POLYPEPTIDES PRESENTANT UNE ACTIVITE DE CELLOBIOHYDROLASE ET POLYNUCLEOTIDES CODANT POUR CEUX-CI

POLYPEPTIDES HAVING CELLOBIOHYDROLASE ACTIVITY AND POLYNUCLEOTIDES ENCODING SAME

Abrégé français

La présente invention concerne des polypeptides isolés présentant une activité de cellobiohydrolase, des domaines catalytiques et des domaines de liaison à la cellulose, et des polynucléotides codant pour les polypeptides, les domaines catalytiques ou les domaines de liaison à la cellulose. Des hybrides d'acide nucléique, des vecteurs et des cellules hôtes comprenant les polynucléotides ainsi que des procédés de préparation et d'utilisation des polypeptides, des domaines catalytiques ou des domaines de liaison à la cellulose sont également décrits.

Abrégé anglais

Provided are isolated polypeptides having cellobiohydrolase activity, catalytic domains and cellulose binding domains, and polynucleotides encoding the polypeptides, catalytic domains or cellulose binding domains. Also provided are nucleic acid constructs, vectors and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides, catalytic domains or cellulose binding domains.

Revendications

103
Claims
What is claimed is:
1. An isolated polypeptide having cellobiohydrolase activity, selected from
the group
consisting of:
(a) a polypeptide having at least 70%, e.g., at least 72%, at least 74%, at
least 75%,
at least 77%, at least 78%, at least 80%, at least 81%, at least 82%, at least
83%, at least 84%,
at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least
90%, at least 91%,
at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least
97%, at least 98%,
at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID
NO: 2; a
polypeptide having at least 88%, e.g., at least 89%, at least 90%, at least
91%, at least 92%, at
least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least
98%, at least 99%, or
100% sequence identity to the mature polypeptide of SEQ ID NO: 4; a
polypeptide having at
least 66%, e.g., at least 68%, at least 70%, at least 75%, at least 78%, at
least 80%, at least
81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at
least 87%, at least
88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%, at least
95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence
identity to the
mature polypeptide of SEQ ID NO: 6; or a polypeptide having at least 81%,
e.g., at least 82%, at
least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least
88%, at least 89%, at
least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least
95%, at least 96%, at
least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature
polypeptide of
SEQ ID NO: 8;
(b) a polypeptide encoded by a polynucleotide that hybridizes under at
least medium
stringency conditions with (i) the mature polypeptide coding sequence of SEQ
ID NO: 1, the
mature polypeptide coding sequence of SEQ ID NO: 3, the mature polypeptide
coding
sequence of SEQ ID NO: 5 or the mature polypeptide coding sequence of SEQ ID
NO: 7, OD the
cDNA sequence of SEQ ID NO: 1, the cDNA sequence of SEQ ID NO: 3, the cDNA
sequence
of SEQ ID NO: 5 or the cDNA sequence of SEQ ID NO: 7, or (iii) the full-length
complement of
(i) or (ii);
(c) a polypeptide encoded by a polynucleotide having at least 70%, e.g., at
least
72%, at least 74%, at least 75%, at least 77%, at least 78%, at least 80%, at
least 81%, at least
82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at
least 88%, at least
89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at
least 95%, at least

104
96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to
the mature
polypeptide coding sequence of SEQ ID NO: 1 or the cDNA sequence thereof; a
polypeptide
encoded by a polynucleotide having at least 88%, e.g., at least 89%, at least
90%, at least 91%,
at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least
97%, at least 98%,
at least 99%, or 100% sequence identity to the mature polypeptide coding
sequence of SEQ ID
NO: 3 or the cDNA sequence thereof; a polypeptide encoded by a polynucleotide
having at least
66%, e.g., at least 68%, at least 70%, at least 75%, at least 78%, at least
80%, at least 81%, at
least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least
87%, at least 88%, at
least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at least 95%, at
least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity
to the mature
polypeptide coding sequence of SEQ ID NO: 5 or the cDNA sequence thereof; or a
polypeptide
encoded by a polynucleotide having at least 81%, e.g., at least 82%, at least
83%, at least 84%,
at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least
90%, at least 91%,
at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least
97%, at least 98%,
at least 99%, or 100% sequence identity to the mature polypeptide coding
sequence of SEQ ID
NO: 7 or the cDNA sequence thereof;
(d) a variant of the mature polypeptide of SEQ ID NO: 2, the mature
polypeptide of
SEQ ID NO: 4, the mature polypeptide of SEQ ID NO: 6 or the mature polypeptide
of SEQ ID
NO: 8 comprising a substitution, deletion, and/or insertion at one or more
(e.g., several)
positions; and
(e) a fragment of the polypeptide of (a), (b), (c), or (d) that has
cellobiohydrolase
activity.
2. The polypeptide of claim 1, comprising or consisting of SEQ ID NO: 2,
SEQ ID NO: 4,
SEQ ID NO: 6 or SEQ ID NO: 8; or comprising or consisting of the mature
polypeptide of SEQ
ID NO: 2, the mature polypeptide of SEQ ID NO: 4, the mature polypeptide of
SEQ ID NO: 6 or
the mature polypeptide of SEQ ID NO: 8.
3. An isolated polypeptide comprising a catalytic domain selected from the
group consisting
of:
(a) a catalytic domain having at least 70%, e.g., at least 72%, at least
74%, at least
75%, at least 77%, at least 78%, at least 80%, at least 81%, at least 82%, at
least 83%, at least
84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at
least 90%, at least
91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at
least 97%, at least

105
98%, at least 99%, or 100% sequence identity to amino acids 18 to 458 of SEQ
ID NO: 2, a
catalytic domain having at least 88%, e.g., at least 89%, at least 90%, at
least 91%, at least
92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at
least 98%, at least
99%, or 100% sequence identity to amino acids 21 to 450 of SEQ ID NO: 4, a
catalytic domain
having at least 66%, e.g., at least 68%, at least 70%, at least 75%, at least
78%, at least 80%,
at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least
86%, at least 87%,
at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least
93%, at least 94%,
at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%
sequence identity
to amino acids 22 to 457 of SEQ ID NO: 6, or a catalytic domain having at
least 81%, e.g., at
least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least
87%, at least 88%, at
least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at least 95%, at
least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity
to amino acids
21 to 461 of SEQ ID NO: 8;
(b) a catalytic domain encoded by a polynucleotide that hybridizes under
medium,
medium-high, high, or very high stringency conditions with (i) nucleotides 52
to 1454 of SEQ ID
NO: 1, nucleotides 61 to 1733 of SEQ ID NO: 3, nucleotides 64 to 1782 of SEQ
ID NO: 5, or
nucleotides 52 to 1460 of SEQ ID NO: 7, (ii) the cDNA sequence thereof, or
(iii) the full-length
complement of (i) or (ii);
(c) a catalytic domain encoded by a polynucleotide having at least 70%,
e.g., at least
72%, at least 74%, at least 75%, at least 77%, at least 78%, at least 80%, at
least 81%, at least
82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at
least 88%, at least
89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at
least 95%, at least
96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to
nucleotides 52 to
1454 of SEQ ID NO: 1 or the cDNA sequence thereof; or a catalytic domain
encoded by a
polynucleotide having at least 88%, e.g., at least 89%, at least 90%, at least
91%, at least 92%,
at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least
98%, at least 99%,
or 100% sequence identity to nucleotides 61 to 1733 of SEQ ID NO: 3 or the
cDNA sequence
thereof; a catalytic domain encoded by a polynucleotide having at least 66%,
e.g., at least 68%,
at least 70%, at least 75%, at least 78%, at least 80%, at least 81%, at least
82%, at least 83%,
at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least
89%, at least 90%,
at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least
96%, at least 97%,
at least 98%, at least 99%, or 100% sequence identity to nucleotides 64 to
1782 of SEQ ID NO:
or the cDNA sequence thereof; or a catalytic domain encoded by a
polynucleotide having at
least 81%, e.g., at least 82%, at least 83%, at least 84%, at least 85%, at
least 86%, at least

106
87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at
least 93%, at least
94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or
100% sequence
identity to nucleotides 52 to 1460 of SEQ ID NO: 7 or the cDNA sequence
thereof;
(d) a variant of amino acids 18 to 458 of SEQ ID NO: 2, amino acids 21 to
450 of
SEQ ID NO: 4, amino acids 22 to 457 of SEQ ID NO: 6, or amino acids 21 to 461
of SEQ ID
NO: 8, comprising a substitution, deletion, and/or insertion at one or more
positions (e.g.,
several) , wherein the variant has cellobiohydrolase activity; and
(e) a fragment of the catalytic domain of (a), (b), (c), or (d) that has
cellobiohydrolyase activity.
4. An isolated polypeptide comprising a carbohydrate binding domain,
selected from the
group consisting of:
(a) a carbohydrate binding domain having at least 81%, e.g., at least 82%,
at least
83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at
least 89%, at least
90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at
least 96%, at least
97%, at least 98%, at least 99%, or 100% sequence identity to amino acids 486
to 521 of SEQ
ID NO: 8;
(b) a carbohydrate binding domain encoded by a polynucleotide that
hybridizes
under medium, medium-high, high, or very high stringency conditions with (i)
nucleotides 1533
to 1640 of SEQ ID NO: 7, (ii) the cDNA sequence thereof, or (iii) the full-
length complement of
(i) or (ii);
(c) a carbohydrate binding domain encoded by a polynucleotide having at
least 81%,
e.g., at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at
least 87%, at least
88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%, at least
95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence
identity to
nucleotides 1533 to 1640 of SEQ ID NO: 7 or the cDNA sequence thereof;
(d) a variant of amino acids 486 to 521 of SEQ ID NO: 8 comprising a
substitution,
deletion, and/or insertion at one or more positions; and
(e) a fragment of (a), (b), (c), (d) or (e) that has carbohydrate binding
activity.
5. A composition comprising the polypeptide of any of claims 1-4.
6. An isolated polynucleotide encoding the polypeptide of any of claims 1-
4.

107
7. A recombinant host cell comprising the polynucleotide of claim 6
operably linked to one
or more control sequences that direct the production of the polypeptide.
8. A method of producing the polypeptide of any of claims 1-4, comprising:
(a) cultivating a cell, which in its wild-type form produces the
polypeptide, under
conditions conducive for production of the polypeptide; and optionally
(b) recovering the polypeptide.
9. A method of producing a polypeptide having cellobiohydrolase activity,
comprising:
(a) cultivating the host cell of claim 7 under conditions conducive for
production of
the polypeptide; and optionally
(b) recovering the polypeptide.
10. A transgenic plant, plant part or plant cell transformed with a
polynucleotide encoding
the polypeptide of any of claims 1-4.
11. A method of producing a polypeptide having cellobiohydrolase activity,
comprising:
(a) cultivating the transgenic plant or plant cell of claim 10 under
conditions
conducive for production of the polypeptide; and optionally
(b) recovering the polypeptide.
12. A method of producing a mutant of a parent cell, comprising
inactivating a polynucleotide
encoding the polypeptide of any of claims 1-4, which results in the mutant
producing less of the
polypeptide than the parent cell.
13. An isolated polynucleotide encoding a signal peptide comprising or
consisting of amino
acids 1 to 17 of SEQ ID NO: 2, amino acids 1 to 20 of SEQ ID NO: 4, amino
acids 1 to 21 of
SEQ ID NO: 6, or amino acids 1 to 17 of SEQ ID NO: 8.

108
14. A method of producing a protein, comprising:
(a) cultivating a recombinant host cell comprising a gene encoding a
protein
operably linked to the polynucleotide of claim 13, wherein the gene is foreign
to the
polynucleotide encoding the signal peptide, under conditions conducive for
production of the
protein; and optionally
(b) recovering the protein.
15. A whole broth formulation or cell culture composition comprising the
polypeptide of any
of claims 1-4.
16. A process for degrading or converting a cellulosic material,
comprising: treating the
cellulosic material with an enzyme composition in the presence of the
polypeptide having
cellobiohydrolase activity of any of claims 1-4.
17. A process for producing a fermentation product, comprising:
(a) saccharifying a cellulosic material with an enzyme composition in the
presence of
the polypeptide having cellobiohydrolase activity of any of claims 1-4;
(b) fermenting the saccharified cellulosic material with one or more
fermenting
microorganisms to produce the fermentation product; and
(c) recovering the fermentation product from the fermentation.
18. A process of fermenting a cellulosic material, comprising: fermenting
the cellulosic
material with one or more fermenting microorganisms, wherein the cellulosic
material is
saccharified with an enzyme composition in the presence of the polypeptide
having
cellobiohydrolase activity of any of claims 1-4.

Description

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POLYPEPTIDES HAVING CELLOBIOHYDROLASE ACTIVITY AND POLYNUCLEOTIDES
ENCODING SAME
Statement as to Rights to Inventions Made Under
Federally Sponsored Research and Development
This invention was made with Government support under Cooperative Agreement DE-
FC36-08G018080 awarded by the Department of Energy. The government has certain
rights in
this invention.
Reference to a Sequence Listing
This application contains a Sequence Listing in computer readable form, which
is
incorporated herein by reference.
Background of the Invention
Field of the Invention
The present invention relates to polypeptides having cellobiohydrolase
activity, catalytic
domains, and carbohydrate binding domains, and polynucleotides encoding the
polypeptides,
catalytic domains, and carbohydrate binding domains. The invention also
relates to nucleic acid
constructs, vectors, and host cells comprising the polynucleotides as well as
methods of
producing and using the polypeptides, catalytic domains, and carbohydrate
binding domains.
Description of the Related Art
Cellulose is a polymer of the simple sugar glucose covalently linked by beta-
1,4-bonds.
Many microorganisms produce enzymes that hydrolyze beta-linked glucans. These
enzymes
include endoglucanases, cellobiohydrolases, and beta-glucosidases.
Endoglucanases digest
the cellulose polymer at random locations, opening it to attack by
cellobiohydrolases.
Cellobiohydrolases sequentially release molecules of cellobiose from the ends
of the cellulose
polymer. Cellobiose is a water-soluble beta-1,4-linked dimer of glucose. Beta-
glucosidases
hydrolyze cellobiose to glucose.
The conversion of lignocellulosic feedstocks into ethanol has the advantages
of the
ready availability of large amounts of feedstock, the desirability of avoiding
burning or land filling
the materials, and the cleanliness of the ethanol fuel. Wood, agricultural
residues, herbaceous
crops, and municipal solid wastes have been considered as feedstocks for
ethanol production.
These materials primarily consist of cellulose, hemicellulose, and lignin.
Once the lignocellulose

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is converted to fermentable sugars, e.g., glucose, the fermentable sugars can
easily be
fermented by yeast into ethanol.
UNIPROT: Q5B2Q4 discloses a probable cellobiohydrolase A protein from
Emericella
nidulans. W02008140749 discloses a Myceliophthora thermophila
cellobiohydrolase I.
W02003070939-A1 discloses a Coriolus hirsutus cellobiohydrolase I protein.
UNIPROT:
Q692I2 discloses a CBHI exoglucanase from Chaetomium the rmophilum var. the
rmophilum.
There is a need in the art for new polypeptides having cellobiohydrolase
activity for use
in the degradation of cellulosic materials.
The present invention provides polypeptides having cellobiohydrolase activity
and
polynucleotides encoding the polypeptides.
Summary of the Invention
The present invention relates to isolated polypeptides having
cellobiohydrolase activity
selected from the group consisting of:
(a) a polypeptide
having at least 70% sequence identity to the mature polypeptide of
SEQ ID NO: 2, a polypeptide having at least 88% sequence identity to the
mature polypeptide of
SEQ ID NO: 4, a polypeptide having at least 66% sequence identity to the
mature polypeptide of
SEQ ID NO: 6, or a polypeptide having at least 81% sequence identity to the
mature
polypeptide of SEQ ID NO: 8;
(b) a polypeptide
encoded by a polynucleotide that hybridizes under medium
stringency conditions, medium-high stringency conditions, high stringency
conditions, or very
high stringency conditions with (i) the mature polypeptide coding sequence of
SEQ ID NO: 1,
the mature polypeptide coding sequence of SEQ ID NO: 3, the mature polypeptide
coding
sequence of SEQ ID NO: 5 or the mature polypeptide coding sequence of SEQ ID
NO: 7, (ii) the
cDNA sequence of SEQ ID NO: 1, the cDNA sequence of SEQ ID NO: 3, the cDNA
sequence
of SEQ ID NO: 5 or the cDNA sequence of SEQ ID NO: 7, or (iii) the full-length
complement of
(i) or (ii);
(c) a
polypeptide encoded by a polynucleotide having at least 70% sequence identity
to the mature polypeptide coding sequence of SEQ ID NO: 1 or the cDNA sequence
thereof; a
polypeptide encoded by a polynucleotide having at least 88% sequence identity
to the mature
polypeptide coding sequence of SEQ ID NO: 3 or the cDNA sequence thereof; a
polypeptide
encoded by a polynucleotide having at least 66% sequence identity to the
mature polypeptide
coding sequence of SEQ ID NO: 5 or the cDNA sequence thereof; or a polypeptide
encoded by

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a polynucleotide having at least 81% sequence identity to the mature
polypeptide coding
sequence of SEQ ID NO: 7 or the cDNA sequence thereof;
(d) a variant of the mature polypeptide of SEQ ID NO: 2, the mature
polypeptide of
SEQ ID NO: 4, the mature polypeptide of SEQ ID NO: 6 or the mature polypeptide
of SEQ ID
NO: 8 comprising a substitution, deletion, and/or insertion at one or more
(e.g., several)
positions; and
(e) a fragment of the polypeptide of (a), (b), (c), or (d) that has
cellobiohydrolase
activity.
The present invention also relates to isolated polypeptides comprising a
catalytic domain
selected from the group consisting of:
(a) a catalytic domain having at least 70% sequence identity to amino acids
18 to
458 of SEQ ID NO: 2, a catalytic domain having at least 88% sequence identity
to amino acids
21 to 450 of SEQ ID NO: 4, a catalytic domain having at least 66% sequence
identity to amino
acids 22 to 457 of SEQ ID NO: 6, or a catalytic domain having at least 81%
sequence identity to
amino acids 21 to 461 of SEQ ID NO: 8;
(b) a catalytic domain encoded by a polynucleotide that hybridizes under
medium
stringency conditions, medium-high stringency conditions, high stringency
conditions, or very
high stringency conditions with (i) nucleotides 52 to 1454 of SEQ ID NO: 1,
nucleotides 61 to
1733 of SEQ ID NO: 3, nucleotides 64 to 1782 of SEQ ID NO: 5, or nucleotides
52 to 1460 of
SEQ ID NO: 7, (ii) the cDNA sequence thereof, or (iii) the full-length
complement of (i) or (ii);
(c) a catalytic domain encoded by a polynucleotide having at least 70%
sequence
identity to nucleotides 52 to 1454 of SEQ ID NO: 1, at least 88% sequence
identity to
nucleotides 61 to 1733 of SEQ ID NO: 3, at least 66% sequence identity to
nucleotides 64 to
1782 of SEQ ID NO: 5, or at least 81% sequence identity to nucleotides 52 to
1460 of SEQ ID
NO: 7, or the cDNA sequence thereof;
(d) a variant of amino acids 18 to 458 of SEQ ID NO: 2, amino acids 21 to
450 of
SEQ ID NO: 4, amino acids 22 to 457 of SEQ ID NO: 6, or amino acids 21 to 461
of SEQ ID
NO: 8 comprising a substitution, deletion, and/or insertion at one or more
(e.g., several)
positions; and
(e) a fragment of the catalytic domain of (a), (b), (c), or (d) that has
cellobiohydrolase
activity.
The present invention also relates to isolated polypeptides comprising a
carbohydrate
binding domain selected from the group consisting of:

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(a) a carbohydrate binding domain having at least 81% sequence identity to
amino
acids 486 to 521 of SEQ ID NO: 8;
(b) a carbohydrate binding domain encoded by a polynucleotide that
hybridizes
under medium stringency conditions, medium-high stringency conditions, high
stringency
conditions, or very high stringency conditions with (i) nucleotides 1533 to
1640 of SEQ ID NO: 7,
(ii) the cDNA sequence thereof, or (iii) the full-length complement of (i) or
(ii);
(c) a carbohydrate binding domain encoded by a polynucleotide having at
least 81%
sequence identity to nucleotides 1533 to 1640 of SEQ ID NO: 7 or the cDNA
sequence thereof;
(d) a variant of amino acids 486 to 521 of SEQ ID NO: 8 comprising a
substitution,
deletion, and/or insertion at one or more (e.g., several) positions; and
(e) a fragment of the carbohydrate binding domain of (a), (b), (c), or (d)
that has
carbohydrate binding activity.
The present invention also relates to isolated polynucleotides encoding the
polypeptides
of the present invention; nucleic acid constructs; recombinant expression
vectors; recombinant
host cells comprising the polynucleotides; and methods of producing the
polypeptides.
The present invention also relates to processes for degrading or converting a
cellulosic
material, comprising: treating the cellulosic material with an enzyme
composition in the
presence of a polypeptide having cellobiohydrolase activity of the present
invention. In one
aspect, the method further comprises recovering the degraded or converted
cellulosic material.
The present invention also relates to processes of producing a fermentation
product,
comprising: (a) saccharifying a cellulosic material with an enzyme composition
in the presence
of a polypeptide having cellobiohydrolase activity of the present invention;
(b) fermenting the
saccharified cellulosic material with one or more (e.g., several) fermenting
microorganisms to
produce the fermentation product; and (c) recovering the fermentation product
from the
fermentation.
The present invention also relates to processes of fermenting a cellulosic
material,
comprising: fermenting the cellulosic material with one or more (e.g.,
several) fermenting
microorganisms, wherein the cellulosic material is saccharified with an enzyme
composition in
the presence of a polypeptide having cellobiohydrolase activity of the present
invention. In one
aspect, the fermenting of the cellulosic material produces a fermentation
product. In another
aspect, the method further comprises recovering the fermentation product from
the fermentation.
The present invention also relates to a polynucleotide encoding a signal
peptide
comprising or consisting of amino acids 1 to 17 of SEQ ID NO: 2, amino acids 1
to 20 of SEQ ID
NO: 4, amino acids 1 to 21 of SEQ ID NO: 6, or amino acids 1 to 17 of SEQ ID
NO: 8, which is

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operably linked to a gene encoding a protein; nucleic acid constructs,
expression vectors, and
recombinant host cells comprising the polynucleotides; and methods of
producing a protein.
Brief Description of the Figures
5 Figure 1
shows the genomic DNA sequence (SEQ ID NO: 1) and the deduced amino
acid sequence (SEQ ID NO: 2) of the gene encoding a Malbranchea cinnamomea
polypeptide
having cellobiohydrolase activity.
Figure 2 shows the genomic DNA sequence (SEQ ID NO: 3) and the deduced amino
acid sequence (SEQ ID NO: 4) of the gene encoding a Corynascus thermophilus
polypeptide
having cellobiohydrolase activity.
Figure 3 shows the genomic DNA sequence (SEQ ID NO: 5) and the deduced amino
acid sequence (SEQ ID NO: 6) of the gene encoding a Corynascus thermophilus
polypeptide
having cellobiohydrolase activity.
Figure 4 shows the genomic DNA sequence (SEQ ID NO: 7) and the deduced amino
acid sequence (SEQ ID NO: 8) of the gene encoding a Corynascus thermophilus
polypeptide
having cellobiohydrolase activity.
Figure 5 shows a restriction map of plasmid pGH7_ZY582279_485.
Figure 6 shows a restriction map of plasmid pGH7_Mf7339.
Figure 7 shows a restriction map of plasmid pGH7_Mf6627.
Figure 8 shows a restriction map of plasmid pGH7_Mf0261.
Definitions
Acetylxylan esterase: The term "acetylxylan esterase" means a carboxylesterase
(EC
3.1.1.72) that catalyzes the hydrolysis of acetyl groups from polymeric xylan,
acetylated xylose,
acetylated glucose, alpha-napthyl acetate, and p-nitrophenyl acetate. For
purposes of the
present invention, acetylxylan esterase activity is determined using 0.5 mM p-
nitrophenylacetate
as substrate in 50 mM sodium acetate pH 5.0 containing 0.01% TWEENTTM 20
(polyor.yethylene
sorbitan monolaurate). One unit of acetylxylan esterase is defined as the
amount of enzyme
capable of releasing 1 pmole of p-nitrophenolate anion per minute at pH 5, 25
C.
Allelic variant: The term "allelic variant" means any of two or more
alternative forms of
a gene occupying the same chromosomal locus. Allelic variation arises
naturally through
mutation, and may result in polymorphism within populations. Gene mutations
can be silent (no
change in the encoded polypeptide) or may encode polypeptides having altered
amino acid

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sequences. An allelic variant of a polypeptide is a polypeptide encoded by an
allelic variant of a
gene.
Alpha-L-arabinofuranosidase: The term "alpha-L-arabinofuranosidase" means an
alpha-L-arabinofuranoside arabinofuranohydrolase (EC 3.2.1.55) that catalyzes
the hydrolysis
of terminal non-reducing alpha-L-arabinofuranoside residues in alpha-L-
arabinosides. The
enzyme acts on alpha-L-arabinofuranosides, alpha-L-arabinans containing (1,3)-
and/or (1,5)-
linkages, arabinoxylans, and arabinogalactans. Alpha-L-arabinofuranosidase is
also known as
arabinosidase, alpha-arabinosidase, alpha-L-arabinosidase, alpha-
arabinofuranosidase,
polysaccharide alpha-L-arabinofuranosidase, alpha-L-arabinofuranoside
hydrolase, L-
arabinosidase, or alpha-L-arabinanase. For purposes of the present invention,
alpha-L-
arabinofuranosidase activity is determined using 5 mg of medium viscosity
wheat arabinoxylan
(Megazyme International Ireland, Ltd., Bray, Co. Wicklow, Ireland) per ml of
100 mM sodium
acetate pH 5 in a total volume of 200 pl for 30 minutes at 40 C followed by
arabinose analysis
by AMINEX HPX-87H column chromatography (Bio-Rad Laboratories, Inc.,
Hercules, CA,
USA).
Alpha-glucuronidase: The term "alpha-glucuronidase" means an alpha-D-
glucosiduronate glucuronohydrolase (EC 3.2.1.139) that catalyzes the
hydrolysis of an alpha-D-
glucuronoside to D-glucuronate and an alcohol. For purposes of the present
invention, alpha-
glucuronidase activity is determined according to de Vries, 1998, J.
Bactetiol. 180: 243-249.
One unit of alpha-glucuronidase equals the amount of enzyme capable of
releasing 1 pmole of
glucuronic or 4-0-methylglucuronic acid per minute at pH 5, 40 C.
Beta-glucosidase: The term "beta-glucosidase" means a beta-D-glucoside
glucohydrolase (E.C. 3.2.1.21) that catalyzes the hydrolysis of terminal non-
reducing beta-D-
glucose residues with the release of beta-D-glucose. For purposes of the
present invention,
beta-glucosidase activity is determined using p-nitrophenyl-beta-D-
glucopyranoside as
substrate according to the procedure of Venturi et al., 2002, Extracellular
beta-D-glucosidase
from Chaetomium the rmophilum var. coprophilum: production, purification and
some
biochemical properties, J. Basic Microbial. 42: 55-66. One unit of beta-
glucosidase is defined as
1.0 pmole of p-nitrophenolate anion produced per minute at 25 C, pH 4.8 from 1
mM p-
nitrophenyl-beta-D-glucopyranoside as substrate in 50 mM sodium citrate
containing 0.01%
TWEEN 20.
Beta-xylosidase: The term "beta-xylosidase" means a beta-D-xyloside
xylohydrolase
(E.C. 3.2.1.37) that catalyzes the exo-hydrolysis of short beta (1¨>4)-
xylooligosaccharides to
remove successive D-xylose residues from non-reducing termini. For purposes of
the present

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invention, one unit of beta-xylosidase is defined as 1.0 pmole of p-
nitrophenolate anion
produced per minute at 40 C, pH 5 from 1 mM p-nitrophenyl-beta-D-xyloside as
substrate in
100 mM sodium citrate containing 0.01% TWEEN 20.
Carbohydrate binding domain: The term "carbohydrate binding domain" means the
region of an enzyme that mediates binding of the enzyme to amorphous regions
of a
carbohydrate substrate, e.g., cellulose. The carbohydrate binding domain
(CBD), also known as
a carbohydrate binding module, is typically found either at the N-terminal or
at the C-terminal
extremity of an enzyme.
Catalytic domain: The term "catalytic domain" means the region of an enzyme
containing the catalytic machinery of the enzyme.
cDNA: The term "cDNA" means a DNA molecule that can be prepared by reverse
transcription from a mature, spliced, mRNA molecule obtained from a eukaryotic
or prokaryotic
cell. cDNA lacks intron sequences that may be present in the corresponding
genomic DNA. The
initial, primary RNA transcript is a precursor to mRNA that is processed
through a series of
steps, including splicing, before appearing as mature spliced mRNA.
Cellobiohydrolase: The term "cellobiohydrolase" means a 1,4-beta-D-glucan
cellobiohydrolase (E.C. 3.2.1.91 and E.C. 3.2.1.176) that catalyzes the
hydrolysis of 1,4-beta-D-
glucosidic linkages in cellulose, cellooligosaccharides, or any beta-1,4-
linked glucose containing
polymer, releasing cellobiose from the reducing end (cellobiohydrolase I) or
non-reducing end
(cellobiohydrolase II) of the chain (Teen, 1997, Crystalline cellulose
degradation: New insight
into the function of cellobiohydrolases, Trends in Biotechnology 15: 160-167;
Teen i etal., 1998,
Trichoderma reesei cellobiohydrolases: why so efficient on crystalline
cellulose?, Biochem. Soc.
Trans. 26: 173-178). Cellobiohydrolase activity is determined according to the
procedures
described by Lever et al., 1972, Anal. Biochem. 47: 273-279; van Tilbeurgh et
al., 1982, FEBS
Letters, 149: 152-156; van Tilbeurgh and Claeyssens, 1985, FEBS Letters, 187:
283-288; and
Tomme etal., 1988, Eur. J. Biochem. 170: 575-581. In the present invention,
the Tomme et al.
method can be used to determine cellobiohydrolase activity. Alternatively, the
cellobiohydrolase
activity can be determined using microcrystalline cellulose according to the
procedure described
in Example 15 of the present invention.
The polypeptides of the present invention have at least 20%, e.g., at least
40%, at least
50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, and
at least 100% of
the cellobiohydrolase activity of the mature polypeptide of SEQ ID NO: 2, the
mature
polypeptide of SEQ ID NO: 4, the mature polypeptide of SEQ ID NO: 6, or the
mature
polypeptide of SEQ ID NO:8.

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Cellulolytic enzyme or cellulase: The term "cellulolytic enzyme" or
"cellulase" means
one or more (e.g., several) enzymes that hydrolyze a cellulosic material. Such
enzymes include
endoglucanase(s), cellobiohydrolase(s), beta-glucosidase(s), or combinations
thereof. The two
basic approaches for measuring cellulolytic activity include: (1) measuring
the total cellulolytic
activity, and (2) measuring the individual cellulolytic activities
(endoglucanases,
cellobiohydrolases, and beta-glucosidases) as reviewed in Zhang et al.,
Outlook for cellulase
improvement: Screening and selection strategies, 2006, Biotechnology Advances
24: 452-481.
Total cellulolytic activity is usually measured using insoluble substrates,
including VVhatman
filter paper, microcrystalline cellulose, bacterial cellulose, algal
cellulose, cotton, pretreated
lignocellulose, etc. The most common total cellulolytic activity assay is the
filter paper assay
using VVhatman N21 filter paper as the substrate. The assay was established by
the
International Union of Pure and Applied Chemistry (IUPAC) (Ghose, 1987,
Measurement of
cellulase activities, Pure App!. Chem. 59: 257-68).
For purposes of the present invention, cellulolytic enzyme activity is
determined by
measuring the increase in hydrolysis of a cellulosic material by cellulolytic
enzyme(s) under the
following conditions: 1-50 mg of cellulolytic enzyme protein/g of cellulose in
PCS (or other
pretreated cellulosic material) for 3-7 days at a suitable temperature, e.g.,
50 C, 55 C, or 60 C,
compared to a control hydrolysis without addition of cellulolytic enzyme
protein. Typical
conditions are 1 ml reactions, washed or unwashed PCS, 5% insoluble solids, 50
mM sodium
acetate pH 5, 1 mM MnSO4, 50 C, 55 C, or 60 C, 72 hours, sugar analysis by
AMINEX HPX-
87H column (Bio-Rad Laboratories, Inc., Hercules, CA, USA).
Cellulosic material: The term "cellulosic material" means any material
containing
cellulose. The predominant polysaccharide in the primary cell wall of biomass
is cellulose, the
second most abundant is hemicellulose, and the third is pectin. The secondary
cell wall,
produced after the cell has stopped growing, also contains polysaccharides and
is strengthened
by polymeric lignin covalently cross-linked to hemicellulose. Cellulose is a
homopolymer of
anhydrocellobiose and thus a linear beta-(1-4)-D-glucan, while hemicelluloses
include a variety
of compounds, such as xylans, xyloglucans, arabinoxylans, and mannans in
complex branched
structures with a spectrum of substituents. Although generally polymorphous,
cellulose is found
in plant tissue primarily as an insoluble crystalline matrix of parallel
glucan chains.
Hemicelluloses usually hydrogen bond to cellulose, as well as to other
hemicelluloses, which
help stabilize the cell wall matrix.
Cellulose is generally found, for example, in the stems, leaves, hulls, husks,
and cobs of
plants or leaves, branches, and wood of trees. The cellulosic material can be,
but is not limited

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to, agricultural residue, herbaceous material (including energy crops),
municipal solid waste,
pulp and paper mill residue, waste paper, and wood (including forestry
residue) (see, for
example, VViselogel et al., 1995, in Handbook on Bioethanol (Charles E. Wyman,
editor),
pp.105-118, Taylor & Francis, Washington D.C.; Wyman, 1994, Bioresource
Technology 50: 3-
16; Lynd, 1990, Applied Biochemistry and Biotechnology 24/25: 695-719; Mosier
et al., 1999,
Recent Progress in Bioconversion of Lignocellulosics, in Advances in
Biochemical
Engineering/Biotechnology, T. Scheper, managing editor, Volume 65, pp.23-40,
Springer-
Verlag, New York). It is understood herein that the cellulose may be in the
form of
lignocellulose, a plant cell wall material containing lignin, cellulose, and
hemicellulose in a mixed
matrix. In a preferred aspect, the cellulosic material is any biomass
material. In another
preferred aspect, the cellulosic material is lignocellulose, which comprises
cellulose,
hemicelluloses, and lignin.
In one aspect, the cellulosic material is agricultural residue. In another
aspect, the
cellulosic material is herbaceous material (including energy crops). In
another aspect, the
cellulosic material is municipal solid waste. In another aspect, the
cellulosic material is pulp and
paper mill residue. In another aspect, the cellulosic material is waste paper.
In another aspect,
the cellulosic material is wood (including forestry residue).
In another aspect, the cellulosic material is arundo. In another aspect, the
cellulosic
material is bagasse. In another aspect, the cellulosic material is bamboo. In
another aspect, the
cellulosic material is corn cob. In another aspect, the cellulosic material is
corn fiber. In another
aspect, the cellulosic material is corn stover. In another aspect, the
cellulosic material is
miscanthus. In another aspect, the cellulosic material is orange peel. In
another aspect, the
cellulosic material is rice straw. In another aspect, the cellulosic material
is switchgrass. In
another aspect, the cellulosic material is wheat straw.
In another aspect, the cellulosic material is aspen. In another aspect, the
cellulosic
material is eucalyptus. In another aspect, the cellulosic material is fir. In
another aspect, the
cellulosic material is pine. In another aspect, the cellulosic material is
poplar. In another aspect,
the cellulosic material is spruce. In another aspect, the cellulosic material
is willow.
In another aspect, the cellulosic material is algal cellulose. In another
aspect, the
cellulosic material is bacterial cellulose. In another aspect, the cellulosic
material is cotton linter.
In another aspect, the cellulosic material is filter paper. In another aspect,
the cellulosic material
is microcrystalline cellulose. In another aspect, the cellulosic material is
phosphoric-acid treated
cellulose.
In another aspect, the cellulosic material is an aquatic biomass. As used
herein the term

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"aquatic biomass" means biomass produced in an aquatic environment by a
photosynthesis
process. The aquatic biomass can be algae, emergent plants, floating-leaf
plants, or submerged
plants.
The cellulosic material may be used as is or may be subjected to pretreatment,
using
5 conventional methods known in the art, as described herein. In a
preferred aspect, the cellulosic
material is pretreated.
Coding sequence: The term "coding sequence" means a polynucleotide, which
directly
specifies the amino acid sequence of a polypeptide. The boundaries of the
coding sequence are
generally determined by an open reading frame, which begins with a start codon
such as ATG,
10 GTG, or TTG and ends with a stop codon such as TAA, TAG, or TGA. The
coding sequence
may be a genomic DNA, cDNA, synthetic DNA, or a combination thereof.
Control sequences: The term "control sequences" means nucleic acid sequences
necessary for expression of a polynucleotide encoding a mature polypeptide of
the present
invention. Each control sequence may be native (i.e., from the same gene) or
foreign (i.e., from
a different gene) to the polynucleotide encoding the polypeptide or native or
foreign to each
other. Such control sequences include, but are not limited to, a leader,
polyadenylation
sequence, propeptide sequence, promoter, signal peptide sequence, and
transcription
terminator. At a minimum, the control sequences include a promoter, and
transcriptional and
translational stop signals. The control sequences may be provided with linkers
for the purpose
of introducing specific restriction sites facilitating ligation of the control
sequences with the
coding region of the polynucleotide encoding a polypeptide.
Endoglucanase: The term "endoglucanase" means an endo-1,4-(1,3;1,4)-beta-D-
glucan 4-glucanohydrolase (E.C. 3.2.1.4) that catalyzes endohydrolysis of 1,4-
beta-D-glycosidic
linkages in cellulose, cellulose derivatives (such as carboxymethyl cellulose
and hydroxyethyl
cellulose), lichenin, beta-1,4 bonds in mixed beta-1,3 glucans such as cereal
beta-D-glucans or
xyloglucans, and other plant material containing cellulosic components.
Endoglucanase activity
can be determined by measuring reduction in substrate viscosity or increase in
reducing ends
determined by a reducing sugar assay (Zhang et at., 2006, Biotechnology
Advances 24: 452-
481). For purposes of the present invention, endoglucanase activity is
determined using
carboxymethyl cellulose (CMC) as substrate according to the procedure of
Ghose, 1987, Pure
and App!. Chem. 59: 257-268, at pH 5, 40 C.
Expression: The term "expression" includes any step involved in the production
of a
polypeptide including, but not limited to, transcription, post-transcriptional
modification,
translation, post-translational modification, and secretion.

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Expression vector: The term "expression vector' means a linear or circular DNA
molecule that comprises a polynucleotide encoding a polypeptide and is
operably linked to
control sequences that provide for its expression.
Family 61 glycoside hydrolase: The term "Family 61 glycoside hydrolase" or
"Family
GH61" or "GH61" means a polypeptide falling into the glycoside hydrolase
Family 61 according
to Henrissat B., 1991, A classification of glycosyl hydrolases based on amino-
acid sequence
similarities, Biochem. J. 280: 309-316, and Henrissat B., and Bairoch A.,
1996, Updating the
sequence-based classification of glycosyl hydrolases, Biochem. J. 316: 695-
696. The enzymes
in this family were originally classified as a glycoside hydrolase family
based on measurement
of very weak endo-1,4-beta-D-glucanase activity in one family member. The
structure and mode
of action of these enzymes are non-canonical and they cannot be considered as
bona fide
glycosidases. However, they are kept in the CAZy classification on the basis
of their capacity to
enhance the breakdown of lignocellulose when used in conjunction with a
cellulase or a mixture
of cellulases.
Feruloyl esterase: The term "feruloyl esterase" means a 4-hydroxy-3-
methoxycinnamoyl-sugar hydrolase (EC 3.1.1.73) that catalyzes the hydrolysis
of 4-hydroxy-3-
methoxycinnamoyl (feruloyl) groups from esterified sugar, which is usually
arabinose in natural
biomass substrates, to produce ferulate (4-hydroxy-3-methmcinnamate). Feruloyl
esterase is
also known as ferulic acid esterase, hydrmcinnamoyl esterase, FAE-III,
cinnamoyl ester
hydrolase, FAEA, cinnAE, FAE-I, or FAE-II. For purposes of the present
invention, feruloyl
esterase activity is determined using 0.5 mM p-nitrophenylferulate as
substrate in 50 mM
sodium acetate pH 5Ø One unit of feruloyl esterase equals the amount of
enzyme capable of
releasing 1 pmole of p-nitrophenolate anion per minute at pH 5, 25 C.
Fragment: The term "fragment" means a polypeptide or a catalytic domain or
carbohydrate binding domain having one or more (e.g., several) amino acids
absent from the
amino and/or carboxyl terminus of a mature polypeptide or domain; wherein the
fragment has
cellobiohydrolase activity or carbohydrate binding activity. In one aspect, a
fragment contains at
least 375 amino acid residues, e.g., at least 397 amino acid residues or at
least 419 amino acid
residues of SEQ ID NO: 2. In another aspect, a fragment contains at least 364
amino acid
residues, e.g., at least 386 amino acid residues or at least 408 amino acid
residues of SEQ ID
NO: 4. In one aspect, a fragment contains at least 370 amino acid residues,
e.g., at least 392
amino acid residues or at least 414 amino acid residues of SEQ ID NO: 6. In
another aspect, a
fragment contains at least 429 amino acid residues, e.g., at least 454 amino
acid residues or at
least 479 amino acid residues of SEQ ID NO: 8.

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Hemicellulolytic enzyme or hemicellulase: The term "hemicellulolytic enzyme"
or
"hemicellulase" means one or more (e.g., several) enzymes that hydrolyze a
hemicellulosic
material. See, for example, Shallom, D. and Shoham, Y. Microbial
hemicellulases. Current
Opinion In Microbiology, 2003, 6(3): 219-228). Hemicellulases are key
components in the
degradation of plant biomass. Examples of hemicellulases include, but are not
limited to, an
acetylmannan esterase, an acetylxylan esterase, an arabinanase, an
arabinofuranosidase, a
coumaric acid esterase, a feruloyl esterase, a galactosidase, a glucuronidase,
a glucuronoyl
esterase, a mannanase, a mannosidase, a xylanase, and a xylosidase. The
substrates of these
enzymes, the hemicelluloses, are a heterogeneous group of branched and linear
polysaccharides that are bound via hydrogen bonds to the cellulose
microfibrils in the plant cell
wall, crosslinking them into a robust network. Hemicelluloses are also
covalently attached to
lignin, forming together with cellulose a highly complex structure. The
variable structure and
organization of hemicelluloses require the concerted action of many enzymes
for its complete
degradation. The catalytic modules of hemicellulases are either glycoside
hydrolases (GHs) that
hydrolyze glycosidic bonds, or carbohydrate esterases (CEs), which hydrolyze
ester linkages of
acetate or ferulic acid side groups. These catalytic modules, based on
homology of their primary
sequence, can be assigned into GH and CE families. Some families, with an
overall similar fold,
can be further grouped into clans, marked alphabetically (e.g., GH-A). A most
informative and
updated classification of these and other carbohydrate active enzymes is
available in the
Carbohydrate-Active Enzymes (CAZy) database. Hemicellulolytic enzyme
activities can be
measured according to Ghose and Bisaria, 1987, Pure & AppL Chem. 59: 1739-
1752, at a
suitable temperature, e.g., 50 C, 55 C, or 60 C, and pH, e.g., 5.0 or 5.5.
High stringency conditions: The term "high stringency conditions" means for
probes of
at least 100 nucleotides in length, prehybridization and hybridization at 42 C
in 5X SSPE, 0.3%
SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 50%
formamide,
following standard Southern blotting procedures for 12 to 24 hours. The
carrier material is finally
washed three times each for 15 minutes using 2X SSC, 0.2% SDS at 65 C.
Host cell: The term "host cell" means any cell type that is susceptible to
transformation,
transfection, transduction, or the like with a nucleic acid construct or
expression vector
comprising a polynucleotide of the present invention. The term "host cell"
encompasses any
progeny of a parent cell that is not identical to the parent cell due to
mutations that occur during
replication.
Isolated: The term "isolated" means a substance in a form or environment that
does not
occur in nature. Non-limiting examples of isolated substances include (1) any
non-naturally

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occurring substance, (2) any substance including, but not limited to, any
enzyme, variant,
nucleic acid, protein, peptide or cofactor, that is at least partially removed
from one or more or
all of the naturally occurring constituents with which it is associated in
nature; (3) any substance
modified by the hand of man relative to that substance found in nature; or (4)
any substance
modified by increasing the amount of the substance relative to other
components with which it is
naturally associated (e.g., recombinant production in a host cell; multiple
copies of a gene
encoding the substance; and use of a stronger promoter than the promoter
naturally associated
with the gene encoding the substance).
Low stringency conditions: The term "low stringency conditions" means for
probes of
at least 100 nucleotides in length, prehybridization and hybridization at 42 C
in 5X SSPE, 0.3%
SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 25%
forrnamide,
following standard Southern blotting procedures for 12 to 24 hours. The
carrier material is finally
washed three times each for 15 minutes using 2X SSC, 0.2% SDS at 50 C.
Mature polypeptide: The term "mature polypeptide" means a polypeptide in its
final
form following translation and any post-translational modifications, such as N-
terminal
processing, C-terminal truncation, glycosylation, phosphorylation, etc. In one
aspect, the mature
polypeptide is amino acids 18 to 458 of SEQ ID NO: 2 (P249XX) based on the
SignalP program
(Nielsen etal., 1997, Protein Engineering 10: 1-6) that predicts amino acids 1
to 17 of SEQ ID
NO: 2 are a signal peptide. In another aspect, the mature polypeptide is amino
acids 21 to 450
of SEQ ID NO: 4 (P24NX2) based on the SignalP program that predicts amino
acids 1 to 20 of
SEQ ID NO: 4 are a signal peptide. In one aspect, the mature polypeptide is
amino acids 22 to
457 of SEQ ID NO: 6 (P24FVN) based on the SignalP program (Nielsen et al.,
1997, Protein
Engineering 10: 1-6) that predicts amino acids 1 to 21 of SEQ ID NO: 6 are a
signal peptide. In
another aspect, the mature polypeptide is amino acids 18 to 521 of SEQ ID NO:
8 (P24FUQ)
based on the SignalP program that predicts amino acids 1 to 17 of SEQ ID NO: 8
are a signal
peptide.
It is known in the art that a host cell may produce a mixture of two of more
different
mature polypeptides (i.e., with a different C-terminal and/or N-terminal amino
acid) expressed
by the same polynucleotide. It is also known in the art that different host
cells process
polypeptides differently, and thus, one host cell expressing a polynucleotide
may produce a
different mature polypeptide (e.g., having a different C-terminal and/or N-
terminal amino acid)
as compared to another host cell expressing the same polynucleotide.
Mature polypeptide coding sequence: The term "mature polypeptide coding
sequence" means a polynucleotide that encodes a mature polypeptide having
cellobiohydrolase

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activity. In one aspect, the mature polypeptide coding sequence is nucleotides
52 to 1454 of
SEQ ID NO: 1 or the cDNA sequence thereof based on the SignalP program
(Nielsen et aL,
1997, supra) that predicts nucleotides 1 to 51 of SEQ ID NO: 1 encode a signal
peptide. In
another aspect, the mature polypeptide coding sequence is nucleotides 61 to
1733 of SEQ ID
NO: 3 or the cDNA sequence thereof based on the SignalP program that predicts
nucleotides 1
to 60 of SEQ ID NO: 3 encode a signal peptide. In one aspect, the mature
polypeptide coding
sequence is nucleotides 64 to 1782 of SEQ ID NO: 5 or the cDNA sequence
thereof based on
the SignalP program (Nielsen et al., 1997, supra) that predicts nucleotides 1
to 63 of SEQ ID
NO: 5 encode a signal peptide. In another aspect, the mature polypeptide
coding sequence is
nucleotides 52 to 1640 of SEQ ID NO: 7 or the cDNA sequence thereof based on
the SignalP
program that predicts nucleotides 1 to 51 of SEQ ID NO: 7 encode a signal
peptide.
Medium stringency conditions: The term "medium stringency conditions" means
for
probes of at least 100 nucleotides in length, prehybridization and
hybridization at 42 C in 5X
SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and
35%
formamide, following standard Southern blotting procedures for 12 to 24 hours.
The carrier
material is finally washed three times each for 15 minutes using 2X SSC, 0.2%
SDS at 55 C.
Medium-high stringency conditions: The term "medium-high stringency
conditions"
means for probes of at least 100 nucleotides in length, prehybridization and
hybridization at
42 C in 5X SSPE, 0.3% SDS, 200 micrograms/m1 sheared and denatured salmon
sperm DNA,
and 35% formamide, following standard Southern blotting procedures for 12 to
24 hours. The
carrier material is finally washed three times each for 15 minutes using 2X
SSC, 0.2% SDS at
60 C.
Nucleic acid construct: The term "nucleic acid construct" means a nucleic acid
molecule, either single- or double-stranded, which is isolated from a
naturally occurring gene or
is modified to contain segments of nucleic acids in a manner that would not
otherwise exist in
nature or which is synthetic, which comprises one or more control sequences.
Operably linked: The term "operably linked" means a configuration in which a
control
sequence is placed at an appropriate position relative to the coding sequence
of a
polynucleotide such that the control sequence directs expression of the coding
sequence.
Polypeptide having cellulolytic enhancing activity: The term "polypeptide
having
cellulolytic enhancing activity" means a GH61 polypeptide that catalyzes the
enhancement of
the hydrolysis of a cellulosic material by enzyme having cellulolytic
activity. For purposes of the
present invention, cellulolytic enhancing activity is determined by measuring
the increase in
reducing sugars or the increase of the total of cellobiose and glucose from
the hydrolysis of a

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cellulosic material by cellulolytic enzyme under the following conditions: 1-
50 mg of total
protein/g of cellulose in pretreated corn stover (PCS), wherein total protein
is comprised of 50-
99.5% w/w cellulolytic enzyme protein and 0.5-50% w/w protein of a GH61
polypeptide having
cellulolytic enhancing activity for 1-7 days at a suitable temperature, e.g.,
50 C, 55 C, or 60 C,
5 and a suitable pH such 4-9, e.g., 5.0 or 5.5, compared to a control
hydrolysis with equal total
protein loading without cellulolytic enhancing activity (1-50 mg of
cellulolytic protein/g of
cellulose in PCS). In a preferred aspect, a mixture of CELLUCLASTO 1.5L
(Novozymes A/S,
Bagsvmrd, Denmark) in the presence of 2-3% of total protein weight Aspergillus
oryzae beta-
glucosidase (recombinantly produced in Aspergillus oryzae according to WO
02/095014) or 2-
10 3% of total protein weight Aspergillus fumigatus beta-glucosidase
(recombinantly produced in
Aspergillus oryzae as described in WO 2002/095014) of cellulase protein
loading is used as the
source of the cellulolytic activity.
The GH61 polypeptides having cellulolytic enhancing activity enhance the
hydrolysis of a
cellulosic material catalyzed by enzyme having cellulolytic activity by
reducing the amount of
15 cellulolytic enzyme required to reach the same degree of hydrolysis
preferably at least 1.01-fold,
e.g., at least 1.05-fold, at least 1.10-fold, at least 1.25-fold, at least 1.5-
fold, at least 2-fold, at
least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, or at least
20-fold.
Pretreated corn stover: The term "PCS" or "Pretreated Corn Stover" means a
cellulosic
material derived from corn stover by treatment with heat and dilute sulfuric
acid, alkaline
pretreatment, or neutral pretreatment.
Sequence identity: The relatedness between two amino acid sequences or between
two nucleotide sequences is described by the parameter "sequence identity".
For purposes of the present invention, the sequence identity between two amino
acid
sequences is determined using the Needleman-Wunsch algorithm (Needleman and
Wunsch,
1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the
EMBOSS
package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et
al., 2000,
Trends Genet. 16: 276-277), preferably version 3Ø0, 5Ø0 or later. The
parameters used are
gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62
(EMBOSS version
of BLOSUM62) substitution matrix. The output of Needle labeled "longest
identity" (obtained
using the ¨nobrief option) is used as the percent identity and is calculated
as follows:
(Identical Residues x 100)/(Length of Alignment ¨ Total Number of Gaps in
Alignment)
For purposes of the present invention, the sequence identity between two
deoxyribonucleotide sequences is determined using the Needleman-Wunsch
algorithm
(Needleman and Wunsch, 1970, supra) as implemented in the Needle program of
the EMBOSS

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WO 2013/071871 PCT/CN2012/084661
package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et
al., 2000,
supra), preferably version 3Ø0, 5Ø0 or later. The parameters used are gap
open penalty of 10,
gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCB! NUC4.4)
substitution matrix. The output of Needle labeled "longest identity" (obtained
using the ¨nobrief
option) is used as the percent identity and is calculated as follows:
(Identical Deoxyribonucleotides x 100)/(Length of Alignment ¨ Total Number of
Gaps in
Alignment)
Subsequence: The term "subsequence" means a polynucleotide having one or more
(e.g., several) nucleotides absent from the 5' and/or 3' end of a mature
polypeptide coding
sequence or domain; wherein the subsequence encodes a fragment having
cellobiohydrolase
activity or carbohydrate binding activity. In one aspect, a subsequence
contains at least 1125
nucleotides, e.g., at least 1191 nucleotides or at least 1257 nucleotides.of
SEQ ID NO: 1. In
another aspect, a subsequence contains at least 1092 nucleotides, e.g., at
least 1158
nucleotides or at least 1224 nucleotides.of SEQ ID NO: 3. In one aspect, a
subsequence
contains at least 1110 nucleotides, e.g., at least 1176 nucleotides or at
least 1242 nucleotides.of
SEQ ID NO: 5. In another aspect, a subsequence contains at least 1287
nucleotides, e.g., at
least 1362 nucleotides or at least 1437 nucleotides.of SEQ ID NO: 7.
Variant: The term "variant" means a polypeptide having cellobiohydrolase
activity
comprising an alteration, i.e., a substitution, insertion, and/or deletion, at
one or more (e.g.,
several) positions. A substitution means replacement of the amino acid
occupying a position
with a different amino acid; a deletion means removal of the amino acid
occupying a position;
and an insertion means adding an amino acid adjacent to and immediately
following the amino
acid occupying a position.
Very high stringency conditions: The term "very high stringency conditions"
means for
probes of at least 100 nucleotides in length, prehybridization and
hybridization at 42 C in 5X
SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and
50%
formamide, following standard Southern blotting procedures for 12 to 24 hours.
The carrier
material is finally washed three times each for 15 minutes using 2X SSC, 0.2%
SDS at 70 C.
Very low stringency conditions: The term "very low stringency conditions"
means for
probes of at least 100 nucleotides in length, prehybridization and
hybridization at 42 C in 5X
SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and
25%
formamide, following standard Southern blotting procedures for 12 to 24 hours.
The carrier
material is finally washed three times each for 15 minutes using 2X SSC, 0.2%
SDS at 45 C.

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Xylan-containing material: The term "xylan-containing material" means any
material
comprising a plant cell wall polysaccharide containing a backbone of beta-(1-
4)-linked xylose
residues. Xylans of terrestrial plants are heteropolymers possessing a beta-(1-
4)-D-
xylopyranose backbone, which is branched by short carbohydrate chains. They
comprise D-
glucuronic acid or its 4-0-methyl ether, L-arabinose, and/or various
oligosaccharides, composed
of D-xylose, L-arabinose, D- or L-galactose, and D-glucose. Xylan-type
polysaccharides can be
divided into hommlans and heteroxylans, which include glucuronoxylans,
(arabino)glucuronoxylans, (glucurono)arabinoxylans, arabinoxylans, and complex
heteroxylans.
See, for example, Ebringerova etal., 2005, Adv. Polym. Sci. 186: 1-67.
In the processes of the present invention, any material containing xylan may
be used. In
a preferred aspect, the xylan-containing material is lignocellulose.
Xylan degrading activity or xylanolytic activity: The term "xylan degrading
activity" or
"xylanolytic activity" means a biological activity that hydrolyzes xylan-
containing material. The
two basic approaches for measuring xylanolytic activity include: (1) measuring
the total
xylanolytic activity, and (2) measuring the individual xylanolytic activities
(e.g., endoxylanases,
beta-xylosidases, arabinofuranosidases, alpha-glucuronidases, acetylxylan
esterases, feruloyl
esterases, and alpha-glucuronyl esterases). Recent progress in assays of
xylanolytic enzymes
was summarized in several publications including Biely and Puchard, 2006,
Recent progress in
the assays of xylanolytic enzymes, Journal of the Science of Food and
Agriculture 86(11): 1636-
1647; Spanikova and Biely, 2006, Glucuronoyl esterase - Novel carbohydrate
esterase
produced by Schizophyllum commune, FEBS Letters 580(19): 4597-4601; Herrmann,
Vrsanska,
Jurickova, Hirsch, Biely, and Kubicek, 1997, The beta-D-xylosidase of
Trichoderma reesei is a
multifunctional beta-D-xylan xylohydrolase, Biochemical Journal 321: 375-381.
Total xylan degrading activity can be measured by determining the reducing
sugars
formed from various types of xylan, including, for example, oat spelt,
beechwood, and
larchwood xylans, or by photometric determination of dyed xylan fragments
released from
various covalently dyed xylans. The most common total xylanolytic activity
assay is based on
production of reducing sugars from polymeric 4-0-methyl glucuronoxylan as
described in Bailey,
Biely, Poutanen, 1992, Interlaboratory testing of methods for assay of
xylanase activity, Journal
of Biotechnology 23(3): 257-270. Xylanase activity can also be determined with
0.2% AZCL-
arabinoxylan as substrate in 0.01% TRITON D X-100 (4-(1,1,3,3-
tetramethylbutyl)phenyl-
polyethylene glycol) and 200 mM sodium phosphate buffer pH 6 at 37 C. One unit
of xylanase
activity is defined as 1.0 gmole of azurine produced per minute at 37 C, pH 6
from 0.2% AZCL-
arabinmlan as substrate in 200 mM sodium phosphate pH 6 buffer.

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For purposes of the present invention, xylan degrading activity is determined
by
measuring the increase in hydrolysis of birchwood xylan (Sigma Chemical Co.,
Inc., St. Louis,
MO, USA) by xylan-degrading enzyme(s) under the following typical conditions:
1 ml reactions,
mg/ml substrate (total solids), 5 mg of xylanolytic protein/g of substrate, 50
mM sodium
5 acetate pH 5, 50 C, 24 hours, sugar analysis using p-hydroxybenzoic acid
hydrazide (PHBAH)
assay as described by Lever, 1972, A new reaction for colorimetric
determination of
carbohydrates, Anal. Biochem 47: 273-279.
Xylanase: The term "xylanase" means a 1,4-beta-D-xylan-xylohydrolase (E.C.
3.2.1.8)
that catalyzes the endohydrolysis of 1,4-beta-D-xylosidic linkages in xylans.
For purposes of the
present invention, xylanase activity is determined with 0.2% AZCL-arabinoxylan
as substrate in
0.01% TRITON X-100 and 200 mM sodium phosphate buffer pH 6 at 37 C. One unit
of
xylanase activity is defined as 1.0 mole of azurine produced per minute at 37
C, pH 6 from
0.2% AZCL-arabinoxylan as substrate in 200 mM sodium phosphate pH 6 buffer.
Detailed Description of the Invention
Polypeptides Having Cellobiohydrolase Activity
In an embodiment, the present invention relates to isolated polypeptides
having a
sequence identity to the mature polypeptide of SEQ ID NO: 2 of at least 70%,
e.g., at least 75%,
at least 78%, at least 80%, at least 81%, at least 82%, at least 83%, at least
84%, at least 85%,
at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least
91%, at least 92%,
at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least
98%, at least 99%,
or 100%, which have cellobiohydrolase activity. In an embodiment, the present
invention relates
to isolated polypeptides having a sequence identity to the mature polypeptide
of SEQ ID NO: 4
of at least 88%, e.g., at least 89%, at least 90%, at least 91%, at least 92%,
at least 93%, at
least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least
99%, or 100%, which
have cellobiohydrolase activity. In an embodiment, the present invention
relates to isolated
polypeptides having a sequence identity to the mature polypeptide of SEQ ID
NO: 6 of at least
66%, e.g., at least 68%, at least 70%, at least 75%, at least 78%, at least
80%, at least 81%, at
least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least
87%, at least 88%, at
least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at least 95%, at
least 96%, at least 97%, at least 98%, at least 99%, or 100%, which have
cellobiohydrolase
activity. In an embodiment, the present invention relates to isolated
polypeptides having a
sequence identity to the mature polypeptide of SEQ ID NO: 8 of at least 81%,
e.g., at least 82%,
at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least
88%, at least 89%,

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at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least
95%, at least 96%,
at least 97%, at least 98%, at least 99%, or 100%, which have
cellobiohydrolase activity. In one
aspect, the polypeptides differ by up to 10 amino acids, e.g., 1, 2, 3, 4, 5,
6, 7, 8, 9, or 10, from
the mature polypeptide of SEQ ID NO: 2, the mature polypeptide of SEQ ID NO:
4, the mature
polypeptide of SEQ ID NO: 6, or the mature polypeptide of SEQ ID NO: 8.
A polypeptide of the present invention preferably comprises or consists of the
amino acid
sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8 or an
allelic variant
thereof; or is a fragment thereof having cellobiohydrolase activity. In
another aspect, the
polypeptide comprises or consists of the mature polypeptide of SEQ ID NO: 2,
the mature
polypeptide of SEQ ID NO: 4, the mature polypeptide of SEQ ID NO: 6 or the
mature
polypeptide of SEQ ID NO: 8. In another aspect, the polypeptide comprises or
consists of amino
acids 18 to 458 of SEQ ID NO: 2, amino acids 21 to 450 of SEQ ID NO: 4, amino
acids 22 to
457 of SEQ ID NO: 6, or amino acids 18 to 521 of SEQ ID NO: 8.
In another embodiment, the present invention relates to isolated polypeptides
having
cellobiohydrolase activity encoded by a polynucleotide that hybridizes under
very low stringency
conditions, low stringency conditions, medium stringency conditions, medium-
high stringency
conditions, high stringency conditions, or very high stringency conditions
with (i) the mature
polypeptide coding sequence of SEQ ID NO: 1, the mature polypeptide coding
sequence of
SEQ ID NO: 3, the mature polypeptide coding sequence of SEQ ID NO: 5 or the
mature
polypeptide coding sequence of SEQ ID NO: 7; (ii) the cDNA sequence of SEQ ID
NO: 1, the
cDNA sequence of SEQ ID NO: 3, the cDNA sequence of SEQ ID NO: 5 or the cDNA
sequence
of SEQ ID NO: 7, or (iii) the full-length complement of (i) or (ii) (Sambrook
et al, 1989,
Molecular Cloning, A Laboratory Manual, 2d edition, Cold Spring Harbor, New
York).
The polynucleotide of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO:
7, or
a subsequence thereof, as well as the polypeptide of SEQ ID NO: 2, SEQ ID NO:
4, SEQ ID
NO: 6 or SEQ ID NO: 8, or a fragment thereof, may be used to design nucleic
acid probes to
identify and clone DNA encoding polypeptides having cellobiohydrolase activity
from strains of
different genera or species according to methods well known in the art. In
particular, such
probes can be used for hybridization with the genomic DNA or cDNA of a cell of
interest,
following standard Southern blotting procedures, in order to identify and
isolate the
corresponding gene therein. Such probes can be considerably shorter than the
entire sequence,
but should be at least 15, e.g., at least 25, at least 35, or at least 70
nucleotides in length.
Preferably, the nucleic acid probe is at least 100 nucleotides in length,
e.g., at least 200
nucleotides, at least 300 nucleotides, at least 400 nucleotides, at least 500
nucleotides, at least

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600 nucleotides, at least 700 nucleotides, at least 800 nucleotides, or at
least 900 nucleotides in
length. Both DNA and RNA probes can be used. The probes are typically labeled
for detecting
the corresponding gene (for example, with 32P, 3H, 35S, biotin, or avidin).
Such probes are
encompassed by the present invention.
5 A genomic DNA or cDNA library prepared from such other strains may be
screened for
DNA that hybridizes with the probes described above and encodes a polypeptide
having
cellobiohydrolase 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
10 suitable carrier material. In order to identify a clone or DNA that
hybridizes with SEQ ID NO: 1,
SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7, or the mature polypeptide coding
sequence
thereof, or a subsequence thereof, the carrier material is used in a Southern
blot.
For purposes of the present invention, hybridization indicates that the
polynucleotides
hybridize to a labeled nucleic acid probe corresponding to (i) SEQ ID NO: 1,
SEQ ID NO: 3,
15 SEQ ID NO: 5 or SEQ ID NO: 7; (ii) the mature polypeptide coding
sequence of SEQ ID NO: 1,
the mature polypeptide coding sequence of SEQ ID NO: 3, the mature polypeptide
coding
sequence of SEQ ID NO: 5 or the mature polypeptide coding sequence of SEQ ID
NO: 7; (iii)
the cDNA sequence of SEQ ID NO: 1, the cDNA sequence of SEQ ID NO: 3, the cDNA
sequence of SEQ ID NO: 5 or the cDNA sequence of SEQ ID NO: 7; (iv) the full-
length
20 complement thereof; or (v) a subsequence thereof; under very low to very
high stringency
conditions. Molecules to which the nucleic acid probe hybridizes under these
conditions can be
detected using, for example, X-ray film or any other detection means known in
the art.
In one aspect, the nucleic acid probe is a polynucleotide that encodes the
polypeptide of
SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8; the mature
polypeptide thereof;
or a fragment thereof. In another aspect, the nucleic acid probe is SEQ ID NO:
1, SEQ ID NO:
3, SEQ ID NO: 5 or SEQ ID NO: 7; or the mature polypeptide coding sequence of
SEQ ID NO:
1, the mature polypeptide coding sequence of SEQ ID NO: 3, the mature
polypeptide coding
sequence of SEQ ID NO: 5 or the mature polypeptide coding sequence of SEQ ID
NO: 7; or the
cDNA sequence of SEQ ID NO: 1, the cDNA sequence of SEQ ID NO: 3, the cDNA
sequence
of SEQ ID NO: 5 or the cDNA sequence of SEQ ID NO: 7.
In another embodiment, the present invention relates to isolated polypeptides
having
cellobiohydrolase activity encoded by polynucleotides having a sequence
identity to the mature
polypeptide coding sequence of SEQ ID NO: 1 or the cDNA sequence thereof, of
at least 70%,
e.g., at least 72%, at least 75%, at least 78%, at least 80%, at least 81%, at
least 82%, at least

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83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at
least 89%, at least
90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at
least 96%, at least
97%, at least 98%, at least 99%, or 100%; isolated polypeptides having
cellobiohydrolase
activity encoded by polynucleotides having a sequence identity to the mature
polypeptide
coding sequence of SEQ ID NO: 3, or the cDNA sequence thereof, of at least
88%, e.g., at least
89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at
least 95%, at least
96%, at least 97%, at least 98%, at least 99%, or 100%; isolated polypeptides
having
cellobiohydrolase activity encoded by polynucleotides having a sequence
identity to the mature
polypeptide coding sequence of SEQ ID NO: 5 or the cDNA sequence thereof, of
at least 66%,
e.g., at least 68%, at least 70%, at least 75%, at least 78%, at least 80%, at
least 81%, at least
82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at
least 88%, at least
89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at
least 95%, at least
96%, at least 97%, at least 98%, at least 99%, or 100%; isolated polypeptides
having
cellobiohydrolase activity encoded by polynucleotides having a sequence
identity to the mature
polypeptide coding sequence of SEQ ID NO: 7 or the cDNA sequence thereof, of
at least 81%,
e.g., at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at
least 87%, at least
88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%, at least
95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%.
In another embodiment, the present invention relates to variants of the mature
polypeptide of SEQ ID NO: 2, the mature polypeptide of SEQ ID NO: 4, the
mature polypeptide
of SEQ ID NO: 6 or the mature polypeptide of SEQ ID NO: 8 comprising a
substitution, deletion,
and/or insertion at one or more (e.g., several) positions. In an aspect, the
number of amino acid
substitutions, deletions and/or insertions introduced into the mature
polypeptide of SEQ ID NO:
2, the mature polypeptide of SEQ ID NO: 4, the mature polypeptide of SEQ ID
NO: 6 or the
mature polypeptide of SEQ ID NO: 8 is up to 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8,
9, or 10. The amino
acid changes may be of a minor nature, that is conservative amino acid
substitutions or
insertions that do not significantly affect the folding and/or activity of the
protein; small deletions,
typically of 1-30 amino acids; small amino- or carboxyl-terminal extensions,
such as an amino-
terminal methionine residue; a small linker peptide of up to 20-25 residues;
or a small extension
that facilitates purification by changing net charge or another function, such
as a poly-histidine
tract, an antigenic epitope or a binding domain.
Examples of conservative substitutions are within the groups of basic amino
acids
(arginine, lysine and histidine), acidic amino acids (glutamic acid and
aspartic acid), polar amino
acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine
and valine),

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aromatic amino acids (phenylalanine, tryptophan and tyrosine), and small amino
acids (glycine,
alanine, serine, threonine and methionine). Amino acid substitutions that do
not generally alter
specific activity are known in the art and are described, for example, by H.
Neurath and R.L. Hill,
1979, In, The Proteins, Academic Press, New York. Common substitutions are
Ala/Ser, Val/Ile,
Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, AlaNal, Ser/Gly, Tyr/Phe,
Ala/Pro, Lys/Arg,
Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly.
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.
Essential amino acids in a polypeptide can be identified according to
procedures known
in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis
(Cunningham
and Wells, 1989, Science 244: 1081-1085). In the latter technique, single
alanine mutations are
introduced at every residue in the molecule, and the resultant mutant
molecules are tested for
cellobiohydrolase activity to identify amino acid residues that are critical
to the activity of the
molecule. See also, Hilton et aL, 1996, J. Biol. Chem. 271: 4699-4708. The
active site of the
enzyme or other biological interaction can also be determined by physical
analysis of structure,
as determined by such techniques as nuclear magnetic resonance,
crystallography, electron
diffraction, or photoaffinity labeling, in conjunction with mutation of
putative contact site amino
acids. See, for example, de Vos et al., 1992, Science 255: 306-312; Smith et
at, 1992, J. Mot
BioL 224: 899-904; VVIodaver et at, 1992, FEBS Lett. 309: 59-64. The identity
of essential
amino acids can also be inferred from an alignment with a related polypeptide.
Single or multiple amino acid substitutions, deletions, and/or insertions can
be made and
tested using known methods of mutagenesis, recombination, and/or shuffling,
followed by a
relevant screening procedure, such as those disclosed by Reidhaar-Olson and
Sauer, 1988,
Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86: 2152-
2156; WO
95/17413; or WO 95/22625. Other methods that can be used include error-prone
PCR, phage
display (e.g., Lowman et at, 1991, Biochemistry 30: 10832-10837; U.S. Patent
No. 5,223,409;
WO 92/06204), and region-directed mutagenesis (Derbyshire etal., 1986, Gene
46: 145; Ner et
at, 1988, DNA 7: 127).
Mutagenesis/shuffling methods can be combined with high-throughput, automated
screening methods to detect activity of cloned, mutagenized polypeptides
expressed by host
cells (Ness etal., 1999, Nature Biotechnology 17: 893-896). Mutagenized DNA
molecules that
encode active polypeptides can be recovered from the host cells and rapidly
sequenced using

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standard methods in the art. These methods allow the rapid determination of
the importance of
individual amino acid residues in a polypeptide.
The polypeptide may be a hybrid polypeptide in which a region of one
polypeptide is
fused at the N-terminus or the C-terminus of a region of another polypeptide.
The polypeptide may be a fusion polypeptide or cleavable fusion polypeptide in
which
another polypeptide is fused at the N-terminus or the C-terminus of the
polypeptide of the
present invention. A fusion polypeptide is produced by fusing a polynucleotide
encoding another
polypeptide to a polynucleotide of the present invention. Techniques for
producing fusion
polypeptides are known in the art, and include ligating the coding sequences
encoding the
polypeptides so that they are in frame and that expression of the fusion
polypeptide is under
control of the same promoter(s) and terminator. Fusion polypeptides may also
be constructed
using intein technology in which fusion polypeptides are created post-
translationally (Cooper et
aL, 1993, EMBO J. 12: 2575-2583; Dawson et a/., 1994, Science 266: 776-779).
A fusion polypeptide can further comprise a cleavage site between the two
polypeptides.
Upon secretion of the fusion protein, the site is cleaved releasing the two
polypeptides.
Examples of cleavage sites include, but are not limited to, the sites
disclosed in Martin et al.,
2003, J. Ind. Microbiol BiotechnoL 3: 568-576; Svetina et aL, 2000, J.
Biotechnol. 76: 245-251;
Rasmussen-Wilson et al., 1997, App!. Environ. MicrobioL 63: 3488-3493; Ward et
aL, 1995,
Biotechnology 13: 498-503; and Contreras et aL, 1991, Biotechnology 9: 378-
381; Eaton etal.,
1986, Biochemistry 25: 505-512; Collins-Racie etal., 1995, Biotechnology 13:
982-987; Carter
et al., 1989, Proteins: Structure, Function, and Genetics 6: 240-248; and
Stevens, 2003, Drug
Discovery World 4: 35-48.
Sources of Polypeptides Having Cellobiohydrolase Activity
A polypeptide having cellobiohydrolase activity of the present invention may
be obtained
from microorganisms of any genus. For purposes of the present invention, the
term "obtained
from" as used herein in connection with a given source shall mean that the
polypeptide encoded
by a polynucleotide is produced by the source or by a strain in which the
polynucleotide from the
source has been inserted. In one aspect, the polypeptide obtained from a given
source is
secreted extracellularly.
The polypeptide may be a fungal polypeptide. In one aspect, the polypeptide is
a
Malbranchea polypeptide. In another aspect, the polypeptide is a Malbranchea
cinnamomea
polypeptide. In one aspect, the polypeptide is a Corynascus polypeptide. In
another aspect, the
polypeptide is a Corynascus thermophilus polypeptide.

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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.
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 und Zellkulturen GmbH (DSMZ), Centraalbureau Voor
Schimmelcultures
(CBS), and Agricultural Research Service Patent Culture Collection, Northern
Regional
Research Center (NRRL).
The polypeptide may be identified and obtained from other sources including
microorganisms isolated from nature (e.g., soil, composts, water, etc.) or DNA
samples obtained
directly from natural materials (e.g., soil, composts, water, etc.) using the
above-mentioned
probes. Techniques for isolating microorganisms and DNA directly from natural
habitats are well
known in the art. A polynucleotide encoding the polypeptide may then be
obtained by similarly
screening a genomic DNA or cDNA library of another microorganism or mixed DNA
sample.
Once a polynucleotide encoding a polypeptide has been detected with the
probe(s), the
polynucleotide can be isolated or cloned by utilizing techniques that are
known to those of
ordinary skill in the art (see, e.g., Sambrook etal., 1989, supra).
Catalytic Domains
In one embodiment, the present invention also relates to catalytic domains
having a
sequence identity to amino acids 18 to 458 of SEQ ID NO: 2 of at least 70%,
e.g., at least 72%,
at least 75%, at least 78%, at least 80%, at least 81%, at least 82%, at least
83%, at least 84%,
at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least
90%, at least 91%,
at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least
97%, at least 98%,
at least 99%, or 100%; to catalytic domains having a sequence identity to
amino acids 21 to 450
of SEQ ID NO: 4 of at least 88%, e.g., at least 89%, at least 90%, at least
91%, at least 92%, at
least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least
98%, at least 99%, or
100%; to catalytic domains having a sequence identity to amino acids 22 to 457
of SEQ ID NO:
6 of at least 66%, e.g., at least 68%, at least 70%, at least 75%, at least
78%, at least 80%, at
least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least
86%, at least 87%, at
least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least
93%, at least 94%, at
least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%; to
catalytic domains
having a sequence identity to amino acids 21 to 461 of SEQ ID NO: 8 of at
least 81%, e.g., at

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least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least
87%, at least 88%, at
least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at least 95%, at
least 96%, at least 97%, at least 98%, at least 99%, or 100%. In one aspect,
the catalytic
domains comprise amino acid sequences that differ by up to 10 amino acids,
e.g., 1, 2, 3, 4, 5,
5 6, 7, 8, 9, or 10, from amino acids 18 to 458 of SEQ ID NO: 2, from amino
acids 21 to 450 of
SEQ ID NO: 4, from amino acids 22 to 457 of SEQ ID NO: 6, or from amino acids
21 to 461 of
SEQ ID NO: 8.
The catalytic domain preferably comprises or consists of amino acids 18 to 458
of SEQ
ID NO: 2, amino acids 21 to 450 of SEQ ID NO: 4, amino acids 22 to 457 of SEQ
ID NO: 6, or
10 amino acids 21 to 461 of SEQ ID NO: 8, or an allelic variant thereof; or
is a fragment thereof
having cellobiohydrolase activity.
In another embodiment, the present invention also relates to catalytic domains
encoded
by polynucleotides that hybridize under medium stringency conditions, medium-
high stringency
conditions, high stringency conditions, or very high stringency conditions (as
defined above)
15 with (i) the nucleotides 52 to 1454 of SEQ ID NO: 1, the nucleotides 61
to 1733 of SEQ ID NO:
3, the nucleotides 64 to 1782 of SEQ ID NO: 5, or the nucleotides 52 to 1460
of SEQ ID NO: 7
(ii) the cDNA sequence thereof, or (iii) the full-length complement of (i) or
(ii) (Sambrook etal.,
1989, supra).
In another embodiment, the present invention also relates to catalytic domains
encoded
20 by polynucleotides having a sequence identity to nucleotides 52 to 1454
of SEQ ID NO: 1 or the
cDNA sequence thereof of at least 70%, e.g., at least 72%, at least 75%, at
least 78%, at least
80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at
least 86%, at least
87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at
least 93%, at least
94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or
100%. In another
25 embodiment, the present invention also relates to catalytic domains
encoded by polynucleotides
having a sequence identity to nucleotides 61 to 1733 of SEQ ID NO: 3 or the
cDNA sequence
thereof of at least 88%, e.g., at least 90%, at least 91%, at least 92%, at
least 93%, at least
94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or
100%. In another
embodiment, the present invention also relates to catalytic domains encoded by
polynucleotides
having a sequence identity to nucleotides 64 to 1782 of SEQ ID NO: 5 or the
cDNA sequence
thereof of at least 66%, e.g., at least 68%, at least 70%, at least 75%, at
least 78%, at least
80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at
least 86%, at least
87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at
least 93%, at least
94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or
100%. In another

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WO 2013/071871 PCT/CN2012/084661
embodiment, the present invention also relates to catalytic domains encoded by
polynucleotides
having a sequence identity to nucleotides 52 to 1460 of SEQ ID NO: 7 or the
cDNA sequence
thereof of at least 81%, e.g., at least 82%, at least 83%, at least 84%, at
least 85%, at least
86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at
least 92%, at least
93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at
least 99%, or
100%.
The polynucleotide encoding the catalytic domain preferably comprises or
consists of
nucleotides 52 to 1454 of SEQ ID NO: 1, nucleotides 61 to 1733 of SEQ ID NO:
3, nucleotides
64 to 1782 of SEQ ID NO: 5 or nucleotides 52 to 1460 of SEQ ID NO: 7.
In another embodiment, the present invention also relates to catalytic domain
variants of
amino acids 18 to 458 of SEQ ID NO: 2, amino acids 21 to 450 of SEQ ID NO: 4,
amino acids
22 to 457 of SEQ ID NO: 6, or amino acids 21 to 461 of SEQ ID NO: 8,
comprising a
substitution, deletion, and/or insertion at one or more (e.g., several)
positions. In one aspect, the
number of amino acid substitutions, deletions and/or insertions introduced
into the sequence of
amino acids 18 to 458 of SEQ ID NO: 2, amino acids 21 to 450 of SEQ ID NO: 4,
amino acids
22 to 457 of SEQ ID NO: 6, or amino acids 21 to 461 of SEQ ID NO: 8 is up to
10, e.g., 1, 2, 3,
4, 5, 6, 8, 9, or 10.
Carbohydrate Binding domains
In one embodiment, the present invention also relates to carbohydrate binding
domains
having a sequence identity to amino acids 486 to 521 of SEQ ID NO: 8 of at
least 81%, e.g., at
least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least
87%, at least 88%, at
least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at least 95%, at
least 96%, at least 97%, at least 98%, at least 99%, or 100%. In one aspect,
the carbohydrate
binding domains comprise amino acid sequences that differ by up to 10 amino
acids, e.g., 1, 2,
3, 4, 5, 6, 7, 8, 9, or 10, from amino acids 486 to 521 of SEQ ID NO: 8.
The carbohydrate binding domain preferably comprises or consists of amino
acids 486
to 521 of SEQ ID NO: 8 or an allelic variant thereof; or is a fragment thereof
having
carbohydrate binding activity.
In another embodiment, the present invention also relates to carbohydrate
binding
domains encoded by polynucleotides that hybridize under medium stringency
conditions,
medium-high stringency conditions, high stringency conditions, or very high
stringency
conditions (as defined above) with (i) the nucleotides 1533 to 1640 of SEQ ID
NO: 7, (ii) the

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cDNA sequence thereof or (iii) the full-length complement of (i) or (ii)
(Sambrook et al., 1989,
supra).
In another embodiment, the present invention also relates to carbohydrate
binding
domains encoded by polynucleotides having a sequence identity to nucleotides
1533 to 1640 of
SEQ ID NO: 7 of at least 81%, e.g., at least 82%, at least 83%, at least 84%,
at least 85%, at
least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least
91%, at least 92%, at
least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least
98%, at least 99%, or
100%.
The polynucleotide encoding the carbohydrate binding domain preferably
comprises or
consists of nucleotides 1533 to 1640 of SEQ ID NO: 7.
In another embodiment, the present invention also relates to carbohydrate
binding
domain variants of amino acids 486 to 521 of SEQ ID NO: 8 comprising a
substitution, deletion,
and/or insertion at one or more (e.g., several) positions. In one aspect, the
number of amino
acid substitutions, deletions and/or insertions introduced into the sequence
of amino acids 486
to 521 of SEQ ID NO: 8 is up to 10, e.g., 1, 2, 3, 4, 5, 6, 8, 9, or 10.
A catalytic domain operably linked to the carbohydrate binding domain may be
from a
hydrolase, isomerase, ligase, lyase, oxidoreductase, or transferase, e.g., an
aminopeptidase,
amylase, carbohydrase, carboxypeptidase, catalase, cellobiohydrolase,
cellulase, chitinase,
cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, endoglucanase,
esterase, alpha-
galactosidase, beta-galactosidase, glucoamylase, alpha-glucosidase, beta-
glucosidase,
invertase, laccase, lipase, mannosidase, mutanase, oxidase, pectinolytic
enzyme, peroxidase,
phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease,
transglutaminase, xylanase, or
beta-xylosidase. The polynucleotide encoding the catalytic domain may be
obtained from any
prokaryotic, eukaryotic, or other source.
Polynucleotides
The present invention also relates to isolated polynucleotides encoding a
polypeptide, a
catalytic domain, or carbohydrate binding domain of the present invention, as
described herein.
The techniques used to isolate or clone a polynucleotide are known in the art
and
include isolation from genomic DNA or cDNA, or a combination thereof. The
cloning of the
polynucleotides from genomic DNA can be effected, e.g., by using the well
known polymerase
chain reaction (PCR) or antibody screening of expression libraries to detect
cloned DNA
fragments with shared structural features. See, e.g., Innis et al., 1990, PCR:
A Guide to
Methods and Application, Academic Press, New York. Other nucleic acid
amplification

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WO 2013/071871 PCT/CN2012/084661
procedures such as ligase chain reaction (LCR), ligation activated
transcription (LAT) and
polynucleotide-based amplification (NASBA) may be used. The polynucleotides
may be cloned
from a strain of Malbranchea or Corynascus, or a related organism and thus,
for example, may
be an allelic or species variant of the polypeptide encoding region of the
polynucleotide.
Modification of a polynucleotide encoding a polypeptide of the present
invention may be
necessary for synthesizing polypeptides substantially similar to the
polypeptide. The term
"substantially similar" to the polypeptide refers to non-naturally occurring
forms of the
polypeptide. These polypeptides may differ in some engineered way from the
polypeptide
isolated from its native source, e.g., variants that differ in specific
activity, thermostability, pH
optimum, or the like. The variants may be constructed on the basis of the
polynucleotide
presented as the mature polypeptide coding sequence of SEQ ID NO: 1, the
mature polypeptide
coding sequence of SEQ ID NO: 3, the mature polypeptide coding sequence of SEQ
ID NO: 5
or the mature polypeptide coding sequence of SEQ ID NO: 7 or the cDNA sequence
thereof, or
a subsequence thereof, by introduction of nucleotide substitutions that do not
result in a change
in the amino acid sequence of the polypeptide, but which correspond to the
codon usage of the
host organism intended for production of the enzyme, or by introduction of
nucleotide
substitutions that may give rise to a different amino acid sequence. For a
general description of
nucleotide substitution, see, e.g., Ford et al., 1991, Protein Expression and
Purification 2: 95-
107.
Nucleic Acid Constructs
The present invention also relates to nucleic acid constructs comprising a
polynucleotide
of the present invention operably linked to one or more (e.g., several)
control sequences that
direct the expression of the coding sequence in a suitable host cell under
conditions compatible
with the control sequences.
The polynucleotide may be manipulated in a variety of ways to provide for
expression of
the polypeptide. Manipulation of the polynucleotide prior to its insertion
into a vector may be
desirable or necessary depending on the expression vector. The techniques for
modifying
polynucleotides utilizing recombinant DNA methods are well known in the art.
The control sequence may be a promoter, a polynucleotide that is recognized by
a host
cell for expression of a polynucleotide encoding a polypeptide of the present
invention. The
promoter contains transcriptional control sequences that mediate the
expression of the
polypeptide. The promoter may be any polynucleotide that shows transcriptional
activity in the
host cell including mutant, truncated, and hybrid promoters, and may be
obtained from genes

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WO 2013/071871 PCT/CN2012/084661
encoding extracellular or intracellular polypeptides either homologous or
heterologous to the
host cell.
Examples of suitable promoters for directing transcription of the nucleic acid
constructs
of the present invention in a bacterial host cell are the promoters obtained
from the Bacillus
amyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformis alpha-
amylase gene
(amyL), Bacillus licheniformis penicillinase gene (penP), Bacillus
stearothermophilus maltogenic
amylase gene (amyM), Bacillus subtilis levansucrase gene (sacB), Bacillus
subtilis xylA and
xylB genes, Bacillus thutingiensis cryllIA gene (Agaisse and Lereclus, 1994,
Molecular
Microbiology 13: 97-107), E. coil iac operon, E. coli trc promoter (Egon et
al., 1988, Gene 69:
301-315), Streptomyces coelicolor agarase gene (dagA), and prokaryotic beta-
lactamase gene
(Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. USA 75: 3727-3731), as
well as the tac
promoter (DeBoer et al., 1983, Proc. Natl. Acad. ScL USA 80: 21-25). Further
promoters are
described in "Useful proteins from recombinant bacteria" in Gilbert et al.,
1980, Scientific
American 242: 74-94; and in Sambrook etal., 1989, supra. Examples of tandem
promoters are
disclosed in WO 99/43835.
Examples of suitable promoters for directing transcription of the nucleic acid
constructs
of the present invention in a filamentous fungal host cell are promoters
obtained from the genes
for Aspergillus nidulans acetamidase, Aspergillus niger neutral alpha-amylase,
Aspergillus niger
acid stable alpha-amylase, Aspergillus niger or Aspergillus awamori
glucoamylase (glaA),
Aspergillus oryzae TAKA amylase, Aspergillus oryzae alkaline protease,
Aspergillus oryzae
triose phosphate isomerase, Fusarium oxysporum trypsin-like protease (WO
96/00787),
Fusarium venenatum amyloglucosidase (WO 00/56900), Fusarium venenatum Dada (WO
00/56900), Fusarium venenatum Quinn (WO 00/56900), Rhizomucor miehei lipase,
Rhizomucor
miehei aspartic proteinase, Trichoderma reesei beta-glucosidase, Trichoderma
reesei
cellobiohydrolase I, Trichoderma reesei cellobiohydrolase II, Trichoderma
reesei endoglucanase
I, Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanase III,
Trichoderma
reesei endoglucanase V, Trichoderma reesei xylanase I, Trichoderma reesei
xylanase II,
Trichoderma reesei xylanase Ill, Trichoderma reesei beta-xylosidase, and
Trichoderma reesei
translation elongation factor, as well as the NA2-tpi promoter (a modified
promoter from an
Aspergillus neutral alpha-amylase gene in which the untranslated leader has
been replaced by
an untranslated leader from an Aspergillus triose phosphate isomerase gene;
non-limiting
examples include modified promoters from an Aspergillus niger neutral alpha-
amylase gene in
which the untranslated leader has been replaced by an untranslated leader from
an Aspergillus

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nidulans or Aspergillus otyzae triose phosphate isomerase gene); and mutant,
truncated, and
hybrid promoters thereof. Other promoters are described in U.S. Patent No.
6,011,147.
In a yeast host, useful promoters are obtained from the genes for
Saccharomyces
cerevisiae enolase (ENO-1), Saccharomyces cerevisiae galactokinase (GAL1),
Saccharomyces
5 cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase
(ADH1,
ADH2/GAP), Saccharomyces cerevisiae triose phosphate isomerase (TPI),
Saccharomyces
cerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae 3-
phosphoglycerate kinase.
Other useful promoters for yeast host cells are described by Romanos et at,
1992, Yeast 8:
423-488.
10 The control sequence may also be a transcription terminator, which
is recognized by a
host cell to terminate transcription. The terminator is operably linked to the
3'-terminus of the
polynucleotide encoding the polypeptide. Any terminator that is functional in
the host cell may
be used in the present invention.
Preferred terminators for bacterial host cells are obtained from the genes for
Bacillus
15 clausii alkaline protease (aprH), Bacillus licheniforrnis alpha-
amylase (amyL), and Escherichia
coli ribosomal RNA (rmB).
Preferred terminators for filamentous fungal host cells are obtained from the
genes for
Aspergillus nidulans acetamidase, Aspergillus nidulans anthranilate synthase,
Aspergillus niger
glucoamylase, Aspergillus niger alpha-glucosidase, Aspergillus oryzae TAKA
amylase,
20
Fusarium oxysporum trypsin-like protease, Trichoderma reesei beta-glucosidase,
Trichoderma
reesei cellobiohydrolase I, Trichoderma reesei cellobiohydrolase II,
Trichoderma reesei
endoglucanase I, Trichoderma reesei endoglucanase II, Trichoderma reesei
endoglucanase III,
Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I, Trichoderma
reesei
xylanase II, Trichoderma reesei xylanase III, Trichoderma reesei beta-
xylosidase, and
25 Trichoderma reesei translation elongation factor.
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.
30 The control sequence may also be an mRNA stabilizer region
downstream of a promoter
and upstream of the coding sequence of a gene which increases expression of
the gene.
Examples of suitable mRNA stabilizer regions are obtained from a Bacillus
thuringiensis
cry//IA gene (WO 94/25612) and a Bacillus subtilis SP82 gene (Hue et at, 1995,
Journal of
Bacteriology 177: 3465-3471).

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The control sequence may also be a leader, a nontranslated region of an mRNA
that is
important for translation by the host cell. The leader is operably linked to
the 5'-terminus of the
polynucleotide encoding the polypeptide. Any leader that is functional in the
host cell may be
used.
Preferred leaders for filamentous fungal host cells are obtained from the
genes for
Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate
isomerase.
Suitable leaders for yeast host cells are obtained from the genes for
Saccharomyces
cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate
kinase,
Saccharomyces cerevisiae alpha-factor, and Saccharomyces cerevisiae alcohol
dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).
The control sequence may also be a polyadenylation sequence, a sequence
operably
linked to the 3'-terminus of the polynucleotide and, when transcribed, is
recognized by the host
cell as a signal to add polyadenosine residues to transcribed mRNA. Any
polyadenylation
sequence that is functional in the host cell may be used.
Preferred polyadenylation sequences for filamentous fungal host cells are
obtained from
the genes for Aspergfflus nidulans anthranilate synthase, Aspergillus niger
glucoamylase,
Aspergillus niger alpha-glucosidase Aspergillus oryzae TAKA amylase, and
Fusarium
oxysporum trypsin-like protease.
Useful polyadenylation sequences for yeast host cells are described by Guo and
Sherman, 1995, Mol. Cellular Biol. 15: 5983-5990.
The control sequence may also be a signal peptide coding region that encodes a
signal
peptide linked to the N-terminus of a polypeptide and directs the polypeptide
into the cell's
secretory pathway. The 5'-end of the coding sequence of the polynucleotide may
inherently
contain a signal peptide coding sequence naturally linked in translation
reading frame with the
segment of the coding sequence that encodes the polypeptide. Alternatively,
the 5'-end of the
coding sequence may contain a signal peptide coding sequence that is foreign
to the coding
sequence. A foreign signal peptide coding sequence may be required where the
coding
sequence does not naturally contain a signal peptide coding sequence.
Alternatively, a foreign
signal peptide coding sequence may simply replace the natural signal peptide
coding sequence
in order to enhance secretion of the polypeptide. However, any signal peptide
coding sequence
that directs the expressed polypeptide into the secretory pathway of a host
cell may be used.
Effective signal peptide coding sequences for bacterial host cells are the
signal peptide
coding sequences obtained from the genes for Bacillus NCIB 11837 maltogenic
amylase,
Bacillus licheniformis subtilisin, Bacillus
lichen iformis beta-lactamase, Bacillus

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stearothermophilus alpha-amylase, Bacillus stearothermophilus neutral
proteases (nprT, nprS,
nprM), and Bacillus subtilis prsA. Further signal peptides are described by
Simonen and PaIva,
1993, Microbiological Reviews 57: 109-137.
Effective signal peptide coding sequences for filamentous fungal host cells
are the signal
peptide coding sequences obtained from the genes for Aspergillus niger neutral
amylase,
Aspergillus niger glucoamylase, Aspergillus otyzae TAKA amylase, Humicola
insolens cellulase,
Humicola insolens endoglucanase V, Humicola lanuginosa lipase, and Rhizomucor
miehei
aspartic proteinase.
Useful signal peptides for yeast host cells are obtained from the genes for
Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiae invertase.
Other useful
signal peptide coding sequences are described by Romanos et al., 1992, supra.
The control sequence may also be a propeptide coding sequence that encodes a
propeptide positioned at the N-terminus of a polypeptide. The resultant
polypeptide is known as
a proenzyme or propolypeptide (or a zymogen in some cases). A propolypeptide
is generally
inactive and can be converted to an active polypeptide by catalytic or
autocatalytic cleavage of
the propeptide from the propolypeptide. The propeptide coding sequence may be
obtained from
the genes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilis
neutral protease (nprT),
Myceliophthora thermophila laccase (WO 95/33836), Rhizomucor miehei aspartic
proteinase,
and Saccharomyces cerevisiae alpha-factor.
Where both signal peptide and propeptide sequences are present, the propeptide
sequence is positioned next to the N-terminus of a polypeptide and the signal
peptide sequence
is positioned next to the N-terminus of the propeptide sequence.
It may also be desirable to add regulatory sequences that regulate expression
of the
polypeptide relative to the growth of the host cell. Examples of regulatory
sequences are those
that cause expression of the gene to be turned on or off in response to a
chemical or physical
stimulus, including the presence of a regulatory compound. Regulatory
sequences in
prokaryotic systems include the lac, tac, and ttp operator systems. In yeast,
the ADH2 system
or GAL1 system may be used. In filamentous fungi, the Aspergillus niger
glucoamylase
promoter, Aspergillus oryzae TAKA alpha-amylase promoter, and Aspergillus
otyzae
glucoamylase promoter, Trichoderma reesei cellobiohydrolase I promoter, and
Ttichoderma
reesei cellobiohydrolase ll promoter may be used. Other examples of regulatory
sequences are
those that allow for gene amplification. In eukaryotic systems, these
regulatory sequences
include the dihydrofolate reductase gene that is amplified in the presence of
methotrexate, and

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the metallothionein genes that are amplified with heavy metals. In these
cases, the
polynucleotide encoding the polypeptide would be operably linked to the
regulatory sequence.
Expression Vectors
The present invention also relates to recombinant expression vectors
comprising a
polynucleotide of the present invention, a promoter, and transcriptional and
translational stop
signals. The various nucleotide and control sequences may be joined together
to produce a
recombinant expression vector that may include one or more (e.g., several)
convenient
restriction sites to allow for insertion or substitution of the polynucleotide
encoding the
polypeptide at such sites. Alternatively, the polynucleotide may be expressed
by inserting the
polynucleotide or a nucleic acid construct comprising the polynucleotide into
an appropriate
vector for expression. In creating the expression vector, the coding sequence
is located in the
vector so that the coding sequence is operably linked with the appropriate
control sequences for
expression.
The recombinant expression vector may be any vector (e.g., a plasmid or virus)
that can
be conveniently subjected to recombinant DNA procedures and can bring about
expression of
the polynucleotide. The choice of the vector will typically depend on the
compatibility of the
vector with the host cell into which the vector is to be introduced. The
vector may be a linear or
closed circular plasmid.
The vector may be an autonomously replicating vector, i.e., a vector that
exists as an
extrachromosomal entity, the replication of which is independent of
chromosomal replication,
e.g., a plasmid, an extrachromosomal element, a minichromosome, or an
artificial chromosome.
The vector may contain any means for assuring self-replication. Alternatively,
the vector may be
one that, when introduced into the host cell, is integrated into the genome
and replicated
together with the chromosome(s) into which it has been integrated.
Furthermore, a single vector
or plasmid or two or more vectors or plasnnids that together contain the total
DNA to be
introduced into the genome of the host cell, or a transposon, may be used.
The vector preferably contains one or more (e.g., several) selectable markers
that permit
easy selection of transformed, transfected, transduced, or the like cells. A
selectable marker is a
gene the product of which provides for biocide or viral resistance, resistance
to heavy metals,
prototrophy to auxotrophs, and the like.
Examples of bacterial selectable markers are Bacillus licheniformis or
Bacillus subtilis
dal genes, or markers that confer antibiotic resistance such as ampicillin,
chloramphenicol,
kanamycin, neomycin, spectinomycin, or tetracycline resistance. Suitable
markers for yeast host

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cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and
URA3.
Selectable markers for use in a filamentous fungal host cell include, but are
not limited to, adeA
(phosphoribosylaminoimidazole-succinocarboxamide synthase), adeB
(phosphoribosyl-
aminoimidazole synthase), amdS (acetamidase), argB (omithine
carbamoyltransferase), bar
(phosphinothricin acetyltransferase), hph (hygromycin phosphotransferase),
niaD (nitrate
reductase), pyrG (orotidine-5'-phosphate decarboxylase), sC (sulfate
adenyltransferase), and
trpC (anthranilate synthase), as well as equivalents thereof. Preferred for
use in an Aspergillus
cell are Aspergillus nidulans or Aspergillus oryzae amdS and pyrG genes and a
Streptomyces
hygroscopicus bar gene. Preferred for use in a Trichoderma cell are adeA,
adeB, amdS, hph,
and pyrG genes.
The selectable marker may be a dual selectable marker system as described in
WO
2010/039889. In one aspect, the dual selectable marker is a hph-tk dual
selectable marker
system.
The vector preferably contains an element(s) that permits integration of the
vector into
the host cell's genome or autonomous replication of the vector in the cell
independent of the
genome.
For integration into the host cell genome, the vector may rely on the
polynucleotide's
sequence encoding the polypeptide or any other element of the vector for
integration into the
genome by homologous or non-homologous recombination. Alternatively, the
vector may
contain additional polynucleotides for directing integration by homologous
recombination into
the genome of the host cell at a precise location(s) in the chromosome(s). To
increase the
likelihood of integration at a precise location, the integrational elements
should contain a
sufficient number of nucleic acids, such as 100 to 10,000 base pairs, 400 to
10,000 base pairs,
and 800 to 10,000 base pairs, which have a high degree of sequence identity to
the
corresponding target sequence to enhance the probability of homologous
recombination. The
integrational elements may be any sequence that is homologous with the target
sequence in the
genome of the host cell. Furthermore, the integrational elements may be non-
encoding or
encoding polynucleotides. On the other hand, the vector may be integrated into
the genome of
the host cell by non-homologous recombination.
For autonomous replication, the vector may further comprise an origin of
replication
enabling the vector to replicate autonomously in the host cell in question.
The origin of
replication may be any plasmid replicator mediating autonomous replication
that functions in a
cell. The term "origin of replication" or "plasmid replicator" means a
polynucleotide that enables
a plasmid or vector to replicate in vivo.

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Examples of bacterial origins of replication are the origins of replication of
plasmids
pBR322, pUC19, pACYC177, and pACYC184 permitting replication in E. coli, and
pUB110,
pE194, pTA1060, and pAM111 permitting replication in Bacillus.
Examples of origins of replication for use in a yeast host cell are the 2
micron origin of
5 replication, ARS1, ARS4, the combination of ARS1 and CEN3, and the
combination of ARS4
and CEN6.
Examples of origins of replication useful in a filamentous fungal cell are
AMA1 and ANSI
(Gems et al., 1991, Gene 98: 61-67; Cullen et aL, 1987, Nucleic Acids Res. 15:
9163-9175;
WO 00/24883). Isolation of the AMA1 gene and construction of plasmids or
vectors comprising
10 the gene can be accomplished according to the methods disclosed in WO
00/24883.
More than one copy of a polynucleotide of the present invention may be
inserted into a
host cell to increase production of a polypeptide. An increase in the copy
number of the
polynucleotide can be obtained by integrating at least one additional copy of
the sequence into
the host cell genome or by including an amplifiable selectable marker gene
with the
15 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.
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
20 (see, e.g., Sambrook et al, 1989, supra).
Host Cells
The present invention also relates to recombinant host cells, comprising a
polynucleotide
of the present invention operably linked to one or more (e.g., several)
control sequences that
25 direct the production of a polypeptide of the present invention. A
construct or vector comprising
a polynucleotide is introduced into a host cell so that the construct or
vector is maintained as a
chromosomal integrant or as a self-replicating extra-chromosomal vector as
described earlier.
The term "host cell" encompasses any progeny of a parent cell that is not
identical to the parent
cell due to mutations that occur during replication. The choice of a host cell
will to a large extent
30 depend upon the gene encoding the polypeptide and its source.
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.
The prokaryotic host cell may be any Gram-positive or Gram-negative bacterium.
Gram-
positive bacteria include, but are not limited to, Bacillus, Clostridium,
Enterococcus, Geobacillus,

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WO 2013/071871 PCT/CN2012/084661
Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, and
Streptomyces. Gram-negative bacteria include, but are not limited to,
Campylobacter, E coil,
Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter, Neissefia,
Pseudomonas, Salmonella,
and Ureaplasma.
The bacterial host cell may be any Bacillus cell including, but not limited
to, Bacillus
alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans,
Bacillus clausfi,
Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus,
Bacillus licheniformis,
Bacillus megatetium, Bacillus pumilus, Bacillus stearothetmophilus, Bacillus
subtilis, and
Bacillus thufingiensis cells.
The bacterial host cell may also be any Streptococcus cell including, but not
limited to,
Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, and
Streptococcus
equi subsp. Zooepidemicus cells.
The bacterial host cell may also be any Streptomyces cell including, but not
limited to,
Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor,
Streptomyces
griseus, and Streptomyces lividans cells.
The introduction of DNA into a Bacillus cell may be effected by protoplast
transformation
(see, e.g., Chang and Cohen, 1979, MoL Gen. Genet. 168: 111-115), competent
cell
transformation (see, e.g., Young and Spizizen, 1961, J. BacterioL 81: 823-829,
or Dubnau and
Davidoff-Abelson, 1971, J. MoL Biol. 56: 209-221), electroporation (see, e.g.,
Shigekawa and
Dower, 1988, Biotechniques 6: 742-751), or conjugation (see, e.g., Koehler and
Thome, 1987,
J. BacterioL 169: 5271-5278). The introduction of DNA into an E. coli cell may
be effected by
protoplast transformation (see, e.g., Hanahan, 1983, J. Mol. Biol. 166: 557-
580) or
electroporation (see, e.g., Dower et al., 1988, Nucleic Acids Res. 16: 6127-
6145). The
introduction of DNA into a Streptomyces cell may be effected by protoplast
transformation,
electroporation (see, e.g., Gong et al., 2004, Folia MicrobioL (Praha) 49: 399-
405), conjugation
(see, e.g., Mazodier et aL, 1989, J. Bacteriol. 171: 3583-3585), or
transduction (see, e.g., Burke
et al., 2001, Proc. NatL Acad. Sci. USA 98: 6289-6294). The introduction of
DNA into a
Pseudomonas cell may be effected by electroporation (see, e.g., Choi et aL,
2006, J. Microbiol
Methods 64: 391-397) or conjugation (see, e.g., Pinedo and Smets, 2005, AppL
Environ.
MicrobioL 71: 51-57). The introduction of DNA into a Streptococcus cell may be
effected by
natural competence (see, e.g., Perry and Kuramitsu, 1981, Infect. Immun. 32:
1295-1297),
protoplast transformation (see, e.g., Catt and Jollick, 1991, Microbios 68:
189-207),
electroporation (see, e.g., Buckley et al., 1999, Appl. Environ. MicrobioL 65:
3800-3804), or

CA 02856083 2014-05-15
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WO 2013/071871 PCT/CN2012/084661
conjugation (see, e.g., Clewell, 1981, Microbiol Rev. 45: 409-436). However,
any method
known in the art for introducing DNA into a host cell can be used.
The host cell may also be a eukaryote, such as a mammalian, insect, plant, or
fungal
cell.
The host cell may be a fungal cell. "Fungi" as used herein includes the phyla
Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota as well as the
Oomycota and
all mitosporic fungi (as defined by Hawksworth et al., In, Ainsworth and
Bisby's Dictionary of
The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge,
UK).
The fungal host cell may be a yeast cell. "Yeast" as used herein includes
ascosporogenous yeast (Endomycetales), basidiosporogenous yeast, and yeast
belonging to
the Fungi Imperfect' (Blastomycetes). Since the classification of yeast may
change in the future,
for the purposes of this invention, yeast shall be defined as described in
Biology and Activities of
Yeast (Skinner, Passmore, and Davenport, editors, Soc. App. Bacteriot
Symposium Series No.
9, 1980).
The yeast host cell may be a Candida, Hansenula, Kluyveromyces, Pichia,
Saccharomyces, Schizosaccharomyces, or Yarrowia cell, such as a Kluyveromyces
lactis,
Saccharomyces carisbergensis, Saccharomyces cerevisiae, Saccharomyces
diastaticus,
Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis,
Saccharomyces oviformis, or Yarrowia lipolytica cell.
The fungal host cell may be a filamentous fungal cell. "Filamentous fungi"
include all
filamentous forms of the subdivision Eumycota and Oomycota (as defined by
Hawksworth etal.,
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 cereyisiae is by budding of
a unicellular
thallus and carbon catabolism may be fermentative.
The filamentous fungal host cell may be an Acremonium, Aspergillus,
Aureobasidium,
Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus,
Filibasidium,
Fusatium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix,
Neurospora,
Paecilomyces, Peniciffium, Phanerochaete, Phlebia, Piromyces, Pleurotus,
Schizophyllum,
Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or Trichoderma
cell.
For example, the filamentous fungal host cell may be an Aspergillus awamoti,
Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus,
Aspergillus nidulans,
Aspergillus niger, Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsis
aneitina, Ceriporiopsis

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WO 2013/071871 PCT/CN2012/084661
carvgiea, Cetipotiopsis gilvescens, Cefiporiopsis pannocinta, Cefipotiopsis
fivulosa,
Ceripotiopsis subrufa, Ceripotiopsis subvermispora, Chtysosporium Mops,
Chtysosporium
keratinophilum, Chrysospofium lucknowense, Chrysospofium mercladum,
Chtysosporium
pannicola, Chrysosporium queenslandicum, Chrysospofium tropicum, Chrysosporium
zonatum,
Coptinus cinereus, Cofiolus hirsutus, Fusarium bactfidioides, Fusarium
cerealis, Fusarium
crookwellense, Fusatium culmorum, Fusarium graminearum, Fusarium graminum,
Fusarium
heterosporum, Fusarium negundi, Fusarium oxysporirm, Fusarium reticulatum,
Fusarium
roseum, Fusarium sambucinum, Fusarium satrochroum, Fusarium sporotfichioides,
Fusarium
sulphureum, Fusarium torulosum, Fusarium ttichothecioides, Fusafium venenatum,
Humicola
insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila,
Neurospora crassa,
Penicililurn putpurogenum, Phanerochaete chtysospofium, Phlebia radiata,
Pleurotus etyngii,
Thiela via terrestfis, Tra metes villosa, Trametes versicolor, Ttichoderma
harzianum,
Trichoderma koningii, Tfichoderma longibrachiatum, Trichoderma reesei, or
Trichoderma viride
cell.
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 Ttichoderma host
cells are described
in EP 238023, YeIton etal., 1984, Proc. Natl. Acad. Sci. USA 81: 1470-1474,
and Christensen
etal., 1988, Bio/Technology 6: 1419-1422. Suitable methods for transforming
Fusarium species
are described by Malardier et at, 1989, Gene 78: 147-156, and WO 96/00787.
Yeast may be
transformed using the procedures described by Becker and Guarente, In Abelson,
J.N. and
Simon, M. I., editors, Guide to Yeast Genetics and Molecular Biology, Methods
in Enzymology,
Volume 194, pp 182-187, Academic Press, Inc., New York; Ito et al., 1983, J.
Bactetiot 153:
163; and Hinnen et at, 1978, Proc. NatL Acad. ScL USA 75: 1920.
Methods of Production
The present invention also relates to methods of producing a polypeptide of
the present
invention, comprising (a) cultivating a cell, which in its wild-type form
produces the polypeptide,
under conditions conducive for production of the polypeptide; and optionally
(b) recovering the
polypeptide. In one aspect, the cell is a Malbranchea cell In another aspect,
the cell is a
Malbranchea cinnamomea cell. In another aspect, the cell is a Malbranchea
cinnamomea
NN044758 cell. In one aspect, the cell is a Corynascus cell In another aspect,
the cell is a
Cotynascus thermophilus cell. In another aspect, the cell is a Corynascus
thermophilus
NN000308 cell.

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WO 2013/071871 PCT/CN2012/084661
The present invention also relates to methods of producing a polypeptide of
the present
invention, comprising (a) cultivating a recombinant host cell of the present
invention under
conditions conducive for production of the polypeptide; and optionally (b)
recovering the
polypeptide.
The host cells are cultivated in a nutrient medium suitable for production of
the
polypeptide using methods known in the art. For example, the cells may be
cultivated by shake
flask cultivation, or small-scale or large-scale fermentation (including
continuous, batch, fed-
batch, or solid state fermentations) in laboratory or industrial fermentors in
a suitable medium
and under conditions allowing the polypeptide to be expressed and/or isolated.
The cultivation
takes place in a suitable nutrient medium comprising carbon and nitrogen
sources and inorganic
salts, using procedures known in the art. Suitable media are available from
commercial
suppliers or may be prepared according to published compositions (e.g., in
catalogues of the
American Type Culture Collection). If the polypeptide is secreted into the
nutrient medium, the
polypeptide can be recovered directly from the medium. If the polypeptide is
not secreted, it can
be recovered from cell lysates.
The polypeptide may be detected using methods known in the art that are
specific for
the polypeptides. These detection methods include, but are not limited to, use
of specific
antibodies, formation of an enzyme product, or disappearance of an enzyme
substrate. For
example, an enzyme assay may be used to determine the activity of the
polypeptide.
The polypeptide may be recovered using methods known in the art. For example,
the
polypeptide may be recovered from the nutrient medium by conventional
procedures including,
but not limited to, collection, centrifugation, filtration, extraction, spray-
drying, evaporation, or
precipitation. In one aspect, a whole fermentation broth comprising the
polypeptide is recovered.
The polypeptide may be purified by a variety of procedures known in the art
including,
but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic,
chromatofocusing,
and size exclusion), electrophoretic procedures (e.g., preparative isoelectric
focusing),
differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, or
extraction (see, e.g.,
Protein Purification, Janson and Ryden, editors, VCH Publishers, New York,
1989) to obtain
substantially pure polypeptides.
In an alternative aspect, the polypeptide is not recovered, but rather a host
cell of the
present invention expressing the polypeptide is used as a source of the
polypeptide.

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Plants
The present invention also relates to isolated plants, e.g., a transgenic
plant, plant part,
or plant cell, comprising a polynucleotide of the present invention so as to
express and produce
a polypeptide or domain in recoverable quantities. The polypeptide or domain
may be recovered
5 from the
plant or plant part. Alternatively, the plant or plant part containing the
polypeptide or
domain may be used as such for improving the quality of a food or feed, e.g.,
improving
nutritional value, palatability, and rheological properties, or to destroy an
antinutritive factor.
The transgenic plant can be dicotyledonous (a dicot) or monocotyledonous (a
monocot).
Examples of monocot plants are grasses, such as meadow grass (blue grass,
Poa), forage
10 grass
such as Festuca, Lolium, temperate grass, such as Agrostis, and cereals, e.g.,
wheat,
oats, rye, barley, rice, sorghum, and maize (corn).
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.
15 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, meristenns. 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.
20 Likewise,
plant parts such as specific tissues and cells isolated to facilitate the
utilization of the
invention are also considered plant parts, e.g., embryos, endosperms, aleurone
and seed coats.
Also included within the scope of the present invention are the progeny of
such plants,
plant parts, and plant cells.
The transgenic plant or plant cell expressing the polypeptide or domain may be
25
constructed in accordance with methods known in the art. In short, the plant
or plant cell is
constructed by incorporating one or more expression constructs encoding the
polypeptide or
domain into the plant host genome or chloroplast genome and propagating the
resulting
modified plant or plant cell into a transgenic plant or plant cell.
The expression construct is conveniently a nucleic acid construct that
comprises a
30
polynucleotide encoding a polypeptide or domain operably linked with
appropriate regulatory
sequences required for expression of the polynucleotide in the plant or plant
part of choice.
Furthermore, the expression construct may comprise a selectable marker useful
for identifying
plant cells into which the expression construct has been integrated and DNA
sequences

CA 02856083 2014-05-15
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WO 2013/071871 PCT/CN2012/084661
necessary for introduction of the construct into the plant in question (the
latter depends on the
DNA introduction method to be used).
The choice of regulatory sequences, such as promoter and terminator sequences
and
optionally signal or transit sequences, is determined, for example, on the
basis of when, where,
and how the polypeptide or domain is desired to be expressed. For instance,
the expression of
the gene encoding a polypeptide or domain may be constitutive or inducible, or
may be
developmental, stage or tissue specific, and the gene product may be targeted
to a specific
tissue or plant part such as seeds or leaves. Regulatory sequences are, for
example, described
by Tague at aL, 1988, Plant Physiology 86: 506.
For constitutive expression, the 35S-CaMV, the maize ubiquitin 1, or the rice
actin 1
promoter may be used (Franck etal., 1980, Cell 21: 285-294; Christensen etal.,
1992, Plant
MoL Biol. 18: 675-689; Zhang et aL, 1991, Plant Cell 3: 1155-1165). Organ-
specific promoters
may be, for example, a promoter from storage sink tissues such as seeds,
potato tubers, and
fruits (Edwards and Coruzzi, 1990, Ann. Rev. Genet. 24: 275-303), or from
metabolic sink
tissues such as meristems (Ito at al., 1994, Plant MoL BioL 24: 863-878), a
seed specific
promoter such as the glutelin, prolamin, globulin, or albumin promoter from
rice (Wu etal., 1998,
Plant Cell PhysioL 39: 885-889), a Vicia faba promoter from the legumin 34 and
the unknown
seed protein gene from Viola faba (Conrad et aL, 1998, J. Plant PhysioL 152:
708-711), a
promoter from a seed oil body protein (Chen et al, 1998, Plant Cell PhysioL
39: 935-941), the
storage protein napA promoter from Brassica napus, or any other seed specific
promoter known
in the art, e.g., as described in WO 91/14772. Furthermore, the promoter may
be a leaf specific
promoter such as the rbcs promoter from rice or tomato (Kyozuka at al., 1993,
Plant Physiol
102: 991-1000), the chlorella virus adenine methyltransferase gene promoter
(Mitra and
Higgins, 1994, Plant MoL Biol. 26: 85-93), the aldP gene promoter from rice
(Kagaya et al.,
1995, MoL Gen. Genet. 248: 668-674), or a wound inducible promoter such as the
potato pin2
promoter (Xu etal., 1993, Plant MoL Biol. 22: 573-588). Likewise, the promoter
may be induced
by abiotic treatments such as temperature, drought, or alterations in salinity
or induced by
exogenously applied substances that activate the promoter, e.g., ethanol,
oestrogens, plant
hormones such as ethylene, abscisic acid, and gibberellic acid, and heavy
metals.
A promoter enhancer element may also be used to achieve higher expression of a
polypeptide or domain in the plant. For instance, the promoter enhancer
element may be an
intron that is placed between the promoter and the polynucleotide encoding a
polypeptide or
domain. For instance, Xu et aL, 1993, supra, disclose the use of the first
intron of the rice actin 1
gene to enhance expression.

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The selectable marker gene and any other parts of the expression construct may
be
chosen from those available in the art.
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,
Bioirechnology 8:
535; Shimamoto et aL, 1989, Nature 338: 274).
Agrobacterium tumefaciens-mediated gene transfer is a method for generating
transgenic dicots (for a review, see Hooykas and Schilperoort, 1992, Plant MoL
Biol. 19: 15-38)
and for transforming monocots, although other transformation methods may be
used for these
plants. A method for generating transgenic monocots is particle bombardment
(microscopic gold
or tungsten particles coated with the transforming DNA) of embryonic calli or
developing
embryos (Christou, 1992, Plant J. 2:275-281; Shimamoto, 1994, Curr. Opin.
BiotechnoL 5: 158-
162; Vasil etal., 1992, Bio/Technology 10: 667-674). An alternative method for
transformation of
monocots is based on protoplast transformation as described by Omirulleh et
al., 1993, Plant
MoL BioL 21: 415-428. Additional transformation methods include those
described in U.S.
Patent Nos. 6,395,966 and 7,151,204 (both of which are herein incorporated by
reference in
their entirety).
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.
In addition to direct transformation of a particular plant genotype with a
construct of the
present invention, transgenic plants may be made by crossing a plant having
the construct to a
second plant lacking the construct. For example, a construct encoding a
polypeptide or domain
can be introduced into a particular plant variety by crossing, without the
need for ever directly
transforming a plant of that given variety. Therefore, the present invention
encompasses not
only a plant directly regenerated from cells which have been transformed in
accordance with the
present invention, but also the progeny of such plants. As used herein,
progeny may refer to the
offspring of any generation of a parent plant prepared in accordance with the
present invention.
Such progeny may include a DNA construct prepared in accordance with the
present invention.
Crossing results in the introduction of a transgene into a plant line by cross
pollinating a starting

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line with a donor plant line. Non-limiting examples of such steps are
described in U.S. Patent
No. 7,151,204.
Plants may be generated through a process of backcross conversion. For
example,
plants include plants referred to as a backcross converted genotype, line,
inbred, or hybrid.
Genetic markers may be used to assist in the introgression of one or more
transgenes of
the invention from one genetic background into another. Marker assisted
selection offers
advantages relative to conventional breeding in that it can be used to avoid
errors caused by
phenotypic variations. Further, genetic markers may provide data regarding the
relative degree
of elite germplasm in the individual progeny of a particular cross. For
example, when a plant
with a desired trait which otherwise has a non-agronomically desirable genetic
background is
crossed to an elite parent, genetic markers may be used to select progeny
which not only
possess the trait of interest, but also have a relatively large proportion of
the desired
gerrnplasm. In this way, the number of generations required to introgress one
or more traits into
a particular genetic background is minimized.
The present invention also relates to methods of producing a polypeptide or
domain of
the present invention comprising (a) cultivating a transgenic plant or a plant
cell comprising a
polynucleotide or domain encoding the polypeptide or domain under conditions
conducive for
production of the polypeptide or domain; and optionally (b) recovering the
polypeptide or
domain.
Removal or Reduction of Cellobiohydrolase Activity
The present invention also relates to methods of producing a mutant of a
parent cell,
which comprises disrupting or deleting a polynucleotide, or a portion thereof,
encoding a
polypeptide of the present invention, which results in the mutant cell
producing less of the
polypeptide than the parent cell when cultivated under the same conditions.
The mutant cell may be constructed by reducing or eliminating expression of
the
polynucleotide using methods well known in the art, for example, insertions,
disruptions,
replacements, or deletions. In a preferred aspect, the polynucleotide is
inactivated. The
polynucleotide to be modified or inactivated may be, for example, the coding
region or a part
thereof essential for activity, or a regulatory element required for
expression of the coding
region. An example of such a regulatory or control sequence may be a promoter
sequence or a
functional part thereof, i.e., a part that is sufficient for affecting
expression of the polynucleotide.
Other control sequences for possible modification include, but are not limited
to, a leader,

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polyadenylation sequence, propeptide sequence, signal peptide sequence,
transcription
terminator, and transcriptional activator.
Modification or inactivation of the polynucleotide may be performed by
subjecting the
parent cell to mutagenesis and selecting for mutant cells in which expression
of the
polynucleotide has been reduced or eliminated. The mutagenesis, which may be
specific or
random, may be performed, for example, by use of a suitable physical or
chemical mutagenizing
agent, by use of a suitable oligonucleotide, or by subjecting the DNA sequence
to PCR
generated mutagenesis. Furthermore, the mutagenesis may be performed by use of
any
combination of these mutagenizing agents.
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), 0-methyl hydroxylamine, nitrous acid, ethyl methane sulphonate (EMS),
sodium
bisulphite, formic acid, and nucleotide analogues.
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.
Modification or inactivation of the polynucleotide may also be accomplished by
insertion,
substitution, or deletion of one or more nucleotides in the gene or a
regulatory element required
for transcription or translation thereof. For example, nucleotides may be
inserted or removed so
as to result in the introduction of a stop codon, the removal of the start
codon, or a change in the
open reading frame. Such modification or inactivation may be accomplished by
site-directed
mutagenesis or PCR generated mutagenesis in accordance with methods known in
the art.
Although, in principle, the modification may be performed in vivo, i.e.,
directly on the cell
expressing the polynucleotide to be modified, it is preferred that the
modification be performed
in vitro as exemplified below.
An example of a convenient way to eliminate or reduce expression of a
polynucleotide is
based on techniques of gene replacement, gene deletion, or gene disruption.
For example, in
the gene disruption method, a nucleic acid sequence corresponding to the
endogenous
polynucleotide is mutagenized in vitro to produce a defective nucleic acid
sequence that is then
transformed into the parent cell to produce a defective gene. By homologous
recombination, the
defective nucleic acid sequence replaces the endogenous polynucleotide. It may
be desirable
that the defective polynucleotide also encodes a marker that may be used for
selection of

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transformants in which the polynucleotide has been modified or destroyed. In
an aspect, the
polynucleotide is disrupted with a selectable marker such as those described
herein.
The present invention also relates to methods of inhibiting the expression of
a
polypeptide having cellobiohydrolase activity in a cell, comprising
administering to the cell or
5 expressing in the cell a double-stranded RNA (dsRNA) molecule, wherein
the dsRNA comprises
a subsequence of a polynucleotide of the present invention. In a preferred
aspect, the dsRNA is
about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more duplex nucleotides in
length.
The dsRNA is preferably a small interfering RNA (siRNA) or a micro RNA
(miRNA). In a
preferred aspect, the dsRNA is small interfering RNA for inhibiting
transcription. In another
10 preferred aspect, the dsRNA is micro RNA for inhibiting translation.
The present invention also relates to such double-stranded RNA (dsRNA)
molecules,
comprising a portion of the mature polypeptide coding sequence of SEQ ID NO:
1, the mature
polypeptide coding sequence of SEQ ID NO: 3, the mature polypeptide coding
sequence of
SEQ ID NO: 5 or the mature polypeptide coding sequence of SEQ ID NO: 7 for
inhibiting
15 expression of the polypeptide in a cell. While the present invention is
not limited by any
particular mechanism of action, the dsRNA can enter a cell and cause the
degradation of a
single-stranded RNA (ssRNA) of similar or identical sequences, including
endogenous mRNAs.
When a cell is exposed to dsRNA, mRNA from the homologous gene is selectively
degraded by
a process called RNA interference (RNAi).
20 The dsRNAs of the present invention can be used in gene-silencing. In
one aspect, the
invention provides methods to selectively degrade RNA using a dsRNAi of the
present
invention. The process may be practiced in vitro, ex vivo or in vivo. In one
aspect, the dsRNA
molecules can be used to generate a loss-of-function mutation in a cell, an
organ or an animal.
Methods for making and using dsRNA molecules to selectively degrade RNA are
well known in
25 the art; see, for example, U.S. Patent Nos. 6,489,127; 6,506,559;
6,511,824; and 6,515,109.
The present invention further relates to a mutant cell of a parent cell that
comprises a
disruption or deletion of a polynucleotide encoding the polypeptide or a
control sequence
thereof or a silenced gene encoding the polypeptide, which results in the
mutant cell producing
less of the polypeptide or no polypeptide compared to the parent cell.
30 The polypeptide-deficient mutant cells are particularly useful as host
cells for expression
of native and heterologous polypeptides. Therefore, the present invention
further relates to
methods of producing a native or heterologous polypeptide, comprising (a)
cultivating the
mutant cell under conditions conducive for production of the polypeptide; and
optionally (b)
recovering the polypeptide. The term "heterologous polypeptides" means
polypeptides that are

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not native to the host cell, e.g., a variant of a native protein. The host
cell may comprise more
than one copy of a polynucleotide encoding the native or heterologous
polypeptide.
The methods used for cultivation and purification of the product of interest
may be
performed by methods known in the art.
The methods of the present invention for producing an essentially
cellobiohydrolase
activity-free product are of particular interest in the production of
eukaryotic polypeptides, in
particular fungal proteins such as enzymes. The cellobiohydrolase activity-
deficient cells may
also be used to express heterologous proteins of pharmaceutical interest such
as hormones,
growth factors, receptors, and the like. The term "eukaryotic polypeptides"
includes not only
native polypeptides, but also those polypeptides, e.g., enzymes, which have
been modified by
amino acid substitutions, deletions or additions, or other such modifications
to enhance activity,
thermostability, pH tolerance and the like.
In a further aspect, the present invention relates to a protein product
essentially free
from cellobiohydrolase activity that is produced by a method of the present
invention.
Fermentation Broth Formulations or Cell Compositions
The present invention also relates to a fermentation broth formulation or a
cell
composition comprising a polypeptide of the present invention. The
fermentation broth product
further comprises additional ingredients used in the fermentation process,
such as, for example,
cells (including, the host cells containing the gene encoding the polypeptide
of the present
invention which are used to produce the polypeptide of interest), cell debris,
biomass,
fermentation media and/or fermentation products. In some embodiments, the
composition is a
cell-killed whole broth containing organic acid(s), killed cells and/or cell
debris, and culture
medium.
The term "fermentation broth" as used herein refers to a preparation produced
by cellular
fermentation that undergoes no or minimal recovery and/or purification. For
example,
fermentation broths are produced when microbial cultures are grown to
saturation, incubated
under carbon-limiting conditions to allow protein synthesis (e.g., expression
of enzymes by host
cells) and secretion into cell culture medium. The fermentation broth can
contain unfractionated
or fractionated contents of the fermentation materials derived at the end of
the fermentation.
Typically, the fermentation broth is unfractionated and comprises the spent
culture medium and
cell debris present after the microbial cells (e.g., filamentous fungal cells)
are removed, e.g., by
centrifugation. In some embodiments, the fermentation broth contains spent
cell culture
medium, extracellular enzymes, and viable and/or nonviable microbial cells.

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In an embodiment, the fermentation broth formulation and cell compositions
comprise a
first organic acid component comprising at least one 1-5 carbon organic acid
and/or a salt
thereof and a second organic acid component comprising at least one 6 or more
carbon organic
acid and/or a salt thereof. In a specific embodiment, the first organic acid
component is acetic
acid, formic acid, propionic acid, a salt thereof, or a mixture of two or more
of the foregoing and
the second organic acid component is benzoic acid, cyclohexanecarboxylic acid,
4-methylvaleric acid, phenylacetic acid, a salt thereof, or a mixture of two
or more of the
foregoing.
In one aspect, the composition contains an organic acid(s), and optionally
further
contains killed cells and/or cell debris. In one embodiment, the killed cells
and/or cell debris are
removed from a cell-killed whole broth to provide a composition that is free
of these
components.
The fermentation broth formulations or cell compositions may further comprise
a
preservative and/or anti-microbial (e.g., bacteriostatic) agent, including,
but not limited to,
sorbitol, sodium chloride, potassium sorbate, and others known in the art.
The fermentation broth formulations or cell compositions may further comprise
multiple
enzymatic activities, such as one or more (e.g., several) enzymes selected
from the group
consisting of a cellulase, a GH61 polypeptide having cellulolytic enhancing
activity, a
hemicellulase, an esterase, an expansin, a laccase, a ligninolytic enzyme, a
pectinase, a
peroxidase, a protease, and a swollenin. The fermentation broth formulations
or cell
compositions may also comprise one or more (e.g., several) enzymes selected
from the group
consisting of a hydrolase, an isomerase, a ligase, a lyase, an oxidoreductase,
or a transferase,
e.g., an alpha-galactosidase, alpha-glucosidase, aminopeptidase, amylase, beta-
galactosidase,
beta-glucosidase, beta-xylosidase, carbohydrase,
carboxypeptidase, catalase,
cellobiohydrolase, cellulase, chitinase, cutinase, cyclodextrin
glycosyltransferase,
deoxyribonuclease, endoglucanase, esterase, glucoamylase, invertase, laccase,
lipase,
mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase, phytase,
polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase, or
xylanase.
The cell-killed whole broth or composition may contain the unfractionated
contents of the
fermentation materials derived at the end of the fermentation. Typically, the
cell-killed whole
broth or composition contains the spent culture medium and cell debris present
after the
microbial cells (e.g., filamentous fungal cells) are grown to saturation,
incubated under carbon-
limiting conditions to allow protein synthesis (e.g., expression of cellulase
and/or glucosidase
enzyme(s)). In some embodiments, the cell-killed whole broth or composition
contains the spent

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cell culture medium, extracellular enzymes, and killed filamentous fungal
cells. In some
embodiments, the microbial cells present in the cell-killed whole broth or
composition can be
permeabilized and/or lysed using methods known in the art.
A whole broth or cell composition as described herein is typically a liquid,
but may
contain insoluble components, such as killed cells, cell debris, culture media
components,
and/or insoluble enzyme(s). In some embodiments, insoluble components may be
removed to
provide a clarified liquid composition.
The whole broth formulations and cell compositions of the present invention
may be
produced by a method described in \NO 90/15861 or WO 2010/096673.
Examples are given below of preferred uses of the compositions of the present
invention. The dosage of the composition and other conditions under which the
composition is
used may be determined on the basis of methods known in the art.
Enzyme Compositions
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 cellobiohydrolase activity of the composition
has been increased,
e.g., with an enrichment factor of at least 1.1.
The compositions may comprise a polypeptide of the present invention as the
major
enzymatic component, e.g., a mono-component composition. Alternatively, the
compositions
may comprise multiple enzymatic activities, such as one or more (e.g.,
several) enzymes
selected from the group consisting of a cellulase, a GH61 polypeptide having
cellulolytic
enhancing activity, a hemicellulase, an esterase, an expansin, a laccase, a
ligninolytic enzyme,
a pectinase, a peroxidase, a protease, and a swollenin. The compositions may
also comprise
one or more (e.g., several) enzymes selected from the group consisting of a
hydrolase, an
isomerase, a ligase, a lyase, an oxidoreductase, or a transferase, e.g., an
alpha-galactosidase,
alpha-glucosidase, aminopeptidase, amylase, beta-galactosidase, beta-
glucosidase, beta-
xylosidase, carbohydrase, carboxpeptidase, catalase, cellobiohydrolase,
cellulase, chitinase,
cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, endoglucanase,
esterase,
glucoamylase, invertase, laccase, lipase, mannosidase, mutanase, oxidase,
pectinolytic
enzyme, peroxidase, phytase, polyphenoloxidase, proteolytic enzyme,
ribonuclease,
transglutaminase, or xylanase. The compositions may be prepared in accordance
with methods
known in the art and may be in the form of a liquid or a dry composition. The
compositions may
be stabilized in accordance with methods known in the art.

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Examples are given below of preferred uses of the compositions of the present
invention. The dosage of the composition and other conditions under which the
composition is
used may be determined on the basis of methods known in the art.
Uses
The present invention is also directed to the following processes for using
the
polypeptides having cellobiohydrolase activity, or compositions thereof.
The present invention also relates to processes for degrading or converting a
cellulosic
material, comprising: treating the cellulosic material with an enzyme
composition in the
presence of a polypeptide having cellobiohydrolase activity of the present
invention. In one
aspect, the processes further comprise recovering the degraded or converted
cellulosic material.
Soluble products of degradation or conversion of the cellulosic material can
be separated from
insoluble cellulosic material using a method known in the art such as, for
example,
centrifugation, filtration, or gravity settling.
The present invention also relates to processes of producing a fermentation
product,
comprising: (a) saccharifying a cellulosic material with an enzyme composition
in the presence
of a polypeptide having cellobiohydrolase activity of the present invention;
(b) fermenting the
saccharified cellulosic material with one or more (e.g., several) fermenting
microorganisms to
produce the fermentation product; and (c) recovering the fermentation product
from the
fermentation.
The present invention also relates to processes of fermenting a cellulosic
material,
comprising: fermenting the cellulosic material with one or more (e.g.,
several) fermenting
microorganisms, wherein the cellulosic material is saccharified with an enzyme
composition in
the presence of a polypeptide having cellobiohydrolase activity of the present
invention. In one
aspect, the fermenting of the cellulosic material produces a fermentation
product. In another
aspect, the processes further comprise recovering the fermentation product
from the
fermentation.
The processes of the present invention can be used to saccharify the
cellulosic material
to fermentable sugars and to convert the fermentable sugars to many useful
fermentation
products, e.g., fuel, potable ethanol, and/or platform chemicals (e.g., acids,
alcohols, ketones,
gases, and the like). The production of a desired fermentation product from
the cellulosic
material typically involves pretreatment, enzymatic hydrolysis
(saccharification), and
fermentation.
The processing of the cellulosic material according to the present invention
can be

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accomplished using methods conventional in the art. Moreover, the processes of
the present
invention can be implemented using any conventional biomass processing
apparatus configured
to operate in accordance with the invention.
Hydrolysis (saccharification) and fermentation, separate or simultaneous,
include, but
5 are not
limited to, separate hydrolysis and fermentation (SHE); simultaneous
saccharification
and fermentation (SSF); simultaneous saccharification and co-fermentation
(SSCF); hybrid
hydrolysis and fermentation (HHF); separate hydrolysis and co-fermentation
(SHCF); hybrid
hydrolysis and co-fermentation (HHCF); and direct microbial conversion (DMC),
also sometimes
called consolidated bioprocessing (CBP). SHE uses separate process steps to
first
10
enzymatically hydrolyze the cellulosic material to fermentable sugars, e.g.,
glucose, cellobiose,
and pentose monomers, and then ferment the fermentable sugars to ethanol. In
SSF, the
enzymatic hydrolysis of the cellulosic material and the fermentation of sugars
to ethanol are
combined in one step (Philippidis, G. P., 1996, Cellulose bioconversion
technology, in
Handbook on Bioethana Production and Utilization, Wyman, C. E., ed., Taylor &
Francis,
15
Washington, DC, 179-212). SSCF involves the co-fermentation of multiple sugars
(Sheehan, J.,
and Himmel, M., 1999, Enzymes, energy and the environment: A strategic
perspective on the
U.S. Department of Energy's research and development activities for
bioethanol, Biotechnol
Prog. 15: 817-827). HHF involves a separate hydrolysis step, and in addition a
simultaneous
saccharification and hydrolysis step, which can be carried out in the same
reactor. The steps in
20 an HI-IF
process can be carried out at different temperatures, i.e., high temperature
enzymatic
saccharification followed by SSF at a lower temperature that the fermentation
strain can
tolerate. DMC combines all three processes (enzyme production, hydrolysis, and
fermentation)
in one or more (e.g., several) steps where the same organism is used to
produce the enzymes
for conversion of the cellulosic material to fermentable sugars and to convert
the fermentable
25 sugars
into a final product (Lynd, L. R., Weimer, P. J., van Zyl, W. H., and
Pretorius, I. S., 2002,
Microbial cellulose utilization: Fundamentals and biotechnology, Microbiol.
MoL BioL Reviews
66: 506-577). It is understood herein that any method known in the art
comprising pretreatment,
enzymatic hydrolysis (saccharification), fermentation, or a combination
thereof, can be used in
the practicing the processes of the present invention.
30 A
conventional apparatus can include a fed-batch stirred reactor, a batch
stirred reactor,
a continuous flow stirred reactor with ultrafiltration, and/or a continuous
plug-flow column reactor
(Fernanda de Castilhos Corazza, Flavio Faria de Moraes, Gisella Maria Zanin
and Ivo Neitzel,
2003, Optimal control in fed-batch reactor for the cellobiose hydrolysis, Acta
Scientiarum.
Technology 25: 33-38; Gusakov, A. V., and Sinitsyn, A. P., 1985, Kinetics of
the enzymatic

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WO 2013/071871 PCT/CN2012/084661
hydrolysis of cellulose: 1. A mathematical model for a batch reactor process,
Enz. Microb.
Technol 7: 346-352), an attrition reactor (Ryu, S. K., and Lee, J. M., 1983,
Bioconversion of
waste cellulose by using an attrition bioreactor, BiotechnoL Bioeng. 25: 53-
65), or a reactor with
intensive stirring induced by an electromagnetic field (Gusakov, A. V.,
Sinitsyn, A. P., Davydkin,
I. Y., Davydkin, V. Y., Protas, 0. V., 1996, Enhancement of enzymatic
cellulose hydrolysis using
a novel type of bioreactor with intensive stirring induced by electromagnetic
field, App!.
Biochem. Biotechnol. 56: 141-153). Additional reactor types include fluidized
bed, upflow
blanket, immobilized, and extruder type reactors for hydrolysis and/or
fermentation.
Pretreatment. In practicing the processes of the present invention, any
pretreatment
process known in the art can be used to disrupt plant cell wall components of
the cellulosic
material (Chandra et aL, 2007, Substrate pretreatment: The key to effective
enzymatic
hydrolysis of lignocellulosics?, Adv. Biochem. Engin./BiotechnoL 108: 67-93;
Galbe and Zacchi,
2007, Pretreatment of lignocellulosic materials for efficient bioethanol
production, Adv. Biochem.
Engin./BiotechnoL 108: 41-65; Hendriks and Zeeman, 2009, Pretreatments to
enhance the
digestibility of lignocellulosic biomass, Bioresource Technol. 100: 10-18;
Mosier et al., 2005,
Features of promising technologies for pretreatment of lignocellulosic
biomass, Bioresource
TechnoL 96: 673-686; Taherzadeh and Karimi, 2008, Pretreatment of
lignocellulosic wastes to
improve ethanol and biogas production: A review, mt. J. of MoL Sci. 9: 1621-
1651; Yang and
Wyman, 2008, Pretreatment: the key to unlocking low-cost cellulosic ethanol,
Biofuels
Bioproducts and Biorefining-Biofpr. 2: 26-40).
The cellulosic material can also be subjected to particle size reduction,
sieving, pre-
soaking, wetting, washing, and/or conditioning prior to pretreatment using
methods known in the
art.
Conventional pretreatments include, but are not limited to, steam pretreatment
(with or
without explosion), dilute acid pretreatment, hot water pretreatment, alkaline
pretreatment, lime
pretreatment, wet oxidation, wet explosion, ammonia fiber explosion,
organosolv pretreatment,
and biological pretreatment. Additional pretreatments include ammonia
percolation, ultrasound,
electroporation, microwave, supercritical CO2, supercritical H20, ozone, ionic
liquid, and gamma
irradiation pretreatments.
The cellulosic material can be pretreated before hydrolysis and/or
fermentation.
Pretreatment is preferably performed prior to the hydrolysis. Alternatively,
the pretreatment can be
carried out simultaneously with enzyme hydrolysis to release fermentable
sugars, such as glucose,
xylose, and/or cellobiose. In most cases the pretreatment step itself results
in some conversion
of biomass to fermentable sugars (even in absence of enzymes).

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Steam Pretreatment. In steam pretreatment, the cellulosic material is heated
to disrupt
the plant cell wall components, including lignin, hemicellulose, and cellulose
to make the
cellulose and other fractions, e.g., hemicellulose, accessible to enzymes. The
cellulosic material
is passed to or through a reaction vessel where steam is injected to increase
the temperature to
the required temperature and pressure and is retained therein for the desired
reaction time.
Steam pretreatment is preferably performed at 140-250 C, e.g., 160-200 C or
170-190 C,
where the optimal temperature range depends on addition of a chemical
catalyst. Residence
time for the steam pretreatment is preferably 1-60 minutes, e.g., 1-30
minutes, 1-20 minutes, 3-
12 minutes, or 4-10 minutes, where the optimal residence time depends on
temperature range
and addition of a chemical catalyst. Steam pretreatment allows for relatively
high solids
loadings, so that the cellulosic material is generally only moist during the
pretreatment. The
steam pretreatment is often combined with an explosive discharge of the
material after the
pretreatment, which is known as steam explosion, that is, rapid flashing to
atmospheric pressure
and turbulent flow of the material to increase the accessible surface area by
fragmentation (Duff
and Murray, 1996, Bioresource Technology 855: 1-33; Galbe and Zacchi, 2002,
Appl Microbiot
BiotechnoL 59: 618-628; U.S. Patent Application No. 20020164730). During steam
pretreatment, hemicellulose acetyl groups are cleaved and the resulting acid
autocatalyzes
partial hydrolysis of the hemicellulose to monosaccharides and
oligosaccharides. Lignin is
removed to only a limited extent.
Chemical Pretreatment: The term "chemical treatment" refers to any chemical
pretreatment
that promotes the separation and/or release of cellulose, hemicellulose,
and/or lignin. Such a
pretreatment can convert crystalline cellulose to amorphous cellulose.
Examples of suitable
chemical pretreatment processes include, for example, dilute acid
pretreatment, lime pretreatment,
wet oxidation, ammonia fiber/freeze explosion (AFEX), ammonia percolation
(APR), ionic liquid,
and organosolv pretreatments.
A catalyst such as H2SO4 or SO2 (typically 0.3 to 5% w/w) is often added prior
to steam
pretreatment, which decreases the time and temperature, increases the
recovery, and improves
enzymatic hydrolysis (Ballesteros et al., 2006, App!. Biochem. BiotechnoL 129-
132: 496-508;
Varga at aL, 2004, App!. Biochem. BiotechnoL 113-116: 509-523; Sassner at aL,
2006, Enzyme
Microb. TechnoL 39: 756-762). In dilute acid pretreatment, the cellulosic
material is mixed with
dilute acid, typically H2SO4, and water to form a slurry, heated by steam to
the desired
temperature, and after a residence time flashed to atmospheric pressure. The
dilute acid
pretreatment can be performed with a number of reactor designs, e.g., plug-
flow reactors, counter-
current reactors, or continuous counter-current shrinking bed reactors (Duff
and Murray, 1996,

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WO 2013/071871 PCT/CN2012/084661
supra; Schell et at, 2004, Bioresource TechnoL 91: 179-188; Lee et at, 1999,
Adv. Biochem. Eng.
Biotechnot 65: 93-115).
Several methods of pretreatment under alkaline conditions can also be used.
These
alkaline pretreatments include, but are not limited to, sodium hydroxide,
lime, wet oxidation,
ammonia percolation (APR), and ammonia fiber/freeze explosion (AFEX).
Lime pretreatment is performed with calcium oxide or calcium hydroxide at
temperatures of
85-150 C and residence times from 1 hour to several days (Wyman et al., 2005,
Bioresource
TechnoL 96: 1959-1966; Mosier et at, 2005, Bioresource TechnoL 96: 673-686).
WO
2006/110891, WO 2006/110899, WO 2006/110900, and WO 2006/110901 disclose
pretreatment
methods using ammonia.
Wet oxidation is a thermal pretreatment performed typically at 180-200 C for 5-
15 minutes
with addition of an oxidative agent such as hydrogen peroxide or over-pressure
of oxygen (Schmidt
and Thomsen, 1998, Bioresource TechnoL 64: 139-151; Palonen et at, 2004, AppL
Biochem.
Biotechnot 117: 1-17; Varga et al., 2004, BiotechnoL Bioeng. 88: 567-574;
Martin et at, 2006, J.
Chem. TechnoL Biotechnot 81: 1669-1677). The pretreatment is performed
preferably at 1-40%
dry matter, e.g., 2-30% dry matter or 5-20% dry matter, and often the initial
p1-1 is increased by the
addition of alkali such as sodium carbonate.
A modification of the wet oxidation pretreatment method, known as wet
explosion
(combination of wet oxidation and steam explosion) can handle dry matter up to
30%. In wet
explosion, the oxidizing agent is introduced during pretreatment after a
certain residence time. The
pretreatment is then ended by flashing to atmospheric pressure MO
2006/032282).
Ammonia fiber explosion (AFEX) involves treating the cellulosic material with
liquid or
gaseous ammonia at moderate temperatures such as 90-150 C and high pressure
such as 17-20
bar for 5-10 minutes, where the dry matter content can be as high as 60%
(Gollapalli et at, 2002,
Appl. Biochem. Biotechnol. 98: 23-35; Chundawat et at, 2007, Biotechnot
Bioeng. 96: 219-231;
Alizadeh et at, 2005, Appl. Biochem. Biotechnot 121: 1133-1141; Teymouri et
at, 2005,
Bioresource TechnoL 96: 2014-2018). During AFEX pretreatment cellulose and
hemicelluloses
remain relatively intact. Lignin-carbohydrate complexes are cleaved.
Organosolv pretreatment delignifies the cellulosic material by extraction
using aqueous
ethanol (40-60% ethanol) at 160-200 C for 30-60 minutes (Pan et at, 2005,
Biotechnot Bioeng.
90: 473-481; Pan et at, 2006, Biotechnot Bioeng. 94: 851-861; Kurabi et at,
2005, Appl. Biochem.
Biotechnot 121: 219-230). Sulphuric acid is usually added as a catalyst. In
organosolv
pretreatment, the majority of hemicellulose and lignin is removed.
Other examples of suitable pretreatment methods are described by Schell et at,
2003,

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Appl Biochem. and Biotechnot Vol. 105-108, p. 69-85, and Mosier et al, 2005,
Bioresource
Technology 96: 673-686, and U.S. Published Application 2002/0164730.
In one aspect, the chemical pretreatment is preferably carried out as a dilute
acid
treatment, and more preferably as a continuous dilute acid treatment. The acid
is typically sulfuric
acid, but other acids can also be used, such as acetic acid, citric acid,
nitric acid, phosphoric acid,
tartaric acid, succinic acid, hydrogen chloride, or mixtures thereof. Mild
acid treatment is conducted
in the pH range of preferably 1-5, e.g., 1-4 or 1-2.5. In one aspect, the acid
concentration is in the
range from preferably 0.01 to 10 wt % acid, e.g., 0.05 to 5 wt % acid or 0.1
to 2 wt % acid. The acid
is contacted with the cellulosic material and held at a temperature in the
range of preferably 140-
200 C, e.g., 165-190 C, for periods ranging from 1 to 60 minutes.
In another aspect, pretreatment takes place in an aqueous slurry. In preferred
aspects,
the cellulosic material is present during pretreatment in amounts preferably
between 10-80
wt %, e.g., 20-70 wt % or 30-60 wt %, such as around 40 wt %. The pretreated
cellulosic
material can be unwashed or washed using any method known in the art, e.g.,
washed with
water.
Mechanical Pretreatment or Physical Pretreatment: The term "mechanical
pretreatment" or
"physical pretreatment" refers to any pretreatment that promotes size
reduction of particles. For
example, such pretreatment can involve various types of grinding or milling
(e.g., dry milling, wet
milling, or vibratory ball milling).
The cellulosic material can be pretreated both physically (mechanically) and
chemically.
Mechanical or physical pretreatment can be coupled with steaming/steam
explosion,
hydrothermolysis, dilute or mild acid treatment, high temperature, high
pressure treatment,
irradiation (e.g., microwave irradiation), or combinations thereof. In one
aspect, high pressure
means pressure in the range of preferably about 100 to about 400 psi, e.g.,
about 150 to about 250
psi. In another aspect, high temperature means temperatures in the range of
about 100 to about
300 C, e.g., about 140 to about 200 C. In a preferred aspect, mechanical or
physical pretreatment
is performed in a batch-process using a steam gun hydrolyzer system that uses
high pressure and
high temperature as defined above, e.g., a Sunds Hydrolyzer available from
Sunds Defibrator AB,
Sweden. The physical and chemical pretreatments can be carried out
sequentially or
simultaneously, as desired.
Accordingly, in a preferred aspect, the cellulosic material is subjected to
physical
(mechanical) or chemical pretreatment, or any combination thereof, to promote
the separation
and/or release of cellulose, hemicellulose, and/or lignin.
Biological Pretreatment: The term "biological pretreatment" refers to any
biological

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pretreatment that promotes the separation and/or release of cellulose,
hemicellulose, and/or
lignin from the cellulosic material. Biological pretreatment techniques can
involve applying
lignin-solubilizing microorganisms and/or enzymes (see, for example, Hsu, T.-
A., 1996,
Pretreatment of biomass, in Handbook on Bioethana Production and Utilization,
Wyman, C. E.,
5 ed., Taylor & Francis, Washington, DC, 179-212; Ghosh and Singh, 1993,
Physicochemical and
biological treatments for enzymatic/microbial conversion of cellulosic
biomass, Adv. AppL
Microbiol. 39: 295-333; McMillan, J. D., 1994, Pretreating lignocellulosic
biomass: a review, in
Enzymatic Conversion of Biomass for Fuels Production, Himmel, M. E., Baker, J.
0., and
Overend, R. P., eds., ACS Symposium Series 566, American Chemical Society,
Washington,
10 DC, chapter 15; Gong, C. S., Cao, N. J., Du, J., and Tsao, G. T., 1999,
Ethanol production from
renewable resources, in Advances in Biochemical Engineering/Biotechnology,
Scheper, T., ed.,
Springer-Verlag Berlin Heidelberg, Germany, 65: 207-241; Olsson and Hahn-
Hagerdal, 1996,
Fermentation of lignocellulosic hydrolysates for ethanol production, Enz.
Microb. Tech. 18: 312-
331; and Vallander and Eriksson, 1990, Production of ethanol from
lignocellulosic materials:
15 State of the art, Adv. Biochem. Eng./Biotechnol. 42: 63-95).
Saccharification. In the hydrolysis step, also known as saccharification, the
cellulosic
material, e.g., pretreated, is hydrolyzed to break down cellulose and/or
hemicellulose to
fermentable sugars, such as glucose, cellobiose, xylose, xylulose, arabinose,
mannose,
galactose, and/or soluble oligosaccharides. The hydrolysis is performed
enzymatically by an
20 enzyme composition as described herein in the presence of a polypeptide
having
cellobiohydrolase activity of the present invention. The enzyme components of
the compositions
can be added simultaneously or sequentially.
Enzymatic hydrolysis is preferably carried out in a suitable aqueous
environment under
conditions that can be readily determined by one skilled in the art. In one
aspect, hydrolysis is
25 performed under conditions suitable for the activity of the enzyme
components, i.e., optimal for the
enzyme components. The hydrolysis can be carried out as a fed batch or
continuous process
where the cellulosic material is fed gradually to, for example, an enzyme
containing hydrolysis
solution.
The saccharification is generally performed in stirred-tank reactors or
fermentors under
30 controlled pH, temperature, and mixing conditions. Suitable process
time, temperature and pH
conditions can readily be determined by one skilled in the art. For example,
the saccharification
can last up to 200 hours, but is typically performed for preferably about 12
to about 120 hours,
e.g., about 16 to about 72 hours or about 24 to about 48 hours. The
temperature is in the range of
preferably about 25 C to about 70 C, e.g., about 30 C to about 65 C, about 40
C to about

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WO 2013/071871 PCT/CN2012/084661
60 C, or about 50 C to about 55 C. The pH is in the range of preferably about
3 to about 8, e.g.,
about 3.5 to about 7, about 4 to about 6, or about 5.0 to about 5.5. The dry
solids content is in
the range of preferably about 5 to about 50 wt %, e.g., about 10 to about 40
wt % or about 20 to
about 30 wt %.
The enzyme compositions can comprise any protein useful in degrading the
cellulosic
material.
In one aspect, the enzyme composition comprises or further comprises one or
more
(e.g., several) proteins/polypeptides selected from the group consisting of a
cellulase, a GH61
polypeptide having cellulolytic enhancing activity, a hemicellulase, an
esterase, an expansin, a
laccase, a ligninolytic enzyme, a pectinase, a peroxidase, a protease, and a
swollenin. In
another aspect, the cellulase is preferably one or more (e.g., several)
enzymes selected from
the group consisting of an endoglucanase, a cellobiohydrolase, and a beta-
glucosidase. In
another aspect, the hemicellulase is preferably one or more (e.g., several)
enzymes selected
from the group consisting of an acetylmannan esterase, an acetylxylan
esterase, an
arabinanase, an arabinofuranosidase, a coumaric acid esterase, a feruloyl
esterase, a
galactosidase, a glucuronidase, a glucuronoyl esterase, a mannanase, a
mannosidase, a
xylanase, and a xylosidase.
In another aspect, the enzyme composition comprises one or more (e.g.,
several)
cellulolytic enzymes. In another aspect, the enzyme composition comprises or
further comprises
one or more (e.g., several) hemicellulolytic enzymes. In another aspect, the
enzyme
composition comprises one or more (e.g., several) cellulolytic enzymes and one
or more (e.g.,
several) hemicellulolytic enzymes. In another aspect, the enzyme composition
comprises one or
more (e.g., several) enzymes selected from the group of cellulolytic enzymes
and
hemicellulolytic enzymes. In another aspect, the enzyme composition comprises
an
endoglucanase. In another aspect, the enzyme composition comprises a
cellobiohydrolase. In
another aspect, the enzyme composition comprises a beta-glucosidase. In
another aspect, the
enzyme composition comprises a polypeptide having cellulolytic enhancing
activity. In another
aspect, the enzyme composition comprises an endoglucanase and a polypeptide
having
cellulolytic enhancing activity. In another aspect, the enzyme composition
comprises a
cellobiohydrolase and a polypeptide having cellulolytic enhancing activity. In
another aspect, the
enzyme composition comprises a beta-glucosidase and a polypeptide having
cellulolytic
enhancing activity. In another aspect, the enzyme composition comprises an
endoglucanase
and a cellobiohydrolase. In another aspect, the enzyme composition comprises
an
endoglucanase and a beta-glucosidase. In another aspect, the enzyme
composition comprises

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WO 2013/071871 PCT/CN2012/084661
a cellobiohydrolase and a beta-glucosidase. In another aspect, the enzyme
composition
comprises an endoglucanase, a cellobiohydrolase, and a polypeptide having
cellulolytic
enhancing activity. In another aspect, the enzyme composition comprises an
endoglucanase, a
beta-glucosidase, and a polypeptide having cellulolytic enhancing activity. In
another aspect, the
enzyme composition comprises a cellobiohydrolase, a beta-glucosidase, and a
polypeptide
having cellulolytic enhancing activity. In another aspect, the enzyme
composition comprises an
endoglucanase, a cellobiohydrolase, and a beta-glucosidase. In another aspect,
the enzyme
composition comprises an endoglucanase, a cellobiohydrolase, a beta-
glucosidase, and a
polypeptide having cellulolytic enhancing activity.
In another aspect, the enzyme composition comprises an acetylmannan esterase.
In
another aspect, the enzyme composition comprises an acetylxylan esterase. In
another aspect,
the enzyme composition comprises an arabinanase (e.g., alpha-L-arabinanase).
In another
aspect, the enzyme composition comprises an arabinofuranosidase (e.g., alpha-L-
arabinofuranosidase). In another aspect, the enzyme composition comprises a
coumaric acid
esterase. In another aspect, the enzyme composition comprises a feruloyl
esterase. In another
aspect, the enzyme composition comprises a galactosidase (e.g., alpha-
galactosidase and/or
beta-galactosidase). In another aspect, the enzyme composition comprises a
glucuronidase
(e.g., alpha-D-glucuronidase). In another aspect, the enzyme composition
comprises a
glucuronoyl esterase. In another aspect, the enzyme composition comprises a
mannanase. In
another aspect, the enzyme composition comprises a mannosidase (e.g., beta-
mannosidase).
In another aspect, the enzyme composition comprises a xylanase. In a preferred
aspect, the
xylanase is a Family 10 xylanase. In another aspect, the enzyme composition
comprises a
xylosidase (e.g., beta-xylosidase).
In another aspect, the enzyme composition comprises an esterase. In another
aspect,
the enzyme composition comprises an expansin. In another aspect, the enzyme
composition
comprises a laccase. In another aspect, the enzyme composition comprises a
ligninolytic
enzyme. In a preferred aspect, the ligninolytic enzyme is a manganese
peroxidase. In another
preferred aspect, the ligninolytic enzyme is a lignin peroxidase. In another
preferred aspect, the
ligninolytic enzyme is a H202-producing enzyme. In another aspect, the enzyme
composition
comprises a pectinase. In another aspect, the enzyme composition comprises a
peroxidase. In
another aspect, the enzyme composition comprises a protease. In another
aspect, the enzyme
composition comprises a swollenin.
In the processes of the present invention, the enzyme(s) can be added prior to
or during
saccharification, saccharification and fermentation, or fermentation.

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One or more (e.g., several) components of the enzyme composition may be wild-
type
proteins, recombinant proteins, or a combination of wild-type proteins and
recombinant proteins.
For example, one or more (e.g., several) components may be native proteins of
a cell, which is
used as a host cell to express recombinantly one or more (e.g., several) other
components of
the enzyme composition. One or more (e.g., several) components of the enzyme
composition
may be produced as monocomponents, which are then combined to form the enzyme
composition. The enzyme composition may be a combination of multicomponent and
monocomponent protein preparations.
The enzymes used in the processes of the present invention may be in any form
suitable
for use, such as, for example, a fermentation broth formulation or a cell
composition, a cell
lysate with or without cellular debris, a semi-purified or purified enzyme
preparation, or a host
cell as a source of the enzymes. The enzyme composition may be a dry powder or
granulate, a
non-dusting granulate, a liquid, a stabilized liquid, or a stabilized
protected enzyme. Liquid
enzyme preparations may, for instance, be stabilized by adding stabilizers
such as a sugar, a
sugar alcohol or another polyol, and/or lactic acid or another organic acid
according to
established processes.
The optimum amounts of the enzymes and a polypeptide having cellobiohydrolase
activity depend on several factors including, but not limited to, the mixture
of cellulolytic and/or
hemicellulolytic enzyme components, the cellulosic material, the concentration
of cellulosic
material, the pretreatment(s) of the cellulosic material, temperature, time,
pH, and inclusion of
fermenting organism (e.g., yeast for Simultaneous Saccharification and
Fermentation).
In one aspect, an effective amount of cellulolytic or hemicellulolytic enzyme
to the
cellulosic material is about 0.5 to about 50 mg, e.g., about 0.5 to about 40
mg, about 0.5 to
about 25 mg, about 0.75 to about 20 mg, about 0.75 to about 15 mg, about 0.5
to about 10 mg,
or about 2.5 to about 10 mg per g of the cellulosic material.
In another aspect, an effective amount of a polypeptide having
cellobiohydrolase activity
to the cellulosic material is about 0.01 to about 50.0 mg, e.g., about 0.01 to
about 40 mg, about
0.01 to about 30 mg, about 0.01 to about 20 mg, about 0.01 to about 10 mg,
about 0.01 to about
5 mg, about 0.025 to about 1.5 mg, about 0.05 to about 1.25 mg, about 0.075 to
about 1.25 mg,
about 0.1 to about 1.25 mg, about 0.15 to about 1.25 mg, or about 0.25 to
about 1.0 mg per g of
the cellulosic material.
In another aspect, an effective amount of a polypeptide having
cellobiohydrolase activity
to cellulolytic or hemicellulolytic enzyme is about 0.005 to about 1.0 g,
e.g., about 0.01 to about
1.0 g, about 0.15 to about 0.75 g, about 0.15 to about 0.5 g, about 0.1 to
about 0.5 g, about 0.1

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to about 0.25 g, or about 0.05 to about 0.2 g per g of cellulolytic or
hemicellulolytic enzyme.
The polypeptides having cellulolytic enzyme activity or hemicellulolytic
enzyme activity
as well as other proteins/polypeptides useful in the degradation of the
cellulosic material , e.g.,
GH61 polypeptides having cellulolytic enhancing activity (collectively
hereinafter "polypeptides
having enzyme activity") can be derived or obtained from any suitable origin,
including,
bacterial, fungal, yeast, plant, or mammalian origin. The term "obtained" also
means herein that
the enzyme may have been produced recombinantly in a host organism employing
methods
described herein, wherein the recombinantly produced enzyme is either native
or foreign to the
host organism or has a modified amino acid sequence, e.g., having one or more
(e.g., several)
amino acids that are deleted, inserted and/or substituted, i.e., a
recombinantly produced
enzyme that is a mutant and/or a fragment of a native amino acid sequence or
an enzyme
produced by nucleic acid shuffling processes known in the art. Encompassed
within the
meaning of a native enzyme are natural variants and within the meaning of a
foreign enzyme
are variants obtained recombinantly, such as by site-directed mutagenesis or
shuffling.
A polypeptide having enzyme activity may be a bacterial polypeptide. For
example, the
polypeptide may be a Gram-positive bacterial polypeptide such as a Bacillus,
Streptococcus,
Streptomyces, Staphylococcus, Enterococcus, Lactobacillus, Lactococcus,
Clostridium,
Geobacillus, Caldicellulosiruptor, Acidothermus, Thermobifidia, or
Oceanobacillus polypeptide
having enzyme activity, or a Gram negative bacterial polypeptide such as an E
coil,
Pseudomonas, Salmonella, Campylobacter, Helicobacter, Flavobactetium,
Fusobacterium,
Ilyobacter, Neissetia, or Urea plasma polypeptide having enzyme activity.
In one aspect, the polypeptide is a Bacillus alkalophilus, Bacillus
amyloliquefaciens,
Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans,
Bacillus firmus, Bacillus
lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus
pumilus, Bacillus
stearothermophilus, Bacillus subtilis, or Bacillus thuringiensis polypeptide
having enzyme
activity.
In another aspect, the polypeptide is a Streptococcus equisimilis,
Streptococcus
pyo genes, Streptococcus ubetis, or Streptococcus equi subsp. Zooepidemicus
polypeptide
having enzyme activity.
In another aspect, the polypeptide is a Streptomyces achromogenes,
Streptomyces
Streptomyces coelicolor, Streptomyces griseus, or Streptomyces lividans
polypeptide having enzyme activity.
The polypeptide having enzyme activity may also be a fungal polypeptide, and
more
preferably a yeast polypeptide such as a Candida, Kluyveromyces, Pichia,
Saccharomyces,

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Schizosaccharomyces, or Yarrowia polypeptide having enzyme activity; or more
preferably a
filamentous fungal polypeptide such as an Acremonium, Agaticus, Altemaria,
Aspergillus,
Aureobasidium, Bottyospaeria, Ceriporiopsis, Chaetomidium, Chrysosporium,
Claviceps,
Cochliobolus, Coprinopsis, Coptotermes, Corynascus, Cryphonectria,
Cryptococcus, Diplodia,
5 Exidia, Filibasidium, Fusarium, Gibberella, Holomastigotoides, Humicola,
hoax, Lentinula,
Leptospaeria, Magnaporthe, Melanocarpus, Meripilus, Mucor, Myceliophthora,
Neocallimastix,
Neurospota, Paecilomyces, Penicillium, Phanerochaete, Piromyces, Poitrasia,
Pseudo plectania,
Pseudottichonympha, Rhizomucor, Schizophyfium, Scytalidium, Talaromyces,
Thermoascus,
Thielavia, Tolypocladium, Trichoderma, Trichophaea, Verticillium, Volvariella,
or Xylaria
10 polypeptide having enzyme activity.
In one aspect, the polypeptide is a Saccharomyces carisbergensis,
Saccharomyces
cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasfi, Saccharomyces
kluyveri,
Saccharomyces norbensis, or Saccharomyces oviformis polypeptide having enzyme
activity.
In another aspect, the polypeptide is an Acremonium cellulolyticus,
Aspergillus
15 aculeatus, Aspergillus awamori, Aspergillus fumigatus, Aspergillus
foetidus, Aspergillus
japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae,
Chrysospodum
keratinophilum, Chtysospotium lucknowense, Chrysospotium tropicum,
Chtysosporium
metriarium, Chtysosporium Mops, Chrysospotium pannicola, Chrysosporium
queenslandicum,
Chtysosporium zona turn, Fusarium bactridioides, Fusatium cerealis, Fusarium
crookwellense,
20 Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium
heterosporum,
Fusarium negundi, Fusarium oxysporum, Fusarium reticula turn, Fusarium roseum,
Fusarium
sambucinum, Fusarium sarcochroum, Fusarium sporottichioides, Fusarium
sulphureum,
Fusarium torulosum, Fusarium trichothecioides, Fusari urn venenatum, Humicola
gtisea,
Humicola insolens, Humicola lanuginosa, lrpex lacteus, Mucor miehei,
Myceliophthora
25 thermophila, Neurospora crassa, Penicillium funiculosum, Penicillium
purpurogenum,
Phanerochaete chrysosporium, Thielavia achromatica, Thielavia albomyces,
Thielavia
albopilosa, Thielavia austtaleinsis, Thielavia firneti, Thielavia microspora,
Thielavia ovispora,
Thielavia peruviana, Thielavia spededonium, Thielavia setosa, Thielavia
subthermophila,
Thielavia terrestris, Trichoderma harzian urn,
Trichoderma koningfi, Trichoderma
30 longibtachiatum, Trichoderma reesei, Trichoderma vitide, or Trichophaea
saccata polypeptide
having enzyme activity.
Chemically modified or protein engineered mutants of polypeptides having
enzyme
activity may also be used.
One or more (e.g., several) components of the enzyme composition may be a

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recombinant component, i.e., produced by cloning of a DNA sequence encoding
the single
component and subsequent cell transformed with the DNA sequence and expressed
in a host
(see, for example, WO 91/17243 and WO 91/17244). The host is preferably a
heterologous host
(enzyme is foreign to host), but the host may under certain conditions also be
a homologous
In one aspect, the one or more (e.g., several) cellulolytic enzymes comprise a
commercial cellulolytic enzyme preparation. Examples of commercial
cellulolytic enzyme
preparations suitable for use in the present invention include, for example,
CELLIC CTec
Examples of bacterial endoglucanases that can be used in the processes of the
present
Examples of fungal endoglucanases that can be used in the present invention,
include,

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endoglucanase (GENBANKTm accession no. L29381), Humicola grisea var.
thermoidea
endoglucanase (GENBANKTM accession no. AB003107), Melanocatpus albomyces
endoglucanase (GENBANKTM accession no. MAL515703), Neurospora crassa
endoglucanase
(GENBANKTM accession no. XM_324477), Humicola insolens endoglucanase V,
Myceliophthora
thermophila CBS 117.65 endoglucanase, basidiomycete CBS 495.95 endoglucanase,
basidiomycete CBS 494.95 endoglucanase, Thielavia terrestris NRRL 8126 CEL6B
endoglucanase, Thielavia terrestris NRRL 8126 CEL6C endoglucanase, Thielavia
terrestris
NRRL 8126 CEL7C endoglucanase, Thielavia terrestris NRRL 8126 CEL7E
endoglucanase,
Thielavia terrestris NRRL 8126 CEL7F endoglucanase, Cladorrhinum
foecundissimum ATCC
62373 CEL7A endoglucanase, and Trichodetma reesei strain No. VTT-D-80133
endoglucanase
(GENBANKTM accession no. M15665).
Examples of cellobiohydrolases useful in the present invention include, but
are not
limited to, Aspergillus aculeatus cellobiohydrolase II (WO 2011/059740),
Chaetomium
thermophilum cellobiohydrolase I, Chaetomium thermophilum cellobiohydrolase
II, Humicola
insolens cellobiohydrolase I, Myceliophthora thermophila cellobiohydrolase II
(WO
2009/042871), Thielavia hyrcanie cellobiohydrolase II (WO 2010/141325),
Thielavia terrestris
cellobiohydrolase II (CEL6A, WO 2006/074435), Trichoderma reesei
cellobiohydrolase I,
Trichoderma reesei cellobiohydrolase II, and Trichophaea saccata
cellobiohydrolase II (WO
2010/057086).
Examples of beta-glucosidases useful in the present invention include, but are
not
limited to, beta-glucosidases from Aspergillus aculeatus (Kawaguchi et aL,
1996, Gene 173:
287-288), Aspergillus fumigatus (WO 2005/047499), Aspergillus niger (Dan et
aL, 2000, J. Biol.
Chem. 275: 4973-4980), Aspergillus oryzae (WO 2002/095014), Penicillium
brasilianum IBT
20888 (VVO 2007/019442 and WO 2010/088387), Thielavia terrestris (WO
2011/035029), and
Trichophaea saccata (WO 2007/019442).
The beta-glucosidase may be a fusion protein. In one aspect, the beta-
glucosidase is an
Aspergillus otyzae beta-glucosidase variant BG fusion protein (WO 2008/057637)
or an
Aspergillus oryzae beta-glucosidase fusion protein (WO 2008/057637).
Other useful endoglucanases, cellobiohydrolases, and beta-glucosidases are
disclosed
in numerous Glycosyl Hydrolase families using the classification according to
Henrissat B.,
1991, A classification of glycosyl hydrolases based on amino-acid sequence
similarities,
Biochem. J. 280: 309-316, and Henrissat B., and Bairoch A., 1996, Updating the
sequence-
based classification of glycosyl hydrolases, Biochem. J. 316: 695-696.
Other cellulolytic enzymes that may be used in the present invention are
described in

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WO 98/13465, WO 98/015619, WO 98/015633, WO 99/06574, WO 99/10481, WO
99/025847,
WO 99/031255, WO 2002/101078, WO 2003/027306, WO 2003/052054, WO 2003/052055,
WO 2003/052056, WO 2003/052057, WO 2003/052118, WO 2004/016760, WO
2004/043980,
WO 2004/048592, WO 2005/001065, WO 2005/028636, WO 2005/093050, WO
2005/093073,
WO 2006/074005, WO 2006/117432, WO 2007/071818, WO 2007/071820, WO
2008/008070,
WO 2008/008793, U.S. Patent No. 5,457,046, U.S. Patent No. 5,648,263, and U.S.
Patent No.
5,686,593.
In the methods of the present invention, any GH61 polypeptide having
cellulolytic
enhancing activity can be used.
In a first aspect, the GH61 polypeptide having cellulolytic enhancing activity
comprises
the following motifs:
[ILMVI-P-X(4,5)-G-X-Y-[ILMA-X-R-X4EQ1-X(4)-[HNQ] (SEQ ID NO: 29 or SEQ ID NO:
30) and [FW1-[TF1-K4A11/],
wherein X is any amino acid, X(4,5) is any amino acid at 4 or 5 contiguous
positions, and
X(4) is any amino acid at 4 contiguous positions.
The isolated polypeptide comprising the above-noted motifs may further
comprise:
H-X(1,2)-G-P-X(3)-[YW]-[AILMV] (SEQ ID NO: 31 or SEQ ID NO: 32),
[EQ)-X-Y-X(2)-C-X-[EHQN[FlLVFX-[ILVI (SEQ ID NO: 33), or
H-X(1,2)-G-P-X(3)-[YW]-[AILMV] (SEQ ID NO: 31 or SEQ ID Na: 32) and [EQ]-X-Y-
X(2)-C-X-FHQNHFILA-X-fILV] (SEQ ID NO: 33),
wherein X is any amino acid, X(1,2) is any amino acid at 1 position or 2
contiguous
positions, X(3) is any amino acid at 3 contiguous positions, and X(2) is any
amino acid at 2
contiguous positions. In the above motifs, the accepted IUPAC single letter
amino acid
abbreviation is employed.
In a preferred embodiment, the isolated GH61 polypeptide having cellulolytic
enhancing
activity further comprises H-X(1,2)-G-P-X(3)-[YWHAILMV] (SEQ ID NO: 31 or SEQ
ID NO: 32).
In another preferred embodiment, the isolated GH61 polypeptide having
cellulolytic enhancing
activity further comprises [EQ]-X-Y-X(2)-C-X-[EHQN]-[FILA-X-[ILV] (SEQ ID NO:
33). In
another preferred embodiment, the isolated GH61 polypeptide having
cellulolytic enhancing
activity further comprises H-X(1,2)-G-P-X(3)-[YW]-[AILMV] (SEQ ID NO: 31 or
SEQ ID NO: 32)
and [EQ]-X-Y-X(2)-C-X4EHQNHFILVI-X-[ILV] (SEQ ID NO: 33).
In a second aspect, isolated polypeptides having cellulolytic enhancing
activity, comprise
the following motif:
[ILMV]-P-X(4,5)-G-X-Y-[ILMA-X-R-X-[EQ]-X(3)-A-[HNQ] (SEQ ID NO: 34 or SEQ ID

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NO: 35),
wherein X is any amino acid, X(4,5) is any amino acid at 4 or 5 contiguous
positions, and
X(3) is any amino acid at 3 contiguous positions. In the above motif, the
accepted IUPAC single
letter amino acid abbreviation is employed.
Examples of GH61 polypeptides having cellulolytic enhancing activity useful in
the
methods of the present invention include, but are not limited to, GH61
polypeptides from
Thiela via tenestris (WO 2005/074647, WO 2008/148131, and WO 2011/035027),
Thermoascus
aurantiacus (WO 2005/074656 and WO 2010/065830), Trichoderma reesei (WO
2007/089290),
Myceliophthora thetmophila (VVO 2009/085935, WO 2009/085859, WO 2009/085864,
WO
2009/085868), Aspergillus fumigatus (WO 2010/138754), GH61 polypeptides from
Penicillium
pinophilum (WO 2011/005867), Thermoascus sp. (WO 2011/039319), Penicillium sp.
(WO
2011/041397), and Thermoascus crustaceous (VVO 2011/041504).
In one aspect, the GH61 polypeptide having cellulolytic enhancing activity is
used in the
presence of a soluble activating divalent metal cation according to WO
2008/151043, e.g.,
manganese or copper.
In another aspect, the GH61 polypeptide having cellulolytic enhancing activity
is used in
the presence of a dioxy compound, a bicylic compound, a heterocyclic compound,
a nitrogen-
containing compound, a quinone compound, a sulfur-containing compound, or a
liquor obtained
from a pretreated cellulosic material such as pretreated corn stover (PCS).
The dioxy compound may include any suitable compound containing two or more
oxygen atoms. In some aspects, the dioxy compounds contain a substituted aryl
moiety as
described herein. The dioxy compounds may comprise one or more (e.g., several)
hydroxyl
and/or hydroxyl derivatives, but also include substituted aryl moieties
lacking hydroxyl and
hydroxyl derivatives. Non-limiting examples of the dioxy compounds include
pyrocatechol or
catechol; caffeic acid; 3,4-dihydroxybenzoic acid; 4-tert-buty1-5-methoxy-1,2-
benzenediol;
pyrogallol; gallic acid; methyl-3,4,5-trihydroxybenzoate; 2,3,4-
trihydroxybenzophenone; 2,6-
dimethoxyphenol; sinapinic acid; 3,5-dihydroxybenzoic acid; 4-chloro-1,2-
benzenediol; 4-nitro-
1,2-benzenediol; tannic acid; ethyl gallate; methyl glycolate;
dihydroxyfumaric acid; 2-butyne-
1,4-diol; (croconic acid; 1,3-propanediol; tartaric acid; 2,4-pentanediol; 3-
ethyoxy-1,2-
propanediol; 2,4,4'-trihydroxybenzophenone; cis-2-butene-1,4-
diol; 3,4-dihydroxy-3-
cyclobutene-1,2-dione; dihydroxyacetone; acrolein acetal; methyl-4-
hydroxybenzoate; 4-
hydroxybenzoic acid; and methyl-3,5-dimethoxy-4-hydroxybenzoate; or a salt or
solvate thereof.
The bicyclic compound may include any suitable substituted fused ring system
as
described herein. The compounds may comprise one or more (e.g., several)
additional rings,

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and are not limited to a specific number of rings unless otherwise stated. In
one aspect, the
bicyclic compound is a flavonoid. In another aspect, the bicyclic compound is
an optionally
substituted isoflavonoid. In another aspect, the bicyclic compound is an
optionally substituted
flavylium ion, such as an optionally substituted anthocyanidin or optionally
substituted
5 anthocyanin, or derivative thereof. Non-limiting examples of the bicyclic
compounds include
epicatechin; quercetin; myricetin; taxifolin; kaempferol; morin; acacetin;
naringenin;
isorhamnetin; apigenin; cyanidin; cyanin; kuromanin; keracyanin; or a salt or
solvate thereof.
The heterocyclic compound may be any suitable compound, such as an optionally
substituted aromatic or non-aromatic ring comprising a heteroatom, as
described herein. In one
10 aspect, the heterocyclic is a compound comprising an optionally
substituted heterocycloalkyl
moiety or an optionally substituted heteroaryl moiety. In another aspect, the
optionally
substituted heterocycloalkyl moiety or optionally substituted heteroaryl
moiety is an optionally
substituted 5-membered heterocycloalkyl or an optionally substituted 5-
membered heteroaryl
moiety. In another aspect, the optionally substituted heterocycloalkyl or
optionally substituted
15 heteroaryl moiety is an optionally substituted moiety selected from
pyrazolyl, furanyl, imidazolyl,
isoxazolyl, oxadiazolyl, oxazolyl, pyrrolyl, pyridyl, pyrimidyl, pyridazinyl,
thiazolyl, triazolyl,
thienyl, dihydrothieno-pyrazolyl, thianaphthenyl, carbazolyl, benzimidazolyl,
benzothienyl,
benzofuranyl, indolyl, quinolinyl, benzotriazolyl, benzothiazolyl,
benzooxazolyl, benzimidazolyl,
isoquinolinyl, isoindolyl, acridinyl, benzoisazolyl, dimethylhydantoin,
pyrazinyl, tetrahydrofuranyl,
20 pyrrolinyl, pyrrolidinyl, morpholinyl, indolyl, diazepinyl, azepinyl,
thiepinyl, piperidinyl, and
oxepinyl. In another aspect, the optionally substituted heterocycloalkyl
moiety or optionally
substituted heteroaryl moiety is an optionally substituted furanyl. Non-
limiting examples of the
heterocyclic compounds include (1,2-dihydroxyethyl)-3,4-dihydroxyfuran-2(5H)-
one; 4-hydroxy-
5-methy1-3-furanone; 5-hydroxy-2(5H)-furanone; [1,2-dihydroxyethyl]furan-
2,3,4(5H)-trione; a-
25 hydroxy-y-butyrolactone; ribonic y-lactone; aldohexuronicaldohexuronic
acid y-lactone; gluconic
acid 5-lactone; 4-hydroxycoumarin; dihydrobenzofuran; 5-
(hydroxymethyl)furfural; furoin; 2(5H)-
furanone; 5,6-dihydro-2H-pyran-2-one; and 5,6-dihydro-4-hydroxy-6-methyl-2H-
pyran-2-one; or
a salt or solvate thereof.
The nitrogen-containing compound may be any suitable compound with one or more
30 nitrogen atoms. In one aspect, the nitrogen-containing compound
comprises an amine, imine,
hydroxylamine, or nitroxide moiety. Non-limiting examples of the nitrogen-
containing
compounds include acetone oxime; violuric acid; pyridine-2-aldoxime; 2-
aminophenol; 1,2-
benzenediamine; 2,2,6,6-tetramethy1-1-piperidinyloxy; 5,6,7,8-
tetrahydrobiopterin; 6,7-dimethy1-
5,6,7,8-tetrahydropterine; and maleamic acid; or a salt or solvate thereof.

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The quinone compound may be any suitable compound comprising a quinone moiety
as
described herein. Non-limiting examples of the quinone compounds include 1,4-
benzoquinone;
1,4-naphthoquinone; 2-hydroxy-1,4-naphthoquinone; 2,3-dimethwry-5-methyl-1,4-
benzoquinone
or coenzyme Q0; 2,3,5,6-tetramethy1-1,4-benzoquinone or duroquinone; 1,4-
dihydroxyanthraquinone; 3-hydroxy-1-methy1-5,6-indolinedione or adrenochrome;
4-tert-buty1-5-
methoxy-1,2-benzoquinone; pyrroloquinoline quinone; or a salt or solvate
thereof.
The sulfur-containing compound may be any suitable compound comprising one or
more
sulfur atoms. In one aspect, the sulfur-containing comprises a moiety selected
from thionyl,
thioether, sulfinyl, sulfonyl, sulfamide, sulfonamide, sulfonic acid, and
sulfonic ester. Non-limiting
examples of the sulfur-containing compounds include ethanethiol; 2-
propanethiol; 2-propene-1-
thiol; 2-mercaptoethanesulfonic acid; benzenethiol; benzene-1,2-dithiol;
cysteine; methionine;
glutathione; cystine; or a salt or solvate thereof.
In one aspect, an effective amount of such a compound described above to
cellulosic
material as a molar ratio to glucosyl units of cellulose is about 10-6 to
about 10, e.g., about 10-6
to about 7.5, about 10-6 to about 5, about 10-6 to about 2.5, about 10-6 to
about 1, about 10-5 to
about 1, about 10-5 to about 10-1, about 10-4 to about 10-1, about 10-3 to
about 101, or about 10 3
to about 10-2. In another aspect, an effective amount of such a compound
described above is
about 0.1 pM to about 1 M, e.g., about 0.5 pM to about 0.75 M, about 0.75 pM
to about 0.5 M,
about 1 pM to about 0.25 M, about 1 pM to about 0.1 M, about 5 pM to about 50
mM, about 10
pM to about 25 mM, about 50 pM to about 25 mM, about 10 pM to about 10 mM,
about 5 pM to
about 5 mM, or about 0.1 mM to about 1 mM.
The term "liquor" means the solution phase, either aqueous, organic, or a
combination
thereof, arising from treatment of a lignocellulose and/or hemicellulose
material in a slurry, or
monosaccharides thereof, e.g., xylose, arabinose, mannose, etc., under
conditions as described
herein, and the soluble contents thereof. A liquor for cellulolytic
enhancement of a GH61
polypeptide can be produced by treating a lignocellulose or hemicellulose
material (or
feedstock) by applying heat and/or pressure, optionally in the presence of a
catalyst, e.g., acid,
optionally in the presence of an organic solvent, and optionally in
combination with physical
disruption of the material, and then separating the solution from the residual
solids. Such
conditions determine the degree of cellulolytic enhancement obtainable through
the combination
of liquor and a GH61 polypeptide during hydrolysis of a cellulosic substrate
by a cellulase
preparation. The liquor can be separated from the treated material using a
method standard in
the art, such as filtration, sedimentation, or centrifugation.
In one aspect, an effective amount of the liquor to cellulose is about 10-6 to
about 10 g

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per g of cellulose, e.g., about 10-6 to about 7.5 g, about 10-6 to about 5,
about 10-5 to about 2.5
g, about 10-6 to about 1 g, about 10-5 to about 1 g, about 10-5 to about 10-
1g, about 10-4 to about
10-1g, about 10-3 to about 10-1g, or about 10-3 to about 10-2 g per g of
cellulose.
In one aspect, the one or more (e.g., several) hemicellulolytic enzymes
comprise a
commercial hemicellulolytic enzyme preparation. Examples of commercial
hemicellulolytic
enzyme preparations suitable for use in the present invention include, for
example,
SHEARZYMETm (Novozymes A/S), CELLIC HTec (Novozymes A/S), CELLIC HTec2
(Novozymes A/S), CELLIC HTec3 (Novozymes A/S), VISCOZYMEC) (Novozymes A/S),
ULTRAFLO (Novozymes A/S), PULPZYME HC (Novozymes A/S), MULTIFECTC) Xylanase
(Genencor), ACCELLERASE XY (Genencor), ACCELLERASE XC (Genencor), ECOPULP
TX-200A (AB Enzymes), HSP 6000 Xylanase (DSM), DEPOLTm 333P (Biocatalysts
Limit, Wales,
UK), DEPOLTm 740L. (Biocatalysts Limit, Wales, UK), and DEPOLTm 762P
(Biocatalysts Limit,
Wales, UK).
Examples of xylanases useful in the processes of the present invention
include, but are
not limited to, xylanases from Aspergillus aculeatus (GeneSeqP:AAR63790; WO
94/21785),
Aspergillus fumigatus (WO 2006/078256), Penicillium pinophilum (WO
2011/041405),
Penicillium sp. (WO 2010/126772), Thielavia ter-rest/is NRRL 8126 ONO
2009/079210), and
Trichophaea saccata GH10 (WO 2011/057083).
Examples of beta-xylosidases useful in the processes of the present invention
include,
but are not limited to, beta-xylosidases from Neurospora crassa (SwissProt
accession number
Q7SOW4), Trichoderma reesei (UniProtKB/TrEMBL accession number Q92458), and
Talaromyces emersonfi (SwissProt accession number Q8X212).
Examples of acetylxylan esterases useful in the processes of the present
invention
include, but are not limited to, acetylxylan esterases from Aspergillus
aculeatus ONO
2010/108918), Chaetomium globosum (Uniprot accession number Q2GVVX4),
Chaetomium
gracile (GeneSeqP accession number AAB82124), Humicola insolens DSM 1800 (WO
2009/073709), Hypocrea jecorina (WO 2005/001036), Myceliophtera thermophila
ONO
2010/014880), Neurospora crassa (UniProt accession number q7s259),
Phaeosphaeria
nodorum (Uniprot accession number QOUHJ1), and Thielavia terrestris NRRL 8126
(VVO
2009/042846).
Examples of feruloyl esterases (ferulic acid esterases) useful in the
processes of the
present invention include, but are not limited to, feruloyl esterases form
Humicola insolens DSM
1800 (WO 2009/076122), Neosartorya fischeri (UniProt Accession number A1D9T4),
Neurospora crassa (UniProt accession number Q9HGR3), Penicillium
aurantiogriseum ono

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2009/127729), and Thielavia terrestris (WO 2010/053838 and WO 2010/065448).
Examples of arabinofuranosidases useful in the processes of the present
invention
include, but are not limited to, arabinofuranosidases from Aspergillus niger
(GeneSeqP
accession number AAR94170), Humicola insolens DSM 1800 (WO 2006/114094 and WO
2009/073383), and M giganteus (WO 2006/114094).
Examples of alpha-glucuronidases useful in the processes of the present
invention
include, but are not limited to, alpha-glucuronidases from Aspergillus
clavatus (UniProt
accession number alcc12), Aspergillus fumigatus (SwissProt accession number
Q4VVW45),
Aspergillus niger (Uniprot accession number Q96WX9), Aspergillus terreus
(SwissProt
accession number Q0CJP9), Humicola insolens (WO 2010/014706), Penicillium
aurantiogriseum (WO 2009/068565), Ta/ammyces emersonii (UniProt accession
number
Q8X211), and Trichoderma reesei (Uniprot accession number Q99024).
The polypeptides having enzyme activity used in the processes of the present
invention
may be produced by fermentation of the above-noted microbial strains on a
nutrient medium
containing suitable carbon and nitrogen sources and inorganic salts, using
procedures known in
the art (see, e.g., Bennett, J.W. and LaSure, L. (eds.), More Gene
Manipulations in Fungi,
Academic Press, CA, 1991). Suitable media are available from commercial
suppliers or may be
prepared according to published compositions (e.g., in catalogues of the
American Type Culture
Collection). Temperature ranges and other conditions suitable for growth and
enzyme
production are known in the art (see, e.g., Bailey, J.E., and 011is, D.F.,
Biochemical Engineering
Fundamentals, McGraw-Hill Book Company, NY, 1986).
The fermentation can be any method of cultivation of a cell resulting in the
expression or
isolation of an enzyme or protein. Fermentation may, therefore, be understood
as comprising
shake flask cultivation, or small- or large-scale fermentation (including
continuous, batch, fed-
batch, or solid state fermentations) in laboratory or industrial ferrnentors
performed in a suitable
medium and under conditions allowing the enzyme to be expressed or isolated.
The resulting
enzymes produced by the methods described above may be recovered from the
fermentation
medium and purified by conventional procedures.
Fermentation. The fermentable sugars obtained from the hydrolyzed cellulosic
material
can be fermented by one or more (e.g., several) fermenting microorganisms
capable of
fermenting the sugars directly or indirectly into a desired fermentation
product. "Fermentation" or
"fermentation process" refers to any fermentation process or any process
comprising a
fermentation step. Fermentation processes also include fermentation processes
used in the
consumable alcohol industry (e.g., beer and wine), dairy industry (e.g.,
fermented dairy

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products), leather industry, and tobacco industry. The fermentation conditions
depend on the
desired fermentation product and fermenting organism and can easily be
determined by one
skilled in the art.
In the fermentation step, sugars, released from the cellulosic material as a
result of the
pretreatment and enzymatic hydrolysis steps, are fermented to a product, e.g.,
ethanol, by a
fermenting organism, such as yeast. Hydrolysis (saccharification) and
fermentation can be
separate or simultaneous, as described herein.
Any suitable hydrolyzed cellulosic material can be used in the fermentation
step in
practicing the present invention. The material is generally selected based on
the desired
fermentation product, i.e., the substance to be obtained from the
fermentation, and the process
employed, as is well known in the art.
The term "fermentation medium" is understood herein to refer to a medium
before the
fermenting microorganism(s) is(are) added, such as, a medium resulting from a
saccharification
process, as well as a medium used in a simultaneous saccharification and
fermentation process
(SSF).
"Fermenting microorganism" refers to any microorganism, including bacterial
and fungal
organisms, suitable for use in a desired fermentation process to produce a
fermentation product.
The fermenting organism can be hexose and/or pentose fermenting organisms, or
a combination
thereof. Both hexose and pentose fermenting organisms are well known in the
art. Suitable
fermenting microorganisms are able to ferment, i.e., convert, sugars, such as
glucose, xylose,
xylulose, arabinose, maltose, mannose, galactose, and/or oligosaccharides,
directly or indirectly
into the desired fermentation product. Examples of bacterial and fungal
fermenting organisms
producing ethanol are described by Lin etal., 2006, App!. Microbiol.
Biotechnol. 69: 627-642.
Examples of fermenting microorganisms that can ferment hexose sugars include
bacterial and fungal organisms, such as yeast. Preferred yeast includes
strains of Candida,
Kluyveromyces, and Saccharomyces, e.g., Candida sonorensis, Kluyveromyces
marxianus, and
Saccharomyces cerevisiae.
Examples of fermenting organisms that can ferment pentose sugars in their
native state
include bacterial and fungal organisms, such as some yeast. Preferred xylose
fermenting yeast
include strains of Candida, preferably C. sheatae or C. sonorensis; and
strains of Pichia, preferably
P. stipitis, such as P. stipitis CBS 5773. Preferred pentose fermenting yeast
include strains of
Pachysolen, preferably P. tannophilus. Organisms not capable of fermenting
pentose sugars, such
as xylose and arabinose, may be genetically modified to do so by methods known
in the art
Examples of bacteria that can efficiently ferment hexose and pentose to
ethanol include,

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for example, Bacillus coagulans, Clostridium acetobutylicum, Clostridium
the rmocellum,
Clostridium phytofermentans, Geobacillus sp., Thermoanaerobacter
saccharolyticum, and
Zymomonas mobfiis (Philippidis, 1996, supra).
Other fermenting organisms include strains of Bacillus, such as Bacillus
coagulans;
5 Candida, such as C. sonorensis, C. methanosorbosa, C. diddensiae, C.
parapsilosis, C.
naedodendra, C. blankii, C. entomophilia, C. brassicae, C. pseudotropicalis,
C. boidinii, C. utills,
and C. scehatae; Clostridium, such as C. acetobutylicum, C. therrnocefium, and
C.
phytofermentans; E. coil, especially E coil strains that have been genetically
modified to improve
the yield of ethanol; Geobacifius sp.; Hansenula, such as Hansenula anomala;
Klebsiella, such
10 as K. oxytoca; Kluyvernmyces, such as K. marxianus, K lactis, K
therrnotolerans, and K. fragilis;
Schizosaccharnmyces, such as S. pombe; Thermoanaerobacter, such as
Thermoanaerobacter
saccharolyticum; and Zymomonas, such as Zymomonas mobil/s.
In a preferred aspect, the yeast is a Bretannomyces. In a more preferred
aspect, the
yeast is Bretannomyces clausenfi. In another preferred aspect, the yeast is a
Candida. In
15 another more preferred aspect, the yeast is Candida sonorensis. In
another more preferred
aspect, the yeast is Candida boidinii. In another more preferred aspect, the
yeast is Candida
blankii. In another more preferred aspect, the yeast is Candida brassicae. In
another more
preferred aspect, the yeast is Candida diddensii. In another more preferred
aspect, the yeast is
Candida entomophiliia. In another more preferred aspect, the yeast is Candida
pseudotropicalis.
20 In another more preferred aspect, the yeast is Candida scehatae. In
another more preferred
aspect, the yeast is Candida utilis. In another preferred aspect, the yeast is
a Clavispora. In
another more preferred aspect, the yeast is Clavispora lusitaniae. In another
more preferred
aspect, the yeast is Clavispora opuntiae. In another preferred aspect, the
yeast is a
Kluyveromyces. In another more preferred aspect, the yeast is Kluyveromyces
fragilis. In
25 another more preferred aspect, the yeast is Kluyveromyces marxianus. In
another more
preferred aspect, the yeast is Kluyveromyces thermotolerans. In another
preferred aspect, the
yeast is a Pachysolen. In another more preferred aspect, the yeast is
Pachysolen tannophilus.
In another preferred aspect, the yeast is a Pichia. In another more preferred
aspect, the yeast is
a Pichia stipitis. In another preferred aspect, the yeast is a Saccharomyces
spp. In another
30 more preferred aspect, the yeast is Saccharomyces cerevisiae. In another
more preferred
aspect, the yeast is Saccharomyces distaticus. In another more preferred
aspect, the yeast is
Saccharomyces uvarum.
In a preferred aspect, the bacterium is a Bacillus. In a more preferred
aspect, the
bacterium is Bacillus coagulans. In another preferred aspect, the bacterium is
a Clostridium. In

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another more preferred aspect, the bacterium is Clostridium acetobutylicum. In
another more
preferred aspect, the bacterium is Clostridium phytofermentans. In another
more preferred
aspect, the bacterium is Clostridium thermocellum. In another more preferred
aspect, the
bacterium is Geobacilus sp. In another more preferred aspect, the bacterium is
a
Thermoanaerobacter. In another more preferred aspect, the bacterium is
Thermoanaerobacter
saccharolyticum. In another preferred aspect, the bacterium is a Zymomonas. In
another more
preferred aspect, the bacterium is Zymomonas mobilis.
Commercially available yeast suitable for ethanol production include, e.g.,
BIOFERMTm
AFT and XR (NABC - North American Bioproducts Corporation, GA, USA), ETHANOL
REDTM
yeast (Fermentis/Lesaffre, USA), FALITm (Fleischmann's Yeast, USA), FERMIOLTm
(DSM
Specialties), GERT STRAND Tm (Gert Strand AB, Sweden), and SUPERSTARTT1A and
THERMOSACCTm fresh yeast (Ethanol Technology, WI, USA).
In a preferred aspect, the fermenting microorganism has been genetically
modified to
provide the ability to ferment pentose sugars, such as xylose utilizing,
arabinose utilizing, and
xylose and arabinose co-utilizing microorganisms.
The cloning of heterologous genes into various fermenting microorganisms has
led to
the construction of organisms capable of converting hexoses and pentoses to
ethanol (co-
fermentation) (Chen and Ho, 1993, Cloning and improving the expression of
Pichia stipitis
xylose reductase gene in Saccharomyces cerevisiae, App!. Biochem. BiotechnoL
39-40: 135-
147; Ho et a/., 1998, Genetically engineered Saccharomyces yeast capable of
effectively
cofermenting glucose and xylose, App!. Environ. MicrobioL 64: 1852-1859;
Kotter and Ciriacy,
1993, Xylose fermentation by Saccharomyces cerevisiae, App!. MicrobioL
Biotechnol 38: 776-
783; Walfridsson et al., 1995, Xylose-metabolizing Saccharomyces cerevisiae
strains
overexpressing the TKL1 and TAL1 genes encoding the pentose phosphate pathway
enzymes
transketolase and transaldolase, AppL Environ. MicrobioL 61: 4184-4190; Kuyper
et al., 2004,
Minimal metabolic engineering of Saccharomyces cerevisiae for efficient
anaerobic xylose
fermentation: a proof of principle, FEMS Yeast Research 4: 655-664; Beall et
aL, 1991,
Parametric studies of ethanol production from xylose and other sugars by
recombinant
Escherichia coli, Biotech. Bioeng. 38: 296-303; Ingram et al., 1998, Metabolic
engineering of
bacteria for ethanol production, BiotechnoL Bioeng. 58: 204-214; Zhang etal.,
1995, Metabolic
engineering of a pentose metabolism pathway in ethanologenic Zymomonas
mobilis, Science
267: 240-243; Deanda et al., 1996, Development of an arabinose-fermenting
Zymomonas
mobilis strain by metabolic pathway engineering, App!. Environ. MicrobioL 62:
4465-4470; WO
2003/062430, xylose isomerase).

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In a preferred aspect, the genetically modified fermenting microorganism is
Candida
sonorensis. In another preferred aspect, the genetically modified fermenting
microorganism is
Escherichia colt. In another preferred aspect, the genetically modified
fermenting microorganism
is Klebsiella oxytoca. In another preferred aspect, the genetically modified
fermenting
microorganism is Kluyverornyces marxianus. In another preferred aspect, the
genetically
modified fermenting microorganism is Saccharomyces cerevisiae. In another
preferred aspect,
the genetically modified fermenting microorganism is Zymomonas mobilis.
It is well known in the art that the organisms described above can also be
used to
produce other substances, as described herein.
The fermenting microorganism is typically added to the degraded cellulosic
material or
hydrolysate and the fermentation is performed for about 8 to about 96 hours,
e.g., about 24 to
about 60 hours. The temperature is typically between about 26 C to about 60 C,
e.g., about
32 C or 50 C, and about pH 3 to about pH 8, e.g., pH 4-5, 6, or 7.
In one aspect, the yeast and/or another microorganism are applied to the
degraded
cellulosic material and the fermentation is performed for about 12 to about 96
hours, such as
typically 24-60 hours. In another aspect, the temperature is preferably
between about 20 C to
about 60 C, e.g., about 25 C to about 50 C, about 32 C to about 50 C, or about
32 C to about
50 C, and the pH is generally from about pH 3 to about pH 7, e.g., about pH 4
to about pH 7.
However, some fermenting organisms, e.g., bacteria, have higher fermentation
temperature
optima. Yeast or another microorganism is preferably applied in amounts of
approximately 105
to 1012, preferably from approximately 107 to 1010, especially approximately 2
x 108 viable cell
count per ml of fermentation broth. Further guidance in respect of using yeast
for fermentation
can be found in, e.g., "The Alcohol Textbook" (Editors K. Jacques, T.P. Lyons
and D.R. Kelsall,
Nottingham University Press, United Kingdom 1999), which is hereby
incorporated by reference.
A fermentation stimulator can be used in combination with any of the processes
described herein to further improve the fermentation process, and in
particular, the performance
of the fermenting microorganism, such as, rate enhancement and ethanol yield.
A "fermentation
stimulator" refers to stimulators for growth of the fermenting microorganisms,
in particular, yeast.
Preferred fermentation stimulators for growth include vitamins and minerals.
Examples of
vitamins include multivitamins, biotin, pantothenate, nicotinic acid, meso-
inositol, thiamine,
pyridoxine, para-aminobenzoic acid, folic acid, riboflavin, and Vitamins A, B,
C, D, and E. See,
for example, Alfenore et at, 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

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salts that can supply nutrients comprising P, K, Mg, S, Ca, Fe, Zn, Mn, and
Cu.
Fermentation products: A fermentation product can be any substance derived
from the
fermentation. The fermentation product can be, without limitation, an alcohol
(e.g., arabinitol, n-
butanol, isobutanol, ethanol, glycerol, methanol, ethylene glycol, 1,3-
propanediol [propylene
glycol], butanediol, glycerin, sorbitol, and xylitol); an alkane (e.g.,
pentane, hexane, heptane,
octane, nonane, decane, undecane, and dodecane), a cycloalkane (e.g.,
cyclopentane,
cyclohexane, cycloheptane, and cyclooctane), an alkene (e.g. pentene, hexene,
heptene, and
octene); an amino acid (e.g., aspartic acid, glutamic acid, glycine, lysine,
serine, and threonine);
a gas (e.g., methane, hydrogen (H2), carbon dioxide (CO2), and carbon monoxide
(CO));
isoprene; a ketone (e.g., acetone); an organic acid (e.g., acetic acid,
acetonic acid, adipic acid,
ascorbic acid, citric acid, 2,5-diketo-D-gluconic acid, formic acid, fumaric
acid, glucaric acid,
gluconic acid, glucuronic acid, glutaric acid, 3-hydroxypropionic acid,
itaconic acid, lactic acid,
malic acid, malonic acid, oxalic acid, oxaloacetic acid, propionic acid,
succinic acid, and xylonic
acid); and polyketide. The fermentation product can also be protein as a high
value product.
In a preferred aspect, the fermentation product is an alcohol. It will be
understood that
the term "alcohol" encompasses a substance that contains one or more hydroxyl
moieties. In a
more preferred aspect, the alcohol is n-butanol. In another more preferred
aspect, the alcohol is
isobutanol. In another more preferred aspect, the alcohol is ethanol. In
another more preferred
aspect, the alcohol is methanol. In another more preferred aspect, the alcohol
is arabinitol. In
another more preferred aspect, the alcohol is butanediol. In another more
preferred aspect, the
alcohol is ethylene glycol. In another more preferred aspect, the alcohol is
glycerin. In another
more preferred aspect, the alcohol is glycerol. In another more preferred
aspect, the alcohol is
1,3-propanediol. In another more preferred aspect, the alcohol is sorbitol. In
another more
preferred aspect, the alcohol is xylitol. See, for example, Gong, C. S., Cao,
N. J., Du, J., and
Tsao, G. T., 1999, Ethanol production from renewable resources, in Advances in
Biochemical
Engineering/Biotechnology, Scheper, T., ed., Springer-Verlag Berlin
Heidelberg, Germany, 65:
207-241; Silveira, M. M., and Jonas, R., 2002, The biotechnological production
of sorbitol, App!.
Microbiol. Biotechnol. 59: 400-408; Nigam, P., and Singh, D., 1995, Processes
for fermentative
production of xylitol ¨ a sugar substitute, Process Biochemistry 30 (2): 117-
124; Ezeji, T. C.,
Qureshi, N. and Blaschek, H. P., 2003, Production of acetone, butanol and
ethanol by
Clostridium beijerinckii BA101 and in situ recovery by gas stripping, World
Journal of
Microbiology and Biotechnology 19 (6): 595-603.
In another preferred aspect, the fermentation product is an alkane. The alkane
can be an
unbranched or a branched alkane. In another more preferred aspect, the alkane
is pentane. In

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another more preferred aspect, the alkane is hexane. In another more preferred
aspect, the
alkane is heptane. In another more preferred aspect, the alkane is octane. In
another more
preferred aspect, the alkane is nonane. In another more preferred aspect, the
alkane is decane.
In another more preferred aspect, the alkane is undecane. In another more
preferred aspect,
the alkane is dodecane.
In another preferred aspect, the fermentation product is a cycloalkane. In
another more
preferred aspect, the cycloalkane is cyclopentane. In another more preferred
aspect, the
cycloalkane is cyclohexane. In another more preferred aspect, the cycloalkane
is cycloheptane.
In another more preferred aspect, the cycloalkane is cyclooctane.
In another preferred aspect, the fermentation product is an alkene. The alkene
can be an
unbranched or a branched alkene. In another more preferred aspect, the alkene
is pentene. In
another more preferred aspect, the alkene is hexene. In another more preferred
aspect, the
alkene is heptene. In another more preferred aspect, the alkene is octene.
In another preferred aspect, the fermentation product is an amino acid. In
another more
preferred aspect, the organic acid is aspartic acid. In another more preferred
aspect, the amino
acid is glutamic acid. In another more preferred aspect, the amino acid is
glycine. In another
more preferred aspect, the amino acid is lysine. In another more preferred
aspect, the amino
acid is serine. In another more preferred aspect, the amino acid is threonine.
See, for example,
Richard, A., and Margaritis, A., 2004, Empirical modeling of batch
fermentation kinetics for
poly(glutamic acid) production and other microbial biopolymers, Biotechnology
and
Bioengineering 87 (4): 501-515.
In another preferred aspect, the fermentation product is a gas. In another
more preferred
aspect, the gas is methane. In another more preferred aspect, the gas is H2.
In another more
preferred aspect, the gas is CO2. In another more preferred aspect, the gas is
CO. See, for
example, Kataoka, N., A. Miya, and K. Kiriyama, 1997, Studies on hydrogen
production by
continuous culture system of hydrogen-producing anaerobic bacteria, Water
Science and
Technology 36 (6-7): 41-47; and Gunaseelan V.N. in Biomass and Bioenergy, Vol.
13 (1-2), pp.
83-114, 1997, Anaerobic digestion of biomass for methane production: A review.
In another preferred aspect, the fermentation product is isoprene.
In another preferred aspect, the fermentation product is a ketone. It will be
understood
that the term "ketone" encompasses a substance that contains one or more
ketone moieties. In
another more preferred aspect, the ketone is acetone. See, for example,
Qureshi and Blaschek,
2003, supra.
In another preferred aspect, the fermentation product is an organic acid. In
another more

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preferred aspect, the organic acid is acetic acid. In another more preferred
aspect, the organic
acid is acetonic acid. In another more preferred aspect, the organic acid is
adipic acid. In
another more preferred aspect, the organic acid is ascorbic acid. In another
more preferred
aspect, the organic acid is citric acid. In another more preferred aspect, the
organic acid is 2,5-
5 diketo-D-gluconic acid. In another more preferred aspect, the organic
acid is formic acid. In
another more preferred aspect, the organic acid is fumaric acid. In another
more preferred
aspect, the organic acid is glucaric acid. In another more preferred aspect,
the organic acid is
gluconic acid. In another more preferred aspect, the organic acid is
glucuronic acid. In another
more preferred aspect, the organic acid is glutaric acid. In another preferred
aspect, the organic
10 acid is 3-hydroxpropionic acid. In another more preferred aspect, the
organic acid is itaconic
acid. In another more preferred aspect, the organic acid is lactic acid. In
another more preferred
aspect, the organic acid is malic acid. In another more preferred aspect, the
organic acid is
malonic acid. In another more preferred aspect, the organic acid is oxalic
acid. In another more
preferred aspect, the organic acid is propionic acid. In another more
preferred aspect, the
15 organic acid is succinic acid. In another more preferred aspect, the
organic acid is xylonic acid.
See, for example, Chen, R., and Lee, Y. Y., 1997, Membrane-mediated extractive
fermentation
for lactic acid production from cellulosic biomass, AppL Biochem. Biotechnol
63-65: 435-448.
In another preferred aspect, the fermentation product is polyketide.
Recovery. The fermentation product(s) can be optionally recovered from the
fermentation
20 medium using any method known in the art including, but not limited to,
chromatography,
electrophoretic procedures, differential solubility, distillation, or
extraction. For example, alcohol
is separated from the fermented cellulosic material and purified by
conventional methods of
distillation. Ethanol with a purity of up to about 96 vol.13/0 can be
obtained, which can be used as,
for example, fuel ethanol, drinking ethanol, i.e., potable neutral spirits, or
industrial ethanol.
Signal Peptides
The present invention also relates to isolated polynucleotides encoding a
signal peptide
comprising or consisting of amino acids 1 to 17 of SEQ ID NO: 2, amino acids 1
to 20 of SEQ ID
NO: 4, amino acids 1 to 21 of SEQ ID NO: 6 or amino acids 1 to 17 of SEQ ID
NO: 8. The
polynucleotide may further comprise a gene encoding a protein, which is
operably linked to the
signal peptide. The protein is preferably foreign to the signal peptide. In
one aspect, the
polynucleotide encoding the signal peptide is nucleotides 1 to 51 of SEQ ID
NO: 1. In another
aspect, the polynucleotide encoding the signal peptide is nucleotides 1 to 60
of SEQ ID NO: 3.
In another aspect, the polynucleotide encoding the signal peptide is
nucleotides 1 to 63 of SEQ

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ID NO: 5. In another aspect, the polynucleotide encoding the signal peptide is
nucleotides 1 to
51 of SEQ ID NO: 7.
The present invention also relates to nucleic acid constructs, expression
vectors and
recombinant host cells comprising such polynucleotides.
The present invention also relates to methods of producing a protein,
comprising (a)
cultivating a recombinant host cell comprising such a polynucleotide operably
linked to a gene
encoding the protein; and optionally (b) recovering the protein.
The protein may be native or heterologous to a host cell. The term "protein"
is not meant
herein to refer to a specific length of the encoded product and, therefore,
encompasses
peptides, oligopeptides, and polypeptides. The term "protein" also encompasses
two or more
polypeptides combined to form the encoded product. The proteins also include
hybrid
polypeptides and fused polypeptides.
Preferably, the protein is a hormone, enzyme, receptor or portion thereof,
antibody or
portion thereof, or reporter. For example, the protein may be a hydrolase,
isomerase, ligase,
lyase, oxidoreductase, or transferase, e.g., an alpha-galactosidase, alpha-
glucosidase,
aminopeptidase, amylase, beta-galactosidase, beta-
glucosidase, beta-xylosidase,
carbohydrase, carboxypeptidase, catalase, cellobiohydrolase, cellulase,
chitinase, cutinase,
cyclodextrin glycosyltransferase, demribonuclease, endoglucanase, esterase,
glucoamylase,
invertase, laccase, lipase, mannosidase, mutanase, oxidase, pectinolytic
enzyme, peroxidase,
phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease,
transglutaminase, or xylanase.
The gene may be obtained from any prokaryotic, eukaryotic, or other source.
The present invention is further described by the following examples that
should not be
construed as limiting the scope of the invention.
Examples
Strain
The fungal strain NN044758 was isolated from a soil sample collected from
China by the
dilution plate method with PDA medium at 45 C. It was then purified by
transferring a single
conidium onto a YG agar plate. The strain NN044758 was identified as
Malbranchea
cinnamomea, based on both morphological characteristics and ITS rDNA sequence.
The fungal strain NN000308 was purchased from Centraalbureau voor
Schimmelcultures named as CBS174.70. The strain NN000308 was identified as
Colynascus

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thermophilus (previously identified as Thielavia thermophila, - syn.
Myceliophthora fergusii),
based on both morphological characteristics and ITS rDNA sequence.
Media
FDA medium was composed of 39 grams of potato dextrose agar and deionized
water
to 1 liter.
YG agar plates were composed of 5.0 g of yeast extract, 10.0 g of glucose,
20.0 g of
agar, and deionized water to 1 liter.
YPG medium was composed of 0.4% of yeast extract, 0.1% of KH2PO4, 0.05% of
MgSO4 =7H20, 1.5% glucose in deionized water.
YPM medium was composed of 1% yeast extract, 2% of peptone, and 2% of maltose
in
deionized water.
Minimal medium plates were composed of 342 g of sucrose, 20 ml of salt
solution, 20 g
of agar, and deionized water to 1 liter. The salt solurtion was composed of
2.6% KCI, 2.6%
MgSO4.7H20, 7.6% KH2PO4, 2 ppm Na2B407.10H20, 20 ppm CuSO4.5H20, 40 ppm
FeSO4=7H20,
40 ppm MnSar2H20, 40 ppm Na2Mo04.2H20, and 400 ppm ZnSO4.7H20.
Example 1: Malbranchea cinnamomea genomic DNA extraction
Malbranchea cinnamomea strain NN044758 was inoculated onto a FDA plate and
incubated for 3 days at 45 C in the darkness. Several mycelia-PDA plugs were
inoculated into
500 ml shake flasks containing 100 ml of YPG medium. The flasks were incubated
for 3 days at
45 C with shaking at 160 rpm. The mycelia were collected by filtration through
MIRACLOTHO
(Calbiochem, La Jolla, CA, USA) and frozen under liquid nitrogen. Frozen
mycelia were ground,
by a mortar and a pestle, to a fine powder, and genomic DNA was isolated using
Large-Scale
Column Fungal DNAout (BAOMAN BIOTECHNOLOGY, Shanghai, China) following the
manufacturer's instruction.
Example 2: Genome sequencing, assembly and annotation
The extracted genomic DNA samples were delivered to Beijing Genome Institute
(BGI,
Shenzhen, China) for genome sequencing using an ILLUMINA@ GA2 System
(Illumine,
Inc., San Diego, CA, USA). The raw reads were assembled at BGI using program
SOAPdenovo
(Li et al., 2010, Genome Research 20(2): 265-72). The assembled sequences were
analyzed
using standard bioinformatics methods for gene identification and functional
prediction. GenelD
(Parra etal., 2000, Genome Research 10(4): 511-515) was used for gene
prediction. Blastall

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version 2.2.10 (Altschul et al., 1990, J. Mol. Biol. 215(3): 403-410, National
Center for
Biotechnology Information (NCB!), Bethesda, MD, USA) and HMMER version 2.1.1
(National
Center for Biotechnology Information (NCB!), Bethesda, MD, USA) were used to
predict function
based on structural homology. The GH7 family cellobiohydrolase polypeptides
were identified
directly by analysis of the Blast results. The Agene program (Munch and Krogh,
2006, BMC
Bioinformatics 7: 263) and SignalP program (Nielsen etal., 1997, Protein
Engineering 10: 1-6)
were used to identify starting codons. The SignalP program was further used to
predict signal
peptides. Pepstats (Rice et al., 2000, Trends Genet. 16(6): 276-277) was used
to predict
isoelectric points and molecular weights of the deduced amino acid sequences.
Example 3: Characterization of a Malbranchea cinnamomea genomic sequence
encoding
a polypeptide having cellobiohydrolase (CBH) activity
The genomic DNA sequence (SEQ ID NO: 1) and deduced amino acid sequence (SEQ
ID NO: 2) of the Malbranchea cinnamomea polypeptide coding sequence is shown
in Figure 1.
The coding sequence is 1457 bp including the stop codon and is interrupted by
1 intron of 80 bp
(nucleotides 613 to 692). The G+C content of the mature polypeptide coding
sequence without
intron and stop condon is 52.38%. The encoded predicted protein is 458 amino
acids. Using the
SignalP program (Nielsen et al., 1997, Protein Engineering 10: 1-6), a signal
peptide of 17
residues was predicted. The predicted mature protein contains 441 amino acids
with a predicted
molecular weight of 47890.24 Dalton and predicted isoelectric point of 4.12.
The
cellobiohydrolase catalytic domain was predicted to be amino acids 18 to 458,
by aligning the
amino acid sequence using BLAST to all CAZY-defined subfamily modules
(Cantarel et al.,
2009, Nucleic Acids Res. 37: D233-238), where the single most significant
alignment within a
subfamily was used to predict the GH7 domain.
A comparative pairwise global alignment of amino acid sequences was determined
using
the Needleman and Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol Biol.
48: 443-
453) with gap open penalty of 10, gap extension penalty of 0.5, and the
EBLOSUM62 matrix.
The alignment showed that the mature part of the amino acid sequence of the
Malbranchea
cinnamomea coding sequence encoding the cellobiohydrolase polypeptide shares
69.23%
identity to the deduced amino acid sequence of a cellobiohydrolase A protein
from Emericella
nidulans (accession number UNIPROT:Q532Q4).
Example 4: Cloning of the Malbranchea cinnamomea GH7 cellobiohydrolase (CBH)
gene
from genomic DNA

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The GH7 CBH gene, GH7_ZY582279_485, was selected for cloning.
Based on DNA information (SEQ ID NO: 1) obtained from genome sequencing,
oligonucleotide primers, shown below in Table 1, were designed to amplify the
CBH gene from
genomic DNA of Malbranchea cinnamomea NN044758. Primers were synthesized by
Invitrogen, Beijing, China.
Table 1: primers
Forward primer ACACAACTGGGGATCCACCatgcatcgccaactcgctc SEQ ID NO: 9
Reverse primer GTCACCCTCTAGATCTgacacgcagcatgctaggagac SEQ ID NO: 10
Lowercase characters represent the coding regions of the genes in forward
primers and
the flanking region of the gene in reverse primers, while capitalized parts
were homologous to
the insertion sites of pPFJ0355 vector which has been described in
W02011005867.
For each gene, 20 picomoles of each forward and reverse primer pair were used
in a
PCR reaction composed of 2 pl of Malbranchea cinnamomea NN044758 genomic DNA,
10 pl of
5X GC Buffer (Finnzymes Oy, Espoo, Finland), 1.5 pl of dimethyl sulphoxide
(DMSO), 2.5 mM
each of dATP, dTTP, dGTP, and dCTP, and 0.6 unit of PHUSIONTm High-Fidelity
DNA
Polymerase (Finnzymes Oy, Espoo, Finland) in a final volume of 50 pl. The
amplification was
performed using a Peltier Thermal Cycler (M J Research Inc., South San
Francisco, CA, USA)
programmed for denaturing at 94 C for 1 minute; 6 cycles of denaturing at 94 C
for 15 seconds,
annealing at 68 C for 30 seconds, with a 1 C decrease per cycle, and
elongation at 72 C for
100 seconds; 23 cycles each at 94 C for 15 seconds, 63 C for 30 seconds, and
72 C for 100
seconds; and a final extension at 72 C for 5 minutes. The heat block then went
to a 4 C soak
cycle.
The PCR products were isolated by 1.0% agarose gel electrophoresis using 90 mM
Tris-
borate and 1 mM EDTA (TBE) buffer where a single product band around the
expected size, 1.5
kb, was visualized under UV light. The PCR products were then purified from
solution by using
an ILLUSTRA GFX PCR DNA and Gel Band Purification Kit (GE Healthcare,
Buckinghamshire, UK) according to the manufacturers instructions.
Plasmid pPFJ0355 was digested with Barn HI and Bgl II, isolated by 1.0%
agarose gel
electrophoresis using TBE buffer, and purified using an ILLUSTRA GFX PCR DNA
and Gel
Band Purification Kit according to the manufacturers instructions.
The PCR product and the digested vector were ligated together using an IN-
FUSIONTm
CF Dry-down PCR Cloning Kit (Clontech Laboratories, Inc., Mountain View, CA,
USA) resulting
in plasmid: pGH7_ZY582279_485 (Figure 5), in which transcription of
Malbranchea
cinnamomea GH7 CBH gene was under the control of a promoter from the gene for
Aspergillus

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oryzae alpha-amylase. The cloning operation was according to the
manufacturer's instruction. In
brief, for each ligation reaction 30 ng of pPFJ0355 digested with Barn HI and
Bgl II, and 60 ng
of the purified Malbranchea cinnamomea GH7 CBH PCR products were added to the
reaction
vials and resuspended in a final volume of 10 pl with addition of deionized
water. The reactions
5 were incubated at 37 C for 15 minutes and then 50 C for 15 minutes. Three
microlitres of the
reaction products were used to transform E coil TOP10 competent cells (TIANGEN
Biotech
(Beijing) Co. Ltd., Beijing, China). E. coli transformants containing
expression constructs were
detected by colony PCR. Colony PCR is a method for quick screening of plasmid
inserts directly
from E. coli colonies. Briefly, in a premixed PCR solution aliquot in each PCR
tube, including
10 PCR buffer, MgC12, dNTPs, and primer pairs from which the PCR fragment
was generated, a
single colony was added by picking with a sterile tip and twirling the tip in
the reaction solution.
Normally 7-10 colonies were screened. After the PCR, reactions were analyzed
by 1.0%
agarose gel electrophoresis using TBE buffer. Plasmid DNA was prepared from
colonies
showing inserts with the expected sizes using a QIAprep Spin Miniprep Kit
(QIAGEN GmbH,
15 Hi!den, Germany). The Malbranchea cinnamomea GH7 CBH gene inserted in
pGH7_ZY582279_485 was confirmed by DNA sequencing using a 3730XL DNA Analyzer
(Applied Biosystems Inc, Foster City, CA, USA).
Example 5: Expression of Malbranchea cinnamomea GH7 CBH gene in Aspergillus
20 oryzae
Aspergillus otyzae HowB101 (WO 95/035385, Example 1) protoplasts were prepared
according to the method of Christensen et aL, 1988, Bio/Technology 6: 1419-
1422 and
transformed with 3 pg of pGH7_ZY582279_485. The transformation of Aspergillus
oryzae
HowB101 with pGH7_ZY582279_485 yielded about 50 transformants. Eight
transformants
25 were isolated to individual Minimal medium plates.
Four transformants were inoculated separately into 3 ml of YPM medium in a 24-
well
plate and incubated at 30 C with mixing at 150 rpm. After 3 days incubation,
20 pl of
supernatant from each culture were analyzed by SDS-PAGE using a NUPAGE NOVEX
4-
12% Bis-Tris Gel with 50 mM MES (lnvitrogen Corporation, Carlsbad, CA, USA)
according to
30 the manufacturer's instructions. The resulting gel was stained with
INSTANTBLUETm
(Expedeon Ltd., Babraham Cambridge, UK). SDS-PAGE profiles of the cultures
showed that all
4 clones expressed a major protein band at about 45 kDa. The expression strain
was
designated Aspergillus oryzae 05XGY.

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Example 6: Fermentation of expression strains
A slant of Aspergillus oryzae 05XGY was washed with 10 ml of YPM medium and
inoculated into six 2-liter flasks containing 400 ml of YPM medium to generate
broth. The culture
was harvested on day 3 and filtered using a 0.45 pm DURAPORE Membrane
(Millipore,
Bedford, MA, USA).
Example 7: Purification of recombinant Malbranchea cinnamomea CBH from
Aspergillus
oryzae 05XGY
A 2400 ml volume of filtered supematant of Aspergillus oryzae 05XGY (Example
6) was
precipitated with ammonium sulfate (80% saturation), re-dissolved in 50 ml 20
mM sodium
acetate pH 5.5, dialyzed against the same buffer, and filtered through a 0.45
pm filter. The final
volume was 80 ml. The solution was applied to a 40 ml Q SEPHAROSES Fast Flow
column
(GE Healthcare, Buckinghamshire, UK) equilibrated in 20 mM sodium acetate pH
5.5. Proteins
were eluted with a linear NaCI gradient (0-0.5 M). Fractions from the column
were analyzed by
SDS-PAGE using a NUPAGE NOVEX 4-12% Bis-Tris Gel with 50 mM MES. Fractions
containing a band at approximately 45 kDa were pooled and concentrated by
ultrafiltration.
Example 8: Corynascus thermophilus genomic DNA extraction
Corynascus thermophilus strain NN000308 was inoculated onto a FDA plate and
incubated for 3 days at 45 C in the darkness. Several mycelia-PDA plugs were
inoculated into
500 ml shake flasks containing 100 ml of YPG medium. The flasks were incubated
for 4 days at
45 C with shaking at 160 rpm. The mycelia were collected by filtration through
MIRACLOTHO
(Calbiochem, La Jolla, CA, USA) and frozen in liquid nitrogen. Frozen mycelia
were ground, by
a mortar and a pestle, to a fine powder, and genomic DNA was isolated using a
DNeasy Plant
Maxi Kit (QIAGEN GmbH, Hi[den, Germany).
Example 9: Genome sequencing, assembly and annotation
The extracted genomic DNA samples were delivered to Beijing Genome Institute
(BGI,
Shenzhen, China) for genome sequencing using an ILLUMINA GA2 System
(IIlumina,
Inc., San Diego, CA, USA). The raw reads were assembled at BGI using program
SOAPdenovo
(Li et al., 2010, Genome Research 20(2): 265-72). The assembled sequences were
analyzed
using standard bioinformatics methods for gene identification and functional
prediction. GenelD
(Parra et al., 2000, Genome Research 10(4):511-515) was used for gene
prediction. Blastall
version 2.2.10 (Altschul et aL, 1990, J. MoL Biol. 215 (3): 403-410, National
Center for

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Biotechnology Information (NCBI), Bethesda, MD, USA) and HMMER version 2.1.1
(National
Center for Biotechnology Information (NCBI), Bethesda, MD, USA) were used to
predict function
based on structural homology. The GH7 family cellobiohydrolase polypeptides
were identified
directly by analysis of the Blast results. The Agene program (Munch and Krogh,
2006, BMC
Bioinformatics 7:263) and SignalP program (Nielsen etal., 1997, Protein
Engineering 10: 1-6)
were used to identify starting codons. The SignalP program was further used to
predict signal
peptides. Pepstats (Rice et al., 2000, Trends Genet 16(6): 276-277) was used
to predict
isoelectric points and molecular weights of the deduced amino acid sequences.
Example 10: Characterization of a Corynascus thermophilus genomic sequence
encoding a polypeptide having cellobiohydrolase activity
The genomic DNA sequence (SEQ ID NO: 3) and deduced amino acid sequence (SEQ
ID NO: 4) of the Corynascus thermophilus polypeptide coding sequence is shown
in Figure 2.
The coding sequence is 1736 bp including the stop codon and is interrupted by
5 introns of 76
bp (nucleotides 568 to 643), 68 bp (nucleotides 781 to 848), 75 bp
(nucleotides 1062 to 1136),
94 bp (nucleotides 1384 to 1477) and 70 bp (nucleotides 1609 to 1678). The G+C
content of the
mature polypeptide coding sequence without introns and stop codon is 65.9%.
The encoded
predicted protein is 450 amino acids. Using the SignalP program (Nielsen
etal., 1997, supra), a
signal peptide of 20 residues was predicted. The predicted mature protein
contains 430 amino
acids with a predicted molecular weight of 46517.43 Dalton and predicted
isoelectric point of
4.88. The cellobiohydrolase catalytic domain was predicted to be amino acids
21 to 450, by
aligning the amino acid sequence using BLAST to all CAZY-defined subfamily
modules
(Cantarel et al., 2009, Nucleic Acids Res. 37: D233-238), where the single
most significant
alignment within a subfamily was used to predict the GH7 domain.
A comparative pairwise global alignment of amino acid sequences was determined
using
the Needleman and Wunsch algorithm (Needleman and Wunsch, 1970, J. Mot BioL
48: 443-
453) with gap open penalty of 10, gap extension penalty of 0.5, and the
EBLOSUM62 matrix.
The alignment showed that the mature part of the amino acid sequence of the
Corynascus
thermophilus coding sequence encoding the cellobiohydrolase polypeptide shared
86.98%
identity to the amino acid sequence of a Myceliophthora thermophila
cellobiohydrolase I
(accession number GENESEQP:ATS95014 and W02008140749).
The genomic DNA sequence (SEQ ID NO: 5) and deduced amino acid sequence (SEQ
ID NO: 6) of the Corynascus thermophilus polypeptide coding sequence is shown
in Figure 3.
The coding sequence is 1785 bp including the stop codon and is interrupted by
4 introns of 131

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bp (nucleotides 160 to 290), 94 bp (nucleotides 750 to 843), 94 bp
(nucleotides 1052 to 1145)
and 92 bp (nucleotides 1501 to 1592). The G+C content of the mature
polypeptide coding
sequence without introns and stop codon is 63.38%. The encoded predicted
protein is 457
amino acids. Using the SignalP program (Nielsen et aL, 1997, supra), a signal
peptide of 21
A comparative pairwise global alignment of amino acid sequences was determined
using
the Needleman and Wunsch algorithm (Needleman and Wunsch, 1970, J. Mot BioL
48: 443-
453) with gap open penalty of 10, gap extension penalty of 0.5, and the
EBLOSUM62 matrix.
The alignment showed that the mature part of the amino acid sequence of the
Corynascus
The genomic DNA sequence (SEQ ID NO: 7) and deduced amino acid sequence (SEQ
ID NO: 8) of the Corynascus thermophilus polypeptide coding sequence is shown
in Figure 4.
The coding sequence is 1643 bp including the stop codon and is interrupted by
1 introns of 77
A comparative pairwise global alignment of amino acid sequences was determined
using

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thermophilum var. thermophilum (accession number UNIPROT:Q69212).
Example 11: Cloning of the Corynascus thermophilus GH7 cellobiohydrolase genes
from
genomic DNA
Three CBH genes were selected for expression cloning as shown in Table 2.
Table 2: CBH genes
Working name DNA sequence Protein sequence
GH7_Mf7339 SEQ ID NO: 3 SEQ ID NO: 4
GH7_Mf6627 SEQ ID NO: 5 SEQ ID NO: 6
GH7_Mf0261 SEQ ID NO: 7 SEQ ID NO: 8
Based on the DNA information obtained from genome sequencing, oligonucleotide
primers, shown below in Table 3, were designed to amplify the genes from
genomic DNA of
Corynascus thermophilus NN000308. Primers were synthesized by lnvitrogen,
Beijing, China.
Table 3: primers
1Jorward ACACAACTGGGGATCCACCatgaagcagtacctccagtacctcg SEQ ID NO: 11
l_reverse GTCACCCTCTAGATCTg a g cccctcg a gccaa ac SEQ ID NO: 12
2 _forward ACACAACTGGGGATCCACCatgatgtaccggcgggtc SEQ ID NO: 13
2_reverse GTCACCCTCTAGATCTacctctcccatcaactaccattcc SEQ ID NO: 14
3Jorward ACACAACTGGGGATCCACCatgtacaccaagttcgcgac SEQ ID NO: 15
3_reverse GTCACCCTCTAGATCTaagaattggcgcttgtcaaagaac SEQ ID NO: 16
Lowercase characters represent the coding regions of the genes in forward
primers and
the flanking region of the gene in reverse primers, while capitalized
characters represent
insertion sites of plasmid pPFJ0355.
For each gene, 20 picomoles of each forward and reverse primer pair were used
in a
PCR reaction composed of 2 pl of Corynascus thermophilus NN000308 genomic DNA,
10 pl of
5X GC Buffer, 1.5 pl of DMSO, 2.5 mM each of dATP, dTTP, dGTP, and dCTP, and
0.6 unit of
PhusionTm High-Fidelity DNA Polymerase in a final volume of 50 pl. The
amplification was
performed using a Peltier Thermal Cycler programmed for denaturing at 98 C for
1 minute; 6
cycles of denaturing at 98 C for 15 seconds, annealing at 67 C 30 seconds,
with a 1 C
decrease per cycle, and elongation at 72 C for 3 minutes; 23 cycles each at 94
C for 15
seconds, 63 C for 30 seconds, and 72 C for 3 minutes; and a final extension at
72 C for 5
minutes. The heat block then went to a 4 C soak cycle.

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The PCR products were isolated by 1.0% agarose gel electrophoresis using TBE
buffer
where a single product band of approximately 1.8kb, 1.8kb, 1.7kb of
GH7_Mf7339,
GH7_Mf6627, and GH7_Mf0261, respectively, was visualized under UV light. The
PCR
products were then purified from solution by using an ILLUSTRA GFX0 PCR DNA
and Gel
5 Band Purification Kit according to the manufacturer's instructions.
Plasmid pPFJ0355 was digested with Barn I and Bgl II, isolated by 1.0% agarose
gel
electrophoresis using TBE buffer, and purified using an ILLUSTRA GFX PCR DNA
and Gel
Band Purification Kit according to the manufacturer's instructions.
Table 4: plasmids
Gene Plasmid DNA map
GH7_Mt7339 pGH7_Mf7339 Figure 6
GH7_Mf6627 pGH7_Mf6627 Figure 7
GH7_Mf0261 pGH7_Mf0261 Figure 8
The PCR products and the digested vector were ligated together using an IN-
FUSIONTm
CF Dry-down FOR Cloning Kit resulting in plasmids (Table 4): pGH7_Mf7339
(Figure 6),
pGH7_Mf6627 (Figure 7) and pGH7_Mf0261 (Figure 8) respectively in which
transcription of
Corynascus thermophilus CBH genes was under the control of a promoter from the
gene for
Aspergillus otyzae alpha-amylase. In brief, for each ligation reaction 30 ng
of pPFJ0355
digested with Barn HI and Bgl II and 60 ng of the purified Corynascus
thermophilus GH7 CBH
PCR product were added to a reaction vial and resuspended the powder in a
final volume of 10
pl with addition of deionized water. The reaction was incubated at 37 C for 15
minutes and then
50 C for 15 minutes. Three pl of the reaction were used to transform E. coli
TOP10 competent
cells. E. coli transformants containing expression constructs were detected by
colony FOR as
described in Example 4. The Corynascus thermophilus GH7 CBH genes inserted in
pGH7_Mf7339, pGH7_Mf6627 and pGH7_Mf0261 were confirmed by DNA sequencing
using a
3730XL DNA Analyzer.
Example 12: Expression of Corynascus thermophilus GH7 CBH gene in Aspergillus
oryzae
Aspergillus otyzae HowB101 protoplasts were prepared according to the method
of
Christensen et al., 1988, Bioffechnology 6: 1419-1422 and transformed with 3
pg of
pGH7_Mf7339, pGH7_Mf6627 and pGH7_Mf0261, respectively.
The transformation of Aspergillus oryzae HowB101 with pGH7_Mf6627 and

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pGH7_Mf0261 yielded about 50 transformants for each transformation. Eight
transformants of
each transformation were isolated to individual Minimal medium plates.
Four transformants of each transformation were inoculated separately into 3 ml
of YPM
medium in a 24-well plate and incubated at 30 C with mixing at 150 rpm. After
3 days
incubation, 20 pl of supematant from each culture were analyzed on NuPAGE
Novex 4-12%
Bis-Tris Gel with MES (Invitrogen Corporation, Carlsbad, CA, USA) according to
the
manufacturer's instructions. The resulting gel was stained with INSTANTBLUETTM
(Expedeon
Ltd., Babraham Cambridge, UK). SDS-PAGE profiles of the cultures demonstrated
the
expression of the GH7 cellobiohydrolase polypeptides. The sizes of major bands
of the GH7
cellobiohydrolase polypeptides are shown below in Table 5. The expression
strains were
designated as shown in the second column.
Table 5: expression
Plas mid Expression strain Size of recombinant protein
pGH7_Mf6627 07J2B 50kDa
pGH7_Mf0261 07J19 60kDa
Example 13: Fermentation of expression strains 07J2B and 07J19
A slant of the transforrnant, 07J2B, was washed with 10 ml of YPM and
inoculated into
eight 2-liter flasks containing 400 ml of YPM medium to generate broth. The
flasks were then
shaking at 80rpm, 30 C, for 3days. The culture was harvested on day 3 and
filtered using a 0.45
pm DURAPORE Membrane (Millipore, Bedford, MA, USA).
A slant of the transformant, 07J19, was washed with 10 ml of YPM and
inoculated into
four 2-liter flasks containing 400 ml of YPM medium to generate broth. The
flasks were then
shaking at 80rpm, 30C, for 3days. The culture was harvested on day 3 and
filtered using a 0.45
pm DURAPORE Membrane (Millipore, Bedford, MA, USA).
Example 14: Purification of recombinant Malbranchea cinnamomea CBH from
Aspergillus on/zee 07J2B and 07J19
A 3200 ml volume of filtered supematant of Aspergillus wyne 07J2B (Example 13)
was
precipitated with ammonium sulfate (80% saturation), re-dissolved in 50 ml of
20mM Bis-Tris
pH6.0, dialyzed against the same buffer, and filtered through a 0.45 pm
filter. The final volume
was 100 ml. The solution was applied to a 40 ml Q SEPHAROSE Fast Flow column
(GE
Healthcare, Buckinghamshire, UK) equilibrated with 20mM Bis-Tris pH6Ø
Proteins were eluted
with a linear NaCI gradient (0-0.25 M). Fractions eluted with 0.15-0.25M NaCI
were collected

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and further purified on the same Q SEPHAROSE Fast Flow column (GE Healthcare,
Buckinghamshire, UK) with NaCI gradients (0.0-0.2M). Fractions were analyzed
by SDS-PAGE
using a NUPAGE NOVEX 4-12% Bis-Tris Gel with 50 mM MES. The resulting gel
was
stained with INSTANTBLUETm. Fractions containing a band at approximately 50
kDa were
pooled and concentrated by ultrafiltration.
A 1600 ml volume of filtered supematant of Aspergillus oryzae 07J19 (Example
13) was
precipitated with ammonium sulfate (80% saturation), re-dissolved in 50 ml of
20 mM Bis-Tris
pH 6.0, dialyzed against the same buffer, and filtered through a 0.45 pm
filter. The final volume
was 60 ml. The solution was applied to a 40 ml Q SEPHAROSEO Fast Flow column
equilibrated with 20 mM Bis-Tris pH 6Ø Proteins were eluted with a linear
NaCI gradient (0-0.5
M). Fractions eluted with 0.1-0.3 M NaCI were collected and further purified
using a 40 ml
Phenyl SEPHAROSE 6 Fast Flow column (GE Healthcare, Buckinghamshire, UK) with
a linear
1.2-0 M (N1-14)2SO4 gradient. Fractions were analyzed by SDS-PAGE using a
NUPAGEO
NOVEX 4-12% Bis-Tris Gel with 50 mM MES. The resulting gel was stained with
INSTANTBLUETm. Fractions containing a band at approximately 60 kDa were pooled
and
concentrated by ultrafiltration.
Example 15: GH7 cellobiohydrolase (CBH) activity assay on phosphoric acid-
swollen
cellulose (PASC)
A PASC stock slurry solution was prepared by moistening 5 g of
microcrystalline
cellulose (AVICEL ; JRS Pharma, Holzmuhle 1, Rosenberg, Germany) with water,
followed by
the addition of 150 ml of ice cold 85% 0-phosphoric acid. The suspension was
slowly stirred in
an ice-bath for 1 hour. Then 500 ml of ice cold acetone were added while
stirring. The slurry
was filtered using Calbiochem MIRACLOTH (EMD Millipore Bioscience,
Billerica, MA, USA)
and then washed three times with 100 ml of ice cold acetone (drained as dry as
possible after
each wash). Finally, the filtered slurry was washed twice with 500 ml of
water, and again
drained as dry as possible after each wash. The PASC was mixed with deionized
water to a
total volume of 500 ml with a concentration of 10 g/liter, blended to
homogeneity (using an
ULTRA-TURRA)( Homogenizer, Cole-Parmer, Vernon Hills, IL, USA), and stored in
a
refrigerator for up to one month.
The PASC stock solution was diluted with 50 mM sodium acetate pH 5.0 buffer to
a
concentration of 2 g/liter, and used as the substrate. To 150 pl of PASC stock
solution, 20 pl of
enzyme sample were added and the reaction mixture was incubated for 60 minutes
with
shaking at 850 rpm. At the end of the incubation, 50 pl of 2% NaOH were added
to stop the

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reaction. The reaction mixture was centrifuged at 1,000 x g. The released
sugars were
measured by first mixing 10 pl of the reaction mixture with 90 pl of 0.4%
NaOH, followed by 50
pl of 1.5% p-hydroxybenzoic acid hydrazide in 2% NaOH (PHBAH, Sigma Chemical
Co., St.
Loius, MO, USA). The mixture was boiled at 100 C for 5 minutes, and then 100
pl were
transferred to a microtiter plate for an absorbance reading at 410 nm (Spectra
Max M2,
Molecular devices Sunnyvale, CA, USA). Blanks were made by omitting PASC in
the hydrolysis
step, and by replacing the hydrolysate with buffer in the sugar determination
step. The
cellobiohydrolase activity was calculated based on the difference between the
absorbance of
the sample and the absorbance of the blanks.
As a result, 05XGY comprising Malbranchea cinnamomea cellobiohydrolases I
(P249)0() and 07J2B comprising Corynascus thermophilus cellobiohydrolases I
(P24FVN)
showed some cellobiohydrolase activity, and 07J19 comprising Cotynascus
thermophilus
cellobiohydrolases I (P24FUQ) showed cellobiohydrolase activity with an
absorbance at 410 nm
of 1.1407.
Example 16: Pretreated corn stover hydrolysis assay
Corn stover was pretreated at the U.S. Department of Energy National Renewable
Energy Laboratory (NREL) using 1.4 wt % sulfuric acid at 165 C and 107 psi for
8 minutes. The
water-insoluble solids in the pretreated corn stover (PCS) contained 56.5%
cellulose, 4.6%
hemicellulose and 28.4% lignin. Cellulose and hemicellulose were determined by
a two¨stage
sulfuric acid hydrolysis with subsequent analysis of sugars by high
performance liquid
chromatography using NREL Standard Analytical Procedure #002. Lignin was
determined
gravimetrically after hydrolyzing the cellulose and hemicellulose fractions
with sulfuric acid using
NREL Standard Analytical Procedure #003.
Unmilled, unwashed PCS (whole slurry PCS) was prepared by adjusting the pH of
the
PCS to 5.0 by addition of 10 M NaOH with extensive mixing, and then
autoclaving for 20
minutes at 120 C. The dry weight of the whole slurry PCS was 29%. Milled
unwashed PCS (dry
weight 32.35%) was prepared by milling whole slurry PCS in a Cosmos ICMG 40
wet multi-
utility grinder (EssEmm Corporation, Tamil Nadu, India).
The hydrolysis of PCS was conducted using 2.2 ml deep-well plates (Axygen,
Union
City, CA, USA) in a total reaction volume of 1.0 ml. The hydrolysis was
performed with 50 mg of
insoluble PCS solids per ml of 50 mM sodium acetate pH 5.0 buffer containing 1
mM
manganese sulfate and various protein loadings of various enzyme compositions
(expressed as
mg protein per gram of cellulose). Enzyme compositions were prepared and then
added

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simultaneously to all wells in a volume ranging from 50 pl to 200 pl, for a
final volume of 1 ml in
each reaction. The plate was then sealed using an ALPS-30O' m plate heat
sealer, mixed
thoroughly, and incubated at a specific temperature for 72 hours. All
experiments reported were
performed in triplicate.
Following hydrolysis, samples were filtered using a 0.45 pm MULTISCREEN 96-
well
filter plate and the filtrates were analyzed for sugar content as described
below. When not used
immediately, filtered aliquots were frozen at -20 C. The sugar concentrations
of samples diluted
in 0.005 M H2SO4 were measured using a 4.6 x 250 mm AMINEX HPX-87H column by
elution
with 0.05% w/w benzoic acid-0.005 M H2SO4 at 65 C at a flow rate of 0.6 ml per
minute, and
quantitation by integration of the glucose, cellobiose, and xylose signals
from refractive index
detection (CHEMSTATION , AGILENT 1100 HPLC) calibrated by pure sugar samples.
The
resultant glucose and cellobiose equivalents were used to calculate the
percentage of cellulose
conversion for each reaction.
Glucose and cellobiose were measured individually. Measured sugar
concentrations
were adjusted for the appropriate dilution factor. The net concentrations of
enzymatically-
produced sugars from the milled unwashed PCS were determined by adjusting the
measured
sugar concentrations for corresponding background sugar concentrations in
unwashed PCS at
zero time point. All HPLC data processing was performed using MICROSOFT
EXCELTm
software.
The degree of cellulose conversion to glucose was calculated using the
following
equation: % conversion = (glucose concentration/glucose concentration in a
limit digest) x 100.
In order to calculate % conversion, a 100% conversion point was set based on a
cellulase
control (100 mg of Trichoderma reeseicellulase per gram cellulose), and all
values were divided
by this number and then multiplied by 100. Triplicate data points were
averaged and standard
deviation was calculated.
Example 17: Preparation of an enzyme composition
The Aspergillus fumigatus GH6A cellobiohydrolase II (SEQ ID NO: 17 [DNA
sequence]
and SEQ ID NO: 18 [deduced amino acid sequence]) was prepared recombinantly in
Aspergillus oryzae as described in WO 2011/057140. The filtered broth of the
A. fumigatus
cellobiohydrolase II was buffer exchanged into 20 mM Tris pH 8.0 using a 400
ml SEPHADEXTm
G-25 column (GE Healthcare, United Kingdom). The fractions were pooled and
adjusted to 1.2
M ammonium sulphate-20 mM Tris pH 8Ø The equilibrated protein was loaded
onto a PHENYL
SEPHAROSETm 6 Fast Flow column (high sub) (GE Healthcare, Piscataway, NJ, USA)

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equilibrated in 20 mM Tris pH 8.0 with 1.2 M ammonium sulphate, and bound
proteins were
eluted with 20 mM Tris pH 8.0 with no ammonium sulphate. The fractions were
pooled.
The Penicillium sp. (emersonii) GH61A polypeptide (SEQ ID NO: 19 [DNA
sequence]
and SEQ ID NO: 20 [deduced amino acid sequence]) was recombinantly prepared
according to
5 WO
2011/041397. The Penicillium sp. (emersonir) GH61A polypeptide gene was
purified
according to WO 2011/041397.
The Trichoderma reesei GH5 endoglucanase II (SEQ ID NO: 21 [DNA sequence] and
SEQ ID NO: 22 [deduced amino acid sequence]) was prepared recombinantly
according to WO
2011/057140 using Aspergillus oryzae as a host. The filtered broth of the T.
reesei
10
endoglucanase II was desalted and buffer-exchanged into 20 mM Tris pH 8.0
using a tangential
flow concentrator (Pall Filtron, Northborough, MA, USA) equipped with a 10 kDa
polyethersulfone membrane (Pall Filtron, Northborough, MA, USA). The protein
concentration
was determined using a Microplate BCATm Protein Assay Kit (Thermo Fischer
Scientific,
Waltham, MA, USA) in which bovine serum albumin was used as a protein
standard.
15 The
Aspergillus fumigatus GH10 xylanase (xyn3) (SEQ ID NO: 23 [DNA sequence] and
SEQ ID NO: 24 [deduced amino acid sequence]) was prepared recombinantly
according to WO
2006/078256 using Aspergillus oryzae BECh2 (WO 2000/39322) as a host. The
filtered broth of
the A. fumigatus xylanase was desalted and buffer-exchanged into 50 mM sodium
acetate pH
5.0 using a HIPREP 26/10 Desalting Column (GE Healthcare, Piscataway, NJ,
USA).
20 The
Aspergillus fumigatus NN055679 Cel3A beta-glucosidase (SEQ ID NO: 25 [DNA
sequence] and SEQ ID NO: 26 [deduced amino acid sequence]) was recombinantly
prepared
according to WO 2005/047499 using Aspergillus oryzae as a host. The filtered
broth was
adjusted to pH 8.0 with 20% sodium acetate, which made the solution turbid. To
remove the
turbidity, the solution was centrifuged at 20,000 x g for 20 minutes, and the
supematant was
25 filtered
through a 0.2 pm filtration unit (Nalgene, Rochester, NY, USA). The filtrate
was diluted
with deionized water to reach the same conductivity as 50 mM Tris-HCI pH 8Ø
The adjusted
enzyme solution was applied to a Q SEPHAROSE Fast Flow column (GE Healthcare,
Piscataway, NJ, USA) equilibrated in 50 mM Tris-HCI pH 8.0 and eluted with a
linear 0 to 500
mM sodium chloride gradient. Fractions were pooled and treated with 1% (why)
activated
30 charcoal
to remove color from the beta-glucosidase pool. The charcoal was removed by
filtration of the suspension through a 0.2 pm filtration unit. The filtrate
was adjusted to pH 5.0
with 20% acetic acid and diluted 10 times with deionized water. The adjusted
filtrate was applied
to a SP SEPHAROSE0 Fast Flow column (GE Healthcare, Piscataway, NJ, USA)
equilibrated
in 10 mM succinic acid pH 5.0 and eluted with a linear 0 to 500 mM sodium
chloride gradient.

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Fractions were collected and analyzed for beta-glucosidase activity using p-
nitrophenyl-beta-D-
glucopyranoside as substrate. A p-nitrophenyl-beta-D-glucopyranoside stock
solution was
prepared by dissolving 50 mg of the substrate in 1.0 ml of DMSO. Just before
use a substrate
solution was prepared by mixing 100 pl of the stock solution with 4900 pl of
100 mM succinic
acid, 100 mM HEPES, 100 mM CHES, 100 mM CABS, 1 mM CaCl2, 150 mM KCI, 0.01%
TRITON X-100, pH 5.0 (assay buffer). A 200 pl volume of the substrate
solution was
dispensed into a tube and placed on ice followed by 20 pl of enzyme sample
(diluted in 0.01%
TRITON X-100). The assay was initiated by transferring the tube to a
thermomixer, which was
set to an assay temperature of 37 C. The tube was incubated for 15 minutes on
the
thermomixer at its highest shaking rate (1400 rpm). The assay was stopped by
transferring the
tube back to the ice bath and adding 600 pl of Stop solution (500 mM
H3603/NaOH pH 9.7).
Then the tube was mixed and allowed to reach room temperature. A 200 pl of
supematant was
transferred to a microtiter plate and the absorbance at 405 nm was read as a
measure of beta-
glucosidase activity. A buffer control was included in the assay (instead of
enzyme). Fractions
with beta-glucosidase activity were further analyzed by SDS-PAGE. Fractions,
where only one
band was seen on a Coomassie blue stained SDS-PAGE gel, were pooled as the
purified
product. The protein concentration was determined using a Microplate BCATM
Protein Assay Kit
in which bovine serum albumin was used as a protein standard.
The Aspergillus fumigatus NN051616 GH3 beta-xylosidase (SEQ ID NO: 27 [DNA
sequence] and SEQ ID NO: 28 [deduced amino acid sequence]) was prepared
recombinantly in
Aspergillus oryzae as described in WO 2011/057140. The filtered broth of the
A. fumigatus
beta-xylosidase was desalted and buffer-exchanged into 50 mM sodium acetate pH
5.0 using a
HI PREP 26/10 Desalting Column.
The protein concentration for each of the monocomponents described above was
determined using a Microplate BCATm Protein Assay Kit in which bovine serum
albumin was
used as a protein standard. An enzyme composition was prepared composed of
each
monocomponent as follows: 25% Aspergillus fumigatus Cel6A cellobiohydrolase
II, 15%
Penicillium emersonii GH61A polypeptide, 10% Trichoderma reesei GH5
endoglucanase II, 5%
Aspergillus fumigatus GH10 xylanase, 5% Aspergillus fumigatus beta-
glucosidase, and 3%
Aspergillus fumigatus beta-xylosidase. The enzyme composition is designated
herein as
"cellulolytic enzyme composition".
Example 18: Effect of the Corynascus thermophilus cellbiohydrolases I on the
hydrolysis of milled unwashed PCS by a cellulolytic enzyme composition

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Both Corynascus thermophilus cellobiohydrolases I (P24FVN and P24FUQ) were
evaluated for the ability to enhance the hydrolysis of milled unwashed PCS
(Example 16) by the
cellulolytic enzyme composition (Example 17) at 1.89 mg total protein per g
cellulose at 50 C,
55 C, 60 C, and 65 C. The Corynascus the rmophilus cellobiohydrolases were
added at 1.11 mg
protein per g cellulose. The cellulolytic enzyme composition was also run
without added
cellobiohydrolase I at 1.89 mg protein per g cellulose or 3 mg protein per g
cellulose.
The assay was performed as described in Example 16. The 1 ml reactions with
milled
unwashed PCS (5% insoluble solids) were conducted for 72 hours in 50 mM sodium
acetate pH
5.0 buffer containing 1 mM manganese sulfate. All reactions were performed in
triplicate and
involved single mixing at the beginning of hydrolysis.
As shown in Table 6, the cellulolytic enzyme composition that included either
Cotynascus thermophilus cellobiohydrolase I (P24FVN or P24FUQ) outperformed
the
cellulolytic enzyme composition (1.89 mg protein per g cellulose) without
cellobiohydrolase I.
The degree of cellulose conversion to glucose for the Corynascus the
rmophilus
cellobiohydrolases I (P24FVN or P24FUQ) added to the cellulolytic enzyme
composition was
significantly higher than the cellulolytic enzyme composition without added
cellobiohydrolase I at
50 C, 55 C, 60 C, and 65 C.
Table 6: the effect of Corynascus the rmophilus cellobiohydrolase I
% Conversion (cellulose to glucose)
Tempertu re Standard Deviation
50 ( C) 55 ( C) 60 (*C) 65 (*C)
50 (CC) 55 (CC) 60 (CC) 65 ( C)
Cellulolytic Enzyme 46.73917 47.76768 30.91584 24.03815 1.234456 0.787361
0.170454 0.783082
Composition no CBH I (1.89mg/g)
Cellulolytic Enzyme 56.44285 57.89507 36.76348 28.51199 0.721591 0.625392
0.5686590.176343
Composition (1.89mg/g)
with Corynascus thermophilus
CBHI P24FVN (1.11mg/g)
Cellulolytic Enzyme 66.45576 66.0967 42.38305 31.84248 1.346328 0.828681
0.201917 0.06523
Composition (1.89mg/g)
with Cotynascus thermophilus
CM! P24FUQ (1.11mg/g)
Cellulolytic Enzyme Composition 54.26059 57.76175 35.473 27.29206 1.510733
2.1599360.0706610.168844
no CBHI (3mg/g)
The present invention is further described by the following numbered
paragraphs:
[1] An isolated polypeptide having cellobiohydrolase activity, selected from
the group
consisting of:

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(a) a polypeptide having at least 70%, e.g., at least 72%, at least 74%, at
least 75%,
at least 77%, at least 78%, at least 80%, at least 81%, at least 82%, at least
83%, at least 84%,
at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least
90%, at least 91%,
at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least
97%, at least 98%,
at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID
NO: 2; a
polypeptide having at least 88%, e.g., at least 89%, at least 90%, at least
91%, at least 92%, at
least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least
98%, at least 99%, or
100% sequence identity to the mature polypeptide of SEQ ID NO: 4; a
polypeptide having at
least 66%, e.g., at least 68%, at least 70%, at least 75%, at least 78%, at
least 80%, at least
81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at
least 87%, at least
88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%, at least
95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence
identity to the
mature polypeptide of SEQ ID NO: 6; or a polypeptide having at least 81%,
e.g., at least 82%, at
least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least
88%, at least 89%, at
least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least
95%, at least 96%, at
least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature
polypeptide of
SEQ ID NO: 8;
(b) a polypeptide encoded by a polynucleotide that hybridizes under medium
stringency conditions, medium-high stringency conditions, high stringency
conditions, or very
high stringency conditions with (i) the mature polypeptide coding sequence of
SEQ ID NO: 1,
the mature polypeptide coding sequence of SEQ ID NO: 3, the mature polypeptide
coding
sequence of SEQ ID NO: 5 or the mature polypeptide coding sequence of SEQ ID
NO: 7, (ii) the
cDNA sequence of SEQ ID NO: 1, the cDNA sequence of SEQ ID NO: 3, the cDNA
sequence
of SEQ ID NO: 5 or the cDNA sequence of SEQ ID NO: 7, or (iii) the full-length
complement of
(i) or (H);
(c) a polypeptide encoded by a polynucleotide having at least 70%, e.g., at
least
72%, at least 74%, at least 75%, at least 77%, at least 78%, at least 80%, at
least 81%, at least
82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at
least 88%, at least
89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at
least 95%, at least
96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to
the mature
polypeptide coding sequence of SEQ ID NO: 1 or the cDNA sequence thereof; a
polypeptide
encoded by a polynucleotide having at least 88%, e.g., at least 89%, at least
90%, at least 91%,
at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least
97%, at least 98%,
at least 99%, or 100% sequence identity to the mature polypeptide coding
sequence of SEQ ID

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NO: 3 or the cDNA sequence thereof; a polypeptide encoded by a polynucleotide
having at least
66%, e.g., at least 68%, at least 70%, at least 75%, at least 78%, at least
80%, at least 81%, at
least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least
87%, at least 88%, at
least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at least 95%, at
least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity
to the mature
polypeptide coding sequence of SEQ ID NO: 5 or the cDNA sequence thereof; or a
polypeptide
encoded by a polynucleotide having at least 81%, e.g., at least 82%, at least
83%, at least 84%,
at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least
90%, at least 91%,
at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least
97%, at least 98%,
at least 99%, or 100% sequence identity to the mature polypeptide coding
sequence of SEQ ID
NO: 7 or the cDNA sequence thereof;
(d) a variant of the mature polypeptide of SEQ ID NO: 2, the mature
polypeptide of
SEQ ID NO: 4, the mature polypeptide of SEQ ID NO: 6 or the mature polypeptide
of SEQ ID
NO: 8 comprising a substitution, deletion, and/or insertion at one or more
(e.g., several)
positions; and
(e) a fragment of the polypeptide of (a), (b), (c), or (d) that has
cellobiohydrolase
activity.
[2] The polypeptide of paragraph 1, having at least 70%, at least 72%, at
least 74%, at
least 75%, at least 77%, at least 78%, at least 80%, at least 81%, at least
82%, at least 83%, at
least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least
89%, at least 90%, at
least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least
96%, at least 97%, at
least 98%, at least 99% or 100% sequence identity to the mature polypeptide of
SEQ ID NO: 2;
having at least 88%, at least 89%, at least 90%, at least 91%, at least 92%,
at least 93%, at
least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least
99% or 100%
sequence identity to the mature polypeptide of SEQ ID NO: 4; having at least
66%, at least
68%, at least 70%, at least 75%, at least 78%, at least 80%, at least 81%, at
least 82%, at least
83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at
least 89%, at least
90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at
least 96%, at least
97%, at least 98%, at least 99%, or 100% sequence identity to the mature
polypeptide of SEQ
ID NO: 6; or having at least 85%, at least 86%, at least 87%, at least 88%, at
least 89%, at least
90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at
least 96%, at least
97%, at least 98%, at least 99%, or 100% sequence identity to the mature
polypeptide of SEQ
ID NO: 8.

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[3] The polypeptide of paragraph 1 or 2, which is encoded by a polynucleotide
that
hybridizes under medium, medium-high, high, or very high stringency conditions
with (i) the
mature polypeptide coding sequence of SEQ ID NO: 1, the mature polypeptide
coding
sequence of SEQ ID NO: 3, the mature polypeptide coding sequence of SEQ ID NO:
5 or the
5 mature
polypeptide coding sequence of SEQ ID NO: 7, (ii) the cDNA sequence of SEQ ID
NO:
1, the cDNA sequence of SEQ ID NO: 3, the cDNA sequence of SEQ ID NO: 5 or the
cDNA
sequence of SEQ ID NO: 7, or (iii) the full-length complement of (i) or (ii).
[4] The polypeptide of paragraph 1, comprising or consisting of SEQ ID NO: 2,
SEQ ID
NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8; or comprising or consisting of the mature
polypeptide of
10 SEQ ID
NO: 2, the mature polypeptide of SEQ ID NO: 4, the mature polypeptide of SEQ
ID NO:
6 or the mature polypeptide of SEQ ID NO: 8.
[5] The polypeptide of paragraph 4, wherein the mature polypeptide is amino
acids 18 to
458 of SEQ ID NO: 2, amino acids 21 to 450 of SEQ ID NO: 4, amino acids 22 to
457 of SEQ ID
NO: 6, or amino acids 18 to 521 of SEQ ID NO: 8.
15 [6] The
polypeptide of any of paragraphs 1-5, which is encoded by a polynucleotide
having at least 70%, at least 72%, at least 74%, at least 75%, at least 77%,
at least 78%, at
least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least
85%, at least 86%, at
least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least
92%, at least 93%, at
least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least
99% or 100%
20 sequence
identity to the mature polypeptide coding sequence of SEQ ID NO: 1, or the
cDNA
sequence thereof; which is encoded by a polynucleotide having at least 88%, at
least 89%, at
least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least
95%, at least 96%, at
least 97%, at least 98%, at least 99% or 100% sequence identity to the mature
polypeptide
coding sequence of SEQ ID NO: 3, or the cDNA sequence thereof; which is
encoded by a
25
polynucleotide having at least 66%, at least 68%, at least 70%, at least 75%,
at least 78%, at
least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least
85%, at least 86%, at
least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least
92%, at least 93%, at
least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least
99% or 100%
sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 5,
or the cDNA
30 sequence
thereof; or which is encoded by a polynucleotide having at least 81%, at least
82%, at
least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least
88%, at least 89%, at
least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least
95%, at least 96%, at
least 97%, at least 98%, at least 99% or 100% sequence identity to the mature
polypeptide
coding sequence of SEQ ID NO: 7, or the cDNA sequence thereof.

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[7] The polypeptide of any of paragraphs 1-6, which is a variant of the mature
polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8
comprising a
substitution, deletion, and/or insertion at one or more (e.g., several)
positions, wherein the
variant has cellobiohydrolase activity.
[8] The polypeptide of paragraph 1, which is a fragment of SEQ ID NO: 2, SEQ
ID NO: 4,
SEQ ID NO: 6 or SEQ ID NO: 8, wherein the fragment has cellobiohydrolase
activity.
[9] An isolated polypeptide comprising a catalytic domain selected from the
group
consisting of:
(a) a catalytic domain having at least 70%, e.g., at least 72%, at least
74%, at least
75%, at least 77%, at least 78%, at least 80%, at least 81%, at least 82%, at
least 83%, at least
84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at
least 90%, at least
91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at
least 97%, at least
98%, at least 99%, or 100% sequence identity to amino acids 18 to 458 of SEQ
ID NO: 2, a
catalytic domain having at least 88%, e.g., at least 89%, at least 90%, at
least 91%, at least
92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at
least 98%, at least
99%, or 100% sequence identity to amino acids 21 to 450 of SEQ ID NO: 4, a
catalytic domain
having at least 66%, e.g., at least 68%, at least 70%, at least 75%, at least
78%, at least 80%,
at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least
86%, at least 87%,
at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least
93%, at least 94%,
at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%
sequence identity
to amino acids 22 to 457 of SEQ ID NO: 6, or a catalytic domain having at
least 81%, e.g., at
least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least
87%, at least 88%, at
least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at least 95%, at
least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity
to amino acids
21 to 461 of SEQ ID NO: 8;
(b) a catalytic domain encoded by a polynucleotide that hybridizes under
medium,
medium-high, high, or very high stringency conditions with (i) nucleotides 52
to 1454 of SEQ ID
NO: 1, nucleotides 61 to 1733 of SEQ ID NO: 3, nucleotides 64 to 1782 of SEQ
ID NO: 5, or
nucleotides 52 to 1460 of SEQ ID NO: 7, (ii) the cDNA sequence thereof, or
(iii) the full-length
complement of (i) or (ii);
(c) a catalytic domain encoded by a polynucleotide having at least 70%,
e.g., at least
72%, at least 74%, at least 75%, at least 77%, at least 78%, at least 80%, at
least 81%, at least
82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at
least 88%, at least
89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at
least 95%, at least

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96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to
nucleotides 52 to
1454 of SEQ ID NO: 1 or the cDNA sequence thereof; or a catalytic domain
encoded by a
polynucleotide having at least 88%, e.g., at least 89%, at least 90%, at least
91%, at least 92%,
at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least
98%, at least 99%,
or 100% sequence identity to nucleotides 61 to 1733 of SEQ ID NO: 3 or the
cDNA sequence
thereof; a catalytic domain encoded by a polynucleotide having at least 66%,
e.g., at least 68%,
at least 70%, at least 75%, at least 78%, at least 80%, at least 81%, at least
82%, at least 83%,
at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least
89%, at least 90%,
at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least
96%, at least 97%,
at least 98%, at least 99%, or 100% sequence identity to nucleotides 64 to
1782 of SEQ ID NO:
5 or the cDNA sequence thereof; or a catalytic domain encoded by a
polynucleotide having at
least 81%, e.g., at least 82%, at least 83%, at least 84%, at least 85%, at
least 86%, at least
87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at
least 93%, at least
94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or
100% sequence
identity to nucleotides 52 to 1460 of SEQ ID NO: 7 or the cDNA sequence
thereof;
(d) a
variant of amino acids 18 to 458 of SEQ ID NO: 2, amino acids 21 to 450 of
SEQ ID NO: 4, amino acids 22 to 457 of SEQ ID NO: 6, or amino acids 21 to 461
of SEQ ID
NO: 8, comprising a substitution, deletion, and/or insertion at one or more
positions (e.g.,
several) , wherein the variant has cellobiohydrolase activity; and
(e) a fragment of
the catalytic domain of (a), (b), (c), or (d) that has
cellobiohydrolyase activity.
[10] The polypeptide of paragraph 9, further comprising a carbohydrate binding
domain.
[11] An isolated polypeptide comprising a carbohydrate binding domain,
selected from
the group consisting of:
(a) a carbohydrate
binding domain having at least 81%, e.g., at least 82%, at least
83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at
least 89%, at least
90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at
least 96%, at least
97%, at least 98%, at least 99%, or 100% sequence identity to amino acids 486
to 521 of SEQ
ID NO: 8;
(b) a carbohydrate
binding domain encoded by a polynucleotide that hybridizes
under medium, medium-high, high, or very high stringency conditions with (i)
nucleotides 1533
to 1640 of SEQ ID NO: 7, (ii) the cDNA sequence thereof, or (iii) the full-
length complement of
(i) or (ii);

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(C) a
carbohydrate binding domain encoded by a polynucleotide having at least 81%,
e.g., at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at
least 87%, at least
88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%, at least
95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence
identity to
nucleotides 1533 to 1640 of SEQ ID NO: 7 or the cDNA sequence thereof;
(d) a variant of amino acids 486 to 521 of SEQ ID NO: 8 comprising a
substitution,
deletion, and/or insertion at one or more positions; and
(e) a fragment of (a), (b), (c), (d) or (e) that has carbohydrate binding
activity.
[12] The polypeptide of paragraph 11, wherein the carbohydrate binding domain
is
operably linked to a catalytic domain, preferably a catalytic domain is
obtained from a
hydrolase, isomerase, ligase, lyase, oxidoreductase, or transferase, e.g., an
aminopeptidase,
amylase, carbohydrase, carboxypeptidase, catalase, cellobiohydrolase,
cellulase, chitinase,
cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, endoglucanase,
esterase, alpha-
galactosidase, beta-galactosidase, glucoamylase, alpha-glucosidase, beta-
glucosidase,
invertase, laccase, lipase, mannosidase, mutanase, oxidase, pectinolytic
enzyme, peroxidase,
phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease,
transglutaminase, xylanase, or
beta-xylosidase.
[13] A composition comprising the polypeptide of any of paragraphs 1-12.
[14] An isolated polynucleotide encoding the polypeptide of any of paragraphs
1-12.
[15] A nucleic acid construct or expression vector comprising the
polynucleotide of
paragraph 14 operably linked to one or more control sequences that direct the
production of the
polypeptide in an expression host.
[16] A recombinant host cell comprising the polynucleotide of paragraph 14
operably
linked to one or more control sequences that direct the production of the
polypeptide.
[17] A method of producing the polypeptide of any of paragraphs 1-12,
comprising:
(a) cultivating a cell, which in its wild-type form produces the
polypeptide, under
conditions conducive for production of the polypeptide; and optionally
(b) recovering the polypeptide.
[18] A method of producing a polypeptide having cellobiohydrolase activity,
comprising:
(a) cultivating the
host cell of paragraph 16 under conditions conducive for
production of the polypeptide; and optionally
(b) recovering the polypeptide.
[19] A transgenic plant, plant pail or plant cell transformed with a
polynucleotide
encoding the polypeptide of any of paragraphs 1-12.

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[20] A method of producing a polypeptide having cellobiohydrolase activity,
comprising:
(a) cultivating the transgenic plant or plant cell of paragraph 19 under
conditions
conducive for production of the polypeptide; and optionally
(b) recovering the polypeptide.
[21] A method of producing a mutant of a parent cell, comprising inactivating
a
polynucleotide encoding the polypeptide of any of paragraphs 1-12, which
results in the mutant
producing less of the polypeptide than the parent cell.
[22] A mutant cell produced by the method of paragraph 21.
[23] The mutant cell of paragraph 22, further comprising a gene encoding a
native or
heterologous protein.
[24] A method of producing a protein, comprising:
(a) cultivating the mutant cell of paragraph 22 or 23 under conditions
conducive for
production of the protein; and optionally
(b) recovering the protein.
[25] A double-stranded inhibitory RNA (dsRNA) molecule comprising a
subsequence of
the polynucleotide of paragraph 14, wherein optionally the dsRNA is an siRNA
or an miRNA
molecule.
[26] The double-stranded inhibitory RNA (dsRNA) molecule of paragraph 25,
which is
about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more duplex nucleotides in
length.
[27] A method of inhibiting the expression of a polypeptide having
cellobiohydrolase
activity in a cell, comprising administering to the cell or expressing in the
cell the double-
stranded inhibitory RNA (dsRNA) molecule of paragraph 25 or 26.
[28] A cell produced by the method of paragraph 27.
[29] The cell of paragraph 28, further comprising a gene encoding a native or
heterologous protein.
[30] A method of producing a protein, comprising:
(a) cultivating the cell of paragraph 28 or 29 under conditions conducive
for
production of the protein; and optionally
(b) recovering the protein.
[31] An isolated polynucleotide encoding a signal peptide comprising or
consisting of
amino acids 1 to 17 of SEQ ID NO: 2, amino acids 1 to 20 of SEQ ID NO: 4,
amino acids 1 to 21
of SEQ ID NO: 6, or amino acids 1 to 17 of SEQ ID NO: 8.

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[32] A nucleic acid construct or expression vector comprising a gene encoding
a protein
operably linked to the polynucleotide of paragraph 31, wherein the gene is
foreign to the
polynucleotide encoding the signal peptide.
[33] A recombinant host cell comprising a gene encoding a protein operably
linked to the
polynucleotide of paragraph 31, wherein the gene is foreign to the
polynucleotide encoding the
signal peptide.
[34] A method of producing a protein, comprising:
(a) cultivating a recombinant host cell comprising a gene encoding a
protein
operably linked to the polynucleotide of paragraph 31, wherein the gene is
foreign to the
polynucleotide encoding the signal peptide, under conditions conducive for
production of the
protein; and optionally
(b) recovering the protein.
[35] A whole broth formulation or cell culture composition comprising the
polypeptide of
any of paragraphs 1-12.
[36] A process for degrading or converting a cellulosic material, comprising:
treating the
cellulosic material with an enzyme composition in the presence of the
polypeptide having
cellobiohydrolase activity of any of paragraphs 1-12.
[37] The process of paragraph 36, wherein the cellulosic material is
pretreated.
[38] The process of paragraph 36 or 37, wherein the enzyme composition
comprises one
or more enzymes selected from the group consisting of a cellulase, a GH61
polypeptide having
cellulolytic enhancing activity, a hemicellulase, an esterase, an expansin, a
laccase, a
ligninolytic enzyme, a pectinase, a peroxidase, a protease, and a swollenin.
[39] The process of paragraph 38, wherein the cellulase is one or more enzymes
selected from the group consisting of an endoglucanase, a cellobiohydrolase,
and a beta-
glucosidase.
[40] The process of paragraph 38, wherein the hemicellulase is one or more
enzymes
selected from the group consisting of a xylanase, an acetylxylan esterase, a
feruloyl esterase, an
arabinofuranosidase, a xylosidase, and a glucuronidase.
[41] The process of any of paragraphs 36-40, further comprising recovering the
degraded or converted cellulosic material.
[42] The process of paragraph 41, wherein the degraded or converted cellulosic
material
is a sugar.
[43] The process of paragraph 42, wherein the sugar is selected from the group
consisting of glucose, xylose, mannose, galactose, and arabinose.

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[44] A process for producing a fermentation product, comprising:
(a) saccharifying a cellulosic material with an enzyme composition in the
presence of
the polypeptide having cellobiohydrolase activity of any of paragraphs 1-12;
(b) fermenting the saccharified cellulosic material with one or more
fermenting
microorganisms to produce the fermentation product; and
(c) recovering the fermentation product from the fermentation.
[45] The process of paragraph 44, wherein the cellulosic material is
pretreated.
[46] The process of paragraph 44 or 45, wherein the enzyme composition
comprises one
or more enzymes selected from the group consisting of a cellulase, a GH61
polypeptide having
cellulolytic enhancing activity, a hemicellulase, an esterase, an expansin, a
laccase, a
ligninolytic enzyme, a pectinase, a peroxidase, a protease, and a swollenin.
[47] The process of paragraph 46, wherein the cellulase is one or more enzymes
selected from the group consisting of an endoglucanase, a cellobiohydrolase,
and a beta-
glucosid ase.
[48] The process of paragraph 46, wherein the hemicellulase is one or more
enzymes
selected from the group consisting of a xylanase, an acetylxylan esterase, a
feruloyl esterase, an
arabinofuranosidase, a xylosidase, and a glucuronidase.
[49] The process of any of paragraphs 44-48, wherein steps (a) and optionally
(b) are
performed simultaneously in a simultaneous saccharification and fermentation.
[50] The process of any of paragraphs 44-49, wherein the fermentation product
is an
alcohol, an alkane, a cycloalkane, an alkene, an amino acid, a gas, isoprene,
a ketone, an
organic acid, or polyketide.
[51] A process of fermenting a cellulosic material, comprising: fermenting the
cellulosic
material with one or more fermenting microorganisms, wherein the cellulosic
material is
saccharified with an enzyme composition in the presence of the polypeptide
having
cellobiohydrolase activity of any of paragraphs 1-12.
[52] The process of paragraph 51, wherein the fermenting of the cellulosic
material
produces a fermentation product.
[53] The process of paragraph 52, further comprising recovering the
fermentation
product from the fermentation.
[54] The process of any of paragraphs 51-53, wherein the cellulosic material
is
pretreated before saccharification.
[55] The process of any of paragraphs 51-54, wherein the enzyme composition
comprises one or more enzymes selected from the group consisting of a
cellulase, a GH61

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polypeptide having cellulolytic enhancing activity, a hemicellulase, an
esterase, an expansin, a
laccase, a ligninolytic enzyme, a pectinase, a peroxidase, a protease, and a
swollen in.
[56] The process of paragraph 55, wherein the cellulase is one or more enzymes
selected from the group consisting of an endoglucanase, a cellobiohydrolase,
and a beta-
glucosidase.
[57] The process of paragraph 55, wherein the hemicellulase is one or more
enzymes
selected from the group consisting of a xylanase, an acetylxylan esterase, a
feruloyl esterase, an
arabinofuranosidase, a xylosidase, and a glucuronidase.
[58] The process of any of paragraphs 52-57, wherein the fermentation product
is an
alcohol, an alkane, a cycloalkane, an alkene, an amino acid, a gas, isoprene,
a ketone, an
organic acid, or polyketide.
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.