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United States Patent Application |
20120090054
|
Kind Code
|
A1
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Harris; Paul
;   et al.
|
April 12, 2012
|
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
for producing and using the polypeptides.
Inventors: |
Harris; Paul; (Carnation, WA)
; Valsenko; Elena; (Davis, CA)
; Zaretsky; Elizabeth; (Reno, NV)
; Kauppinnen; Marcus Sakari; (Smorum, DK)
|
Assignee: |
Novozymes, Inc.
Davis
CA
Novozymes A/S
Bagsvaerd
|
Serial No.:
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330523 |
Series Code:
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13
|
Filed:
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December 19, 2011 |
Current U.S. Class: |
800/298; 435/201; 435/252.3; 435/252.31; 435/252.33; 435/252.34; 435/252.35; 435/254.11; 435/254.3; 435/254.4; 435/254.5; 435/254.6; 435/254.7; 435/254.8; 435/255.1; 435/255.2; 435/255.4; 435/255.5; 435/255.6; 435/320.1; 435/325; 435/348; 435/419; 536/23.2 |
Class at Publication: |
800/298; 536/23.2; 435/320.1; 435/252.31; 435/252.3; 435/252.35; 435/252.33; 435/252.34; 435/325; 435/348; 435/419; 435/254.11; 435/255.1; 435/255.4; 435/255.6; 435/255.5; 435/255.2; 435/254.3; 435/254.7; 435/254.8; 435/254.4; 435/254.5; 435/254.6; 435/201 |
International Class: |
C12N 9/26 20060101 C12N009/26; C12N 15/63 20060101 C12N015/63; C12N 1/21 20060101 C12N001/21; A01H 5/00 20060101 A01H005/00; C12N 5/04 20060101 C12N005/04; C12N 1/15 20060101 C12N001/15; C12N 1/19 20060101 C12N001/19; C12N 15/56 20060101 C12N015/56; C12N 5/07 20100101 C12N005/07 |
Goverment Interests
REFERENCE TO A DEPOSIT OF BIOLOGICAL MATERIAL
[0002] This application contains a reference to deposits of biological
material which have been made at the Northern Regional Research Center
(NRRL) under the Budapest Treaty and assigned accession numbers
NRRLB-30898 and NRRLB-30901, which microbial deposits are incorporated
herein by reference.
Claims
1. An isolated polynucleotide encoding a polypeptide having endoglucanase
activity, selected from the group consisting of: (a) a polynucleotide
encoding a polypeptide comprising an amino acid sequence having at least
90% identity with the mature polypeptide of SEQ ID NO: 2 or the mature
polypeptide of SEQ ID NO: 4; (b) a polynucleotide encoding a polypeptide
which hybridizes under at least high stringency conditions with (i) the
mature polypeptide coding sequence of SEQ ID NO: 1 or the mature
polypeptide coding sequence of SEQ ID NO: 3, (ii) the cDNA sequence of
the mature polypeptide coding sequence of SEQ ID NO: 1 or the genomic DNA
sequence of the mature polypeptide coding sequence of SEQ ID NO: 3, or
(iii) the full-length complementary strand 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 polynucleotide encoding a polypeptide comprising a nucleotide sequence
having at least 90% identity with the mature polypeptide coding sequence
of SEQ ID NO: 1 or the mature polypeptide coding sequence of SEQ ID NO:
3.
2. The polynucleotide of claim 1, wherein the polypeptide having
endoglucanase activity comprises an amino acid sequence having at least
90% identity with the mature polypeptide of SEQ ID NO: 2 or the mature
polypeptide of SEQ ID NO: 4.
3. The polynucleotide of claim 2, wherein the polypeptide having
endoglucanase activity comprises an amino acid sequence having at least
95% identity with the mature polypeptide of SEQ ID NO: 2 or the mature
polypeptide of SEQ ID NO: 4.
4. The polynucleotide of claim 3, wherein the polypeptide having
endoglucanase activity comprises an amino acid sequence having at least
97% identity with the mature polypeptide of SEQ ID NO: 2 or the mature
polypeptide of SEQ ID NO: 4.
5. The polynucleotide of claim 1, which hybridizes under at least high
stringency conditions with (i) the mature polypeptide coding sequence of
SEQ ID NO: 1 or the mature polypeptide coding sequence of SEQ ID NO: 3,
(ii) the cDNA sequence of the mature polypeptide coding sequence of SEQ
ID NO: 1 or the genomic DNA sequence of the mature polypeptide coding
sequence of SEQ ID NO: 3, or (iii) the full-length complementary strand
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.
6. The polynucleotide of claim 5, which hybridizes under at least very
high stringency conditions with (i) the mature polypeptide coding
sequence of SEQ ID NO: 1 or the mature polypeptide coding sequence of SEQ
ID NO: 3, (ii) the cDNA sequence of the mature polypeptide coding
sequence of SEQ ID NO: 1 or the genomic DNA sequence of the mature
polypeptide coding sequence of SEQ ID NO: 3, or (iii) the full-length
complementary strand 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.
7. The polynucleotide of claim 1, which comprises a nucleotide sequence
having at least 90% identity with the mature polypeptide coding sequence
of SEQ ID NO: 1 or the mature polypeptide of SEQ ID NO: 3.
8. The polynucleotide of claim 7, which comprises a nucleotide sequence
having at least 95% identity with the mature polypeptide coding sequence
of SEQ ID NO: 1 or the mature polypeptide of SEQ ID NO: 3.
9. The polynucleotide of claim 8, which comprises a nucleotide sequence
having at least 97% identity with the mature polypeptide coding sequence
of SEQ ID NO: 1 or the mature polypeptide of SEQ ID NO: 3.
10. The polynucleotide of claim 1, which encodes a polypeptide having
endoglucanase activity comprising or consisting of the amino acid
sequence of SEQ ID NO: 2 or SEQ ID NO: 4.
11. The polynucleotide of claim 1, which encodes a polypeptide having
endoglucanase activity comprising or consisting of the mature polypeptide
of SEQ ID NO: 2 or the mature polypeptide of SEQ ID NO: 4.
12. The polynucleotide of claim 1, which is contained in plasmid pPH47
which is contained in E. coli NRRL B-30898 or plasmid pCIBG146 which is
contained in E. coli NRRL B-30901.
13. A nucleic acid construct comprising the polynucleotide of claim 1
operably linked to one or more control sequences that direct the
production of the polypeptide in an expression host.
14. A recombinant expression vector comprising the nucleic acid construct
of claim 13.
15. A recombinant host cell comprising the nucleic acid construct of
claim 13.
16. A method for producing a polypeptide having endoglucanase activity,
comprising: (a) cultivating the recombinant host cell of claim 15 under
conditions conducive for production of the polypeptide; and (b)
recovering the polypeptide.
17. 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.
18. A transgenic plant, plant part or plant cell, which has been
transformed with the polynucleotide of claim 1.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application Ser.
No. 12/294,506 filed on Sep. 25, 2008, which is a 35 U.S.C. 371 national
application of PCT/US07/65639 filed on Mar. 30, 2007 and claims priority
from U.S. provisional application Ser. No. 60/788,523 filed on Mar. 30,
2006, which applications are fully 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.
BACKGROUND OF THE INVENTION
[0004] 1. Field of the Invention
[0005] 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
for producing and using the polypeptides.
[0006] 2. Description of the Related Art
[0007] Cellulose is a polymer of the simple sugar glucose covalently
bonded by beta-1,4-linkages. 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. Celiobiohydrolases sequentially release molecules of
cellobiose from the ends of the cellulose polymer. Cellobiohydrolase I is
a 1,4-beta-D-glucan cellobiohydrolase (E.C. 3.2.1.91) activity that
catalyzes the hydrolysis of 1,4-beta-D-glucosidic linkages in cellulose,
cellotetriose, or any beta-1,4-linked glucose containing polymer,
releasing cellobiose from the reducing ends of the chain.
Cellobiohydrolase II is a 1,4-beta-D-glucan cellobiohydrolase (E.C.
3.2.1.91) activity that catalyzes the hydrolysis of 1,4-beta-D-glucosidic
linkages in cellulose, cellotetriose, or any beta-1,4-linked glucose
containing polymer, releasing cellobiose from the non-reducing ends of
the chain. Cellobiose is a water-soluble beta-1,4-linked dimer of
glucose. Beta-glucosidases hydrolyze cellobiose to glucose.
[0008] The conversion of cellulosic feedstocks into ethanol has the
advantages of the ready availability of large amounts of feedstock, the
desirability of avoiding burning or land filling the materials, and the
cleanliness of the ethanol fuel. Wood, agricultural residues, herbaceous
crops, and municipal solid wastes have been considered as feedstocks for
ethanol production. These materials primarily consist of cellulose,
hemicellulose, and lignin. Once the cellulose is converted to glucose,
the glucose is easily fermented by yeast into ethanol.
[0009] Kvesitadaze at 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 cellulose system of
Thielavia terrestris 255B. Breuil at al., 1986, Biotechnology Letters 8:
673-676, describe production and localization of cellulases and
beta-glucosidases from Thielavia terrestris strains C464 and NRRL 8126.
[0010] It would be an advantage in the art to identify new endoglucanases
having improved properties, such as improved hydrolysis rate, better
thermal stability, reduced adsorption to lignin, and ability to hydrolyze
non-cellulosic components of biomass, such as hemicellulose, in addition
to hydrolyzing cellulose. Endoglucanases with a broad range of side
activities on hemicellulose can be especially beneficial for improving
the overall hydrolysis yield of complex, hemicellulose-rich biomass
substrates.
[0011] It is an object of the present invention to provide improved
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
90% identity with the mature polypeptide of SEQ ID NO: 2 or at least 60%
identity with the mature polypeptide of SEQ ID NO: 4;
[0014] (b) a polypeptide which 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 cDNA sequence
contained in the mature polypeptide coding sequence of SEQ ID NO: 1, or
(iii) a complementary strand of (1) or (ii), or under at least medium
stringency conditions with (i) the mature polypeptide coding sequence of
SEQ ID NO: 3, (ii) the genomic DNA sequence comprising the mature
polypeptide coding sequence of SEQ ID NO: 3, or (iii) a complementary
strand of (i) or (ii);
[0015] (c) a polypeptide which is encoded by a polynucleotide comprising a
nucleotide sequence having at least 90% identity with the mature
polypeptide coding sequence of SEQ ID NO: 1 or at least 60% identity with
the mature polypeptide coding sequence of SEQ ID NO: 3; and
[0016] (d) a variant comprising a substitution, deletion, and/or insertion
of one or more 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 90% identity with the mature polypeptide of
SEQ ID NO: 2 or at least 60% identity with the mature polypeptide of SEQ
ID NO: 4;
[0019] (b) a polynucleotide which hybridizes under at least high
stringency conditions with (i) the mature polypeptide coding sequence of
SEQ ID NO: 1, (ii) the cDNA sequence contained in the mature polypeptide
coding sequence of SEQ ID NO: 1, or (iii) a complementary strand of (i)
or (ii), or under at least medium stringency conditions with (i) the
mature polypeptide coding sequence of SEQ ID NO: 3, (ii) the genomic DNA
sequence comprising the mature polypeptide coding sequence of SEQ ID NO:
3, or (iii) a complementary strand of (i) flr (ii):
[0020] (c) a polynucleotide comprising a nucleotide sequence having at
least 90% identity with the mature polypeptide coding sequence of SEQ ID
NO: 1 or at least 60% identity with the mature polypeptide coding
sequence of SEQ ID NO: 3; and
[0021] (d) a polynucleotide encoding a variant comprising a substitution,
deletion, and/or insertion of one or more amino acids of the mature
polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4.
[0022] In a preferred aspect, the mature polypeptide is amino acids 19 to
396 of SEQ ID NO: 2. In another preferred aspect, the mature polypeptide
is amino acids 20 to 400 of SEQ ID NO: 4. In another preferred aspect,
the mature polypeptide coding sequence is nucleotides 110 to 1557 of SEQ
ID NO: 1. In another preferred aspect, the mature polypeptide coding
sequence is nucleotides 58 to 1200 of SEQ ID NO: 3.
[0023] 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.
[0024] The present invention also relates to methods of inhibiting the
expression of a polypeptide 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 such a double-stranded
inhibitory RNA (dsRNA) molecule, wherein optionally the dsRNA is an siRNA
or an miRNA molecule.
[0025] The present invention also relates to methods of using the
polypeptides having endoglucanase activity in the conversion of cellulose
to glucose and various substances.
[0026] The present invention also relates to plants comprising an isolated
polynucleotide encoding such a polypeptide having endoglucanase activity.
[0027] The present invention also relates to methods for producing such a
polypeptide having endoglucanase activity, comprising: (a) cultivating a
transgenic plant or a plant cell comprising a polynucleotide encoding
such a polypeptide having endoglucanase activity under conditions
conducive for production of the polypeptide; and (b) recovering the
polypeptide.
[0028] 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 18 of SEQ ID NO: 2 or amino acids 1 to 19
of SEQ ID NO: 4, wherein the gene is foreign to the first and second
nucleotide sequences
BRIEF DESCRIPTION OF THE FIGURES
[0029] FIG. 1 shows a restriction map of pPH23.
[0030] FIGS. 2A and 2B show the genomic DNA sequence and the deduced amino
acid sequence of a Thielavia terrestris NRRL 8126 CEL6B endoglucanase
(SEQ ID NOs: 1 and 2, respectively).
[0031] FIG. 3 shows a restriction map of pCIBG146.
[0032] FIGS. 4A and 4B show the cDNA sequence and the deduced amino acid
sequence of a Thielavia terrestris NRRL 8126 CEL6C endoglucanase (SEQ ID
NOs: 3 and 4, respectively).
[0033] FIG. 5 shows a restriction map of pAlLo1.
[0034] FIG. 6 shows a restriction map of pBANe10.
[0035] FIG. 7 shows a restriction map of pAlLo2.
[0036] FIG. 8 shows a restriction map of pEJG105.
[0037] FIG. 9 shows a restriction map of pA2BG146.
DEFINITIONS
[0038] Endoglucanase activity: The term "endoglucanase activity" is
defined herein as an endo-1,4-beta-D-glucan 4-glucanohydrolase (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), lignocellulose, lignocellulose
derivatives, 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 5.0.
[0039] In a preferred aspect, the polypeptides of the present invention
having endogiucanase activity further have enzyme activity toward one or
more substrates selected from the group consisting of xylan, xyloglucan,
arabinoxylan, 1,4-beta-D-mannan, and galactomannan. The activity of the
polypeptides having endoglucanase activity on these polysaccharide
substrates is determined as percent of the substrate hydrolyzed to
reducing sugars after incubating the substrate (5 mg per ml) with a
polypeptide having endoglucanase activity of the present invention (5 mg
protein per g of substrate) for 24 hours with intermittent stirring at pH
5.0 (50 mM sodium acetate) and 50.degree. C. Reducing sugars in
hydrolysis mixtures are determined by the p-hydroxybenzoic acid hydrazide
(PHBAH) assay.
[0040] In a more preferred aspect, the polypeptides of the present
invention having endoglucanase activity further have enzyme activity
toward xylan. In another more preferred aspect, the polypeptides of the
present invention having endoglucanase activity further have enzyme
activity toward xyloglucan. In another more preferred aspect, the
polypeptides of the present invention having endoglucanase activity
further have enzyme activity toward arabinoxylan. In another more
preferred aspect, the polypeptides of the present invention having
endoglucanase activity further have enzyme activity toward
1,4-beta-D-mannan. In another more preferred aspect, the polypeptides of
the present invention having endoglucanase activity further have enzyme
activity toward galactomannan. In another more preferred aspect, the
polypeptides of the present invention having endoglucanase activity
further have enzyme activity toward xylan, xyloglucan, arabinoxylan,
1,4-beta-D-mannan, and/or galactomannan.
[0041] 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 mature
polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4.
[0042] Family 6 glycoside hydrolase or Family GH6: The term "Family 6
glycoside hydrolase" or "Family GH6" is defined herein as a polypeptide
falling into the glycoside hydrolase Family 6 according to Henrissat B.,
1991, A classification of glycosyl hydrolases based on amino-acid
sequence similarities, Biochem. J. 280: 309-316, and Henrissat B., and
Bairoch A., 1996, Updating the sequence-based classification of glycosyl
hydrolases, Biochem. J. 316: 695-696.
[0043] Isolated polypeptide: The term "isolated polypeptide" as used
herein refers to a polypeptide which is at least 20% pure, preferably at
least 40% pure, more preferably at least 60% pure, even more preferably
at least 80% pure, most preferably at least 90% pure, and even most
preferably at least 95% pure, as determined by SDS-PAGE.
[0044] Substantially pure polypeptide: The term "substantially pure
polypeptide" denotes herein a polypeptide preparation which 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 96% pure, more
preferably at least 97% pure, more preferably at least 98% pure, even
more preferably at least 99%, most preferably at least 99.5% pure, and
even most preferably 100% pure by weight of the total polypeptide
material present in the preparation.
[0045] The polypeptides of the present invention are preferably in a
substantially pure form. In particular, it is preferred that the
polypeptides are in "essentially 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 means of well-known recombinant
methods or by classical purification methods.
[0046] Herein, the term "substantially pure polypeptide" is synonymous
with the terms "isolated polypeptide" and "polypeptide in isolated form."
[0047] Mature polypeptide: The term "mature polypeptide" is defined herein
as a polypeptide having endoglucanase activity that is in its final form
following translation and any post-translational modifications, such as
N-terminal processing, C-terminal truncation, glycosylation, etc.
[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.
[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 EMBOSS with gap open penalty of 10,
gap extension penalty of 0.5, and the EBLOSUM62 matrix. The output of
Needle labeled "longest identity" is used as the percent identity and is
calculated as follows:
(Identical Residues.times.100)/(Length of Alignment-Number of Gaps in
Alignment)
[0051] For purposes of the present invention, the degree of identity
between two nucleotide 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 EMBOSS with gap open penalty of 10,
gap extension penalty of 0.5, and the EDNAFULL matrix. The output of
Needle labeled "longest identity" is used as the percent identity and is
calculated as follows:
(Identical Residues.times.100)/(Length of Alignment-Number of Gaps in
Alignment)
[0052] Homologous sequence: The term "homologous sequence" is defined
herein as a predicted protein which gives an E value (or expectancy
score) of less than 0.001 in a fasta search (Pearson, W. R., 1999, in
Bioinformatics Methods and Protocols, S. Misener and S. A. Krawetz, ed.,
pp. 185-219) with SEQ ID NO: 2 or SEQ ID NO: 4.
[0053] Polypeptide fragment: The term "polypeptide fragment" is defined
herein as a polypeptide having one or more 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 320 amino acid residues, more preferably at least 340 amino acid
residues, and most preferably at least 360 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 320 amino acid
residues, more preferably at least 340 amino acid residues, and most
preferably at least 360 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 nucleotides deleted from the 5'
and/or 3' end of the mature polypeptide coding sequence of SEQ ID NO: 1
or SEQ ID NO: 3; or a homologous sequence thereof; wherein the
subsequence encodes a polypeptide fragment having endoglucanase activity.
In a preferred aspect, a subsequence contains at least 960 nucleotides,
more preferably at least 1020 nucleotides, and most preferably at least
1080 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 960 nucleotides, more preferably at least
1020 nucleotides, and most preferably at least 1080 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 which is at least 20% pure, preferably
at least 40% pure, more preferably at least 60% pure, even more
preferably at least 80% pure, most preferably at least 90% pure, and even
most preferably at least 95% 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%, and even
most preferably at least 99.5% pure by weight. The polynucleotides of the
present invention are preferably in a substantially pure form. In
particular, it is preferred that the polynucleotides disclosed herein are
in "essentially pure form", i.e., that the polynucleotide preparation is
essentially free of other polynucleotide material with which it is
natively or recombinantly associated. Herein, the term "substantially
pure polynucleotide" is synonymous with the terms "isolated
polynucleotide" and "polynucleotide in isolated form." 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, or recombinant nucleotide sequence.
[0059] 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.
[0060] cDNA: The term "cDNA" is defined herein as a DNA molecule which can
be prepared by reverse transcription from a mature, spliced, mRNA
molecule obtained from a eukaryotic cell. cDNA lacks intron sequences
that are usually present in the corresponding genomic DNA. The initial,
primary RNA transcript is a precursor to mRNA which 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.
[0061] 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
which is modified to contain segments of nucleic acids in a manner that
would not otherwise exist in nature. 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.
[0062] Control sequence: 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.
[0063] 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.
[0064] 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.
[0065] 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 which is operably
linked to additional nucleotides that provide for its expression.
[0066] Host cell: The term "host cell", as used herein, includes any cell
type which is susceptible to transformation, transfection, transduction,
and the like with a nucleic acid construct or expression vector
comprising a polynucleotide of the present invention.
[0067] 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 substitutions, deletions and/or insertions of one or
more amino acids as well as replacements of one or more amino acid side
chains.
[0068] Artificial variant: When used herein, the term "artificial variant"
means a polypeptide having endoglucanase activity produced by an organism
expressing a modified nucleotide 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 nucleotide 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
[0069] 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 at
least 60%, 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%, 97%, 98%, or 99%, which have
endoglucanase activity (hereinafter "homologous polypeptides"). In a
preferred aspect, the homologous polypeptides have an amino acid sequence
which 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.
[0070] 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 that has endoglucanase activity. In a preferred aspect,
a polypeptide comprises the amino acid sequence of SEQ ID NO: 2. In
another preferred aspect, a polypeptide comprises the mature polypeptide
of SEQ ID NO: 2. In another preferred aspect, a polypeptide comprises
amino acids 19 to 396 of SEQ ID NO: 2, or an allelic variant thereof; or
a fragment thereof that has endoglucanase activity. In another preferred
aspect, a polypeptide comprises amino acids 19 to 396 of SEQ ID NO: 2. In
another preferred aspect, a polypeptide consists of the amino acid
sequence of SEQ ID NO: 2 or an allelic variant thereof; or a fragment
thereof that has endoglucanase activity. In another preferred aspect, a
polypeptide consists of the amino acid sequence of SEQ ID NO: 2. In
another preferred aspect, a polypeptide consists of the mature
polypeptide of SEQ ID NO: 2. In another preferred aspect, a polypeptide
consists of amino acids 19 to 396 of SEQ ID NO: 2 or an allelic variant
thereof; or a fragment thereof that has endoglucanase activity. In
another preferred aspect, a polypeptide consists of amino acids 19 to 396
of SEQ ID NO: 2.
[0071] A polypeptide of the present invention preferably comprises the
amino acid sequence of SEQ ID NO: 4 or an allelic variant thereof; or a
fragment thereof that has endoglucanase activity. In a preferred aspect,
a polypeptide comprises the amino acid sequence of SEQ ID NO: 4. In
another preferred aspect, a polypeptide comprises the mature polypeptide
of SEQ ID NO: 4. In another preferred aspect, a polypeptide comprises
amino acids 20 to 400 of SEQ ID NO: 4, or an allelic variant thereof; or
a fragment thereof that has endoglucanase activity. In another preferred
aspect, a polypeptide comprises amino acids 20 to 400 of SEQ ID NO: 4. In
another preferred aspect, a polypeptide consists of the amino acid
sequence of SEQ ID NO: 4 or an allelic variant thereof; or a fragment
thereof that has endoglucanase activity. In another preferred aspect, a
polypeptide consists of the amino acid sequence of SEQ ID NO: 4. In
another preferred aspect, a polypeptide consists of the mature
polypeptide of SEQ ID NO: 4. In another preferred aspect, a polypeptide
consists of amino acids 20 to 400 of SEQ ID NO: 4 or an allelic variant
thereof; or a fragment thereof that has endoglucanase activity. In
another preferred aspect, a polypeptide consists of amino acids 20 to 400
of SEQ ID NO: 4.
[0072] In a second aspect, the present invention relates to isolated
polypeptides having endoglucanase activity which are encoded by
polynucleotides 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 cDNA sequence contained in the
mature polypeptide coding sequence of SEQ ID NO: 1 or the genomic DNA
sequence comprising the mature polypeptide coding sequence of SEQ ID NO:
3, (iii) a subsequence of (i) or (ii), or (iv) a 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,
N.Y.). 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 which has endoglucanase activity. In a
preferred aspect, the mature polypeptide coding sequence is nucleotides
110 to 1557 of SEQ ID NO: 1. In another preferred aspect, the mature
polypeptide coding sequence is nucleotides 58 to 1200 of SEQ ID NO: 3. In
another 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.
[0073] 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 a nucleic
acid probe 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 which are at least
600 nucleotides, at least preferably at least 700 nucleotides, more
preferably at least 800 nucleotides, or most preferably at least 900
nucleotides in length. Both DNA and RNA probes can be used. The probes
are typically labeled for detecting the corresponding gene (for example,
with .sup.32P, .sup.3H, .sup.35S, biotin, or avidin). Such probes are
encompassed by the present invention.
[0074] A genomic DNA or cDNA library prepared from such other strains may,
therefore, be screened for DNA which hybridizes with the probes described
above and which 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 which 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.
[0075] 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 cDNA sequence contained in the mature polypeptide
coding sequence of SEQ ID NO: 1 or the genomic DNA sequence comprising
the mature polypeptide coding sequence of SEQ ID NO: 3, its 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.
[0076] 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 110 to 1557 of SEQ ID NO: 1. In
another preferred aspect, the nucleic acid probe is a polynucleotide
sequence which 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 pPH47 which is contained in
E. coli NRRL B-30898, 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 pPH47 which is contained in E. coli NRRL B-30898.
[0077] In a 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 58 to 1200 of SEQ ID NO: 3. In
another preferred aspect, the nucleic acid probe is a polynucleotide
sequence which 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 pCIBG146 which is contained
in E. coli NRRL B-30901, 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 pCIBG146 which is contained in E. coli NRRL B-30901.
[0078] 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.
[0079] For long probes of at least 100 nucleotides in length, the carrier
material is filially washed three times each for 15 minutes using
2.times.SSC, 0.2% SDS preferably at least at 45.degree. C. (very low
stringency), more preferably at least at 50.degree. C. (low stringency),
more preferably at least at 55.degree. C. (medium stringency), more
preferably at least at 60.degree. C. (medium-high stringency), even more
preferably at least at 65.degree. C. (high stringency), and most
preferably at least at 70.degree. C. (very high stringency).
[0080] For short probes which are 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.
[0081] For short probes which are 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.
[0082] In a third aspect, the present invention relates to isolated
polypeptides encoded by polynucleotides comprising or consisting of
nucleotide sequences which have a degree of identity to the mature
polypeptide coding sequence of SEQ ID NO: 1 or SEQ ID NO: 3 of at least
60%, preferably at least 65%, more preferably at least 70%, more
preferably at least 75%, more preferably at least 80%, more preferably at
least 85%, more preferably at least 90%, even more preferably at least
95%, and most preferably at least 97% identity, which encode an active
polypeptide. In a preferred aspect, the mature polypeptide coding
sequence is nucleotides 110 to 1557 of SEQ ID NO: 1. In another preferred
aspect, the mature polypeptide coding sequence is nucleotides 58 to 1200
of SEQ ID NO: 3. See polynucleotide section herein.
[0083] 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.
[0084] 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 which 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.
[0085] 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.
[0086] 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.
[0087] 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., 1995, 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 which are related to a
polypeptide according to the invention.
[0088] 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. Sal. 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).
[0089] 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.
[0090] 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,
such as amino acids 19 to 396 of SEQ ID NO: 2 or amino acids 20 to 400 of
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
[0091] 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.
[0092] 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.
[0093] 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.
[0094] In another preferred aspect, the polypeptide is a Streptococcus
equisimilis, Streptococcus pyoaenes, Streptococcus uberis, or
Streptococcus equi subsp. Zooepidemicus polypeptide having endoglucanase
activity.
[0095] In another preferred aspect, the polypeptide is a Streptomyces
achrotnogenes, Streptomyces avermitilis, Streptomyces coelicolor,
Streptomyces griseus, or Streptomyces lividans polypeptide having
endoglucanase activity.
[0096] 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, Aspergillus, Aureobasidium, Chtysosporium, Cryptococcus,
Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora,
Neocallimastix, Neurospora, Paecilomyces, Penicillium, Piromyces,
Schizophylium, Talaromyces, Thertnoascus, Thielavia, Tolypocladium, or
Trichoderma polypeptide having endoglucanase activity.
[0097] In a preferred aspect, the polypeptide is a Saccharomyces
carisbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus,
Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis,
or Saccharomyces oviformis polypeptide having endoglucanase activity.
[0098] In another preferred aspect, the polypeptide is an Aspergillus
aculeates, Aspergillus awamori, Aspergillus fumigatus, Aspergillus
foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus nicer,
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 insolens, Humicola lanuginosa, Mucor miehei,
Myceliophthora thermophila, Neurospora crassa, Penicillium brasilianum,
Penicillium camembertii, Penicillium capsulatum, Penicillium chrysogenum,
Penicillium citreonigrum, Penicillium citrinum, Penicillium claviforme,
Penicillium corylophilum, Penicillium crustosum, Penicillium digitatum,
Penicillium expansum, Penicillium funiculosum, Penicillium glabrum,
Penicillium granulatum, Penicillium griseofulvum, Penicillium islandicum,
Penicillium italicum, Penicillium janthinellum, Penicillium lividum,
Penicillium megasporum, melinii, Penicillium notatum, Penicillium
oxalicum, Penicillium puberulum, Penicillium purpurescens, Penicillium
purpurogenum, Penicillium roquefortii, Penicillium rugulosum, Penicillium
spinulosum, Penicillium waksmanii, Trichoderma harzianum, Trichoderma
koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma
viride polypeptide having endoglucanase activity.
[0099] 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 subthermophda, Thielavia terrestris, Thielavia terricola,
Thielavia thermophila, Thielavia variospora, or Thielavia wareingii
polypeptide having endoglucanase activity.
[0100] In a more preferred aspect, the polypeptide is a Thielavia
terrestris polypeptide, and most preferably a Thielavia terrestris NRRL
8126 polypeptide, e.g., the polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4,
or the mature polypeptide thereof.
[0101] 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.
[0102] 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).
[0103] 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 which
are well known to those of ordinary skill in the art (see, e.g., Sambrook
et al., 1989, supra).
[0104] 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.
[0105] 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.
[0106] Examples of cleavage sites include, but are not limited to, a Kex2
site which encodes the dipeptide Lys-Arg (Martin at al., 2003, J. Ind.
Microbiol. Biotechnol. 3: 568-76: Svetina at al., 2000, J. Biatechnol.
76: 245-251; Rasmussen-Wilson et al., 1997, Appl. Environ. Microbial. 63:
3488-3493; Ward et al., 1995, Biotechnology 13: 498-503; and Contreras at
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 at 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 30 protease after the Gln (Stevens,
2003, supra).
Polynucleotides
[0107] The present invention also relates to an isolated polynucleotide
comprising or consisting of a nucleotide sequence which encodes a
polypeptide of the present invention having endoglucanase activity.
[0108] 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 pPH47 which is contained in E. coli NRRL B-30898. In another
preferred aspect, the nucleotide sequence comprises or consists of the
mature polypeptide coding region of SEQ ID NO: 1. In another preferred
aspect, the nucleotide sequence comprises or consists of nucleotides 110
to 1557 of SEQ ID NO: 1. In another more preferred aspect, the nucleotide
sequence comprises or consists of the mature polypeptide coding region
contained in plasmid pPH47 which is contained in E. coli NRRL B-30898.
The present invention also encompasses nucleotide sequences which 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 which encode fragments of SEQ ID NO: 2 that
have endoglucanase activity.
[0109] 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 pCIBG146 which is contained in E. coli NRRL B-30901. In another
preferred aspect, the nucleotide sequence comprises or consists of the
mature polypeptide coding region of SEQ ID NO: 3. In another preferred
aspect, the nucleotide sequence comprises or consists of nucleotides 58
to 1200 of SEQ ID NO: 3. In another more preferred aspect, the nucleotide
sequence comprises or consists of the mature polypeptide coding region
contained in plasmid pCIBG146 which is contained in E. coli NRRL B-30901.
The present invention also encompasses nucleotide sequences which 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 which encode fragments of SEQ ID NO: 4 that
have endoglucanase activity.
[0110] 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. In a preferred aspect, the mature polypeptide is amino
acids 19 to 396 of SEQ ID NO: 2. In another preferred aspect, the mature
polypeptide is amino acids 20 to 400 of SEQ ID NO: 4.
[0111] 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 Myceliophthora thermophila CBS 117.65,
basidiomycete CBS 494.95, or basidiomycete CBS 495.95, 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.
[0112] The present invention also relates to isolated polynucleotides
comprising or consisting of nucleotide sequences which have a degree of
identity to the mature polypeptide coding sequence of SEQ ID NO: 1 or SEQ
ID NO: 3 of at least 60%, preferably at least 65%, more preferably at
least 70%, more preferably at least 75%, more preferably at least 80%,
more preferably at least 85%, more preferably at least 90%, even more
preferably at least 95%, and most preferably at least 97% identity, which
encode an active polypeptide. In a preferred aspect, the mature
polypeptide coding sequence is nucleotides 110 to 1557 of SEQ ID NO: 1.
In another preferred aspect, the mature polypeptide coding sequence is
nucleotides 58 to 1200 of SEQ ID NO: 3.
[0113] 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 polypeptide encoding region of SEQ
ID NO: 1 or SEQ ID NO: 3, e.g., a subsequence thereof, and/or by
introduction of nucleotide substitutions which 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 which 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.
[0114] 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).
[0115] The present invention also relates to isolated polynucleotides
encoding a polypeptide 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 under at least
high stringency conditions with (i) the mature polypeptide coding
sequence of SEQ ID NO: 1 or SEQ ID NO: 3, (ii) the cDNA sequence
contained in the mature polypeptide coding sequence of SEQ ID NO: 1 or
the genomic DNA sequence comprising the mature polypeptide coding
sequence of SEQ ID NO: 3, (iii) a subsequence of (i) or (ii), or (iv) a
complementary strand of (i), (ii), or (iii); or allelic variants thereof
(Sambrook et al., 1989, supra), as defined herein. In a preferred aspect,
the mature polypeptide coding sequence is nucleotides 110 to 1557 of SEQ
ID NO: 1. In another preferred aspect, the mature polypeptide coding
sequence is nucleotides 58 to 1200 of SEQ ID NO: 3. In another 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.
[0116] 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 cDNA sequence contained in the mature polypeptide coding
sequence of SEQ ID NO: 1 or the genomic DNA sequence comprising the
mature polypeptide coding sequence of SEQ ID NO: 3, or (iii) a
complementary strand of (i) or (ii); and (b) isolating the hybridizing
polynucleotide, which encodes a polypeptide having endoglucanase
activity. In a preferred aspect, the mature polypeptide coding sequence
is nucleotides 110 to 1557 of SEQ ID NO: 1. In another preferred aspect,
the mature polypeptide coding sequence is nucleotides 58 to 1200 of SEQ
ID NO: 3. In another 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
[0117] The present invention also relates to nucleic acid constructs
comprising an isolated polynucleotide of the present invention operably
linked to one or more control sequences that direct the expression of the
coding sequence in a suitable host cell under conditions compatible with
the control sequences.
[0118] 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.
[0119] The control sequence may be an appropriate promoter sequence, a
nucleotide sequence which 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 which
mediate the expression of the polypeptide. The promoter may be any
nucleotide sequence which 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.
[0120] 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 (amyl),
Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacillus
licheniformis penicillinase gene (penP), Bacillus subtilis xylA and xylB
genes, and prokaryotic beta-lactamase gene (Villa-Kamaroff at al., 1978,
Proceedings of the National Academy of Sciences USA 75: 3727-3731), as
well as the tac promoter (DeBoer at 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.
[0121] 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 xylanase I, Trichoderma reesei xylanase 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.
[0122] 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 (TP1), Saccharomyces
cerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae
3-phosphoglycerate kinase.
[0123] Other useful promoters for yeast host cells are described by
Romanos et al., 1992, Yeast 8: 423-488.
[0124] 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 which is functional in the host cell of choice may be used in
the present invention.
[0125] 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.
[0126] 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.
[0127] The control sequence may also be a suitable leader sequence, a
nontranslated region of an mRNA which 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.
[0128] Preferred leaders for filamentous fungal host cells are obtained
from the genes for Aspergillus oryzae TAKA amylase and Aspergillus
nidulans triose phosphate isomerase.
[0129] 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).
[0130] The control sequence may also be a polyadenylation sequence, a
sequence operably linked to the 3' terminus of the nucleotide sequence
and which, when transcribed, is recognized by the host cell as a signal
to add polyadenosine residues to transcribed mRNA. Any polyadenylation
sequence which is functional in the host cell of choice may be used in
the present invention.
[0131] 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.
[0132] Useful polyadenylation sequences for yeast host cells are described
by Guo and Sherman, 1995, Molecular Cellular Biology 15: 5983-5990.
[0133] The control sequence may also be a signal peptide coding region
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 region naturally linked in
translation reading frame with the segment of the coding region which
encodes the secreted polypeptide. Alternatively, the 5' end of the coding
sequence may contain a signal peptide coding region which is foreign to
the coding sequence. The foreign signal peptide coding region may be
required where the coding sequence does not naturally contain a signal
peptide coding region. Alternatively, the foreign signal peptide coding
region may simply replace the natural signal peptide coding region in
order to enhance secretion of the polypeptide. However, any signal
peptide coding region which 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.
[0134] Effective signal peptide coding regions for bacterial host cells
are the signal peptide coding regions 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.
[0135] Effective signal peptide coding regions for filamentous fungal host
cells are the signal peptide coding regions 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.
[0136] 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 regions are
described by Romanos et al., 1992, supra.
[0137] In a preferred aspect, the signal peptide comprises or consists of
amino acids 1 to 18 of SEQ ID NO: 2. In another preferred aspect, the
signal peptide coding region is nucleotides 56 to 109 of SEQ ID NO: 1.
[0138] In another preferred aspect, the signal peptide comprises or
consists of amino acids 1 to 19 of SEQ ID NO: 4. In another preferred
aspect, the signal peptide coding region comprises or consists of
nucleotides 1 to 57 of SEQ ID NO: 3.
[0139] The control sequence may also be a propeptide coding region 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 region 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).
[0140] Where both signal peptide and propeptide regions are present at the
amino terminus of a polypeptide, the propeptide region is positioned next
to the amino terminus of a polypeptide and the signal peptide region is
positioned next to the amino terminus of the propeptide region.
[0141] It may also be desirable to add regulatory sequences which allow
the regulation of the expression of the polypeptide relative to the
growth of the host cell. Examples of regulatory systems are those which
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, lac,
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 which allow for gene amplification. In eukaryotic
systems, these include the dihydrofolate reductase gene which is
amplified in the presence of methotrexate, and the metallothionein genes
which are amplified with heavy metals. In these cases, the nucleotide
sequence encoding the polypeptide would be operably linked with the
regulatory sequence.
Expression Vectors
[0142] 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 which may include one or more
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.
[0143] The recombinant expression vector may be any vector (e.g., a
plasmid or virus) which 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.
[0144] The vector may be an autonomously replicating vector, i.e., a
vector which 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 which, 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 which together
contain the total DNA to be introduced into the genome of the host cell,
or a transposon may be used.
[0145] The vectors of the present invention preferably contain one or more
selectable markers which 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.
[0146] Examples of bacterial selectable markers are the dal genes from
Bacillus subtilis or Bacillus licheniformis, or markers which 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.
[0147] 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.
[0148] 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 with 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.
[0149] 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 which 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.
[0150] 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.
[0151] 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.
[0152] Examples of origins of replication useful in a filamentous fungal
cell are AMA1 and ANS1 (Gems et al., 1991, Gene 98: 61-67; Cullen et al.,
1987, Nucleic Acids 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.
[0153] 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.
[0154] 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
[0155] 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.
[0156] 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.
[0157] 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 circulars, Bacillus clausii, Bacillus coagulans,
Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus
licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus
stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.
[0158] 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.
[0159] 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.
[0160] 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.
[0161] 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, Streptotnyces coelicolor, Streptomyces griseus, and
Streptomyces lividans.
[0162] 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.
[0163] 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., Koehier 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 at 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., Cott 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 for
introducing DNA into a host cell can be used.
[0164] The host cell may also be a eukaryote, such as a mammalian, insect,
plant, or fungal cell.
[0165] 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).
[0166] 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. Bacterial, Symposium Series
No. 9, 1980).
[0167] In an even more preferred aspect, the yeast host cell is a Candida,
Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or
Yarrowia cell.
[0168] In a most preferred aspect, the yeast host cell is a Saccharomyces
carisbergensis, 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.
[0169] 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.
[0170] In an even more preferred aspect, the filamentous fungal host cell
is an Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis,
Chtysosporium, Coprinus, Conelus, Cryptococcus, Filibasidium, Fusarium,
Humicola, Magnaperthe, Mucor, Myceliophthora, Neocallimastix, Neurospora,
Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus,
Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium,
Trametes, or Trichoderma cell.
[0171] 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
suiphureum, 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 brasilianum, 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.
[0172] 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 at al., 1984, Proceedings of the National Academy
of Sciences USA 81: 1470-1474. Suitable methods for transforming Fusarium
species are described by Malardier at 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
[0173] The present invention also relates to methods for 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.
[0174] The present invention also relates to methods for producing a
polypeptide of the present invention, comprising: (a) cultivating a host
cell under conditions conducive for production of the polypeptide; and
(b) recovering the polypeptide.
[0175] The present invention also relates to methods for producing a
polypeptide of the present invention, comprising: (a) cultivating a 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 which comprises or consists of the mature polypeptide of SEQ
ID NO: 2 or SEQ ID NO: 4, and (b) recovering the polypeptide.
[0176] In a preferred aspect, the mature polypeptide is amino acids 19 to
396 of SEQ ID NO: 2. In another preferred aspect, the mature polypeptide
is amino acids 20 to 400 of SEQ ID NO: 4. In another preferred aspect,
the mature polypeptide coding sequence is nucleotides 110 to 1557 of SEQ
ID NO: 1. In another preferred aspect, the mature polypeptide coding
sequence is nucleotides 58 to 1200 of SEQ ID NO: 3.
[0177] 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.
[0178] 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.
[0179] 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-d ring, evaporation, or
precipitation.
[0180] 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
[0181] 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.
[0182] 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).
[0183] 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.
[0184] 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.
[0185] Also included within the scope of the present invention are the
progeny of such plants, plant parts, and plant cells.
[0186] 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 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.
[0187] The expression construct is conveniently a nucleic acid construct
which 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).
[0188] 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 at al., 1988,
Plant Physiology 86: 506.
[0189] 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 Vida 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 (Mitre 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 inducibie
promoter such as the potato pint 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 add, and heavy metals.
[0190] 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 which 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.
[0191] The selectable marker gene and any other parts of the expression
construct may be chosen from those available in the art.
[0192] 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).
[0193] Presently, Agrobacterium tumefaciens-mediated gene transfer is the
method of choice for generating transgenic divots (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.
[0194] 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.
[0195] The present invention also relates to methods for producing a
polypeptide of the present invention comprising: (a) cultivating a
transgenic plant or a plant cell comprising a polynucleotide encoding a
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
[0196] The present invention also relates to methods for 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.
[0197] 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.
[0198] 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.
[0199] 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.
[0200] 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.
[0201] Modification or inactivation of the nucleotide sequence may be
accomplished by introduction, substitution, or removal of one or more
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.
[0202] 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 which 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.
[0203] 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.
[0204] The present invention further relates to a mutant cell of a parent
cell which 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.
[0205] The polypeptide-deficient mutant cells so created are particularly
useful as host cells for the expression of homologous and/or heterologous
polypeptides. Therefore, the present invention further relates to methods
for producing a homologous 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 which 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.
[0206] In a further aspect, the present invention relates to a method for
producing a protein product essentially free of endoglucanase activity by
fermentation of a cell which 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.
[0207] In a further aspect, the present invention relates to a method for
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.
[0208] 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.
[0209] The combined pH and temperature treatment is preferably carried out
at a pH in the range of 2-3 or 10-11 and a temperature in the range of at
least 75-85.degree. C. for a sufficient period of time to attain the
desired effect, where typically, 1 to 3 hours is sufficient.
[0210] The methods used for cultivation and purification of the product of
interest may be performed by methods known in the art.
[0211] 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, cellulytic enzyme, oxidoreductase, or plant
cell-wall degrading enzyme. Examples of such enzymes include an
aminopeptidase, amylase, amyloglucosidase, carbohydrase,
carboxypeptidase, catalase, cellulase, chitinase, cutinase, cyclodextrin
glycosyltransferase, deoxyribonuclease, 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 xylanase. The
endoglucanase-deficient cells may also be used to express heterologous
proteins of pharmaceutical interest such as hormones, growth factors,
receptors, and the like.
[0212] 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.
[0213] In a further aspect, the present invention relates to a protein
product essentially free from endoglucanase activity which is produced by
a method of the present invention.
Methods of Inhibiting Expression of a Polypeptide
[0214] The present invention also relates to methods of inhibiting
expression of a polypeptide 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 or portion 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. In another preferred aspect, the polypeptide has endoglucanase
activity.
[0215] The dsRNA is preferably a small interfering RNA (siRNA) 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.
[0216] The present invention also relates to such double-stranded RNA
(dsRNA) molecules for inhibiting expression of a polypeptide in a cell,
wherein the dsRNA comprises a subsequence or portion of a polynucleotide
encoding the mature polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4. 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).
[0217] 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
[0218] 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.
[0219] 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
xylanase. 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.
[0220] 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.
[0221] 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
[0222] The present invention also relates to methods for degrading or
converting a cellulosic material, comprising: treating the cellulosic
material with a composition comprising an effective amount 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.
[0223] The polypeptides and host cells of the present invention may be
used in the production of monosaccharides, disaccharides, and
polysaccharides as chemical or fermentation feedstocks from cellulosic
biomass for the production of ethanol, plastics, other products or
intermediates. The composition comprising the polypeptide having
endoglucanase activity may 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. Alternatively, the composition may comprise
a host cell of the present invention as a source of the polypeptide
having endoglucanase activity in a fermentation process with the biomass.
The host cell may also contain native or heterologous genes that encode
other proteins and enzymes, mentioned above, useful in the processing of
biomass. In particular, the polypeptides and host cells of the present
invention may be used to increase the value of processing residues (dried
distillers grain, spent grains from brewing, sugarcane bagasse, etc.) by
partial or complete degradation of cellulose or hemicellulose. The
compositions can also comprise other proteins and enzymes useful in the
processing of biomass, e.g., cellobiohydrolase, beta-glucosidase,
hemicellulolytic enzymes, enhancers (WO 2005/074647, WO 2005/074656),
etc.
[0224] In the methods of the present invention, any cellulosic material,
such as biomass, can be used. It is understood herein that the term
"cellulosic material" encompasses lignocellulose. Biomass can include,
but is 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).
[0225] 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 hemiceliuloses, which help stabilize the
cell wall matrix.
[0226] Three major classes of enzymes are used to breakdown cellulosic
biomass: [0227] (1) The "endo-1,4-beta-glucanases" or
1,4-beta-D-glucan-4-glucanohydrolases (EC 3.2.1.4), which act randomly on
soluble and insoluble 1,4-beta-glucan substrates. [0228] (2) The
"exo-1,4-beta-D-glucanases" including both the 1,4-beta-D-glucan
glucohydrolases (EC 3.2.1.74), which liberate D-glucose from
1,4-beta-D-glucans and hydrolyze D-cellobiose slowly, and
cellobiohydrolases (1,4-beta-D-glucan cellobiohydrolases, EC 3.2.1.91),
which liberate D-cellobiose from 1,4-beta-glucans. [0229] (3) The
"beta-D-glucosidases" or beta-D-glucoside glucohydrolases (EC 32.1.21),
which act to release D-glucose units from cellobiose and soluble
cellodextrins, as well as an array of glycosides.
[0230] The polypeptides having endoglucanase activity of the present
invention are preferably used in conjunction with other cellulolytic
proteins, e.g., exo-1,4-beta-D-glucanases and beta-D-glucosidases, to
degrade the cellulose component of the biomass substrate, (see, for
example, Brigham et al., 1995, in Handbook on Bioethanol (Charles E.
Wyman, editor), pp. 119-141, Taylor & Francis, Washington D.C.; Lee,
1997, Journal of Biotechnology 56: 1-24). The term "cellulolytic
proteins" is defined herein as those proteins or mixtures of proteins
shown as being capable of hydrolyzing or converting or degrading
cellulose under the conditions tested.
[0231] The exo-1,4-beta-D-glucanases and beta-D-glucosidases may be
produced by any known method known in the art (see, e.g., Bennett, J. W.
and LaSure, L. (eds.), More Gene Manipulations in Fungi, Academic Press,
CA, 1991).
[0232] The optimum amounts of a polypeptide having endoglucanase activity
and other cellulolytic proteins depends 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).
[0233] In a preferred aspect, the amount of polypeptide having
endoglucanase activity per g of cellulosic material is about 0.5 to about
50 mg, preferably about 0.5 to about 40 mg, more preferably about 0.5 to
about 25 mg, more preferably about 0.75 to about 20 mg, more preferably
about 0.75 to about 15 mg, even more preferably about 0.5 to about 10 mg,
and most preferably about 2.5 to about 10 mg per g of cellulosic
material.
[0234] In another preferred aspect, the amount of cellulolytic proteins
per g of cellulosic material is about 0.5 to about 50 mg, preferably
about 0.5 to about 40 mg, more preferably about 0.5 to about 25 mg, more
preferably about 0.75 to about 20 mg, more preferably about 0.75 to about
15 mg, even more preferably about 0.5 to about 10 mg, and most preferably
about 2.5 to about 10 mg per g of cellulosic material.
[0235] In the methods of the present invention, the composition may be
supplemented by one or more additional enzyme activities to improve the
degradation of the cellulosic material. Preferred additional enzymes are
hemicellulases, esterases (e.g., lipases, phospholipases, and/or
cutinases), proteases, laccases, peroxidases, or mixtures thereof.
[0236] In the methods of the present invention, the additional enzyme(s)
may be added prior to or during fermentation, including during or after
the propagation of the fermenting microorganism(s).
[0237] The enzymes may be derived or obtained from any suitable origin,
including, bacterial, fungal, yeast or mammalian origin. The term
"obtained" means herein that the enzyme may have been isolated from an
organism which 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, 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 which are
deleted, inserted and/or substituted, i.e., a recombinantly produced
enzyme which 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.
[0238] The enzymes may also be purified. The term "purified" as used
herein covers enzymes free from other components from the organism from
which it is derived. The term "purified" also covers enzymes free from
components from the native organism from which it is obtained. The
enzymes may be purified, with only minor amounts of other proteins being
present. The expression "other proteins" relate in particular to other
enzymes. The term "purified" as used herein also refers to removal of
other components, particularly other proteins and most particularly other
enzymes present in the cell of origin of the enzyme of the invention. The
enzyme may be "substantially pure," that is, free from other components
from the organism in which it is produced, that is, for example, a host
organism for recombinantly produced enzymes. In a preferred aspect, the
enzymes are at least 75% (w/w), preferably at least 80%, more preferably
at least 85%, more preferably at least 90%, more preferably at least 95%,
more preferably at least 96%, more preferably at least 97%, even more
preferably at least 98%, or most preferably at least 99% pure. In another
preferred aspect, the enzyme is 100% pure.
[0239] The enzymes used in the present invention may be in any form
suitable for use in the processes described herein, such as, for example,
a crude fermentation broth with or without cells, a dry powder or
granulate, a non-dusting granulate, a liquid, a stabilized liquid, or a
protected enzyme. Granulates may be produced, e.g., as disclosed in U.S.
Pat. Nos. 4,106,991 and 4,661,452, and may optionally be coated by
process known in the art. Liquid enzyme preparations may, for instance,
be stabilized by adding stabilizers such as a sugar, a sugar alcohol or
another polyol, and/or lactic acid or another organic acid according to
established process. Protected enzymes may be prepared according to the
process disclosed in EP 238,216.
[0240] The methods of the present invention may be used to process a
cellulosic material to many useful organic products, chemicals and fuels.
In addition to ethanol, some commodity and specialty chemicals that can
be produced from cellulose include xylose, acetone, acetate, glycine,
lysine, organic acids (e.g., lactic acid), 1,3-propanediol, butanediol,
glycerol, ethylene glycol, furfural, polyhydroxyalkanoates, ds,
cis-muconic acid, and animal feed (Lynd, L. R., Wyman, C. E., and
Gerngross, T. U., 1999, Biocommodity Engineering, Biotechnol. Prog., 15:
777-793; Philippidis, G. P., 1996, Cellulose bioconversion technology, in
Handbook on Bloethanol: Production and Utilization, Wyman, C. E., ed.,
Taylor & Francis, Washington, D.C., 179-212; and Ryu, D. D. Y., and
Mandels, M., 1980, Cellulases: biosynthesis and applications, Enz.
Microb. Technol., 2: 91-102). Potential coproduction benefits extend
beyond the synthesis of multiple organic products from fermentable
carbohydrate. Lignin-rich residues remaining after biological processing
can be converted to lignin-derived chemicals, or used for power
production.
[0241] Conventional methods used to process the cellulosic material in
accordance with the methods of the present invention are well understood
to those skilled in the art. The methods of the present invention may be
implemented using any conventional biomass processing apparatus
configured to operate in accordance with the invention.
[0242] Such an apparatus may include a batch-stirred reactor, a continuous
flow stirred reactor with ultrafiltration, a continuous plug-flow column
reactor (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).
[0243] The conventional methods include, but are not limited to,
saccharification, fermentation, separate hydrolysis and fermentation
(SHF), simultaneous saccharification and fermentation (SSF), simultaneous
saccharification and cofermentation (SSCF), hybrid hydrolysis and
fermentation (HHF), and direct microbial conversion (DMC).
[0244] SHF uses separate process steps to first enzymatically hydrolyze
cellulose to glucose and then ferment glucose to ethanol. In SSF, the
enzymatic hydrolysis of cellulose and the fermentation of glucose to
ethanol is 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 includes 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 includes two separate steps carried out in the same reactor but 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 (cellulase production,
cellulose hydrolysis, and fermentation) in one step (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: 596-577).
[0245] "Fermentation" or "fermentation process" refers to any fermentation
process or any process comprising a fermentation step. A fermentation
process includes, without limitation, fermentation processes used to
produce fermentation products including alcohols (e.g., arabinitol,
butanol, ethanol, glycerol, methanol, 1,3-propanediol, sorbitol, and
xylitol); organic acids (e.g., acetic acid, acetonic acid, adipic acid,
ascorbic acid, citric acid, 2,5-diketo-D-&conic 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); ketones (e.g., acetone); amino acids (e.g., aspartic acid,
glutamic acid, glycine, lysine, serine, and threonine); gases (e.g.,
methane, hydrogen (H.sub.2), carbon dioxide (CO.sub.2), and carbon
monoxide (CO)). 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.
[0246] The present invention further relates to methods of producing a
substance, comprising: (a) saccharifying a cellulosic material with a
composition comprising an effective amount of a polypeptide having
endoglucanase activity; (b) fermenting the saccharified cellulosic
material of step (a) with one or more fermentating microorganisms; and
(c) recovering the substance from the fermentation. The composition
comprising the polypeptide having endoglucanase activity may 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 may comprise a host cell of the present invention as a source
of the polypeptide having endoglucanase activity in a fermentation
process with the biomass.
[0247] The substance can be any substance derived from the fermentation.
In a preferred embodiment, the substance 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 embodiment, the
alcohol is arabinitol. In another more preferred embodiment, the alcohol
is butanol. In another more preferred embodiment, the alcohol is ethanol.
In another more preferred embodiment, the alcohol is glycerol. In another
more preferred embodiment, the alcohol is methanol. In another more
preferred embodiment, the alcohol is 1,3-propanediol. In another more
preferred embodiment, the alcohol is sorbitol. In another more preferred
embodiment, 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, 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.
[0248] In another preferred embodiment, the substance is an organic acid.
In another more preferred embodiment, the organic acid is acetic acid. In
another more preferred embodiment, the organic acid is acetonic acid. In
another more preferred embodiment, the organic acid is adipic acid. In
another more preferred embodiment, the organic acid is ascorbic acid. In
another more preferred embodiment, the organic acid is citric acid. In
another more preferred embodiment, the organic acid is
2,5-diketo-D-gluconic acid. In another more preferred embodiment, the
organic acid is formic acid. In another more preferred embodiment, the
organic acid is fumaric acid. In another more preferred embodiment, the
organic acid is glucaric acid. In another more preferred embodiment, the
organic acid is gluconic acid. In another more preferred embodiment, the
organic acid is glucuronic acid. In another more preferred embodiment,
the organic acid is glutaric acid. In another preferred embodiment, the
organic acid is 3-hydroxypropionic acid. In another more preferred
embodiment, the organic acid is itaconic acid. In another more preferred
embodiment, the organic acid is lactic acid. In another more preferred
embodiment, the organic acid is malic acid. In another more preferred
embodiment, the organic acid is malonic acid. In another more preferred
embodiment, the organic acid is oxalic acid. In another more preferred
embodiment, the organic acid is propionic acid. In another more preferred
embodiment, the organic acid is succinic acid. In another more preferred
embodiment, 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.
[0249] In another preferred embodiment, the substance 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
embodiment, the ketone is acetone. See, for example, Qureshi and
Blaschek, 2003, supra.
[0250] In another preferred embodiment, the substance is an amino acid. In
another more preferred embodiment, the organic acid is aspartic acid. In
another more preferred embodiment, the amino acid is glutamic acid. In
another more preferred embodiment, the amino acid is glycine. In another
more preferred embodiment, the amino acid is lysine. In another more
preferred embodiment, the amino acid is serine. In another more preferred
embodiment, 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.
[0251] In another preferred embodiment, the substance is a gas. In another
more preferred embodiment, the gas is methane. In another more preferred
embodiment, the gas is H.sub.2. In another more preferred embodiment, the
gas is CO.sub.2. In another more preferred embodiment, 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.
[0252] Production of a substance from cellulosic material typically
requires four major steps. These four steps are pretreatment, enzymatic
hydrolysis, fermentation, and recovery. Exemplified below is a process
for producing ethanol, but it will be understood that similar processes
can be used to produce other substances, for example, the substances
described above.
[0253] Pretreatment. In the pretreatment or pre-hydrolysis step, the
cellulosic material is heated to break down the lignin and carbohydrate
structure, solubilize most of the hemicellulose, and make the cellulose
fraction accessible to cellulolytic enzymes. The heating is performed
either directly with steam or in slurry where a catalyst may also be
added to the material to speed up the reactions. Catalysts include strong
acids, such as sulfuric acid and SO.sub.2, or alkali, such as sodium
hydroxide. The purpose of the pre-treatment stage is to facilitate the
penetration of the enzymes and microorganisms. Cellulosic biomass may
also be subject to a hydrothermal steam explosion pre-treatment (See U.S.
Patent Application No. 20020164730).
[0254] Saccharification. In the enzymatic hydrolysis step, also known as
saccharification, enzymes as described herein are added to the pretreated
material to convert the cellulose fraction to glucose and/or other
sugars. The saccharification is generally performed in stirred-tank
reactors or fermentors under controlled pH, temperature, and mixing
conditions. A saccharification step may last up to 200 hours.
Saccharification may be carried out at temperatures from about 30.degree.
C. to about 65.degree. C., in particular around 50.degree. C., and at a
pH in the range between about 4 and about 5, especially around pH 4.5. To
produce glucose that can be metabolized by yeast, the hydrolysis is
typically performed in the presence of a beta-glucosidase.
[0255] Fermentation. In the fermentation step, sugars, released from the
cellulosic material as a result of the pretreatment and enzymatic
hydrolysis steps, are fermented to ethanol by a fermenting organism, such
as yeast. The fermentation can also be carried out simultaneously with
the enzymatic hydrolysis in the same vessel, again under controlled pH,
temperature, and mixing conditions. When saccharification and
fermentation are performed simultaneously in the same vessel, the process
is generally termed simultaneous saccharification and fermentation or
SSF.
[0256] Any suitable cellulosic substrate or raw material may be used in a
fermentation process of the present invention. The substrate 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 may be supplied by direct addition to the
fermentation medium.
[0257] The term "fermentation medium" will be understood 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).
[0258] "Fermenting microorganism" refers to any microorganism suitable for
use in a desired fermentation process. Suitable fermenting microorganisms
according to the invention are able to ferment, i.e., convert, sugars,
such as glucose, xylose, arabinose, mannose, galactose, or
oligosaccharides directly or indirectly into the desired fermentation
product. Examples of fermenting microorganisms include fungal organisms,
such as yeast. Preferred yeast includes strains of the Saccharomyces
spp., and in particular, Saccharomyces cerevisiae. Commercially available
yeast include, e.g., Red Star.RTM./.TM./Lesaffre Ethanol Red (available
from Red Startesaffre, USA) FALI (available from Fleischmann's Yeast, a
division of Burns Philp Food Inc., USA), SUPERSTART (available from
Alltech), GERT STRAND (available from Gert Strand AB, Sweden) and FERMIOL
(available from DSM Specialties).
[0259] In a preferred embodiment, the yeast is a Saccharomyces spp. In a
more preferred embodiment, the yeast is Saccharomyces cerevisiae. In
another more preferred embodiment, the yeast is Saccharomyces distaticus.
In another more preferred embodiment, the yeast is Saccharomyces uvarum.
In another preferred embodiment, the yeast is a Kluyveromyces. In another
more preferred embodiment, the yeast is Kluyveromyces marxianus. In
another more preferred embodiment, the yeast is Kluyveromyces fragilis.
In another preferred embodiment, the yeast is a Candida. In another more
preferred embodiment, the yeast is Candida pseudotropicalis. In another
more preferred embodiment, the yeast is Candida brassicae. In another
preferred embodiment, the yeast is a Clavispora. In another more
preferred embodiment, the yeast is Clavispora lusitaniae. In another more
preferred embodiment, the yeast is Clavispora opuntiae. In another
preferred embodiment, the yeast is a Pachysolen. In another more
preferred embodiment, the yeast is Pachysolen tannophilus. In another
preferred embodiment, the yeast is a Bretannomyces. In another more
preferred embodiment, 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).
[0260] Bacteria that can efficiently ferment glucose to ethanol include,
for example, Zymomonas mobilis and Clostridium thermocellum (Philippidis,
1996, supra).
[0261] It is well known in the art that the organisms described above can
also be used to produce other substances, as described herein.
[0262] The cloning of heterologous genes in Saccharomyces cerevisiae
(Chen, Z., Ho, N. W. Y., 1993, Cloning and improving the expression of
Pichia stipitis xylose reductase gene in Saccharomyces cerevisiae, App.
Biochem. Biotechnol. 39-40: 135-147: Ho, N. W. Y., Chen, Z, Brainard, A.
P., 1998, Genetically engineered Saccharomyces yeast capable of
effectively cofermenting glucose and xylose, Appl. Environ. Microbiol,
64: 1852-1859), or in bacteria such as Escherichia coli (Beall, D. S.,
Ohta, K., Ingram, L. O., 1991, Parametric studies of ethanol production
from xylose and other sugars by recombinant Escherichia coli, Biotech.
Bioeng. 38: 296-303), Kiebsiella oxytoca (Ingram, L. O., Gomes, P. F.,
Lai, X., Moniruzzaman, M., Wood, B. E., Yomano, L. P., York, S. W., 1998,
Metabolic engineering of bacteria for ethanol production, Biotechnol.
Bioeng. 58: 204-214), and Zymomonas mobilis (Zhang, M., Eddy, C., Deanda,
K., Finkelstein, M., and Picataggio, S., 1995, Metabolic engineering of a
pentose metabolism pathway in ethanologenic Zymomonas mobilis, Science
267: 240-243; Deanda, K., Zhang, M., Eddy, C., and Picataggio, S., 1996,
Development of an arabinose-fermenting Zymomonas mobilis strain by
metabolic pathway engineering, Appl. Environ. Microbiol. 62: 4465-4470)
has led to the construction of organisms capable of converting hexoses
and pentoses to ethanol (cofermentation).
[0263] Yeast or another microorganism typically is added to the degraded
cellulose or hydrolysate and the fermentation is ongoing for about 24 to
about 96 hours, such as about 35 to about 60 hours. The temperature is
typically between about 26.degree. C. to about 40.degree. C., in
particular at about 32.degree. C., and at about pH 3 to about pH 6, in
particular around pH 4-5.
[0264] In a preferred embodiment, yeast or another microorganism is
applied to the degraded cellulose or hydrolysate and the fermentation is
ongoing for about 24 to about 96 hours, such as typically 35-60 hours. In
a preferred embodiments, the temperature is generally between about 26 to
about 40.degree. C., in particular about 32.degree. C., and the pH is
generally from about pH 3 to about pH 6, preferably around pH 4-5. 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 5.times.10.sup.7 viable
count per ml of fermentation broth. During an ethanol producing phase the
yeast cell count should preferably be in the range from approximately
10.sup.7 to 10.sup.10, especially around approximately 2.times.10.sup.8.
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.
[0265] 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.
[0266] For ethanol production, following the fermentation the mash is
distilled to extract the ethanol. The ethanol obtained according to the
process of the invention may be used as, e.g., fuel ethanol; drinking
ethanol, i.e., potable neutral spirits, or industrial ethanol.
[0267] A fermentation stimulator may 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, e.g., 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.
[0268] Recovery. The 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. % ethanol can be obtained, which can
be used as, for example, fuel ethanol, drinking ethanol, i.e., potable
neutral spirits, or industrial ethanol.
[0269] For other substances, any method known in the art can be used
including, but not limited to, chromatography (e.g., on exchange,
affinity, hydrophobic, chromatofocusing, and size exclusion),
electrophoretic procedures (e.g., preparative isoelectric focusing),
differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE,
distillation, or extraction.
[0270] In the methods of the present invention, the polypeptide having
endoglucanase activity and other cellulolytic protein(s) may be
supplemented by one or more additional enzyme activities to improve the
degradation of the cellulosic material. Preferred additional enzymes are
hemicellulases, esterases (e.g., lipases, phospholipases, and/or
cutinases), proteases, laccases, peroxidases, or mixtures thereof.
[0271] In the methods of the present invention, the additional enzyme(s)
may be added prior to or during fermentation, including during or after
the propagation of the fermenting microorganism(s).
Signal Peptides
[0272] 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 18 of SEQ ID NO: 2 or amino acids 1 to 19
of SEQ ID NO: 4, wherein the gene is foreign to the first and second
nucleotide sequences
[0273] In a preferred aspect, the nucleotide sequence comprises or
consists of nucleotides 56 to 109 of SEQ ID NO: 1. In another preferred
aspect, the nucleotide sequence comprises or consists of nucleotides 1 to
57 of SEQ ID NO: 3.
[0274] The present invention also relates to recombinant expression
vectors and recombinant host cells comprising such nucleic acid
constructs.
[0275] The present invention also relates to methods for producing a
protein comprising (a) cultivating such a recombinant host cell under
conditions suitable for production of the protein; and (b) recovering the
protein.
[0276] 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 which comprise a combination of partial or complete
polypeptide sequences obtained from at least two different proteins
wherein one or more 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.
[0277] 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 xylanase.
[0278] The gene may be obtained from any prokaryotic, eukaryotic, or other
source.
[0279] The present invention is further described by the following
examples which should not be construed as limiting the scope of the
invention.
EXAMPLES
Materials
[0280] Chemicals used as buffers and substrates were commercial products
of at least reagent grade.
Strains
[0281] Thielavia terrestris NRRL 8126 was used as the source of the Family
6 polypeptides having endoglucanase activity. Saccharomyces cerevisiae
strain W3124 (MATa; ura 3-52; leu 2-3, 112; his 3-D200; pep 4-1137;
prc1::HIS3; prb1:: LEU2; cir.sup.+) was used for screening of Thielavia
terrestris NRRL 8126 expression libraries for endoglucanase activity.
Aspergillus oryzae HowB104 strain (alpha-amylase negative) was used for
expression of the Thielavia terrestris NRRL 8126 CEL6B polypeptide having
endoglucanase activity. Aspergillus oryzae JaL250 strain (WO 99/61651)
was used for expression of the Thielavia terrestris NRRL 8126 CEL6C
polypeptide having endoglucanase activity.
Media and Solutions
[0282] LB medium was composed per liter of 10 g of tryptone, 5 g of yeast
extract, and 5 g of sodium chloride.
[0283] LB ampicillin medium was composed per liter of 10 g of tryptone, 5
g of yeast extract, 5 g of sodium chloride, and 50 .mu.g of ampicillin
per ml (filter sterilized, added after autoclaving).
[0284] LB ampicillin plates were composed per liter of LB ampicillin
medium and 15 g of bacto agar.
[0285] YEG medium was composed of 0.5% yeast extract and 2% glucose.
[0286] YPD medium was composed of 1% yeast extract, 2% peptone, and
filter-sterilized 2% glucose added after autoclaving.
[0287] YPM medium was composed of 1% yeast extract, 2% peptone, and
filter-sterilized 2% maltodextrin added after autoclaving.
[0288] SC-URA medium with galactose was composed per liter of 100 ml of
10.times. Basal salts, 28 ml of 20% casamino acids without vitamins, 10
ml of 1% tryptophan, 3.6 ml of 5% threonine (filter sterilized, added
after autoclaving), and 100 ml of 20% galactose (filter sterilized, added
after autoclaving).
[0289] SC-URA medium with glucose was composed per liter of 100 ml of
10.times. Basal salts solution, 28 ml of 20% casamino acids without
vitamins, 10 ml of 1% tryptophan, 3.6 ml of 5% threonine (filter
sterilized, added after autoclaving), and 100 ml of 20% glucose (filter
sterilized, added after autoclaving).
[0290] 10.times. Basal salts solution was composed per liter of 75 g of
yeast nitrogen base, 113 g of succinic acid, and 68 g of NaOH.
[0291] SC-agar was composed per liter of SC-URA medium (with glucose or
galactose as indicated) and 20 g of agar.
[0292] 0.1% AZCL HE cellulose SC agar plates with galactose were composed
per liter of SC-URA medium with galactose, 20 g of agar, and 0.1% AZCL HE
cellulose (Megazyme, Wicklow, Ireland).
[0293] PD medium with cellulose was composed per liter of 24 grams potato
dextrose (Difco) and 30 grams of Solcafloc (Diacel available from
Dicalie-Europe-Nord, Gent, Belgium)
[0294] Potato dextrose medium was composed per liter of 39 grams of potato
dextrose (Difco).
[0295] PDA plates were composed per liter of 39 grams of potato dextrose
agar.
[0296] 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.2SO.sub.4, 12 g of
KH.sub.2PO.sub.4, 7 g of yeast extract, 2 g of urea, and 0.5 ml of AMG
trace metals solution, pH adjusted to 5.0.
[0297] 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.H.sub.2O, and 3 g of citric acid.
[0298] COVE plates were composed per liter of 342.3 g of sucrose, 20 ml of
COVE salt solution, 10 ml of 1 M acetamide, 10 ml of 1.5 M CsCl.sub.2,
and 25 g of Noble agar.
[0299] COVE salt solution was composed per liter of 26 g of KCl, 26 g of
MgSO.sub.4.7H.sub.2O, 76 g of KH.sub.2PO.sub.4, and 50 ml of COVE trace
metals solution.
[0300] 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.H.sub.2O and 10 g of ZnSO.sub.4.7H.sub.2O.
[0301] Trace metals solution was composed per liter of 41.2 mg of
FeCl.sub.3.6H.sub.2O, 11.6 mg of ZnSO.sub.4.7H.sub.2O, 5.4 mg of
MnSO.sub.4.H.sub.2O, 2.0 mg of CuSO.sub.4.5H.sub.2O, 0.48 mg of
H.sub.3BO.sub.3, and 67.2 mg of citric acid.
[0302] SOC medium was composed of 2% tryptone, 0.5% yeast extract, 10 mM
NaCl, 2.5 mM KCl, 10 mM MgCl.sub.2: 10 mM MgSO.sub.4, and
filter-sterilized glucose to 20 mM, added after autoclaving.
[0303] Freezing medium was composed of 60% SOC medium and 40% glycerol.
[0304] 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.
[0305] TE was composed of 10 mM Tris pH 7.4 and 0.1 mM EDTA.
Example 1
Thielavia terrestris NRRL 8126 Genomic DNA Extraction
[0306] Thielavia terrestris NRRL 8126 was grown in 25 ml of YEG medium at
37.degree. C. and 250 rpm for 24 hours. Mycelia were then collected by
filtration through Miracloth.TM. (CalBiochem, La Jolla, Calif., USA) and
washed once with 25 ml of 10 mM Tris-1 mM EDTA (TE) buffer. Excess buffer
was drained from the mycelia preparation, which was subsequently frozen
in liquid nitrogen. The frozen mycelia preparation was ground to a fine
powder in an electric coffee grinder, and the powder was added to a
disposable plastic centrifuge tube containing 20 ml of TE buffer and 5 ml
of 20% w/v sodium dodecylsulfate (SDS). The mixture was gently inverted
several times to ensure mixing, and extracted twice with an equal volume
of phenol:chloroform:isoamyl alcohol (25:24:1 v/v/v). Sodium acetate (3 M
solution) was added to the extracted sample to a final concentration of
0.3 M followed by 2.5 volumes of ice cold ethanol to precipitate the DNA.
The tube was centrifuged at 15,000.times.g for 30 minutes to pellet the
DNA. The DNA pellet was allowed to air-dry for 30 minutes before
resuspension in 0.5 ml of TE buffer. DNase-free ribonuclease A was added
to the resuspended DNA pellet to a concentration of 100 .mu.g per ml and
the mixture was then incubated at 37.degree. C. for 30 minutes.
Proteinase K (200 .mu.g/ml) was added and the tube was incubated an
additional one hour at 37.degree. C. Finally, the sample was centrifuged
for 15 minutes at 12,000.times.g, and the supernatant was applied to a
Qiaprep.RTM. 8 manifold (QIAGEN Inc., Valencia, Calif., USA). The columns
were washed twice with 1 ml of PB (QIAGEN Inc. Valencia, Calif., USA) and
1 ml of PE (QIAGEN Inc., Valencia, Calif., USA) under vacuum. The
isolated DNA was eluted with 100 .mu.l of TE, precipitated with ethanol,
washed with 70% ethanol, dried under vacuum, resuspended in TE buffer,
and stored at 4.degree. C.
[0307] To generate genomic DNA for PCR amplification, Thielavia terrestris
NRRL 8126 was grown in 50 ml of NNCYP medium supplemented with 1% glucose
in a baffled shake flask at 42.degree. C. and 200 rpm for 24 hours.
Mycelia were harvested by filtration, washed twice in TE (10 mM Tris-1 mM
EDTA), and frozen under liquid nitrogen. A pea-size, piece of frozen
mycelia was suspended in 0.7 ml of 1% lithium dodecyl sulfate in TE and
disrupted by agitation with an equal volume of 0.1 mm zirconia/silica
beads (Biospec Products, Inc., Bartlesville, Okla., USA) for 45 seconds
in a FastPrep FP120 (ThermoSavant, Holbrook, N.Y., USA). Debris was
removed by centrifugation at 13,000.times.g for 10 minutes and the
cleared supernatant was brought to 2.5 M ammonium acetate and incubated
on ice for 20 minutes. After the incubation period, the nucleic acids
were precipitated by addition of 2 volumes of ethanol. After
centrifugation for 15 minutes in a microfuge at 4.degree. C., the pellet
was washed in 70% ethanol and air dried. The DNA was resuspended in 120
.mu.l of 0.1.times.TE and incubated with 1 .mu.l of DNase-free RNase A at
37.degree. C. for 20 minutes. Ammonium acetate was added to 2.5 M and the
DNA was precipitated with 2 volumes of ethanol. The pellet was washed in
70% ethanol, air dried, and resuspended in TE buffer.
Example 2
PCR Amplification of a cel6b Gene Fragment from Thielavia terrestris NRRL
8126 Genomic DNA
[0308] Primers were designed based upon conserved motifs found in other
Family 6 glycosyl hydrolases. The specific peptide sequences used for
primer design were:
TABLE-US-00001
EPDSLANLVT (corresponding to amino acids 167 to 176 of SEQ ID NO: 2)
W[I,V]KPGGE[C,S] (amino acids 343 to 350 of SEQ ID NO: 2)
The CODEHOP strategy was employed (Rose at al., 1998, Nucleic Acids Res.
26: 1628-1635' to design the following primers:
TABLE-US-00002
Sense Primer:
(SEQ ID NO: 5)
5'-AGCCCGACTCCCTGGCNAAYCTGGTNAC-3'
Antisense Primer:
(SEQ ID NO: 6)
5'-GCACTCGCCGCCNGGYTTNAYCCA-3'
[0309] PCR amplification was performed in a volume of 30 .mu.l containing
1.times. AmpliTaq.RTM. buffer (Applied Biosystems, Foster City, Calif.,
USA), 2.5 units of AmpliTaq'., DNA polymerase (Applied Biosystems, Foster
City, Calif., USA), 1 .mu.M of each sense and antisense primer, and
approximately 1 .mu.g of genomic DNA from Thielavia terrestris.
Amplification was performed in a Robocycler.RTM. (Stratagene, La Jolla,
Calif., USA) programmed for 1 cycle at 96.degree. C. for 3 minutes and
72.degree. C. for 3 minutes (during which DNA polymerase was added); and
35 cycles each at 94.degree. C. for 45 seconds, 58.degree. C. for 45
seconds, and 72.degree. C. for 1 minute; followed by a final extension at
72.degree. C. for 7 minutes.
[0310] The reaction products were fractionated on a 1.6% agarose gel using
40 mM Tris base-20 mM sodium acetate-1 mM disodium EDTA (TAE) buffer, and
a band of approximately 800 bp was excised, purified using a QIAEX.RTM.
II Gel Extraction Kit (QIAGEN Inc., Valencia, Calif., USA), and subcloned
using a TOPO TA Kit (Invitrogen, Carlsbad, Calif., USA). The plasmid from
one E. coli transformant was sequenced and found to contain an insert of
789 bp coding for a Family 6 protein (CEL6B). This plasmid was designated
pPH23 (FIG. 1).
Example 3
Thielavia terrestris NRRL 8126 Genomic DNA Library Construction and
Screening
[0311] A genomic DNA library of Thielavia terrestris NRRL 8126 was
constructed using the bacteriophage cloning vector .lamda.ZipLox (Life
Technologies, Gaithersburg, Md., USA) with E. coli Y1090ZL cells (Life
Technologies, Gaithersburg, Md., USA) as a host for plating and
purification of recombinant bacteriophage and E. coli DH10Bzip (Life
Technologies, Gaithersburg, Md., USA) for excision of individual pZL1
clones containing the cel6b gene.
[0312] Thielavia terrestris NRRL 8126 genomic DNA prepared as described in
Example 1 was partially digested with Tsp 5091 and size-fractionated on
1% agarose gels using TAE buffer. DNA fragments migrating in the size
range 3-7 kb were excised and eluted from the gel using Prep-a-Gene
reagents (BioRad Laboratories, Hercules, Calif., USA). The eluted DNA
fragments were ligated with Eco RI-cleaved and dephosphorylated
.lamda.ZipLox vector arms (Life Technologies, Gaithersburg, Md., USA),
and the ligation mixtures were packaged using commercial packaging
extracts (Stratagene, La Jolla, Calif., USA). The packaged DNA libraries
were plated and amplified in E. coli Y1090ZL cells. The unamplifled
genomic DNA library contained 3.1.times.10.sup.6 pfu/ml (background
titers with no DNA were 2.0.times.10.sup.4 pfu/ml.
[0313] A Thielavia terrestris cel6b probe fragment was amplified from
pPH23 using primers homologous to the TOPO vector and Herculase.RTM. DNA
Polymerase (Stratagene, La Jolla, Calif., USA), as shown below.
TABLE-US-00003
(SEQ ID NO: 7)
5'-CTTGGTACCGAGCTCGGATCCACTA-3'
(SEQ ID NO: 8)
5'-ATAGGGCGAATTGGGCCCTCTAGAT-3'
[0314] Fifty picomoles of each of the primers were used in a PCR reaction
containing 10 ng of pPH23, 1.times. Herculase.RTM. Amplification Buffer
(Stratagene, La Jolla, Calif., USA), 1 .mu.l of 10 mM blend of dATP,
dTTP, dGTP, and dCTP, and 2.5 units of Herculase.RTM. DNA Polymerase in a
final volume of 50 .mu.l. Amplification was performed in a
Robocycler.RTM. programmed for 1 cycle at 94.degree. C. for 1 minute; and
20 cycles each at 94.degree. C. for 30 seconds, 55.degree. C. for 30
seconds, and 72.degree. C. for 1 minute. The heat block then went to a
4.degree. C. soak cycle.
[0315] The reaction product was isolated on a 1.0% agarose gel using TAE
buffer where a 0.8 kb product band was excised from the gel and purified
using a QIAquick.RTM. Gel Extraction Kit (QIAGEN Inc., Valencia, Calif.,
USA) according to the manufacturer's instructions. A twenty five ng
sample of the fragment was radiolabeled with .sup.32P using a
Prime-It.RTM. II Kit (Stratagene, La Jolla, Calif., USA).
[0316] Approximately 90,000 plaques from the library were screened by
plaque-hybridization using the labeled PCR fragment as the probe. The DNA
was cross-linked onto membranes (Hybond N+, Amersham, Arlington Heights,
Ill., USA) using a UV Stratalinkerr.RTM. (Stratagene, La Jolla, Calif.,
USA). The .sup.32P-radiolabeled gene fragment was denatured by adding
sodium hydroxide to a final concentration of 0.1 M, and added to a
hybridization solution containing 6.times.SSPE, 7% SDS at an activity of
approximately 1.times.10.sup.6 cpm per ml of hybridization solution. The
mixture was incubated overnight at 65.degree. C. in a shaking water bath.
Following incubation, the membranes were washed three times for fifteen
minutes in 0.2.times.SSC with 0.1% SDS at 65.degree. C. The membranes
were dried on blotting paper for 15 minutes, wrapped in SaranWrap.TM.,
and exposed to X-ray film overnight at 70.degree. C. with intensifying
screens (Kodak, Rochester, N.Y., USA).
[0317] Based on the production of strong hybridization signals with the
cel6b probe described above, several plaques were chosen for further
study. The plaques were purified twice in E. coli Y1090ZL cells and the
inserted genes and pZL1 plasmid were subsequently excised from the
.lamda.ZipLox vector as pZL1-derivatives (D'Alessio et al., 1992,
Focus.RTM. 14:76) using in vivo excision by infection of E. coli DH10BZL
cells (Life Technologies, Gaithersburg, Md., USA). The colonies were
inoculated into three ml of LB ampicillin medium and grown overnight at
37.degree. C. Miniprep DNA was prepared from each of these cultures using
a BioRobot 9600 (QIAGEN Inc., Valencia, Calif., USA). A clone designated
pPH47 was shown by DNA sequencing to contain the full-length gene for
cel6b.
[0318] The E. coli strain PaHa47 containing plasmid pPH47 was deposited
with the Agricultural Research Service Patent Culture Collection,
Northern Regional Research Center, 1815 University Street, Peoria, Ill.,
61604, as NRRL B-30898, with a deposit date of Feb. 23, 2006.
Example 4
Characterization of the Thielavia terrestris NRRL 8126 Genomic Sequence
Encoding a CEL6B Endoglucanase
[0319] DNA sequencing of the Thielavia terrestris NRRL 8126 cel6b genomic
clone was performed with an Applied Biosystems Model 3700 Automated DNA
Sequencer using version 3.1 Big-Dye.TM. terminator chemistry (Applied
Biosystems, Inc., Foster City, Calif., USA) and dGTP chemistry (Applied
Biosystems, Inc., Foster City, Calif., USA) and primer walking strategy.
Nucleotide sequence data were scrutinized for quality and all sequences
were compared to each other with assistance of PHRED/PHRAP software
(University of Washington, Seattle, Wash., USA).
[0320] A gene model for the Thielavia terrestris cel6b genomic DNA
sequence was constructed based on similarity to homologous endoglucanase
genes from Fusarium oxysporum and Chrysosporium lucknowense (Accession
numbers XP383804 and AAQ38151.1, respectively).
[0321] The nucleotide sequence (SEQ ID NO: 1) and deduced amino acid
sequence (SEQ ID NO: 2) of the Thielavia terrestris cel6b gene are shown
in FIG. 2. The coding sequence is 1505 bp including the stop codon and is
interrupted by introns of 77, 127 and 110 bp. The encoded predicted
protein is 396 amino acids. The % G+C of the coding sequence of the gene
(including introns) is 64.9% G+C and the mature polypeptide coding
sequence is 64.6%. Using the SignalP program (Nielsen et al., 1997,
Protein Engineering 10:1-6), a signal peptide of 18 residues was
predicted. The predicted mature protein contains 378 amino acids with a
molecular mass of 40.6 kDa and an isoelectric pH of 5.01.
[0322] 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 gene encoding the CEL6B polypeptide
having endoglucanase activity shares 85% and 82% identity (excluding
gaps) to the deduced amino acid sequences of two Family 6 glycosyl
hydrolase proteins from Chrysosporium lucknowense and Neurospora crassa,
respectively (accession numbers AAQ38151 and Q7RXI7, respectively).
Example 5
Cloning and Expression of a Thielavia terrestris NRRL 8126 cDNA Encoding a
CEL6C Endoglucanase
[0323] Thielavia terrestris NRRL 8126 was cultivated in 200 ml of PD
medium with cellulose at 30.degree. C. for five days at 200 rpm. Mycelia
from the shake flask culture were harvested by filtering the contents
through a funnel lined with Miracloth.TM.. The mycelia were then
sandwiched between two Miracloth.TM. pieces and blotted dry with
absorbent paper towels. The mycelial mass was then transferred to
Falcon.RTM. 1059 plastic centrifuge tubes and frozen in liquid nitrogen.
Frozen mycelia were stored in a -80.degree. C. freezer until use.
[0324] The extraction of total RNA was performed with guanidinium
thiocyanate followed by ultracentrifugation through a 5.7 M CsCl cushion,
and isolation of poly(A)+RNA was carried out by oligo(dT)-cellulose
affinity chromatography, using the procedures described in WO 94/14953.
[0325] Double-stranded cDNA was synthesized from 5 .mu.g of poly(A)+RNA by
the RNase H method (Gubler and Hoffman, 1983, Gene 25: 263-269, Sambrook
et al., 1989, Molecular cloning: A laboratory manual, Cold Spring Harbor
lab., Cold Spring Harbor, N.Y., USA). The poly(A).sup.+ RNA (5 .mu.g in 5
.mu.l of DEPC (0.1% diethylpyrocarbonate)-treated water) was heated at
70.degree. C. for 8 minutes in a pre-siliconized, RNase-free
Eppendorf.RTM. tube, quenched on ice, and combined in a final volume of
50 .mu.l with reverse transcriptase buffer composed of 50 mM Tris-HCl, pH
8.3, 75 mM KCl, 3 mM MgCl.sub.2, 10 mM dithiothreitol (DTT) (Bethesda
Research Laboratories, Bethesda, Md., USA), 1 mM of dATP, dGTP and dTTP,
and 0.5 mM 5-methyl-dCTP (Pharmacia, Uppsala, Sweden), 40 units of human
placental ribonuclease inhibitor (RNasin, Promega, Madison, Wis., USA),
1.45 .mu.g of oligo(dT).sub.18-Not I primer (Pharmacia, Uppsala, Sweden),
and 1000 units of SuperScript.RTM. II RNase H reverse transcriptase
(Bethesda Research Laboratories, Bethesda, Md., USA). First-strand cDNA
was synthesized by incubating the reaction mixture at 45.degree. C. for 1
hour. After synthesis, the mRNA:cDNA hybrid mixture was gel filtrated
through a MicroSpin S-400 HR spin column (Pharmacia, Uppsala, Sweden)
according to the manufacturer's instructions.
[0326] After gel filtration, the hybrids were diluted in 250 .mu.l of
second strand buffer (20 mM Tris-HCl, pH 7.4, 90 mM KCl, 4.6 mM
MgCl.sub.2, 10 mM (NH.sub.4) SO.sub.4, 0.16 mM NAD) containing 200 .mu.M
of each dNTP, 60 units of E. coli DNA polymerase I (Pharmacia, Uppsala,
Sweden), 5.25 units of RNase H (Promega, Madison, Wis., USA), and 15
units of E. coli DNA ligase (Boehringer Mannheim, Manheim, Germany),
Second strand cDNA synthesis was performed by incubating the reaction
tube at 16.degree. C. for 2 hours and an additional 15 minutes at
25.degree. C. The reaction was stopped by addition of EDTA to a final
concentration of 20 mM followed by phenol and chloroform extractions.
[0327] The double-stranded cDNA was precipitated at -20.degree. C. for 12
hours by addition of 2 volumes of 96% ethanol and 0.2 volume of 10 M
ammonium acetate, recovered by centrifugation at 13,000.times.g, washed
in 70% ethanol, dried, and resuspended in 30 .mu.l of Mung bean nuclease
buffer (30 mM sodium acetate pH 4.6, 300 mM NaCl, 1 mM ZnSO.sub.4, 0.35
mM DTT, 2% glycerol) containing 25 units of Mung bean nuclease
(Pharmacia, Uppsala, Sweden). The single-stranded hair-pin DNA was
clipped by incubating the reaction at 30.degree. C. for 30 minutes,
followed by addition of 70 .mu.l of 10 mM Tris-HCl-1 mM EDTA pH 7.5,
phenol extraction, and precipitation with 2 volumes of 96% ethanol and
0.1 volume of 3 M sodium acetate pH 5.2 on ice for 30 minutes.
[0328] The double-stranded cDNAs were recovered by centrifugation at
13,000.times.g and blunt-ended in 30 .mu.l of T4 DNA polymerase buffer
(20 mM Tris-acetate, pH 7.9, 10 mM magnesium acetate, 50 mM potassium
acetate, 1 mM DTT) containing 0.5 mM of each dNTP and 5 units of T4 DNA
polymerase (New England Biolabs, Ipswich, Mass., USA) by incubating the
reaction mixture at 16.degree. C. for 1 hour. The reaction was stopped by
addition of EDTA to a final concentration of 20 mM, followed by phenol
and chloroform extractions, and precipitation for 12 hours at -20.degree.
C. by adding 2 volumes of 96% ethanol and 0.1 volume of 3 M sodium
acetate pH 5.2.
[0329] After the fill-in reaction the cDNAs were recovered by
centrifugation at 13,000.times.g, washed in 70% ethanol, and dried. The
cDNA pellet was resuspended in 25 .mu.l of ligation buffer (30 mM
Tris-HCl, pH 7.8, 10 mM MgCl.sub.2, 10 mM DTT, 0.5 mM ATP) containing 2.5
.mu.g of non-palindromic Bst XI adaptors (Invitrogen, Carlsbad, Calif.,
USA), shown below, and 30 units of T4 ligase (Promega, Madison, Wis.,
USA), and then incubated at 16.degree. C. for 12 hours. The reaction was
stopped by heating at 65.degree. C. for 20 minutes and then cooled on ice
for 5 minutes.
5-CTTTCCAGCACA-3' (SEG ID NO: 9)
3'-GAAAGGTC-5'
[0330] The adapted cDNA was digested with Not I, followed by incubation
for 2.5 hours at 37.degree. C. The reaction was stopped by heating at
65.degree. C. for 10 minutes. The cDNAs were size-fractionated by gel
electrophoresis on a 0.8% SeaPlaque.RTM. GTG low melting temperature
agarose gel (Cambrex Corporation, East Rutherford, N.J., USA) in 44 mM
Tris Base, 44 mM boric acid, 0.5 mM EDTA (TBE) buffer to separate
unligated adaptors and small cDNAs. The cDNA was size-selected with a
cut-off at 0.7 kb and rescued from the gel by use of beta-agarase (New
England Biolabs, Ipswich, Mass., USA) according to the manufacturer's
instructions and precipitated for 12 hours at -20.degree. C. by adding
two volumes of 96% ethanol and 0.1 volume of 3 M sodium acetate pH 5.2.
[0331] The directional, size-selected cDNA was recovered by centrifugation
at 13,000.times.g, washed in 70% ethanol, dried, and then resuspended in
30 .mu.l of 10 mM Tris-HCl-1 mM EDTA pH 7.5. The cDNAs were desalted by
gel filtration through a MicroSpin S-300 HR spin column according to the
manufacturer's instructions. Three test ligations were carried out in 10
.mu.l of ligation buffer (30 mM Tris-HCl, pH 7.8, 10 mM MgCl.sub.2, 10 mM
DTT, 0.5 mM ATP) containing 5 .mu.l of double-stranded cDNA (reaction
tubes #1 and #2), 15 units of T4 ligase (Promega, Madison, Wis., USA),
and 30 ng (tube #1), 40 ng (tube #2), and 40 ng (tube #3, the vector
background control) of Bst XI-Not I cleaved pYES2.0 vector (invitrogen,
Carlsbad, Calif., USA). The ligation reactions were performed by
incubation at 16.degree. C. for 12 hours, then heating at 70.degree. C.
for 20 minutes, and finally adding 10 .mu.l of water to each tube. One
.mu.l of each ligation mixture was electroporated into 40 .mu.l of
electrocompetent E. coli DH10B cells (Bethesda Research Laboratories,
Bethesda, Md., USA) as described by Sambrook et al., 1989, supra.
[0332] The Thielavia terrestris NRRL 8126 cDNA library was established as
pools in E. coli DH10B. Each pool was made by spreading transformed E.
coli on LB ampicillin plates, yielding 15,000-30,000 colonies/plate after
incubation at 37.degree. C. for 24 hours. Twenty ml of LB ampicillin
medium was added to the plate and the cells were suspended therein. The
cell suspension was shaken at 100 rpm in a 50 ml tube for 1 hour at
37.degree. C.
[0333] The resulting Thielavia terrestris NRRL 8126 cDNA library consisted
of approximately 10.sup.6 individual clones, with a vector background of
1%. Plasmid DNA from some of the library pools was isolated using a
Plasmid Midi Kit (QIAGEN Inc., Valencia, Calif., USA) according to the
manufacturer's instructions, and stored at -20.degree. C.
[0334] One ml aliquots of purified plasmid DNA (100 ng/ml) from some of
the library pools (Example 1) were transformed into Saccharomyces
cerevisiae W3124 by electroporation (Becker and Guarante, 1991, Methods
Enzymol, 194: 182-187) and the transformants were plated on SC agar
containing 2% glucose and incubated at 30.degree. C. In total, 50-100
plates containing 250-400 yeast colonies were obtained from each pool.
[0335] After 3-5 days of incubation, the SC agar plates were replica
plated onto a set of 0.1% AZCL HE cellulose SC URA agar plates with
galactose. The plates were incubated for 2-4 days at 30.degree. C. and
endoglucanase positive colonies were identified as colonies surrounded by
a blue halo.
Example 6
Characterization of the Thielavia terrestris NRRL 8126 cDNA Sequence
Encoding a CEL6C Endoglucanase
[0336] Endoglucanase-expressing yeast colonies were inoculated into 20 ml
of YPD medium in 50 ml glass test tubes. The tubes were shaken at 200 rpm
for 2 days at 30.degree. C. The cells were harvested by centrifugation
for 10 minutes at 3000 rpm in a Heraeus Megafuge 1.0R centrifuge with a
75002252 rotor (Hanau, Germany).
[0337] DNA was isolated according to WO 94/14953 and dissolved in 50 .mu.l
of deionized water. The DNA was transformed into E. coli DH10B cells by
standard procedures according to Sambrook et al., 1989, supra. One E.
coli transformant subsequently shown to contain the Thielavia terrestris
NRRL 8126 cel6c gene was designated pCIBG146 (FIG. 3) and used as
material for deposit of biological material, E. coli strain pCIBG146 was
deposited as E. coli NRRL B-30901 on Feb. 23, 2006.
[0338] Plasmid DNA was isolated from the E. coli transformants using
standard procedures according to Sambrook et al., 1989, supra. The full
length cDNA sequence of the cel6c gene from Thielavia terrestris NRRL
8126 was sequenced with a Taq DyeDeoxy Terminator Cycle Sequencing Kit
(Perkin Elmer, Wellesley, Mass., USA) and synthetic oligonucleotide
primers using an Applied Biosystems ABI PRISM.TM. 377 DNA Sequencer (ABI,
Foster City, Calif., USA) according to the manufacturer's instructions.
[0339] The nucleotide sequence (SEQ ID NO: 3) and deduced amino acid
sequence (SEQ ID NO: 4) of the Thielavia terrestris cel6c gene are shown
in FIG. 4. The coding sequence is 1203 bp including the stop codon. The
encoded predicted protein is 400 amino acids. The % G+C of the coding
sequence of the gene is 60.0% and the mature polypeptide coding sequence
is 60.0%. Using the SignalP program (Nielsen et al., 1997, supra), a
signal peptide of 19 residues was predicted. The predicted mature protein
contains 381 amino acids with a molecular mass of 42.1 kDa and pl of
6.76.
[0340] A comparative pairwise global alignment of amino acid sequences was
determined using the Needleman-Wunsch algorithm (Needleman and Wunsch,
1970, supra) 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 gene encoding the CEL6C polypeptide shares 50%, 49%
and 48% identity (excluding gaps) to the deduced amino acid sequences of
three Family 6 glycosyl hydrolase proteins from Neurospora crassa,
Magnaporthe grisea, and Agaricus bisporus, respectively (accession
numbers Q87B5, XP 368004, and P49075, respectively).
Example 7
Construction of pAlLo2 Expression Vector
[0341] Expression vector pAlLo1 was constructed by modifying pBANe6 (U.S.
Pat. No. 6,461,837), which comprises a hybrid of the promoters from the
genes for Aspergillus niger neutral alpha-amylase and Aspergillus oryzae
triose phosphate isomerase (NA2-tpi promoter), Aspergillus niger
amyloglucosidase terminator sequence (AMG terminator), and Aspergillus
nidulans acetamidase gene (amdS). All mutagenesis steps were verified by
sequencing using Big-Dye.TM. terminator chemistry (Applied Biosystems,
Inc., Foster City, Calif., USA). Modification of pBANe6 was performed by
first eliminating three Nco I restriction sites at positions 2051, 2722,
and 3397 bp from the amdS selection marker by site-directed mutagenesis.
All changes were designed to be "silent" leaving the actual protein
sequence of the amdS gene product unchanged. Removal of these three sites
was performed simultaneously with a GeneEditor.TM. in vitro Site-Directed
Mutagenesis Kit (Promega, Madison, Wis., USA) according to the
manufacturer's instructions using the following primers (underlined
nucleotide represents the changed base):
TABLE-US-00004
AMDS3NcoMut (2050):
(SEQ ID NO: 10)
5'-GTGCCCCATGATACGCCTCCGG-3'
AMDS2NcoMut (2721):
(SEQ ID NO: 11)
5'-GAGTCGTATTTCCAAGGCTCCTGACC-3'
AMDS1NcoMut (3396):
(SEQ ID NO: 12)
5'-GGAGGCCATGAAGTGGACCAACGG-3'
[0342] A plasmid comprising all three expected sequence changes was then
submitted to site-directed mutagenesis, using a QuickChangen.RTM.
Site-Directed Mutagenesis Kit (Stratagene, La Jolla, Calif., USA), to
eliminate the Nco I restriction site at the end of the AMG terminator at
position 1643. The following primers (underlined nucleotide represents
the changed base) were used for mutagenesis:
TABLE-US-00005
Upper Primer to mutagenize the AMG
terminator sequence:
(SEQ ID NO: 13)
5'-CACCGTGAAAGCCATGCTCTTTCCTTCGTGTAGAAGACCAGACAG-3'
Lower Primer to mutagenize the AMG
terminator sequence:
(SEQ ID NO: 14)
5'-CTGGTCTTCTACACGAAGGAAAGAGCATGGCTTTCACGGTGTCTG-3'
[0343] The last step in the modification of pBANe6 was the addition of a
new Nco I restriction site at the beginning of the polylinker using a
QuickChange.TM. Site-Directed Mutagenesis Kit and the following primers
(underlined nucleotides represent the changed bases) to yield pAlLo1
(FIG. 5).
TABLE-US-00006
Upper Primer to mutagenize the NA2-tpi promoter:
(SEQ ID NO: 15)
5'-CTATATACACAACTGGATTTACCATGGGCCCGCGGCCGCAGATC-3'
Lower Primer to mutegenize the NA2-tpi promoter:
(SEQ ID NO: 16)
5'-GATCTGCGGCCGCGGGCCCATGGTAAATCCAGTTGTGTATATAG-3'
[0344] The amdS gene of pAlLo1 was swapped with the Aspergillus nidulans
pyrG gene. Plasmid pBANe10 (FIG. 6) was used as a source for the pyrG
gene as a selection marker, Analysis of the sequence of pBANe10 showed
that the pyrG marker was contained within an Nsi I restriction fragment
and does not contain either Nco I or Pac I restriction sites. Since the
amdS is also flanked by Nsi I restriction sites the strategy to switch
the selection marker was a simple swap of Nsi I restriction fragments.
Plasmid DNA from pAlLo1 and pBANe10 were digested with the restriction
enzyme Nsi I and the products purified by agarose gel electrophoresis.
The Nsi I fragment from pBANe 10 containing the pyrG gene was ligated to
the backbone of pAlLo1 to replace the original Nsi I DNA fragment
containing the amdS gene, Recombinant clones were analyzed by restriction
enzyme digestion to determine that they had the correct insert and also
its orientation. A clone with the pyrG gene transcribed in the
counterclockwise direction was selected. The new plasmid was designated
pAlLo2 (FIG. 7).
Example 8
Expression of Thielavia terrestris NRRL 8126 cel6b Endoglucanase Gene in
Aspergillus oryzae
[0345] Two synthetic oligonucleotide primers, shown below, were designed
to PCR amplify the full-length open reading frame from plasmid pPH47
encoding a CEL6B endoglucanase. An In-Fusion Cloning Kit (BD Biosciences,
Palo Alto, Calif., USA) was used to clone the fragment directly into
pAlLo2.
TABLE-US-00007
In-Fusion Forward primer:
(SEQ ID NO: 17)
5'-ACTGGATTACCATGAAGCTCTCGCAGTCG-3'
In-Fusion Reverse primer:
(SEQ ID NO: 18)
5'-AGTCACCTCTAGTTAGAACGACGGCACGGC-3'
Bold letters represent coding sequence. The remain ng sequence contains
sequence identity compared with the insertion sites of pAlLo2.
[0346] Fifty picomoles of each of the primers above were used in a PCR
reaction containing 50 ng of pPH47 DNA, 1.times.Pfx Amplification Buffer
(Invitrogen, Carlsbad, Calif., USA), 6 .mu.l of 10 mM blend of dATP,
dTTP, dGTP, and dCTP, 2.5 units of Platinum Pfx DNA Polymerase
(Invitrogen, Carisbad, Calif.), 1 .mu.l of 50 mM MgSO.sub.4, and 5 .mu.l
of 10.times.pCRx Enhancer solution (Invitrogen, Carlsbad, Calif.) in a
final volume of 50 .mu.l. The amplification was performed in an
Eppendorf.RTM. Mastercycler.RTM. 5333 (Eppendorf Scientific, Inc.,
Westbury, N.Y., USA) programmed for 1 cycle at 98.degree. C. for 2
minutes; and 35 cycles each at 94.degree. C. for 30 seconds, 65.degree.
C. for 30 seconds, and 68'C for 1.5 minutes. After the 35 cycles, the
reaction was incubated at 68.degree. C. for 10 minutes and then cooled at
10.degree. C. until further processed. A 1.5 kb PCR reaction product was
isolated on a 0.8% GTG-agarose gel (Cambrex Bioproducts, East Rutherford,
N.J., USA) using TAE buffer and 0.1 .mu.g of ethidium bromide per ml. The
DNA band was visualized with the aid of a Dark Reader.TM. (Clare Chemical
Research, Dolores, Colo., USA) to avoid UV-induced mutations. The 1.5 kb
DNA band was excised with a disposable razor blade and purified using a
QIAquick.RTM. Gel Extraction Kit (QIAGEN Inc., Valencia, Calif., USA)
according to the manufacturer's instructions.
[0347] The vector pAlLo2 was linearized by digestion with Nco I and Pac I
(using conditions specified by the manufacturer). The fragment was
purified by gel electrophoresis and a QIAquick Gel Extraction Kit as
described above. Cloning of the purified PCR fragment into the purified
linearized pAlLo2 vector was performed with an In-Fusion Cloning Kit. The
reaction (20 .mu.l) contained 1.times. In-Fusion Buffer (BD Biosciences,
Palo Alto, Calif., USA), 1.times.BSA (BD Biosciences, Palo Alto, Calif.,
USA), 1 .mu.l of In-Fusion enzyme (diluted 1:10) (BD Biosciences, Palo
Alto, Calif., USA), 160 ng of pAlLo2 digested with Nco I and Pac I, and
50 ng of the Thielavia terrestris cel6b purified PCR product. The
reaction was incubated at room temperature for 30 minutes. A 1 .mu.l
sample of the reaction were used to transform E. coli XL10 SoloPac.RTM.
Gold cells (Stratagene, La Jolla, Calif.) according to the manufacturer's
instructions. After the recovery period, two 100 .mu.l aliquots from the
transformation reaction were plated onto 150 mm 2.times.YT plates
supplemented with 100 .mu.g of ampicillin per ml. The plates were
incubated overnight at 37.degree. C. A set of eight putative recombinant
clones was selected at random from the selection plates and plasmid DNA
was prepared from each one using a BioRobot 9600. Clones were analyzed by
Eco RI restriction digest. Two clones that had the expected restriction
digest pattern were then sequenced to confirm that there were no
mutations in the cloned insert. One of the clones had the correct
sequence and was selected and designated pEJG105 (FIG. 8).
[0348] Aspergillus oryzae Jal250 (WO 99/61651) protoplasts were prepared
according to the method of Christensen et al., 1988, Bio/Technology 6:
1419-1422. Five micrograms of pEJG105 (as well as pAlLo2 as a vector
control) were used to transform Aspergillus oryzae JAL250 protoplasts.
[0349] The transformation of Aspergillus oryzae Jal250 with pEJG105
yielded about 100 transformants. Five transformants were isolated to
individual PDA plates and incubated for five days at 34.degree. C.
[0350] Confluent spore plates were washed with 5 ml of 0.01% Tween 80 and
the spore suspension was used to inoculate 25 ml of MDU2BP medium in 125
ml glass shake flasks. Transformant cultures were incubated at 34.degree.
C. with constant shaking at 200 rpm. At day five post-inoculation, an
aliquot of each culture was centrifuged at 12000.times.g. Five .mu.l of
each supernatant were mixed with an equal volume of 2.times. loading
buffer (10% beta-mercaptoethanol) and loaded onto a 1.5 mm 8%-16%
Tris-Glycine SDS-PAGE gel and stained with SimplyBlue.RTM. SafeStain
(Invitrogen, Carlsbad, Calif.). SDS-PAGE profiles of the culture broths
showed that four out of five transformants had a new protein band of
approximately 40 kDa. Transformant number 1 was selected for further
studies and designated Aspergillus oryzae EJG105.
Example 9
Expression of Thielavia terrestris NRRL 8126 cel6c Endoglucanase Gene in
Aspergillus oryzae
[0351] The Thielavia terrestris NRRL 8126 cel6c gene was excised from the
pYES2.0 vector using Bam HI and Xba I, and ligated into the Aspergillus
expression vector pHD414 (EP 238 023, WO 93/11249) using standard methods
(Sambrook et al., 1989, supra). The Aspergillus expression vector pHD414
is a derivative of p775 (EP 238 023). The resulting plasmid was
designated pA2BG146 (FIG. 9).
[0352] Protoplasts of Aspergillus oryzae HowB104 were prepared as
described in WO 95/02043. One hundred microliters of protoplast
suspension were mixed with 5-25 .mu.g of pA2BG146 in 10 .mu.l of STC
composed of 1.2 M sorbitol, 10 mM Tris-HCl, pH 7.5, 10 mM CaCl.sub.2) and
further mixed with 5-25 .mu.g of p3SR2, an Aspergillus nidulans amdS gene
carrying plasmid (Christensen at al., 1988, Bio/Technology 6: 1419-1422).
The mixture was left at room temperature for 25 minutes. Two hundred
microliters of 60% PEG 4000 (BDH, Poole, England) (polyethylene glycol,
molecular weight 4,000), 10 mM CaCl.sub.2, and 10 mM Tris-HCl pH 7.5 was
added and gently mixed and finally 0.85 ml of the same solution was added
and gently mixed. The mixture was left at room temperature for 25
minutes, centrifuged at 2,500.times.g for 15 minutes, and the pellet was
resuspended in 2 ml of 1.2 M sorbitol. This sedimentation process was
repeated, and the protoplasts were spread on COVE plates. After
incubation for 4-7 days at 37.degree. C. spores were picked and spread in
order to isolate single colonies. This procedure was repeated and spores
of a single colony after the second reisolation were stored.
[0353] Each of the transformants was inoculated in 10 ml of YPM medium.
After 2-5 days of incubation at 30.degree. C., 200 rpm, the supernatant
was removed. Endoglucanase activity was identified by applying 20 .mu.l
of culture broth to 4 mm diameter holes punched out in a 0.1% AZCL HE
cellulose SC-agar plate and incubation overnight at 30.degree. C. The
presence of endoglucanase activity produced a blue halo around a colony.
Several transformant broths had endoglucanase activity that was
significantly greater than broth from an untransformed Aspergillus oryzae
background control, which demonstrated efficient expression of the CEL6C
endoglucanase from Thielavia terrestris NRRL 8126 in Aspergillus oryzae.
Example 10
Large Shake Flask Cultures of Aspergillus oryzae Jal250 Containing cel6b
Gene
[0354] Aspergillus oryzae Jal250 containing pEJG105 spores were spread
onto a PDA plate and incubated for five days at 34.degree. C. The
confluent spore plate was washed twice with 5 ml of 0.01% Tween 80 to
maximize the number of spores collected. The spore suspension was then
used to inoculate 500 ml of MDU2BP medium in a two-liter Fernbach flask.
The culture was incubated at 34.degree. C. with constant shaking (200
rpm). At day five post inoculum, the culture broth was collected by
filtration on a 500 milliliter, 75 mm Nylon filter unit with a pore size
of 0.45 .mu.m with a glass-fiber pre-filter (Nalgene Nunc International,
Rochester, N.Y., USA). A 5 .mu.l sample of the broth was analyzed by
SDS-PAGE as described in Example 9 to confirm that the protein pattern
was the same as the one obtained before. The broth was shown to contain a
40 kDa protein band.
Example 11
Hydrolysis of Carboxymethylcellulose by Thielavia terrestris CEL6B and
Thielavia terrestris CEL6C Endoglucanases
[0355] Sodium salt of carboxymethylcellulose (CMC, type 7L2) with average
degree of substitution (DS) of 0.7 was obtained from Aqualon Division of
Hercules Inc. (Wilmington, Del., USA). A 6.25 mg per ml solution of CMC
in 50 mM sodium acetate pH 5.0 was prepared by slowly adding CMC to a
vigorously agitated buffer, followed by heating to approximately
60.degree. C. under continuous stirring until complete dissolution.
[0356] The Thielavia terrestris CEL6B and Thielavia terrestris CEL6C
endoglucanases (both expressed in Aspergillus oryzae Jal250 as described
in Examples 8 and 9, respectively) were concentrated and
desalted/exchanged to 50 mM sodium acetate pH 5.0 using a Centricon.RTM.
Plus-20 centrifugal filter with a 5000 NMWL Biomax-5.RTM. membrane
(Millipore, Bedford, Mass., USA). The concentrated and desalted enzyme
solutions were stored at -20.degree. C. The protein concentration in the
enzyme solutions was determined by a BCA Microplate Assay using a BCA
Protein Assay Reagent Kit according to the manufacturer's instructions
(Pierce Chemical Co., Rockford, Ill., USA).
[0357] Hydrolysis of CMC was carried out in Eppendorf.RTM. DeepWell Plates
96 (1.2 ml, Eppendorf North America, Inc., Westbury, N.Y., USA) sealed by
a ALPS-300''; plate sealer (ABgene Inc., Rochester, N.Y., USA). All
reactions were run in 50 mM sodium acetate pH 5.0 in a 1 ml volume
without stirring at 50.degree. C. The initial substrate concentration was
5 mg per ml. Both enzymes were used at four different concentrations.
Corresponding enzyme dilutions in 50 mM sodium acetate pH 5.0 were
prepared fresh before enzymatic hydrolysis from stock enzyme solutions.
[0358] After 70 hours of hydrolysis, 40 d aliquots were transferred from
the hydrolysis reactions to a flat-bottomed 96-well microplate (Corning
Inc., Corning, N.Y., USA) and mixed with 160 .mu.l of 128 mM
Na.sub.2CO.sub.3-72.5 M NaHCO.sub.3 to terminate the reactions. The
diluted samples were analyzed for reducing sugars (RS) using a microplate
p-hydroxybenzoic acid hydrazide (PHBAH) assay as described below.
[0359] A 90-.mu.l aliquot of the diluted sample was placed into each well
of a 96-well conical-bottomed microplate (Corning Inc., Corning, N.Y.,
USA). The assay was initiated by adding 60 .mu.l of 1.25% PHBAH in 2%
sodium hydroxide to each well. The uncovered plate was heated on a
custom-made heating block for 10 minutes at 95.degree. C. After the
microplate was cooled to room temperature, 35 .mu.l of deionized water
was added to each well. A 100-.mu.l aliquot was removed from each well
and transferred to a flat-bottomed 96-well microplate (Corning Inc.,
Corning, N.Y., USA). The absorbance at 410 nm (A.sub.410) was measured
using a SpectraMAX.RTM. Microplate Reader (Molecular Devices, Sunnyvale,
Calif., USA). The A.sub.410 value was translated into glucose equivalents
using a standard curve.
[0360] The standard curve was obtained using six glucose standards (0.005,
0.010, 0.025, 0.050, 0.075, and 0.100 mg/ml), which were treated
similarly to the samples. Glucose standards were prepared by diluting 10
mg/ml stock glucose solution with deionized water.
[0361] The degree of carboxymethylcellulose conversion to reducing sugars
(hydrolysis yield, %) was calculated using the following equation:
RS Yield.sub.(%)=RS.sub.(mg/ml).times.100.times.162/(CMC.sub.(mg/ml).tim-
es.180)=RS.sub.(mg/ml).times.100/(CMC.sub.(mg/ml).times.1.111)
[0362] In this equation, RS is the concentration of reducing sugars in
solution measured in glucose equivalents (mg/ml), and the factor 1.111
reflects the weight gain in converting cellulose to glucose.
[0363] The assay results demonstrated that after 70 hours of hydrolysis
the Thielavia terrestris CEL6B and Thielavia terrestris CEL6C
endoglucanases were able to hydrolyze from 2 to 4% of available
glycosidic bonds in carboxymethylcellulose.
DEPOSIT OF BIOLOGICAL MATERIAL
[0364] The following biological material have been deposited under the
terms of the Budapest Treaty with the Agricultural Research Service
Patent Culture Collection, Northern Regional Research Center, 1815
University Street, Peoria, Ill., 61604, and given the following accession
numbers:
TABLE-US-00008
Deposit Accession Number Date of Deposit
E. coli PaHa47 (pPH47) NRRL B-30898 Feb. 23, 2006
E. coli (pCIBG146) NRRL B-30901 Feb. 23, 2006
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 the Commissioner of Patents and
Trademarks to be entitled thereto under 37 C.F.R. .sctn.1.14 and 35
U.S.C. .sctn.122. 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.
[0365] 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.
[0366] Various references are cited herein, the disclosures of which are
incorporated by reference in their entireties.
Sequence CWU
1
1811580DNAThielavia terrestris 1agccccccgt tcaggcacac ttggcatcag
atcagcttag cagcgcctgc acagcatgaa 60gctctcgcag tcggccgcgc tggcggcact
caccgcgacg gcgctcgccg ccccctcgcc 120cacgacgccg caggcgccga ggcaggcttc
agccggctgc tcgtctgcgg tcacgctcga 180cgccagcacc aacgtttgga agaagtacac
gctgcacccc aacagctact accgcaagga 240ggttgaggcc gcggtggcgc agatctcgga
cccggacctc gccgccaagg ccaagaaggt 300ggccgacgtc ggcaccttcc tgtggctcga
ctcgatcgag aacatcggca agctggagcc 360ggcgatccag gacgtgccct gcgagaacat
cctgggcctg gtcatctacg acctgccggg 420ccgcgactgc gcggccaagg cgtccaacgg
cgagctcaag gtcggcgaga tcgaccgcta 480caagaccgag tacatcgaca gtgagtgctg
ccccccgggt tcgagaagag cgtgggggaa 540agggaaaggg ttgactgact gacacggcgc
actgcagaga tcgtgtcgat cctcaaggca 600caccccaaca cggcgttcgc gctggtcatc
gagccggact cgctgcccaa cctggtgacc 660aacagcaact tggacacgtg ctcgagcagc
gcgtcgggct accgcgaagg cgtggcttac 720gccctcaaga acctcaacct gcccaacgtg
atcatgtacc tcgacgccgg ccacggcggc 780tggctcggct gggacgccaa cctgcagccc
ggcgcgcagg agctagccaa ggcgtacaag 840aacgccggct cgcccaagca gctccgcggc
ttctcgacca acgtggccgg ctggaactcc 900tggtgagctt ttttccattc catttcttct
tcctcttctc tcttcgctcc cactctgcag 960ccccccctcc cccaagcacc cactggcgtt
ccggcttgct gactcggcct ccctttcccc 1020gggcaccagg gatcaatcgc ccggcgaatt
ctcccaggcg tccgacgcca agtacaacaa 1080gtgccagaac gagaagatct acgtcagcac
cttcggctcc gcgctccagt cggccggcat 1140gcccaaccac gccatcgtcg acacgggccg
caacggcgtc accggcctgc gcaaggagtg 1200gggtgactgg tgcaacgtca acggtgcagg
ttcgttgtct tctttttctc ctcttttgtt 1260tgcacgtcgt ggtccttttc aagcagccgt
gtttggttgg gggagatgga ctccggctga 1320tgttctgctt cctctctagg cttcggcgtg
cgcccgacga gcaacacggg cctcgagctg 1380gccgacgcgt tcgtgtgggt caagcccggc
ggcgagtcgg acggcaccag cgacagctcg 1440tcgccgcgct acgacagctt ctgcggcaag
gacgacgcct tcaagccctc gcccgaggcc 1500ggcacctgga acgaggccta cttcgagatg
ctgctcaaga acgccgtgcc gtcgttctaa 1560gacggtccag catcatccgg
15802396PRTThielavia terrestris 2Met Lys
Leu Ser Gln Ser Ala Ala Leu Ala Ala Leu Thr Ala Thr Ala1 5
10 15Leu Ala Ala Pro Ser Pro Thr Thr
Pro Gln Ala Pro Arg Gln Ala Ser 20 25
30Ala Gly Cys Ser Ser Ala Val Thr Leu Asp Ala Ser Thr Asn Val
Trp 35 40 45Lys Lys Tyr Thr Leu
His Pro Asn Ser Tyr Tyr Arg Lys Glu Val Glu 50 55
60Ala Ala Val Ala Gln Ile Ser Asp Pro Asp Leu Ala Ala Lys
Ala Lys65 70 75 80Lys
Val Ala Asp Val Gly Thr Phe Leu Trp Leu Asp Ser Ile Glu Asn
85 90 95Ile Gly Lys Leu Glu Pro Ala
Ile Gln Asp Val Pro Cys Glu Asn Ile 100 105
110Leu Gly Leu Val Ile Tyr Asp Leu Pro Gly Arg Asp Cys Ala
Ala Lys 115 120 125Ala Ser Asn Gly
Glu Leu Lys Val Gly Glu Ile Asp Arg Tyr Lys Thr 130
135 140Glu Tyr Ile Asp Lys Ile Val Ser Ile Leu Lys Ala
His Pro Asn Thr145 150 155
160Ala Phe Ala Leu Val Ile Glu Pro Asp Ser Leu Pro Asn Leu Val Thr
165 170 175Asn Ser Asn Leu Asp
Thr Cys Ser Ser Ser Ala Ser Gly Tyr Arg Glu 180
185 190Gly Val Ala Tyr Ala Leu Lys Asn Leu Asn Leu Pro
Asn Val Ile Met 195 200 205Tyr Leu
Asp Ala Gly His Gly Gly Trp Leu Gly Trp Asp Ala Asn Leu 210
215 220Gln Pro Gly Ala Gln Glu Leu Ala Lys Ala Tyr
Lys Asn Ala Gly Ser225 230 235
240Pro Lys Gln Leu Arg Gly Phe Ser Thr Asn Val Ala Gly Trp Asn Ser
245 250 255Trp Asp Gln Ser
Pro Gly Glu Phe Ser Gln Ala Ser Asp Ala Lys Tyr 260
265 270Asn Lys Cys Gln Asn Glu Lys Ile Tyr Val Ser
Thr Phe Gly Ser Ala 275 280 285Leu
Gln Ser Ala Gly Met Pro Asn His Ala Ile Val Asp Thr Gly Arg 290
295 300Asn Gly Val Thr Gly Leu Arg Lys Glu Trp
Gly Asp Trp Cys Asn Val305 310 315
320Asn Gly Ala Gly Phe Gly Val Arg Pro Thr Ser Asn Thr Gly Leu
Glu 325 330 335Leu Ala Asp
Ala Phe Val Trp Val Lys Pro Gly Gly Glu Ser Asp Gly 340
345 350Thr Ser Asp Ser Ser Ser Pro Arg Tyr Asp
Ser Phe Cys Gly Lys Asp 355 360
365Asp Ala Phe Lys Pro Ser Pro Glu Ala Gly Thr Trp Asn Glu Ala Tyr 370
375 380Phe Glu Met Leu Leu Lys Asn Ala
Val Pro Ser Phe385 390
39531203DNAThielavia terrestris 3atgaagtacc tcaacctcct cgcagctctc
ctcgccgtcg ctcctctctc cctcgctgca 60cccagcatcg aggccagaca gtcgaacgtc
aacccataca tcggcaagag cccgctcgtt 120attaggtcgt acgcccaaaa gcttgaggag
accgtcagga ccttccagca acgtggcgac 180cagctcaacg ctgcgaggac acggacggtg
cagaacgttg cgactttcgc ctggatctcg 240gataccaatg gtattggagc cattcgacct
ctcatccaag atgctctcgc ccagcaggct 300cgcactggac agaaggtcat cgtccaaatc
gtcgtctaca acctcccaga tcgcgactgc 360tctgccaacg cctcgactgg agagttcacc
gtaggaaacg acggtctcaa ccgatacaag 420aactttgtca acaccatcgc ccgcgagctc
tcgactgctg acgctgacaa gctccacttt 480gccctcctcc tcgaacccga cgcacttgcc
aacctcgtca ccaacgcgaa tgcccccagg 540tgccgaatcg ccgctcccgc ttacaaggag
ggtatcgcct acaccctcgc caccttgtcc 600aagcccaacg tcgacgtcta catcgacgcc
gccaacggtg gctggctcgg ctggaacgac 660aacctccgcc ccttcgccga actcttcaag
gaagtctacg acctcgcccg ccgcatcaac 720cccaacgcca aggtccgcgg cgtccccgtc
aacgtctcca actacaacca gtaccgcgct 780gaagtccgcg agcccttcac cgagtggaag
gacgcctggg acgagagccg ctacgtcaac 840gtcctcaccc cgcacctcaa cgccgtcggc
ttctccgcgc acttcatcgt tgaccaggga 900cgcggtggca agggcggtat caggacggag
tggggccagt ggtgcaacgt taggaacgct 960gggttcggta tcaggcctac tgcggatcag
ggcgtgctcc agaacccgaa tgtggatgcg 1020attgtgtggg ttaagccggg tggagagtcg
gatggcacga gtgatttgaa ctcgaacagg 1080tatgatccta cgtgcaggag tccggtggcg
catgttcccg ctcctgaggc tggccagtgg 1140ttcaacgagt atgttgttaa cctcgttttg
aacgctaacc cccctcttga gcctacctgg 1200taa
12034400PRTThielavia terrestris 4Met Lys
Tyr Leu Asn Leu Leu Ala Ala Leu Leu Ala Val Ala Pro Leu1 5
10 15Ser Leu Ala Ala Pro Ser Ile Glu
Ala Arg Gln Ser Asn Val Asn Pro 20 25
30Tyr Ile Gly Lys Ser Pro Leu Val Ile Arg Ser Tyr Ala Gln Lys
Leu 35 40 45Glu Glu Thr Val Arg
Thr Phe Gln Gln Arg Gly Asp Gln Leu Asn Ala 50 55
60Ala Arg Thr Arg Thr Val Gln Asn Val Ala Thr Phe Ala Trp
Ile Ser65 70 75 80Asp
Thr Asn Gly Ile Gly Ala Ile Arg Pro Leu Ile Gln Asp Ala Leu
85 90 95Ala Gln Gln Ala Arg Thr Gly
Gln Lys Val Ile Val Gln Ile Val Val 100 105
110Tyr Asn Leu Pro Asp Arg Asp Cys Ser Ala Asn Ala Ser Thr
Gly Glu 115 120 125Phe Thr Val Gly
Asn Asp Gly Leu Asn Arg Tyr Lys Asn Phe Val Asn 130
135 140Thr Ile Ala Arg Glu Leu Ser Thr Ala Asp Ala Asp
Lys Leu His Phe145 150 155
160Ala Leu Leu Leu Glu Pro Asp Ala Leu Ala Asn Leu Val Thr Asn Ala
165 170 175Asn Ala Pro Arg Cys
Arg Ile Ala Ala Pro Ala Tyr Lys Glu Gly Ile 180
185 190Ala Tyr Thr Leu Ala Thr Leu Ser Lys Pro Asn Val
Asp Val Tyr Ile 195 200 205Asp Ala
Ala Asn Gly Gly Trp Leu Gly Trp Asn Asp Asn Leu Arg Pro 210
215 220Phe Ala Glu Leu Phe Lys Glu Val Tyr Asp Leu
Ala Arg Arg Ile Asn225 230 235
240Pro Asn Ala Lys Val Arg Gly Val Pro Val Asn Val Ser Asn Tyr Asn
245 250 255Gln Tyr Arg Ala
Glu Val Arg Glu Pro Phe Thr Glu Trp Lys Asp Ala 260
265 270Trp Asp Glu Ser Arg Tyr Val Asn Val Leu Thr
Pro His Leu Asn Ala 275 280 285Val
Gly Phe Ser Ala His Phe Ile Val Asp Gln Gly Arg Gly Gly Lys 290
295 300Gly Gly Ile Arg Thr Glu Trp Gly Gln Trp
Cys Asn Val Arg Asn Ala305 310 315
320Gly Phe Gly Ile Arg Pro Thr Ala Asp Gln Gly Val Leu Gln Asn
Pro 325 330 335Asn Val Asp
Ala Ile Val Trp Val Lys Pro Gly Gly Glu Ser Asp Gly 340
345 350Thr Ser Asp Leu Asn Ser Asn Arg Tyr Asp
Pro Thr Cys Arg Ser Pro 355 360
365Val Ala His Val Pro Ala Pro Glu Ala Gly Gln Trp Phe Asn Glu Tyr 370
375 380Val Val Asn Leu Val Leu Asn Ala
Asn Pro Pro Leu Glu Pro Thr Trp385 390
395 400528DNAThielavia
terrestrismisc_feature(17)..(17)N=A,C,G, OR T 5agcccgactc cctggcnaay
ctggtnac 28624DNAThielavia
terrestrismisc_feature(13)..(13)N=A,C,G, OR T 6gcactcgccg ccnggyttna ycca
24725DNAThielavia terrestris
7cttggtaccg agctcggatc cacta
25825DNAThielavia terrestris 8atagggcgaa ttgggccctc tagat
25912DNAThielavia terrestris 9ctttccagca ca
121022DNAAspergillus
nidulans 10gtgccccatg atacgcctcc gg
221126DNAAspergillus nidulans 11gagtcgtatt tccaaggctc ctgacc
261224DNAAspergillus nidulans
12ggaggccatg aagtggacca acgg
241345DNAAspergillus niger 13caccgtgaaa gccatgctct ttccttcgtg tagaagacca
gacag 451445DNAAspergillus niger 14ctggtcttct
acacgaagga aagagcatgg ctttcacggt gtctg
451544DNAAspergillus niger 15ctatatacac aactggattt accatgggcc cgcggccgca
gatc 441644DNAAspergillus niger 16gatctgcggc
cgcgggccca tggtaaatcc agttgtgtat atag
441729DNAThielavia terrestris 17actggattac catgaagctc tcgcagtcg
291830DNAThielavia terrestris 18agtcacctct
agttagaacg acggcacggc 30
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