U.S. patent application number 14/642268 was filed with the patent office on 2015-06-25 for polypeptides having endoglucanase activity and polynucleotides encoding same.
The applicant listed for this patent is Novozymes A/S. Invention is credited to Alfredo Lopez de Leon, Michael Rey.
Application Number | 20150175990 14/642268 |
Document ID | / |
Family ID | 40722067 |
Filed Date | 2015-06-25 |
United States Patent
Application |
20150175990 |
Kind Code |
A1 |
Lopez de Leon; Alfredo ; et
al. |
June 25, 2015 |
Polypeptides Having Endoglucanase Activity And Polynucleotides
Encoding Same
Abstract
The present invention relates to isolated polypeptides having
endoglucanase activity and isolated polynucleotides encoding the
polypeptides. The invention also relates to nucleic acid
constructs, vectors, and host cells comprising the polynucleotides
as well as methods of producing and using the polypeptides.
Inventors: |
Lopez de Leon; Alfredo;
(Davis, CA) ; Rey; Michael; (Davis, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Novozymes A/S |
Bagsvaerd |
|
DK |
|
|
Family ID: |
40722067 |
Appl. No.: |
14/642268 |
Filed: |
March 9, 2015 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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13912915 |
Jun 7, 2013 |
8975059 |
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14642268 |
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13600071 |
Aug 30, 2012 |
8465958 |
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13912915 |
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12763578 |
Apr 20, 2010 |
8268606 |
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13600071 |
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12327509 |
Dec 3, 2008 |
7741074 |
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12763578 |
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60992576 |
Dec 5, 2007 |
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Current U.S.
Class: |
800/298 ;
435/209; 435/252.3; 435/252.31; 435/252.33; 435/252.34; 435/252.35;
435/254.11; 435/254.2; 435/254.21; 435/254.22; 435/254.23;
435/254.3; 435/254.4; 435/254.5; 435/254.6; 435/254.7; 435/254.8;
435/320.1; 435/419 |
Current CPC
Class: |
C12N 9/2437 20130101;
C12N 15/8246 20130101; C12Y 302/01004 20130101; C12N 9/2434
20130101; Y02E 50/10 20130101; Y02E 50/16 20130101; C12P 19/14
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 with Government support under NREL
Subcontract No. ZCO-30017-02, Prime Contract DE-AC36-98GO10337
awarded by the Department of Energy. The government has certain
rights in this invention.
Claims
1. A nucleic acid construct comprising a polynucleotide encoding a
polypeptide having endoglucanase activity, wherein the
polynucleotide is operably linked to one or more heterologous
control sequences that direct production of the polypeptide in an
expression host, and wherein the polypeptide is selected from: (a)
a polypeptide comprising an amino acid sequence having at least 90%
sequence identity to the mature polypeptide of SEQ ID NO: 2; (b) a
polypeptide encoded by a polynucleotide which hybridizes under at
least high stringency conditions with (i) the mature polypeptide
coding sequence of SEQ ID NO: 1, (ii) the genomic DNA sequence of
the mature polypeptide coding sequence of SEQ ID NO: 1, or (iii)
the full-length complement of (i) or (ii), wherein high stringency
conditions are defined as prehybridization and hybridization at
42.degree. C. in 5.times.SSPE, 0.3% SDS, 200 .mu.g/ml sheared and
denatured salmon sperm DNA, and 50% formamide and washing three
times each for 15 minutes using 2.times.SSC, 0.2% SDS at 65.degree.
C.; and (c) a polypeptide encoded by a polynucleotide comprising a
nucleotide sequence having at least 90% sequence identity to the
mature polypeptide coding sequence of SEQ ID NO: 1.
2. The nucleic acid construct of claim 1, wherein the polypeptide
having endoglucanase activity comprises an amino acid sequence
having at least 90% sequence identity to the mature polypeptide of
SEQ ID NO: 2.
3. The nucleic acid construct of claim 1, wherein the polypeptide
having endoglucanase activity comprises an amino acid sequence
having at least 95% sequence identity to the mature polypeptide of
SEQ ID NO: 2.
4. The nucleic acid construct of claim 1, wherein the polypeptide
having endoglucanase activity comprises an amino acid sequence
having at least 97% sequence identity to the mature polypeptide of
SEQ ID NO: 2.
5. The nucleic acid construct of claim 1, wherein the polypeptide
having endoglucanase activity comprises an amino acid sequence
having at least 99% sequence identity to the mature polypeptide of
SEQ ID NO: 2.
6. The nucleic acid construct of claim 1, wherein the polypeptide
having endoglucanase activity comprises or consists of the mature
polypeptide of SEQ ID NO: 2.
7. The nucleic acid construct of claim 1, wherein the polypeptide
having endoglucanase activity is a variant comprising a
substitution, deletion, or insertion of one or more amino acids of
the mature polypeptide of SEQ ID NO: 2.
8. The nucleic acid construct of claim 1, wherein the polypeptide
having endoglucanase activity comprises or consists of the amino
acid sequence of SEQ ID NO: 2, or a fragment thereof having
endoglucanase activity.
9. The nucleic acid construct of claim 1, wherein the polypeptide
having endoglucanase activity is encoded by a polynucleotide which
hybridizes under at least high stringency conditions with (i) the
mature polypeptide coding sequence of SEQ ID NO: 1, (ii) the
genomic DNA sequence of the mature polypeptide coding sequence of
SEQ ID NO: 1, or (iii) the full-length complement of (i) or (ii),
wherein high stringency conditions are defined as prehybridization
and hybridization at 42.degree. C. in 5.times.SSPE, 0.3% SDS, 200
.mu.g/ml sheared and denatured salmon sperm DNA, and 50% formamide
and washing three times each for 15 minutes using 2.times.SSC, 0.2%
SDS at 65.degree. C.
10. The nucleic acid construct of claim 1, wherein the polypeptide
having endoglucanase activity is encoded by a polynucleotide which
hybridizes under at least very high stringency conditions with (i)
the mature polypeptide coding sequence of SEQ ID NO: 1, (ii) the
genomic DNA sequence of the mature polypeptide coding sequence of
SEQ ID NO: 1, or (iii) the full-length complement of (i) or (ii),
wherein very high stringency conditions are defined as
prehybridization and hybridization at 42.degree. C. in
5.times.SSPE, 0.3% SDS, 200 .mu.g/ml sheared and denatured salmon
sperm DNA, and 50% formamide and washing three times each for 15
minutes using 2.times.SSC, 0.2% SDS at 70.degree. C.
11. The nucleic acid construct of claim 1, wherein the polypeptide
having endoglucanase activity is encoded by a polynucleotide which
comprises a nucleotide sequence having at least 90% sequence
identity to the mature polypeptide coding sequence of SEQ ID NO:
1.
12. The nucleic acid construct of claim 1, wherein the polypeptide
having endoglucanase activity is encoded by a polynucleotide which
comprises a nucleotide sequence having at least 95% sequence
identity to the mature polypeptide coding sequence of SEQ ID NO:
1.
13. The nucleic acid construct of claim 1, wherein the polypeptide
having endoglucanase activity is encoded by a polynucleotide which
comprises a nucleotide sequence having at least 97% sequence
identity to the mature polypeptide coding sequence of SEQ ID NO:
1.
14. The nucleic acid construct of claim 1, wherein the polypeptide
having endoglucanase activity is encoded by a polynucleotide which
comprises or consists of the nucleotide sequence of SEQ ID NO: 1;
or a subsequence thereof encoding a polypeptide fragment having
endoglucanase activity.
15. The nucleic acid construct of claim 1, wherein the polypeptide
having endoglucanase activity is encoded by a polynucleotide which
is contained in plasmid pTter16A which is contained in E. coli NRRL
B-50081.
16. A recombinant expression vector comprising the nucleic acid
construct of claim 1.
17. An isolated recombinant host cell comprising the nucleic acid
construct of claim 1.
18. A method for producing a polypeptide having endoglucanase
activity, comprising: (a) cultivating the recombinant host cell of
claim 16 under conditions conducive for production of the
polypeptide; and (b) recovering the polypeptide.
19. A method for producing a polypeptide having endoglucanase
activity, comprising: (a) cultivating a transgenic plant or a plant
cell comprising the polynucleotide of claim 1 under conditions
conducive for production of the polypeptide; and (b) recovering the
polypeptide.
20. A transgenic plant, plant part or plant cell, which has been
transformed with the nucleic acid construct of claim 1.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a divisional application of U.S. patent
application Ser. No. 13/912,915, now U.S. Pat. No. 8,975,059, which
is a divisional application of U.S. patent application Ser. No.
13/600,071, filed Aug. 30, 2012, which is a divisional application
of U.S. patent application Ser. No. 12/763,578, filed Apr. 20,
2010, now U.S. Pat. No. 8,268,606, which is a divisional
application of U.S. patent application Ser. No. 12/327,509, filed
Dec. 3, 2008, now U.S. Pat. No. 7,741,074, which claims the benefit
of U.S. Provisional Application No. 60/992,576, filed Dec. 5, 2007,
which applications are incorporated herein by reference.
REFERENCE TO A SEQUENCE LISTING
[0003] This application contains a Sequence Listing in computer
readable form. The computer readable form is incorporated herein by
reference.
REFERENCE TO DEPOSITS OF BIOLOGICAL MATERIAL
[0004] This application contains a reference to deposits of
biological material, which deposits are incorporated herein by
reference.
BACKGROUND OF THE INVENTION
[0005] 1. Field of the Invention
[0006] The present invention relates to isolated polypeptides
having endoglucanase activity and isolated polynucleotides encoding
the polypeptides. The invention also relates to nucleic acid
constructs, vectors, and host cells comprising the polynucleotides
as well as methods of producing and using the polypeptides.
[0007] 2. Description of the Related Art
[0008] Cellulose is a polymer of the simple sugar glucose linked by
beta-1,4-bonds. Many microorganisms produce enzymes that hydrolyze
beta-linked glucans. These enzymes include endoglucanases,
cellobiohydrolases, and beta-glucosidases. Endoglucanases digest
the cellulose polymer at random locations, opening it to attack by
cellobiohydrolases. Cellobiohydrolases sequentially release
molecules of cellobiose from the ends of the cellulose polymer.
Cellobiose is a water-soluble beta-1,4-linked dimer of glucose.
Beta-glucosidases hydrolyze cellobiose to glucose.
[0009] The conversion of lignocellulosic feedstocks into ethanol
has the advantages of the ready availability of large amounts of
feedstock, the desirability of avoiding burning or land filling the
materials, and the cleanliness of the ethanol fuel. Wood,
agricultural residues, herbaceous crops, and municipal solid wastes
have been considered as feedstocks for ethanol production. These
materials primarily consist of cellulose, hemicellulose, and
lignin. Once the cellulose is converted to glucose, the glucose can
be easily fermented by yeast into ethanol.
[0010] Kvesitadaze et al., 1995, Applied Biochemistry and
Biotechnology 50: 137-143, describe the isolation and properties of
a thermostable endoglucanase from a thermophilic mutant strain of
Thielavia terrestris. Gilbert et al., 1992, Bioresource Technology
39: 147-154, describe the characterization of the enzymes present
in the cellulase system of Thielavia terrestris 255B. Breuil et
al., 1986, Biotechnology Letters 8: 673-676, describe production
and localization of cellulases and beta-glucosidases from Thielavia
terrestris strains C464 and NRRL 8126.
[0011] The present invention relates to polypeptides having
endoglucanase activity and polynucleotides encoding the
polypeptides.
SUMMARY OF THE INVENTION
[0012] The present invention relates to isolated polypeptides
having endoglucanase activity selected from the group consisting
of:
[0013] (a) a polypeptide comprising an amino acid sequence having
at least 60% identity to the mature polypeptide of SEQ ID NO: 2 or
SEQ ID NO: 4;
[0014] (b) a polypeptide encoded by a polynucleotide that
hybridizes under at least medium stringency conditions with (i) the
mature polypeptide coding sequence of SEQ ID NO: 1 or SEQ ID NO: 3,
(ii) the genomic DNA sequence comprising the mature polypeptide
coding sequence of SEQ ID NO: 1 or SEQ ID NO: 3, or (iii) a
full-length complementary strand of (i) or (ii);
[0015] (c) a polypeptide encoded by a polynucleotide comprising a
nucleotide sequence having at least 60% identity to the mature
polypeptide coding sequence of SEQ ID NO: 1 or SEQ ID NO: 3;
and
[0016] (d) a variant comprising a substitution, deletion, and/or
insertion of one or more (several) amino acids of the mature
polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4.
[0017] The present invention also relates to isolated
polynucleotides encoding polypeptides having endoglucanase
activity, selected from the group consisting of:
[0018] (a) a polynucleotide encoding a polypeptide comprising an
amino acid sequence having at least 60% identity to the mature
polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4;
[0019] (b) a polynucleotide that hybridizes under at least medium
stringency conditions with (i) the mature polypeptide coding
sequence of SEQ ID NO: 1 or SEQ ID NO: 3, (ii) the genomic DNA
sequence comprising the mature polypeptide coding sequence of SEQ
ID NO: 1 or SEQ ID NO: 3, or (iii) a full-length complementary
strand of (i) or (ii);
[0020] (c) a polynucleotide comprising a nucleotide sequence having
at least 60% identity to the mature polypeptide coding sequence of
SEQ ID NO: 1 or SEQ ID NO: 3; and
[0021] (d) a polynucleotide encoding a variant comprising a
substitution, deletion, and/or insertion of one or more (several)
amino acids of the mature polypeptide of SEQ ID NO: 2 or SEQ ID NO:
4.
[0022] The present invention also relates to nucleic acid
constructs, recombinant expression vectors, recombinant host cells
comprising the polynucleotides, and methods of producing a
polypeptide having endoglucanase activity.
[0023] 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. The present also relates to a double-stranded inhibitory
RNA (dsRNA) molecule, wherein optionally the dsRNA is a siRNA or a
miRNA molecule.
[0024] The present invention also relates to methods of using the
polypeptides having endoglucanase activity in the degradation or
conversion of cellulosic material.
[0025] The present invention also relates to plants comprising an
isolated polynucleotide encoding a polypeptide having endoglucanase
activity.
[0026] The present invention also relates to methods of producing a
polypeptide having endoglucanase, comprising: (a) cultivating a
transgenic plant or a plant cell comprising a polynucleotide
encoding the polypeptide having endoglucanase activity under
conditions conducive for production of the polypeptide; and (b)
recovering the polypeptide.
[0027] The present invention further relates to nucleic acid
constructs comprising a gene encoding a protein, wherein the gene
is operably linked to a nucleotide sequence encoding a signal
peptide comprising or consisting of amino acids 1 to 20 of SEQ ID
NO: 2 or amino acids 1 to 18 of SEQ ID NO: 4, wherein the gene is
foreign to the nucleotide sequence.
BRIEF DESCRIPTION OF THE FIGURES
[0028] FIGS. 1A and 1B show the cDNA sequence and the deduced amino
acid sequence of a Thielavia terrestris NRRL 8126 CEL16A
endoglucanase (SEQ ID NOs: 1 and 2, respectively).
[0029] FIG. 2 shows the cDNA sequence and the deduced amino acid
sequence of a Thielavia terrestris NRRL 8126 CEL16B endoglucanase
(SEQ ID NOs: 3 and 4, respectively).
[0030] FIG. 3 shows a restriction map of pTter16A.
[0031] FIG. 4 shows a restriction map of pTter16B.
DEFINITIONS
[0032] Endoglucanase activity: The term "endoglucanase activity" is
defined herein as an endo-1,4-beta-D-glucan 4-glucanohydrolase
activity (E.C. No. 3.2.1.4) that catalyses the 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. For purposes of the present invention,
endoglucanase activity is determined using carboxymethyl cellulose
(CMC) hydrolysis according to the procedure of Ghose, 1987, Pure
and Appl. Chem. 59: 257-268. One unit of endoglucanase activity is
defined as 1.0 .mu.mole of reducing sugars produced per minute at
50.degree. C., pH 4.8.
[0033] The polypeptides of the present invention have at least 20%,
preferably at least 40%, more preferably at least 50%, more
preferably at least 60%, more preferably at least 70%, more
preferably at least 80%, even more preferably at least 90%, most
preferably at least 95%, and even most preferably at least 100% of
the endoglucanase activity of the mature polypeptide of SEQ ID NO:
2 or SEQ ID NO: 4.
[0034] Family 16 or Family GH16 or CEL16: The term "Family 16" or
"Family GH16" or "CEL16" is defined herein as a polypeptide falling
into the glycoside hydrolase Family 16 according to Henrissat B.,
1991, A classification of glycosyl hydrolases based on amino-acid
sequence similarities, Biochem. J. 280: 309-316, and Henrissat and
Bairoch, 1996, Updating the sequence-based classification of
glycosyl hydrolases, Biochem. J. 316: 695-696.
[0035] Cellulolytic activity: The term "cellulolytic activity" is
defined herein as a biological activity that hydrolyzes a
cellulosic material. Cellulolytic protein may hydrolyze or
hydrolyzes carboxymethyl cellulose (CMC), thereby decreasing the
viscosity of the incubation mixture. The resulting reduction in
viscosity may be determined by a vibration viscosimeter (e.g., MIVI
3000 from Sofraser, France). Determination of cellulase activity,
measured in terms of Cellulase Viscosity Unit (CEVU), quantifies
the amount of catalytic activity present in a sample by measuring
the ability of the sample to reduce the viscosity of a solution of
carboxymethyl cellulose (CMC). The assay is performed at the
temperature and pH suitable for the cellulolytic protein and
substrate. For CELLUCLAST.TM. (Trichoderma reesei cellulase;
Novozymes A/S, Bagsv.ae butted.rd, Denmark) the assay is carried
out at 40.degree. C. in 0.1 M phosphate pH 9.0 buffer for 30
minutes with CMC as substrate (33.3 g/L carboxymethyl cellulose
Hercules 7 LFD) and an enzyme concentration of approximately
3.3-4.2 CEVU/ml. The CEVU activity is calculated relative to a
declared enzyme standard, such as CELLUZYME.TM. Standard 17-1194
(obtained from Novozymes A/S, Bagsv.ae butted.rd, Denmark).
[0036] For purposes of the present invention, cellulolytic activity
can be determined by measuring the increase in hydrolysis of a
cellulosic material by a cellulolytic mixture under the following
conditions: 1-10 mg of cellulolytic protein/g of cellulose in PCS
for 5-7 day at 50.degree. C. compared to a control hydrolysis
without addition of cellulolytic protein.
[0037] Cellobiohydrolase: The term "cellobiohydrolase" is defined
herein as a 1,4-beta-D-glucan cellobiohydrolase (E.C. 3.2.1.91),
which 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. For purposes of the present
invention, cellobiohydrolase activity is determined according to
the procedures described by Lever et al., 1972, Anal. Biochem. 47:
273-279 and by van Tilbeurgh et al., 1982, FEBS Letters 149:
152-156; van Tilbeurgh and Claeyssens, 1985, FEBS Letters 187:
283-288. In the present invention, the Lever et al. method can be
employed to assess hydrolysis of cellulose in corn stover, while
the method of van Tilbeurgh et al. can be used to determine
cellobiohydrolase activity on a fluorescent disaccharide
derivative.
[0038] Beta-glucosidase: The term "beta-glucosidase" is defined
herein as a beta-D-glucoside glucohydrolase (E.C. 3.2.1.21), which
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
according to the basic procedure described by Venturi et al., 2002,
J. Basic Microbiol. 42: 55-66, except different conditions were
employed as described herein. One unit of beta-glucosidase activity
is defined as 1.0 .mu.mole of p-nitrophenol produced per minute at
50.degree. C., pH 5 from 4 mM p-nitrophenyl-beta-D-glucopyranoside
as substrate in 100 mM sodium citrate, 0.01% TWEEN.RTM. 20.
[0039] Cellulosic material: The predominant polysaccharide in the
primary cell wall of biomass is cellulose, the second most abundant
is hemi-cellulose, 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.
[0040] The cellulosic material can be any material containing
cellulose. 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, herbaceous material, agricultural residue, forestry residue,
municipal solid waste, waste paper, and pulp and paper mill residue
The cellulosic material can be any type of biomass including, but
not limited to, wood resources, municipal solid waste, wastepaper,
crops, and crop residues (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.
[0041] In one aspect, the cellulosic material is herbaceous
material. In another aspect, the cellulosic material is
agricultural residue. In another aspect, the cellulosic material is
forestry residue. In another aspect, the cellulosic material is
municipal solid waste. In another aspect, the cellulosic material
is waste paper. In another aspect, the cellulosic material is pulp
and paper mill residue.
[0042] In another aspect, the cellulosic material is corn stover.
In another preferred aspect, the cellulosic material is corn fiber.
In another aspect, the cellulosic material is corn cob. 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 wheat straw. In another aspect, the
cellulosic material is switch grass. In another aspect, the
cellulosic material is miscanthus. In another aspect, the
cellulosic material is bagasse.
[0043] The cellulosic material may be used as is or may be
subjected to pretreatment, using conventional methods known in the
art. For example, physical pretreatment techniques can include
various types of milling, irradiation, steaming/steam explosion,
and hydrothermolysis; chemical pretreatment techniques can include
dilute acid, alkaline, organic solvent, ammonia, sulfur dioxide,
carbon dioxide, and pH-controlled hydrothermolysis; and biological
pretreatment techniques can involve applying lignin-solubilizing
microorganisms (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, P., and Singh, A., 1993, Physicochemical and biological
treatments for enzymatic/microbial conversion of lignocellulosic
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, L., and Hahn-Hagerdal,
B., 1996, Fermentation of lignocellulosic hydrolysates for ethanol
production, Enz. Microb. Tech. 18: 312-331; and Vallander, L., and
Eriksson, K.-E. L., 1990, Production of ethanol from
lignocellulosic materials: State of the art, Adv. Biochem.
Eng./Biotechnol. 42: 63-95).
[0044] Pretreated corn stover: The term "PCS" or "Pretreated Corn
Stover" is defined herein as a cellulosic material derived from
corn stover by treatment with heat and dilute acid.
[0045] Isolated polypeptide: The term "isolated polypeptide" as
used herein refers to a polypeptide that is isolated from a source.
In a preferred aspect, the polypeptide is at least 1% pure,
preferably at least 5% pure, more preferably at least 10% pure,
more preferably at least 20% pure, more preferably at least 40%
pure, more preferably at least 60% pure, even more preferably at
least 80% pure, and most preferably at least 90% pure, as
determined by SDS-PAGE.
[0046] Substantially pure polypeptide: The term "substantially pure
polypeptide" denotes herein a polypeptide preparation that contains
at most 10%, preferably at most 8%, more preferably at most 6%,
more preferably at most 5%, more preferably at most 4%, more
preferably at most 3%, even more preferably at most 2%, most
preferably at most 1%, and even most preferably at most 0.5% by
weight of other polypeptide material with which it is natively or
recombinantly associated. It is, therefore, preferred that the
substantially pure polypeptide is at least 92% pure, preferably at
least 94% pure, more preferably at least 95% pure, more preferably
at least 96% pure, more preferably at least 97% pure, more
preferably at least 98% pure, even more preferably at least 99%
pure, most preferably at least 99.5% pure, and even most preferably
100% pure by weight of the total polypeptide material present in
the preparation. The polypeptides of the present invention are
preferably in a substantially pure form, i.e., that the polypeptide
preparation is essentially free of other polypeptide material with
which it is natively or recombinantly associated. This can be
accomplished, for example, by preparing the polypeptide by
well-known recombinant methods or by classical purification
methods.
[0047] Mature polypeptide: The term "mature polypeptide" is defined
herein as 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 a preferred aspect, the mature polypeptide is amino acids
21 to 797 of SEQ ID NO: 2 based on the SignalP software program
(Nielsen et al., 1997, Protein Engineering 10: 1-6) that predicts
amino acids 1 to 20 of SEQ ID NO: 2 are a signal peptide. In
another preferred aspect, the mature polypeptide is amino acids 19
to 285 of SEQ ID NO: 4 based on the SignalP software program that
predicts amino acids 1 to 18 of SEQ ID NO: 4 are a signal
peptide.
[0048] Mature polypeptide coding sequence: The term "mature
polypeptide coding sequence" is defined herein as a nucleotide
sequence that encodes a mature polypeptide having endoglucanase
activity. In a preferred aspect, the mature polypeptide coding
sequence is nucleotides 61 to 1428 of SEQ ID NO: 1 based on the
SignalP software program that predicts nucleotides 1 to 60 encode a
signal peptide. In another preferred aspect, the mature polypeptide
coding sequence is nucleotides 55 to 855 of SEQ ID NO: 3 based on
the SignalP software program that predicts nucleotides 1 to 54
encode a signal peptide.
[0049] Identity: The relatedness between two amino acid sequences
or between two nucleotide sequences is described by the parameter
"identity".
[0050] For purposes of the present invention, the degree of
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 in Genetics 16: 276-277),
preferably version 3.0.0 or later. The optional 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)
[0051] For purposes of the present invention, the degree of
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 3.0.0 or later. The
optional 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)
[0052] Homologous sequence: The term "homologous sequence" is
defined herein as a predicted protein that has an E value (or
expectancy score) of less than 0.001 in a tfasty search (Pearson,
W. R., 1999, in Bioinformatics Methods and Protocols, S. Misener
and S. A. Krawetz, ed., pp. 185-219) with the Thielavia terrestris
endoglucanase of SEQ ID NO: 2 or SEQ ID NO: 4, or the mature
polypeptides thereof.
[0053] Polypeptide fragment: The term "polypeptide fragment" is
defined herein as a polypeptide having one or more (several) amino
acids deleted from the amino and/or carboxyl terminus of the mature
polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4; or a homologous
sequence thereof; wherein the fragment has endoglucanase activity.
In a preferred aspect, a fragment contains at least 660 amino acid
residues, more preferably at least 700 amino acid residues, and
most preferably at least 740 amino acid residues, of the mature
polypeptide of SEQ ID NO: 2 or a homologous sequence thereof. In
another preferred aspect, a fragment contains at least 225 amino
acid residues, more preferably at least 240 amino acid residues,
and most preferably at least 255 amino acid residues, of the mature
polypeptide of SEQ ID NO: 4 or a homologous sequence thereof.
[0054] Subsequence: The term "subsequence" is defined herein as a
nucleotide sequence having one or more (several) nucleotides
deleted from the 5' and/or 3' end of the mature polypeptide coding
sequence of SEQ ID NO: 1; or a homologous sequence thereof; wherein
the subsequence encodes a polypeptide fragment having endoglucanase
activity. In a preferred aspect, a subsequence contains at least
1980 nucleotides, more preferably at least 2100 nucleotides, and
most preferably at least 2220 nucleotides of the mature polypeptide
coding sequence of SEQ ID NO: 1 or a homologous sequence thereof.
In another preferred aspect, a subsequence contains at least 675
nucleotides, more preferably at least 720 nucleotides, and most
preferably at least 765 nucleotides of the mature polypeptide
coding sequence of SEQ ID NO: 3 or a homologous sequence
thereof.
[0055] Allelic variant: The term "allelic variant" denotes herein
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.
[0056] Isolated polynucleotide: The term "isolated polynucleotide"
as used herein refers to a polynucleotide that is isolated from a
source. In a preferred aspect, the polynucleotide is at least 1%
pure, preferably at least 5% pure, more preferably at least 10%
pure, more preferably at least 20% pure, more preferably at least
40% pure, more preferably at least 60% pure, even more preferably
at least 80% pure, and most preferably at least 90% pure, as
determined by agarose electrophoresis.
[0057] Substantially pure polynucleotide: The term "substantially
pure polynucleotide" as used herein refers to a polynucleotide
preparation free of other extraneous or unwanted nucleotides and in
a form suitable for use within genetically engineered protein
production systems. Thus, a substantially pure polynucleotide
contains at most 10%, preferably at most 8%, more preferably at
most 6%, more preferably at most 5%, more preferably at most 4%,
more preferably at most 3%, even more preferably at most 2%, most
preferably at most 1%, and even most preferably at most 0.5% by
weight of other polynucleotide material with which it is natively
or recombinantly associated. A substantially pure polynucleotide
may, however, include naturally occurring 5' and 3' untranslated
regions, such as promoters and terminators. It is preferred that
the substantially pure polynucleotide is at least 90% pure,
preferably at least 92% pure, more preferably at least 94% pure,
more preferably at least 95% pure, more preferably at least 96%
pure, more preferably at least 97% pure, even more preferably at
least 98% pure, most preferably at least 99% pure, and even most
preferably at least 99.5% pure by weight. The polynucleotides of
the present invention are preferably in a substantially pure form,
i.e., that the polynucleotide preparation is essentially free of
other polynucleotide material with which it is natively or
recombinantly associated. The polynucleotides may be of genomic,
cDNA, RNA, semisynthetic, synthetic origin, or any combinations
thereof.
[0058] Coding sequence: When used herein the term "coding sequence"
means a nucleotide sequence, which directly specifies the amino
acid sequence of its protein product. The boundaries of the coding
sequence are generally determined by an open reading frame, which
usually begins with the ATG start codon or alternative start codons
such as GTG and TTG and ends with a stop codon such as TAA, TAG,
and TGA. The coding sequence may be a DNA, cDNA, synthetic, or
recombinant nucleotide sequence.
[0059] cDNA: The term "cDNA" is defined herein as a DNA molecule
that can be prepared by reverse transcription from a mature,
spliced, mRNA molecule obtained from a eukaryotic 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 before appearing as
mature spliced mRNA. These steps include the removal of intron
sequences by a process called splicing. cDNA derived from mRNA
lacks, therefore, any intron sequences.
[0060] Nucleic acid construct: The term "nucleic acid construct" as
used herein refers to 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 is synthetic. The term
nucleic acid construct is synonymous with the term "expression
cassette" when the nucleic acid construct contains the control
sequences required for expression of a coding sequence of the
present invention.
[0061] Control sequences: The term "control sequences" is defined
herein to include all components, which are necessary or
advantageous for the expression of a polynucleotide encoding a
polypeptide of the present invention. Each control sequence may be
native or foreign to the nucleotide sequence 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 nucleotide sequence encoding a
polypeptide.
[0062] Operably linked: The term "operably linked" denotes herein a
configuration in which a control sequence is placed at an
appropriate position relative to the coding sequence of the
polynucleotide sequence such that the control sequence directs the
expression of the coding sequence of a polypeptide.
[0063] Expression: The term "expression" includes any step involved
in the production of the polypeptide including, but not limited to,
transcription, post-transcriptional modification, translation,
post-translational modification, and secretion.
[0064] Expression vector: The term "expression vector" is defined
herein as a linear or circular DNA molecule that comprises a
polynucleotide encoding a polypeptide of the present invention and
is operably linked to additional nucleotides that provide for its
expression.
[0065] Host cell: The term "host cell", as used herein, includes
any cell type that is susceptible to transformation, transfection,
transduction, and the like with a nucleic acid construct or
expression vector comprising a polynucleotide of the present
invention.
[0066] Modification: The term "modification" means herein any
chemical modification of the polypeptide consisting of the mature
polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4; or a homologous
sequence thereof; as well as genetic manipulation of the DNA
encoding such a polypeptide. The modification can be a
substitution, a deletion and/or an insertion of one or more
(several) amino acids as well as replacements of one or more
(several) amino acid side chains.
[0067] Artificial variant: When used herein, the term "artificial
variant" means a polypeptide having endoglucanase activity produced
by an organism expressing a modified polynucleotide sequence of the
mature polypeptide coding sequence of SEQ ID NO: 1 or SEQ ID NO: 3;
or a homologous sequence thereof. The modified nucleotide sequence
is obtained through human intervention by modification of the
polynucleotide sequence disclosed in SEQ ID NO: 1 or SEQ ID NO: 3;
or a homologous sequence thereof.
DETAILED DESCRIPTION OF THE INVENTION
Polypeptides Having Endoglucanase Activity
[0068] In a first aspect, the present invention relates to isolated
polypeptides comprising an amino acid sequence having a degree of
identity to the mature polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4
of preferably at least 60%, more preferably at least 65%, more
preferably at least 70%, more preferably at least 75%, more
preferably at least 80%, more preferably at least 85%, even more
preferably at least 90%, most preferably at least 95%, and even
most preferably at least 96%, at least 97%, at least 98%, or at
least 99%, which have endoglucanase activity (hereinafter
"homologous polypeptides"). In a preferred aspect, the homologous
polypeptides have an amino acid sequence that differs by ten amino
acids, preferably by five amino acids, more preferably by four
amino acids, even more preferably by three amino acids, most
preferably by two amino acids, and even most preferably by one
amino acid from the mature polypeptide of SEQ ID NO: 2 or SEQ ID
NO: 4.
[0069] A polypeptide of the present invention preferably comprises
the amino acid sequence of SEQ ID NO: 2 or an allelic variant
thereof; or a fragment thereof having endoglucanase activity. In a
preferred aspect, the polypeptide comprises the amino acid sequence
of SEQ ID NO: 2. In another preferred aspect, the polypeptide
comprises the mature polypeptide of SEQ ID NO: 2. In another
preferred aspect, the polypeptide comprises amino acids 21 to 797
of SEQ ID NO: 2, or an allelic variant thereof; or a fragment
thereof having endoglucanase activity. In another preferred aspect,
the polypeptide comprises amino acids 21 to 797 of SEQ ID NO: 2. In
another preferred aspect, the polypeptide consists of the amino
acid sequence of SEQ ID NO: 2 or an allelic variant thereof; or a
fragment thereof having endoglucanase activity. In another
preferred aspect, the polypeptide consists of the amino acid
sequence of SEQ ID NO: 2. In another preferred aspect, the
polypeptide consists of the mature polypeptide of SEQ ID NO: 2. In
another preferred aspect, the polypeptide consists of amino acids
21 to 797 of SEQ ID NO: 2 or an allelic variant thereof; or a
fragment thereof having endoglucanase activity. In another
preferred aspect, the polypeptide consists of amino acids 21 to 797
of SEQ ID NO: 2.
[0070] A polypeptide of the present invention also preferably
comprises the amino acid sequence of SEQ ID NO: 4 or an allelic
variant thereof; or a fragment thereof having endoglucanase
activity. In a preferred aspect, the polypeptide comprises the
amino acid sequence of SEQ ID NO: 4. In another preferred aspect,
the polypeptide comprises the mature polypeptide of SEQ ID NO: 4.
In another preferred aspect, the polypeptide comprises amino acids
19 to 285 of SEQ ID NO: 4, or an allelic variant thereof; or a
fragment thereof having endoglucanase activity. In another
preferred aspect, the polypeptide comprises amino acids 19 to 285
of SEQ ID NO: 4. In another preferred aspect, the polypeptide
consists of the amino acid sequence of SEQ ID NO: 4 or an allelic
variant thereof; or a fragment thereof having endoglucanase
activity. In another preferred aspect, the polypeptide consists of
the amino acid sequence of SEQ ID NO: 4. In another preferred
aspect, the polypeptide consists of the mature polypeptide of SEQ
ID NO: 4. In another preferred aspect, the polypeptide consists of
amino acids 19 to 285 of SEQ ID NO: 4 or an allelic variant
thereof; or a fragment thereof having endoglucanase activity. In
another preferred aspect, the polypeptide consists of amino acids
19 to 285 of SEQ ID NO: 4.
[0071] In a second aspect, the present invention relates to
isolated polypeptides having endoglucanase activity that are
encoded by polynucleotides that hybridize under preferably very low
stringency conditions, more preferably low stringency conditions,
more preferably medium stringency conditions, more preferably
medium-high stringency conditions, even more preferably high
stringency conditions, and most preferably very high stringency
conditions with (i) the mature polypeptide coding sequence of SEQ
ID NO: 1 or SEQ ID NO: 3, (ii) the genomic DNA sequence comprising
the mature polypeptide coding sequence of SEQ ID NO: 1 or SEQ ID
NO: 3, (iii) a subsequence of (i) or (ii), or (iv) a full-length
complementary strand of (i), (ii), or (iii) (J. Sambrook, E. F.
Fritsch, and T. Maniatis, 1989, Molecular Cloning, A Laboratory
Manual, 2d edition, Cold Spring Harbor, New York). A subsequence of
the mature polypeptide coding sequence of SEQ ID NO: 1 or SEQ ID
NO: 3 contains at least 100 contiguous nucleotides or preferably at
least 200 contiguous nucleotides. Moreover, the subsequence may
encode a polypeptide fragment having endoglucanase activity. In a
preferred aspect, the complementary strand is the full-length
complementary strand of the mature polypeptide coding sequence of
SEQ ID NO: 1 or SEQ ID NO: 3.
[0072] The nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3; or
a subsequence thereof; as well as the amino acid sequence of SEQ ID
NO: 2 or SEQ ID NO: 4; 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 or cDNA
of the genus or species 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 14, preferably at
least 25, more preferably at least 35, and most preferably at least
70 nucleotides in length. It is, however, preferred that the
nucleic acid probe is at least 100 nucleotides in length. For
example, the nucleic acid probe may be at least 200 nucleotides,
preferably at least 300 nucleotides, more preferably at least 400
nucleotides, or most preferably at least 500 nucleotides in length.
Even longer probes may be used, e.g., nucleic acid probes that are
preferably at least 600 nucleotides, more preferably at least 700
nucleotides, or most preferably at least 800 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.
[0073] A genomic DNA or cDNA library prepared from such other
strains may, therefore, 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 is homologous with SEQ ID
NO: 1 or SEQ ID NO: 3; or a subsequence thereof; the carrier
material is preferably used in a Southern blot.
[0074] For purposes of the present invention, hybridization
indicates that the nucleotide sequence hybridizes to a labeled
nucleic acid probe corresponding to the mature polypeptide coding
sequence of SEQ ID NO: 1 or SEQ ID NO: 3; the genomic DNA sequence
comprising the mature polypeptide coding sequence of SEQ ID NO: 1
or SEQ ID NO: 3; its full-length complementary strand; or 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.
[0075] In a preferred aspect, the nucleic acid probe is the mature
polypeptide coding sequence of SEQ ID NO: 1. In another preferred
aspect, the nucleic acid probe is nucleotides 61 to 1428 of SEQ ID
NO: 1. In another preferred aspect, the nucleic acid probe is a
polynucleotide sequence that encodes the polypeptide of SEQ ID NO:
2, or a subsequence thereof. In another preferred aspect, the
nucleic acid probe is SEQ ID NO: 1. In another preferred aspect,
the nucleic acid probe is the polynucleotide sequence contained in
plasmid pTter16A which is contained in E. coli NRRL B-50081,
wherein the polynucleotide sequence thereof encodes a polypeptide
having endoglucanase activity. In another preferred aspect, the
nucleic acid probe is the mature polypeptide coding region
contained in plasmid pTter16A which is contained in E. coli NRRL
B-50081.
[0076] In another preferred aspect, the nucleic acid probe is the
mature polypeptide coding sequence of SEQ ID NO: 3. In another
preferred aspect, the nucleic acid probe is nucleotides 55 to 855
of SEQ ID NO: 3. In another preferred aspect, the nucleic acid
probe is a polynucleotide sequence that encodes the polypeptide of
SEQ ID NO: 4, or a subsequence thereof. In another preferred
aspect, the nucleic acid probe is SEQ ID NO: 3. In another
preferred aspect, the nucleic acid probe is the polynucleotide
sequence contained in plasmid pTter16B which is contained in E.
coli NRRL B-50082, wherein the polynucleotide sequence thereof
encodes a polypeptide having endoglucanase activity. In another
preferred aspect, the nucleic acid probe is the mature polypeptide
coding region contained in plasmid pTter16B which is contained in
E. coli NRRL B-50082.
[0077] For long probes of at least 100 nucleotides in length, very
low to very high stringency conditions are defined as
prehybridization and hybridization at 42.degree. C. in
5.times.SSPE, 0.3% SDS, 200 .mu.g/ml sheared and denatured salmon
sperm DNA, and either 25% formamide for very low and low
stringencies, 35% formamide for medium and medium-high
stringencies, or 50% formamide for high and very high stringencies,
following standard Southern blotting procedures for 12 to 24 hours
optimally.
[0078] For long probes of at least 100 nucleotides in length, the
carrier material is finally washed three times each for 15 minutes
using 2.times.SSC, 0.2% SDS preferably at 45.degree. C. (very low
stringency), more preferably at 50.degree. C. (low stringency),
more preferably at 55.degree. C. (medium stringency), more
preferably at 60.degree. C. (medium-high stringency), even more
preferably at 65.degree. C. (high stringency), and most preferably
at 70.degree. C. (very high stringency).
[0079] For short probes of about 15 nucleotides to about 70
nucleotides in length, stringency conditions are defined as
prehybridization, hybridization, and washing post-hybridization at
about 5.degree. C. to about 10.degree. C. below the calculated
T.sub.m using the calculation according to Bolton and McCarthy
(1962, Proceedings of the National Academy of Sciences USA 48:1390)
in 0.9 M NaCl, 0.09 M Tris-HCl pH 7.6, 6 mM EDTA, 0.5% NP-40,
1.times.Denhardt's solution, 1 mM sodium pyrophosphate, 1 mM sodium
monobasic phosphate, 0.1 mM ATP, and 0.2 mg of yeast RNA per ml
following standard Southern blotting procedures for 12 to 24 hours
optimally. For short probes of about 15 nucleotides to about 70
nucleotides in length, the carrier material is washed once in
6.times.SCC plus 0.1% SDS for 15 minutes and twice each for 15
minutes using 6.times.SSC at 5.degree. C. to 10.degree. C. below
the calculated T.sub.m.
[0080] In a third aspect, the present invention relates to isolated
polypeptides having endoglucanase activity encoded by
polynucleotides comprising or consisting of nucleotide sequences
that have a degree of identity to the mature polypeptide coding
sequence of SEQ ID NO: 1 or SEQ ID NO: 3 of preferably at least
60%, more preferably at least 65%, more preferably at least 70%,
more preferably at least 75%, more preferably at least 80%, more
preferably at least 85%, even more preferably at least 90%, most
preferably at least 95%, and even most preferably at least 96%, at
least 97%, at least 98%, or at least 99%, which encode a
polypeptide having endoglucanase activity. See polynucleotide
section herein.
[0081] In a fourth aspect, the present invention relates to
artificial variants comprising a substitution, deletion, and/or
insertion of one or more (or several) amino acids of the mature
polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4; or a homologous
sequence thereof. Preferably, amino acid changes are 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 one to about 30 amino acids;
small amino- or carboxyl-terminal extensions, such as an
amino-terminal methionine residue; a small linker peptide of up to
about 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.
[0082] Examples of conservative substitutions are within the group
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. The most commonly occurring
exchanges 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.
[0083] In addition to the 20 standard amino acids, non-standard
amino acids (such as 4-hydroxyproline, 6-N-methyl lysine,
2-aminoisobutyric acid, isovaline, and alpha-methyl serine) may be
substituted for amino acid residues of a wild-type polypeptide. A
limited number of non-conservative amino acids, amino acids that
are not encoded by the genetic code, and unnatural amino acids may
be substituted for amino acid residues. "Unnatural amino acids"
have been modified after protein synthesis, and/or have a chemical
structure in their side chain(s) different from that of the
standard amino acids. Unnatural amino acids can be chemically
synthesized, and preferably, are commercially available, and
include pipecolic acid, thiazolidine carboxylic acid,
dehydroproline, 3- and 4-methylproline, and
3,3-dimethylproline.
[0084] 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.
[0085] Essential amino acids in the parent 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
biological activity (i.e., 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 identities of essential
amino acids can also be inferred from analysis of identities with
polypeptides that are related to a polypeptide according to the
invention.
[0086] 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, Biochem. 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).
[0087] 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 of interest, and
can be applied to polypeptides of unknown structure.
[0088] The total number of amino acid substitutions, deletions
and/or insertions of the mature polypeptide of SEQ ID NO: 2 or SEQ
ID NO: 4 is 10, preferably 9, more preferably 8, more preferably 7,
more preferably at most 6, more preferably 5, more preferably 4,
even more preferably 3, most preferably 2, and even most preferably
1.
Sources of Polypeptides Having Endoglucanase Activity
[0089] A polypeptide 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 nucleotide
sequence is produced by the source or by a strain in which the
nucleotide sequence from the source has been inserted. In a
preferred aspect, the polypeptide obtained from a given source is
secreted extracellularly.
[0090] A polypeptide having endoglucanase activity of the present
invention 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,
or Oceanobacillus polypeptide having endoglucanase activity, or a
Gram negative bacterial polypeptide such as an E. coli,
Pseudomonas, Salmonella, Campylobacter, Helicobacter,
Flavobacterium, Fusobacterium, Ilyobacter, Neisseria, or Ureaplasma
polypeptide having endoglucanase activity.
[0091] In a preferred 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
endoglucanase activity.
[0092] In another preferred aspect, the polypeptide is a
Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus
uberis, or Streptococcus equi subsp. Zooepidemicus polypeptide
having endoglucanase activity.
[0093] In another preferred aspect, the polypeptide is a
Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces
coelicolor, Streptomyces griseus, or Streptomyces lividans
polypeptide having endoglucanase activity.
[0094] A polypeptide having endoglucanase activity of the present
invention may also be a fungal polypeptide, and more preferably a
yeast polypeptide such as a Candida, Kluyveromyces, Pichia,
Saccharomyces, Schizosaccharomyces, or Yarrowia polypeptide having
endoglucanase activity; or more preferably a filamentous fungal
polypeptide such as an Acremonium, Agaricus, Alternaria,
Aspergillus, Aureobasidium, Botryosphaeria, Ceriporiopsis,
Chaetomidium, Chrysosporium, Claviceps, Cochliobolus, Coprinopsis,
Coptotermes, Corynascus, Cryphonectria, Cryptococcus, Diplodia,
Exidia, Filibasidium, Fusarium, Gibberella, Holomastigotoides,
Humicola, Irpex, Lentinula, Leptospaeria, Magnaporthe,
Melanocarpus, Meripilus, Mucor, Myceliophthora, Neocallimastix,
Neurospora, Paecilomyces, Penicillium, Phanerochaete, Piromyces,
Poitrasia, Pseudoplectania, Pseudotrichonympha, Rhizomucor,
Schizophyllum, Scytalidium, Talaromyces, Thermoascus, Thielavia,
Tolypocladium, Trichoderma, Trichophaea, Verticillium, Volvariella,
or Xylaria polypeptide having endoglucanase activity.
[0095] In a preferred aspect, the polypeptide is a Saccharomyces
carlsbergensis, Saccharomyces cerevisiae, Saccharomyces
diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri,
Saccharomyces norbensis, or Saccharomyces oviformis polypeptide
having endoglucanase activity.
[0096] In another preferred 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, Trichoderma
harzianum, Trichoderma koningii, Trichoderma longibrachiatum,
Trichoderma reesei, or Trichoderma viride polypeptide having
endoglucanase activity.
[0097] In another preferred aspect, the polypeptide is a Thielavia
achromatica, Thielavia albomyces, Thielavia albopilosa, Thielavia
australeinsis, Thielavia fimeti, Thielavia microspora, Thielavia
ovispora, Thielavia peruviana, Thielavia spededonium, Thielavia
setosa, Thielavia subthermophila, or Thielavia terrestris
polypeptide.
[0098] In a more preferred aspect, the polypeptide is a Thielavia
terrestris polypeptide having endoglucanase activity. In a most
preferred aspect, the polypeptide is a Thielavia terrestris NRRL
8126 polypeptide having endoglucanase activity, e.g., the
polypeptide comprising the mature polypeptide of SEQ ID NO: 2 or
SEQ ID NO: 4.
[0099] 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.
[0100] 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 (DSM), Centraalbureau Voor
Schimmelcultures (CBS), and Agricultural Research Service Patent
Culture Collection, Northern Regional Research Center (NRRL).
[0101] Furthermore, such polypeptides may be identified and
obtained from other sources including microorganisms isolated from
nature (e.g., soil, composts, water, etc.) using the
above-mentioned probes. Techniques for isolating microorganisms
from natural habitats are well known in the art. The polynucleotide
may then be obtained by similarly screening a genomic or cDNA
library of such a microorganism. Once a polynucleotide sequence
encoding a polypeptide has been detected with the probe(s), the
polynucleotide can be isolated or cloned by utilizing techniques
that are well known to those of ordinary skill in the art (see,
e.g., Sambrook et al., 1989, supra).
[0102] Polypeptides of the present invention also include fused
polypeptides or cleavable fusion polypeptides in which another
polypeptide is fused at the N-terminus or the C-terminus of the
polypeptide or fragment thereof. A fused polypeptide is produced by
fusing a nucleotide sequence (or a portion thereof) encoding
another polypeptide to a nucleotide sequence (or a portion thereof)
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 fused polypeptide is under control of the
same promoter(s) and terminator.
[0103] A fusion polypeptide can further comprise a cleavage site.
Upon secretion of the fusion protein, the site is cleaved releasing
the polypeptide having endoglucanase activity from the fusion
protein. Examples of cleavage sites include, but are not limited
to, a Kex2 site that encodes the dipeptide Lys-Arg (Martin et al.,
2003, J. Ind. Microbiol. Biotechnol. 3: 568-76; 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), an Ile-(Glu or Asp)-Gly-Arg site, which
is cleaved by a Factor Xa protease after the arginine residue
(Eaton et al., 1986, Biochem. 25: 505-512); a Asp-Asp-Asp-Asp-Lys
site, which is cleaved by an enterokinase after the lysine
(Collins-Racie et al., 1995, Biotechnology 13: 982-987); a
His-Tyr-Glu site or His-Tyr-Asp site, which is cleaved by Genenase
I (Carter et al., 1989, Proteins: Structure, Function, and Genetics
6: 240-248); a Leu-Val-Pro-Arg-Gly-Ser site, which is cleaved by
thrombin after the Arg (Stevens, 2003, Drug Discovery World 4:
35-48); a Glu-Asn-Leu-Tyr-Phe-Gln-Gly site, which is cleaved by TEV
protease after the Gln (Stevens, 2003, supra); and a
Leu-Glu-Val-Leu-Phe-Gln-Gly-Pro site, which is cleaved by a
genetically engineered form of human rhinovirus 3C protease after
the Gln (Stevens, 2003, supra).
Polynucleotides
[0104] The present invention also relates to isolated
polynucleotides comprising or consisting of nucleotide sequences
that encode polypeptides having endoglucanase activity of the
present invention.
[0105] In a preferred aspect, the nucleotide sequence comprises or
consists of SEQ ID NO: 1. In another more preferred aspect, the
nucleotide sequence comprises or consists of the sequence contained
in plasmid pTter16A which is contained in E. coli NRRL B-50081. In
another preferred aspect, the nucleotide sequence comprises or
consists of the mature polypeptide coding sequence of SEQ ID NO: 1.
In another preferred aspect, the nucleotide sequence comprises or
consists of nucleotides 61 to 1428 of SEQ ID NO: 1. In another more
preferred aspect, the nucleotide sequence comprises or consists of
the mature polypeptide coding sequence contained in plasmid
pTter16A which is contained in E. coli NRRL B-50081. The present
invention also encompasses nucleotide sequences that encode
polypeptides comprising or consisting of the amino acid sequence of
SEQ ID NO: 2 or the mature polypeptide thereof, which differ from
SEQ ID NO: 1 or the mature polypeptide coding sequence thereof by
virtue of the degeneracy of the genetic code. The present invention
also relates to subsequences of SEQ ID NO: 1 that encode fragments
of SEQ ID NO: 2 that have endoglucanase activity.
[0106] In another preferred aspect, the nucleotide sequence
comprises or consists of SEQ ID NO: 3. In another more preferred
aspect, the nucleotide sequence comprises or consists of the
sequence contained in plasmid pTter16B which is contained in E.
coli NRRL B-50082. In another preferred aspect, the nucleotide
sequence comprises or consists of the mature polypeptide coding
sequence of SEQ ID NO: 3. In another preferred aspect, the
nucleotide sequence comprises or consists of nucleotides 55 to 855
of SEQ ID NO: 3. In another more preferred aspect, the nucleotide
sequence comprises or consists of the mature polypeptide coding
sequence contained in plasmid pTter16B which is contained in E.
coli NRRL B-50082. The present invention also encompasses
nucleotide sequences that encode polypeptides comprising or
consisting of the amino acid sequence of SEQ ID NO: 4 or the mature
polypeptide thereof, which differ from SEQ ID NO: 3 or the mature
polypeptide coding sequence thereof by virtue of the degeneracy of
the genetic code. The present invention also relates to
subsequences of SEQ ID NO: 3 that encode fragments of SEQ ID NO: 4
that have endoglucanase activity.
[0107] The present invention also relates to mutant polynucleotides
comprising or consisting of at least one mutation in the mature
polypeptide coding sequence of SEQ ID NO: 1 or SEQ ID NO: 3, in
which the mutant nucleotide sequence encodes the mature polypeptide
of SEQ ID NO: 2 or SEQ ID NO: 4, respectively.
[0108] The techniques used to isolate or clone a polynucleotide
encoding a polypeptide are known in the art and include isolation
from genomic DNA, preparation from cDNA, or a combination thereof.
The cloning of the polynucleotides of the present invention from
such 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),
ligated activated transcription (LAT) and nucleotide sequence-based
amplification (NASBA) may be used. The polynucleotides may be
cloned from a strain of Thielavia, or another or related organism
and thus, for example, may be an allelic or species variant of the
polypeptide encoding region of the nucleotide sequence.
[0109] The present invention also relates to isolated
polynucleotides comprising or consisting of nucleotide sequences
that have a degree of identity to the mature polypeptide coding
sequence of SEQ ID NO: 1 or SEQ ID NO: 3 of preferably at least
60%, more preferably at least 65%, more preferably at least 70%,
more preferably at least 75%, more preferably at least 80%, more
preferably at least 85%, even more preferably at least 90%, most
preferably at least 95%, and even most preferably at least 96%, at
least 97%, at least 98%, or at least 99% identity, which encode an
active polypeptide.
[0110] Modification of a nucleotide sequence encoding a polypeptide
of the present invention may be necessary for the synthesis of
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., artificial variants that differ in specific
activity, thermostability, pH optimum, or the like. The variant
sequence may be constructed on the basis of the nucleotide sequence
presented as the mature polypeptide coding sequence of SEQ ID NO: 1
or SEQ ID NO: 3, e.g., a subsequence thereof, and/or by
introduction of nucleotide substitutions that do not give rise to
another amino acid sequence of the polypeptide encoded by the
nucleotide sequence, 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.
[0111] It will be apparent to those skilled in the art that such
substitutions can be made outside the regions critical to the
function of the molecule and still result in an active polypeptide.
Amino acid residues essential to the activity of the polypeptide
encoded by an isolated polynucleotide of the invention, and
therefore preferably not subject to substitution, may be identified
according to procedures known in the art, such as site-directed
mutagenesis or alanine-scanning mutagenesis (see, e.g., Cunningham
and Wells, 1989, supra). In the latter technique, mutations are
introduced at every positively charged 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. Sites of substrate-enzyme interaction can
also be determined by analysis of the three-dimensional structure
as determined by such techniques as nuclear magnetic resonance
analysis, crystallography or photoaffinity labeling (see, e.g., de
Vos et al., 1992, supra; Smith et al., 1992, supra; Wlodaver et
al., 1992, supra).
[0112] The present invention also relates to isolated
polynucleotides encoding polypeptides of the present invention,
which hybridize under very low stringency conditions, preferably
low stringency conditions, more preferably medium stringency
conditions, more preferably medium-high stringency conditions, even
more preferably high stringency conditions, and most preferably
very high stringency conditions with (i) the mature polypeptide
coding sequence of SEQ ID NO: 1 or SEQ ID NO: 3, (ii) the genomic
DNA sequence comprising the mature polypeptide coding sequence of
SEQ ID NO: 1 or SEQ ID NO: 3, or (iii) a full-length complementary
strand of (i) or (ii); or allelic variants and subsequences thereof
(Sambrook et al., 1989, supra), as defined herein. In a preferred
aspect, the complementary strand is the full-length complementary
strand of the mature polypeptide coding sequence of SEQ ID NO: 1 or
SEQ ID NO: 3.
[0113] The present invention also relates to isolated
polynucleotides obtained by (a) hybridizing a population of DNA
under very low, low, medium, medium-high, high, or very high
stringency conditions with (i) the mature polypeptide coding
sequence of SEQ ID NO: 1 or SEQ ID NO: 3, (ii) the genomic DNA
sequence comprising the mature polypeptide coding sequence of SEQ
ID NO: 1 or SEQ ID NO: 3, or (iii) a full-length complementary
strand of (i) or (ii); and (b) isolating the hybridizing
polynucleotide, which encodes a polypeptide having endoglucanase
activity. In a preferred aspect, the complementary strand is the
full-length complementary strand of the mature polypeptide coding
sequence of SEQ ID NO: 1 or SEQ ID NO: 3.
Nucleic Acid Constructs
[0114] The present invention also relates to nucleic acid
constructs comprising an isolated polynucleotide of the present
invention operably linked to one or more (several) control
sequences that direct the expression of the coding sequence in a
suitable host cell under conditions compatible with the control
sequences.
[0115] An isolated polynucleotide encoding a polypeptide of the
present invention may be manipulated in a variety of ways to
provide for expression of the polypeptide. Manipulation of the
polynucleotide's sequence prior to its insertion into a vector may
be desirable or necessary depending on the expression vector. The
techniques for modifying polynucleotide sequences utilizing
recombinant DNA methods are well known in the art.
[0116] The control sequence may be an appropriate promoter
sequence, a nucleotide sequence that is recognized by a host cell
for expression of a polynucleotide encoding a polypeptide of the
present invention. The promoter sequence contains transcriptional
control sequences that mediate the expression of the polypeptide.
The promoter may be any nucleotide sequence that shows
transcriptional activity in the host cell of choice 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.
[0117] Examples of suitable promoters for directing the
transcription of the nucleic acid constructs of the present
invention, especially in a bacterial host cell, are the promoters
obtained from the E. coli lac operon, Streptomyces coelicolor
agarase gene (dagA), Bacillus subtilis levansucrase gene (sacB),
Bacillus licheniformis alpha-amylase gene (amyL), Bacillus
stearothermophilus maltogenic amylase gene (amyM), Bacillus
amyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformis
penicillinase gene (penP), Bacillus subtilis xylA and xylB genes,
and prokaryotic beta-lactamase gene (Villa-Kamaroff et al., 1978,
Proceedings of the National Academy of Sciences USA 75: 3727-3731),
as well as the tac promoter (DeBoer et al., 1983, Proceedings of
the National Academy of Sciences USA 80: 21-25). Further promoters
are described in "Useful proteins from recombinant bacteria" in
Scientific American, 1980, 242: 74-94; and in Sambrook et al.,
1989, supra.
[0118] Examples of suitable promoters for directing the
transcription of the nucleic acid constructs of the present
invention in a filamentous fungal host cell are promoters obtained
from the genes for Aspergillus oryzae TAKA amylase, Rhizomucor
miehei aspartic proteinase, Aspergillus niger neutral
alpha-amylase, Aspergillus niger acid stable alpha-amylase,
Aspergillus niger or Aspergillus awamori glucoamylase (glaA),
Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease,
Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans
acetamidase, Fusarium venenatum amyloglucosidase (WO 00/56900),
Fusarium venenatum Daria (WO 00/56900), Fusarium venenatum Quinn
(WO 00/56900), Fusarium oxysporum trypsin-like protease (WO
96/00787), Trichoderma reesei beta-glucosidase, Trichoderma reesei
cellobiohydrolase I, Trichoderma reesei cellobiohydrolase II,
Trichoderma reesei endoglucanase I, Trichoderma reesei
endoglucanase II, Trichoderma reesei endoglucanase III, Trichoderma
reesei endoglucanase IV, Trichoderma reesei endoglucanase V,
Trichoderma reesei endoglucanase I, Trichoderma reesei
endoglucanase II, Trichoderma reesei beta-xylosidase, as well as
the NA2-tpi promoter (a hybrid of the promoters from the genes for
Aspergillus niger neutral alpha-amylase and Aspergillus oryzae
triose phosphate isomerase); and mutant, truncated, and hybrid
promoters thereof.
[0119] 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.
[0120] The control sequence may also be a suitable transcription
terminator sequence, a sequence recognized by a host cell to
terminate transcription. The terminator sequence is operably linked
to the 3' terminus of the nucleotide sequence encoding the
polypeptide. Any terminator that is functional in the host cell of
choice may be used in the present invention.
[0121] Preferred terminators for filamentous fungal host cells are
obtained from the genes for Aspergillus oryzae TAKA amylase,
Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate
synthase, Aspergillus niger alpha-glucosidase, and Fusarium
oxysporum trypsin-like protease.
[0122] 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.
[0123] The control sequence may also be a suitable leader sequence,
a nontranslated region of an mRNA that is important for translation
by the host cell. The leader sequence is operably linked to the 5'
terminus of the nucleotide sequence encoding the polypeptide. Any
leader sequence that is functional in the host cell of choice may
be used in the present invention.
[0124] Preferred leaders for filamentous fungal host cells are
obtained from the genes for Aspergillus oryzae TAKA amylase and
Aspergillus nidulans triose phosphate isomerase.
[0125] 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).
[0126] The control sequence may also be a polyadenylation sequence,
a sequence operably linked to the 3' terminus of the nucleotide
sequence 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 of
choice may be used in the present invention.
[0127] Preferred polyadenylation sequences for filamentous fungal
host cells are obtained from the genes for Aspergillus oryzae TAKA
amylase, Aspergillus niger glucoamylase, Aspergillus nidulans
anthranilate synthase, Fusarium oxysporum trypsin-like protease,
and Aspergillus niger alpha-glucosidase.
[0128] Useful polyadenylation sequences for yeast host cells are
described by Guo and Sherman, 1995, Molecular Cellular Biology 15:
5983-5990.
[0129] The control sequence may also be a signal peptide coding
sequence that codes for an amino acid sequence linked to the amino
terminus of a polypeptide and directs the encoded polypeptide into
the cell's secretory pathway. The 5' end of the coding sequence of
the nucleotide sequence may inherently contain a signal peptide
coding sequence naturally linked in translation reading frame with
the segment of the coding sequence that encodes the secreted
polypeptide. Alternatively, the 5' end of the coding sequence may
contain a signal peptide coding sequence that is foreign to the
coding sequence. The foreign signal peptide coding sequence may be
required where the coding sequence does not naturally contain a
signal peptide coding sequence. Alternatively, the 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 of choice, i.e., secreted into a culture medium, may be
used in the present invention.
[0130] 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
stearothermophilus alpha-amylase, Bacillus licheniformis
subtilisin, Bacillus licheniformis beta-lactamase, 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.
[0131] Effective signal peptide coding sequences for filamentous
fungal host cells are the signal peptide coding sequences obtained
from the genes for Aspergillus oryzae TAKA amylase, Aspergillus
niger neutral amylase, Aspergillus niger glucoamylase, Rhizomucor
miehei aspartic proteinase, Humicola insolens cellulase, Humicola
insolens endoglucanase V, and Humicola lanuginosa lipase.
[0132] 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.
[0133] In a preferred aspect, the signal peptide comprises or
consists of amino acids 1 to 20 of SEQ ID NO: 2. In another
preferred aspect, the signal peptide coding sequence comprises or
consists of nucleotides 1 to 60 of SEQ ID NO: 1.
[0134] In another preferred aspect, the signal peptide comprises or
consists of amino acids 1 to 18 of SEQ ID NO: 4. In another
preferred aspect, the signal peptide coding sequence comprises or
consists of nucleotides 1 to 54 of SEQ ID NO: 3.
[0135] The control sequence may also be a propeptide coding
sequence that codes for an amino acid sequence positioned at the
amino terminus of a polypeptide. The resultant polypeptide is known
as a proenzyme or propolypeptide (or a zymogen in some cases). A
propeptide is generally inactive and can be converted to a mature
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),
Saccharomyces cerevisiae alpha-factor, Rhizomucor miehei aspartic
proteinase, and Myceliophthora thermophila laccase (WO
95/33836).
[0136] Where both signal peptide and propeptide sequences are
present at the amino terminus of a polypeptide, the propeptide
sequence is positioned next to the amino terminus of a polypeptide
and the signal peptide sequence is positioned next to the amino
terminus of the propeptide sequence.
[0137] It may also be desirable to add regulatory sequences that
allow the regulation of the expression of the polypeptide relative
to the growth of the host cell. Examples of regulatory systems are
those that cause the expression of the gene to be turned on or off
in response to a chemical or physical stimulus, including the
presence of a regulatory compound. Regulatory systems in
prokaryotic systems include the lac, tac, and trp operator systems.
In yeast, the ADH2 system or GAL1 system may be used. In
filamentous fungi, the TAKA alpha-amylase promoter, Aspergillus
niger glucoamylase promoter, and Aspergillus oryzae glucoamylase
promoter may be used as regulatory sequences. 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 nucleotide sequence encoding the
polypeptide would be operably linked with the regulatory
sequence.
Expression Vectors
[0138] 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 nucleic acids and control sequences described herein may be
joined together to produce a recombinant expression vector that may
include one or more (several) convenient restriction sites to allow
for insertion or substitution of the nucleotide sequence encoding
the polypeptide at such sites. Alternatively, a polynucleotide
sequence of the present invention may be expressed by inserting the
nucleotide sequence or a nucleic acid construct comprising the
sequence 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.
[0139] 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 nucleotide
sequence. 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 vectors may be linear or closed
circular plasmids.
[0140] 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.
[0141] The vectors of the present invention preferably contain one
or more (several) 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.
[0142] Examples of bacterial selectable markers are the dal genes
from Bacillus subtilis or Bacillus licheniformis, or markers that
confer antibiotic resistance such as ampicillin, kanamycin,
chloramphenicol, or tetracycline resistance. Suitable markers for
yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3.
Selectable markers for use in a filamentous fungal host cell
include, but are not limited to, amdS (acetamidase), argB
(ornithine carbamoyltransferase), bar (phosphinothricin
acetyltransferase), hph (hygromycin phosphotransferase), niaD
(nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase),
sC (sulfate adenyltransferase), and trpC (anthranilate synthase),
as well as equivalents thereof. Preferred for use in an Aspergillus
cell are the amdS and pyrG genes of Aspergillus nidulans or
Aspergillus oryzae and the bar gene of Streptomyces
hygroscopicus.
[0143] The vectors of the present invention preferably contain 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.
[0144] 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 nonhomologous recombination. Alternatively, the
vector may contain additional nucleotide sequences 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 preferably contain a sufficient number of nucleic
acids, such as 100 to 10,000 base pairs, preferably 400 to 10,000
base pairs, and most preferably 800 to 10,000 base pairs, which
have a high degree of 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
nucleotide sequences. On the other hand, the vector may be
integrated into the genome of the host cell by non-homologous
recombination.
[0145] 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" is defined herein as a
nucleotide sequence that enables a plasmid or vector to replicate
in vivo.
[0146] Examples of bacterial origins of replication are the origins
of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184
permitting replication in E. coli, and pUB110, pE194, pTA1060, and
pAM.beta.1 permitting replication in Bacillus.
[0147] 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.
[0148] Examples of origins of replication useful in a filamentous
fungal cell are AMA1 and ANSI (Gems et al., 1991, Gene 98: 61-67;
Cullen et al., 1987, Nucleic Acids Research 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.
[0149] More than one copy of a polynucleotide of the present
invention may be inserted into a host cell to increase production
of the gene product. 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.
[0150] 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
[0151] The present invention also relates to recombinant host
cells, comprising an isolated polynucleotide of the present
invention, which are advantageously used in the recombinant
production of the polypeptides. A vector comprising a
polynucleotide of the present invention is introduced into a host
cell so that the 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.
[0152] 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.
[0153] The prokaryotic host cell may be any Gram positive bacterium
or a Gram negative bacterium. Gram positive bacteria include, but
not limited to, Bacillus, Streptococcus, Streptomyces,
Staphylococcus, Enterococcus, Lactobacillus, Lactococcus,
Clostridium, Geobacillus, and Oceanobacillus. Gram negative
bacteria include, but not limited to, E. coli, Pseudomonas,
Salmonella, Campylobacter, Helicobacter, Flavobacterium,
Fusobacterium, Ilyobacter, Neisseria, and Ureaplasma.
[0154] The bacterial host cell may be any Bacillus cell. Bacillus
cells useful in the practice of the present invention include, but
are 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.
[0155] In a preferred aspect, the bacterial host cell is a Bacillus
amyloliquefaciens, Bacillus lentus, Bacillus licheniformis,
Bacillus stearothermophilus or Bacillus subtilis cell. In a more
preferred aspect, the bacterial host cell is a Bacillus
amyloliquefaciens cell. In another more preferred aspect, the
bacterial host cell is a Bacillus clausii cell. In another more
preferred aspect, the bacterial host cell is a Bacillus
licheniformis cell. In another more preferred aspect, the bacterial
host cell is a Bacillus subtilis cell.
[0156] The bacterial host cell may also be any Streptococcus cell.
Streptococcus cells useful in the practice of the present invention
include, but are not limited to, Streptococcus equisimilis,
Streptococcus pyogenes, Streptococcus uberis, and Streptococcus
equi subsp. Zooepidemicus cells.
[0157] In a preferred aspect, the bacterial host cell is a
Streptococcus equisimilis cell. In another preferred aspect, the
bacterial host cell is a Streptococcus pyogenes cell. In another
preferred aspect, the bacterial host cell is a Streptococcus uberis
cell. In another preferred aspect, the bacterial host cell is a
Streptococcus equi subsp. Zooepidemicus cell.
[0158] The bacterial host cell may also be any Streptomyces cell.
Streptomyces cells useful in the practice of the present invention
include, but are not limited to, Streptomyces achromogenes,
Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces
griseus, and Streptomyces lividans cells.
[0159] In a preferred aspect, the bacterial host cell is a
Streptomyces achromogenes cell. In another preferred aspect, the
bacterial host cell is a Streptomyces avermitilis cell. In another
preferred aspect, the bacterial host cell is a Streptomyces
coelicolor cell. In another preferred aspect, the bacterial host
cell is a Streptomyces griseus cell. In another preferred aspect,
the bacterial host cell is a Streptomyces lividans cell.
[0160] The introduction of DNA into a Bacillus cell may, for
instance, be effected by protoplast transformation (see, e.g.,
Chang and Cohen, 1979, Molecular General Genetics 168: 111-115), by
using competent cells (see, e.g., Young and Spizizen, 1961, Journal
of Bacteriology 81: 823-829, or Dubnau and Davidoff-Abelson, 1971,
Journal of Molecular Biology 56: 209-221), by electroporation (see,
e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), or by
conjugation (see, e.g., Koehler and Thorne, 1987, Journal of
Bacteriology 169: 5271-5278). The introduction of DNA into an E
coli cell may, for instance, 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, for instance, be effected by protoplast
transformation and electroporation (see, e.g., Gong et al., 2004,
Folia Microbiol. (Praha) 49: 399-405), by conjugation (see, e.g.,
Mazodier et al., 1989, J. Bacteriol. 171: 3583-3585), or by
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, for instance, be effected by electroporation (see, e.g., Choi
et al., 2006, J. Microbiol. Methods 64: 391-397) or by conjugation
(see, e.g., Pinedo and Smets, 2005, Appl. Environ. Microbiol. 71:
51-57). The introduction of DNA into a Streptococcus cell may, for
instance, be effected by natural competence (see, e.g., Perry and
Kuramitsu, 1981, Infect. Immun. 32: 1295-1297), by protoplast
transformation (see, e.g., Catt and Jollick, 1991, Microbios. 68:
189-2070, by electroporation (see, e.g., Buckley et al., 1999,
Appl. Environ. Microbiol. 65: 3800-3804) or by 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.
[0161] The host cell may also be a eukaryote, such as a mammalian,
insect, plant, or fungal cell.
[0162] In a preferred aspect, the host cell is a fungal cell.
"Fungi" as used herein includes the phyla Ascomycota,
Basidiomycota, Chytridiomycota, and Zygomycota (as defined by
Hawksworth et al., In, Ainsworth and Bisby's Dictionary of The
Fungi, 8th edition, 1995, CAB International, University Press,
Cambridge, UK) as well as the Oomycota (as cited in Hawksworth et
al., 1995, supra, page 171) and all mitosporic fungi (Hawksworth et
al., 1995, supra).
[0163] In a more preferred aspect, the fungal host cell is 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, F. A., Passmore, S. M., and Davenport, R. R., eds,
Soc. App. Bacteriol. Symposium Series No. 9, 1980).
[0164] In an even more preferred aspect, the yeast host cell is a
Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces,
Schizosaccharomyces, or Yarrowia cell.
[0165] In a most preferred aspect, the yeast host cell is a
Saccharomyces carlsbergensis, Saccharomyces cerevisiae,
Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces
kluyveri, Saccharomyces norbensis, or Saccharomyces oviformis cell.
In another most preferred aspect, the yeast host cell is a
Kluyveromyces lactis cell. In another most preferred aspect, the
yeast host cell is a Yarrowia lipolytica cell.
[0166] In another more preferred aspect, the fungal host cell is 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.
[0167] In an even more preferred aspect, the filamentous fungal
host cell is an Acremonium, Aspergillus, Aureobasidium,
Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus,
Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor,
Myceliophthora, Neocallimastix, Neurospora, Paecilomyces,
Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus,
Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium,
Trametes, or Trichoderma cell.
[0168] In a most preferred aspect, the filamentous fungal host cell
is an Aspergillus awamori, Aspergillus fumigatus, Aspergillus
foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus
niger or Aspergillus oryzae cell. In another most preferred aspect,
the filamentous fungal host cell is a 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, or Fusarium venenatum cell. In another
most preferred aspect, the filamentous fungal host cell is a
Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis aneirina,
Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis
pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa,
Ceriporiopsis subvermispora, Chrysosporium keratinophilum,
Chrysosporium lucknowense, Chrysosporium tropicum, Chrysosporium
merdarium, Chrysosporium inops, Chrysosporium pannicola,
Chrysosporium queenslandicum, Chrysosporium zonatum, Coprinus
cinereus, Coriolus hirsutus, 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.
[0169] 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 238 023 and Yelton et al., 1984,
Proceedings of the National Academy of Sciences USA 81: 1470-1474.
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,
Journal of Bacteriology 153: 163; and Hinnen et al., 1978,
Proceedings of the National Academy of Sciences USA 75: 1920.
Methods of Production
[0170] The present invention also relates to methods of producing a
polypeptide of the present invention, comprising: (a) cultivating a
cell, which in its wild-type form produces the polypeptide, under
conditions conducive for production of the polypeptide; and (b)
recovering the polypeptide. In a preferred aspect, the cell is of
the genus Thielavia. In a more preferred aspect, the cell is
Thielavia terrestris. In a most preferred aspect, the cell is
Thielavia terrestris NRRL 8126.
[0171] The present invention also relates to methods of producing a
polypeptide of the present invention, comprising: (a) cultivating a
recombinant host cell, as described herein, under conditions
conducive for production of the polypeptide; and (b) recovering the
polypeptide.
[0172] The present invention also relates to methods of producing a
polypeptide of the present invention, comprising: (a) cultivating a
recombinant host cell under conditions conducive for production of
the polypeptide, wherein the host cell comprises a mutant
nucleotide sequence having at least one mutation in the mature
polypeptide coding sequence of SEQ ID NO: 1 or SEQ ID NO: 3,
wherein the mutant nucleotide sequence encodes a polypeptide that
comprises or consists of the mature polypeptide of SEQ ID NO: 2 or
SEQ ID NO: 4, respectively; and (b) recovering the polypeptide.
[0173] In the production methods of the present invention, the
cells are cultivated in a nutrient medium suitable for production
of the polypeptide using methods well known in the art. For
example, the cell may be cultivated by shake flask cultivation, and
small-scale 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 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 into the medium, it can be
recovered from cell lysates.
[0174] The polypeptides may be detected using methods known in the
art that are specific for the polypeptides. These detection methods
may include 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 as described herein.
[0175] The resulting 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, centrifugation, filtration, extraction,
spray-drying, evaporation, or precipitation.
[0176] The polypeptides of the present invention 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, J.-C. Janson and Lars Ryden,
editors, VCH Publishers, New York, 1989) to obtain substantially
pure polypeptides.
Plants
[0177] The present invention also relates to plants, e.g., a
transgenic plant, plant part, or plant cell, comprising an isolated
polynucleotide encoding a polypeptide having endoglucanase activity
of the present invention so as to express and produce the
polypeptide in recoverable quantities. The polypeptide may be
recovered from the plant or plant part. Alternatively, the plant or
plant part containing the recombinant polypeptide 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.
[0178] 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).
[0179] 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.
[0180] 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 utilisation of the invention are also
considered plant parts, e.g., embryos, endosperms, aleurone and
seeds coats.
[0181] Also included within the scope of the present invention are
the progeny of such plants, plant parts, and plant cells.
[0182] The transgenic plant or plant cell expressing a polypeptide
of the present invention 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 (several) expression
constructs encoding a polypeptide of the present invention into the
plant host genome or chloroplast genome and propagating the
resulting modified plant or plant cell into a transgenic plant or
plant cell.
[0183] The expression construct is conveniently a nucleic acid
construct that comprises a polynucleotide encoding a polypeptide of
the present invention operably linked with appropriate regulatory
sequences required for expression of the nucleotide sequence in the
plant or plant part of choice. Furthermore, the expression
construct may comprise a selectable marker useful for identifying
host 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).
[0184] 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 is desired to be expressed. For instance, the
expression of the gene encoding a polypeptide of the present
invention 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.
[0185] For constitutive expression, the 35S-CaMV, the maize
ubiquitin 1, and the rice actin 1 promoter may be used (Franck et
al., 1980, Cell 21: 285-294, Christensen et al., 1992, Plant Mo.
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 & 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 and Cell Physiology 39: 885-889), a Vicia faba
promoter from the legumin B4 and the unknown seed protein gene from
Vicia faba (Conrad et al., 1998, Journal of Plant Physiology 152:
708-711), a promoter from a seed oil body protein (Chen et al.,
1998, Plant and Cell Physiology 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
Physiology 102: 991-1000, the chlorella virus adenine
methyltransferase gene promoter (Mitra and Higgins, 1994, Plant
Molecular Biology 26: 85-93), or the aldP gene promoter from rice
(Kagaya et al., 1995, Molecular and General Genetics 248: 668-674),
or a wound inducible promoter such as the potato pin2 promoter (Xu
et al., 1993, Plant Molecular Biology 22: 573-588). Likewise, the
promoter may inducible 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.
[0186] A promoter enhancer element may also be used to achieve
higher expression of a polypeptide of the present invention in the
plant. For instance, the promoter enhancer element may be an intron
that is placed between the promoter and the nucleotide sequence
encoding a polypeptide of the present invention. For instance, Xu
et al., 1993, supra, disclose the use of the first intron of the
rice actin 1 gene to enhance expression.
[0187] The selectable marker gene and any other parts of the
expression construct may be chosen from those available in the
art.
[0188] 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).
[0189] Presently, Agrobacterium tumefaciens-mediated gene transfer
is the method of choice for generating transgenic dicots (for a
review, see Hooykas and Schilperoort, 1992, Plant Molecular Biology
19: 15-38) and can also be used for transforming monocots, although
other transformation methods are often used for these plants.
Presently, the method of choice 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 Journal 2: 275-281; Shimamoto, 1994,
Current Opinion Biotechnology 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 Molecular Biology 21:
415-428.
[0190] 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.
[0191] The present invention also relates to methods of producing a
polypeptide of the present invention comprising: (a) cultivating a
transgenic plant or a plant cell comprising a polynucleotide
encoding the polypeptide having endoglucanase activity of the
present invention under conditions conducive for production of the
polypeptide; and (b) recovering the polypeptide.
Removal or Reduction of Endoglucanase Activity
[0192] The present invention also relates to methods of producing a
mutant of a parent cell, which comprises disrupting or deleting a
polynucleotide sequence, 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.
[0193] The mutant cell may be constructed by reducing or
eliminating expression of a nucleotide sequence encoding a
polypeptide of the present invention using methods well known in
the art, for example, insertions, disruptions, replacements, or
deletions. In a preferred aspect, the nucleotide sequence is
inactivated. The nucleotide sequence 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 the 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
nucleotide sequence. 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.
[0194] Modification or inactivation of the nucleotide sequence may
be performed by subjecting the parent cell to mutagenesis and
selecting for mutant cells in which expression of the nucleotide
sequence 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.
[0195] 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.
[0196] 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.
[0197] Modification or inactivation of the nucleotide sequence may
be accomplished by introduction, substitution, or removal of one or
more (several) nucleotides in the gene or a regulatory element
required for the 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 nucleotide sequence to be
modified, it is preferred that the modification be performed in
vitro as exemplified below.
[0198] An example of a convenient way to eliminate or reduce
expression of a nucleotide sequence by a cell 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 nucleotide sequence 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 nucleotide sequence. It may be desirable
that the defective nucleotide sequence also encodes a marker that
may be used for selection of transformants in which the nucleotide
sequence has been modified or destroyed. In a particularly
preferred aspect, the nucleotide sequence is disrupted with a
selectable marker such as those described herein.
[0199] Alternatively, modification or inactivation of the
nucleotide sequence may be performed by established anti-sense or
RNAi techniques using a sequence complementary to the nucleotide
sequence. More specifically, expression of the nucleotide sequence
by a cell may be reduced or eliminated by introducing a sequence
complementary to the nucleotide sequence of the gene that may be
transcribed in the cell and is capable of hybridizing to the mRNA
produced in the cell. Under conditions allowing the complementary
anti-sense nucleotide sequence to hybridize to the mRNA, the amount
of protein translated is thus reduced or eliminated.
[0200] The present invention further relates to a mutant cell of a
parent cell that comprises a disruption or deletion of a nucleotide
sequence encoding the polypeptide or a control sequence thereof,
which results in the mutant cell producing less of the polypeptide
or no polypeptide compared to the parent cell.
[0201] The polypeptide-deficient mutant cells so created are
particularly useful as host cells for the expression of native
and/or 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" is
defined herein as polypeptides that are not native to the host
cell, a native protein in which modifications have been made to
alter the native sequence, or a native protein whose expression is
quantitatively altered as a result of a manipulation of the host
cell by recombinant DNA techniques.
[0202] In a further aspect, the present invention relates to a
method of producing a protein product essentially free of
endoglucanase activity by fermentation of a cell that produces both
a polypeptide of the present invention as well as the protein
product of interest by adding an effective amount of an agent
capable of inhibiting endoglucanase activity to the fermentation
broth before, during, or after the fermentation has been completed,
recovering the product of interest from the fermentation broth, and
optionally subjecting the recovered product to further
purification.
[0203] In a further aspect, the present invention relates to a
method of producing a protein product essentially free of
endoglucanase activity by cultivating the cell under conditions
permitting the expression of the product, subjecting the resultant
culture broth to a combined pH and temperature treatment so as to
reduce the endoglucanase activity substantially, and recovering the
product from the culture broth. Alternatively, the combined pH and
temperature treatment may be performed on an enzyme preparation
recovered from the culture broth. The combined pH and temperature
treatment may optionally be used in combination with a treatment
with an endoglucanase inhibitor.
[0204] In accordance with this aspect of the invention, it is
possible to remove at least 60%, preferably at least 75%, more
preferably at least 85%, still more preferably at least 95%, and
most preferably at least 99% of the endoglucanase activity.
Complete removal of endoglucanase activity may be obtained by use
of this method.
[0205] The combined pH and temperature treatment is preferably
carried out at a pH in the range of 2-4 or 9-11 and a temperature
in the range of at least 60-70.degree. C. for a sufficient period
of time to attain the desired effect, where typically, 30 to 60
minutes is sufficient.
[0206] The methods used for cultivation and purification of the
product of interest may be performed by methods known in the
art.
[0207] The methods of the present invention for producing an
essentially endoglucanase-free product is of particular interest in
the production of eukaryotic polypeptides, in particular fungal
proteins such as enzymes. The enzyme may be selected from, e.g., an
amylolytic enzyme, lipolytic enzyme, proteolytic enzyme,
cellulolytic enzyme, oxidoreductase, or plant cell-wall degrading
enzyme. Examples of such enzymes include an aminopeptidase,
amylase, amyloglucosidase, carbohydrase, carboxypeptidase,
catalase, cellobiohydrolase, cellulase, chitinase, cutinase,
cyclodextrin glycosyltransferase, deoxyribonuclease, endoglucanase,
esterase, galactosidase, beta-galactosidase, glucoamylase, glucose
oxidase, glucosidase, haloperoxidase, hemicellulase, invertase,
isomerase, laccase, ligase, lipase, lyase, mannosidase, oxidase,
pectinolytic enzyme, peroxidase, phytase, phenoloxidase,
polyphenoloxidase, proteolytic enzyme, ribonuclease, transferase,
transglutaminase, or endoglucanase. The endoglucanase-deficient
cells may also be used to express heterologous proteins of
pharmaceutical interest such as hormones, growth factors,
receptors, and the like.
[0208] It will be understood that 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.
[0209] 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.
Methods of Inhibiting Expression of a Polypeptide Having
Endoglucanase Activity
[0210] 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.
[0211] The dsRNA is preferably a small interfering RNA (sRNA) or a
micro RNA (miRNA). In a preferred aspect, the dsRNA is small
interfering RNA (siRNAs) for inhibiting transcription. In another
preferred aspect, the dsRNA is micro RNA (miRNAs) for inhibiting
translation.
[0212] 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 or SEQ ID NO: 3 for
inhibiting expression of a 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).
[0213] The dsRNAs of the present invention can be used in
gene-silencing therapeutics. In one aspect, the invention provides
methods to selectively degrade RNA using the dsRNAis 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. No. 6,506,559;
U.S. Pat. No. 6,511,824; U.S. Pat. No. 6,515,109; and U.S. Pat. No.
6,489,127.
Compositions
[0214] 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.
[0215] The composition may comprise a polypeptide of the present
invention as the major enzymatic component, e.g., a mono-component
composition. Alternatively, the composition may comprise multiple
enzymatic activities, such as an aminopeptidase, amylase,
carbohydrase, carboxypeptidase, catalase, cellulase, chitinase,
cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease,
esterase, alpha-galactosidase, beta-galactosidase, glucoamylase,
alpha-glucosidase, beta-glucosidase, haloperoxidase, invertase,
laccase, lipase, mannosidase, oxidase, pectinolytic enzyme,
peptidoglutaminase, peroxidase, phytase, polyphenoloxidase,
proteolytic enzyme, ribonuclease, transglutaminase, or
endoglucanase. The additional enzyme(s) may be produced, for
example, by a microorganism belonging to the genus Aspergillus,
preferably Aspergillus aculeatus, Aspergillus awamori, Aspergillus
fumigatus, Aspergillus foetidus, Aspergillus japonicus, Aspergillus
nidulans, Aspergillus niger, or Aspergillus oryzae; Fusarium,
preferably 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 sulphureum, Fusarium
toruloseum, Fusarium trichothecioides, or Fusarium venenatum;
Humicola, preferably Humicola insolens or Humicola lanuginosa; or
Trichoderma, preferably Trichoderma harzianum, Trichoderma
koningii, Trichoderma longibrachiatum, Trichoderma reesei, or
Trichoderma viride.
[0216] The polypeptide compositions may be prepared in accordance
with methods known in the art and may be in the form of a liquid or
a dry composition. For instance, the polypeptide composition may be
in the form of a granulate or a microgranulate. The polypeptide to
be included in the composition may be stabilized in accordance with
methods known in the art.
[0217] Examples are given below of preferred uses of the
polypeptide compositions of the invention. The dosage of the
polypeptide composition of the invention and other conditions under
which the composition is used may be determined on the basis of
methods known in the art.
Uses
[0218] The present invention also relates to methods for degrading
or converting a cellulosic material, comprising: treating the
cellulosic material with a composition comprising one or more
cellulolytic proteins in the presence of a polypeptide having
endoglucanase activity of the present invention. In a preferred
aspect, the method further comprises recovering the degraded or
converted cellulosic material.
[0219] The present invention further relates to methods of
producing a fermentation product, comprising: (a) saccharifying a
cellulosic material with a composition comprising one or more
cellulolytic proteins in the presence of a polypeptide having
endoglucanase activity of the present invention; (b) fermenting the
saccharified cellulosic material of step (a) with one or more
fermenting microorganisms to produce the fermentation product; and
(c) recovering the fermentation product from the fermentation.
[0220] The composition comprising the polypeptide having
endoglucanase activity can be in the form of a crude fermentation
broth with or without the cells removed or in the form of a
semi-purified or purified enzyme preparation or the composition can
comprise a host cell of the present invention as a source of the
polypeptide having endoglucanase activity in a fermentation process
with the biomass.
[0221] The methods of the present invention can be used to
saccharify a cellulosic material to fermentable sugars and convert
the fermentable sugars to many useful substances, e.g., chemicals
and fuels. The production of a desired fermentation product from
cellulosic material typically involves pretreatment, enzymatic
hydrolysis (saccharification), and fermentation.
[0222] The processing of 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.
[0223] 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
cofermentation (SSCF); hybrid hydrolysis and fermentation (HHF);
SHCF (separate hydrolysis and co-fermentation), HHCF (hybrid
hydrolysis and fermentation), and direct microbial conversion
(DMC). SHF uses separate process steps to first enzymatically
hydrolyze lignocellulose to fermentable sugars, e.g., glucose,
cellobiose, cellotriose, and pentose sugars, and then ferment the
fermentable sugars to ethanol. In SSF, the enzymatic hydrolysis of
lignocellulose 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 cofermentation 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
separate 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,
lignocellulose hydrolysis, and fermentation) in one or more steps
where the same organism is used to produce the enzymes for
conversion of the lignocellulose 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 practicing the methods of the present
invention.
[0224] 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.
[0225] Pretreatment.
[0226] In practicing the methods of the present invention, any
pretreatment process known in the art can be used to disrupt the
plant cell wall components. The cellulosic material can also be
subjected to pre-soaking, wetting, or conditioning prior to
pretreatment using methods known in the art. Conventional
pretreatments include, but are not limited to, steam pretreatment
(with or without explosion), dilute acid pretreatment, hot water
pretreatment, lime pretreatment, wet oxidation, wet explosion,
ammonia fiber explosion, organosolv pretreatment, and biological
pretreatment. Additional pretreatments include ultrasound,
electroporation, microwave, supercritical CO.sub.2, supercritical
H.sub.2O, and ammonia percolation pretreatments.
[0227] 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 hydrolysis, such as simultaneously with
treatment of the cellulosic material with one or more cellulolytic
enzymes, or other enzyme activities, to release fermentable sugars,
such as glucose and/or maltose. In most cases the pretreatment step
itself results in some conversion of biomass to fermentable sugars
(even in absence of enzymes).
[0228] 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., hemicellulase, accessible to
enzymes. The lignocellulose 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
done at 140-230.degree. C., more preferably 160-200.degree. C., and
most preferably 170-190.degree. C., where the optimal temperature
range depends on any addition of a chemical catalyst. Residence
time for the steam pretreatment is preferably 1-15 minutes, more
preferably 3-12 minutes, and most preferably 4-10 minutes, where
the optimal residence time depends on temperature range and any
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.
[0229] A catalyst such as H.sub.2SO.sub.4 or SO.sub.2 (typically
0.3 to 3% 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).
[0230] Chemical Pretreatment: The term "chemical treatment" refers
to any chemical pretreatment that promotes the separation and/or
release of cellulose, hemicellulose, and/or lignin. Examples of
suitable chemical pretreatment processes include, for example,
dilute acid pretreatment, lime pretreatment, wet oxidation, ammonia
fiber/freeze explosion (AFEX), ammonia percolation (APR), and
organosolv pretreatments.
[0231] 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).
[0232] Several methods of pretreatment under alkaline conditions
can also be used. These alkaline pretreatments include, but are not
limited to, lime pretreatment, wet oxidation, ammonia percolation
(APR), and ammonia fiber/freeze explosion (AFEX).
[0233] Lime pretreatment is performed with calcium carbonate,
sodium hydroxide, or ammonia at low 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 2006/11899,
WO 2006/11900, and WO 2006/110901 disclose pretreatment methods
using ammonia.
[0234] 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 at preferably 1-40% dry matter, more
preferably 2-30% dry matter, and most preferably 5-20% dry matter,
and often the initial pH is increased by the addition of alkali
such as sodium carbonate.
[0235] 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).
[0236] Ammonia fiber explosion (AFEX) involves treating cellulosic
material with liquid or gaseous ammonia at moderate temperatures
such as 90-100.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). AFEX pretreatment
results in the depolymerization of cellulose and partial hydrolysis
of hemicellulose. Lignin-carbohydrate complexes are cleaved.
[0237] Organosolv pretreatment delignifies 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 the hemicellulose is
removed.
[0238] 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.
[0239] In one aspect, the chemical pretreatment is preferably
carried out as an acid treatment, and more preferably as a
continuous dilute and/or mild 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, more
preferably 1-4, and most preferably 1-3. In one aspect, the acid
concentration is in the range from preferably 0.01 to 20 wt % acid,
more preferably 0.05 to 10 wt % acid, even more preferably 0.1 to 5
wt % acid, and most preferably 0.2 to 2.0 wt % acid. The acid is
contacted with the cellulosic material and held at a temperature in
the range of preferably 160-220.degree. C., and more preferably
165-195.degree. C., for periods ranging from seconds to minutes to,
e.g., 1 second to 60 minutes.
[0240] In another aspect, pretreatment is carried out as an ammonia
fiber explosion step (AFEX pretreatment step).
[0241] 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 %, more
preferably between 20-70 wt %, and most preferably between 30-60 wt
%, such as around 50 wt %. The pretreated cellulosic material can
be unwashed or washed using any method known in the art, e.g.,
washed with water.
[0242] Mechanical Pretreatment: The term "mechanical pretreatment"
refers to various types of grinding or milling (e.g., dry milling,
wet milling, or vibratory ball milling).
[0243] Physical Pretreatment: The term "physical pretreatment"
refers to any pretreatment that promotes the separation and/or
release of cellulose, hemicellulose, and/or lignin from cellulosic
material. For example, physical pretreatment can involve
irradiation (e.g., microwave irradiation), steaming/steam
explosion, hydrothermolysis, and combinations thereof.
[0244] Physical pretreatment can involve high pressure and/or high
temperature (steam explosion). In one aspect, high pressure means
pressure in the range of preferably about 300 to about 600 psi,
more preferably about 350 to about 550 psi, and most preferably
about 400 to about 500 psi, such as around 450 psi. In another
aspect, high temperature means temperatures in the range of about
100 to about 300.degree. C., preferably about 140 to about
235.degree. C. In a preferred aspect, mechanical pretreatment is
performed in a batch-process, 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.
[0245] Combined Physical and Chemical Pretreatment: The cellulosic
material can be pretreated both physically and chemically. For
instance, the pretreatment step can involve dilute or mild acid
treatment and high temperature and/or pressure treatment. The
physical and chemical pretreatments can be carried out sequentially
or simultaneously, as desired. A mechanical pretreatment can also
be included.
[0246] Accordingly, in a preferred aspect, the cellulosic material
is subjected to mechanical, chemical, or physical pretreatment, or
any combination thereof to promote the separation and/or release of
cellulose, hemicellulose and/or lignin.
[0247] 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 (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 lignocellulosic 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).
[0248] Saccharification.
[0249] In the hydrolysis step, also known as saccharification, the
pretreated cellulosic material is hydrolyzed to break down
cellulose and alternatively also hemicellulose to fermentable
sugars, such as glucose, xylose, xylulose, arabinose, maltose,
mannose, galactose, or soluble oligosaccharides. The hydrolysis is
performed enzymatically by a cellulolytic enzyme composition
comprising an effective amount of a polypeptide having
endoglucanase activity of the present invention, which can further
comprise one or more hemicellulolytic enzymes. The enzymes of the
compositions can also be added sequentially.
[0250] 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 a preferred 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
pretreated cellulosic material (substrate) is fed gradually to, for
example, an enzyme containing hydrolysis solution.
[0251] 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 96 hours, more
preferably about 16 to about 72 hours, and most preferably about 24
to about 48 hours. The temperature is in the range of preferably
about 25.degree. C. to about 70.degree. C., more preferably about
30.degree. C. to about 65.degree. C., and more preferably about
40.degree. C. to 60.degree. C., in particular about 50.degree. C.
The pH is in the range of preferably about 3 to about 8, more
preferably about 3.5 to about 7, and most preferably about 4 to
about 6, in particular about pH 5. The dry solids content is in the
range of preferably about 5 to about 50 wt %, more preferably about
10 to about 40 wt %, and most preferably about 20 to about 30 wt
%.
[0252] In addition to a polypeptide having endoglucanase activity
of the present invention, the cellulolytic enzyme components of the
composition are preferably enzymes having endoglucanase,
cellobiohydrolase, and beta-glucosidase activities. In a preferred
aspect, the cellulolytic enzyme composition further comprises one
or more polypeptides having cellulolytic enhancing activity (see,
for example, WO 2005/074647, WO 2005/074656, and U.S. Published
Application Serial No. 2007/0077630, which are incorporated herein
by reference). In another preferred aspect, the cellulolytic enzyme
preparation is supplemented with one or more additional enzyme
activities selected from the group consisting of hemicellulases,
esterases (e.g., lipases, phospholipases, and/or cutinases),
proteases, laccases, peroxidases, or mixtures thereof. In the
methods of the present invention, the additional enzyme(s) can be
added prior to or during fermentation, including during or after
propagation of the fermenting microorganism(s).
[0253] The enzymes can be derived or obtained from any suitable
origin, including, bacterial, fungal, yeast, plant, or mammalian
origin. The term "obtained" means herein that the enzyme may have
been isolated from an organism that naturally produces the enzyme
as a native enzyme. 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 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.
[0254] The enzymes used in the present invention can be in any form
suitable for use in the methods described herein, such as a crude
fermentation broth with or without cells or substantially pure
polypeptides. The enzyme(s) can be a dry powder or granulate, a
non-dusting granulate, a liquid, a stabilized liquid, or a
protected enzyme(s). Granulates can be produced, e.g., as disclosed
in U.S. Pat. Nos. 4,106,991 and 4,661,452, and can optionally be
coated by process known in the art. Liquid enzyme preparations can,
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 process. Protected enzymes
can be prepared according to the process disclosed in EP
238,216.
[0255] The optimum amounts of the enzymes and polypeptides having
cellulolytic enhancing activity depend on several factors
including, but not limited to, the mixture of component
cellulolytic proteins, the cellulosic substrate, the concentration
of cellulosic substrate, the pretreatment(s) of the cellulosic
substrate, temperature, time, pH, and inclusion of fermenting
organism (e.g., yeast for Simultaneous Saccharification and
Fermentation).
[0256] In a preferred aspect, an effective amount of cellulolytic
protein(s) to cellulosic material is about 0.5 to about 50 mg,
preferably at about 0.5 to about 40 mg, more preferably at about
0.5 to about 25 mg, more preferably at about 0.75 to about 20 mg,
more preferably at about 0.75 to about 15 mg, even more preferably
at about 0.5 to about 10 mg, and most preferably at about 2.5 to
about 10 mg per g of cellulosic material.
[0257] In another preferred aspect, an effective amount of a
polypeptide having endoglucanase activity to cellulosic material is
about 0.01 to about 50 mg, preferably at about 0.5 to about 40 mg,
more preferably at about 0.5 to about 25 mg, more preferably at
about 0.75 to about 20 mg, more preferably at about 0.75 to about
15 mg, even more preferably at about 0.5 to about 10 mg, and most
preferably at about 2.5 to about 10 mg per g of cellulosic
material.
[0258] In another preferred aspect, an effective amount of
polypeptide(s) having cellulolytic enhancing activity to cellulosic
material is about 0.01 to about 50.0 mg, preferably about 0.01 to
about 40 mg, more preferably about 0.01 to about 30 mg, more
preferably about 0.01 to about 20 mg, more preferably about 0.01 to
about 10 mg, more preferably about 0.01 to about 5 mg, more
preferably at about 0.025 to about 1.5 mg, more preferably at about
0.05 to about 1.25 mg, more preferably at about 0.075 to about 1.25
mg, more preferably at about 0.1 to about 1.25 mg, even more
preferably at about 0.15 to about 1.25 mg, and most preferably at
about 0.25 to about 1.0 mg per g of cellulosic material.
[0259] In another preferred aspect, an effective amount of
polypeptide(s) having cellulolytic enhancing activity to
cellulolytic protein(s) is about 0.005 to about 1.0 g, preferably
at about 0.01 to about 1.0 g, more preferably at about 0.15 to
about 0.75 g, more preferably at about 0.15 to about 0.5 g, more
preferably at about 0.1 to about 0.5 g, even more preferably at
about 0.1 to about 0.5 g, and most preferably at about 0.05 to
about 0.2 g per g of cellulolytic protein(s).
[0260] Fermentation.
[0261] The fermentable sugars obtained from the pretreated and
hydrolyzed cellulosic material can be fermented by one or more
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.
[0262] 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. Such methods
include, but are not limited to, separate hydrolysis and
fermentation (SHF); simultaneous saccharification and fermentation
(SSF); simultaneous saccharification and cofermentation (SSCF);
hybrid hydrolysis and fermentation (HHF); SHCF (separate hydrolysis
and co-fermentation), HHCF (hybrid hydrolysis and fermentation),
and direct microbial conversion (DMC).
[0263] 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. Examples of
substrates suitable for use in the methods of present invention,
include cellulose-containing materials, such as wood or plant
residues or low molecular sugars DP1-3 obtained from processed
cellulosic material that can be metabolized by the fermenting
microorganism, and which can be supplied by direct addition to the
fermentation medium.
[0264] 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).
[0265] "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 C.sub.6 and/or C.sub.5 fermenting
organisms, or a combination thereof. Both C.sub.6 and C.sub.5
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,
or oligosaccharides, directly or indirectly into the desired
fermentation product.
[0266] Examples of bacterial and fungal fermenting organisms
producing ethanol are described by Lin et al., 2006, Appl.
Microbiol. Biotechnol. 69: 627-642.
[0267] Examples of fermenting microorganisms that can ferment C6
sugars include bacterial and fungal organisms, such as yeast.
Preferred yeast includes strains of the Saccharomyces spp.,
preferably Saccharomyces cerevisiae.
[0268] Examples of fermenting organisms that can ferment C5 sugars
include bacterial and fungal organisms, such as yeast. Preferred C5
fermenting yeast include strains of Pichia, preferably Pichia
stipitis, such as Pichia stipitis CBS 5773; strains of Candida,
preferably Candida boidinii, Candida brassicae, Candida sheatae,
Candida diddensii, Candida pseudotropicalis, or Candida utilis.
[0269] Other fermenting organisms include strains of Zymomonas,
such as Zymomonas mobilis; Hansenula, such as Hansenula anomala;
Klyveromyces, such as K. fragilis; Schizosaccharomyces, such as S.
pombe; and E. coli, especially E. coli strains that have been
genetically modified to improve the yield of ethanol.
[0270] In a preferred aspect, the yeast is a Saccharomyces spp. In
a more preferred aspect, the yeast is Saccharomyces cerevisiae. In
another more preferred aspect, the yeast is Saccharomyces
distaticus. In another more preferred aspect, the yeast is
Saccharomyces uvarum. In another preferred aspect, the yeast is a
Kluyveromyces. In another more preferred aspect, the yeast is
Kluyveromyces marxianus. In another more preferred aspect, the
yeast is Kluyveromyces fragilis. In another preferred aspect, the
yeast is a Candida. In another more preferred aspect, the yeast is
Candida boidinii. 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 pseudotropicalis. 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
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
Bretannomyces. In another more preferred aspect, the yeast is
Bretannomyces clausenii (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).
[0271] Bacteria that can efficiently ferment hexose and pentose to
ethanol include, for example, Zymomonas mobilis and Clostridium
thermocellum (Philippidis, 1996, supra).
[0272] In a preferred aspect, the bacterium is a Zymomonas. In a
more preferred aspect, the bacterium is Zymomonas mobilis. In
another preferred aspect, the bacterium is a Clostridium. In
another more preferred aspect, the bacterium is Clostridium
thermocellum.
[0273] Commercially available yeast suitable for ethanol production
includes, e.g., ETHANOL RED.TM. yeast (available from
Fermentis/Lesaffre, USA), FALI.TM. (available from Fleischmann's
Yeast, USA), SUPERSTART.TM. and THERMOSACC.TM. fresh yeast
(available from Ethanol Technology, WI, USA), BIOFERM.TM. AFT and
XR (available from NABC--North American Bioproducts Corporation,
GA, USA), GERT STRAND.TM. (available from Gert Strand AB, Sweden),
and FERMIOL.TM. (available from DSM Specialties).
[0274] 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.
[0275] The cloning of heterologous genes into various fermenting
microorganisms has led to the construction of organisms capable of
converting hexoses and pentoses to ethanol (cofermentation) (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).
[0276] In a preferred aspect, the genetically modified fermenting
microorganism is Saccharomyces cerevisiae. In another preferred
aspect, the genetically modified fermenting microorganism is
Zymomonas mobilis. In another preferred aspect, the genetically
modified fermenting microorganism is Escherichia coli. In another
preferred aspect, the genetically modified fermenting microorganism
is Klebsiella oxytoca.
[0277] It is well known in the art that the organisms described
above can also be used to produce other substances, as described
herein.
[0278] The fermenting microorganism is typically added to the
degraded lignocellulose or hydrolysate and the fermentation is
performed for about 8 to about 96 hours, such as about 24 to about
60 hours. The temperature is typically between about 26.degree. C.
to about 60.degree. C., in particular about 32.degree. C. or
50.degree. C., and at about pH 3 to about pH 8, such as around pH
4-5, 6, or 7.
[0279] In a preferred aspect, the yeast and/or another
microorganism is applied to the degraded lignocellulose or
hydrolysate and the fermentation is performed for about 12 to about
96 hours, such as typically 24-60 hours. In a preferred aspect, the
temperature is preferably between about 20.degree. C. to about
60.degree. C., more preferably about 25.degree. C. to about
50.degree. C., and most preferably about 32.degree. C. to about
50.degree. C., in particular about 32.degree. C. or 50.degree. C.,
and the pH is generally from about pH 3 to about pH 7, preferably
around pH 4-7. However, some, e.g., bacterial fermenting organisms
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.
[0280] The most widely used process in the art is the simultaneous
saccharification and fermentation (SSF) process where there is no
holding stage for the saccharification, meaning that yeast and
enzyme are added together.
[0281] For ethanol production, following the fermentation the
fermented slurry is distilled to extract the ethanol. The ethanol
obtained according to the methods of the invention can be used as,
e.g., fuel ethanol, drinking ethanol, i.e., potable neutral
spirits, or industrial ethanol.
[0282] A fermentation stimulator can be used in combination with
any of the enzymatic processes described herein to further improve
the fermentation process, and in particular, the performance of the
fermenting microorganism, such as, rate enhancement and ethanol
yield. A "fermentation stimulator" refers to stimulators for growth
of the fermenting microorganisms, in particular, yeast. Preferred
fermentation stimulators for growth include vitamins and minerals.
Examples of vitamins include multivitamins, biotin, pantothenate,
nicotinic acid, meso-inositol, thiamine, pyridoxine,
para-aminobenzoic acid, folic acid, riboflavin, and Vitamins A, B,
C, D, and E. See, for example, Alfenore et al., Improving ethanol
production and viability of Saccharomyces cerevisiae by a vitamin
feeding strategy during fed-batch process, Springer-Verlag (2002),
which is hereby incorporated by reference. Examples of minerals
include minerals and mineral salts that can supply nutrients
comprising P, K, Mg, S, Ca, Fe, Zn, Mn, and Cu.
[0283] Fermentation Products:
[0284] A fermentation product can be any substance derived from the
fermentation. The fermentation product can be, without limitation,
an alcohol (e.g., arabinitol, butanol, ethanol, glycerol, methanol,
1,3-propanediol, sorbitol, and xylitol); 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, propionic acid, succinic acid, and
xylonic acid); a ketone (e.g., acetone); an amino acid (e.g.,
aspartic acid, glutamic acid, glycine, lysine, serine, and
threonine); and a gas (e.g., methane, hydrogen (H.sub.2), carbon
dioxide (CO.sub.2), and carbon monoxide (CO)). The fermentation
product can also be protein as a high value product.
[0285] 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 arabinitol. In another more
preferred aspect, the alcohol is butanol. In another more preferred
aspect, the alcohol is ethanol. In another more preferred aspect,
the alcohol is glycerol. In another more preferred aspect, the
alcohol is methanol. 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.
[0286] 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.
[0287] 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.
[0288] 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.
[0289] 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.
[0290] Recovery.
[0291] 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.
Signal Peptide
[0292] The present invention also relates to nucleic acid
constructs comprising a gene encoding a protein, wherein the gene
is operably linked to a nucleotide sequence encoding a signal
peptide comprising or consisting of amino acids 1 to 20 of SEQ ID
NO: 2 or amino acids 1 to 18 of SEQ ID NO: 4, wherein the gene is
foreign to the nucleotide sequence.
[0293] In a preferred aspect, the nucleotide sequence comprises or
consists of nucleotides 1 to 60 of SEQ ID NO: 1. In another
preferred aspect, the nucleotide sequence comprises or consists of
nucleotides 1 to 54 of SEQ ID NO: 3.
[0294] The present invention also relates to recombinant expression
vectors and recombinant host cells comprising such nucleic acid
constructs.
[0295] The present invention also relates to methods of producing a
protein comprising (a) cultivating such a recombinant host cell
under conditions suitable for production of the protein; and (b)
recovering the protein.
[0296] 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 proteins. The term "protein" also encompasses
two or more polypeptides combined to form the encoded product. The
proteins also include hybrid polypeptides that comprise a
combination of partial or complete polypeptide sequences obtained
from at least two different proteins wherein one or more (several)
may be heterologous or native to the host cell. Proteins further
include naturally occurring allelic and engineered variations of
the above mentioned proteins and hybrid proteins.
[0297] Preferably, the protein is a hormone or variant thereof,
enzyme, receptor or portion thereof, antibody or portion thereof,
or reporter. In a more preferred aspect, the protein is an
oxidoreductase, transferase, hydrolase, lyase, isomerase, or
ligase. In an even more preferred aspect, the protein is an
aminopeptidase, amylase, carbohydrase, carboxypeptidase, catalase,
cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase,
deoxyribonuclease, esterase, alpha-galactosidase,
beta-galactosidase, glucoamylase, alpha-glucosidase,
beta-glucosidase, invertase, laccase, another lipase, mannosidase,
mutanase, oxidase, pectinolytic enzyme, peroxidase, phytase,
polyphenoloxidase, proteolytic enzyme, ribonuclease,
transglutaminase or endoglucanase.
[0298] The gene may be obtained from any prokaryotic, eukaryotic,
or other source.
[0299] The present invention is further described by the following
examples that should not be construed as limiting the scope of the
invention.
EXAMPLES
Materials
[0300] Chemicals used as buffers and substrates were commercial
products of at least reagent grade.
Strain
[0301] Thielavia terrestris NRRL 8126 was used as the source of
genes encoding Family 16 polypeptides having endoglucanase
activity.
Media
[0302] PDA plates were composed per liter of 39 grams of potato
dextrose agar.
[0303] NNCYP medium was composed per liter of 5.0 g of
NH.sub.4NO.sub.3, 0.5 g of MgSO.sub.4.7H.sub.2O, 0.3 g of
CaCl.sub.2, 2.5 g of citric acid, 1.0 g of Bacto Peptone, 5.0 g of
yeast extract, 1 ml of COVE trace metals solution, and sufficient
K.sub.2HPO.sub.4 to achieve a final pH of 5.4.
[0304] NNCYPmod medium was composed per liter of 1.0 g of NaCl, 5.0
g of NH.sub.4NO.sub.3, 0.2 g of MgSO.sub.4.7H.sub.2O, 0.2 g of
CaCl.sub.2, 2.0 g of citric acid, 1.0 g of Bacto Peptone, 5.0 g of
yeast extract, 1 ml of COVE trace metals solution, and sufficient
K.sub.2HPO.sub.4 to achieve a final pH of approximately 5.4.
[0305] COVE trace metals solution was composed per liter 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.2.2H.sub.2O, and 10
g of ZnSO.sub.4.7H.sub.2O.
[0306] LB plates were composed per liter of 10 g of tryptone, 5 g
of yeast extract, 5 g of sodium chloride, and 15 g of Bacto
Agar.
[0307] MDU2BP medium was composed per liter of 45 g of maltose, 1 g
of MgSO.sub.4.7H.sub.2O, 1 g of NaCl, 2 g of K.sub.2HSO.sub.4, 12 g
of KH.sub.2PO.sub.4, 2 g of urea, and 500 .mu.l of AMG trace metals
solution; the pH was adjusted to 5.0 and then filter sterilized
with a 0.22 .mu.m filtering unit.
[0308] AMG trace metals solution was composed per liter 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, and 3 g of citric acid.
[0309] SOC medium was composed of 2% tryptone, 0.5% yeast extract,
10 mM NaCl, 2.5 mM KCl, 10 mM MgCl.sub.2, and 10 mM MgSO.sub.4,
sterilized by autoclaving and then filter-sterilized glucose was
added to 20 mM.
[0310] Freezing medium was composed of 60% SOC medium and 40%
glycerol.
[0311] 2.times.YT medium was composed per liter of 16 g of
tryptone, 10 g of yeast extract, 5 g of NaCl, and 15 g of Bacto
agar.
Example 1
Expressed Sequence Tags (EST) cDNA Library Construction
[0312] Thielavia terrestris NRRL 8126 was cultivated in 50 ml of
NNCYPmod medium supplemented with 1% glucose in a 250 ml flask at
45.degree. C. for 24 hours with shaking at 200 rpm. A two ml
aliquot from the 24-hour liquid culture of Thielavia terrestris
NRRL 8126 was used to seed a 500 ml flask containing 100 ml of
NNCYPmod medium supplemented with 2% SIGMACELL.RTM. 20 (Sigma
Chemical Co., St. Louis, Mo., USA). The culture was incubated at
45.degree. C. for 3 days with shaking at 200 rpm. The mycelia were
harvested by filtration through a funnel with a glass fiber
prefilter (Nalgene, Rochester, N.Y., USA), washed twice with 10 mM
Tris-HCl-1 mM EDTA pH 8 (TE), and quick frozen in liquid
nitrogen.
[0313] Total RNA was isolated using the following method. Frozen
mycelia of Thielavia terrestris NRRL 8126 were ground in an
electric coffee grinder. The ground material was mixed 1:1 v/v with
20 ml of FENAZOL.TM. (Ambion, Inc., Austin, Tex., USA) in a 50 ml
FALCON.RTM. tube. Once the mycelia were suspended, they were
extracted with chloroform and three times with a mixture of
phenol-chloroform-isoamyl alcohol 25:24:1 v/v/v. From the resulting
aqueous phase, the RNA was precipitated by adding 1/10 volume of 3
M sodium acetate pH 5.2 and 1.25 volumes of isopropanol. The
precipitated RNA was recovered by centrifugation at 12,000.times.g
for 30 minutes at 4.degree. C. The final pellet was washed with
cold 70% ethanol, air dried, and resuspended in 500 ml of
diethylpyrocarbonate treated water (DEPC-water).
[0314] The quality and quantity of the purified RNA was assessed
with an AGILENT.RTM. 2100 Bioanalyzer (Agilent Technologies, Inc.,
Palo Alto, Calif., USA). Polyadenylated mRNA was isolated from 360
.mu.g of total RNA with the aid of a POLY(A)PURIST.TM. Magnetic Kit
(Ambion, Inc., Austin, Tex., USA) according to the manufacturer's
instructions.
[0315] To create the cDNA library, a CLONEMINER.TM. Kit (Invitrogen
Corp., Carlsbad, Calif., USA) was employed to construct a
directional library that does not require the use of restriction
enzyme cloning, thereby reducing the number of chimeric clones and
size bias.
[0316] To insure the successful synthesis of the cDNA, two
reactions were performed in parallel with two different
concentrations of mRNA (2.2 and 4.4 .mu.g of poly (A).sup.+ mRNA).
The mRNA samples were mixed with a Biotin-attB2-Oligo(dt) primer
(Invitrogen Corp., Carlsbad, Calif., USA), 1.times. first strand
buffer (Invitrogen Corp., Carlsbad, Calif., USA), 2 .mu.l of 0.1 M
dithiothreitol (DTT), 10 mM of each dNTP, and water to a final
volume of 18 and 16 .mu.l, respectively.
[0317] The reaction mixtures were mixed and then 2 and 4 .mu.l of
SUPERSCRIPT.TM. reverse transcriptase (Invitrogen Corp., Carlsbad,
Calif., USA) were added. The reaction mixtures were incubated at
45.degree. C. for 60 minutes to synthesize the first complementary
strand. For second strand synthesis, to each first strand reaction
was added 30 .mu.l of 5.times. second strand buffer (Invitrogen
Corp., Carlsbad, Calif., USA), 3 .mu.l of 10 mM of each dNTP, 10
units of E. coli DNA ligase (Invitrogen Corp., Carlsbad, Calif.,
USA), 40 units of E. coli DNA polymerase I (Invitrogen Corp.,
Carlsbad, Calif., USA), and 2 units of E. coli RNase H (Invitrogen
Corp., Carlsbad, Calif., USA) in a total volume of 150 .mu.l. The
mixtures were then incubated at 16.degree. C. for two hours. After
the two-hour incubation 2 .mu.l of T4 DNA polymerase (Invitrogen
Corp., Carlsbad, Calif., USA) were added to each reaction and
incubated at 16.degree. C. for 5 minutes to create a bunt-ended
cDNA. The cDNA reactions were extracted with a mixture of
phenol-chloroform-isoamyl alcohol 25:24:1 v/v/v and precipitated in
the presence of 20 .mu.g of glycogen, 120 .mu.l of 5 M ammonium
acetate, and 660 .mu.l of ethanol. After centrifugation at
12,000.times.g for 30 minutes at 4.degree. C., the cDNA pellets
were washed with cold 70% ethanol, dried under vacuum for 2-3
minutes, and resuspended in 18 .mu.l of DEPC-water. To each
resuspended cDNA sample were added 10 .mu.l of 5.times. adapted
buffer (Invitrogen, Carlsbad, Calif., USA), 10 .mu.g of each attB1
adapter (Invitrogen, Carlsbad, Calif., USA), 7 .mu.l of 0.1 M DTT,
and 5 units of T4 DNA ligase (Invitrogen, Carlsbad, Calif.,
USA).
[0318] Ligation reactions were incubated overnight at 16.degree. C.
Excess adapters were removed by size-exclusion chromatography in 1
ml of SEPHACRYL.TM. S-500 HR resin (Amersham Biosciences,
Piscataway, N.J., USA). Column fractions were collected according
to the CLONEMINER.TM. Kit's instructions and fractions 3 to 14 were
analyzed with an AGILENT.RTM. 2100 Bioanalyzer to determine the
fraction at which the attB1 adapters started to elute. This
analysis showed that the adapters began eluting around fraction 10
or 11. For the first library fractions 6-11 were pooled and for the
second library fractions 4-11 were pooled.
[0319] Cloning of the cDNA was performed by homologous DNA
recombination according to the GATEWAY.RTM. System protocol
(Invitrogen Corp., Carlsbad, Calif., USA) using BP CLONASE.TM.
(Invitrogen Corp., Carlsbad, Calif., USA) as the recombinase. Each
BP CLONASE.TM. recombination reaction contained approximately 70 ng
of attB-flanked-cDNA, 250 ng of pDONR.TM.222, 2 .mu.l of 5.times.BP
CLONASE.TM. buffer, 2 .mu.l of TE, and 3 .mu.l of BP CLONASE.TM.
All reagents were obtained from Invitrogen, Carlsbad, Calif., USA.
Recombination reactions were incubated at 25.degree. C.
overnight.
[0320] Heat-inactivated BP recombination reactions were then
divided into 6 aliquots and electroporated into ELECTROMAX.TM. E.
coli DH10B electrocompetent cells (Invitrogen Corp., Carlsbad,
Calif., USA) using a GENE PULSER.TM. (Bio-Rad Laboratories, Inc.,
Hercules, Calif., USA) with the following parameters: Voltage: 2.0
kV; Resistance: 200 SI; and Capacity: 25 .mu.F. Electrophorated
cells were resuspended in 1 ml of SOC medium and incubated at
37.degree. C. for 60 minutes with constant shaking at 200 rpm.
After the incubation period, the transformed cells were pooled and
mixed 1:1 with freezing medium. A 200 .mu.l aliquot was removed for
library titration and then the rest of each library was aliquoted
into 1.8 ml cryovials (Wheaton Science Products, Millville, N.J.,
USA) and stored frozen at -80.degree. C.
[0321] Four serial dilutions of each library were prepared: 1/100,
1/1000, 1/10.sup.4, and 1/10.sup.5. From each dilution 100 .mu.l
were plated onto 150 mm LB plates supplemented with 50 .mu.g of
kanamycin per ml and incubated at 37.degree. C. overnight. The
number of colonies on each dilution plate was counted and used to
calculate the total number of transformants in each library.
[0322] The first library contained approximately 5.4 million
independent clones and the second library contained approximately 9
million independent clones.
Example 2
Template Preparation and Nucleotide Sequencing of cDNA Clones
[0323] Aliquots from both libraries described in Example 1 were
mixed and plated onto 25.times.25 cm LB plates supplemented with 50
.mu.g of kanamycin per ml. Individual colonies were arrayed onto
96-well plates containing 100 .mu.l of LB medium supplemented with
50 .mu.g of kanamycin per ml with the aid of a QPix Robot (Genetix
Inc., Boston, Mass., USA). Forty-five 96-well plates were obtained
for a total of 4320 individual clones. The plates were incubated
overnight at 37.degree. C. with shaking at 200 rpm. After
incubation, 100 .mu.l of sterile 50% glycerol was added to each
well. The transformants were replicated with the aid of a 96-pin
tool (Boekel, Feasterville, Pa., USA) into secondary, deep-dish
96-well microculture plates (Advanced Genetic Technologies
Corporation, Gaithersburg, Md., USA) containing 1 ml of MAGNIFICENT
BROTH.TM. (MacConnell Research, San Diego, Calif., USA)
supplemented with 50 .mu.g of kanamycin per ml in each well. The
primary microtiter plates were stored frozen at -80.degree. C. The
secondary deep-dish plates were incubated at 37.degree. C.
overnight with vigorous agitation at 300 rpm on a rotary shaker. To
prevent spilling and cross-contamination, and to allow sufficient
aeration, each secondary culture plate was covered with a
polypropylene pad (Advanced Genetic Technologies Corporation,
Gaithersburg, Md., USA) and a plastic microtiter dish cover.
Plasmid DNA was prepared with a Robot-Smart 384 (MWG Biotech Inc.,
High Point, N.C., USA) and a MONTAGE.TM. Plasmid Miniprep Kit
(Millipore, Billerica, Mass., USA).
[0324] Sequencing reactions were performed using BIGDYE.RTM.
(Applied Biosystems, Inc., Foster City, Calif., USA) terminator
chemistry (Giesecke et al., 1992, Journal of Virology Methods 38:
47-60) and a M13 Forward (-20) sequencing primer:
TABLE-US-00001 (SEQ ID NO: 7) 5'-GTAAAACGACGGCCAG-3'
[0325] The sequencing reactions were performed in a 384-well format
with a Robot-Smart 384. Terminator removal was performed with a
MULTISCREEN.RTM. Seq384 Sequencing Clean-up Kit (Millipore,
Billerica, Mass., USA). Reactions contained 6 .mu.l of plasmid DNA
and 4 .mu.l of sequencing master-mix (Applied Biosystems, Foster
City, Calif., USA) containing 1 .mu.l of 5.times. sequencing buffer
(Millipore, Billerica, Mass., USA), 1 .mu.l of BIGDYE.RTM.
terminator (Applied Biosystems, Inc., Foster City, Calif., USA),
1.6 .mu.moles of M13 Forward primer, and 1 .mu.l of water.
Single-pass DNA sequencing was performed with an ABI PRISM
Automated DNA Sequencer Model 3700 (Applied Biosystems, Foster
City, Calif., USA).
Example 3
Analysis of DNA Sequence Data of cDNA Clones
[0326] Base calling, quality value assignment, and vector trimming
were performed with the assistance of PHRED/PHRAP software
(University of Washington, Seattle, Wash., USA). Clustering
analysis of the ESTs was performed with a Transcript Assembler v.
2.6.2. (Paracel, Inc., Pasadena, Calif., USA). Analysis of the EST
clustering indicated the presence of 395 independent clusters.
[0327] Sequence homology analysis of the assembled EST sequences
against the PIR and other databases was performed with the Blastx
program (Altschul et. al., 1990, J. Mol. Biol. 215:403-410) on a
32-node Linux cluster (Paracel, Inc., Pasadena, Calif., USA) using
the BLOSUM 62 matrix (Henikoff, 1992, Proc. Natl. Acad. Sci. USA
89: 10915-10919) From these, 246 had hits to known genes in various
protein databases and 149 had no significant hits against these
databases. Among these 246 genes, 13 had hits against well
characterized homologues of glycosyl hydrolase genes.
Example 4
Identification of cDNA Clones Encoding Thielavia terrestris Family
16A and 16B Endoglucanases
[0328] A cDNA clone encoding a Thielavia terrestris Family 16A
endoglucanase was initially identified by sequence homology to a
putative glycosyl hydrolase Family 16 from Schizosaccharolyces
pombe (GENBANK.RTM. accession number NP.sub.--595680).
[0329] After this initial identification, a clone designated
Tter30G12 was retrieved from the original frozen stock plate and
streaked onto a LB plate supplemented with 50 .mu.g of kanamycin
per ml. The plate was incubated overnight at 37.degree. C. and a
single colony from the plate was used to inoculate 3 ml of LB
medium supplemented with 150 .mu.g of kanamycin per ml. The liquid
culture was incubated overnight at 37.degree. C. and plasmid DNA
was prepared with a BIOROBOT.RTM. 9600 (QIAGEN Inc., Valencia,
Calif., USA). Clone Tter30G12 plasmid DNA was sequenced again with
BIGDYE.RTM. terminator chemistry as described above, using the M13
forward primer, the M13 reverse primer, and a Poly-T primer shown
below to sequence the 3' end of the clone.
TABLE-US-00002 (SEQ ID NO: 8) 5'-TTTTTTTTTTTTTTTTTTTTTTTVN-3',
where V = G, A, C and N = G, A, C, T.
[0330] Analysis of the deduced amino acid sequence of clone 30G12
with the Interproscan program (Zdobnov and Apweiler, 2001,
Bioinformatics 17: 847-8) showed that the amino acid sequence
contained the sequence signature of the concanavalin A-like
glucanase. This sequence signature known as the InterPro:IPR008985
was found 88 amino acids from the starting amino acid methionine.
This region also contained the signature sequence for the active
site of the glycosyl hydrolase Family 16, InterPro:IPR008263
confirming that clone Tter30G12 encoded a Family 16
endoglucanase.
[0331] The cDNA sequence (SEQ ID NO: 1) and deduced amino acid
sequence (SEQ ID NO: 2) are shown in FIGS. 1A and 1B. The cDNA
clone encodes a polypeptide of 797 amino acids. The % G+C content
of the full-length coding region is 64.5% and of the mature protein
coding region (nucleotides 61 to 1428 of SEQ ID NO: 1) is 65%.
Using the SignalP software program (Nielsen et al., 1997, Protein
Engineering 10: 1-6), a signal peptide of 20 residues was
predicted. The predicted mature protein contains 777 amino acids
with a molecular mass of 80.7 kDa.
[0332] A comparative pairwise global alignment of amino acid
sequences 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
Thielavia terrestris endoglucanase gene shared 34% identity
(excluding gaps) to the deduced amino acid sequence of identity to
Phaffia rhodozyma beta-glucanase Family 16 sequence
(GENESEQP:AAW77311; WO 98/36056).
[0333] A cDNA clone encoding a Thielavia terrestris Family 16B
endoglucanase was initially identified by sequence homology to a
glycosyl hydrolase Family 16 from Rhodothermus marinus
(UNIPROT.RTM. accession number P45798).
[0334] After this initial identification, a clone designated
Tter07B12 was retrieved from the original frozen stock plate and
streaked onto a LB plate supplemented with 50 .mu.g of kanamycin
per ml. Plasmid DNA was prepared from a culture of this E. coli
strain and sequenced to completion using the same procedure
described above.
[0335] Analysis of the deduced amino acid sequence of clone 07B12
with the Interproscan program (Zdobnov and Apweiler, 2001, supra)
showed that the amino acid sequence contained the sequence
signature of the concanavalin A-like glucanase. This sequence
signature known as the InterPro:IPR008985 was found 58 amino acids
from the starting amino acid methionine. This region also contained
the signature sequence for the active site of the glycosyl
hydrolase Family 16, InterPro:IPR008263 confirming that clone
Tter07B12 encoded a Family 16 endoglucanase.
[0336] The cDNA sequence (SEQ ID NO: 3) and deduced amino acid
sequence (SEQ ID NO: 4) are shown in FIG. 2. The cDNA clone encodes
a polypeptide of 285 amino acids. The % G+C content of the
full-length coding region is 68.4% and of the mature protein coding
region (nucleotides 55 to 855 of SEQ ID NO: 3) is 68.7%. Using the
SignalP software program (Nielsen et al., 1997, supra), a signal
peptide of 18 residues was predicted. The predicted mature protein
contains 267 amino acids with a molecular mass of 28.2 kDa.
[0337] A comparative pairwise global alignment of amino acid
sequences was determined using the Clustal W method (Higgins, 1989,
supra) with the AlignX module of Vector NTI Advance 10.3 software
(Invitrogen, Carlsbad, Calif., USA) and a blosum62mt2 scoring
matrix and the following multiple alignment parameters: K-tuple
size 1; best diagonals 5; window size 5; gap penalty 5; gap opening
penalty 10; gap extension penalty 0.1. The alignment showed that
the deduced mature amino acid sequence of the Thielavia terrestris
GH16B gene shared 58% identity Trichoderma harzianum
endo-1,3(4)-beta-glucanase Family 16 sequence (GENESEQP: AAR88406;
WO 95/31533).
[0338] Once the identities of clones Tter30G12 and Tter07B12 were
confirmed, a 0.5 .mu.l aliquot of plasmid DNA from each clone
designated pTter16A (FIG. 3) and pTter16B (FIG. 4) was transferred
into a separate vial of E. coli TOP10 cells (Invitrogen Corp.,
Carlsbad, Calif., USA), gently mixed, and incubated on ice for 10
minutes. The cells were then heat-shocked at 42.degree. C. for 30
seconds and incubated again on ice for 2 minutes. The cells were
resuspended in 250 .mu.l of SOC medium and incubated at 37.degree.
C. for 60 minutes with constant shaking at 200 rpm. After the
incubation period, two 30 .mu.l aliquots were plated onto LB plates
supplemented with 50 .mu.g of kanamycin per ml and incubated
overnight at 37.degree. C. The next day a single colony was picked
from each transformation and streaked onto three 1.8 ml cryovials
containing about 1.5 mls of LB agarose supplemented with 50 .mu.g
of kanamycin per ml. The vials were sealed with PETRISEAL.TM.
(Diversified Biotech, Boston Mass., USA) and deposited with the
Agricultural Research Service Patent Culture Collection, Northern
Regional Research Center, Peoria, Ill., USA, as NRRL B-50081
(pTter16A) and NRRL B-50082 (pTter16B) with a deposit date of Nov.
30, 2007.
Deposits of Biological Material
[0339] The following biological materials have been deposited under
the terms of the Budapest Treaty with the Agricultural Research
Service Patent Culture Collection (NRRL), Northern Regional
Research Center, 1815 University Street, Peoria, Ill., USA, and
given the following accession numbers:
TABLE-US-00003 Deposit Accession Number Date of Deposit E. coli
pTter16A NRRL B-50081 Nov. 30, 2007 E. coli pTter16B NRRL B-50082
Nov. 30, 2007
[0340] The strains have been deposited under conditions that assure
that access to the cultures will be available during the pendency
of this patent application to one determined by foreign patent laws
to be entitled thereto. The deposits represent substantially pure
cultures of the deposited strains. The deposits are available as
required by foreign patent laws in countries wherein counterparts
of the subject application, or its progeny are filed. However, it
should be understood that the availability of a deposit does not
constitute a license to practice the subject invention in
derogation of patent rights granted by governmental action.
[0341] 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
812394DNAThielavia terrestris 1atggcctcct ccatcgtccg gctgggcgcc
gcggctgccc tagcctacgg ctccggtgct 60gccgccctcc agtcctacca actctcggag
tcatacaacc ccaccaactt ctttgacaag 120ttcaacttct tcgatggcaa
ggaccccaac agtggattcg ttcagtacag aaacagaacc 180tacgccagct
cccatggcct catccagacg gggaccaacg acgtgaccat ccgcgtcgat
240acgaccggca ccgaccagaa cggacgcagc agcgtccggc tggagagtat
caacacgtac 300aattcgggcc tgttcatcgc cgatttcacc cacctgccct
acgctccctg tggtgcttgg 360ccagccttct ggatggtggg ccccaactgg
cccacggatg gcgagatcga catctacgag 420ggctggaacc tggccccgac
gaacaaggtt gtgctgcaca cggatagccc gaccatctcg 480ggcacgtgcc
ggatccagca gggcgacttc actggcaccc tggagtaccc cgactgctgg
540accaacgacc cgacccagcc cagcaatgcg ggttgtgccg ttgaggagaa
gaacggcctg 600ttcgggaatg ccgccggcgg cgtctacgct accgagtggc
aggaagacag catcaaggtc 660tggagctggt ctcataacag cgtccccgct
gatgtcacga gtggcaaacc caacccggcg 720aactggggta agcccactct
cgccgcgggc agccagtgcg atgtcaagtc ggcattccgc 780gacatgcgct
tgatcctgaa tatcaacttc tgcggtgacg ctgccggcaa cacctggccc
840ggatctgcgt gccagaagag taccaatgtc cccgcgtgct atacctacgt
gcagtggaac 900ccgcacgtct acaacagcac gttctggtcc gtccggagca
tcaaggtgta ccagctcggg 960gagtcgcaga ccacgaccac cacgaccagg
tcctcgactt cgactacgac gtcgaccagg 1020tcctcgactt cgactacgac
gtcggccagg tcctcgactt cgaccacgac gtcggccagg 1080tcctcgactt
cgaccacgac tacctctttg acttcgacca cgtcttcaat ttcgaccacg
1140accacgtcct cgacctcgac cagcacgtcg acggcgtccc ccacctccac
gtctaccagc 1200acctcggccg ctgtgacctc ggccatcact tcagagactg
ccacggcgac tgccacggag 1260accgccacgg agaccgagag cgactgccct
gatgacaacg ccacctccac cacgaccggt 1320gctgccacca cggagactgg
gtcgagcaca agcacgacgg ctaccgcgac tgccactgag 1380accgagagcg
actgccctga cgacaacgcc acctccacca cgaccggtgc tgccaccacg
1440gagactgggt caagcacgac ggctaccgcg actgccactg agaccgagag
cgactgcccc 1500gatgacaacg ccacctccac cacgaccggt gctgccacca
cggagactgg gtcgagcacg 1560acggctaccg cgaccgggac cgctgcgcct
tccagctcgg tccccgacgt gaccgatatt 1620gtcactgcta ccgcgaccgc
gaccggaggc ttcaccacgt cgaccattta caccacgatc 1680acgtcgacca
ttacgtcgtg cgcgccgacc gtgaccaact gccccgcgcg caccgttacg
1740tcggtcatcc ccatcggcac gaccgtctgc cccgtgaccg aggccagctc
ggcgccggtg 1800acgacggaga cggccaccct gccggagggc tggaccacgt
cgaccgtcta ctccaccatc 1860acgtacacga tcacgtcgtg cgcggcgtcg
gtcaccaact gcccggcccg cgtgaccacg 1920tcggtggttg ccgtcggcac
caccgtctgc ccgatcgcgt cgttcagcgc cagccccagc 1980accagcgccg
gcgtcaaccc cagcgaggtc gtcagcacgg tgcgctcctt caccaccgtc
2040tccaagacga cgaccgtcgt gctgccccgc cccagcgggt cggccgccgg
gtcgtcgtca 2100tcgccggtcc tcgtcgccag cccgggcccc ctcctcctcc
gccggctcct ctcccgcctc 2160cccctccgtg ccgagcgtgg tggtcggtgg
caacaacggc accgcctccg tgccgccggt 2220ccaggtcgtt accgccagcc
agccgggcgc cagcaccagc ctgtcgcagc cggtgactgc 2280cggcggcggc
cgcatgatgg cgggcagcgg gctgatggcg ctgctgcttg gtgcggccgc
2340ggcgatgttg atctaaggtg tgttgctgtt gcttgcttgc gggggtgggt gtag
23942797PRTThielavia terrestris 2Met Ala Ser Ser Ile Val Arg Leu
Gly Ala Ala Ala Ala Leu Ala Tyr 1 5 10 15 Gly Ser Gly Ala Ala Ala
Leu Gln Ser Tyr Gln Leu Ser Glu Ser Tyr 20 25 30 Asn Pro Thr Asn
Phe Phe Asp Lys Phe Asn Phe Phe Asp Gly Lys Asp 35 40 45 Pro Asn
Ser Gly Phe Val Gln Tyr Arg Asn Arg Thr Tyr Ala Ser Ser 50 55 60
His Gly Leu Ile Gln Thr Gly Thr Asn Asp Val Thr Ile Arg Val Asp 65
70 75 80 Thr Thr Gly Thr Asp Gln Asn Gly Arg Ser Ser Val Arg Leu
Glu Ser 85 90 95 Ile Asn Thr Tyr Asn Ser Gly Leu Phe Ile Ala Asp
Phe Thr His Leu 100 105 110 Pro Tyr Ala Pro Cys Gly Ala Trp Pro Ala
Phe Trp Met Val Gly Pro 115 120 125 Asn Trp Pro Thr Asp Gly Glu Ile
Asp Ile Tyr Glu Gly Trp Asn Leu 130 135 140 Ala Pro Thr Asn Lys Val
Val Leu His Thr Asp Ser Pro Thr Ile Ser 145 150 155 160 Gly Thr Cys
Arg Ile Gln Gln Gly Asp Phe Thr Gly Thr Leu Glu Tyr 165 170 175 Pro
Asp Cys Trp Thr Asn Asp Pro Thr Gln Pro Ser Asn Ala Gly Cys 180 185
190 Ala Val Glu Glu Lys Asn Gly Leu Phe Gly Asn Ala Ala Gly Gly Val
195 200 205 Tyr Ala Thr Glu Trp Gln Glu Asp Ser Ile Lys Val Trp Ser
Trp Ser 210 215 220 His Asn Ser Val Pro Ala Asp Val Thr Ser Gly Lys
Pro Asn Pro Ala 225 230 235 240 Asn Trp Gly Lys Pro Thr Leu Ala Ala
Gly Ser Gln Cys Asp Val Lys 245 250 255 Ser Ala Phe Arg Asp Met Arg
Leu Ile Leu Asn Ile Asn Phe Cys Gly 260 265 270 Asp Ala Ala Gly Asn
Thr Trp Pro Gly Ser Ala Cys Gln Lys Ser Thr 275 280 285 Asn Val Pro
Ala Cys Tyr Thr Tyr Val Gln Trp Asn Pro His Val Tyr 290 295 300 Asn
Ser Thr Phe Trp Ser Val Arg Ser Ile Lys Val Tyr Gln Leu Gly 305 310
315 320 Glu Ser Gln Thr Thr Thr Thr Thr Thr Arg Ser Ser Thr Ser Thr
Thr 325 330 335 Thr Ser Thr Arg Ser Ser Thr Ser Thr Thr Thr Ser Ala
Arg Ser Ser 340 345 350 Thr Ser Thr Thr Thr Ser Ala Arg Ser Ser Thr
Ser Thr Thr Thr Thr 355 360 365 Ser Leu Thr Ser Thr Thr Ser Ser Ile
Ser Thr Thr Thr Thr Ser Ser 370 375 380 Thr Ser Thr Ser Thr Ser Thr
Ala Ser Pro Thr Ser Thr Ser Thr Ser 385 390 395 400 Thr Ser Ala Ala
Val Thr Ser Ala Ile Thr Ser Glu Thr Ala Thr Ala 405 410 415 Thr Ala
Thr Glu Thr Ala Thr Glu Thr Glu Ser Asp Cys Pro Asp Asp 420 425 430
Asn Ala Thr Ser Thr Thr Thr Gly Ala Ala Thr Thr Glu Thr Gly Ser 435
440 445 Ser Thr Ser Thr Thr Ala Thr Ala Thr Ala Thr Glu Thr Glu Ser
Asp 450 455 460 Cys Pro Asp Asp Asn Ala Thr Ser Thr Thr Thr Gly Ala
Ala Thr Thr 465 470 475 480 Glu Thr Gly Ser Ser Thr Thr Ala Thr Ala
Thr Ala Thr Glu Thr Glu 485 490 495 Ser Asp Cys Pro Asp Asp Asn Ala
Thr Ser Thr Thr Thr Gly Ala Ala 500 505 510 Thr Thr Glu Thr Gly Ser
Ser Thr Thr Ala Thr Ala Thr Gly Thr Ala 515 520 525 Ala Pro Ser Ser
Ser Val Pro Asp Val Thr Asp Ile Val Thr Ala Thr 530 535 540 Ala Thr
Ala Thr Gly Gly Phe Thr Thr Ser Thr Ile Tyr Thr Thr Ile 545 550 555
560 Thr Ser Thr Ile Thr Ser Cys Ala Pro Thr Val Thr Asn Cys Pro Ala
565 570 575 Arg Thr Val Thr Ser Val Ile Pro Ile Gly Thr Thr Val Cys
Pro Val 580 585 590 Thr Glu Ala Ser Ser Ala Pro Val Thr Thr Glu Thr
Ala Thr Leu Pro 595 600 605 Glu Gly Trp Thr Thr Ser Thr Val Tyr Ser
Thr Ile Thr Tyr Thr Ile 610 615 620 Thr Ser Cys Ala Ala Ser Val Thr
Asn Cys Pro Ala Arg Val Thr Thr 625 630 635 640 Ser Val Val Ala Val
Gly Thr Thr Val Cys Pro Ile Ala Ser Phe Ser 645 650 655 Ala Ser Pro
Ser Thr Ser Ala Gly Val Asn Pro Ser Glu Val Val Ser 660 665 670 Thr
Val Arg Ser Phe Thr Thr Val Ser Lys Thr Thr Thr Val Val Leu 675 680
685 Pro Arg Pro Ser Gly Ser Ala Ala Gly Ser Ser Ser Ser Pro Val Leu
690 695 700 Val Ala Ser Pro Gly Pro Leu Leu Leu Arg Arg Leu Leu Ser
Arg Leu 705 710 715 720 Pro Leu Arg Ala Glu Arg Gly Gly Arg Trp Gln
Gln Arg His Arg Leu 725 730 735 Arg Ala Ala Gly Pro Gly Arg Tyr Arg
Gln Pro Ala Gly Arg Gln His 740 745 750 Gln Pro Val Ala Ala Gly Asp
Cys Arg Arg Arg Pro His Asp Gly Gly 755 760 765 Gln Arg Ala Asp Gly
Ala Ala Ala Trp Cys Gly Arg Gly Asp Val Asp 770 775 780 Leu Arg Cys
Val Ala Val Ala Cys Leu Arg Gly Trp Val 785 790 795
3858DNAThielavia terrestris 3atgcgtgcca gcagcctcac cgcgctgtcc
ttcttttcgt cccttgttgc ggctctcccg 60gccccagcct acccgggata cacactcctc
tggtccgatg aattcgcggg ccccgccggc 120tcgagccccg accagagccg
ctggaacatc atcaccaatg tccacaccaa ccacgaggtc 180gagacctaca
ccacgtcgaa ccaaaacctc cagatctccg gcggcggcac cgtccagatc
240gtcccgcgca agagcccctc ggggcagtgg acgtcggcgc gcatcgagtc
caaggcgacc 300ttcacccccg ccgcgggcaa ggtgaccatg ttcgaggcgg
cgatccggtt cggcacgaac 360ccggcgtcgc agaagcaggg catctggccg
gcgttctggc tgctgggcga cgcgatccac 420cacggcaccg cctggccgct
gtgcggcgag ctcgacatca tggagaccgt caacggcctg 480ccgaccggcc
acggcaccct gcactgcggc gccaccacca gcggcgggcc ctgcgccgag
540cccaccggcc gcaccggcgc cgtcgccctg cccgacgacg gctggcacac
ctggtcgctg 600caaatcgacc gcacgaatgc cgccggcggc tgggccggcg
aggtcatcaa gttcctcaag 660gacggccagg tgtttttcca ggtctccggc
gcgcagctcg gggacgaggg catctgggcc 720accctcgcgc actcgccgct
gtttatcatc ttgaacgtgg cggttggcgg tgactggcct 780ggtgctccca
acgccgcgac tgcggacggc tacggcagta tgatggaggt cgagtacgtc
840gccgtctact cgtcgtaa 8584285PRTThielavia terrestris 4Met Arg Ala
Ser Ser Leu Thr Ala Leu Ser Phe Phe Ser Ser Leu Val 1 5 10 15 Ala
Ala Leu Pro Ala Pro Ala Tyr Pro Gly Tyr Thr Leu Leu Trp Ser 20 25
30 Asp Glu Phe Ala Gly Pro Ala Gly Ser Ser Pro Asp Gln Ser Arg Trp
35 40 45 Asn Ile Ile Thr Asn Val His Thr Asn His Glu Val Glu Thr
Tyr Thr 50 55 60 Thr Ser Asn Gln Asn Leu Gln Ile Ser Gly Gly Gly
Thr Val Gln Ile 65 70 75 80 Val Pro Arg Lys Ser Pro Ser Gly Gln Trp
Thr Ser Ala Arg Ile Glu 85 90 95 Ser Lys Ala Thr Phe Thr Pro Ala
Ala Gly Lys Val Thr Met Phe Glu 100 105 110 Ala Ala Ile Arg Phe Gly
Thr Asn Pro Ala Ser Gln Lys Gln Gly Ile 115 120 125 Trp Pro Ala Phe
Trp Leu Leu Gly Asp Ala Ile His His Gly Thr Ala 130 135 140 Trp Pro
Leu Cys Gly Glu Leu Asp Ile Met Glu Thr Val Asn Gly Leu 145 150 155
160 Pro Thr Gly His Gly Thr Leu His Cys Gly Ala Thr Thr Ser Gly Gly
165 170 175 Pro Cys Ala Glu Pro Thr Gly Arg Thr Gly Ala Val Ala Leu
Pro Asp 180 185 190 Asp Gly Trp His Thr Trp Ser Leu Gln Ile Asp Arg
Thr Asn Ala Ala 195 200 205 Gly Gly Trp Ala Gly Glu Val Ile Lys Phe
Leu Lys Asp Gly Gln Val 210 215 220 Phe Phe Gln Val Ser Gly Ala Gln
Leu Gly Asp Glu Gly Ile Trp Ala 225 230 235 240 Thr Leu Ala His Ser
Pro Leu Phe Ile Ile Leu Asn Val Ala Val Gly 245 250 255 Gly Asp Trp
Pro Gly Ala Pro Asn Ala Ala Thr Ala Asp Gly Tyr Gly 260 265 270 Ser
Met Met Glu Val Glu Tyr Val Ala Val Tyr Ser Ser 275 280 285
532DNAThielavia terrestris 5tcgtcgggga caactttgta caaaaaagtt gg
32627DNAThielavia terrestris 6cccctgttga aacatgtttt ttcaacc
27716DNAThielavia terrestris 7gtaaaacgac ggccag 16825DNAThielavia
terrestrismisc_feature(24)..(24)V = A, C, OR G 8tttttttttt
tttttttttt tttvn 25
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