with the aid of an EAGLE EYE.RTM. Imaging System
(Stratagene, La Jolla, Calif., USA) and a DARKREADER.RTM.
Transilluminator (Clare Chemical Research, Dolores, Colo., USA).
The 1.34 kbp DNA band was excised from the gel and purified using a
GFX.RTM. PCR DNA and Gel Band Purification Kit (GE Healthcare Life
Sciences, Piscataway, N.J., USA) according to the manufacturer's
instructions. The 1.34 kbp fragment was cleaved with Bam HI and Xho
I and purified using a GFX.RTM. PCR DNA and Gel Band Purification
Kit according to the manufacturer's instructions. An IN-FUSION.TM.
Cloning Kit (BD Biosciences, Palo Alto, Calif., USA) was used to
clone the fragment directly into the expression vector pDau109
digested with Bam HI and Xho I.
[0432] The cloning mixture was transformed into E. coli TOP10F
competent cells (Invitrogen Corp., Carlsbad, Calif., USA) according
to the manufacturer's instructions. The transformation mixture was
plated onto LB plates supplemented with 100 .mu.g of ampicillin per
ml. Plasmid minipreps were prepared from several transformants and
sequenced. One plasmid with the correct C. virescens GH5 coding
sequence (SEQ ID NO: 1) was chosen. The plasmid was designated
pDAU222-PE03860005710 (FIG. 1). Cloning of the Chaetomium virescens
GH5 gene into Bam HI-Xho I digested pDau109 resulted in
transcription of the C. virescens GH5 gene under the control of a
NA2-tpi double promoter. NA2-tpi is a modified promoter from the
Aspergillus niger neutral alpha-amylase gene in which the
untranslated leader has been replaced by the untranslated leader
from the Aspergillus nidulans triose phosphate isomerase gene.
[0433] The expression plasmid pDAU222-PE03860005710 was transformed
into protoplasts of Aspergillus oryzae MT3568 according to the
method of European Patent EP 0238023, pages 14-15. Aspergillus
oryzae MT3568 is an amdS (acetamidase) disrupted derivative of A.
oryzae JaL355 (WO 2002/40694) in which pyrG auxotrophy was restored
in the process of inactivating the A. oryzae amdS gene.
[0434] Transformants were purified on COVE sucrose plates through
single conidia prior to sporulating them on PDA plates. Production
of the Chaetomium virescens GH5 polypeptide by the transformants
was analyzed from culture supernatants of 1 ml 96 deep well
stationary cultivations at 26.degree. C. in YP+2% glucose medium.
Expression was verified by SDS-PAGE using a NU-PAGE.RTM. 10%
Bis-Tris SDS-PAGE gel (Invitrogen, Carlsbad, Calif., USA) and
Coomassie blue staining where a band of approximately 50 kDa was
observed. One transformant was selected and designated Aspergillus
oryzae 17.4.
[0435] For larger scale production, A. oryzae 17.4 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 cultures were incubated at
30.degree. C. with constant shaking at 85 rpm. At day four
post-inoculation, the culture broths were collected by filtration
through a bottle top MF75.TM. SUPOR.RTM. MachV 0.2 .mu.m PES filter
(Thermo Fisher Scientific, Roskilde, Denmark). The culture broths
from this transformant produced a band of GH5 protein of
approximately 50 kDa (designated EXP03745). The identity of this
band as the C. virescens GH5 polypeptide was verified by peptide
sequencing.
[0436] Alternative Method for Producing Cv1 GH5 from Chaetomium
Virescens.
[0437] 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.
[0438] Using the two synthetic oligonucleotide primers F-Cv1 and
R-Cv1 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 2
Characterization of the Chaetomium Virescens GH5 Endoglucanase
Gene
[0439] The genomic DNA sequence and deduced amino acid sequence of
the Chaetomium virescens GH5 endoglucanase encoding sequence are
shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively. The genomic
DNA sequence of 1320 bp (including the stop codon) contains one
intron located at nucleotides 1119 to 1178 of SEQ ID NO: 1. The
genomic DNA sequence encodes a polypeptide of 419 amino acids. The
% G+C content of the mature polypeptide coding sequence is 58.7%.
Using the SignalP software program (Nielsen et al., 1997, Protein
Engineering 10: 1-6), a signal peptide of 17 residues was
predicted. The predicted mature protein contains 402 amino acids
with a predicted molecular mass of 43.1 kDa and a predicted
isoelectric point of 5.5. The protein contains a cellulose binding
module of the CBM1 type at the N-terminus (amino acids 23 to 58 of
SEQ ID NO: 2). The catalytic domain is amino acids 93 to 419.
[0440] A comparative alignment of mature endoglucanase amino acid
sequences, without the signal peptides, was determined using the
Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol.
Biol. 48: 443-453) as implemented in the Needle program of EMBOSS
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 Chaetomium virescens GH5 endoglucanase (mature
polypeptide) shares 75.4% sequence identity (excluding gaps) to the
deduced amino acid sequence of an endoglucanase from Hypocrea
jecorina (GENESEQP:ASQ45591).
Example 3
Purification of Chaetomium Virescens GH5 Polypeptide Having
Endoglucanase Activity
[0441] Filtered broth (Example 1) was adjusted to pH 7.0 and
filtered using a 0.22 .mu.m PES filter (Thermo Fisher Scientific,
Roskilde, Denmark). To the filtrate was added ammonium sulphate to
1.0 M pH 7.0. The filtrate was loaded onto a PHENYL SEPHAROSE.TM. 6
Fast Flow column (high sub) (GE Healthcare, Piscataway, N.J., USA)
equilibrated with 1.0 M ammonium sulphate pH 7.0, and bound
proteins were eluted with 25 mM HEPES pH 7.0. The fractions were
pooled and applied to a SEPHADEX.TM. G-25 (medium) column (GE
Healthcare, Piscataway, N.J., USA) equilibrated in 50 mM HEPES pH
7.0. The fractions were applied to a SOURCE.TM. 15Q column (GE
Healthcare, Piscataway, N.J., USA) equilibrated in 50 mM HEPES pH
7.0. The protein eluted in the flow-through. The flow-through was
concentrated 5-fold using a VIVASPIN.RTM. 20 centrifugal
concentrator with a 10,000 MWCO PES membrane (Sartorius Stedim
Biotech GmbH, 37070 Goettingen, Germany).
Example 4
Pretreated Corn Stover Hydrolysis Assay
[0442] Corn stover was pretreated at the U.S. Department of Energy
National Renewable Energy Laboratory (NREL) using 1.4 wt % sulfuric
acid at 165.degree. C. and 107 psi for 8 minutes. The
water-insoluble solids in the pretreated corn stover (PCS)
contained 56.5% cellulose, 4.6% hemicellulose, and 28.4% lignin.
Cellulose and hemicellulose were determined by a two-stage sulfuric
acid hydrolysis with subsequent analysis of sugars by high
performance liquid chromatography using NREL Standard Analytical
Procedure #002. Lignin was determined gravimetrically after
hydrolyzing the cellulose and hemicellulose fractions with sulfuric
acid using NREL Standard Analytical Procedure #003.
[0443] Unmilled, unwashed PCS (whole slurry PCS) was prepared by
adjusting the pH of PCS to 5.0 by addition of 10 M NaOH with
extensive mixing, and then autoclaving for 20 minutes at
120.degree. C. The dry weight of the whole slurry PCS was 29%.
Milled unwashed PCS (dry weight 32.35%) was prepared by milling
whole slurry PCS in a Cosmos ICMG 40 wet multi-utility grinder
(EssEmm Corporation, Tamil Nadu, India).
[0444] The hydrolysis of PCS was conducted using 2.2 ml deep-well
plates (Axygen, Union City, Calif., USA) in a total reaction volume
of 1.0 ml. The hydrolysis was performed with 50 mg of insoluble PCS
solids per ml of 50 mM sodium acetate pH 5.0 buffer containing 1 mM
manganese sulfate and various protein loadings of various enzyme
compositions (expressed as mg protein per gram of cellulose).
Enzyme compositions were prepared and then added simultaneously to
all wells in a volume ranging from 50 .mu.l to 200 .mu.l, for a
final volume of 1 ml in each reaction. The plate was then sealed
using an ALPS-300.TM. plate heat sealer (Abgene, Epsom, United
Kingdom), mixed thoroughly, and incubated at a specific temperature
for 72 hours. All experiments reported were performed in
triplicate.
[0445] Following hydrolysis, samples were filtered using a 0.45
.mu.m MULTISCREEN.RTM. 96-well filter plate (Millipore, Bedford,
Mass., USA) and filtrates analyzed for sugar content as described
below. When not used immediately, filtered aliquots were frozen at
-20.degree. C. The sugar concentrations of samples diluted in 0.005
M H.sub.2SO.sub.4 were measured using a 4.6.times.250 mm
AMINEX.RTM. HPX-87H column (Bio-Rad Laboratories, Inc., Hercules,
Calif., USA) by elution with 0.05% w/w benzoic acid-0.005 M
H.sub.2SO.sub.4 at 65.degree. C. at a flow rate of 0.6 ml per
minute, and quantitation by integration of the glucose, cellobiose,
and xylose signals using refractive index detection
(CHEMSTATION.RTM., AGILENT.RTM. 1100 HPLC, Agilent Technologies,
Santa Clara, Calif., USA) calibrated by pure sugar samples. The
resultant glucose and cellobiose equivalents were used to calculate
the percentage of cellulose conversion for each reaction.
[0446] Glucose, cellobiose, and xylose were measured individually.
Measured sugar concentrations were adjusted for the appropriate
dilution factor. In case of unwashed PCS, the net concentrations of
enzymatically-produced sugars were determined by adjusting the
measured sugar concentrations for corresponding background sugar
concentrations in unwashed PCS at zero time. All HPLC data
processing was performed using MICROSOFT EXCEL.TM. software
(Microsoft, Richland, Wash., USA).
[0447] The degree of cellulose conversion to glucose was calculated
using the following equation: % conversion=(glucose
concentration/glucose concentration in a limit digest).times.100.
To calculate total conversion the glucose and cellobiose values
were combined. Cellobiose concentration was multiplied by 1.053 in
order to convert to glucose equivalents and added to the glucose
concentration. The degree of total cellulose conversion was
calculated using the following equation: % conversion=([glucose
concentration+1.053.times.(cellobiose concentration)]/[(glucose
concentration+1.053.times.(cellobiose concentration) in a limit
digest]).times.100. The 1.053 factor for cellobiose takes into
account the increase in mass when cellobiose is converted to
glucose. In order to calculate % conversion, a 100% conversion
point was set based on a cellulase control (100 mg of Trichoderma
reesei cellulase per gram cellulose), and all values were divided
by this number and then multiplied by 100. Triplicate data points
were averaged and standard deviation was calculated.
Example 5
Preparation of Aspergillus Fumigatus NN055679 Cel7A
Cellobiohydrolase I
[0448] A tfasty search (Pearson et al., 1997, Genomics 46:24-36) of
the Aspergillus fumigatus partial genome sequence (The Institute
for Genomic Research, Rockville, Md.) was performed using as query
a Cel7 cellobiohydrolase protein sequence from Trichoderma reesei
(Accession No. P00725). Several genes were identified as putative
Family GH7 homologs based upon a high degree of similarity to the
query sequence at the amino acid level. One genomic region with
significant identity to the query sequence was chosen, and the
corresponding gene was designated cel7A.
[0449] Two synthetic oligonucleotide primers shown below were
designed to PCR amplify the Aspergillus fumigatus NN055679 cel7A
cellobiohydrolase I gene (SEQ ID NO: 5 [DNA sequence] and SEQ ID
NO: 6 [deduced amino acid sequence]) from genomic DNA of
Aspergillus fumigatus prepared as described in WO 2005/047499.
TABLE-US-00005 Forward primer: 5'-gggcATGCTGGCCTCCACCTTCTCC-3' (SEQ
ID NO: 7) Reverse primer: 5'-gggttaattaaCTACAGGCACTGAGAGTAA-3' (SEQ
ID NO: 8)
[0450] Upper case letters represent the coding sequence. The
remainder of the sequence provides restriction endonuclease sites
for Sph I and Pac I in the forward and reverse sequences,
respectively. Using these primers, the Aspergillus fumigatus cel7A
gene was amplified using standard PCR methods and the reaction
product isolated by 1% agarose gel electrophoresis using TAE buffer
and purified using a QIAQUICK.RTM. Gel Extraction Kit (QIAGEN Inc.,
Valencia, Calif., USA) according to the manufacturer's
instructions.
[0451] The fragment was digested with Sph I and Pac I and ligated
into the expression vector pAILo2 (WO 2004/099228) also digested
with Sph I and Pac I according to standard procedures. The ligation
products were transformed into E. coli XL10 SOLOPACK.RTM. cells
(Stratagene, La Jolla, Calif., USA) according to the manufacturer's
instructions. An E. coli transformant containing a plasmid of the
correct size was detected by restriction digestion and plasmid DNA
was prepared using a BIOROBOT.RTM. 9600 (QIAGEN Inc., Valencia,
Calif., USA). DNA sequencing of the insert from this plasmid was
performed with an Applied Biosystems Model 377 XL Automated DNA
Sequencer (Perkin-Elmer/Applied Biosystems, Inc., Foster City,
Calif., USA) using dye-terminator chemistry (Giesecke et al., 1992,
Journal of Virology Methods 38: 47-60) 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 nucleotide sequence was shown to match the genomic
sequence determined by TIGR. The resulting plasmid was designated
pEJG93.
[0452] Aspergillus oryzae JaL250 (WO 99/61651) protoplasts were
prepared according to the method of Christensen et al., 1988,
Bio/Technology 6: 1419-1422, and transformed with 5 .mu.g of pEJG93
(as well as pAILo2 as a vector control). The transformation yielded
about 100 transformants. Ten transformants were isolated to
individual PDA plates.
[0453] Confluent PDA plates of five of the ten transformants were
washed with 5 ml of 0.01% TWEEN.RTM. 20 and inoculated separately
into 25 ml of MDU2BP medium in 125 ml glass shake flasks and
incubated at 34.degree. C., 250 rpm. Five days after incubation,
0.5 .mu.l of supernatant from each culture was analyzed using 8-16%
Tris-Glycine SDS-PAGE gels (Invitrogen, Carlsbad, Calif., USA)
according to the manufacturer's instructions. SDS-PAGE profiles of
the cultures showed that one of the transformants had a major band
of approximately 70 kDa. This transformant was named Aspergillus
oryzae JaL250EJG93.
[0454] One hundred ml of shake flask medium were added to a 500 ml
shake flask. The shake flask medium was composed of 50 g of
sucrose, 10 g of KH.sub.2PO.sub.4, 0.5 g of CaCl.sub.2, 2 g of
MgSO.sub.4.7H.sub.2O, 2 g of K.sub.2SO.sub.4, 2 g of urea, 10 g of
yeast extract, 2 g of citric acid, 0.5 ml of trace metals solution,
and deionized water to 1 liter. The trace metals solution was
composed of 13.8 g of FeSO.sub.4.7H.sub.2O, 14.3 g of
ZnSO.sub.4.7H.sub.2O, 8.5 g of MnSO.sub.4.H.sub.2O, 2.5 g of
CuSO.sub.4.5H.sub.2O, 3 g of citric acid, and deionized water to 1
liter. The shake flask was inoculated with two plugs of Aspergillus
oryzae JaL250EJG93 from a PDA plate and incubated at 34.degree. C.
on an orbital shaker at 200 rpm for 24 hours. Fifty ml of the shake
flask broth were used to inoculate a 3 liter fermentation
vessel.
[0455] A total of 1.8 liters of the fermentation batch medium was
added to a three liter glass jacketed fermentor (Applikon
Biotechnology, Schiedam, Netherlands). The fermentation batch
medium was composed per liter of 10 g of yeast extract, 24 g of
sucrose, 5 g of (NH.sub.4).sub.2SO.sub.4, 2 g of KH.sub.2PO.sub.4,
0.5 g of CaCl.sub.2.2H.sub.2O, 2 g of MgSO.sub.4.7H.sub.2O, 1 g of
citric acid, 2 g of K.sub.2SO.sub.4, 0.5 ml of anti-foam, and 0.5
ml of trace metals solution. The trace metals solution was composed
of 13.8 g of FeSO.sub.4.7H.sub.2O, 14.3 g of ZnSO.sub.4.7H.sub.2O,
8.5 g of MnSO.sub.4.H.sub.2O, 2.5 g of CuSO.sub.4.5H.sub.2O, 3 g of
citric acid, and deionized water to 1 liter. Fermentation feed
medium was composed of maltose. The fermentation feed medium was
dosed at a rate of 0 to 4.4 g/l/hr for a period of 185 hours. The
fermentation vessel was maintained at a temperature of 34.degree.
C. and pH was controlled using an Applikon 1030 control system
(Applikon Biotechnology, Schiedam, Netherlands) to a set-point of
6.1+/-0.1. Air was added to the vessel at a rate of 1 vvm and the
broth was agitated by a Rushton impeller rotating at 1100 to 1300
rpm. At the end of the fermentation, whole broth was harvested from
the vessel and centrifuged at 3000.times.g to remove the biomass.
The supernatant was sterile filtered and stored at 5 to 10.degree.
C.
[0456] The filtered broth was concentrated and buffer exchanged
with 20 mM Tris-HCl pH 8.5 using a tangential flow concentrator
(Pall Filtron, Northborough, Mass., USA) equipped with a 10 kDa
polyethersulfone membrane (Pall Filtron, Northborough, Mass., USA).
The sample was loaded onto a Q SEPHAROSE.RTM. High Performance
column (GE Healthcare, Piscataway, N.J., USA) equilibrated in 20 mM
Tris pH 8.0, and bound proteins were eluted with a linear gradient
from 0-600 mM sodium chloride. Protein concentration was determined
using a Microplate BCA.TM. Protein Assay Kit (Thermo Fischer
Scientific, Waltham, Mass., USA) in which bovine serum albumin was
used as a protein standard.
Example 6
Preparation of Aspergillus Fumigatus Cellobiohydrolase II
[0457] Aspergillus fumigatus NN055679 GH6A cellobiohydrolase II
(CBHII) (SEQ ID NO: 9 [DNA sequence] and SEQ ID NO: 10 [deduced
amino acid sequence]) was prepared according to the following
procedure.
[0458] Two synthetic oligonucleotide primers, shown below, were
designed to PCR amplify the full-length open reading frame of the
Aspergillus fumigatus cellobiohydrolase II gene from genomic DNA. A
TOPO.RTM. Cloning Kit (Invitrogen Corp., Carlsbad, Calif., USA) was
used to clone the PCR product. An IN-FUSION.TM. Cloning Kit was
used to clone the fragment into pAILo2.
TABLE-US-00006 Forward primer: (SEQ ID NO: 11)
5'-ACTGGATTTACCATGAAGCACCTTGCATCTTCCATCG-3' Reverse primer: (SEQ ID
NO: 12) 5'-TCACCTCTAGTTAATTAAAAGGACGGGTTAGCGT-3'
Bold letters represent coding sequence. The remaining sequence
contains sequence identity compared with the insertion sites of
pAILo2.
[0459] Fifty picomoles of each of the primers above were used in a
PCR reaction containing 500 ng of Aspergillus fumigatus genomic
DNA, 1.times. ThermoPol Taq reaction buffer (New England Biolabs,
Ipswich, Mass., USA), 6 .mu.l of a 10 mM blend of dATP, dTTP, dGTP,
and dCTP, and 0.1 unit of Taq DNA polymerase (New England Biolabs,
Ipswich, Mass., USA), in a final volume of 50 .mu.l. An
EPPENDORF.RTM. MASTERCYCLER.RTM. 5333 (Eppendorf Scientific, Inc.,
Westbury, N.Y., USA) was used to amplify the fragment programmed
for 1 cycle at 98.degree. C. for 2 minutes; and 35 cycles each at
96.degree. C. for 30 seconds, 61.degree. C. for 30 seconds, and
72.degree. C. for 2 minutes. After the 35 cycles, the reaction was
incubated at 72.degree. C. for 10 minutes and then cooled at
10.degree. C. until further processed. To remove the A-tails
produced by the Taq DNA polymerase the reaction was incubated for
10 minutes at 68.degree. C. in the presence of 1 unit of Pfx DNA
polymerase (Invitrogen, Carlsbad, Calif., USA).
[0460] A 1.3 kb PCR reaction product was isolated by 0.8%
GTG-agarose gel electrophoresis (Cambrex Bioproducts, East
Rutherford, N.J., USA) using TAE buffer and 0.1 .mu.g of ethidium
bromide per ml. The DNA band was visualized with the aid of a
DARKREADER.TM. Transilluminator to avoid UV-induced mutations. The
1.3 kb DNA band was excised with a disposable razor blade and
purified using an ULTRAFREE.RTM.-DA spin cup (Millipore, Billerica,
Mass.) according to the manufacturer's instructions.
[0461] The purified 1.3 kb PCR product was cloned into vector
pCR.RTM.4Blunt-TOPO.RTM. (Invitrogen, Carlsbad, Calif., USA). Two
.mu.l of the purified PCR product were mixed with 1 .mu.l of a 2 M
sodium chloride solution and 1 .mu.l of the vector. The reaction
was incubated at room temperature for 15 minutes and then 2 .mu.l
of the reaction were transformed into E. coli TOP10 competent cells
(Invitrogen Corp., Carlsbad, Calif., USA) according to the
manufacturer's instructions. Two aliquots of 100 .mu.l each of the
transformation reaction were spread onto two 150 mm 2XYT plates
supplemented with 100 .mu.g of ampicillin per ml and incubated
overnight at 37.degree. C.
[0462] Eight recombinant colonies were used to inoculate liquid
cultures containing 3 ml of LB medium supplemented with 100 .mu.g
of ampicillin per ml. Plasmid DNA was prepared from these cultures
using a BIOROBOT.RTM. 9600. Clones were analyzed by restriction
digestion. Plasmid DNA from each clone was digested with Eco RI and
analyzed by agarose gel electrophoresis as above. Six out of eight
clones had the expected restriction digestion pattern and from
these, clones 2, 4, 5, 6, 7 and 8 were selected to be sequenced to
confirm that there were no mutations in the cloned insert. Sequence
analysis of their 5-prime and 3-prime ends indicated that clones 2,
6 and 7 had the correct sequence. These three clones were selected
for re-cloning into pAILo2. One microliter aliquot of each clone
was mixed with 17 .mu.l of 10-fold diluted 0.1 M EDTA-10 mM Tris
(TE) and 1 .mu.l of this mix was used to re-amplify the Aspergillus
fumigatus GH6A coding region.
[0463] Fifty picomoles of each of the primers above were used in a
PCR reaction containing 1 .mu.l of the diluted mix of clones 2, 6
and 7, 1.times. Pfx Amplification Buffer (Invitrogen, Carlsbad,
Calif., USA), 6 .mu.l of a 10 mM blend of dATP, dTTP, dGTP, and
dCTP, 2.5 units of PLATINUM.RTM. Pfx DNA polymerase (Invitrogen,
Carlsbad, Calif., USA), and 1 .mu.l of 50 mM MgSO.sub.4, in a final
volume of 50 .mu.l. An EPPENDORF.RTM. MASTERCYCLER.RTM. 5333 was
used to amplify the fragment programmed for 1 cycle at 98.degree.
C. for 2 minutes; and 35 cycles each at 94.degree. C. for 30
seconds, 61.degree. C. for 30 seconds, and 68.degree. C. for 1.5
minutes. After the 35 cycles, the reaction was incubated at
68.degree. C. for 10 minutes and then cooled to 10.degree. C. until
further processed. A 1.3 kb PCR reaction product was isolated by
0.8% GTG-agarose gel electrophoresis using TAE buffer and 0.1 .mu.g
of ethidium bromide per ml. The DNA band was visualized with the
aid of a DARKREADER.TM. Transilluminator to avoid UV-induced
mutations. The 1.0 kb DNA band was excised with a disposable razor
blade and purified with an ULTRAFREE.RTM.-DA spin cup according to
the manufacturer's instructions.
[0464] The vector pAILo2 was linearized by digestion with Nco I and
Pac I. The fragment was purified by gel electrophoresis and
ultrafiltration as described above. Cloning of the purified PCR
fragment into the linearized and purified pAILo2 vector was
performed with an IN-FUSION.TM. Cloning Kit. The reaction (20
.mu.l) contained 2 .mu.l of 1.times. IN-FUSION.TM. Buffer, 1.times.
BSA, 1 .mu.l of IN-FUSION.TM. enzyme (diluted 1:10), 100 ng of
pAILo2 digested with Nco I and Pac I, and 50 ng of the Aspergillus
fumigatus GH6A purified PCR product. The reaction was incubated at
room temperature for 30 minutes. A 2 .mu.l sample of the reaction
was used to transform E. coli TOP10 competent cells according to
the manufacturer's instructions. After the recovery period, two 100
.mu.l aliquots from the transformation reaction were plated onto
150 mm 2XYT plates supplemented with 100 .mu.g of ampicillin per
ml. The plates were incubated overnight at 37.degree. C. A set of
eight putative recombinant clones was selected at random from the
selection plates and plasmid DNA was prepared from each one using a
BIOROBOT.RTM. 9600. Clones were analyzed by Pst I restriction
digest. Seven out of eight clones had the expected restriction
digestion pattern. Clones 1, 2, and 3 were then sequenced to
confirm that there were no mutations in the cloned insert. Clone #2
was selected and designated pAILo33.
[0465] Aspergillus oryzae JaL355 protoplasts were prepared
according to the method of Christensen et al., 1988, Bio/Technology
6: 1419-1422, which were transformed with 5 .mu.g of plasmid
pAILo33. The transformation yielded about 30 transformants.
Twenty-six transformants were isolated to individual PDA
plates.
[0466] Confluent PDA plates of four of the transformants were
washed with 8 ml of 0.01% TWEEN.RTM. 20 and inoculated separately
into 1 ml of MDU2BP medium in sterile 24 well tissue culture plates
and incubated at 34.degree. C. Three days after incubation, 20
.mu.l of harvested broth from each culture were analyzed using
8-16% Tris-Glycine SDS-PAGE gels (Invitrogen, Carlsbad, Calif.,
USA) according to the manufacturer's instructions. SDS-PAGE
profiles of the cultures showed that several transformants had a
new major band of approximately 75 kDa. One transformant was
designated Aspergillus oryzae JaL355 ALLO33 (EXP03191).
[0467] One hundred ml of shake flask medium were added to a 500 ml
shake flask. The shake flask medium was composed of 50 g of
sucrose, 10 g of KH.sub.2PO.sub.4, 0.5 g of CaCl.sub.2, 2 g of
MgSO.sub.4.7H.sub.2O, 2 g of K.sub.2SO.sub.4, 2 g of urea, 10 g of
yeast extract, 2 g of citric acid, 0.5 ml of trace metals solution,
and deionized water to 1 liter. The trace metals solution was
composed of 13.8 g of FeSO.sub.4.7H.sub.2O, 14.3 g of
ZnSO.sub.4.7H.sub.2O, 8.5 g of MnSO.sub.4.H.sub.2O, 2.5 g of
CuSO.sub.4.5H.sub.2O, 3 g of citric acid, and deionized water to 1
liter. The shake flask was inoculated with two plugs of Aspergillus
oryzae JaL355 ALLO33 (EXP03191) from a PDA plate and incubated at
34.degree. C. on an orbital shaker at 200 rpm for 24 hours. Fifty
ml of the shake flask broth were used to inoculate a 3 liter
fermentation vessel.
[0468] A total of 1.8 liters of the fermentation batch medium was
added to a three liter glass jacketed fermentor. The fermentation
batch medium was composed per liter of 10 g of yeast extract, 24 g
of sucrose, 5 g of (NH.sub.4).sub.2SO.sub.4, 2 g of
KH.sub.2PO.sub.4, 0.5 g of CaCl.sub.2.2H.sub.2O, 2 g of
MgSO.sub.4.7H.sub.2O, 1 g of citric acid, 2 g of K.sub.2SO.sub.4,
0.5 ml of anti-foam, and 0.5 ml of trace metals solution. The trace
metals solution was composed of 13.8 g of FeSO.sub.4.7H.sub.2O,
14.3 g of ZnSO.sub.4.7H.sub.2O, 8.5 g of MnSO.sub.4.H.sub.2O, 2.5 g
of CuSO.sub.4.5H.sub.2O, 3 g of citric acid, and deionized water to
1 liter. Fermentation feed medium was composed of maltose. The
fermentation feed medium was dosed at a rate of 0 to 4.4 g/l/hr for
a period of 185 hours. The fermentation vessel was maintained at a
temperature of 34.degree. C. and pH was controlled using an
Applikon 1030 control system to a set-point of 6.1+/-0.1. Air was
added to the vessel at a rate of 1 vvm and the broth was agitated
by a Rushton impeller rotating at 1100 to 1300 rpm. At the end of
the fermentation, whole broth was harvested from the vessel and
centrifuged at 3000.times.g to remove the biomass. The supernatant
was sterile filtered and stored at 5-10.degree. C. The broth was
filtered using a 0.22 .mu.m EXPRESS.TM. Plus Membrane (Millipore,
Bedford, Mas., USA).
[0469] A 100 ml volume of the filtered broth was buffer exchanged
into 20 mM Tris pH 8.0 using a 400 ml SEPHADEX.TM. G-25 column (GE
Healthcare, United Kingdom) according to the manufacturer's
instructions. The fractions were pooled and adjusted to 1.2 M
ammonium sulphate-20 mM Tris pH 8.0. The equilibrated protein was
loaded onto a PHENYL SEPHAROSE.TM. 6 Fast Flow (high sub) column
equilibrated in 20 mM Tris pH 8.0 with 1.2 M ammonium sulphate, and
bound proteins were eluted with 20 mM Tris pH 8.0 with no ammonium
sulphate. The fractions were pooled and protein concentration was
determined using a Microplate BCA.TM. Protein Assay Kit in which
bovine serum albumin was used as a protein standard.
Example 7
Preparation of Thermoascus Aurantiacus CGMCC 0583 GH61A Polypeptide
Having Cellulolytic Enhancing Activity
[0470] Thermoascus aurantiacus CGMCC 0583 GH61A polypeptide having
cellulolytic enhancing activity (SEQ ID NO: 13 [DNA sequence] and
SEQ ID NO: 14 [deduced amino acid sequence]) was recombinantly
prepared according to WO 2005/074656 using Aspergillus oryzae
JaL250 as a host. The recombinantly produced Thermoascus
aurantiacus GH61A polypeptide was first concentrated by
ultrafiltration using a 10 kDa membrane, buffer exchanged into 20
mM Tris-HCl pH 8.0, and then purified using 320 ml SUPERDEX.RTM. 75
SEC column (GE Healthcare, Piscataway, N.J., USA) with isocratic
elution of approximately 1.3 liters of 150 mM NaCl-20 mM Tris pH
8.0. Protein concentration was determined using a Microplate
BCA.TM. Protein Assay Kit in which bovine serum albumin was used as
a protein standard.
Example 8
Preparation of Aspergillus Fumigatus NN055679 Cel3A
Beta-Glucosidase
[0471] Aspergillus fumigatus NN055679 Cel3A beta-glucosidase (SEQ
ID NO: 15 [DNA sequence] and SEQ ID NO: 16 [deduced amino acid
sequence]) was recombinantly prepared according to WO 2005/047499
using Trichoderma reesei RutC30 as a host.
[0472] The broth was filtered using a 0.22 .mu.m EXPRESS.TM. Plus
Membrane. The filtered broth was concentrated and buffer exchanged
into 20 mM Tris-HCl pH 8.5 using a tangential flow concentrator
equipped with a 10 kDa polyethersulfone membrane. The sample was
loaded onto a Q SEPHAROSE.RTM. High Performance column equilibrated
in 20 mM Tris pH 8.0, and bound proteins were eluted with a linear
gradient from 0-600 mM sodium chloride. The fractions were
concentrated and loaded onto a SUPERDEX.RTM. 75 HR 26/60 column (GE
Healthcare, Piscataway, N.J., USA) equilibrated with 20 mM Tris-150
mM sodium chloride pH 8.5. Protein concentration was determined
using a Microplate BCA.TM. Protein Assay Kit in which bovine serum
albumin was used as a protein standard.
Example 9
Preparation of Aspergillus Fumigatus NN055679 GH10 Xylanase
[0473] Aspergillus fumigatus NN055679 GH10 xylanase (xyn3) (SEQ ID
NO: 17 [DNA sequence] and SEQ ID NO: 18 [deduced amino acid
sequence]) was prepared recombinantly according to WO 2006/078256
using Aspergillus oryzae BECh2 (WO 2000/039322) as a host.
[0474] The broth was filtered using a 0.22 .mu.m EXPRESS.TM. Plus
Membrane. A 100 ml volume of filtered broth was buffer exchanged
into 50 mM sodium acetate pH 5.0 using a 400 ml SEPHADEX.TM. G-25
column according to the manufacturer's instructions. Protein
concentration was determined using a Microplate BCA.TM. Protein
Assay Kit with bovine serum albumin as a protein standard.
Example 10
Preparation of Talaromyces Emersonii CBS 393.64 GH3
Beta-Xylosidase
[0475] Talaromyces emersonii CBS 393.64 (NN005049) beta-xylosidase
(SEQ ID NO: 19 [DNA sequence] and SEQ ID NO: 20 [deduced amino acid
sequence]) was prepared recombinantly according to Rasmussen et
al., 2006, Biotechnology and Bioengineering 94: 869-876 using
Aspergillus oryzae JaL355 (WO 2003/070956) as a host.
[0476] The broth was filtered using a 0.22 .mu.m EXPRESS.TM. Plus
Membrane. A 100 ml volume of filtered broth was buffer exchanged
into 50 mM sodium acetate pH 5.0 using a 400 ml SEPHADEX.TM. G-25
column according to the manufacturer's instructions. Protein
concentration was determined using a Microplate BCA.TM. Protein
Assay Kit with bovine serum albumin as a protein standard.
Example 11
Effect of Chaetomium Virescens GH5 Endoglucanase on a
High-Temperature Enzyme Composition using Milled Unwashed PCS at
50-65.degree. C.
[0477] The Chaetomium virescens GH5 endoglucanase (EXP03745) was
evaluated in a high-temperature enzyme composition at 50.degree.
C., 55.degree. C., 60.degree. C., and 65.degree. C. using milled
unwashed PCS as a substrate. The high-temperature enzyme
composition included 37% Aspergillus fumigatus Cel7A
cellobiohydrolase I, 25% Aspergillus fumigatus Cel6A
cellobiohydrolase II, 10% Chaetomium virescens Family GH5
endoglucanase II, 15% Thermoascus aurantiacus GH61A polypeptide
having cellulolytic enhancing activity, 5% Aspergillus fumigatus
Cel3A beta-glucosidase, 5% Aspergillus fumigatus GH10 xyn3
xylanase, and 3% Talaromyces emersonii GH3 beta-xylosidase. The
high-temperature enzyme composition was added to PCS hydrolysis
reactions at 3.5 mg total protein per g cellulose, and the
hydrolysis results were compared with the results for a similar
high-temperature enzyme composition without the C. virescens GH5
endoglucanase (3.15 mg protein per g cellulose).
[0478] The assay was performed as described in Example 4. The 1 ml
reactions with milled unwashed PCS (5% insoluble solids) were
conducted for 72 hours in 50 mM sodium acetate pH 5.0 buffer
containing 1 mM manganese sulfate. All reactions were performed in
triplicate and involved single mixing at the beginning of
hydrolysis.
[0479] The results shown in FIG. 2 demonstrated that at 50.degree.
C., 55.degree. C., 60.degree. C., and 65.degree. C. the
high-temperature enzyme composition that included 10% C. virescens
GH5 endoglucanase significantly outperformed the enzyme composition
containing no C. virescens GH5 endoglucanase.
Example 12
Effect of Chaetomium Virescens GH5 Endoglucanase on Unbleached
Eucalyptus Kraft Pulp
[0480] The Chaetomium virescens GH5 endoglucanase was assessed for
its ability to improve the strength properties of paper made from
eucalyptus Kraft pulp.
[0481] Brazilian unbleached eucalyptus Kraft pulp was treated with
the C. virescens GH5 endoglucanase in a 1000 ml Lab-O-Mat beaker
(Werner Mathis AG, Zurich, Switzerland) at 4% consistency, pH 6 and
50.degree. C. for 1 hour. The amount of pulp was 15 g and the
enzyme dosage was 6 mg of C. virescens GH5 endoglucanase per kg dry
pulp. After the enzyme treatment, the sample was diluted to 2 liter
with deionized water and the slurry was disintegrated for 15,000
revolutions in a standard Pulp Disintegrator (Type 8-3; Lorentzen
& Wettre, Kista, Sweden). The reference pulp (negative control)
was treated in the same way but without enzyme addition. Handsheets
were prepared according to TAPPI test method, "Forming handsheets
for physical testing of pulp" T-205 sp-95. For the determination of
tensile index (Instron Model 5564) and tear index (Digital
Elemendorf Tear Tester) tests were conducted according to TAPPI
Test Methods T494 om-96 and T414 om-98, respectively.
[0482] FIGS. 3 and 4 present the physical data obtained from
handsheets made from C. virescens GH5 endoglucanase treatment of
unbleached eucalyptus Kraft pulp. As shown in FIG. 3, treatment of
the unbleached eucalyptus Kraft pulp with the C. virescens GH5
endoglucanase improved the handsheet tensile index by 33% relative
to the control. The C. virescens GH5 endoglucanase treatment
resulted in approximately 17% increase in handsheet tear index
(FIG. 4).
Example 13
Preparation of Trichoderma Reesei RutC30 GH3 Beta-Xylosidase
[0483] A Trichoderma reesei RutC30 GH3 beta-xylosidase gene (SEQ ID
NO: 31 [DNA sequence] and SEQ ID NO: 32) was prepared in
Trichoderma reesei and purified according to WO 2011/057140.
Example 14
Preparation of Trichoderma Reesei GH5 Endoglucanase II
[0484] The Trichoderma reesei GH5 endoglucanase II (SEQ ID NO: 33
[DNA sequence] and SEQ ID NO: 34 [deduced amino acid sequence]) was
prepared recombinantly according to WO 2011/057140 using
Aspergillus oryzae as a host. The filtered broth was concentrated
and buffer exchanged using a tangential flow concentrator equipped
with a 10 kDa polyethersulfone membrane with 20 mM Tris-HCl pH 8.0.
The buffer-exchanged protein was loaded onto a 20 ml MONO Q.RTM.
column (GE Healthcare, Piscataway, N.J., USA) equilibrated in 20 mM
Tris pH 8.0, and bound proteins were eluted with a linear gradient
from 0-600 mM sodium chloride. The fractions were pooled based on
SDS-PAGE analysis using a 8-16% Tris HCl CRITERION.RTM. Stain Free
gels (Bio-Rad Laboratories, Inc., Hercules, Calif., USA) with
Precision Plus Protein.TM. Unstained Standards (Bio-Rad
Laboratories, Inc., Hercules, Calif., USA). Protein concentration
was determined using a Microplate BCA.TM. Protein Assay Kit in
which bovine serum albumin was used as a protein standard.
Example 15
Comparison of the Effect of Chaetomium Virescens GH5 Endoglucanase
and Trichoderma Reesei GH5 Endoglucanase II in the Hydrolysis of
Milled Unwashed PCS by a Cellulase Enzyme Composition
[0485] Chaetomium virescens GH5 endoglucanase and Trichoderma
reesei GH5 endoglucanase II were evaluated in a cellulase enzyme
composition at 50.degree. C. and 55.degree. C. using milled
unwashed PCS as a substrate. The cellulase enzyme composition
included 37% Aspergillus fumigatus Cel7A cellobiohydrolase I, 25%
Aspergillus fumigatus Cel6A cellobiohydrolase II, 10%
endoglucanase, 15% Thermoascus aurantiacus GH61A polypeptide having
cellulolytic enhancing activity, 5% Aspergillus fumigatus Cel3A
beta-glucosidase, 5% Aspergillus fumigatus GH10 xyn3 xylanase, and
3% Trichoderma reesei GH3 beta-xylosidase. Each endoglucanase was
added individually at 0.35 mg enzyme protein per g cellulose to
3.15 mg enzyme protein of the cellulase enzyme composition without
endoglucanase per g cellulose.
[0486] The assay was performed as described in Example 4. The 1 ml
reactions with milled unwashed PCS (5% insoluble solids) were
conducted for 72 hours in 50 mM sodium acetate pH 5.0 buffer
containing 1 mM manganese sulfate. All reactions were performed in
triplicate and involved single mixing at the beginning of
hydrolysis.
[0487] The results shown in FIG. 5 demonstrated that at 50.degree.
C. and 55.degree. C., the cellulase enzyme composition that
included 10% Chaetomium virescens GH5 endoglucanase had
significantly higher glucose conversion than the cellulase enzyme
composition with 10% Trichoderma reesei GH5 endoglucanase II.
Example 16
Specific Activity of Chaetomium Virescens GH5 Endoglucanase on
Carboxymethyl Cellulose
[0488] The specific activity of the Chaetomium virescens GH5
endoglucanase was determined using carboxymethyl cellulose (CMC;
Hercules Chemical Company, Inc., Passaic, N.J., USA) as substrate
at 10 g per liter of 50 mM sodium acetate pH 5.0. To 190 .mu.l of
the CMC solution, 10 .mu.l of Chaetomium virescens GH5
endoglucanase (at different loadings) were added. Protein
concentration was determined using a Microplate BCA.TM. Protein
Assay Kit in which bovine serum albumin was used as a protein
standard. Substrate control and enzyme control were included. The
reaction was incubated at 50.degree. C. for 30 minutes followed by
addition of 50 .mu.l of 0.5 M sodium hydroxide to stop the
reaction. The reducing sugars produced were determined using a
para-hydroxybenzoic acid hydrazide (PHBAH, Sigma, St. Louis, Mo.)
assay adapted to a 96 well microplate format as described below.
Briefly, a 100 .mu.l aliquot of an appropriately diluted sample was
placed in a 96-well conical bottomed microplate. Reactions were
initiated by adding 50 .mu.l of 1.5% (w/v) PHBAH in 2% sodium
hydroxide to each well. Plates were heated uncovered at 95.degree.
C. for 10 minutes. Plates were allowed to cool to room temperature
and 50 .mu.l of deionized water were added to each well. A 100
.mu.l aliquot from each well was transferred to a flat bottomed 96
well plate and the absorbance at A.sub.410 nm measured using a
SPECTRAMAX.RTM. Microplate Reader (Molecular Devices, Sunnyvale,
Calif.). Glucose standards (0.1-0.0125 mg/ml diluted with 0.4%
sodium hydroxide) were used to prepare a standard curve to
translate the obtained A.sub.410 nm values into glucose
equivalents. The enzyme loading versus the reducing sugars produced
was plotted and the linear range was used to calculate the specific
activity of Chaetomium virescens GH5 endoglucanase, as expressed as
.mu.mol glucose equivalent produced per minute per mg enzyme, or
IU/mg.
[0489] The results indicated that the specific activity of the
Chaetomium virescens GH5 endoglucanase on CMC was 25.1 IU/mg
protein.
[0490] The present invention is further described by the following
numbered paragraphs:
[0491] [1] An isolated polypeptide having endoglucanase activity,
selected from the group consisting of: (a) a polypeptide having at
least 80%, e.g., at least 81%, at least 82%, at least 83%, at least
84%, 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: 2; (b) a polypeptide encoded by a
polynucleotide that hybridizes under high stringency conditions or
very high stringency conditions with (i) the mature polypeptide
coding sequence of SEQ ID NO: 1, (ii) the cDNA sequence thereof, or
(iii) the full-length complement of (i) or (ii); (c) a polypeptide
encoded by a polynucleotide having at least 80%, e.g., at least
81%, at least 82%, at least 83%, at least 84%, 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 coding sequence of
SEQ ID NO: 1 or the cDNA sequence thereof; (d) a variant of the
mature polypeptide of SEQ ID NO: 2 comprising a substitution,
deletion, and/or insertion at one or more positions; and (e) a
fragment of the polypeptide of (a), (b), (c), or (d) that has
endoglucanase activity.
[0492] [2] The polypeptide of paragraph 1, having at least 80%, at
least 81%, at least 82%, at least 83%, at least 84%, 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:
2.
[0493] [3] The polypeptide of paragraph 1 or 2, which is encoded by
a polynucleotide that hybridizes under high stringency conditions
or very high stringency conditions with (i) the mature polypeptide
coding sequence of SEQ ID NO: 1, (ii) the cDNA sequence thereof, or
(iii) the full-length complement of (i) or (ii).
[0494] [4] The polypeptide of any of paragraphs 1-3, which is
encoded by a polynucleotide having at least 80%, at least 81%, at
least 82%, at least 83%, at least 84%, 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 coding sequence of SEQ
ID NO: 1 or the cDNA sequence thereof.
[0495] [5] The polypeptide of any of paragraphs 1-4, comprising or
consisting of SEQ ID NO: 2.
[0496] [6] The polypeptide of any of paragraphs 1-4, comprising or
consisting of the mature polypeptide of SEQ ID NO: 2.
[0497] [7] The polypeptide of paragraph 6, wherein the mature
polypeptide is amino acids 18 to 419 of SEQ ID NO: 2.
[0498] [8] The polypeptide of any of paragraphs 1-7, which is a
variant of the mature polypeptide of SEQ ID NO: 2 comprising a
substitution, deletion, and/or insertion at one or more
positions.
[0499] [9] The polypeptide of any of paragraphs 1-8, which is a
fragment of SEQ ID NO: 2, wherein the fragment has endoglucanase
activity.
[0500] [10] An isolated polypeptide comprising a catalytic domain
selected from the group consisting of: (a) a catalytic domain
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 amino
acids 93 to 419 of SEQ ID NO: 2; (b) a catalytic domain encoded by
a polynucleotide that hybridizes under high stringency conditions
or very high stringency conditions with (i) nucleotides 277 to 1317
of SEQ ID NO: 1, (ii) the cDNA sequence thereof, or (iii) the
full-length complement of (i) or (ii); (c) a catalytic domain
encoded by a polynucleotide 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 catalytic domain of SEQ ID NO: 1 or the
cDNA sequence thereof; (d) a variant of amino acids 93 to 419 of
SEQ ID NO: 2 comprising a substitution, deletion, and/or insertion
at one or more positions; and (e) a fragment of the catalytic
domain of (a), (b), (c), or (d) that has endoglucanase
activity.
[0501] [11] The polypeptide of paragraph 10, further comprising a
cellulose binding domain.
[0502] [12] An isolated polypeptide comprising a cellulose binding
domain operably linked to a catalytic domain, wherein the binding
domain is selected from the group consisting of: (a) a cellulose
binding domain having at least 80%, e.g., at least 81%, at least
82%, at least 83%, at least 84%, 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 amino acids 23 to 58 of SEQ ID NO: 2; (b) a cellulose
binding domain encoded by a polynucleotide that hybridizes under
high stringency conditions or very high stringency conditions with
nucleotides 67 to 174 of SEQ ID NO: 1 or the full-length complement
thereof; (c) a cellulose binding domain encoded by a polynucleotide
having at least 80%, e.g., at least 81%, at least 82%, at least
83%, at least 84%, 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
nucleotides 67 to 174 of SEQ ID NO: 1; (d) a variant of amino acids
23 to 58 of SEQ ID NO: 2 comprising a substitution, deletion,
and/or insertion at one or more positions; and (e) a fragment of
(a), (b), (c), or (d) that has cellulose binding activity.
[0503] [13] The polypeptide of paragraph 12, wherein the catalytic
domain is obtained from 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.
[0504] [14] A composition comprising the polypeptide of any of
paragraphs 1-13.
[0505] [15] An isolated polynucleotide encoding the polypeptide of
any of paragraphs 1-13.
[0506] [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.
[0507] [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.
[0508] [18] A method of producing the polypeptide of any of
paragraphs 1-13, comprising: cultivating a cell, which in its
wild-type form produces the polypeptide, under conditions conducive
for production of the polypeptide.
[0509] [19] The method of paragraph 18, further comprising
recovering the polypeptide.
[0510] [20] A method of producing a polypeptide having
endoglucanase activity, comprising: cultivating the host cell of
paragraph 17 under conditions conducive for production of the
polypeptide.
[0511] [21] The method of paragraph 20, further comprising
recovering the polypeptide.
[0512] [22] A transgenic plant, plant part or plant cell
transformed with a polynucleotide encoding the polypeptide of any
of paragraphs 1-13.
[0513] [23] A method of producing a polypeptide having
endoglucanase activity, comprising: cultivating the transgenic
plant or plant cell of paragraph 22 under conditions conducive for
production of the polypeptide.
[0514] [24] The method of paragraph 18, further comprising
recovering the polypeptide.
[0515] [25] A method of producing a mutant of a parent cell,
comprising inactivating a polynucleotide encoding the polypeptide
of any of paragraphs 1-13, which results in the mutant producing
less of the polypeptide than the parent cell.
[0516] [26] A mutant cell produced by the method of paragraph
25.
[0517] [27] The mutant cell of paragraph 26, further comprising a
gene encoding a native or heterologous protein.
[0518] [28] A method of producing a protein, comprising:
cultivating the mutant cell of paragraph 26 or 27 under conditions
conducive for production of the protein.
[0519] [29] The method of paragraph 28, further comprising
recovering the polypeptide.
[0520] [30] A double-stranded inhibitory RNA (dsRNA) molecule
comprising a subsequence of the polynucleotide of paragraph 15,
wherein optionally the dsRNA is an siRNA or an miRNA molecule.
[0521] [31] The double-stranded inhibitory RNA (dsRNA) molecule of
paragraph 30, which is about 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 25 or more duplex nucleotides in length.
[0522] [32] A method of inhibiting the expression of a polypeptide
having endoglucanase activity in a cell, comprising administering
to the cell or expressing in the cell the double-stranded
inhibitory RNA (dsRNA) molecule, wherein the dsRNA of paragraph 30
or 31.
[0523] [33] A cell produced by the method of paragraph 32.
[0524] [34] The cell of paragraph 33, further comprising a gene
encoding a native or heterologous protein.
[0525] [35] A method of producing a protein, comprising:
cultivating the cell of paragraph 33 or 34 under conditions
conducive for production of the protein.
[0526] [36] The method of paragraph 35, further comprising
recovering the polypeptide.
[0527] [37] An isolated polynucleotide encoding a signal peptide
comprising or consisting of amino acids 1 to 17 of SEQ ID NO:
2.
[0528] [38] A nucleic acid construct or expression vector
comprising a gene encoding a protein operably linked to the
polynucleotide of paragraph 37, wherein the gene is foreign to the
polynucleotide encoding the signal peptide.
[0529] [39] A recombinant host cell comprising a gene encoding a
protein operably linked to the polynucleotide of paragraph 37,
wherein the gene is foreign to the polynucleotide encoding the
signal peptide.
[0530] [40] A method of producing a protein, comprising:
cultivating a recombinant host cell comprising a gene encoding a
protein operably linked to the polynucleotide of paragraph 37,
wherein the gene is foreign to the polynucleotide encoding the
signal peptide, under conditions conducive for production of the
protein.
[0531] [41] The method of paragraph 40, further comprising
recovering the polypeptide.
[0532] [42] A method for degrading or converting a cellulosic
material, comprising: treating the cellulosic material with an
enzyme composition in the presence of the polypeptide having
endoglucanase activity of any of paragraphs 1-13.
[0533] [43] The method of paragraph 42, wherein the cellulosic
material is pretreated.
[0534] [44] The method of paragraph 42 or 43, 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.
[0535] [45] The method of paragraph 44, wherein the cellulase is
one or more enzymes selected from the group consisting of an
endoglucanase, a cellobiohydrolase, and a beta-glucosidase.
[0536] [46] The method of paragraph 44, 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.
[0537] [47] The method of any of paragraphs 42-46, further
comprising recovering the degraded cellulosic material.
[0538] [48] The method of paragraph 47, wherein the degraded
cellulosic material is a sugar.
[0539] [49] The method of paragraph 48, wherein the sugar is
selected from the group consisting of glucose, xylose, mannose,
galactose, and arabinose.
[0540] [50] A method for producing a fermentation product,
comprising: (a) saccharifying a cellulosic material with an enzyme
composition in the presence of the polypeptide having endoglucanase
activity of any of paragraphs 1-13; (b) fermenting the saccharified
cellulosic material with one or more fermenting microorganisms to
produce the fermentation product; and (c) recovering the
fermentation product from the fermentation.
[0541] [51] The method of paragraph 50, wherein the cellulosic
material is pretreated.
[0542] [52] The method of paragraph 50 or 51, wherein the enzyme
composition comprises 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.
[0543] [53] The method of paragraph 52, wherein the cellulase is
one or more enzymes selected from the group consisting of an
endoglucanase, a cellobiohydrolase, and a beta-glucosidase.
[0544] [54] The method of paragraph 52, 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.
[0545] [55] The method of any of paragraphs 50-54, wherein steps
(a) and (b) are performed simultaneously in a simultaneous
saccharification and fermentation.
[0546] [56] The method of any of paragraphs 50-55, wherein the
fermentation product is an alcohol, an organic acid, a ketone, an
amino acid, an alkane, a cycloalkane, an alkene, isoprene,
polyketide, or a gas.
[0547] [57] A method of fermenting a cellulosic material,
comprising: fermenting the cellulosic material with one or more
fermenting microorganisms, wherein the cellulosic material is
saccharified with an enzyme composition in the presence of the
polypeptide having endoglucanase activity of any of paragraphs
1-13.
[0548] [58] The method of paragraph 57, wherein the fermenting of
the cellulosic material produces a fermentation product.
[0549] [59] The method of paragraph 58, further comprising
recovering the fermentation product from the fermentation.
[0550] [60] The method of any of paragraphs 57-59, wherein the
cellulosic material is pretreated before saccharification.
[0551] [61] The method of any of paragraphs 57-60, 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.
[0552] [62] The method of paragraph 61, wherein the cellulase is
one or more enzymes selected from the group consisting of an
endoglucanase, a cellobiohydrolase, and a beta-glucosidase.
[0553] [63] The method of paragraph 61, 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.
[0554] [64] The method of any of paragraphs 59-63, wherein the
fermentation product is an alcohol, an organic acid, a ketone, an
amino acid, an alkane, a cycloalkane, an alkene, isoprene,
polyketide, or a gas.
[0555] [65] A process for manufacturing a paper material, which
process comprises treating a paper-making pulp and/or process water
with the polypeptide of any of paragraphs 1-13.
[0556] [66] The process of paragraph 65, further comprising forming
and drying the enzyme-treated pulp.
[0557] [67] The process of paragraph 65 or 66, in which the
enzyme-treatment results in (a) an increase in handsheet tensile
index; and/or (b) an increase in handsheet tear index, relative to
no treatment with the polypeptide having endoglucanase
activity.
[0558] [68] The process of any of paragraphs 65-67, in which a
chemical pulp forms part of the pulp used for the manufacture of
the paper material.
[0559] [69] The process of any of paragraphs 65-68, wherein the
paper-making pulp comprises pulp from recycled printed paper
materials, and wherein the enzyme-treatment results in a bleaching
of the resulting paper material which is at least partly due to a
deinking effect of the enzyme.
[0560] [70] The process of any of paragraphs 65-69, further
comprising treating the paper-making pulp and/or process water with
an additional enzyme having protease, lipase, xylanase, cutinase,
oxidoreductase, cellulase, endoglucanase, amylase, mannanase,
steryl esterase, fatty acid oxidizing enzyme, and/or cholesterol
esterase activity.
[0561] [71] The process of paragraph 70, wherein the additional
oxidoreductase enzyme has laccase, and/or peroxidase activity.
[0562] [72] The process of any of paragraphs 70 or 71, wherein the
additional enzyme has lipase activity.
[0563] [73] The process of any of paragraphs 70-72, wherein the
further treating with the additional enzyme occurs before,
concomitantly with, and/or after the treatment with the polypeptide
having endoglucanase activity.
[0564] [74] A whole broth formulation or cell culture composition
comprising the polypeptide of any of paragraphs 1-13.
[0565] The invention described and claimed herein is not to be
limited in scope by the specific aspects herein disclosed, since
these aspects are intended as illustrations of several aspects of
the invention. Any equivalent aspects are intended to be within the
scope of this invention. Indeed, various modifications of the
invention in addition to those shown and described herein will
become apparent to those skilled in the art from the foregoing
description. Such modifications are also intended to fall within
the scope of the appended claims. In the case of conflict, the
present disclosure including definitions will control.
Sequence CWU 1
1
3411320DNAChaetophractus villosus 1atgccttcag tcgtcctttc tcttgctttc
ttggcgggca gtgcccttgc ccagcagagt 60gtctggggac aatgcggtgg aataggctgg
attgggccaa caaactgcgc ggctgggagc 120tgctgcgcta cccagaacgc
ctggtatgca caatgtacgc ccggggtggc atgcggtggt 180ggtggtagtg
gtacgacttt gacaaccatg acaacccgga ccacgactgt tcaggccacg
240actacgaccg ttacatcgac ccccaccagc aacggcaagg ttcgcttcgc
cggcgtcaac 300attgcaggct tcgactttgg gtgcgggacc gatggcaact
gtaataccgc aaaggtgtgg 360ccgccagtga agaactaccc cccagactac
aaccaccccg acggcgccgg tcagatgcag 420cacttctaca aggatgacca
catgaacatc ttccgcctgc ccgtgggttg gcaatacctg 480gtcaacaaca
acctcggcgg caacttggac agcaccaact tcggctacta cgaccagctg
540gtccaggcct gcctcaacac gggcgcatac tgcatcatcg acatccacaa
ctacgcgcgg 600tggaatgggg ccattatcgg ccagggcggg cccacaaacg
agcagttcgt cagcatatgg 660aagcagctgg cgagcaagta cgccagccag
tcgcgcatat ggttcggcat catgaacgag 720ccccacgaca tacccaatat
caacacctgg gcggccacgg ttcaggccgt cgtcacagcc 780atccgcaatg
ccggcgccac cagccagttc atctcgctgc ccggcaacgg gtggcagtcc
840gcgggcacct tcatcagcga cggaagcgct gcggctctgt ctaccgtcag
gaacccggat 900ggatcgacga cgaacctcat ttttgatgtg cacaagtacc
tcgattctga caactcgggg 960acgcataccg agtgtgtcaa gaataatatc
gacgacgcgt ttgcgccgct tgccgcctgg 1020ctcaggcaaa ataacagaca
ggcgatcctc actgagacgg gaggcggcaa cacggcttca 1080tgccagactt
atgtgtgcca gcagattgcg tacctgaagt aagtattatc gacatcgttt
1140ttcctttgta ggacaacttg ctcaccttca tgctttagtc aaaactcgga
cgtctatctc 1200ggctatgtcg gctgggctgc tggttcgttc gacaatacgt
acgagctggt cgagacgccg 1260acctggaatg gtaactcgtt tagtgatacg
gcccttgtca ggtcttgcct tgctcgttaa 13202419PRTChaetophractus villosus
2Met Pro Ser Val Val Leu Ser Leu Ala Phe Leu Ala Gly Ser Ala Leu 1
5 10 15 Ala Gln Gln Ser Val Trp Gly Gln Cys Gly Gly Ile Gly Trp Ile
Gly 20 25 30 Pro Thr Asn Cys Ala Ala Gly Ser Cys Cys Ala Thr Gln
Asn Ala Trp 35 40 45 Tyr Ala Gln Cys Thr Pro Gly Val Ala Cys Gly
Gly Gly Gly Ser Gly 50 55 60 Thr Thr Leu Thr Thr Met Thr Thr Arg
Thr Thr Thr Val Gln Ala Thr 65 70 75 80 Thr Thr Thr Val Thr Ser Thr
Pro Thr Ser Asn Gly Lys Val Arg Phe 85 90 95 Ala Gly Val Asn Ile
Ala Gly Phe Asp Phe Gly Cys Gly Thr Asp Gly 100 105 110 Asn Cys Asn
Thr Ala Lys Val Trp Pro Pro Val Lys Asn Tyr Pro Pro 115 120 125 Asp
Tyr Asn His Pro Asp Gly Ala Gly Gln Met Gln His Phe Tyr Lys 130 135
140 Asp Asp His Met Asn Ile Phe Arg Leu Pro Val Gly Trp Gln Tyr Leu
145 150 155 160 Val Asn Asn Asn Leu Gly Gly Asn Leu Asp Ser Thr Asn
Phe Gly Tyr 165 170 175 Tyr Asp Gln Leu Val Gln Ala Cys Leu Asn Thr
Gly Ala Tyr Cys Ile 180 185 190 Ile Asp Ile His Asn Tyr Ala Arg Trp
Asn Gly Ala Ile Ile Gly Gln 195 200 205 Gly Gly Pro Thr Asn Glu Gln
Phe Val Ser Ile Trp Lys Gln Leu Ala 210 215 220 Ser Lys Tyr Ala Ser
Gln Ser Arg Ile Trp Phe Gly Ile Met Asn Glu 225 230 235 240 Pro His
Asp Ile Pro Asn Ile Asn Thr Trp Ala Ala Thr Val Gln Ala 245 250 255
Val Val Thr Ala Ile Arg Asn Ala Gly Ala Thr Ser Gln Phe Ile Ser 260
265 270 Leu Pro Gly Asn Gly Trp Gln Ser Ala Gly Thr Phe Ile Ser Asp
Gly 275 280 285 Ser Ala Ala Ala Leu Ser Thr Val Arg Asn Pro Asp Gly
Ser Thr Thr 290 295 300 Asn Leu Ile Phe Asp Val His Lys Tyr Leu Asp
Ser Asp Asn Ser Gly 305 310 315 320 Thr His Thr Glu Cys Val Lys Asn
Asn Ile Asp Asp Ala Phe Ala Pro 325 330 335 Leu Ala Ala Trp Leu Arg
Gln Asn Asn Arg Gln Ala Ile Leu Thr Glu 340 345 350 Thr Gly Gly Gly
Asn Thr Ala Ser Cys Gln Thr Tyr Val Cys Gln Gln 355 360 365 Ile Ala
Tyr Leu Asn Gln Asn Ser Asp Val Tyr Leu Gly Tyr Val Gly 370 375 380
Trp Ala Ala Gly Ser Phe Asp Asn Thr Tyr Glu Leu Val Glu Thr Pro 385
390 395 400 Thr Trp Asn Gly Asn Ser Phe Ser Asp Thr Ala Leu Val Arg
Ser Cys 405 410 415 Leu Ala Arg 341DNAChaetophractus villosus
3acacaactgg ggatccacca tgccttcagt cgtcctttct c
41437DNAChaetophractus villosus 4ccctctagat ctcgagcaac aatgccgaca
cactcca 3751599DNAAspergillus fumigatus 5atgctggcct ccaccttctc
ctaccgcatg tacaagaccg cgctcatcct ggccgccctt 60ctgggctctg gccaggctca
gcaggtcggt acttcccagg cggaagtgca tccgtccatg 120acctggcaga
gctgcacggc tggcggcagc tgcaccacca acaacggcaa ggtggtcatc
180gacgcgaact ggcgttgggt gcacaaagtc ggcgactaca ccaactgcta
caccggcaac 240acctgggaca cgactatctg ccctgacgat gcgacctgcg
catccaactg cgcccttgag 300ggtgccaact acgaatccac ctatggtgtg
accgccagcg gcaattccct ccgcctcaac 360ttcgtcacca ccagccagca
gaagaacatt ggctcgcgtc tgtacatgat gaaggacgac 420tcgacctacg
agatgtttaa gctgctgaac caggagttca ccttcgatgt cgatgtctcc
480aacctcccct gcggtctcaa cggtgctctg tactttgtcg ccatggacgc
cgacggtggc 540atgtccaagt acccaaccaa caaggccggt gccaagtacg
gtactggata ctgtgactcg 600cagtgccctc gcgacctcaa gttcatcaac
ggtcaggcca acgtcgaagg gtggcagccc 660tcctccaacg atgccaatgc
gggtaccggc aaccacgggt cctgctgcgc ggagatggat 720atctgggagg
ccaacagcat ctccacggcc ttcacccccc atccgtgcga cacgcccggc
780caggtgatgt gcaccggtga tgcctgcggt ggcacctaca gctccgaccg
ctacggcggc 840acctgcgacc ccgacggatg tgatttcaac tccttccgcc
agggcaacaa gaccttctac 900ggccctggca tgaccgtcga caccaagagc
aagtttaccg tcgtcaccca gttcatcacc 960gacgacggca cctccagcgg
caccctcaag gagatcaagc gcttctacgt gcagaacggc 1020aaggtgatcc
ccaactcgga gtcgacctgg accggcgtca gcggcaactc catcaccacc
1080gagtactgca ccgcccagaa gagcctgttc caggaccaga acgtcttcga
aaagcacggc 1140ggcctcgagg gcatgggtgc tgccctcgcc cagggtatgg
ttctcgtcat gtccctgtgg 1200gatgatcact cggccaacat gctctggctc
gacagcaact acccgaccac tgcctcttcc 1260accactcccg gcgtcgcccg
tggtacctgc gacatctcct ccggcgtccc tgcggatgtc 1320gaggcgaacc
accccgacgc ctacgtcgtc tactccaaca tcaaggtcgg ccccatcggc
1380tcgaccttca acagcggtgg ctcgaacccc ggtggcggaa ccaccacgac
aactaccacc 1440cagcctacta ccaccacgac cacggctgga aaccctggcg
gcaccggagt cgcacagcac 1500tatggccagt gtggtggaat cggatggacc
ggacccacaa cctgtgccag cccttatacc 1560tgccagaagc tgaatgatta
ttactctcag tgcctgtag 15996532PRTAspergillus fumigatus 6Met Leu Ala
Ser Thr Phe Ser Tyr Arg Met Tyr Lys Thr Ala Leu Ile 1 5 10 15 Leu
Ala Ala Leu Leu Gly Ser Gly Gln Ala Gln Gln Val Gly Thr Ser 20 25
30 Gln Ala Glu Val His Pro Ser Met Thr Trp Gln Ser Cys Thr Ala Gly
35 40 45 Gly Ser Cys Thr Thr Asn Asn Gly Lys Val Val Ile Asp Ala
Asn Trp 50 55 60 Arg Trp Val His Lys Val Gly Asp Tyr Thr Asn Cys
Tyr Thr Gly Asn 65 70 75 80 Thr Trp Asp Thr Thr Ile Cys Pro Asp Asp
Ala Thr Cys Ala Ser Asn 85 90 95 Cys Ala Leu Glu Gly Ala Asn Tyr
Glu Ser Thr Tyr Gly Val Thr Ala 100 105 110 Ser Gly Asn Ser Leu Arg
Leu Asn Phe Val Thr Thr Ser Gln Gln Lys 115 120 125 Asn Ile Gly Ser
Arg Leu Tyr Met Met Lys Asp Asp Ser Thr Tyr Glu 130 135 140 Met Phe
Lys Leu Leu Asn Gln Glu Phe Thr Phe Asp Val Asp Val Ser 145 150 155
160 Asn Leu Pro Cys Gly Leu Asn Gly Ala Leu Tyr Phe Val Ala Met Asp
165 170 175 Ala Asp Gly Gly Met Ser Lys Tyr Pro Thr Asn Lys Ala Gly
Ala Lys 180 185 190 Tyr Gly Thr Gly Tyr Cys Asp Ser Gln Cys Pro Arg
Asp Leu Lys Phe 195 200 205 Ile Asn Gly Gln Ala Asn Val Glu Gly Trp
Gln Pro Ser Ser Asn Asp 210 215 220 Ala Asn Ala Gly Thr Gly Asn His
Gly Ser Cys Cys Ala Glu Met Asp 225 230 235 240 Ile Trp Glu Ala Asn
Ser Ile Ser Thr Ala Phe Thr Pro His Pro Cys 245 250 255 Asp Thr Pro
Gly Gln Val Met Cys Thr Gly Asp Ala Cys Gly Gly Thr 260 265 270 Tyr
Ser Ser Asp Arg Tyr Gly Gly Thr Cys Asp Pro Asp Gly Cys Asp 275 280
285 Phe Asn Ser Phe Arg Gln Gly Asn Lys Thr Phe Tyr Gly Pro Gly Met
290 295 300 Thr Val Asp Thr Lys Ser Lys Phe Thr Val Val Thr Gln Phe
Ile Thr 305 310 315 320 Asp Asp Gly Thr Ser Ser Gly Thr Leu Lys Glu
Ile Lys Arg Phe Tyr 325 330 335 Val Gln Asn Gly Lys Val Ile Pro Asn
Ser Glu Ser Thr Trp Thr Gly 340 345 350 Val Ser Gly Asn Ser Ile Thr
Thr Glu Tyr Cys Thr Ala Gln Lys Ser 355 360 365 Leu Phe Gln Asp Gln
Asn Val Phe Glu Lys His Gly Gly Leu Glu Gly 370 375 380 Met Gly Ala
Ala Leu Ala Gln Gly Met Val Leu Val Met Ser Leu Trp 385 390 395 400
Asp Asp His Ser Ala Asn Met Leu Trp Leu Asp Ser Asn Tyr Pro Thr 405
410 415 Thr Ala Ser Ser Thr Thr Pro Gly Val Ala Arg Gly Thr Cys Asp
Ile 420 425 430 Ser Ser Gly Val Pro Ala Asp Val Glu Ala Asn His Pro
Asp Ala Tyr 435 440 445 Val Val Tyr Ser Asn Ile Lys Val Gly Pro Ile
Gly Ser Thr Phe Asn 450 455 460 Ser Gly Gly Ser Asn Pro Gly Gly Gly
Thr Thr Thr Thr Thr Thr Thr 465 470 475 480 Gln Pro Thr Thr Thr Thr
Thr Thr Ala Gly Asn Pro Gly Gly Thr Gly 485 490 495 Val Ala Gln His
Tyr Gly Gln Cys Gly Gly Ile Gly Trp Thr Gly Pro 500 505 510 Thr Thr
Cys Ala Ser Pro Tyr Thr Cys Gln Lys Leu Asn Asp Tyr Tyr 515 520 525
Ser Gln Cys Leu 530 725DNAAspergillus fumigatus 7gggcatgctg
gcctccacct tctcc 25830DNAAspergillus fumigatus 8gggttaatta
actacaggca ctgagagtaa 3091713DNAAspergillus fumigatus 9atgaagcacc
ttgcatcttc catcgcattg actctactgt tgcctgccgt gcaggcccag 60cagaccgtat
ggggccaatg tatgttctgg ctgtcactgg aataagactg tatcaactgc
120tgatatgctt ctaggtggcg gccaaggctg gtctggcccg acgagctgtg
ttgccggcgc 180agcctgtagc acactgaatc cctgtatgtt agatatcgtc
ctgagtggag acttatactg 240acttccttag actacgctca gtgtatcccg
ggagccaccg cgacgtccac caccctcacg 300acgacgacgg cggcgacgac
gacatcccag accaccacca aacctaccac gactggtcca 360actacatccg
cacccaccgt gaccgcatcc ggtaaccctt tcagcggcta ccagctgtat
420gccaacccct actactcctc cgaggtccat actctggcca tgccttctct
gcccagctcg 480ctgcagccca aggctagtgc tgttgctgaa gtgccctcat
ttgtttggct gtaagtggcc 540ttatcccaat actgagacca actctctgac
agtcgtagcg acgttgccgc caaggtgccc 600actatgggaa cctacctggc
cgacattcag gccaagaaca aggccggcgc caaccctcct 660atcgctggta
tcttcgtggt ctacgacttg ccggaccgtg actgcgccgc tctggccagt
720aatggcgagt actcaattgc caacaacggt gtggccaact acaaggcgta
cattgacgcc 780atccgtgctc agctggtgaa gtactctgac gttcacacca
tcctcgtcat cggtaggccg 840tacacctccg ttgcgcgccg cctttctctg
acatcttgca gaacccgaca gcttggccaa 900cctggtgacc aacctcaacg
tcgccaaatg cgccaatgcg cagagcgcct acctggagtg 960tgtcgactat
gctctgaagc agctcaacct gcccaacgtc gccatgtacc tcgacgcagg
1020tatgcctcac ttcccgcatt ctgtatccct tccagacact aactcatcag
gccatgcggg 1080ctggctcgga tggcccgcca acttgggccc cgccgcaaca
ctcttcgcca aagtctacac 1140cgacgcgggt tcccccgcgg ctgttcgtgg
cctggccacc aacgtcgcca actacaacgc 1200ctggtcgctc agtacctgcc
cctcctacac ccagggagac cccaactgcg acgagaagaa 1260gtacatcaac
gccatggcgc ctcttctcaa ggaagccggc ttcgatgccc acttcatcat
1320ggatacctgt aagtgcttat tccaatcgcc gatgtgtgcc gactaatcaa
tgtttcagcc 1380cggaatggcg tccagcccac gaagcaaaac gcctggggtg
actggtgcaa cgtcatcggc 1440accggcttcg gtgttcgccc ctcgactaac
accggcgatc cgctccagga tgcctttgtg 1500tggatcaagc ccggtggaga
gagtgatggc acgtccaact cgacttcccc ccggtatgac 1560gcgcactgcg
gatatagtga tgctctgcag cctgctcctg aggctggtac ttggttccag
1620gtatgtcatc cattagccag atgagggata agtgactgac ggacctaggc
ctactttgag 1680cagcttctga ccaacgctaa cccgtccttt taa
171310454PRTAspergillus fumigatus 10Met Lys His Leu Ala Ser Ser Ile
Ala Leu Thr Leu Leu Leu Pro Ala 1 5 10 15 Val Gln Ala Gln Gln Thr
Val Trp Gly Gln Cys Gly Gly Gln Gly Trp 20 25 30 Ser Gly Pro Thr
Ser Cys Val Ala Gly Ala Ala Cys Ser Thr Leu Asn 35 40 45 Pro Tyr
Tyr Ala Gln Cys Ile Pro Gly Ala Thr Ala Thr Ser Thr Thr 50 55 60
Leu Thr Thr Thr Thr Ala Ala Thr Thr Thr Ser Gln Thr Thr Thr Lys 65
70 75 80 Pro Thr Thr Thr Gly Pro Thr Thr Ser Ala Pro Thr Val Thr
Ala Ser 85 90 95 Gly Asn Pro Phe Ser Gly Tyr Gln Leu Tyr Ala Asn
Pro Tyr Tyr Ser 100 105 110 Ser Glu Val His Thr Leu Ala Met Pro Ser
Leu Pro Ser Ser Leu Gln 115 120 125 Pro Lys Ala Ser Ala Val Ala Glu
Val Pro Ser Phe Val Trp Leu Asp 130 135 140 Val Ala Ala Lys Val Pro
Thr Met Gly Thr Tyr Leu Ala Asp Ile Gln 145 150 155 160 Ala Lys Asn
Lys Ala Gly Ala Asn Pro Pro Ile Ala Gly Ile Phe Val 165 170 175 Val
Tyr Asp Leu Pro Asp Arg Asp Cys Ala Ala Leu Ala Ser Asn Gly 180 185
190 Glu Tyr Ser Ile Ala Asn Asn Gly Val Ala Asn Tyr Lys Ala Tyr Ile
195 200 205 Asp Ala Ile Arg Ala Gln Leu Val Lys Tyr Ser Asp Val His
Thr Ile 210 215 220 Leu Val Ile Glu Pro Asp Ser Leu Ala Asn Leu Val
Thr Asn Leu Asn 225 230 235 240 Val Ala Lys Cys Ala Asn Ala Gln Ser
Ala Tyr Leu Glu Cys Val Asp 245 250 255 Tyr Ala Leu Lys Gln Leu Asn
Leu Pro Asn Val Ala Met Tyr Leu Asp 260 265 270 Ala Gly His Ala Gly
Trp Leu Gly Trp Pro Ala Asn Leu Gly Pro Ala 275 280 285 Ala Thr Leu
Phe Ala Lys Val Tyr Thr Asp Ala Gly Ser Pro Ala Ala 290 295 300 Val
Arg Gly Leu Ala Thr Asn Val Ala Asn Tyr Asn Ala Trp Ser Leu 305 310
315 320 Ser Thr Cys Pro Ser Tyr Thr Gln Gly Asp Pro Asn Cys Asp Glu
Lys 325 330 335 Lys Tyr Ile Asn Ala Met Ala Pro Leu Leu Lys Glu Ala
Gly Phe Asp 340 345 350 Ala His Phe Ile Met Asp Thr Ser Arg Asn Gly
Val Gln Pro Thr Lys 355 360 365 Gln Asn Ala Trp Gly Asp Trp Cys Asn
Val Ile Gly Thr Gly Phe Gly 370 375 380 Val Arg Pro Ser Thr Asn Thr
Gly Asp Pro Leu Gln Asp Ala Phe Val 385 390 395 400 Trp Ile Lys Pro
Gly Gly Glu Ser Asp Gly Thr Ser Asn Ser Thr Ser 405 410 415 Pro Arg
Tyr Asp Ala His Cys Gly Tyr Ser Asp Ala Leu Gln Pro Ala 420 425 430
Pro Glu Ala Gly Thr Trp Phe Gln Ala Tyr Phe Glu Gln Leu Leu Thr 435
440 445 Asn Ala Asn Pro Ser Phe 450 1137DNAAspergillus fumigatus
11actggattta ccatgaagca ccttgcatct tccatcg 371234DNAAspergillus
fumigatus 12tcacctctag ttaattaaaa ggacgggtta gcgt
3413799DNAThermoascus aurantiacus 13atgtcctttt ccaagataat
tgctactgcc ggcgttcttg cctctgcttc tctagtggct 60ggccatggct tcgttcagaa
catcgtgatt gatggtaaaa agtatgtcat tgcaagacgc 120acataagcgg
caacagctga caatcgacag ttatggcggg tatctagtga accagtatcc
180atacatgtcc aatcctccag aggtcatcgc ctggtctact acggcaactg
atcttggatt 240tgtggacggt actggatacc aaaccccaga tatcatctgc
cataggggcg ccaagcctgg 300agccctgact gctccagtct ctccaggagg
aactgttgag cttcaatgga ctccatggcc 360tgattctcac catggcccag
ttatcaacta ccttgctccg tgcaatggtg attgttccac 420tgtggataag
acccaattag aattcttcaa
aattgccgag agcggtctca tcaatgatga 480caatcctcct gggatctggg
cttcagacaa tctgatagca gccaacaaca gctggactgt 540caccattcca
accacaattg cacctggaaa ctatgttctg aggcatgaga ttattgctct
600tcactcagct cagaaccagg atggtgccca gaactatccc cagtgcatca
atctgcaggt 660cactggaggt ggttctgata accctgctgg aactcttgga
acggcactct accacgatac 720cgatcctgga attctgatca acatctatca
gaaactttcc agctatatca tccctggtcc 780tcctctgtat actggttaa
79914249PRTThermoascus aurantiacus 14Met Ser Phe Ser Lys Ile Ile
Ala Thr Ala Gly Val Leu Ala Ser Ala 1 5 10 15 Ser Leu Val Ala Gly
His Gly Phe Val Gln Asn Ile Val Ile Asp Gly 20 25 30 Lys Tyr Tyr
Gly Gly Tyr Leu Val Asn Gln Tyr Pro Tyr Met Ser Asn 35 40 45 Pro
Pro Glu Val Ile Ala Trp Ser Thr Thr Ala Thr Asp Leu Gly Phe 50 55
60 Val Asp Gly Thr Gly Tyr Gln Thr Pro Asp Ile Ile Cys His Arg Gly
65 70 75 80 Ala Lys Pro Gly Ala Leu Thr Ala Pro Val Ser Pro Gly Gly
Thr Val 85 90 95 Glu Leu Gln Trp Thr Pro Trp Pro Asp Ser His His
Gly Pro Val Ile 100 105 110 Asn Tyr Leu Ala Pro Cys Asn Gly Asp Cys
Ser Thr Val Asp Lys Thr 115 120 125 Gln Leu Glu Phe Phe Lys Ile Ala
Glu Ser Gly Leu Ile Asn Asp Asp 130 135 140 Asn Pro Pro Gly Ile Trp
Ala Ser Asp Asn Leu Ile Ala Ala Asn Asn 145 150 155 160 Ser Trp Thr
Val Thr Ile Pro Thr Thr Ile Ala Pro Gly Asn Tyr Val 165 170 175 Leu
Arg His Glu Ile Ile Ala Leu His Ser Ala Gln Asn Gln Asp Gly 180 185
190 Ala Gln Asn Tyr Pro Gln Cys Ile Asn Leu Gln Val Thr Gly Gly Gly
195 200 205 Ser Asp Asn Pro Ala Gly Thr Leu Gly Thr Ala Leu Tyr His
Asp Thr 210 215 220 Asp Pro Gly Ile Leu Ile Asn Ile Tyr Gln Lys Leu
Ser Ser Tyr Ile 225 230 235 240 Ile Pro Gly Pro Pro Leu Tyr Thr Gly
245 153060DNAAspergillus fumigatus 15atgagattcg gttggctcga
ggtggccgct ctgacggccg cttctgtagc caatgcccag 60gtttgtgatg ctttcccgtc
attgtttcgg atatagttga caatagtcat ggaaataatc 120aggaattggc
tttctctcca ccattctacc cttcgccttg ggctgatggc cagggagagt
180gggcagatgc ccatcgacgc gccgtcgaga tcgtttctca gatgacactg
gcggagaagg 240ttaaccttac aacgggtact gggtgggttg cgactttttt
gttgacagtg agctttcttc 300actgaccatc tacacagatg ggaaatggac
cgatgcgtcg gtcaaaccgg cagcgttccc 360aggtaagctt gcaattctgc
aacaacgtgc aagtgtagtt gctaaaacgc ggtggtgcag 420acttggtatc
aactggggtc tttgtggcca ggattcccct ttgggtatcc gtttctgtga
480gctatacccg cggagtcttt cagtccttgt attatgtgct gatgattgtc
tctgtatagc 540tgacctcaac tccgccttcc ctgctggtac taatgtcgcc
gcgacatggg acaagacact 600cgcctacctt cgtggcaagg ccatgggtga
ggaattcaac gacaagggcg tggacatttt 660gctggggcct gctgctggtc
ctctcggcaa atacccggac ggcggcagaa tctgggaagg 720cttctctcct
gatccggttc tcactggtgt acttttcgcc gaaactatca agggtatcca
780agacgcgggt gtgattgcta ctgccaagca ttacattctg aatgaacagg
agcatttccg 840acaggttggc gaggcccagg gatatggtta caacatcacg
gagacgatca gctccaacgt 900ggatgacaag accatgcacg agttgtacct
ttggtgagta gttgacactg caaatgagga 960ccttgattga tttgactgac
ctggaatgca ggccctttgc agatgctgtg cgcggtaaga 1020ttttccgtag
acttgacctc gcgacgaaga aatcgctgac gaaccatcgt agctggcgtt
1080ggcgctgtca tgtgttccta caatcaaatc aacaacagct acggttgtca
aaacagtcaa 1140actctcaaca agctcctcaa ggctgagctg ggcttccaag
gcttcgtcat gagtgactgg 1200agcgctcacc acagcggtgt cggcgctgcc
ctcgctgggt tggatatgtc gatgcctgga 1260gacatttcct tcgacgacgg
actctccttc tggggcacga acctaactgt cagtgttctt 1320aacggcaccg
ttccagcctg gcgtgtcgat gacatggctg ttcgtatcat gaccgcgtac
1380tacaaggttg gtcgtgaccg tcttcgtatt ccccctaact tcagctcctg
gacccgggat 1440gagtacggct gggagcattc tgctgtctcc gagggagcct
ggaccaaggt gaacgacttc 1500gtcaatgtgc agcgcagtca ctctcagatc
atccgtgaga ttggtgccgc tagtacagtg 1560ctcttgaaga acacgggtgc
tcttcctttg accggcaagg aggttaaagt gggtgttctc 1620ggtgaagacg
ctggttccaa cccgtggggt gctaacggct gccccgaccg cggctgtgat
1680aacggcactc ttgctatggc ctggggtagt ggtactgcca acttccctta
ccttgtcacc 1740cccgagcagg ctatccagcg agaggtcatc agcaacggcg
gcaatgtctt tgctgtgact 1800gataacgggg ctctcagcca gatggcagat
gttgcatctc aatccaggtg agtgcgggct 1860cttagaaaaa gaacgttctc
tgaatgaagt tttttaacca ttgcgaacag cgtgtctttg 1920gtgtttgtca
acgccgactc tggagagggt ttcatcagtg tcgacggcaa cgagggtgac
1980cgcaaaaatc tcactctgtg gaagaacggc gaggccgtca ttgacactgt
tgtcagccac 2040tgcaacaaca cgattgtggt tattcacagt gttgggcccg
tcttgatcga ccggtggtat 2100gataacccca acgtcactgc catcatctgg
gccggcttgc ccggtcagga gagtggcaac 2160tccctggtcg acgtgctcta
tggccgcgtc aaccccagcg ccaagacccc gttcacctgg 2220ggcaagactc
gggagtctta cggggctccc ttgctcaccg agcctaacaa tggcaatggt
2280gctccccagg atgatttcaa cgagggcgtc ttcattgact accgtcactt
tgacaagcgc 2340aatgagaccc ccatttatga gtttggccat ggcttgagct
acaccacctt tggttactct 2400caccttcggg ttcaggccct caatagttcg
agttcggcat atgtcccgac tagcggagag 2460accaagcctg cgccaaccta
tggtgagatc ggtagtgccg ccgactacct gtatcccgag 2520ggtctcaaaa
gaattaccaa gtttatttac ccttggctca actcgaccga cctcgaggat
2580tcttctgacg acccgaacta cggctgggag gactcggagt acattcccga
aggcgctagg 2640gatgggtctc ctcaacccct cctgaaggct ggcggcgctc
ctggtggtaa ccctaccctt 2700tatcaggatc ttgttagggt gtcggccacc
ataaccaaca ctggtaacgt cgccggttat 2760gaagtccctc aattggtgag
tgacccgcat gttccttgcg ttgcaatttg gctaactcgc 2820ttctagtatg
tttcactggg cggaccgaac gagcctcggg tcgttctgcg caagttcgac
2880cgaatcttcc tggctcctgg ggagcaaaag gtttggacca cgactcttaa
ccgtcgtgat 2940ctcgccaatt gggatgtgga ggctcaggac tgggtcatca
caaagtaccc caagaaagtg 3000cacgtcggca gctcctcgcg taagctgcct
ctgagagcgc ctctgccccg tgtctactag 306016863PRTAspergillus fumigatus
16Met Arg Phe Gly Trp Leu Glu Val Ala Ala Leu Thr Ala Ala Ser Val 1
5 10 15 Ala Asn Ala Gln Glu Leu Ala Phe Ser Pro Pro Phe Tyr Pro Ser
Pro 20 25 30 Trp Ala Asp Gly Gln Gly Glu Trp Ala Asp Ala His Arg
Arg Ala Val 35 40 45 Glu Ile Val Ser Gln Met Thr Leu Ala Glu Lys
Val Asn Leu Thr Thr 50 55 60 Gly Thr Gly Trp Glu Met Asp Arg Cys
Val Gly Gln Thr Gly Ser Val 65 70 75 80 Pro Arg Leu Gly Ile Asn Trp
Gly Leu Cys Gly Gln Asp Ser Pro Leu 85 90 95 Gly Ile Arg Phe Ser
Asp Leu Asn Ser Ala Phe Pro Ala Gly Thr Asn 100 105 110 Val Ala Ala
Thr Trp Asp Lys Thr Leu Ala Tyr Leu Arg Gly Lys Ala 115 120 125 Met
Gly Glu Glu Phe Asn Asp Lys Gly Val Asp Ile Leu Leu Gly Pro 130 135
140 Ala Ala Gly Pro Leu Gly Lys Tyr Pro Asp Gly Gly Arg Ile Trp Glu
145 150 155 160 Gly Phe Ser Pro Asp Pro Val Leu Thr Gly Val Leu Phe
Ala Glu Thr 165 170 175 Ile Lys Gly Ile Gln Asp Ala Gly Val Ile Ala
Thr Ala Lys His Tyr 180 185 190 Ile Leu Asn Glu Gln Glu His Phe Arg
Gln Val Gly Glu Ala Gln Gly 195 200 205 Tyr Gly Tyr Asn Ile Thr Glu
Thr Ile Ser Ser Asn Val Asp Asp Lys 210 215 220 Thr Met His Glu Leu
Tyr Leu Trp Pro Phe Ala Asp Ala Val Arg Ala 225 230 235 240 Gly Val
Gly Ala Val Met Cys Ser Tyr Asn Gln Ile Asn Asn Ser Tyr 245 250 255
Gly Cys Gln Asn Ser Gln Thr Leu Asn Lys Leu Leu Lys Ala Glu Leu 260
265 270 Gly Phe Gln Gly Phe Val Met Ser Asp Trp Ser Ala His His Ser
Gly 275 280 285 Val Gly Ala Ala Leu Ala Gly Leu Asp Met Ser Met Pro
Gly Asp Ile 290 295 300 Ser Phe Asp Asp Gly Leu Ser Phe Trp Gly Thr
Asn Leu Thr Val Ser 305 310 315 320 Val Leu Asn Gly Thr Val Pro Ala
Trp Arg Val Asp Asp Met Ala Val 325 330 335 Arg Ile Met Thr Ala Tyr
Tyr Lys Val Gly Arg Asp Arg Leu Arg Ile 340 345 350 Pro Pro Asn Phe
Ser Ser Trp Thr Arg Asp Glu Tyr Gly Trp Glu His 355 360 365 Ser Ala
Val Ser Glu Gly Ala Trp Thr Lys Val Asn Asp Phe Val Asn 370 375 380
Val Gln Arg Ser His Ser Gln Ile Ile Arg Glu Ile Gly Ala Ala Ser 385
390 395 400 Thr Val Leu Leu Lys Asn Thr Gly Ala Leu Pro Leu Thr Gly
Lys Glu 405 410 415 Val Lys Val Gly Val Leu Gly Glu Asp Ala Gly Ser
Asn Pro Trp Gly 420 425 430 Ala Asn Gly Cys Pro Asp Arg Gly Cys Asp
Asn Gly Thr Leu Ala Met 435 440 445 Ala Trp Gly Ser Gly Thr Ala Asn
Phe Pro Tyr Leu Val Thr Pro Glu 450 455 460 Gln Ala Ile Gln Arg Glu
Val Ile Ser Asn Gly Gly Asn Val Phe Ala 465 470 475 480 Val Thr Asp
Asn Gly Ala Leu Ser Gln Met Ala Asp Val Ala Ser Gln 485 490 495 Ser
Ser Val Ser Leu Val Phe Val Asn Ala Asp Ser Gly Glu Gly Phe 500 505
510 Ile Ser Val Asp Gly Asn Glu Gly Asp Arg Lys Asn Leu Thr Leu Trp
515 520 525 Lys Asn Gly Glu Ala Val Ile Asp Thr Val Val Ser His Cys
Asn Asn 530 535 540 Thr Ile Val Val Ile His Ser Val Gly Pro Val Leu
Ile Asp Arg Trp 545 550 555 560 Tyr Asp Asn Pro Asn Val Thr Ala Ile
Ile Trp Ala Gly Leu Pro Gly 565 570 575 Gln Glu Ser Gly Asn Ser Leu
Val Asp Val Leu Tyr Gly Arg Val Asn 580 585 590 Pro Ser Ala Lys Thr
Pro Phe Thr Trp Gly Lys Thr Arg Glu Ser Tyr 595 600 605 Gly Ala Pro
Leu Leu Thr Glu Pro Asn Asn Gly Asn Gly Ala Pro Gln 610 615 620 Asp
Asp Phe Asn Glu Gly Val Phe Ile Asp Tyr Arg His Phe Asp Lys 625 630
635 640 Arg Asn Glu Thr Pro Ile Tyr Glu Phe Gly His Gly Leu Ser Tyr
Thr 645 650 655 Thr Phe Gly Tyr Ser His Leu Arg Val Gln Ala Leu Asn
Ser Ser Ser 660 665 670 Ser Ala Tyr Val Pro Thr Ser Gly Glu Thr Lys
Pro Ala Pro Thr Tyr 675 680 685 Gly Glu Ile Gly Ser Ala Ala Asp Tyr
Leu Tyr Pro Glu Gly Leu Lys 690 695 700 Arg Ile Thr Lys Phe Ile Tyr
Pro Trp Leu Asn Ser Thr Asp Leu Glu 705 710 715 720 Asp Ser Ser Asp
Asp Pro Asn Tyr Gly Trp Glu Asp Ser Glu Tyr Ile 725 730 735 Pro Glu
Gly Ala Arg Asp Gly Ser Pro Gln Pro Leu Leu Lys Ala Gly 740 745 750
Gly Ala Pro Gly Gly Asn Pro Thr Leu Tyr Gln Asp Leu Val Arg Val 755
760 765 Ser Ala Thr Ile Thr Asn Thr Gly Asn Val Ala Gly Tyr Glu Val
Pro 770 775 780 Gln Leu Tyr Val Ser Leu Gly Gly Pro Asn Glu Pro Arg
Val Val Leu 785 790 795 800 Arg Lys Phe Asp Arg Ile Phe Leu Ala Pro
Gly Glu Gln Lys Val Trp 805 810 815 Thr Thr Thr Leu Asn Arg Arg Asp
Leu Ala Asn Trp Asp Val Glu Ala 820 825 830 Gln Asp Trp Val Ile Thr
Lys Tyr Pro Lys Lys Val His Val Gly Ser 835 840 845 Ser Ser Arg Lys
Leu Pro Leu Arg Ala Pro Leu Pro Arg Val Tyr 850 855 860
171415DNAAspergillus fumigatus 17atggtccatc tatcttcatt ggcagcagcc
ctggctgctc tgcctctgta tgtttaccca 60ctcacgagag gaggaacagc tttgacattg
ctatagtgta tatggagctg gcctgaacac 120agcagccaaa gccaaaggac
taaagtactt tggttccgcc acggacaatc cagagctcac 180ggactctgcg
tatgtcgcgc aactgagcaa caccgatgat tttggtcaaa tcacacccgg
240aaactccatg aaggtttgct tacgtctgcc tccctggagc attgcctcaa
aagctaattg 300gttgttttgt ttggatagtg ggatgccacc gagccttctc
agaattcttt ttcgttcgca 360aatggagacg ccgtggtcaa tctggcgaac
aagaatggcc agctgatgcg atgccatact 420ctggtctggc acagtcagct
accgaactgg ggtatgtaaa cgtcttgtct attctcaaat 480actctctaac
agttgacagt ctctagcggg tcatggacca atgcgaccct tttggcggcc
540atgaagaatc atatcaccaa tgtggttact cactacaagg ggaagtgcta
cgcctgggat 600gttgtcaatg aaggtttgtt gctccatcta tcctcaatag
ttcttttgaa actgacaagc 660ctgtcaatct agccctgaac gaggacggta
ctttccgtaa ctctgtcttc taccagatca 720tcggcccagc atacattcct
attgcgttcg ccacggctgc tgccgcagat cccgacgtga 780aactctacta
caacgactac aacattgaat actcaggcgc caaagcgact gctgcgcaga
840atatcgtcaa gatgatcaag gcctacggcg cgaagatcga cggcgtcggc
ctccaggcac 900actttatcgt cggcagcact ccgagtcaat cggatctgac
gaccgtcttg aagggctaca 960ctgctctcgg cgttgaggtg gcctataccg
aacttgacat ccgcatgcag ctgccctcga 1020ccgccgcaaa gctggcccag
cagtccactg acttccaagg cgtggccgca gcatgcgtta 1080gcaccactgg
ctgcgtgggt gtcactatct gggactggac cgacaagtac tcctgggtcc
1140ccagcgtgtt ccaaggctac ggcgccccat tgccttggga tgagaactat
gtgaagaagc 1200cagcgtacga tggcctgatg gcgggtcttg gagcaagcgg
ctccggcacc acaacgacca 1260ctactactac ttctactacg acaggaggta
cggaccctac tggagtcgct cagaaatggg 1320gacagtgtgg cggtattggc
tggaccgggc caacaacttg tgtcagtggt accacttgcc 1380aaaagctgaa
tgactggtac tcacagtgcc tgtaa 141518397PRTAspergillus fumigatus 18Met
Val His Leu Ser Ser Leu Ala Ala Ala Leu Ala Ala Leu Pro Leu 1 5 10
15 Val Tyr Gly Ala Gly Leu Asn Thr Ala Ala Lys Ala Lys Gly Leu Lys
20 25 30 Tyr Phe Gly Ser Ala Thr Asp Asn Pro Glu Leu Thr Asp Ser
Ala Tyr 35 40 45 Val Ala Gln Leu Ser Asn Thr Asp Asp Phe Gly Gln
Ile Thr Pro Gly 50 55 60 Asn Ser Met Lys Trp Asp Ala Thr Glu Pro
Ser Gln Asn Ser Phe Ser 65 70 75 80 Phe Ala Asn Gly Asp Ala Val Val
Asn Leu Ala Asn Lys Asn Gly Gln 85 90 95 Leu Met Arg Cys His Thr
Leu Val Trp His Ser Gln Leu Pro Asn Trp 100 105 110 Val Ser Ser Gly
Ser Trp Thr Asn Ala Thr Leu Leu Ala Ala Met Lys 115 120 125 Asn His
Ile Thr Asn Val Val Thr His Tyr Lys Gly Lys Cys Tyr Ala 130 135 140
Trp Asp Val Val Asn Glu Ala Leu Asn Glu Asp Gly Thr Phe Arg Asn 145
150 155 160 Ser Val Phe Tyr Gln Ile Ile Gly Pro Ala Tyr Ile Pro Ile
Ala Phe 165 170 175 Ala Thr Ala Ala Ala Ala Asp Pro Asp Val Lys Leu
Tyr Tyr Asn Asp 180 185 190 Tyr Asn Ile Glu Tyr Ser Gly Ala Lys Ala
Thr Ala Ala Gln Asn Ile 195 200 205 Val Lys Met Ile Lys Ala Tyr Gly
Ala Lys Ile Asp Gly Val Gly Leu 210 215 220 Gln Ala His Phe Ile Val
Gly Ser Thr Pro Ser Gln Ser Asp Leu Thr 225 230 235 240 Thr Val Leu
Lys Gly Tyr Thr Ala Leu Gly Val Glu Val Ala Tyr Thr 245 250 255 Glu
Leu Asp Ile Arg Met Gln Leu Pro Ser Thr Ala Ala Lys Leu Ala 260 265
270 Gln Gln Ser Thr Asp Phe Gln Gly Val Ala Ala Ala Cys Val Ser Thr
275 280 285 Thr Gly Cys Val Gly Val Thr Ile Trp Asp Trp Thr Asp Lys
Tyr Ser 290 295 300 Trp Val Pro Ser Val Phe Gln Gly Tyr Gly Ala Pro
Leu Pro Trp Asp 305 310 315 320 Glu Asn Tyr Val Lys Lys Pro Ala Tyr
Asp Gly Leu Met Ala Gly Leu 325 330 335 Gly Ala Ser Gly Ser Gly Thr
Thr Thr Thr Thr Thr Thr Thr Ser Thr 340 345 350 Thr Thr Gly Gly Thr
Asp Pro Thr Gly Val Ala Gln Lys Trp Gly Gln 355 360 365 Cys Gly Gly
Ile Gly Trp Thr Gly Pro Thr Thr Cys Val Ser Gly Thr 370 375 380 Thr
Cys Gln Lys Leu Asn Asp Trp Tyr Ser Gln Cys Leu 385 390 395
192388DNATalaromyces emersonii 19atgatgactc ccacggcgat tctcaccgca
gtggcggcgc tcctgcccac cgcgacatgg 60gcacaggata accaaaccta tgccaattac
tcgtcgcagt ctcagccgga cctgtttccc 120cggaccgtcg cgaccatcga
cctgtccttc cccgactgtg agaatggccc gctcagcacg 180aacctggtgt
gcaacaaatc ggccgatccc tgggcccgag ctgaggccct catctcgctc
240tttaccctcg aagagctgat taacaacacc cagaacaccg ctcctggcgt
gccccgtttg 300ggtctgcccc agtatcaggt gtggaatgaa gctctgcacg
gactggaccg cgccaatttc 360tcccattcgg gcgaatacag ctgggccacg
tccttcccca tgcccatcct gtcgatggcg 420tccttcaacc ggaccctcat
caaccagatt gcctccatca ttgcaacgca agcccgtgcc 480ttcaacaacg
ccggccgtta cggccttgac agctatgcgc ccaacatcaa tggcttccgc
540agtcccctct ggggccgtgg acaggagacg cctggtgagg atgcgttctt
cttgagttcc 600acctatgcgt acgagtacat cacaggcctg cagggcggtg
tcgacccaga gcatgtcaag 660atcgtcgcga cggcgaagca cttcgccggc
tatgatctgg agaactgggg caacgtctct 720cggctggggt tcaatgctat
catcacgcag caggatctct ccgagtacta cacccctcag 780ttcctggcgt
ctgctcgata cgccaagacg cgcagcatca tgtgctccta caatgcagtg
840aatggagtcc caagctgtgc caactccttc ttcctccaga cgcttctccg
agaaaacttt 900gacttcgttg acgacgggta cgtctcgtcg gattgcgacg
ccgtctacaa cgtcttcaac 960ccacacggtt acgcccttaa ccagtcggga
gccgctgcgg actcgctcct agcaggtacc 1020gatatcgact gtggtcagac
cttgccgtgg cacctgaatg agtccttcgt agaaggatac 1080gtctcccgcg
gtgatatcga gaaatccctc acccgtctct actcaaacct ggtgcgtctc
1140ggctactttg acggcaacaa cagcgagtac cgcaacctca actggaacga
cgtcgtgact 1200acggacgcct ggaacatctc gtacgaggcc gcggtggaag
gtatcaccct gctcaagaac 1260gacggaacgc tgccgctgtc caagaaggtc
cgcagcattg cgctcatcgg tccttgggcc 1320aatgccacgg tgcagatgca
gggtaactac tatggaacgc caccgtatct gatcagtccg 1380ctggaagccg
ccaaggccag tgggttcacg gtcaactatg cattcggtac caacatctcg
1440accgattcta cccagtggtt cgcggaagcc atcgcggcgg cgaagaagtc
ggacgtgatc 1500atctacgccg gtggtattga caacacgatc gaggcagagg
gacaggaccg cacggatctc 1560aagtggccgg ggaaccagct ggatctgatc
gagcagctca gccaggtggg caagcccttg 1620gtcgtcctgc agatgggcgg
tggccaggtg gattcgtcgt cactcaaggc caacaagaat 1680gtcaacgctc
tggtgtgggg tggctatccc ggacagtcgg gtggtgcggc cctgtttgac
1740atccttacgg gcaagcgtgc gccggccggt cgtctggtga gcacgcagta
cccggccgag 1800tatgcgacgc agttcccggc caacgacatg aacctgcgtc
cgaacggcag caacccggga 1860cagacataca tctggtacac gggcacgccc
gtgtatgagt tcggccacgg tctgttctac 1920acggagttcc aggagtcggc
tgcggcgggc acgaacaaga cgtcgacttt cgacattctg 1980gaccttttct
ccacccctca tccgggatac gagtacatcg agcaggttcc gttcatcaac
2040gtgactgtgg acgtgaagaa cgtcggccac acgccatcgc cgtacacggg
tctgttgttc 2100gcgaacacga cagccgggcc caagccgtac ccgaacaaat
ggctcgtcgg gttcgactgg 2160ctgccgacga tccagccggg cgagactgcc
aagttgacga tcccggtgcc gttgggcgcg 2220attgcgtggg cggacgagaa
cggcaacaag gtggtcttcc cgggcaacta cgaattggca 2280ctgaacaatg
agcgatcggt agtggtgtcg ttcacgctga cgggcgatgc ggcgactcta
2340gagaaatggc ctttgtggga gcaggcggtt ccgggggtgc tgcagcaa
238820796PRTTalaromyces emersonii 20Met Met Thr Pro Thr Ala Ile Leu
Thr Ala Val Ala Ala Leu Leu Pro 1 5 10 15 Thr Ala Thr Trp Ala Gln
Asp Asn Gln Thr Tyr Ala Asn Tyr Ser Ser 20 25 30 Gln Ser Gln Pro
Asp Leu Phe Pro Arg Thr Val Ala Thr Ile Asp Leu 35 40 45 Ser Phe
Pro Asp Cys Glu Asn Gly Pro Leu Ser Thr Asn Leu Val Cys 50 55 60
Asn Lys Ser Ala Asp Pro Trp Ala Arg Ala Glu Ala Leu Ile Ser Leu 65
70 75 80 Phe Thr Leu Glu Glu Leu Ile Asn Asn Thr Gln Asn Thr Ala
Pro Gly 85 90 95 Val Pro Arg Leu Gly Leu Pro Gln Tyr Gln Val Trp
Asn Glu Ala Leu 100 105 110 His Gly Leu Asp Arg Ala Asn Phe Ser His
Ser Gly Glu Tyr Ser Trp 115 120 125 Ala Thr Ser Phe Pro Met Pro Ile
Leu Ser Met Ala Ser Phe Asn Arg 130 135 140 Thr Leu Ile Asn Gln Ile
Ala Ser Ile Ile Ala Thr Gln Ala Arg Ala 145 150 155 160 Phe Asn Asn
Ala Gly Arg Tyr Gly Leu Asp Ser Tyr Ala Pro Asn Ile 165 170 175 Asn
Gly Phe Arg Ser Pro Leu Trp Gly Arg Gly Gln Glu Thr Pro Gly 180 185
190 Glu Asp Ala Phe Phe Leu Ser Ser Thr Tyr Ala Tyr Glu Tyr Ile Thr
195 200 205 Gly Leu Gln Gly Gly Val Asp Pro Glu His Val Lys Ile Val
Ala Thr 210 215 220 Ala Lys His Phe Ala Gly Tyr Asp Leu Glu Asn Trp
Gly Asn Val Ser 225 230 235 240 Arg Leu Gly Phe Asn Ala Ile Ile Thr
Gln Gln Asp Leu Ser Glu Tyr 245 250 255 Tyr Thr Pro Gln Phe Leu Ala
Ser Ala Arg Tyr Ala Lys Thr Arg Ser 260 265 270 Ile Met Cys Ser Tyr
Asn Ala Val Asn Gly Val Pro Ser Cys Ala Asn 275 280 285 Ser Phe Phe
Leu Gln Thr Leu Leu Arg Glu Asn Phe Asp Phe Val Asp 290 295 300 Asp
Gly Tyr Val Ser Ser Asp Cys Asp Ala Val Tyr Asn Val Phe Asn 305 310
315 320 Pro His Gly Tyr Ala Leu Asn Gln Ser Gly Ala Ala Ala Asp Ser
Leu 325 330 335 Leu Ala Gly Thr Asp Ile Asp Cys Gly Gln Thr Leu Pro
Trp His Leu 340 345 350 Asn Glu Ser Phe Val Glu Gly Tyr Val Ser Arg
Gly Asp Ile Glu Lys 355 360 365 Ser Leu Thr Arg Leu Tyr Ser Asn Leu
Val Arg Leu Gly Tyr Phe Asp 370 375 380 Gly Asn Asn Ser Glu Tyr Arg
Asn Leu Asn Trp Asn Asp Val Val Thr 385 390 395 400 Thr Asp Ala Trp
Asn Ile Ser Tyr Glu Ala Ala Val Glu Gly Ile Thr 405 410 415 Leu Leu
Lys Asn Asp Gly Thr Leu Pro Leu Ser Lys Lys Val Arg Ser 420 425 430
Ile Ala Leu Ile Gly Pro Trp Ala Asn Ala Thr Val Gln Met Gln Gly 435
440 445 Asn Tyr Tyr Gly Thr Pro Pro Tyr Leu Ile Ser Pro Leu Glu Ala
Ala 450 455 460 Lys Ala Ser Gly Phe Thr Val Asn Tyr Ala Phe Gly Thr
Asn Ile Ser 465 470 475 480 Thr Asp Ser Thr Gln Trp Phe Ala Glu Ala
Ile Ala Ala Ala Lys Lys 485 490 495 Ser Asp Val Ile Ile Tyr Ala Gly
Gly Ile Asp Asn Thr Ile Glu Ala 500 505 510 Glu Gly Gln Asp Arg Thr
Asp Leu Lys Trp Pro Gly Asn Gln Leu Asp 515 520 525 Leu Ile Glu Gln
Leu Ser Gln Val Gly Lys Pro Leu Val Val Leu Gln 530 535 540 Met Gly
Gly Gly Gln Val Asp Ser Ser Ser Leu Lys Ala Asn Lys Asn 545 550 555
560 Val Asn Ala Leu Val Trp Gly Gly Tyr Pro Gly Gln Ser Gly Gly Ala
565 570 575 Ala Leu Phe Asp Ile Leu Thr Gly Lys Arg Ala Pro Ala Gly
Arg Leu 580 585 590 Val Ser Thr Gln Tyr Pro Ala Glu Tyr Ala Thr Gln
Phe Pro Ala Asn 595 600 605 Asp Met Asn Leu Arg Pro Asn Gly Ser Asn
Pro Gly Gln Thr Tyr Ile 610 615 620 Trp Tyr Thr Gly Thr Pro Val Tyr
Glu Phe Gly His Gly Leu Phe Tyr 625 630 635 640 Thr Glu Phe Gln Glu
Ser Ala Ala Ala Gly Thr Asn Lys Thr Ser Thr 645 650 655 Phe Asp Ile
Leu Asp Leu Phe Ser Thr Pro His Pro Gly Tyr Glu Tyr 660 665 670 Ile
Glu Gln Val Pro Phe Ile Asn Val Thr Val Asp Val Lys Asn Val 675 680
685 Gly His Thr Pro Ser Pro Tyr Thr Gly Leu Leu Phe Ala Asn Thr Thr
690 695 700 Ala Gly Pro Lys Pro Tyr Pro Asn Lys Trp Leu Val Gly Phe
Asp Trp 705 710 715 720 Leu Pro Thr Ile Gln Pro Gly Glu Thr Ala Lys
Leu Thr Ile Pro Val 725 730 735 Pro Leu Gly Ala Ile Ala Trp Ala Asp
Glu Asn Gly Asn Lys Val Val 740 745 750 Phe Pro Gly Asn Tyr Glu Leu
Ala Leu Asn Asn Glu Arg Ser Val Val 755 760 765 Val Ser Phe Thr Leu
Thr Gly Asp Ala Ala Thr Leu Glu Lys Trp Pro 770 775 780 Leu Trp Glu
Gln Ala Val Pro Gly Val Leu Gln Gln 785 790 795 2119PRTThielavia
terrestrisMISC_FEATURE(1)..(1)X=I,L,M, OR Vmisc_feature(3)..(6)Xaa
can be any naturally occurring amino acidmisc_feature(8)..(8)Xaa
can be any naturally occurring amino
acidMISC_FEATURE(10)..(10)X=I,L,M, OR Vmisc_feature(11)..(11)Xaa
can be any naturally occurring amino acidmisc_feature(13)..(13)Xaa
can be any naturally occurring amino acidMISC_FEATURE(14)..(14)X=E
OR Qmisc_feature(15)..(18)Xaa can be any naturally occurring amino
acidMISC_FEATURE(19)..(19)X=H,N, OR Q 21Xaa Pro Xaa Xaa Xaa Xaa Gly
Xaa Tyr Xaa Xaa Arg Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa
2220PRTThielavia terrestrisMISC_FEATURE(1)..(1)X=I,L,M, OR
Vmisc_feature(3)..(7)Xaa can be any naturally occurring amino
acidmisc_feature(9)..(9)Xaa can be any naturally occurring amino
acidMISC_FEATURE(11)..(11)X=I,L,M, OR Vmisc_feature(12)..(12)Xaa
can be any naturally occurring amino acidmisc_feature(14)..(14)Xaa
can be any naturally occurring amino acidMISC_FEATURE(15)..(15)X=E
OR Qmisc_feature(16)..(19)Xaa can be any naturally occurring amino
acidMISC_FEATURE(20)..(20)X=H,N, OR Q 22Xaa Pro Xaa Xaa Xaa Xaa Xaa
Gly Xaa Tyr Xaa Xaa Arg Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa 20
239PRTThielavia terrestrismisc_feature(2)..(2)Xaa can be any
naturally occurring amino acidmisc_feature(5)..(7)Xaa can be any
naturally occurring amino acidMISC_FEATURE(8)..(8)X= Y OR
WMISC_FEATURE(9)..(9)X= A,I,L,M OR VMISC_FEATURE(9)..(9)X= A,I,L,M
OR V 23His Xaa Gly Pro Xaa Xaa Xaa Xaa Xaa 1 5 2410PRTThielavia
terrestrismisc_feature(2)..(3)Xaa can be any naturally occurring
amino acidmisc_feature(6)..(8)Xaa can be any naturally occurring
amino acidMISC_FEATURE(9)..(9)X= Y OR WMISC_FEATURE(10)..(10)X=
A,I,L,M OR VMISC_FEATURE(10)..(10)X= A,I,L,M OR V 24His Xaa Xaa Gly
Pro Xaa Xaa Xaa Xaa Xaa 1 5 10 2511PRTThielavia
terrestrisMISC_FEATURE(1)..(1)X= E OR Qmisc_feature(2)..(2)Xaa can
be any naturally occurring amino acidmisc_feature(4)..(5)Xaa can be
any naturally occurring amino acidmisc_feature(7)..(7)Xaa can be
any naturally occurring amino acidMISC_FEATURE(8)..(8)X= E,H,Q OR
NMISC_FEATURE(9)..(9)X=F,I,L, OR Vmisc_feature(10)..(10)Xaa can be
any naturally occurring amino acidMISC_FEATURE(11)..(11)X=I,L,OR
VMISC_FEATURE(11)..(11)X=I,L,OR V 25Xaa Xaa Tyr Xaa Xaa Cys Xaa Xaa
Xaa Xaa Xaa 1 5 10 269PRTThielavia
terrestrismisc_feature(2)..(2)Xaa can be any naturally occurring
amino acidmisc_feature(5)..(7)Xaa can be any naturally occurring
amino acidMISC_FEATURE(8)..(8)X= Y OR WMISC_FEATURE(9)..(9)X=
A,I,L,M OR V 26His Xaa Gly Pro Xaa Xaa Xaa Xaa Xaa 1 5
2710PRTThielavia terrestrismisc_feature(2)..(3)Xaa can be any
naturally occurring amino acidmisc_feature(6)..(8)Xaa can be any
naturally occurring amino acidMISC_FEATURE(9)..(9)X= Y OR
WMISC_FEATURE(10)..(10)X= A,I,L,M OR VMISC_FEATURE(10)..(10)X=
A,I,L,M OR V 27His Xaa Xaa Gly Pro Xaa Xaa Xaa Xaa Xaa 1 5 10
2811PRTThielavia terrestrisMISC_FEATURE(1)..(1)X= E OR
Qmisc_feature(2)..(2)Xaa can be any naturally occurring amino
acidmisc_feature(4)..(5)Xaa can be any naturally occurring amino
acidmisc_feature(7)..(7)Xaa can be any naturally occurring amino
acidMISC_FEATURE(8)..(8)X= E,H,Q OR NMISC_FEATURE(9)..(9)X=F,I,L,
OR Vmisc_feature(10)..(10)Xaa can be any naturally occurring amino
acidMISC_FEATURE(11)..(11)X=I,L,OR V 28Xaa Xaa Tyr Xaa Xaa Cys Xaa
Xaa Xaa Xaa Xaa 1 5 10 2919PRTThielavia
terrestrisMISC_FEATURE(1)..(1)X=I,L,M OR Vmisc_feature(3)..(6)Xaa
can be any naturally occurring amino acidmisc_feature(8)..(8)Xaa
can be any naturally occurring amino
acidMISC_FEATURE(10)..(10)X=I,L,M OR Vmisc_feature(11)..(11)Xaa can
be any naturally occurring amino acidmisc_feature(13)..(13)Xaa can
be any naturally occurring amino acidMISC_FEATURE(14)..(14)X= E OR
Qmisc_feature(15)..(17)Xaa can be any naturally occurring amino
acidMISC_FEATURE(19)..(19)X= H,N, OR Q 29Xaa Pro Xaa Xaa Xaa Xaa
Gly Xaa Tyr Xaa Xaa Arg Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Ala Xaa
3020PRTThielavia terrestrisMISC_FEATURE(1)..(1)X=I,L,M OR
Vmisc_feature(3)..(7)Xaa can be any naturally occurring amino
acidmisc_feature(9)..(9)Xaa can be any naturally occurring amino
acidMISC_FEATURE(11)..(11)X=I,L,M OR Vmisc_feature(12)..(12)Xaa can
be any naturally occurring amino acidmisc_feature(14)..(14)Xaa can
be any naturally occurring amino acidMISC_FEATURE(15)..(15)X=E OR
Qmisc_feature(16)..(18)Xaa can be any naturally occurring amino
acidMISC_FEATURE(20)..(20)X= H,N, OR Q 30Xaa Pro Xaa Xaa Xaa Xaa
Xaa Gly Xaa Tyr Xaa Xaa Arg Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Ala Xaa
20 312394DNATrichoderma reesei 31atggtgaata acgcagctct tctcgccgcc
ctgtcggctc tcctgcccac ggccctggcg 60cagaacaatc aaacatacgc caactactct
gctcagggcc agcctgatct ctaccccgag 120acacttgcca cgctcacact
ctcgttcccc gactgcgaac atggccccct caagaacaat 180ctcgtctgtg
actcatcggc cggctatgta gagcgagccc aggccctcat ctcgctcttc
240accctcgagg agctcattct caacacgcaa aactcgggcc ccggcgtgcc
tcgcctgggt 300cttccgaact accaagtctg gaatgaggct ctgcacggct
tggaccgcgc caacttcgcc 360accaagggcg gccagttcga atgggcgacc
tcgttcccca tgcccatcct cactacggcg 420gccctcaacc gcacattgat
ccaccagatt gccgacatca tctcgaccca agctcgagca 480ttcagcaaca
gcggccgtta cggtctcgac gtctatgcgc caaacgtcaa tggcttccga
540agccccctct ggggccgtgg ccaggagacg cccggcgaag acgccttttt
cctcagctcc 600gcctatactt acgagtacat cacgggcatc cagggtggcg
tcgaccctga gcacctcaag 660gttgccgcca cggtgaagca ctttgccgga
tacgacctcg agaactggaa caaccagtcc 720cgtctcggtt tcgacgccat
cataactcag caggacctct ccgaatacta cactccccag 780ttcctcgctg
cggcccgtta tgcaaagtca cgcagcttga tgtgcgcata caactccgtc
840aacggcgtgc ccagctgtgc caacagcttc ttcctgcaga cgcttttgcg
cgagagctgg 900ggcttccccg aatggggata cgtctcgtcc gattgcgatg
ccgtctacaa cgttttcaac 960cctcatgact acgccagcaa ccagtcgtca
gccgccgcca gctcactgcg agccggcacc 1020gatatcgact gcggtcagac
ttacccgtgg cacctcaacg agtcctttgt ggccggcgaa 1080gtctcccgcg
gcgagatcga gcggtccgtc acccgtctgt acgccaacct cgtccgtctc
1140ggatacttcg acaagaagaa ccagtaccgc tcgctcggtt ggaaggatgt
cgtcaagact 1200gatgcctgga acatctcgta cgaggctgct gttgagggca
tcgtcctgct caagaacgat 1260ggcactctcc ctctgtccaa gaaggtgcgc
agcattgctc tgatcggacc atgggccaat 1320gccacaaccc aaatgcaagg
caactactat ggccctgccc catacctcat cagccctctg 1380gaagctgcta
agaaggccgg ctatcacgtc aactttgaac tcggcacaga gatcgccggc
1440aacagcacca ctggctttgc caaggccatt gctgccgcca agaagtcgga
tgccatcatc 1500tacctcggtg gaattgacaa caccattgaa caggagggcg
ctgaccgcac ggacattgct 1560tggcccggta atcagctgga tctcatcaag
cagctcagcg aggtcggcaa accccttgtc 1620gtcctgcaaa tgggcggtgg
tcaggtagac tcatcctcgc tcaagagcaa caagaaggtc 1680aactccctcg
tctggggcgg atatcccggc cagtcgggag gcgttgccct cttcgacatt
1740ctctctggca agcgtgctcc tgccggccga ctggtcacca ctcagtaccc
ggctgagtat 1800gttcaccaat tcccccagaa tgacatgaac ctccgacccg
atggaaagtc aaaccctgga 1860cagacttaca tctggtacac cggcaaaccc
gtctacgagt ttggcagtgg tctcttctac 1920accaccttca aggagactct
cgccagccac cccaagagcc tcaagttcaa cacctcatcg 1980atcctctctg
ctcctcaccc cggatacact tacagcgagc agattcccgt cttcaccttc
2040gaggccaaca tcaagaactc gggcaagacg gagtccccat atacggccat
gctgtttgtt 2100cgcacaagca acgctggccc agccccgtac ccgaacaagt
ggctcgtcgg attcgaccga 2160cttgccgaca tcaagcctgg tcactcttcc
aagctcagca tccccatccc tgtcagtgct 2220ctcgcccgtg ttgattctca
cggaaaccgg attgtatacc ccggcaagta tgagctagcc 2280ttgaacaccg
acgagtctgt gaagcttgag tttgagttgg tgggagaaga ggtaacgatt
2340gagaactggc cgttggagga gcaacagatc aaggatgcta cacctgacgc ataa
239432797PRTTrichoderma reesei 32Met Val Asn Asn Ala Ala Leu Leu
Ala Ala Leu Ser Ala Leu Leu Pro 1 5 10 15 Thr Ala Leu Ala Gln Asn
Asn Gln Thr Tyr Ala Asn Tyr Ser Ala Gln 20 25 30 Gly Gln Pro Asp
Leu Tyr Pro Glu Thr Leu Ala Thr Leu Thr Leu Ser 35 40 45 Phe Pro
Asp Cys Glu His Gly Pro Leu Lys Asn Asn Leu Val Cys Asp 50 55 60
Ser Ser Ala Gly Tyr Val Glu Arg Ala Gln Ala Leu Ile Ser Leu Phe 65
70 75 80 Thr Leu Glu Glu Leu Ile Leu Asn Thr Gln Asn Ser Gly Pro
Gly Val 85 90 95 Pro Arg Leu Gly Leu Pro Asn Tyr Gln Val Trp Asn
Glu Ala Leu His 100 105
110 Gly Leu Asp Arg Ala Asn Phe Ala Thr Lys Gly Gly Gln Phe Glu Trp
115 120 125 Ala Thr Ser Phe Pro Met Pro Ile Leu Thr Thr Ala Ala Leu
Asn Arg 130 135 140 Thr Leu Ile His Gln Ile Ala Asp Ile Ile Ser Thr
Gln Ala Arg Ala 145 150 155 160 Phe Ser Asn Ser Gly Arg Tyr Gly Leu
Asp Val Tyr Ala Pro Asn Val 165 170 175 Asn Gly Phe Arg Ser Pro Leu
Trp Gly Arg Gly Gln Glu Thr Pro Gly 180 185 190 Glu Asp Ala Phe Phe
Leu Ser Ser Ala Tyr Thr Tyr Glu Tyr Ile Thr 195 200 205 Gly Ile Gln
Gly Gly Val Asp Pro Glu His Leu Lys Val Ala Ala Thr 210 215 220 Val
Lys His Phe Ala Gly Tyr Asp Leu Glu Asn Trp Asn Asn Gln Ser 225 230
235 240 Arg Leu Gly Phe Asp Ala Ile Ile Thr Gln Gln Asp Leu Ser Glu
Tyr 245 250 255 Tyr Thr Pro Gln Phe Leu Ala Ala Ala Arg Tyr Ala Lys
Ser Arg Ser 260 265 270 Leu Met Cys Ala Tyr Asn Ser Val Asn Gly Val
Pro Ser Cys Ala Asn 275 280 285 Ser Phe Phe Leu Gln Thr Leu Leu Arg
Glu Ser Trp Gly Phe Pro Glu 290 295 300 Trp Gly Tyr Val Ser Ser Asp
Cys Asp Ala Val Tyr Asn Val Phe Asn 305 310 315 320 Pro His Asp Tyr
Ala Ser Asn Gln Ser Ser Ala Ala Ala Ser Ser Leu 325 330 335 Arg Ala
Gly Thr Asp Ile Asp Cys Gly Gln Thr Tyr Pro Trp His Leu 340 345 350
Asn Glu Ser Phe Val Ala Gly Glu Val Ser Arg Gly Glu Ile Glu Arg 355
360 365 Ser Val Thr Arg Leu Tyr Ala Asn Leu Val Arg Leu Gly Tyr Phe
Asp 370 375 380 Lys Lys Asn Gln Tyr Arg Ser Leu Gly Trp Lys Asp Val
Val Lys Thr 385 390 395 400 Asp Ala Trp Asn Ile Ser Tyr Glu Ala Ala
Val Glu Gly Ile Val Leu 405 410 415 Leu Lys Asn Asp Gly Thr Leu Pro
Leu Ser Lys Lys Val Arg Ser Ile 420 425 430 Ala Leu Ile Gly Pro Trp
Ala Asn Ala Thr Thr Gln Met Gln Gly Asn 435 440 445 Tyr Tyr Gly Pro
Ala Pro Tyr Leu Ile Ser Pro Leu Glu Ala Ala Lys 450 455 460 Lys Ala
Gly Tyr His Val Asn Phe Glu Leu Gly Thr Glu Ile Ala Gly 465 470 475
480 Asn Ser Thr Thr Gly Phe Ala Lys Ala Ile Ala Ala Ala Lys Lys Ser
485 490 495 Asp Ala Ile Ile Tyr Leu Gly Gly Ile Asp Asn Thr Ile Glu
Gln Glu 500 505 510 Gly Ala Asp Arg Thr Asp Ile Ala Trp Pro Gly Asn
Gln Leu Asp Leu 515 520 525 Ile Lys Gln Leu Ser Glu Val Gly Lys Pro
Leu Val Val Leu Gln Met 530 535 540 Gly Gly Gly Gln Val Asp Ser Ser
Ser Leu Lys Ser Asn Lys Lys Val 545 550 555 560 Asn Ser Leu Val Trp
Gly Gly Tyr Pro Gly Gln Ser Gly Gly Val Ala 565 570 575 Leu Phe Asp
Ile Leu Ser Gly Lys Arg Ala Pro Ala Gly Arg Leu Val 580 585 590 Thr
Thr Gln Tyr Pro Ala Glu Tyr Val His Gln Phe Pro Gln Asn Asp 595 600
605 Met Asn Leu Arg Pro Asp Gly Lys Ser Asn Pro Gly Gln Thr Tyr Ile
610 615 620 Trp Tyr Thr Gly Lys Pro Val Tyr Glu Phe Gly Ser Gly Leu
Phe Tyr 625 630 635 640 Thr Thr Phe Lys Glu Thr Leu Ala Ser His Pro
Lys Ser Leu Lys Phe 645 650 655 Asn Thr Ser Ser Ile Leu Ser Ala Pro
His Pro Gly Tyr Thr Tyr Ser 660 665 670 Glu Gln Ile Pro Val Phe Thr
Phe Glu Ala Asn Ile Lys Asn Ser Gly 675 680 685 Lys Thr Glu Ser Pro
Tyr Thr Ala Met Leu Phe Val Arg Thr Ser Asn 690 695 700 Ala Gly Pro
Ala Pro Tyr Pro Asn Lys Trp Leu Val Gly Phe Asp Arg 705 710 715 720
Leu Ala Asp Ile Lys Pro Gly His Ser Ser Lys Leu Ser Ile Pro Ile 725
730 735 Pro Val Ser Ala Leu Ala Arg Val Asp Ser His Gly Asn Arg Ile
Val 740 745 750 Tyr Pro Gly Lys Tyr Glu Leu Ala Leu Asn Thr Asp Glu
Ser Val Lys 755 760 765 Leu Glu Phe Glu Leu Val Gly Glu Glu Val Thr
Ile Glu Asn Trp Pro 770 775 780 Leu Glu Glu Gln Gln Ile Lys Asp Ala
Thr Pro Asp Ala 785 790 795 331849DNATrichoderma reesei
33tgccatttct gacctggata ggttttccta tggtcattcc tataagagac acgctctttc
60gtcggcccgt agatatcaga ttggtattca gtcgcacaga cgaaggtgag ttgatcctcc
120aacatgagtt ctatgagccc cccccttgcc cccccccgtt caccttgacc
tgcaatgaga 180atcccacctt ttacaagagc atcaagaagt attaatggcg
ctgaatagcc tctgctcgat 240aatatctccc cgtcatcgac aatgaacaag
tccgtggctc cattgctgct tgcagcgtcc 300atactatatg gcggcgccgt
cgcacagcag actgtctggg gccagtgtgg aggtattggt 360tggagcggac
ctacgaattg tgctcctggc tcagcttgtt cgaccctcaa tccttattat
420gcgcaatgta ttccgggagc cactactatc accacttcga cccggccacc
atccggtcca 480accaccacca ccagggctac ctcaacaagc tcatcaactc
cacccacgag ctctggggtc 540cgatttgccg gcgttaacat cgcgggtttt
gactttggct gtaccacaga gtgagtaccc 600ttgtttcctg gtgttgctgg
ctggttgggc gggtatacag cgaagcggac gcaagaacac 660cgccggtccg
ccaccatcaa gatgtgggtg gtaagcggcg gtgttttgta caactacctg
720acagctcact caggaaatga gaattaatgg aagtcttgtt acagtggcac
ttgcgttacc 780tcgaaggttt atcctccgtt gaagaacttc accggctcaa
acaactaccc cgatggcatc 840ggccagatgc agcacttcgt caacgaggac
gggatgacta ttttccgctt acctgtcgga 900tggcagtacc tcgtcaacaa
caatttgggc ggcaatcttg attccacgag catttccaag 960tatgatcagc
ttgttcaggg gtgcctgtct ctgggcgcat actgcatcgt cgacatccac
1020aattatgctc gatggaacgg tgggatcatt ggtcagggcg gccctactaa
tgctcaattc 1080acgagccttt ggtcgcagtt ggcatcaaag tacgcatctc
agtcgagggt gtggttcggc 1140atcatgaatg agccccacga cgtgaacatc
aacacctggg ctgccacggt ccaagaggtt 1200gtaaccgcaa tccgcaacgc
tggtgctacg tcgcaattca tctctttgcc tggaaatgat 1260tggcaatctg
ctggggcttt catatccgat ggcagtgcag ccgccctgtc tcaagtcacg
1320aacccggatg ggtcaacaac gaatctgatt tttgacgtgc acaaatactt
ggactcagac 1380aactccggta ctcacgccga atgtactaca aataacattg
acggcgcctt ttctccgctt 1440gccacttggc tccgacagaa caatcgccag
gctatcctga cagaaaccgg tggtggcaac 1500gttcagtcct gcatacaaga
catgtgccag caaatccaat atctcaacca gaactcagat 1560gtctatcttg
gctatgttgg ttggggtgcc ggatcatttg atagcacgta tgtcctgacg
1620gaaacaccga ctggcagtgg taactcatgg acggacacat ccttggtcag
ctcgtgtctc 1680gcaagaaagt agcactctga gctgaatgca gaagcctcgc
caacgtttgt atctcgctat 1740caaacatagt agctactcta tgaggctgtc
tgttctcgat ttcagcttta tatagtttca 1800tcaaacagta catattccct
ctgtggccac gcaaaaaaaa aaaaaaaaa 184934418PRTTrichoderma reesei
34Met Asn Lys Ser Val Ala Pro Leu Leu Leu Ala Ala Ser Ile Leu Tyr 1
5 10 15 Gly Gly Ala Val Ala Gln Gln Thr Val Trp Gly Gln Cys Gly Gly
Ile 20 25 30 Gly Trp Ser Gly Pro Thr Asn Cys Ala Pro Gly Ser Ala
Cys Ser Thr 35 40 45 Leu Asn Pro Tyr Tyr Ala Gln Cys Ile Pro Gly
Ala Thr Thr Ile Thr 50 55 60 Thr Ser Thr Arg Pro Pro Ser Gly Pro
Thr Thr Thr Thr Arg Ala Thr 65 70 75 80 Ser Thr Ser Ser Ser Thr Pro
Pro Thr Ser Ser Gly Val Arg Phe Ala 85 90 95 Gly Val Asn Ile Ala
Gly Phe Asp Phe Gly Cys Thr Thr Asp Gly Thr 100 105 110 Cys Val Thr
Ser Lys Val Tyr Pro Pro Leu Lys Asn Phe Thr Gly Ser 115 120 125 Asn
Asn Tyr Pro Asp Gly Ile Gly Gln Met Gln His Phe Val Asn Glu 130 135
140 Asp Gly Met Thr Ile Phe Arg Leu Pro Val Gly Trp Gln Tyr Leu Val
145 150 155 160 Asn Asn Asn Leu Gly Gly Asn Leu Asp Ser Thr Ser Ile
Ser Lys Tyr 165 170 175 Asp Gln Leu Val Gln Gly Cys Leu Ser Leu Gly
Ala Tyr Cys Ile Val 180 185 190 Asp Ile His Asn Tyr Ala Arg Trp Asn
Gly Gly Ile Ile Gly Gln Gly 195 200 205 Gly Pro Thr Asn Ala Gln Phe
Thr Ser Leu Trp Ser Gln Leu Ala Ser 210 215 220 Lys Tyr Ala Ser Gln
Ser Arg Val Trp Phe Gly Ile Met Asn Glu Pro 225 230 235 240 His Asp
Val Asn Ile Asn Thr Trp Ala Ala Thr Val Gln Glu Val Val 245 250 255
Thr Ala Ile Arg Asn Ala Gly Ala Thr Ser Gln Phe Ile Ser Leu Pro 260
265 270 Gly Asn Asp Trp Gln Ser Ala Gly Ala Phe Ile Ser Asp Gly Ser
Ala 275 280 285 Ala Ala Leu Ser Gln Val Thr Asn Pro Asp Gly Ser Thr
Thr Asn Leu 290 295 300 Ile Phe Asp Val His Lys Tyr Leu Asp Ser Asp
Asn Ser Gly Thr His 305 310 315 320 Ala Glu Cys Thr Thr Asn Asn Ile
Asp Gly Ala Phe Ser Pro Leu Ala 325 330 335 Thr Trp Leu Arg Gln Asn
Asn Arg Gln Ala Ile Leu Thr Glu Thr Gly 340 345 350 Gly Gly Asn Val
Gln Ser Cys Ile Gln Asp Met Cys Gln Gln Ile Gln 355 360 365 Tyr Leu
Asn Gln Asn Ser Asp Val Tyr Leu Gly Tyr Val Gly Trp Gly 370 375 380
Ala Gly Ser Phe Asp Ser Thr Tyr Val Leu Thr Glu Thr Pro Thr Gly 385
390 395 400 Ser Gly Asn Ser Trp Thr Asp Thr Ser Leu Val Ser Ser Cys
Leu Ala 405 410 415 Arg Lys
* * * * *