U.S. patent application number 15/078705 was filed with the patent office on 2016-08-25 for polypeptides having glucuronyl esterase activity and polynucleotides encoding same.
The applicant listed for this patent is Novoyzmes A/S. Invention is credited to Johan Borjesson, Kristian Krogh, Nikolaj Spodsberg, Anders Viksoe-Nielsen.
Application Number | 20160244732 15/078705 |
Document ID | / |
Family ID | 48224807 |
Filed Date | 2016-08-25 |
United States Patent
Application |
20160244732 |
Kind Code |
A1 |
Borjesson; Johan ; et
al. |
August 25, 2016 |
Polypeptides Having Glucuronyl Esterase Activity and
Polynucleotides Encoding Same
Abstract
The present invention relates to isolated polypeptides having
glucuronyl esterase activity, catalytic domains and polynucleotides
encoding the polypeptides or catalytic 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 or catalytic domains.
Inventors: |
Borjesson; Johan; (Malmo,
SE) ; Viksoe-Nielsen; Anders; (Slangerup, DK)
; Spodsberg; Nikolaj; (Bagsvaerd, DK) ; Krogh;
Kristian; (Bagsvaerd, DK) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Novoyzmes A/S |
Bagsvaerd |
|
DK |
|
|
Family ID: |
48224807 |
Appl. No.: |
15/078705 |
Filed: |
March 23, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14395945 |
Oct 21, 2014 |
|
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|
PCT/EP2013/058292 |
Apr 22, 2013 |
|
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15078705 |
|
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61638699 |
Apr 26, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Y 204/01017 20130101;
C12P 7/14 20130101; C12P 19/14 20130101; Y02P 20/52 20151101; C12N
9/2434 20130101; C12N 9/24 20130101; C12P 19/02 20130101; C12N 1/22
20130101; C12N 9/1051 20130101 |
International
Class: |
C12N 9/10 20060101
C12N009/10 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 23, 2012 |
EP |
12165165.7 |
Claims
1-15. (canceled)
16. A nucleic acid construct comprising a polynucleotide encoding a
polypeptide having glucuronyl esterase activity operably linked to
one or more heterologous control sequences that direct the
expression of the polypeptide in an expression host, wherein the
polypeptide having glucuronyl esterase activity is selected from
the group consisting of: (a) a polypeptide comprising an amino acid
sequence having at least 90% sequence identity to the amino acid
sequence of amino acids 21 to 392 of SEQ ID NO: 6; (b) a
polypeptide encoded by a polynucleotide that hybridizes under high
stringency conditions with the full-length complement of the
nucleotide sequence of nucleotides 235 to 1491 of SEQ ID NO: 5,
wherein the high stringency conditions are defined as
prehybridization and hybridization at 42.degree. C. in
5.times.SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured
salmon sperm DNA, and 50% formamide, and washing three times each
for 15 minutes using 2.times.SSC, 0.2% SDS at 65.degree. C.; (c) a
polypeptide encoded by a polynucleotide comprising a nucleotide
sequence having at least 90% sequence identity to the nucleotide
sequence of nucleotides 235 to 1491 of SEQ ID NO: 5; and (d) a
fragment of the amino acid sequence of amino acids 21 to 392 of SEQ
ID NO: 6, wherein the fragment has glucuronyl esterase
activity.
17. The nucleic acid construct of claim 16, wherein the polypeptide
having glucuronyl esterase activity comprises an amino acid
sequence that has at least 95% sequence identity to the amino acid
sequence of amino acids 21 to 392 of SEQ ID NO: 6.
18. The nucleic acid construct of claim 16, wherein the polypeptide
having glucuronyl esterase activity comprises an amino acid
sequence that has at least 97% sequence identity to the amino acid
sequence of amino acids 21 to 392 of SEQ ID NO: 6.
19. The nucleic acid construct of claim 16, wherein the polypeptide
having glucuronyl esterase activity comprises the amino acid
sequence of SEQ ID NO: 6 or the amino acid sequence of amino acids
21 to 392 of SEQ ID NO: 6.
20. The nucleic acid construct of claim 16, wherein the polypeptide
having glucuronyl esterase activity consists of the amino acid
sequence of SEQ ID NO: 6 or the amino acid sequence of amino acids
21 to 392 of SEQ ID NO: 6.
21. The nucleic acid construct of claim 16, wherein the polypeptide
is a fragment of the amino acid sequence of SEQ ID NO: 6, wherein
the fragment has glucuronyl esterase activity.
22. The nucleic acid construct of claim 16, wherein the polypeptide
having glucuronyl esterase activity is encoded by a polynucleotide
that hybridizes under very high stringency conditions with the
full-length complement of the nucleotide sequence of nucleotides
235 to 1491 of SEQ ID NO: 5, wherein the very high stringency
conditions are defined as prehybridization and hybridization at
42.degree. C. in 5.times.SSPE, 0.3% SDS, 200 micrograms/ml sheared
and denatured salmon sperm DNA, and 50% formamide, and washing
three times each for 15 minutes using 2.times.SSC, 0.2% SDS at
70.degree. C.
23. An isolated recombinant host cell transformed with the nucleic
acid construct of claim 16.
24. A method of producing a polypeptide having glucuronyl esterase
activity, comprising: (a) cultivating the isolated recombinant host
cell of claim 23 under conditions conducive for production of the
polypeptide; and (b) recovering the polypeptide.
25. An isolated recombinant host cell transformed with a nucleic
acid construct comprising a polynucleotide encoding a polypeptide
having glucuronyl esterase activity operably linked to one or more
control sequences that direct the expression of the polypeptide in
the host cell, wherein the polypeptide having glucuronyl esterase
activity is selected from the group consisting of: (a) a
polypeptide comprising an amino acid sequence having at least 90%
sequence identity to the amino acid sequence of amino acids 21 to
392 of SEQ ID NO: 6; (b) a polypeptide encoded by a polynucleotide
that hybridizes under high stringency conditions with the
full-length complement of the nucleotide sequence of nucleotides
235 to 1491 of SEQ ID NO: 5, wherein the high stringency conditions
are defined as prehybridization and hybridization at 42.degree. C.
in 5.times.SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured
salmon sperm DNA, and 50% formamide, and washing three times each
for 15 minutes using 2.times.SSC, 0.2% SDS at 65.degree. C.; (c) a
polypeptide encoded by a polynucleotide comprising a nucleotide
sequence having at least 90% sequence identity to the nucleotide
sequence of nucleotides 235 to 1491 of SEQ ID NO: 5; and (d) a
fragment of the amino acid sequence of amino acids 21 to 392 of SEQ
ID NO: 6, wherein the fragment has glucuronyl esterase
activity.
26. The recombinant host cell of claim 25, wherein the polypeptide
having glucuronyl esterase activity comprises an amino acid
sequence that has at least 95% sequence identity to the amino acid
sequence of amino acids 21 to 392 of SEQ ID NO: 6.
27. The recombinant host cell of claim 25, wherein the polypeptide
having glucuronyl esterase activity comprises an amino acid
sequence that has at least 97% sequence identity to the amino acid
sequence of amino acids 21 to 392 of SEQ ID NO: 6.
28. The recombinant host cell of claim 25, wherein the polypeptide
having glucuronyl esterase activity comprises the amino acid
sequence of SEQ ID NO: 6 or the amino acid sequence of amino acids
21 to 392 of SEQ ID NO: 6.
29. The recombinant host cell of claim 25, wherein the polypeptide
having glucuronyl esterase activity consists of the amino acid
sequence of SEQ ID NO: 6 or the amino acid sequence of amino acids
21 to 392 of SEQ ID NO: 6.
30. The recombinant host cell of claim 25, wherein the polypeptide
is a fragment of the amino acid sequence of SEQ ID NO: 6, wherein
the fragment has glucuronyl esterase activity.
31. The recombinant host cell of claim 25, wherein the polypeptide
having glucuronyl esterase activity is encoded by a polynucleotide
that hybridizes under very high stringency conditions with the
full-length complement of the nucleotide sequence of nucleotides
235 to 1491 of SEQ ID NO: 5, wherein the very high stringency
conditions are defined as prehybridization and hybridization at
42.degree. C. in 5.times.SSPE, 0.3% SDS, 200 micrograms/ml sheared
and denatured salmon sperm DNA, and 50% formamide, and washing
three times each for 15 minutes using 2.times.SSC, 0.2% SDS at
70.degree. C.
32. A method of producing a polypeptide having glucuronyl esterase
activity, comprising: (a) cultivating the isolated recombinant host
cell of claim 22 under conditions conducive for production of the
polypeptide; and (b) recovering the polypeptide.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is divisional application of U.S.
application Ser. No. 14/395,945, filed on Apr. 22, 2013, which is a
35 U.S.C. .sctn.371 national application of PCT/EP2013/058292,
filed on Apr. 22, 2013, which claims priority or the benefit under
35 U.S.C. .sctn.119 of European application No. 12165165.7, filed
Apr. 23, 2012, and U.S. Provisional Application Ser. No.
61/638,699, filed on Apr. 26, 2012. The contents of these
applications are fully incorporated herein by reference.
REFERENCE TO A SEQUENCE LISTING
[0002] This application contains a Sequence Listing in computer
readable form, which is incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] The present invention relates to polypeptides having
glucuronyl esterase activity, catalytic domains, binding domains
and polynucleotides encoding the polypeptides, catalytic domains or
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 binding domains.
[0005] 2. Description of the Related Art
[0006] Cellulosic or xylan-containing material can be pretreated
before hydrolysis and/or fermentation. Pretreatment is preferably
performed prior to the hydrolysis. Alternatively, the pretreatment
can be carried out simultaneously with enzyme hydrolysis to release
fermentable sugars, such as glucose, xylose, and/or cellobiose. In
most cases the pretreatment step itself results in some conversion
of biomass to fermentable sugars (even in absence of enzymes).
[0007] The purpose of the pretreatment is to improve the rate of
production as well as the total yield of liberated sugars in the
hydrolysis step. In case of chemical pretreatment, like e.g. acid
pretreatment or alkali pretreatment, the type of pretreatment will
have different effects on lignocelluloses structural components and
thus the enzyme composition used for the hydrolysis step may differ
dependent on the pretreatment method. The aim of the present method
is to improve hydrolysis of pretreated xylan containing
material.
[0008] The present invention provides polypeptides having
glucuronyl esterase activity and polynucleotides encoding the
polypeptides. The use of polypeptides having glucuronyl esterase
activity provides methods for improved hydrolysis of in particular
xylan containing material.
SUMMARY OF THE INVENTION
[0009] The present invention relates to polypeptides having
glucuronyl esterase activity, selected from the group consisting
of:
[0010] (a) a polypeptide having at least 80%, at least 85%, at
least 90%, at least 91%, at least 92%, at least 93%, at least 94%,
at least 95%, at least 96%, at least 97%, at least 98%, at least
99%, or 100% sequence identity to the mature polypeptide of SEQ ID
NO:2; or
[0011] at least 95%, at least 96%, at least 97%, at least 98%, at
least 99%, or 100% sequence identity to the mature polypeptide of
SEQ ID NO:4 or
[0012] 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;
[0013] (b) a polypeptide encoded by a polynucleotide that
hybridizes under high stringency conditions, or very high
stringency conditions with (i) the mature polypeptide coding
sequence of SEQ ID NO:1, (ii) the cDNA sequence thereof, or (iii)
the full-length complement of (i) or (ii); or
[0014] or under very high stringency conditions with (iv) the
mature polypeptide coding sequence of SEQ ID NO:3, (v) the cDNA
sequence thereof, or (vi) the full-length complement of (iv) or
(v);
[0015] or under very high stringency conditions with (vii) the
mature polypeptide coding sequence of SEQ ID NO:5, (viii) the cDNA
sequence thereof, or (ix) the full-length complement of (vii) or
(viii);
[0016] (c) a polypeptide encoded by a polynucleotide having at
least 80%, at least 85%, at least 85%, at least 90%, at least 91%,
at least 92%, at least 93%, at least 94%, at least 95%, at least
96%, at least 97%, at least 98%, at least 99%, or 100% sequence
identity to the mature polypeptide coding sequence of SEQ ID NO:1
or the cDNA sequence thereof or
[0017] having 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 or
[0018] having at least 85%, at least 90%, at least 91%, at least
92%, at least 93%, at least 94%, at least 95%, at least 96%, at
least 97%, at least 98%, at least 99%, or 100% sequence identity to
the mature polypeptide coding sequence of SEQ ID NO:5 or the cDNA
sequence thereof or
[0019] (d) a variant of the mature polypeptide of SEQ ID NO:2, SEQ
ID NO:4 or SEQ ID NO:6 comprising a substitution, deletion, and/or
insertion at one or more positions; and
[0020] (e) a fragment of the polypeptide of (a), (b), (c) or (d)
that has glucuronyl esterase activity.
[0021] In a related aspect, the present invention relates to
compositions comprising the peptides having glucuronyl esterase
activity as described above.
[0022] In yet another related aspect, the present invention relates
to a method of producing the peptides having glucuronyl esterase
activity as described above and or compositions and/or recombinant
host cells comprising the peptides having glucuronyl esterase
activity as described above.
[0023] Further, the present invention relates to a transgenic
plant, plant part or plant cell transformed with a polynucleotide
encoding the polypeptide having glucuronyl esterase activity as
described above.
[0024] Yet, an additional related aspect of the invention relates
to a method of producing a mutant of a parent cell, comprising
inactivating a polynucleotide encoding the polypeptide having
glucuronyl esterase activity as described above, which results in
the mutant producing less of the polypeptide than the parent
cell.
[0025] The present invention also relates to a double-stranded
inhibitory RNA (dsRNA) molecule comprising a subsequence of an
isolated polynucleotide encoding the polypeptide having glucuronyl
esterase activity as described above, wherein optionally the dsRNA
is an siRNA or an miRNA molecule and methods of inhibiting the
expression of a polypeptide having glucuronyl esterase activity in
a cell, comprising administering to the cell or expressing in the
cell the double-stranded inhibitory RNA (dsRNA) molecule and cells
produced by said method.
[0026] In a related aspect, present invention relates to an
isolated polynucleotide encoding a signal peptides, recombinant
host cells comprising the signal peptide and methods of producing a
protein wherein a recombinant host cell comprising the signal
peptide is cultivated and the protein is recovered. In particular,
the signal peptides of present invention comprise or consist of
amino acids 1 to 17 of SEQ ID NO:2 or amino acids 1 to 17 of SEQ ID
NO:4 or amino acids 1 to 20 of SEQ ID NO:6.
[0027] The present invention also relates to methods for degrading
or converting a cellulosic material, comprising: treating the
cellulosic material with an enzyme composition in the presence of
the polypeptide having glucuronyl esterase activity.
[0028] The present invention also relates to methods for producing
a fermentation product, comprising: a) saccharifying a cellulosic
material with an enzyme composition in the presence of the
polypeptide having glucuronyl esterase activity 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.
[0029] The present invention also relates to methods 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 a polypeptide having glucuronyl esterase
activity.
BRIEF DESCRIPTION OF THE FIGURES
[0030] FIG. 1 shows a comparative study of the effect on percentage
conversion of pretreated corn fiber after addition four glucuronyl
esterases on an enzymatic background of a beta-glucanase and
.beta.-xylosidase. Samples: Sample A, C. unicolor (SEQ ID NO:2);
Sample B, T. reesei (SEQ ID NO:4); Sample C, C. globosum (SEQ ID
NO:6).
[0031] FIG. 2 shows a comparative study of the effect on the
release of glucuronic acid (g/kg DM) after addition four glucuronyl
esterases on an enzymatic background of beta-glucanase and
.beta.-xylosidase. Samples: Sample A, C. unicolor (SEQ ID NO:2);
Sample B, T. reesei (SEQ ID NO:4); Sample C, C. globosum (SEQ ID
NO:6).
DEFINITIONS
[0032] Cellulolytic activity: The term "cellulolytic activity"
means a biological activity that hydrolyzes a cellulosic material.
The two basic approaches for measuring cellulolytic activity
include: (1) measuring the total cellulolytic activity, and (2)
measuring the individual cellulolytic activities (endoglucanases,
cellobiohydrolases, and beta-glucosidases) as reviewed in Zhang et
al., Outlook for cellulase improvement: Screening and selection
strategies, 2006, Biotechnology Advances 24: 452-481. Total
cellulolytic activity is usually measured using insoluble
substrates, including Whatman No 1 filter paper, microcrystalline
cellulose, bacterial cellulose, algal cellulose, cotton, pretreated
lignocellulose, etc. The most common total cellulolytic activity
assay is the filter paper assay using Whatman No 1 filter paper as
the substrate. The assay was established by the International Union
of Pure and Applied Chemistry (IUPAC) (Ghose, 1987, Measurement of
cellulase activities, Pure Appl. Chem. 59: 257-68).
[0033] For purposes of the present invention, cellulolytic activity
is determined by measuring the increase in hydrolysis of a
cellulosic material by cellulolytic enzyme(s) under the following
conditions: 1-20 mg of cellulolytic protein/g of cellulose in PCS
for 3-7 days at 50-65.degree. C. compared to a control hydrolysis
without addition of cellulolytic protein. Typical conditions are 1
ml reactions, washed or unwashed PCS, 5% insoluble solids, 50 mM
sodium acetate pH 5, 1 mM MnSO.sub.4, 50-65.degree. C., 72 hours,
sugar analysis by AMINEX.RTM. HPX-87H column (Bio-Rad Laboratories,
Inc., Hercules, Calif., USA).
[0034] Endoglucanase: The term "endoglucanase" means an
endo-1,4-(1,3; 1,4)-beta-D-glucan 4-glucanohydrolase (E.C.
3.2.1.4), which catalyses endohydrolysis of 1,4-beta-D-glycosidic
linkages in cellulose, cellulose derivatives (such as carboxymethyl
cellulose and hydroxyethyl cellulose), lichenin, beta-1,4 bonds in
mixed beta-1,3 glucans such as cereal beta-D-glucans or
xyloglucans, and other plant material containing cellulosic
components. Endoglucanase activity can be determined by measuring
reduction in substrate viscosity or increase in reducing ends
determined by a reducing sugar assay (Zhang et al., 2006,
Biotechnology Advances 24: 452-481). For purposes of the present
invention, endoglucanase activity is determined using carboxymethyl
cellulose (CMC) as substrate according to the procedure of Ghose,
1987, Pure and Appl. Chem. 59: 257-268, at pH 5, 40.degree. C.
[0035] Cellobiohydrolase: The term "cellobiohydrolase" means a
1,4-beta-D-glucan cellobiohydrolase (E.C. 3.2.1.91), which
catalyzes the hydrolysis of 1,4-beta-D-glucosidic linkages in
cellulose, cellooligosaccharides, or any beta-1,4-linked glucose
containing polymer, releasing cellobiose from the reducing or
non-reducing ends of the chain (Teeri, 1997, Crystalline cellulose
degradation: New insight into the function of cellobiohydrolases,
Trends in Biotechnology 15: 160-167; Teeri et al., 1998,
Trichoderma reesei cellobiohydrolases: why so efficient on
crystalline cellulose?, Biochem. Soc. Trans. 26: 173-178). For
purposes of the present invention, cellobiohydrolase activity is
determined using a fluorescent disaccharide derivative
4-methylumbelliferyl-.beta.-D-lactoside according to the procedures
described by van Tilbeurgh et al., 1982, FEBS Letters 149: 152-156
and van Tilbeurgh and Claeyssens, 1985, FEBS Letters 187: 283-288,
at pH 5, 40.degree. C.
[0036] Beta-glucosidase: The term "beta-glucosidase" means a
beta-D-glucoside glucohydrolase (E.C. 3.2.1.21), which catalyzes
the hydrolysis of terminal non-reducing beta-D-glucose residues
with the release of beta-D-glucose. For purposes of the present
invention, beta-glucosidase activity is determined according to the
basic procedure described by Venturi et al., 2002, Extracellular
beta-D-glucosidase from Chaetomium thermophilum var. coprophilum:
production, purification and some biochemical properties, J. Basic
Microbiol. 42: 55-66. One unit of beta-glucosidase is defined as
1.0 .mu.mole of p-nitrophenol produced per minute at 40.degree. C.,
pH 5 from 1 mM p-nitrophenyl-beta-D-glucopyranoside as substrate in
100 mM sodium citrate containing 0.01% TWEEN.RTM. 20.
[0037] Cellulolytic enhancing activity: The term "cellulolytic
enhancing activity" means a biological activity catalyzed by a GH61
polypeptide that enhances the hydrolysis of a cellulosic material
by enzyme having cellulolytic activity. For purposes of the present
invention, cellulolytic enhancing activity is determined by
measuring the increase in reducing sugars or the increase of the
total of cellobiose and glucose from the hydrolysis of a cellulosic
material by cellulolytic enzyme under the following conditions:
1-50 mg of total protein/g of cellulose in PCS, wherein total
protein is comprised of 50-99.5% w/w cellulolytic protein and
0.5-50% w/w protein of a GH61 polypeptide having cellulolytic
enhancing activity for 1-7 day at 50-65.degree. C. compared to a
control hydrolysis with equal total protein loading without
cellulolytic enhancing activity (1-50 mg of cellulolytic protein/g
of cellulose in PCS). In a preferred aspect, a mixture of
CELLUCLAST.RTM. 1.5 L (Novozymes A/S, Bagsvaerd, Denmark) in the
presence of 3% of total protein weight Aspergillus oryzae
beta-glucosidase (recombinantly produced in Aspergillus oryzae
according to WO 02/095014) or 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.
[0038] The GH61 polypeptides having cellulolytic enhancing activity
enhance the hydrolysis of a cellulosic material catalyzed by enzyme
having cellulolytic activity by reducing the amount of cellulolytic
enzyme required to reach the same degree of hydrolysis preferably
at least 1.01-fold, more preferably at least 1.05-fold, more
preferably at least 1.10-fold, more preferably at least 1.25-fold,
more preferably at least 1.5-fold, more preferably at least 2-fold,
more preferably at least 3-fold, more preferably at least 4-fold,
more preferably at least 5-fold, even more preferably at least
10-fold, and most preferably at least 20-fold.
[0039] 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.
[0040] Xylan degrading activity: The terms "xylan degrading
activity" or "xylanolytic activity" mean 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 (endoxylanases, beta-xylosidases, arabinofuranosidases,
alpha-glucuronidases, acetylxylan esterases, feruloyl esterases,
and alpha-glucuronyl esterases). Recent progress in assays of
xylanolytic enzymes was summarized in several publications
including Biely and Puchard, Recent progress in the assays of
xylanolytic enzymes, 2006, Journal of the Science of Food and
Agriculture 86(11): 1636-1647; Spanikova and Biely, 2006,
Glucuronoyl esterase--Novel carbohydrate esterase produced by
Schizophyllum commune, FEBS Letters 580(19): 4597-4601; Herrmann,
Vrsanska, Jurickova, Hirsch, Biely, and Kubicek, 1997, The
beta-D-xylosidase of Trichoderma reesei is a multifunctional
beta-D-xylan xylohydrolase, Biochemical Journal 321: 375-381.
[0041] Total xylan degrading activity can be measured by
determining the reducing sugars formed from various types of xylan,
including, for example, oat spelt, beechwood, and larchwood xylans,
or by photometric determination of dyed xylan fragments released
from various covalently dyed xylans. The most common total
xylanolytic activity assay is based on production of reducing
sugars from polymeric 4-O-methyl glucuronoxylan as described in
Bailey, Biely, Poutanen, 1992, Interlaboratory testing of methods
for assay of xylanase activity, Journal of Biotechnology 23(3):
257-270.
[0042] For purposes of the present invention, xylan degrading
activity is determined by measuring the increase in hydrolysis of
birchwood xylan (Sigma Chemical Co., Inc., St. Louis, Mo., USA) by
xylan-degrading enzyme(s) under the following typical conditions: 1
ml reactions, 5 mg/ml substrate (total solids), 5 mg of xylanolytic
protein/g of substrate, 50 mM sodium acetate pH 5, 50.degree. C.,
24 hours, sugar analysis using p-hydroxybenzoic acid hydrazide
(PHBAH) assay as described by Lever, 1972, A new reaction for
colorimetric determination of carbohydrates, Anal. Biochem 47:
273-279.
[0043] Xylanase: The term "xylanase" means a
1,4-beta-D-xylan-xylohydrolase (E.C. 3.2.1.8) that catalyzes the
endo-hydrolysis of 1,4-beta-D-xylosidic linkages in xylans. For
purposes of the present invention, xylanase activity is determined
using birchwood xylan as substrate. One unit of xylanase is defined
as 1.0 .mu.mole of reducing sugar (measured in glucose equivalents
as described by Lever, 1972, A new reaction for colorimetric
determination of carbohydrates, Anal. Biochem 47: 273-279) produced
per minute during the initial period of hydrolysis at 50.degree.
C., pH 5 from 2 g of birchwood xylan per liter as substrate in 50
mM sodium acetate containing 0.01% TWEEN.RTM. 20.
[0044] Beta-xylosidase: The term "beta-xylosidase" means a
beta-D-xyloside xylohydrolase (E.C. 3.2.1.37) that catalyzes the
exo-hydrolysis of short beta (1.fwdarw.4)-xylooligosaccharides, to
remove successive D-xylose residues from the non-reducing termini.
For purposes of the present invention, one unit of beta-xylosidase
is defined as 1.0 .mu.mole of p-nitrophenol produced per minute at
40.degree. C., pH 5 from 1 mM p-nitrophenyl-beta-D-xyloside as
substrate in 100 mM sodium citrate containing 0.01% TWEEN.RTM.
20.
[0045] Acetylxylan esterase: The term "acetylxylan esterase" means
a carboxylesterase (EC 3.1.1.72) that catalyzes the hydrolysis of
acetyl groups from polymeric xylan, acetylated xylose, acetylated
glucose, alpha-napthyl acetate, and p-nitrophenyl acetate. For
purposes of the present invention, acetylxylan esterase activity is
determined using 0.5 mM p-nitrophenylacetate as substrate in 50 mM
sodium acetate pH 5.0 containing 0.01% TWEEN.TM. 20. One unit of
acetylxylan esterase is defined as the amount of enzyme capable of
releasing 1 .mu.mole of p-nitrophenolate anion per minute at pH 5,
25.degree. C.
[0046] Feruloyl esterase: The term "feruloyl esterase" means a
4-hydroxy-3-methoxycinnamoyl-sugar hydrolase (EC 3.1.1.73) that
catalyzes the hydrolysis of the 4-hydroxy-3-methoxycinnamoyl
(feruloyl) group from an esterified sugar, which is usually
arabinose in "natural" substrates, to produce ferulate
(4-hydroxy-3-methoxycinnamate). Feruloyl esterase is also known as
ferulic acid esterase, hydroxycinnamoyl esterase, FAE-III,
cinnamoyl ester hydrolase, FAEA, cinnAE, FAE-I, or FAE-II. For
purposes of the present invention, feruloyl esterase activity is
determined using 0.5 mM p-nitrophenylferulate as substrate in 50 mM
sodium acetate pH 5.0. One unit of feruloyl esterase equals the
amount of enzyme capable of releasing 1 .mu.mole of
p-nitrophenolate anion per minute at pH 5, 25.degree. C.
[0047] Alpha-glucuronidase: The term "alpha-glucuronidase" means an
alpha-D-glucosiduronate glucuronohydrolase (EC 3.2.1.139) that
catalyzes the hydrolysis of an alpha-D-glucuronoside to
D-glucuronate and an alcohol. For purposes of the present
invention, alpha-glucuronidase activity is determined according to
de Vries, 1998, J. Bacteriol. 180: 243-249. One unit of
alpha-glucuronidase equals the amount of enzyme capable of
releasing 1 .mu.mole of glucuronic or 4-O-methylglucuronic acid per
minute at pH 5, 40.degree. C.
[0048] The polypeptides of the present invention have at least 60%,
e.g. at least 70%, at least 80%, at least 90%, 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 at least 100% of the glucuronyl
esterase activity of the mature polypeptide of SEQ ID NO:2, SEQ ID
NO:4 or SEQ ID NO:6.
[0049] Alpha-L-arabinofuranosidase: The term
"alpha-L-arabinofuranosidase" means an alpha-L-arabinofuranoside
arabinofuranohydrolase (EC 3.2.1.55) that catalyzes the hydrolysis
of terminal non-reducing alpha-L-arabinofuranoside residues in
alpha-L-arabinosides. The enzyme acts on
alpha-L-arabinofuranosides, alpha-L-arabinans containing (1,3)-
and/or (1,5)-linkages, arabinoxylans, and arabinogalactans.
Alpha-L-arabinofuranosidase is also known as arabinosidase,
alpha-arabinosidase, alpha-L-arabinosidase,
alpha-arabinofuranosidase, polysaccharide
alpha-L-arabinofuranosidase, alpha-L-arabinofuranoside hydrolase,
L-arabinosidase, or alpha-L-arabinanase. For purposes of the
present invention, alpha-L-arabinofuranosidase activity is
determined using 5 mg of medium viscosity wheat arabinoxylan
(Megazyme International Ireland, Ltd., Bray, Co. Wicklow, Ireland)
per ml of 100 mM sodium acetate pH 5 in a total volume of 200 .mu.l
for 30 minutes at 40.degree. C. followed by arabinose analysis by
AMINEX.RTM. HPX-87H column chromatography (Bio-Rad Laboratories,
Inc., Hercules, Calif., USA).
[0050] Cellulosic material: The cellulosic material can be 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.
[0051] Cellulose is generally found, for example, in the stems,
leaves, hulls, husks, and cobs of plants or leaves, branches, and
wood of trees. The cellulosic material can be, but is not limited
to, herbaceous material, agricultural residue, forestry residue,
municipal solid waste, waste paper, and pulp and paper mill residue
(see, for example, Wiselogel et al., 1995, in Handbook on
Bioethanol (Charles E. Wyman, editor), pp. 105-118, Taylor &
Francis, Washington D.C.; Wyman, 1994, Bioresource Technology 50:
3-16; Lynd, 1990, Applied Biochemistry and Biotechnology 24/25:
695-719; Mosier et al., 1999, Recent Progress in Bioconversion of
Lignocellulosics, in Advances in Biochemical
Engineering/Biotechnology, T. Scheper, managing editor, Volume 65,
pp. 23-40, Springer-Verlag, New York). It is understood herein that
the cellulose may be in the form of lignocellulose, a plant cell
wall material containing lignin, cellulose, and hemicellulose in a
mixed matrix. In a preferred aspect, the cellulosic material is
lignocellulose.
[0052] In one aspect, the cellulosic material is herbaceous
material. In another aspect, the cellulosic material is
agricultural residue. In another aspect, the cellulosic material is
forestry residue. In another aspect, the cellulosic material is
municipal solid waste. In another aspect, the cellulosic material
is waste paper. In another aspect, the cellulosic material is pulp
and paper mill residue.
[0053] In another aspect, the cellulosic material is corn stover.
In another aspect, the cellulosic material is corn fiber. In
another aspect, the cellulosic material is corn cob. In another
aspect, the cellulosic material is orange peel. In another aspect,
the cellulosic material is rice straw. In another aspect, the
cellulosic material is wheat straw. In another aspect, the
cellulosic material is switch grass. In another aspect, the
cellulosic material is miscanthus. In another aspect, the
cellulosic material is bagasse.
[0054] In another aspect, the cellulosic material is
microcrystalline cellulose. In another aspect, the cellulosic
material is bacterial cellulose. In another aspect, the cellulosic
material is algal cellulose. In another aspect, the cellulosic
material is cotton linter. In another aspect, the cellulosic
material is amorphous phosphoric-acid treated cellulose. In another
aspect, the cellulosic material is filter paper.
[0055] The cellulosic material may be used as is or may be
subjected to pretreatment, using conventional methods known in the
art, as described herein. In a preferred aspect, the cellulosic
material is pretreated.
[0056] 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.
[0057] Xylan-containing material: The term "xylan-containing
material" means any material comprising a plant cell wall
polysaccharide containing a backbone of beta-(1-4)-linked xylose
residues. Xylans of terrestrial plants are heteropolymers
possessing a beta-(1-4)-D-xylopyranose backbone, which is branched
by short carbohydrate chains. They comprise D-glucuronic acid or
its 4-O-methyl ether, L-arabinose, and/or various oligosaccharides,
composed of D-xylose, L-arabinose, D- or L-galactose, and
D-glucose. Xylan-type polysaccharides can be divided into
homoxylans and heteroxylans, which include glucuronoxylans,
(arabino)glucuronoxylans, (glucurono)arabinoxylans, arabinoxylans,
and complex heteroxylans. See, for example, Ebringerova et al.,
2005, Adv. Polym. Sci. 186: 1-67.
[0058] In the methods of the present invention, any material
containing xylan may be used. In a preferred aspect, the
xylan-containing material is lignocellulose.
[0059] Allelic variant: The term "allelic variant" means any of two
or more alternative forms of a gene occupying the same chromosomal
locus. Allelic variation arises naturally through mutation, and may
result in polymorphism within populations. Gene mutations can be
silent (no change in the encoded polypeptide) or may encode
polypeptides having altered amino acid sequences. An allelic
variant of a polypeptide is a polypeptide encoded by an allelic
variant of a gene.
[0060] Binding domain: The term "binding domain" e.g., "cellulose
binding domain" means the region of an enzyme that mediates binding
of the enzyme to amorphous regions of a cellulose substrate. The
cellulose binding domain (CBD) is typically found either at the
N-terminal or at the C-terminal extremity of an glucuronyl
esterase.
[0061] Catalytic domain: The term "catalytic domain" means the
region of an enzyme containing the catalytic machinery of the
enzyme.
[0062] 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.
[0063] Coding sequence: The term "coding sequence" means a
polynucleotide, which directly specifies the amino acid sequence of
a polypeptide. The boundaries of the coding sequence are generally
determined by an open reading frame, which begins with a start
codon such as ATG, GTG, or TTG and ends with a stop codon such as
TAA, TAG, or TGA. The coding sequence may be a genomic DNA, cDNA,
synthetic DNA, or a combination thereof.
[0064] 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.
[0065] 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.
[0066] 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.
[0067] Fragment: The term "fragment" means a polypeptide or a
catalytic or cellulose binding domain having one or more (e.g.,
several) amino acids deleted from the amino and/or carboxyl
terminus of a mature polypeptide or domain; wherein the fragment
has glucuronyl esterase or cellulose binding activity. In one
aspect, a fragment contains at least 85%, 90%, and 95% of the
number of amino acids of the mature polypeptide of SEQ ID NO:2, SEQ
ID NO:4 or SEQ ID NO:6.
[0068] Host cell: The term "host cell" means any cell type that is
susceptible to transformation, transfection, transduction, or the
like with a nucleic acid construct or expression vector comprising
a polynucleotide of the present invention. The term "host cell"
encompasses any progeny of a parent cell that is not identical to
the parent cell due to mutations that occur during replication.
[0069] Isolated or purified: The term "isolated" or "purified"
means a polypeptide or polynucleotide that is removed from at least
one component with which it is naturally associated. For example, a
polypeptide may be at least 1% pure, e.g., at least 5% pure, at
least 10% pure, at least 20% pure, at least 40% pure, at least 60%
pure, at least 80% pure, at least 90% pure, or at least 95% pure,
as determined by SDS-PAGE, and a polynucleotide may be at least 1%
pure, e.g., at least 5% pure, at least 10% pure, at least 20% pure,
at least 40% pure, at least 60% pure, at least 80% pure, at least
90% pure, or at least 95% pure, as determined by agarose
electrophoresis.
[0070] 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 1 to 474 of SEQ ID
NO:2, amino acids 1 to 460 of SEQ ID NO:4 or amino acids 1 to 392
of SEQ ID NO:6 as predicted using SignalP (Nielsen et al., 1997,
Protein Engineering 10: 1-6) that predicts amino acids 1 to 17 of
SEQ ID NO:2, 1 to 17 of SEQ ID NO:4, amino acids 21 to 690 of SEQ
ID NO:4 or amino acids 1 to 2O of SEQ ID NO:6; are a signal
peptides. 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.
[0071] Mature polypeptide coding sequence: The term "mature
polypeptide coding sequence" means a polynucleotide that encodes a
mature polypeptide having glucuronyl esterase activity. In one
aspect, the mature polypeptide coding sequence is nucleotides 1 to
2544 of SEQ ID NO:1, nucleotides 1 to 2526 of SEQ ID NO:3,
nucleotides 1 to 2508 of SEQ ID NO:5, nucleotides 1 to 2124 of SEQ
ID NO:7 or the cDNA sequence thereof; based on the SignalP program
(Nielsen et al., 1997, supra)] that predicts nucleotides 1 to 66 of
SEQ ID NO:1, nucleotides 1 to 60 of SEQ ID NO:3, nucleotides 1 to
45 of SEQ ID NO:5, nucleotides 1 to 81 of SEQ ID NO:7, encode a
signal peptide.
[0072] 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.
[0073] 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 the
expression of the coding sequence.
[0074] Sequence identity: The relatedness between two amino acid
sequences or between two nucleotide sequences is described by the
parameter "sequence identity".
[0075] For purposes of the present invention, the sequence identity
between two amino acid sequences is determined using the
Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol.
Biol. 48: 443-453) as implemented in the Needle program of the
EMBOSS package (EMBOSS: The European Molecular Biology Open
Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277),
preferably version 5.0.0 or later. The parameters used are gap open
penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62
(EMBOSS version of BLOSUM62) substitution matrix. The output of
Needle labeled "longest identity" (obtained using the--nobrief
option) is used as the percent identity and is calculated as
follows:
(Identical Residues.times.100)/(Length of Alignment-Total Number of
Gaps in Alignment)
[0076] For purposes of the present invention, the sequence identity
between two deoxyribonucleotide sequences is determined using the
Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as
implemented in the Needle program of the EMBOSS package (EMBOSS:
The European Molecular Biology Open Software Suite, Rice et al.,
2000, supra), preferably version 5.0.0 or later. The parameters
used are gap open penalty of 10, gap extension penalty of 0.5, and
the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix.
The output of Needle labeled "longest identity" (obtained using
the--nobrief option) is used as the percent identity and is
calculated as follows:
(Identical Deoxyribonucleotides.times.100)/(Length of
Alignment-Total Number of Gaps in Alignment)
[0077] Subsequence: The term "subsequence" means a polynucleotide
having one or more (e.g., several) nucleotides deleted from the 5'
and/or 3' end of a mature polypeptide coding sequence; wherein the
subsequence encodes a fragment having glucuronyl esterase activity.
In one aspect, a subsequence contains at least 85%, 90%, and 95% of
the number of amino acids of the mature polypeptide of SEQ ID NO:2,
SEQ ID NO:4 or SEQ ID NO:6.
[0078] Variant: The term "variant" means a polypeptide having
glucuronyl esterase activity comprising an alteration, i.e., a
substitution, insertion, and/or deletion of one or more (e.g.,
several) amino acid residues at one or more positions. A
substitution means a 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
one or more (e.g., several) amino acids, e.g., 1-5 amino acids,
adjacent to the amino acid occupying a position).
DETAILED DESCRIPTION OF THE INVENTION
Polypeptides Having Glucuronyl Esterase Activity
[0079] The present invention relates to isolated polypeptides
having glucuronyl esterase activity selected from the group
consisting of:
[0080] (a) a polypeptide having at least 80% sequence identity to
the mature polypeptide of SEQ ID NO:2;
[0081] (b) a polypeptide encoded by a polynucleotide that
hybridizes under high, or very high stringency conditions with (i)
the mature polypeptide coding sequence of SEQ ID NO:1, (ii) the
cDNA sequence thereof, or (iii) the full-length complement of (i)
or (ii);
[0082] (c) a polypeptide encoded by a polynucleotide having at
least 80% sequence identity to the mature polypeptide coding
sequence of SEQ ID NO:1 or the cDNA sequence thereof;
[0083] (d) a variant of the mature polypeptide of SEQ ID NO:2
comprising a substitution, deletion, and/or insertion at one or
more (e.g., several) positions; and
[0084] (e) a fragment of the polypeptide of (a), (b), (c) or (d)
that has glucuronyl esterase activity.
[0085] 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.
[0086] 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, a polynucleotide encoding a propeptide
comprising or consisting of amino acids 101 to 474 of SEQ ID NO:2,
or a polynucleotide encoding a signal peptide and a propeptide
comprising or consisting of amino acids 1 to 474 of SEQ ID NO:2,
each of which is operably linked to a gene encoding a protein;
nucleic acid constructs, expression vectors, and recombinant host
cells comprising the polynucleotides; and methods of producing a
protein.
[0087] The present invention relates to isolated polypeptides
having glucuronyl esterase activity selected from the group
consisting of:
[0088] (a) a polypeptide having at least 80% sequence identity to
the mature polypeptide of SEQ ID NO:4;
[0089] (b) a polypeptide encoded by a polynucleotide that
hybridizes under high, or very high stringency conditions with (i)
the mature polypeptide coding sequence of SEQ ID NO:3, (ii) the
cDNA sequence thereof, or (iii) the full-length complement of (i)
or (ii);
[0090] (c) a polypeptide encoded by a polynucleotide having at
least 80% sequence identity to the mature polypeptide coding
sequence of SEQ ID NO:4 or the cDNA sequence thereof;
[0091] (d) a variant of the mature polypeptide of SEQ ID NO:4
comprising a substitution, deletion, and/or insertion at one or
more (e.g., several) positions; and
[0092] (e) a fragment of the polypeptide of (a), (b), (c) or (d)
that has glucuronyl esterase activity.
[0093] 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.
[0094] 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:4, a polynucleotide encoding a propeptide
comprising or consisting of amino acids 17 to 460 of SEQ ID NO:4,
or a polynucleotide encoding a signal peptide and a propeptide
comprising or consisting of amino acids 1 to 460 of SEQ ID NO:4,
each of which is operably linked to a gene encoding a protein;
nucleic acid constructs, expression vectors, and recombinant host
cells comprising the polynucleotides; and methods of producing a
protein.
[0095] The present invention relates to isolated polypeptides
having glucuronyl esterase activity selected from the group
consisting of:
[0096] (a) a polypeptide having at least 80% sequence identity to
the mature polypeptide of SEQ ID NO:6;
[0097] (b) a polypeptide encoded by a polynucleotide that
hybridizes under high, or very high stringency conditions with (i)
the mature polypeptide coding sequence of SEQ ID NO:6, (ii) the
cDNA sequence thereof, or (iii) the full-length complement of (i)
or (ii);
[0098] (c) a polypeptide encoded by a polynucleotide having at
least 80% sequence identity to the mature polypeptide coding
sequence of SEQ ID NO:6 or the cDNA sequence thereof;
[0099] (d) a variant of the mature polypeptide of SEQ ID NO:6
comprising a substitution, deletion, and/or insertion at one or
more (e.g., several) positions; and
[0100] (e) a fragment of the polypeptide of (a), (b), (c) or (d)
that has glucuronyl esterase activity.
[0101] 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.
[0102] The present invention also relates to a polynucleotide
encoding a signal peptide comprising or consisting of amino acids 1
to 20 of SEQ ID NO:6, a polynucleotide encoding a propeptide
comprising or consisting of amino acids 21 to 392 of SEQ ID NO:6,
or a polynucleotide encoding a signal peptide and a propeptide
comprising or consisting of amino acids 1 to 392 of SEQ ID NO:6,
each of which is operably linked to a gene encoding a protein;
nucleic acid constructs, expression vectors, and recombinant host
cells comprising the polynucleotides; and methods of producing a
protein.
[0103] The present invention also relates to methods of inhibiting
expression or producing one or more of the peptides having at least
68% such as e.g. 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% sequence
identity to any of the sequences SEQ ID: NO2, SEQ ID NO: 4, or SEQ
ID NO: 6.
[0104] Furthermore, present invention relates to a method for
degrading or converting a cellulosic material, comprising: treating
the cellulosic material with an enzyme composition in the presence
of the polypeptide having glucuronyl esterase activity having at
least 68% such as e.g. 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100%
sequence identity to any of the sequences SEQ ID: NO2, SEQ ID NO: 4
or SEQ ID NO: 6.
[0105] In an embodiment, the present invention relates to isolated
polypeptides having a sequence identity to the mature polypeptide
of SEQ ID NO:2 of at least 80%, e.g. at least 85%, at least 90%, at
least 91%, at least 92%, at least 93%, at least 94%, at least 95%,
at least 96%, at least 97%, at least 98%, at least 99%, or 100%,
which have glucuronyl esterase activity. In one aspect, the
polypeptides differ by no more than ten amino acids, e.g., nine
amino acids, eight amino acids, seven amino acids, six amino acids,
five amino acids, four amino acids, three amino acids, two amino
acids, or one amino acid from the mature polypeptide of SEQ ID
NO:2.
[0106] A polypeptide of the present invention preferably comprises
or consists of the amino acid sequence of SEQ ID NO:2 or an allelic
variant thereof; or is a fragment thereof having glucuronyl
esterase activity. In another aspect, the polypeptide comprises or
consists of the mature polypeptide of SEQ ID NO:2. In another
preferred aspect, the polypeptide comprises or consists of amino
acids 101 to 474 of SEQ ID NO:2.
[0107] 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 95%, e.g. at least 96%, at least 97%, at
least 98%, at least 99%, or 100%, which have glucuronyl esterase
activity. In one aspect, the polypeptides differ by no more than
ten amino acids, e.g., nine amino acids, eight amino acids, seven
amino acids, six amino acids, five amino acids, four amino acids,
three amino acids, two amino acids, or one amino acid from the
mature polypeptide of SEQ ID NO:4.
[0108] A polypeptide of the present invention preferably comprises
or consists of the amino acid sequence of SEQ ID NO:4 or an allelic
variant thereof; or is a fragment thereof having glucuronyl
esterase activity. In another aspect, the polypeptide comprises or
consists of the mature polypeptide of SEQ ID NO:4. In another
preferred aspect, the polypeptide comprises or consists of amino
acids 94 to 460 of SEQ ID NO:4.
[0109] 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 92%, e.g. 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 glucuronyl esterase activity. In one aspect,
the polypeptides differ by no more than ten amino acids, e.g., nine
amino acids, eight amino acids, seven amino acids, six amino acids,
five amino acids, four amino acids, three amino acids, two amino
acids, or one amino acid from the mature polypeptide of SEQ ID
NO:6.
[0110] A polypeptide of the present invention preferably comprises
or consists of the amino acid sequence of SEQ ID NO:6 or an allelic
variant thereof; or is a fragment thereof having glucuronyl
esterase activity. In another aspect, the polypeptide comprises or
consists of the mature polypeptide of SEQ ID NO:6. In another
preferred aspect, the polypeptide comprises or consists of amino
acids 21 to 392 of SEQ ID NO:6.
[0111] In another embodiment, the present invention relates to
isolated polypeptides having glucuronyl esterase activity that are
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, SEQ ID NO:3, SEQ ID
NO:5 (ii) the cDNA sequence thereof, or (iii) the full-length
complement of (i) or (ii) (Sambrook et al., 1989, Molecular
Cloning, A Laboratory Manual, 2d edition, Cold Spring Harbor,
N.Y.).
[0112] The polynucleotide of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5
or a subsequence thereof, as well as the polypeptide of SEQ ID
NO:2, SEQ ID NO:4, SEQ ID NO:6 or a fragment thereof, may be used
to design nucleic acid probes to identify and clone DNA encoding
polypeptides having glucuronyl esterase activity from strains of
different genera or species according to methods well known in the
art. In particular, such probes can be used for hybridization with
the genomic DNA or cDNA of a cell of interest, following standard
Southern blotting procedures, in order to identify and isolate the
corresponding gene therein. Such probes can be considerably shorter
than the entire sequence, but should be at least 15, e.g., at least
25, at least 35, or at least 70 nucleotides in length. Preferably,
the nucleic acid probe is at least 100 nucleotides in length, e.g.,
at least 200 nucleotides, at least 300 nucleotides, at least 400
nucleotides, at least 500 nucleotides, at least 600 nucleotides, at
least 700 nucleotides, at least 800 nucleotides, or at least 900
nucleotides in length. Both DNA and RNA probes can be used. The
probes are typically labeled for detecting the corresponding gene
(for example, with .sup.32P, .sup.3H, .sup.35S, biotin, or avidin).
Such probes are encompassed by the present invention.
[0113] 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 glucuronyl
esterase activity. Genomic or other DNA from such other strains may
be separated by agarose or polyacrylamide gel electrophoresis, or
other separation techniques. DNA from the libraries or the
separated DNA may be transferred to and immobilized on
nitrocellulose or other suitable carrier material. In order to
identify a clone or DNA that is homologous with SEQ ID NO:1, SEQ ID
NO:3, SEQ ID NO:5 or a subsequence thereof, the carrier material is
preferably used in a Southern blot.
[0114] For purposes of the present invention, hybridization
indicates that the polynucleotide hybridizes to a labeled nucleic
acid probe corresponding to (i) SEQ ID NO:1, SEQ ID NO:3, SEQ ID
NO:5; (ii) the mature polypeptide coding sequence of SEQ ID NO:1,
SEQ ID NO:3, SEQ ID NO:5; (iii) the cDNA sequence; (iv) the
full-length complement thereof; or (v) a subsequence thereof; under
very low to very high stringency conditions. Molecules to which the
nucleic acid probe hybridizes under these conditions can be
detected using, for example, X-ray film.
[0115] In another 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; 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 the cDNA sequence thereof.
[0116] For probes of at least 100 nucleotides in length, very low
stringency conditions are defined as prehybridization and
hybridization at 42.degree. C. in 5.times.SSPE, 0.3% SDS, 200
micrograms/ml sheared and denatured salmon sperm DNA, and 25%
formamide, following standard Southern blotting procedures for 12
to 24 hours optimally. The carrier material is finally washed three
times each for 15 minutes using 2.times.SSC, 0.2% SDS at 45.degree.
C.
[0117] For probes of at least 100 nucleotides in length, low
stringency conditions are defined as prehybridization and
hybridization at 42.degree. C. in 5.times.SSPE, 0.3% SDS, 200
micrograms/ml sheared and denatured salmon sperm DNA, and 25%
formamide, following standard Southern blotting procedures for 12
to 24 hours optimally. The carrier material is finally washed three
times each for 15 minutes using 2.times.SSC, 0.2% SDS at 50.degree.
C.
[0118] For probes of at least 100 nucleotides in length, medium
stringency conditions are defined as prehybridization and
hybridization at 42.degree. C. in 5.times.SSPE, 0.3% SDS, 200
micrograms/ml sheared and denatured salmon sperm DNA, and 35%
formamide, following standard Southern blotting procedures for 12
to 24 hours optimally. The carrier material is finally washed three
times each for 15 minutes using 2.times.SSC, 0.2% SDS at 55.degree.
C.
[0119] For probes of at least 100 nucleotides in length,
medium-high stringency conditions are defined as prehybridization
and hybridization at 42.degree. C. in 5.times.SSPE, 0.3% SDS, 200
micrograms/ml sheared and denatured salmon sperm DNA, and either
35% formamide, following standard Southern blotting procedures for
12 to 24 hours optimally. The carrier material is finally washed
three times each for 15 minutes using 2.times.SSC, 0.2% SDS at
60.degree. C.
[0120] For probes of at least 100 nucleotides in length, high
stringency conditions are defined as prehybridization and
hybridization at 42.degree. C. in 5.times.SSPE, 0.3% SDS, 200
micrograms/ml sheared and denatured salmon sperm DNA, and 50%
formamide, following standard Southern blotting procedures for 12
to 24 hours optimally. The carrier material is finally washed three
times each for 15 minutes using 2.times.SSC, 0.2% SDS at 65.degree.
C.
[0121] For probes of at least 100 nucleotides in length, very high
stringency conditions are defined as prehybridization and
hybridization at 42.degree. C. in 5.times.SSPE, 0.3% SDS, 200
micrograms/ml sheared and denatured salmon sperm DNA, and 50%
formamide, following standard Southern blotting procedures for 12
to 24 hours optimally. The carrier material is finally washed three
times each for 15 minutes using 2.times.SSC, 0.2% SDS at 70.degree.
C.
[0122] In another embodiment, the present invention relates to
isolated polypeptides having glucuronyl esterase activity encoded
by polynucleotides having a sequence identity to the mature
polypeptide coding sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID
NO:5 of at least 60%, e.g., at least 65%, at least 70%, at least
75%, at least 80%, at least 85%, at least 90%, at least 91%, at
least 92%, at least 93%, at least 94%, at least 95%, at least 96%,
at least 97%, at least 98%, at least 99%, or 100%.
[0123] In another embodiment, the present invention relates to
variants of the mature polypeptide of SEQ ID NO:2, SEQ ID NO:4, SEQ
ID NO:6 comprising a substitution, deletion, and/or insertion at
one or more (e.g., several) positions. Preferably, amino acid
changes are of a minor nature, that is conservative amino acid
substitutions or insertions that do not significantly affect the
folding and/or activity of the protein; small deletions, typically
of one to about 30 amino acids; small amino- or carboxyl-terminal
extensions, such as an amino-terminal methionine residue; a small
linker peptide of up to about 20-25 residues; or a small extension
that facilitates purification by changing net charge or another
function, such as a poly-histidine tract, an antigenic epitope or a
binding domain.
[0124] Examples of conservative substitutions are within the groups
of basic amino acids (arginine, lysine and histidine), acidic amino
acids (glutamic acid and aspartic acid), polar amino acids
(glutamine and asparagine), hydrophobic amino acids (leucine,
isoleucine and valine), aromatic amino acids (phenylalanine,
tryptophan and tyrosine), and small amino acids (glycine, alanine,
serine, threonine and methionine). Amino acid substitutions that do
not generally alter specific activity are known in the art and are
described, for example, by H. Neurath and R. L. Hill, 1979, In, The
Proteins, Academic Press, New York. Common substitutions are
Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn,
Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile,
Leu/Val, Ala/Glu, and Asp/Gly.
[0125] 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.
[0126] 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 glucuronyl
esterase activity to identify amino acid residues that are critical
to the activity of the molecule. See also, Hilton et al., 1996, J.
Biol. Chem. 271: 4699-4708. The active site of the enzyme or other
biological interaction can also be determined by physical analysis
of structure, as determined by such techniques as nuclear magnetic
resonance, crystallography, electron diffraction, or photoaffinity
labeling, in conjunction with mutation of putative contact site
amino acids. See, for example, de Vos et al., 1992, Science 255:
306-312; Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver
et al., 1992, FEBS Lett. 309: 59-64. The identity of essential
amino acids can also be inferred from an alignment with a related
polypeptide.
[0127] Single or multiple amino acid substitutions, deletions,
and/or insertions can be made and tested using known methods of
mutagenesis, recombination, and/or shuffling, followed by a
relevant screening procedure, such as those disclosed by
Reidhaar-Olson and Sauer, 1988, Science 241: 53-57; Bowie and
Sauer, 1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95/17413;
or WO 95/22625. Other methods that can be used include error-prone
PCR, phage display (e.g., Lowman et al., 1991, Biochemistry 30:
10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204), and
region-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145;
Ner et al., 1988, DNA 7: 127).
[0128] Mutagenesis/shuffling methods can be combined with
high-throughput, automated screening methods to detect activity of
cloned, mutagenized polypeptides expressed by host cells (Ness et
al., 1999, Nature Biotechnology 17: 893-896). Mutagenized DNA
molecules that encode active polypeptides can be recovered from the
host cells and rapidly sequenced using standard methods in the art.
These methods allow the rapid determination of the importance of
individual amino acid residues in a polypeptide.
[0129] In an embodiment, the number of amino acid substitutions,
deletions and/or insertions introduced into the mature polypeptide
of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6 is not more than 10, e.g.,
1, 2, 3, 4, 5, 6, 7, 8 or 9.
[0130] 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.
[0131] The polypeptide may be a fusion polypeptide or cleavable
fusion polypeptide in which another polypeptide is fused at the
N-terminus or the C-terminus of the polypeptide of the present
invention. A fusion polypeptide is produced by fusing a
polynucleotide encoding another polypeptide to a polynucleotide of
the present invention. Techniques for producing fusion polypeptides
are known in the art, and include ligating the coding sequences
encoding the polypeptides so that they are in frame and that
expression of the fusion polypeptide is under control of the same
promoter(s) and terminator. Fusion polypeptides may also be
constructed using intein technology in which fusion polypeptides
are created post-translationally (Cooper et al., 1993, EMBO J. 12:
2575-2583; Dawson et al., 1994, Science 266: 776-779).
[0132] A fusion polypeptide can further comprise a cleavage site
between the two polypeptides. Upon secretion of the fusion protein,
the site is cleaved releasing the two polypeptides. Examples of
cleavage sites include, but are not limited to, the sites disclosed
in Martin et al., 2003, J. Ind. Microbiol. Biotechnol. 3: 568-576;
Svetina et al., 2000, J. Biotechnol. 76: 245-251; Rasmussen-Wilson
et al., 1997, Appl. Environ. Microbiol. 63: 3488-3493; Ward et al.,
1995, Biotechnology 13: 498-503; and Contreras et al., 1991,
Biotechnology 9: 378-381; Eaton et al., 1986, Biochemistry 25:
505-512; Collins-Racie et al., 1995, Biotechnology 13: 982-987;
Carter et al., 1989, Proteins: Structure, Function, and Genetics 6:
240-248; and Stevens, 2003, Drug Discovery World 4: 35-48.
Sources of Polypeptides Having Glucuronyl Esterase Activity
[0133] A polypeptide having glucuronyl esterase 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.
[0134] The polypeptide may be a bacterial polypeptide. For example,
the polypeptide may be a Gram-positive bacterial polypeptide such
as a Bacillus, Clostridium, Enterococcus, Geobacillus,
Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus,
Streptococcus, or Streptomyces polypeptide having glucuronyl
esterase activity, or a Gram-negative bacterial polypeptide such as
a Campylobacter, E. coli, Flavobacterium e.g. Flavobacterium
johnsoniae, Fusobacterium, Helicobacter, Ilyobacter, Neisseria,
Pseudomonas, Salmonella, or Ureaplasma polypeptide.
[0135] 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.
[0136] In another aspect, the polypeptide is a Streptococcus
equisimilis, Streptococcus pyogenes, Streptococcus uberis, or
Streptococcus equi subsp. Zooepidemicus polypeptide.
[0137] In another aspect, the polypeptide is a Streptomyces
achromogenes, Streptomyces avermitilis, Streptomyces coelicolor,
Streptomyces griseus, or Streptomyces lividans polypeptide.
[0138] The polypeptide may also be a fungal polypeptide. For
example, the polypeptide may be a yeast polypeptide such as a
Candida, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces,
or Yarrowia polypeptide; or a filamentous fungal polypeptide such
as an Acremonium, Agaricus, Alternaria, Aspergillus, Aureobasidium,
Botryosphaeria, Ceriporiopsis, Chaetomidium, Chrysosporium,
Claviceps, Cochliobolus, Coprinopsis, Coptotermes, Corynascus,
Cryphonectria, Cryptococcus, Diplodia, Exidia, Filibasidium,
Fusarium, Gibberella, Holomastigotoides, Humicola, Irpex,
Lentinula, Leptospaeria, Magnaporthe, Melanocarpus, Meripilus,
Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces,
Penicillium, Phanerochaete, Piromyces, Poitrasia, Pseudoplectania,
Pseudotrichonympha, Rhizomucor, Schizophyllum, Scytalidium,
Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trichoderma,
Trichophaea, Verticillium, Volvariella, or Xylaria polypeptide.
[0139] In another aspect, the polypeptide is a Saccharomyces
carlsbergensis, Saccharomyces cerevisiae, Saccharomyces
diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri,
Saccharomyces norbensis, or Saccharomyces oviformis
polypeptide.
[0140] In another aspect, the polypeptide is an Acremonium
cellulolyticus, Aspergillus aculeatus, Aspergillus awamori,
Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus,
Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae,
Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium
lucknowense, Chrysosporium merdarium, Chrysosporium pannicola,
Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium
zonatum, Fusarium bactridioides, Fusarium cerealis, Fusarium
crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium
graminum, Fusarium heterosporum, Fusarium negundi, Fusarium
oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium
sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides,
Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides,
Fusarium venenatum, Humicola grisea, Humicola insolens, Humicola
lanuginosa, Irpex lacteus, Mucor miehei, Myceliophthora
thermophila, Neurospora crassa, Penicillium aurantiogriseum,
Penicillium funiculosum, Penicillium purpurogenum, Phanerochaete
chrysosporium, Thielavia achromatica, Thielavia albomyces,
Thielavia albopilosa, Thielavia australeinsis, Thielavia fimeti,
Thielavia microspora, Thielavia ovispora, Thielavia peruviana,
Thielavia setosa, Thielavia spededonium, Thielavia subthermophila,
Thielavia terrestris, Trichoderma harzianum, Trichoderma koningii,
Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma
viride polypeptide.
[0141] 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.
[0142] Strains of these species are readily accessible to the
public in a number of culture collections, such as the American
Type Culture Collection (ATCC), Deutsche Sammlung von
Mikroorganismen and Zellkulturen GmbH (DSMZ), Centraalbureau Voor
Schimmelcultures (CBS), and Agricultural Research Service Patent
Culture Collection, Northern Regional Research Center (NRRL).
[0143] The polypeptide may be identified and obtained from other
sources including microorganisms isolated from nature (e.g., soil,
composts, water, etc.) using the above-mentioned probes. Techniques
for isolating microorganisms from natural habitats are well known
in the art. 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 well known to those of ordinary skill in the art (see,
e.g., Sambrook et al., 1989, supra).
Domains
[0144] The present invention also relates to catalytic domains.
[0145] In an embodiment, the catalytic domain has a sequence
identity to amino acids 101 to 474 of SEQ ID NO:2 of at least 80%,
e.g. at least 85%, at least 90%, at least 95%, at least 96%, at
least 97%, at least 98%, at least 99%, or 100%. In an aspect, the
catalytic domain comprises an amino acid sequence that differs by
ten amino acids, e.g., nine amino acids, eight amino acids, seven
amino acids, six amino acids, five amino acids, four amino acids,
three amino acids, two amino acids, or one amino acid from amino
acids 101 to 474 of SEQ ID NO:2.
[0146] The catalytic domain preferably comprises or consists of
amino acids 101 to 474 of SEQ ID NO:2 or an allelic variant
thereof; or is a fragment thereof having glucuronyl esterase
activity.
[0147] In another embodiment, the catalytic domain is 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 (as defined above)
with (i) the nucleotides 33 to 1457 OF SEQ ID NO:1, (ii) the cDNA
sequence thereof, or (iii) the full-length complement of (i) or
(ii) (Sambrook et al., 1989, supra).
[0148] In another embodiment, the catalytic domain is encoded by a
polynucleotide having a sequence identity to nucleotides 33 to 1457
OF SEQ ID NO:1 or the cDNA sequence thereof at least 60%, e.g., at
least 65%, at least 70%, at least 75%, at least 80%, at least 85%,
at least 90%, at least 95%, at least 96%, at least 97%, at least
98%, at least 99%, or 100%.
[0149] In another aspect, the polynucleotide encoding the catalytic
domain comprises or consists of nucleotides 33 to 1457 OF SEQ ID
NO:1.
[0150] In another embodiment, the catalytic domain is a variant of
amino acids 101 to 474 of SEQ ID NO:2 comprising a substitution,
deletion, and/or insertion at one or more (e.g., several)
positions. In an embodiment, the number of amino acid
substitutions, deletions and/or insertions introduced into the
sequence of amino acids 101 to 474 of SEQ ID NO:2 is 10, e.g., 1,
2, 3, 4, 5, 6, 8, or 9.
[0151] In an embodiment, the catalytic domain has a sequence
identity to amino acids 94 to 460 of SEQ ID NO:4 of at least 95%,
e.g. at least 96%, at least 97%, at least 98%, at least 99%, or
100%. In an aspect, the catalytic domain comprises an amino acid
sequence that differs by ten amino acids, e.g., nine amino acids,
eight amino acids, seven amino acids, six amino acids, five amino
acids, four amino acids, three amino acids, two amino acids, or one
amino acid from amino acids 94 to 460 of SEQ ID NO:4.
[0152] The catalytic domain preferably comprises or consists of
amino acids 94 to 460 of SEQ ID NO:4 or an allelic variant thereof;
or is a fragment thereof having glucuronyl esterase activity.
[0153] In another embodiment, the catalytic domain is 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 (as defined above)
with (i) the nucleotides 81 to 1463 of SEQ ID NO:3, (ii) the cDNA
sequence thereof, or (iii) the full-length complement of (i) or
(ii) (Sambrook et al., 1989, supra).
[0154] In another embodiment, the catalytic domain is encoded by a
polynucleotide having a sequence identity to nucleotides 81 to 1463
of SEQ ID NO:3 or the cDNA sequence thereof at least 60%, e.g., at
least 65%, at least 70%, at least 75%, at least 80%, at least 85%,
at least 90%, at least 95%, at least 96%, at least 97%, at least
98%, at least 99%, or 100%.
[0155] In another aspect, the polynucleotide encoding the catalytic
domain comprises or consists of nucleotides 81 to 1463 of SEQ ID
NO:3.
[0156] In another embodiment, the catalytic domain is a variant of
amino acids 94 to 460 of SEQ ID NO:4 comprising a substitution,
deletion, and/or insertion at one or more (e.g., several)
positions. In an embodiment, the number of amino acid
substitutions, deletions and/or insertions introduced into the
sequence of amino acids 94 to 460 of SEQ ID NO:4 is 10, e.g., 1, 2,
3, 4, 5, 6, 8, or 9.
[0157] In an embodiment, the catalytic domain has a sequence
identity to amino acids 26 to 392 of SEQ ID NO:6 of at least 90%,
e.g. at least 95%, at least 96%, at least 97%, at least 98%, at
least 99%, or 100%. In an aspect, the catalytic domain comprises an
amino acid sequence that differs by ten amino acids, e.g., nine
amino acids, eight amino acids, seven amino acids, six amino acids,
five amino acids, four amino acids, three amino acids, two amino
acids, or one amino acid from amino acids 26 to 392 of SEQ ID
NO:6.
[0158] The catalytic domain preferably comprises or consists of
amino acids 26 to 392 of SEQ ID NO:6 or an allelic variant thereof;
or is a fragment thereof having glucuronyl esterase activity. In
another embodiment, the catalytic domain is 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 (as defined above)
with (i) the nucleotides 235 to 1491 of SEQ ID NO:5, (ii) the cDNA
sequence thereof, or (iii) the full-length complement of (i) or
(ii) (Sambrook et al., 1989, supra). In another embodiment, the
catalytic domain is encoded by a polynucleotide having a sequence
identity to nucleotides 235 to 1491 of SEQ ID NO:5 or the cDNA
sequence thereof at least 60%, e.g., at least 65%, at least 70%, at
least 75%, at least 80%, at least 85%, at least 90%, at least 95%,
at least 96%, at least 97%, at least 98%, at least 99%, or
100%.
[0159] In another aspect, the polynucleotide encoding the catalytic
domain comprises or consists of nucleotides 235 to 1491 of SEQ ID
NO:5.
[0160] In another embodiment, the catalytic domain is a variant of
amino acids 25 to 392 of SEQ ID NO:6 comprising a substitution,
deletion, and/or insertion at one or more (e.g., several)
positions. In an embodiment, the number of amino acid
substitutions, deletions and/or insertions introduced into the
sequence of amino acids 26 to 392 of SEQ ID NO:6 is 10, e.g., 1, 2,
3, 4, 5, 6, 8, or 9.
[0161] The present invention also relates to isolated polypeptides
comprising a catalytic domain selected from the group consisting
of: glucuronyl esterases (EC 2.4.1.17):
[0162] (a) a catalytic domain having at least 80% sequence identity
to amino acids 101 to 474 of SEQ ID NO:2;
[0163] (b) a catalytic domain encoded by a polynucleotide that
hybridizes under high, or very high stringency conditions with (i)
nucleotides 33 to 1457 OF SEQ ID NO:1, (ii) the cDNA sequence
thereof; or (iii) the full-length complement of (i) or (ii);
[0164] (c) a catalytic domain encoded by a polynucleotide having at
least 80% sequence identity to nucleotides 33 to 1457 OF SEQ ID
NO:1 or the cDNA sequence thereof;
[0165] (e) a variant of amino acids 101 to 474 of SEQ ID NO:2
comprising a substitution, deletion, and/or insertion at one or
more (e.g., several) positions; and
[0166] (f) a fragment of the catalytic domain of (a), (b), (c), (d)
or (e) that has glucuronyl esterase activity.
[0167] The present invention also relates to isolated polypeptides
comprising a catalytic domain selected from the group consisting
of: glucuronyl esterases (EC 2.4.1.17)
[0168] (a) a catalytic domain having at least 80% sequence identity
to amino acids 94 to 460 of SEQ ID NO:4;
[0169] (b) a catalytic domain encoded by a polynucleotide that
hybridizes under high, or very high stringency conditions with (i)
nucleotides 81 to 1463 of SEQ ID NO:3, (ii) the cDNA sequence
thereof; or (iii) the full-length complement of (i) or (ii);
[0170] (c) a catalytic domain encoded by a polynucleotide having at
least 80% sequence identity to nucleotides 81 to 1463 of SEQ ID
NO:3 or the cDNA sequence thereof;
[0171] (e) a variant of amino acids 94 to 460 of SEQ ID NO:4
comprising a substitution, deletion, and/or insertion at one or
more (e.g., several) positions; and
[0172] (f) a fragment of the catalytic domain of (a), (b), (c), (d)
or (e) that has glucuronyl esterase activity.
[0173] The present invention also relates to isolated polypeptides
comprising a catalytic domain selected from the group consisting
of: glucuronyl esterases (EC 2.4.1.17)
[0174] (a) a catalytic domain having at least 80% sequence identity
to amino acids 48 to 392 of SEQ ID NO:6;
[0175] (b) a catalytic domain encoded by a polynucleotide that
hybridizes under high, or very high stringency conditions with (i)
nucleotides 235 to 1491 of SEQ ID NO:5, (ii) the cDNA sequence
thereof; or (iii) the full-length complement of (i) or (ii);
[0176] (c) a catalytic domain encoded by a polynucleotide having at
least 80% sequence identity to nucleotides 235 to 1491 of SEQ ID
NO:5 or the cDNA sequence thereof;
[0177] d) a variant of the mature polypeptide of SEQ ID NO:6
comprising a substitution, deletion, and/or insertion at one or
more (e.g., several) positions; and
[0178] (e) a variant of amino acids 21 to 392 of SEQ ID NO:6
comprising a substitution, deletion, and/or insertion at one or
more (e.g., several) positions; and
[0179] (f) a fragment of the catalytic domain of (a), (b), (c), (d)
or (e) that has glucuronyl esterase activity.
The Present Invention Also Relates to Cellulose Binding
Domains.
[0180] In another embodiment, the cellulose binding domain is
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 (as defined above)
with (i) the nucleotides of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5
(ii) the cDNA sequence thereof, or (iii) the full-length complement
of (i) or (ii) (Sambrook et al., 1989, supra).
[0181] In another embodiment, the cellulose binding domain is a
variant of SEQ ID NO:2 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 SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6 is 10,
e.g., 1, 2, 3, 4, 5, 6, 8, or 9.
[0182] A catalytic domain operably linked to the cellulose binding
domain may be from a hydrolase, isomerase, ligase, lyase,
oxidoreductase, or transferase, e.g., an aminopeptidase, amylase,
carbohydrase, carboxypeptidase, catalase, cellobiohydrolase,
cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase,
deoxyribonuclease, endoglucanase, esterase, alpha-galactosidase,
beta-galactosidase, glucoamylase, alpha-glucosidase,
beta-glucosidase, invertase, laccase, lipase, mannosidase,
mutanase, oxidase, pectinolytic enzyme, peroxidase, phytase,
polyphenoloxidase, proteolytic enzyme, ribonuclease,
transglutaminase, xylanase, or beta-xylosidase. The polynucleotide
encoding the catalytic domain may be obtained from any prokaryotic,
eukaryotic, or other source.
Polynucleotides
[0183] The present invention also relates to isolated
polynucleotides encoding a polypeptide, a catalytic domain, or
cellulose binding domain of the present invention, as described
above.
[0184] The techniques used to isolate or clone a polynucleotide
encoding a polypeptide are known in the art and include isolation
from genomic DNA or cDNA, or a combination thereof. The cloning of
the polynucleotides from genomic DNA can be effected, e.g., by
using the well known polymerase chain reaction (PCR) or antibody
screening of expression libraries to detect cloned DNA fragments
with shared structural features. See, e.g., Innis et al., 1990,
PCR: A Guide to Methods and Application, Academic Press, New York.
Other nucleic acid amplification procedures such as ligase chain
reaction (LCR), ligation activated transcription (LAT) and
polynucleotide-based amplification (NASBA) may be used. The
polynucleotides may be cloned from any relevant microorganism and
thus, for example, may be an allelic or species variant of the
polypeptide encoding region of the polynucleotide.
[0185] Modification of a polynucleotide encoding a polypeptide of
the present invention may be necessary for synthesizing
polypeptides substantially similar to the polypeptide. The term
"substantially similar" to the polypeptide refers to non-naturally
occurring forms of the polypeptide. These polypeptides may differ
in some engineered way from the polypeptide isolated from its
native source, e.g., variants that differ in specific activity,
thermostability, pH optimum, or the like. The variants may be
constructed on the basis of the polynucleotide presented as the
mature polypeptide coding sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ
ID NO:5 or the cDNA sequence thereof, e.g., a subsequence thereof,
and/or by introduction of nucleotide substitutions that do not
result in a change in the amino acid sequence of the polypeptide,
but which correspond to the codon usage of the host organism
intended for production of the enzyme, or by introduction of
nucleotide substitutions that may give rise to a different amino
acid sequence. For a general description of nucleotide
substitution, see, e.g., Ford et al., 1991, Protein Expression and
Purification 2: 95-107.
Nucleic Acid Constructs
[0186] The present invention also relates to nucleic acid
constructs comprising a polynucleotide of the present invention
operably linked to one or more control sequences that direct the
expression of the coding sequence in a suitable host cell under
conditions compatible with the control sequences.
[0187] A polynucleotide may be manipulated in a variety of ways to
provide for expression of the polypeptide. Manipulation of the
polynucleotide prior to its insertion into a vector may be
desirable or necessary depending on the expression vector. The
techniques for modifying polynucleotides utilizing recombinant DNA
methods are well known in the art.
[0188] The control sequence may be a promoter sequence, a
polynucleotide that is recognized by a host cell for expression of
a polynucleotide encoding a polypeptide of the present invention.
The promoter sequence contains transcriptional control sequences
that mediate the expression of the polypeptide. The promoter may be
any polynucleotide that shows transcriptional activity in the host
cell of choice including mutant, truncated, and hybrid promoters,
and may be obtained from genes encoding extracellular or
intracellular polypeptides either homologous or heterologous to the
host cell.
[0189] 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, E. coli lac operon, E. coli trc
promoter (Egon et al., 1988, Gene 69: 301-315), Streptomyces
coelicolor agarase gene (dagA), and prokaryotic beta-lactamase gene
(Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. USA 75:
3727-3731), as well as the tac promoter (DeBoer et al., 1983, Proc.
Natl. Acad. Sci. USA 80: 21-25). Further promoters are described in
"Useful proteins from recombinant bacteria" in Gilbert et al.,
1980, Scientific American, 242: 74-94; and in Sambrook et al.,
1989, supra.
[0190] Examples of suitable promoters for directing transcription
of the nucleic acid constructs of the present invention in a
filamentous fungal host cell are promoters obtained from the genes
for Aspergillus nidulans acetamidase, Aspergillus niger neutral
alpha-amylase, Aspergillus niger acid stable alpha-amylase,
Aspergillus niger or Aspergillus awamori glucoamylase (glaA),
Aspergillus oryzae TAKA amylase, Aspergillus oryzae alkaline
protease, Aspergillus oryzae triose phosphate isomerase, Fusarium
oxysporum trypsin-like protease (WO 96/00787), Fusarium venenatum
amyloglucosidase (WO 00/56900), Fusarium venenatum Daria (WO
00/56900), Fusarium venenatum Quinn (WO 00/56900), Rhizomucor
miehei lipase, Rhizomucor miehei aspartic proteinase, Trichoderma
reesei beta-glucosidase, Trichoderma reesei cellobiohydrolase I,
Trichoderma reesei cellobiohydrolase II, Trichoderma reesei
endoglucanase I, Trichoderma reesei endoglucanase II, Trichoderma
reesei endoglucanase III, Trichoderma reesei endoglucanase IV,
Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I,
Trichoderma reesei xylanase II, Trichoderma reesei beta-xylosidase,
as well as the NA2-tpi promoter (a modified promoter from an
Aspergillus gene encoding a neutral alpha-amylase in which the
untranslated leader has been replaced by an untranslated leader
from an Aspergillus gene encoding a triose phosphate isomerase;
non-limiting examples include modified promoters from an
Aspergillus niger gene encoding neutral alpha-amylase in which the
untranslated leader has been replaced by an untranslated leader
from an Aspergillus nidulans or Aspergillus oryzae gene encoding a
triose phosphate isomerase); and mutant, truncated, and hybrid
promoters thereof.
[0191] In a yeast host, useful promoters are obtained from the
genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces
cerevisiae galactokinase (GAL1), Saccharomyces cerevisiae alcohol
dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1,
ADH2/GAP), Saccharomyces cerevisiae triose phosphate isomerase
(TPI), Saccharomyces cerevisiae metallothionein (CUP1), and
Saccharomyces cerevisiae 3-phosphoglycerate kinase. Other useful
promoters for yeast host cells are described by Romanos et al.,
1992, Yeast 8: 423-488.
[0192] The control sequence may also be a suitable transcription
terminator sequence, which is recognized by a host cell to
terminate transcription. The terminator sequence is operably linked
to the 3'-terminus of the polynucleotide encoding the polypeptide.
Any terminator that is functional in the host cell of choice may be
used in the present invention.
[0193] Preferred terminators for filamentous fungal host cells are
obtained from the genes for Aspergillus nidulans anthranilate
synthase, Aspergillus niger glucoamylase, Aspergillus niger
alpha-glucosidase, Aspergillus oryzae TAKA amylase, and Fusarium
oxysporum trypsin-like protease.
[0194] 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.
[0195] The control sequence may also be a suitable leader sequence,
when transcribed is a nontranslated region of an mRNA that is
important for translation by the host cell. The leader sequence is
operably linked to the 5'-terminus of the polynucleotide encoding
the polypeptide. Any leader sequence that is functional in the host
cell of choice may be used.
[0196] Preferred leaders for filamentous fungal host cells are
obtained from the genes for Aspergillus oryzae TAKA amylase and
Aspergillus nidulans triose phosphate isomerase.
[0197] 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).
[0198] 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 of
choice may be used.
[0199] Preferred polyadenylation sequences for filamentous fungal
host cells are obtained from the genes for Aspergillus oryzae TAKA
amylase, Aspergillus niger glucoamylase, Aspergillus nidulans
anthranilate synthase, Fusarium oxysporum trypsin-like protease,
and Aspergillus niger alpha-glucosidase.
[0200] Useful polyadenylation sequences for yeast host cells are
described by Guo and Sherman, 1995, Mol. Cellular Biol. 15:
5983-5990.
[0201] 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 of choice may be used.
[0202] Effective signal peptide coding sequences for bacterial host
cells are the signal peptide coding sequences obtained from the
genes for Bacillus NCIB 11837 maltogenic amylase, Bacillus
licheniformis subtilisin, Bacillus licheniformis beta-lactamase,
Bacillus stearothermophilus alpha-amylase, Bacillus
stearothermophilus neutral proteases (nprT, nprS, nprM), and
Bacillus subtilis prsA. Further signal peptides are described by
Simonen and Palva, 1993, Microbiological Reviews 57: 109-137.
[0203] Effective signal peptide coding sequences for filamentous
fungal host cells are the signal peptide coding sequences obtained
from the genes for Aspergillus niger neutral amylase, Aspergillus
niger glucoamylase, Aspergillus oryzae TAKA amylase, Humicola
insolens cellulase, Humicola insolens endoglucanase V, Humicola
lanuginosa lipase, and Rhizomucor miehei aspartic proteinase.
[0204] 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.
[0205] 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.
[0206] Where both signal peptide and propeptide sequences are
present at the N-terminus of a polypeptide, 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.
[0207] It may also be desirable to add regulatory sequences that
regulate expression of the polypeptide relative to the growth of
the host cell. Examples of regulatory systems are those that cause
expression of the gene to be turned on or off in response to a
chemical or physical stimulus, including the presence of a
regulatory compound. Regulatory systems in prokaryotic systems
include the lac, tac, and trp operator systems. In yeast, the ADH2
system or GAL1 system may be used. In filamentous fungi, the
Aspergillus niger glucoamylase promoter, Aspergillus oryzae TAKA
alpha-amylase promoter, and Aspergillus oryzae glucoamylase
promoter may be used. Other examples of regulatory sequences are
those that allow for gene amplification. In eukaryotic systems,
these regulatory sequences include the dihydrofolate reductase gene
that is amplified in the presence of methotrexate, and the
metallothionein genes that are amplified with heavy metals. In
these cases, the polynucleotide encoding the polypeptide would be
operably linked with the regulatory sequence.
Expression Vectors
[0208] The present invention also relates to recombinant expression
vectors comprising a polynucleotide of the present invention, a
promoter, and transcriptional and translational stop signals. The
various nucleotide and control sequences may be joined together to
produce a recombinant expression vector that may include one or
more convenient restriction sites to allow for insertion or
substitution of the polynucleotide encoding the polypeptide at such
sites. Alternatively, the polynucleotide may be expressed by
inserting the polynucleotide or a nucleic acid construct comprising
the sequence into an appropriate vector for expression. In creating
the expression vector, the coding sequence is located in the vector
so that the coding sequence is operably linked with the appropriate
control sequences for expression.
[0209] 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.
[0210] The vector may be an autonomously replicating vector, i.e.,
a vector that exists as an extrachromosomal entity, the replication
of which is independent of chromosomal replication, e.g., a
plasmid, an extrachromosomal element, a minichromosome, or an
artificial chromosome. The vector may contain any means for
assuring self-replication. Alternatively, the vector may be one
that, when introduced into the host cell, is integrated into the
genome and replicated together with the chromosome(s) into which it
has been integrated. Furthermore, a single vector or plasmid or two
or more vectors or plasmids that together contain the total DNA to
be introduced into the genome of the host cell, or a transposon,
may be used.
[0211] The vector preferably contains one or more selectable
markers that permit easy selection of transformed, transfected,
transduced, or the like cells. A selectable marker is a gene the
product of which provides for biocide or viral resistance,
resistance to heavy metals, prototrophy to auxotrophs, and the
like.
[0212] Examples of bacterial selectable markers are the dal genes
from Bacillus subtilis or Bacillus licheniformis, or markers that
confer antibiotic resistance such as ampicillin, chloramphenicol,
kanamycin, or tetracycline resistance. Suitable markers for yeast
host cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3.
Selectable markers for use in a filamentous fungal host cell
include, but are not limited to, amdS (acetamidase), argB
(ornithine carbamoyltransferase), bar (phosphinothricin
acetyltransferase), hph (hygromycin phosphotransferase), niaD
(nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase),
sC (sulfate adenyltransferase), and trpC (anthranilate synthase),
as well as equivalents thereof. Preferred for use in an Aspergillus
cell are Aspergillus nidulans or Aspergillus oryzae amdS and pyrG
genes and a Streptomyces hygroscopicus bar gene.
[0213] 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.
[0214] 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.
[0215] 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.
[0216] Examples of bacterial origins of replication are the origins
of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184
permitting replication in E. coli, and pUB110, pE194, pTA1060, and
pAM.beta.1 permitting replication in Bacillus.
[0217] Examples of origins of replication for use in a yeast host
cell are the 2 micron origin of replication, ARS1, ARS4, the
combination of ARS1 and CEN3, and the combination of ARS4 and
CEN6.
[0218] Examples of origins of replication useful in a filamentous
fungal cell are AMA1 and ANS1 (Gems et al., 1991, Gene 98: 61-67;
Cullen et al., 1987, Nucleic Acids Res. 15: 9163-9175; WO
00/24883). Isolation of the AMA1 gene and construction of plasmids
or vectors comprising the gene can be accomplished according to the
methods disclosed in WO 00/24883.
[0219] More than one copy of a polynucleotide of the present
invention may be inserted into a host cell to increase production
of a polypeptide. An increase in the copy number of the
polynucleotide can be obtained by integrating at least one
additional copy of the sequence into the host cell genome or by
including an amplifiable selectable marker gene with the
polynucleotide where cells containing amplified copies of the
selectable marker gene, and thereby additional copies of the
polynucleotide, can be selected for by cultivating the cells in the
presence of the appropriate selectable agent.
[0220] The procedures used to ligate the elements described above
to construct the recombinant expression vectors of the present
invention are well known to one skilled in the art (see, e.g.,
Sambrook et al., 1989, supra).
Host Cells
[0221] The present invention also relates to recombinant host
cells, comprising a polynucleotide of the present invention
operably linked to one or more control sequences that direct the
production of a polypeptide of the present invention. A construct
or vector comprising a polynucleotide is introduced into a host
cell so that the construct or vector is maintained as a chromosomal
integrant or as a self-replicating extra-chromosomal vector as
described earlier. The term "host cell" encompasses any progeny of
a parent cell that is not identical to the parent cell due to
mutations that occur during replication. The choice of a host cell
will to a large extent depend upon the gene encoding the
polypeptide and its source.
[0222] 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.
[0223] The prokaryotic host cell may be any Gram-positive or
Gram-negative bacterium. Gram-positive bacteria include, but not
limited to, Bacillus, Clostridium, Enterococcus, Geobacillus,
Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus,
Streptococcus, and Streptomyces. Gram-negative bacteria include,
but not limited to, Campylobacter, E. coli, Flavobacterium,
Fusobacterium, Helicobacter, Ilyobacter, Neisseria, Pseudomonas,
Salmonella, and Ureaplasma.
[0224] The bacterial host cell may be any Bacillus cell including,
but not limited to, Bacillus alkalophilus, Bacillus
amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus
clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus,
Bacillus lentus, Bacillus licheniformis, Bacillus megaterium,
Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis,
and Bacillus thuringiensis cells.
[0225] 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.
[0226] 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.
[0227] 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), using competent cells (see, e.g.,
Young and Spizizen, 1961, J. Bacteriol. 81: 823-829, or Dubnau and
Davidoff-Abelson, 1971, J. Mol. Biol. 56: 209-221), electroporation
(see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751),
or conjugation (see, e.g., Koehler and Thorne, 1987, J. Bacteriol.
169: 5271-5278). The introduction of DNA into an E. coli cell may
be effected by protoplast transformation (see, e.g., Hanahan, 1983,
J. Mol. Biol. 166: 557-580) or electroporation (see, e.g., Dower et
al., 1988, Nucleic Acids Res. 16: 6127-6145). The introduction of
DNA into a Streptomyces cell may be effected by protoplast
transformation and electroporation (see, e.g., Gong et al., 2004,
Folia Microbiol. (Praha) 49: 399-405), conjugation (see, e.g.,
Mazodier et al., 1989, J. Bacteriol. 171: 3583-3585), or
transduction (see, e.g., Burke et al., 2001, Proc. Natl. Acad. Sci.
USA 98: 6289-6294). The introduction of DNA into a Pseudomonas cell
may be effected by electroporation (see, e.g., Choi et al., 2006,
J. Microbiol. Methods 64: 391-397) or conjugation (see, e.g.,
Pinedo and Smets, 2005, Appl. Environ. Microbiol. 71: 51-57). The
introduction of DNA into a Streptococcus cell may be effected by
natural competence (see, e.g., Perry and Kuramitsu, 1981, Infect.
Immun. 32: 1295-1297), protoplast transformation (see, e.g., Catt
and Jollick, 1991, Microbios 68: 189-207), electroporation (see,
e.g., Buckley et al., 1999, Appl. Environ. Microbiol. 65:
3800-3804) or conjugation (see, e.g., Clewell, 1981, Microbiol.
Rev. 45: 409-436). However, any method known in the art for
introducing DNA into a host cell can be used.
[0228] The host cell may also be a eukaryote, such as a mammalian,
insect, plant, or fungal cell.
[0229] The host cell may be a fungal cell. "Fungi" as used herein
includes the phyla Ascomycota, Basidiomycota, Chytridiomycota, and
Zygomycota (as defined by Hawksworth et al., In, Ainsworth and
Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB
International, University Press, Cambridge, UK) as well as the
Oomycota (as cited in Hawksworth et al., 1995, supra, page 171) and
all mitosporic fungi (Hawksworth et al., 1995, supra).
[0230] The fungal host cell may be a yeast cell. "Yeast" as used
herein includes ascosporogenous yeast (Endomycetales),
basidiosporogenous yeast, and yeast belonging to the Fungi
Imperfecti (Blastomycetes). Since the classification of yeast may
change in the future, for the purposes of this invention, yeast
shall be defined as described in Biology and Activities of Yeast
(Skinner, F. A., Passmore, S. M., and Davenport, R. R., eds, Soc.
App. Bacteriol. Symposium Series No. 9, 1980).
[0231] The yeast host cell may be a Candida, Hansenula,
Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or
Yarrowia cell such as a Kluyveromyces lactis, Saccharomyces
carlsbergensis, Saccharomyces cerevisiae, Saccharomyces
diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri,
Saccharomyces norbensis, Saccharomyces oviformis, or Yarrowia
lipolytica cell.
[0232] The fungal host cell may be a filamentous fungal cell.
"Filamentous fungi" include all filamentous forms of the
subdivision Eumycota and Oomycota (as defined by Hawksworth et al.,
1995, supra). The filamentous fungi are generally characterized by
a mycelial wall composed of chitin, cellulose, glucan, chitosan,
mannan, and other complex polysaccharides. Vegetative growth is by
hyphal elongation and carbon catabolism is obligately aerobic. In
contrast, vegetative growth by yeasts such as Saccharomyces
cerevisiae is by budding of a unicellular thallus and carbon
catabolism may be fermentative.
[0233] The filamentous fungal host cell may be an Acremonium,
Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis,
Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium,
Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora,
Neocallimastix, Neurospora, Paecilomyces, Penicillium,
Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum,
Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or
Trichoderma cell.
[0234] For example, the filamentous fungal host cell may be an
Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus,
Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger,
Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsis aneirina,
Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis
pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa,
Ceriporiopsis subvermispora, Chrysosporium inops, Chrysosporium
keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium,
Chrysosporium pannicola, Chrysosporium queenslandicum,
Chrysosporium tropicum, Chrysosporium zonatum, Coprinus cinereus,
Coriolus hirsutus, Fusarium bactridioides, Fusarium cerealis,
Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum,
Fusarium graminum, Fusarium heterosporum, Fusarium negundi,
Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium
sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides,
Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides,
Fusarium venenatum, Humicola insolens, Humicola lanuginosa, Mucor
miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium
purpurogenum, Phanerochaete chrysosporium, Phlebia radiata,
Pleurotus eryngii, Thielavia terrestris, Trametes villosa, Trametes
versicolor, Trichoderma harzianum, Trichoderma koningii,
Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma
viride cell.
[0235] Fungal cells may be transformed by a process involving
protoplast formation, transformation of the protoplasts, and
regeneration of the cell wall in a manner known per se. Suitable
procedures for transformation of Aspergillus and Trichoderma host
cells are described in EP 238023, Yelton et al., 1984, Proc. Natl.
Acad. Sci. USA 81: 1470-1474, and Christensen et al., 1988,
Bio/Technology 6: 1419-1422. Suitable methods for transforming
Fusarium species are described by Malardier et al., 1989, Gene 78:
147-156, and WO 96/00787. Yeast may be transformed using the
procedures described by Becker and Guarente, In Abelson, J. N. and
Simon, M. I., editors, Guide to Yeast Genetics and Molecular
Biology, Methods in Enzymology, Volume 194, pp 182-187, Academic
Press, Inc., New York; Ito et al., 1983, J. Bacteriol. 153: 163;
and Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA 75: 1920.
Methods of Production
[0236] The present invention also relates to methods of producing a
polypeptide of the present invention, comprising: (a) cultivating a
cell, which in its wild-type form produces the polypeptide, under
conditions conducive for production of the polypeptide; and (b)
recovering the polypeptide. In a preferred aspect, the cell is an
Cerrena (for SEQ ID NO:2), Trichoderma (for SEQ ID NO:4) or
Chaetonium (for SEQ ID NO:6) cell. In a more preferred aspect, the
cell is an Cerrena unicolor for (SEQ ID NO:2), Trichoderma reesei
(for SEQ ID NO:4) or Chaetomium globosum (for SEQ ID NO:6).
[0237] The present invention also relates to methods of producing a
polypeptide of the present invention, comprising: (a) cultivating a
recombinant host cell of the present invention under conditions
conducive for production of the polypeptide; and (b) recovering the
polypeptide.
[0238] The host cells are cultivated in a nutrient medium suitable
for production of the polypeptide using methods well known in the
art. For example, the cell may be cultivated by shake flask
cultivation, and small-scale or large-scale fermentation (including
continuous, batch, fed-batch, or solid state fermentations) in
laboratory or industrial fermentors performed in a suitable medium
and under conditions allowing the polypeptide to be expressed
and/or isolated. The cultivation takes place in a suitable nutrient
medium comprising carbon and nitrogen sources and inorganic salts,
using procedures known in the art. Suitable media are available
from commercial suppliers or may be prepared according to published
compositions (e.g., in catalogues of the American Type Culture
Collection). If the polypeptide is secreted into the nutrient
medium, the polypeptide can be recovered directly from the medium.
If the polypeptide is not secreted, it can be recovered from cell
lysates.
[0239] The polypeptide may be detected using methods known in the
art that are specific for the polypeptides. These detection methods
may include use of specific antibodies, formation of an enzyme
product, or disappearance of an enzyme substrate. For example, an
enzyme assay may be used to determine the activity of the
polypeptide.
[0240] 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, centrifugation, filtration, extraction, spray-drying,
evaporation, or precipitation.
[0241] 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, J.-C. Janson and Lars Ryden, editors, VCH Publishers,
New York, 1989) to obtain substantially pure polypeptides.
[0242] In an alternative aspect, the polypeptide is not recovered,
but rather a host cell of the present invention expressing the
polypeptide is used as a source of the polypeptide.
Plants
[0243] The present invention also relates to isolated plants, e.g.,
a transgenic plant, plant part, or plant cell, comprising a
polynucleotide of the present invention so as to express and
produce a polypeptide or domain in recoverable quantities. The
polypeptide or domain may be recovered from the plant or plant
part. Alternatively, the plant or plant part containing the
polypeptide or domain may be used as such for improving the quality
of a food or feed, e.g., improving nutritional value, palatability,
and rheological properties, or to destroy an antinutritive
factor.
[0244] The transgenic plant can be dicotyledonous (a dicot) or
monocotyledonous (a monocot). Examples of monocot plants are
grasses, such as meadow grass (blue grass, Poa), forage grass such
as Festuca, Lolium, temperate grass, such as Agrostis, and cereals,
e.g., wheat, oats, rye, barley, rice, sorghum, and maize
(corn).
[0245] 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.
[0246] Examples of plant parts are stem, callus, leaves, root,
fruits, seeds, and tubers as well as the individual tissues
comprising these parts, e.g., epidermis, mesophyll, parenchyme,
vascular tissues, meristems. Specific plant cell compartments, such
as chloroplasts, apoplasts, mitochondria, vacuoles, peroxisomes and
cytoplasm are also considered to be a plant part. Furthermore, any
plant cell, whatever the tissue origin, is considered to be a plant
part. Likewise, plant parts such as specific tissues and cells
isolated to facilitate the utilization of the invention are also
considered plant parts, e.g., embryos, endosperms, aleurone and
seed coats.
[0247] Also included within the scope of the present invention are
the progeny of such plants, plant parts, and plant cells.
[0248] The transgenic plant or plant cell expressing the
polypeptide or domain may be constructed in accordance with methods
known in the art. In short, the plant or plant cell is constructed
by incorporating one or more expression constructs encoding the
polypeptide or domain into the plant host genome or chloroplast
genome and propagating the resulting modified plant or plant cell
into a transgenic plant or plant cell.
[0249] The expression construct is conveniently a nucleic acid
construct that comprises a polynucleotide encoding a polypeptide or
domain operably linked with appropriate regulatory sequences
required for expression of the polynucleotide in the plant or plant
part of choice. Furthermore, the expression construct may comprise
a selectable marker useful for identifying host cells into which
the expression construct has been integrated and DNA sequences
necessary for introduction of the construct into the plant in
question (the latter depends on the DNA introduction method to be
used).
[0250] The choice of regulatory sequences, such as promoter and
terminator sequences and optionally signal or transit sequences, is
determined, for example, on the basis of when, where, and how the
polypeptide or domain is desired to be expressed. For instance, the
expression of the gene encoding a polypeptide or domain may be
constitutive or inducible, or may be developmental, stage or tissue
specific, and the gene product may be targeted to a specific tissue
or plant part such as seeds or leaves. Regulatory sequences are,
for example, described by Tague et al., 1988, Plant Physiology 86:
506.
[0251] For constitutive expression, the 35S-CaMV, the maize
ubiquitin 1, or the rice actin 1 promoter may be used (Franck et
al., 1980, Cell 21: 285-294; Christensen et al., 1992, Plant Mol.
Biol. 18: 675-689; Zhang et al., 1991, Plant Cell 3: 1155-1165).
Organ-specific promoters may be, for example, a promoter from
storage sink tissues such as seeds, potato tubers, and fruits
(Edwards and Coruzzi, 1990, Ann. Rev. Genet. 24: 275-303), or from
metabolic sink tissues such as meristems (Ito et al., 1994, Plant
Mol. Biol. 24: 863-878), a seed specific promoter such as the
glutelin, prolamin, globulin, or albumin promoter from rice (Wu et
al., 1998, Plant Cell Physiol. 39: 885-889), a Vicia faba promoter
from the legumin B4 and the unknown seed protein gene from Vicia
faba (Conrad et al., 1998, J. Plant Physiol. 152: 708-711), a
promoter from a seed oil body protein (Chen et al., 1998, Plant
Cell Physiol. 39: 935-941), the storage protein napA promoter from
Brassica napus, or any other seed specific promoter known in the
art, e.g., as described in WO 91/14772. Furthermore, the promoter
may be a leaf specific promoter such as the rbcs promoter from rice
or tomato (Kyozuka et al., 1993, Plant Physiol. 102: 991-1000), the
chlorella virus adenine methyltransferase gene promoter (Mitra and
Higgins, 1994, Plant Mol. Biol. 26: 85-93), the aldP gene promoter
from rice (Kagaya et al., 1995, Mol. Gen. Genet. 248: 668-674), or
a wound inducible promoter such as the potato pin2 promoter (Xu et
al., 1993, Plant Mol. Biol. 22: 573-588). Likewise, the promoter
may be induced by abiotic treatments such as temperature, drought,
or alterations in salinity or induced by exogenously applied
substances that activate the promoter, e.g., ethanol, oestrogens,
plant hormones such as ethylene, abscisic acid, and gibberellic
acid, and heavy metals.
[0252] 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.
[0253] The selectable marker gene and any other parts of the
expression construct may be chosen from those available in the
art.
[0254] The nucleic acid construct is incorporated into the plant
genome according to conventional techniques known in the art,
including Agrobacterium-mediated transformation, virus-mediated
transformation, microinjection, particle bombardment, biolistic
transformation, and electroporation (Gasser et al., 1990, Science
244: 1293; Potrykus, 1990, Bio/Technology 8: 535; Shimamoto et al.,
1989, Nature 338: 274).
[0255] Presently, Agrobacterium tumefaciens-mediated gene transfer
is the method of choice for generating transgenic dicots (for a
review, see Hooykas and Schilperoort, 1992, Plant Mol. Biol. 19:
15-38) and can also be used for transforming monocots, although
other transformation methods are often used for these plants.
Presently, the method of choice for generating transgenic monocots
is particle bombardment (microscopic gold or tungsten particles
coated with the transforming DNA) of embryonic calli or developing
embryos (Christou, 1992, Plant J. 2: 275-281; Shimamoto, 1994,
Curr. Opin. Biotechnol. 5: 158-162; Vasil et al., 1992,
Bio/Technology 10: 667-674). An alternative method for
transformation of monocots is based on protoplast transformation as
described by Omirulleh et al., 1993, Plant Mol. Biol. 21: 415-428.
Additional transformation methods for use in accordance with the
present disclosure include those described in U.S. Pat. Nos.
6,395,966 and 7,151,204 (both of which are herein incorporated by
reference in their entirety).
[0256] 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.
[0257] In addition to direct transformation of a particular plant
genotype with a construct of the present invention, transgenic
plants may be made by crossing a plant having the construct to a
second plant lacking the construct. For example, a construct
encoding a polypeptide or domain can be introduced into a
particular plant variety by crossing, without the need for ever
directly transforming a plant of that given variety. Therefore, the
present invention encompasses not only a plant directly regenerated
from cells which have been transformed in accordance with the
present invention, but also the progeny of such plants. As used
herein, progeny may refer to the offspring of any generation of a
parent plant prepared in accordance with the present invention.
Such progeny may include a DNA construct prepared in accordance
with the present invention. Crossing results in the introduction of
a transgene into a plant line by cross pollinating a starting line
with a donor plant line. Non-limiting examples of such steps are
described in U.S. Pat. No. 7,151,204.
[0258] 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.
[0259] Genetic markers may be used to assist in the introgression
of one or more transgenes of the invention from one genetic
background into another. Marker assisted selection offers
advantages relative to conventional breeding in that it can be used
to avoid errors caused by phenotypic variations. Further, genetic
markers may provide data regarding the relative degree of elite
germplasm in the individual progeny of a particular cross. For
example, when a plant with a desired trait which otherwise has a
non-agronomically desirable genetic background is crossed to an
elite parent, genetic markers may be used to select progeny which
not only possess the trait of interest, but also have a relatively
large proportion of the desired germplasm. In this way, the number
of generations required to introgress one or more traits into a
particular genetic background is minimized.
[0260] The present invention also relates to methods of producing a
polypeptide or domain of the present invention comprising: (a)
cultivating a transgenic plant or a plant cell comprising a
polynucleotide encoding the polypeptide or domain under conditions
conducive for production of the polypeptide or domain; and (b)
recovering the polypeptide or domain.
Removal or Reduction of Glucuronyl Esterase Activity
[0261] 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.
[0262] The mutant cell may be constructed by reducing or
eliminating expression of the polynucleotide using methods well
known in the art, for example, insertions, disruptions,
replacements, or deletions. In a preferred aspect, the
polynucleotide is inactivated. The polynucleotide to be modified or
inactivated may be, for example, the coding region or a part
thereof essential for activity, or a regulatory element required
for expression of the coding region. An example of such a
regulatory or control sequence may be a promoter sequence or a
functional part thereof, i.e., a part that is sufficient for
affecting expression of the polynucleotide. Other control sequences
for possible modification include, but are not limited to, a
leader, polyadenylation sequence, propeptide sequence, signal
peptide sequence, transcription terminator, and transcriptional
activator.
[0263] 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.
[0264] Examples of a physical or chemical mutagenizing agent
suitable for the present purpose include ultraviolet (UV)
irradiation, hydroxylamine, N-methyl-N'-nitro-N-nitrosoguanidine
(MNNG), O-methyl hydroxylamine, nitrous acid, ethyl methane
sulphonate (EMS), sodium bisulphite, formic acid, and nucleotide
analogues.
[0265] 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.
[0266] Modification or inactivation of the polynucleotide may be
accomplished by insertion, substitution, or deletion of one or more
nucleotides in the gene or a regulatory element required for
transcription or translation thereof. For example, nucleotides may
be inserted or removed so as to result in the introduction of a
stop codon, the removal of the start codon, or a change in the open
reading frame. Such modification or inactivation may be
accomplished by site-directed mutagenesis or PCR generated
mutagenesis in accordance with methods known in the art. Although,
in principle, the modification may be performed in vivo, i.e.,
directly on the cell expressing the polynucleotide to be modified,
it is preferred that the modification be performed in vitro as
exemplified below.
[0267] An example of a convenient way to eliminate or reduce
expression of a polynucleotide is based on techniques of gene
replacement, gene deletion, or gene disruption. For example, in the
gene disruption method, a nucleic acid sequence corresponding to
the endogenous polynucleotide is mutagenized in vitro to produce a
defective nucleic acid sequence that is then transformed into the
parent cell to produce a defective gene. By homologous
recombination, the defective nucleic acid sequence replaces the
endogenous polynucleotide. It may be desirable that the defective
polynucleotide also encodes a marker that may be used for selection
of transformants in which the polynucleotide has been modified or
destroyed. In an aspect, the polynucleotide is disrupted with a
selectable marker such as those described herein.
[0268] The present invention also relates to methods of inhibiting
the expression of a polypeptide having glucuronyl esterase activity
in a cell, comprising administering to the cell or expressing in
the cell a double-stranded RNA (dsRNA) molecule, wherein the dsRNA
comprises a subsequence of a polynucleotide of the present
invention. In a preferred aspect, the dsRNA is about 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, 25 or more duplex nucleotides in
length.
[0269] The dsRNA is preferably a small interfering RNA (siRNA) or a
micro RNA (miRNA). In a preferred aspect, the dsRNA is small
interfering RNA for inhibiting transcription. In another preferred
aspect, the dsRNA is micro RNA for inhibiting translation.
[0270] The present invention also relates to such double-stranded
RNA (dsRNA) molecules, comprising a portion of the mature
polypeptide coding sequence of SEQ ID NO:1 for inhibiting
expression of the polypeptide in a cell. While the present
invention is not limited by any particular mechanism of action, the
dsRNA can enter a cell and cause the degradation of a
single-stranded RNA (ssRNA) of similar or identical sequences,
including endogenous mRNAs. When a cell is exposed to dsRNA, mRNA
from the homologous gene is selectively degraded by a process
called RNA interference (RNAi).
[0271] The dsRNAs of the present invention can be used in
gene-silencing. In one aspect, the invention provides methods to
selectively degrade RNA using a dsRNAi of the present invention.
The process may be practiced in vitro, ex vivo or in vivo. In one
aspect, the dsRNA molecules can be used to generate a
loss-of-function mutation in a cell, an organ or an animal. Methods
for making and using dsRNA molecules to selectively degrade RNA are
well known in the art; see, for example, U.S. Pat. Nos. 6,489,127;
6,506,559; 6,511,824; and 6,515,109.
[0272] 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.
[0273] The polypeptide-deficient mutant cells are particularly
useful as host cells for expression of native and heterologous
polypeptides. Therefore, the present invention further relates to
methods of producing a native or heterologous polypeptide,
comprising: (a) cultivating the mutant cell under conditions
conducive for production of the polypeptide; and (b) recovering the
polypeptide. The term "heterologous polypeptides" means
polypeptides that are not native to the host cell, e.g., a variant
of a native protein. The host cell may comprise more than one copy
of a polynucleotide encoding the native or heterologous
polypeptide.
[0274] The methods used for cultivation and purification of the
product of interest may be performed by methods known in the
art.
[0275] The methods of the present invention for producing an
essentially glucuronyl esterase-free product is of particular
interest in the production of eukaryotic polypeptides, in
particular fungal proteins such as enzymes. The glucuronyl
esterase-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.
[0276] In a further aspect, the present invention relates to a
protein product essentially free from glucuronyl esterase activity
that is produced by a method of the present invention.
Methods of Processing Cellulosic Material
[0277] The present invention also relates to methods for degrading
or converting a cellulosic material, comprising: treating the
cellulosic material with an enzyme composition in the presence of a
polypeptide having glucuronyl esterase activity of the present
invention. In a preferred aspect, the method further comprises
recovering the degraded or converted cellulosic material.
[0278] The present invention also relates to methods of producing a
fermentation product, comprising: (a) saccharifying a cellulosic
material with an enzyme composition in the presence of a
polypeptide having glucuronyl esterase activity of the present
invention; (b) fermenting the saccharified cellulosic material with
one or more (several) fermenting microorganisms to produce the
fermentation product; and (c) recovering the fermentation product
from the fermentation.
[0279] The present invention also relates to methods of fermenting
a cellulosic material, comprising: fermenting the cellulosic
material with one or more (several) fermenting microorganisms,
wherein the cellulosic material is saccharified with an enzyme
composition in the presence of a polypeptide having glucuronyl
esterase activity of the present invention. In a preferred aspect,
the fermenting of the cellulosic material produces a fermentation
product. In another preferred aspect, the method further comprises
recovering the fermentation product from the fermentation.
[0280] The methods of the present invention can be used to
saccharify a cellulosic material to fermentable sugars and convert
the fermentable sugars to many useful substances, e.g., fuel,
potable ethanol, and/or fermentation products (e.g., acids,
alcohols, ketones, gases, and the like). The production of a
desired fermentation product from cellulosic material typically
involves pretreatment, enzymatic hydrolysis (saccharification), and
fermentation.
[0281] The processing of cellulosic material according to the
present invention can be accomplished using processes conventional
in the art. Moreover, the methods of the present invention can be
implemented using any conventional biomass processing apparatus
configured to operate in accordance with the invention.
[0282] Hydrolysis (saccharification) and fermentation, separate or
simultaneous, include, but are not limited to, separate hydrolysis
and fermentation (SHF); simultaneous saccharification and
fermentation (SSF); simultaneous saccharification and
cofermentation (SSCF); hybrid hydrolysis and fermentation (HHF);
separate hydrolysis and co-fermentation (SHCF); hybrid hydrolysis
and co-fermentation (HHCF); and direct microbial conversion (DMC).
SHF uses separate process steps to first enzymatically hydrolyze
cellulosic material to fermentable sugars, e.g., glucose,
cellobiose, cellotriose, and pentose sugars, and then ferment the
fermentable sugars to ethanol. In SSF, the enzymatic hydrolysis of
cellulosic material and the fermentation of sugars to ethanol are
combined in one step (Philippidis, G. P., 1996, Cellulose
bioconversion technology, in Handbook on Bioethanol: Production and
Utilization, Wyman, C. E., ed., Taylor & Francis, Washington,
DC, 179-212). SSCF involves the cofermentation of multiple sugars
(Sheehan, J., and Himmel, M., 1999, Enzymes, energy and the
environment: A strategic perspective on the U.S. Department of
Energy's research and development activities for bioethanol,
Biotechnol. Prog. 15: 817-827). HHF involves a separate hydrolysis
step, and in addition a simultaneous saccharification and
hydrolysis step, which can be carried out in the same reactor. The
steps in an HHF process can be carried out at different
temperatures, i.e., high temperature enzymatic saccharification
followed by SSF at a lower temperature that the fermentation strain
can tolerate. DMC combines all three processes (enzyme production,
hydrolysis, and fermentation) in one or more (several) steps where
the same organism is used to produce the enzymes for conversion of
the cellulosic material to fermentable sugars and to convert the
fermentable sugars into a final product (Lynd, L. R., Weimer, P.
J., van Zyl, W. H., and Pretorius, I. S., 2002, Microbial cellulose
utilization: Fundamentals and biotechnology, Microbiol. Mol. Biol.
Reviews 66: 506-577). It is understood herein that any method known
in the art comprising pretreatment, enzymatic hydrolysis
(saccharification), fermentation, or a combination thereof, can be
used in the practicing the methods of the present invention.
[0283] A conventional apparatus can include a fed-batch stirred
reactor, a batch stirred reactor, a continuous flow stirred reactor
with ultrafiltration, and/or a continuous plug-flow column reactor
(Fernanda de Castilhos Corazza, Flavio Faria de Moraes, Gisella
Maria Zanin and Ivo Neitzel, 2003, Optimal control in fed-batch
reactor for the cellobiose hydrolysis, Acta Scientiarum. Technology
25: 33-38; Gusakov, A. V., and Sinitsyn, A. P., 1985, Kinetics of
the enzymatic hydrolysis of cellulose: 1. A mathematical model for
a batch reactor process, Enz. Microb. Technol. 7: 346-352), an
attrition reactor (Ryu, S. K., and Lee, J. M., 1983, Bioconversion
of waste cellulose by using an attrition bioreactor, Biotechnol.
Bioeng. 25: 53-65), or a reactor with intensive stirring induced by
an electromagnetic field (Gusakov, A. V., Sinitsyn, A. P.,
Davydkin, I. Y., Davydkin, V. Y., Protas, O. V., 1996, Enhancement
of enzymatic cellulose hydrolysis using a novel type of bioreactor
with intensive stirring induced by electromagnetic field, Appl.
Biochem. Biotechnol. 56: 141-153). Additional reactor types
include: fluidized bed, upflow blanket, immobilized, and extruder
type reactors for hydrolysis and/or fermentation.
[0284] Pretreatment.
[0285] In practicing the methods of the present invention, any
pretreatment process known in the art can be used to disrupt plant
cell wall components of cellulosic material (Chandra et al., 2007,
Substrate pretreatment: The key to effective enzymatic hydrolysis
of lignocellulosics? Adv. Biochem. Engin./Biotechnol. 108: 67-93;
Galbe and Zacchi, 2007, Pretreatment of lignocellulosic materials
for efficient bioethanol production, Adv. Biochem.
Engin./Biotechnol. 108: 41-65; Hendriks and Zeeman, 2009,
Pretreatments to enhance the digestibility of lignocellulosic
biomass, Bioresource Technol. 100: 10-18; Mosier et al., 2005,
Features of promising technologies for pretreatment of
lignocellulosic biomass, Bioresource Technol. 96: 673-686;
Taherzadeh and Karimi, 2008, Pretreatment of lignocellulosic wastes
to improve ethanol and biogas production: A review, Int. J. of Mol.
Sci. 9: 1621-1651; Yang and Wyman, 2008, Pretreatment: the key to
unlocking low-cost cellulosic ethanol, Biofuels Bioproducts and
Biorefining-Biofpr. 2: 26-40).
[0286] The cellulosic material can also be subjected to particle
size reduction, pre-soaking, wetting, washing, or conditioning
prior to pretreatment using methods known in the art.
[0287] Conventional pretreatments include, but are not limited to,
steam pretreatment (with or without explosion), dilute acid
pretreatment, hot water pretreatment, alkaline pretreatment, lime
pretreatment, wet oxidation, wet explosion, ammonia fiber
explosion, organosolv pretreatment, and biological pretreatment.
Additional pretreatments include ammonia percolation, ultrasound,
electroporation, microwave, supercritical CO.sub.2, supercritical
H.sub.2O, ozone, and gamma irradiation pretreatments.
[0288] 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).
[0289] Steam Pretreatment. In steam pretreatment, 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. 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 done at
140-230.degree. C., more preferably 160-200.degree. C., and most
preferably 170-190.degree. C., where the optimal temperature range
depends on any addition of a chemical catalyst. Residence time for
the steam pretreatment is preferably 1-15 minutes, more preferably
3-12 minutes, and most preferably 4-10 minutes, where the optimal
residence time depends on temperature range and any addition of a
chemical catalyst. Steam pretreatment allows for relatively high
solids loadings, so that cellulosic material is generally only
moist during the pretreatment. The steam pretreatment is often
combined with an explosive discharge of the material after the
pretreatment, which is known as steam explosion, that is, rapid
flashing to atmospheric pressure and turbulent flow of the material
to increase the accessible surface area by fragmentation (Duff and
Murray, 1996, Bioresource Technology 855: 1-33; Galbe and Zacchi,
2002, Appl. Microbiol. Biotechnol. 59: 618-628; U.S. Patent
Application No. 20020164730). During steam pretreatment,
hemicellulose acetyl groups are cleaved and the resulting acid
autocatalyzes partial hydrolysis of the hemicellulose to
monosaccharides and oligosaccharides. Lignin is removed to only a
limited extent.
[0290] A catalyst such as H.sub.2SO.sub.4 or SO.sub.2 (typically
0.3 to 3% w/w) is often added prior to steam pretreatment, which
decreases the time and temperature, increases the recovery, and
improves enzymatic hydrolysis (Ballesteros et al., 2006, Appl.
Biochem. Biotechnol. 129-132: 496-508; Varga et al., 2004, Appl.
Biochem. Biotechnol. 113-116: 509-523; Sassner et al., 2006, Enzyme
Microb. Technol. 39: 756-762).
[0291] Chemical Pretreatment: The term "chemical treatment" refers
to any chemical pretreatment that promotes the separation and/or
release of cellulose, hemicellulose, and/or lignin. Examples of
suitable chemical pretreatment processes include, for example,
dilute acid pretreatment, lime pretreatment, wet oxidation, ammonia
fiber/freeze explosion (AFEX), ammonia percolation (APR), and
organosolv pretreatments.
[0292] In dilute acid pretreatment, cellulosic material is mixed
with dilute acid, typically H.sub.2SO.sub.4, and water to form a
slurry, heated by steam to the desired temperature, and after a
residence time flashed to atmospheric pressure. The dilute acid
pretreatment can be performed with a number of reactor designs,
e.g., plug-flow reactors, counter-current reactors, or continuous
counter-current shrinking bed reactors (Duff and Murray, 1996,
supra; Schell et al., 2004, Bioresource Technol. 91: 179-188; Lee
et al., 1999, Adv. Biochem. Eng. Biotechnol. 65: 93-115).
[0293] Several methods of pretreatment under alkaline conditions
can also be used. These alkaline pretreatments include, but are not
limited to, lime pretreatment, wet oxidation, ammonia percolation
(APR), and ammonia fiber/freeze explosion (AFEX).
[0294] Lime pretreatment is performed with calcium carbonate,
sodium hydroxide, or ammonia at low temperatures of 85-150.degree.
C. and residence times from 1 hour to several days (Wyman et al.,
2005, Bioresource Technol. 96: 1959-1966; Mosier et al., 2005,
Bioresource Technol. 96: 673-686). WO 2006/110891, WO 2006/11899,
WO 2006/11900, and WO 2006/110901 disclose pretreatment methods
using ammonia.
[0295] Wet oxidation is a thermal pretreatment performed typically
at 180-200.degree. C. for 5-15 minutes with addition of an
oxidative agent such as hydrogen peroxide or over-pressure of
oxygen (Schmidt and Thomsen, 1998, Bioresource Technol. 64:
139-151; Palonen et al., 2004, Appl. Biochem. Biotechnol. 117:
1-17; Varga et al., 2004, Biotechnol. Bioeng. 88: 567-574; Martin
et al., 2006, J. Chem. Technol. Biotechnol. 81: 1669-1677). The
pretreatment is performed at preferably 1-40% dry matter, more
preferably 2-30% dry matter, and most preferably 5-20% dry matter,
and often the initial pH is increased by the addition of alkali
such as sodium carbonate.
[0296] A modification of the wet oxidation pretreatment method,
known as wet explosion (combination of wet oxidation and steam
explosion), can handle dry matter up to 30%. In wet explosion, the
oxidizing agent is introduced during pretreatment after a certain
residence time. The pretreatment is then ended by flashing to
atmospheric pressure (WO 2006/032282).
[0297] Ammonia fiber explosion (AFEX) involves treating cellulosic
material with liquid or gaseous ammonia at moderate temperatures
such as 90-100.degree. C. and high pressure such as 17-20 bar for
5-10 minutes, where the dry matter content can be as high as 60%
(Gollapalli et al., 2002, Appl. Biochem. Biotechnol. 98: 23-35;
Chundawat et al., 2007, Biotechnol. Bioeng. 96: 219-231; Alizadeh
et al., 2005, Appl. Biochem. Biotechnol. 121: 1133-1141; Teymouri
et al., 2005, Bioresource Technol. 96: 2014-2018). AFEX
pretreatment results in the depolymerization of cellulose and
partial hydrolysis of hemicellulose. Lignin-carbohydrate complexes
are cleaved.
[0298] Organosolv pretreatment delignifies cellulosic material by
extraction using aqueous ethanol (40-60% ethanol) at
160-200.degree. C. for 30-60 minutes (Pan et al., 2005, Biotechnol.
Bioeng. 90: 473-481; Pan et al., 2006, Biotechnol. Bioeng. 94:
851-861; Kurabi et al., 2005, Appl. Biochem. Biotechnol. 121:
219-230). Sulphuric acid is usually added as a catalyst. In
organosolv pretreatment, the majority of hemicellulose is
removed.
[0299] Other examples of suitable pretreatment methods are
described by Schell et al., 2003, Appl. Biochem. and Biotechnol.
Vol. 105-108, p. 69-85, and Mosier et al., 2005, Bioresource
Technology 96: 673-686, and U.S. Published Application
2002/0164730.
[0300] In one aspect, the chemical pretreatment is preferably
carried out as an acid treatment, and more preferably as a
continuous dilute and/or mild acid treatment. The acid is typically
sulfuric acid, but other acids can also be used, such as acetic
acid, citric acid, nitric acid, phosphoric acid, tartaric acid,
succinic acid, hydrogen chloride, or mixtures thereof. Mild acid
treatment is conducted in the pH range of preferably 1-5, more
preferably 1-4, and most preferably 1-3. In one aspect, the acid
concentration is in the range from preferably 0.01 to 20 wt % acid,
more preferably 0.05 to 10 wt % acid, even more preferably 0.1 to 5
wt % acid, and most preferably 0.2 to 2.0 wt % acid. The acid is
contacted with cellulosic material and held at a temperature in the
range of preferably 160-220.degree. C., and more preferably
165-195.degree. C., for periods ranging from seconds to minutes to,
e.g., 1 second to 60 minutes.
[0301] In another aspect, pretreatment is carried out as an ammonia
fiber explosion step (AFEX pretreatment step).
[0302] In another aspect, pretreatment takes place in an aqueous
slurry. In preferred aspects, cellulosic material is present during
pretreatment in amounts preferably between 10-80 wt %, more
preferably between 20-70 wt %, and most preferably between 30-60 wt
%, such as around 50 wt %. The pretreated cellulosic material can
be unwashed or washed using any method known in the art, e.g.,
washed with water.
[0303] Mechanical Pretreatment: The term "mechanical pretreatment"
refers to various types of grinding or milling (e.g., dry milling,
wet milling, or vibratory ball milling).
[0304] Physical Pretreatment: The term "physical pretreatment"
refers to any pretreatment that promotes the separation and/or
release of cellulose, hemicellulose, and/or lignin from cellulosic
material. For example, physical pretreatment can involve
irradiation (e.g., microwave irradiation), steaming/steam
explosion, hydrothermolysis, and combinations thereof.
[0305] Physical pretreatment can involve high pressure and/or high
temperature (steam explosion). In one aspect, high pressure means
pressure in the range of preferably about 300 to about 600 psi,
more preferably about 350 to about 550 psi, and most preferably
about 400 to about 500 psi, such as around 450 psi. In another
aspect, high temperature means temperatures in the range of about
100 to about 300.degree. C., preferably about 140 to about
235.degree. C. In a preferred aspect, mechanical pretreatment is
performed in a batch-process, steam gun hydrolyzer system that uses
high pressure and high temperature as defined above, e.g., a Sunds
Hydrolyzer available from Sunds Defibrator AB, Sweden.
[0306] Combined Physical and Chemical Pretreatment: Cellulosic
material can be pretreated both physically and chemically. For
instance, the pretreatment step can involve dilute or mild acid
treatment and high temperature and/or pressure treatment. The
physical and chemical pretreatments can be carried out sequentially
or simultaneously, as desired. A mechanical pretreatment can also
be included.
[0307] Accordingly, in a preferred aspect, cellulosic material is
subjected to mechanical, chemical, or physical pretreatment, or any
combination thereof, to promote the separation and/or release of
cellulose, hemicellulose, and/or lignin.
[0308] Biological Pretreatment: The term "biological pretreatment"
refers to any biological pretreatment that promotes the separation
and/or release of cellulose, hemicellulose, and/or lignin from
cellulosic material. Biological pretreatment techniques can involve
applying lignin-solubilizing microorganisms (see, for example, Hsu,
T.-A., 1996, Pretreatment of biomass, in Handbook on Bioethanol:
Production and Utilization, Wyman, C. E., ed., Taylor &
Francis, Washington, 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. O., and Overend, R. P., eds.,
ACS Symposium Series 566, American Chemical Society, Washington,
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: State of the art, Adv. Biochem.
Eng./Biotechnol. 42: 63-95).
[0309] Saccharification.
[0310] In the hydrolysis step, also known as saccharification, the
cellulosic material, e.g., pretreated, is hydrolyzed to break down
cellulose and alternatively also hemicellulose to fermentable
sugars, such as glucose, cellobiose, xylose, xylulose, arabinose,
mannose, galactose, and/or soluble oligosaccharides. The hydrolysis
is performed enzymatically by an enzyme composition in the presence
of a polypeptide having glucuronyl esterase activity of the present
invention. The composition can further comprise one or more
(several) hemicellulolytic or xylan degrading enzymes. The enzymes
of the compositions can also be added sequentially.
[0311] Enzymatic hydrolysis is preferably carried out in a suitable
aqueous environment under conditions that can be readily determined
by one skilled in the art. In a preferred aspect, hydrolysis is
performed under conditions suitable for the activity of the
enzyme(s), i.e., optimal for the enzyme(s). The hydrolysis can be
carried out as a fed batch or continuous process where the
pretreated cellulosic material (substrate) is fed gradually to, for
example, an enzyme containing hydrolysis solution.
[0312] The saccharification is generally performed in stirred-tank
reactors or fermentors under controlled pH, temperature, and mixing
conditions. Suitable process time, temperature and pH conditions
can readily be determined by one skilled in the art. For example,
the saccharification can last up to 200 hours, but is typically
performed for preferably about 12 to about 96 hours, more
preferably about 16 to about 72 hours, and most preferably about 24
to about 48 hours. The temperature is in the range of preferably
about 25.degree. C. to about 70.degree. C., more preferably about
30.degree. C. to about 65.degree. C., and more preferably about
40.degree. C. to 60.degree. C., in particular about 50.degree. C.
The pH is in the range of preferably about 3 to about 8, more
preferably about 3.5 to about 7, and most preferably about 4 to
about 6, in particular about pH 5. The dry solids content is in the
range of preferably about 5 to about 50 wt %, more preferably about
10 to about 40 wt %, and most preferably about 20 to about 30 wt
%.
[0313] The enzyme composition preferably comprises enzymes having
cellulolytic activity and/or xylan degrading activity. In one
aspect, the enzyme composition comprises one or more (several)
cellulolytic enzymes. In another aspect, the enzyme composition
comprises one or more (several) xylan degrading enzymes. In another
aspect, the enzyme composition comprises one or more (several)
cellulolytic enzymes and one or more (several) xylan degrading
enzymes.
[0314] The one or more (several) cellulolytic enzymes are
preferably selected from the group consisting of an endoglucanase,
a cellobiohydrolase, and a beta-glucosidase. The one or more
(several) xylan degrading enzymes are preferably selected from the
group consisting of a xylanase, an acetyxylan esterase, a feruloyl
esterase, an arabinofuranosidase, a xylosidase, and a
glucuronidase.
[0315] In another aspect, the enzyme composition further or even
further comprises a polypeptide having cellulolytic enhancing
activity (see, for example, WO 2005/074647, WO 2005/074656, and WO
2007/089290). In another aspect, the enzyme composition may further
or even further comprise one or more (several) additional enzyme
activities to improve the degradation of the cellulose-containing
material. Preferred additional enzymes are hemicellulases (e.g.,
alpha-D-glucuronidases, alpha-L-arabinofuranosidases,
endo-mannanases, beta-mannosidases, alpha-galactosidases,
endo-alpha-L-arabinanases, beta-galactosidases),
carbohydrate-esterases (e.g., acetyl-xylan esterases, acetyl-mannan
esterases, ferulic acid esterases, coumaric acid esterases,
glucuronoyl esterases), pectinases, proteases, ligninolytic enzymes
(e.g., laccases, manganese peroxidases, lignin peroxidases,
H.sub.2O.sub.2-producing enzymes, oxidoreductases), expansins,
swollenins, or mixtures thereof. In the methods of the present
invention, the additional enzyme(s) can be added prior to or during
fermentation, e.g., during saccharification or during or after
propagation of the fermenting microorganism(s).
[0316] One or more (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 (several) components may be native proteins of a cell, which
is used as a host cell to express recombinantly one or more
(several) other components of the enzyme composition. One or more
(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.
[0317] The enzymes used in the methods of the present invention may
be in any form suitable for use in the processes described herein,
such as, for example, a crude fermentation broth with or without
cells removed, a cell lysate with or without cellular debris, a
semi-purified or purified enzyme preparation, or a host cell as a
source of the enzymes. The enzyme composition may be a dry powder
or granulate, a non-dusting granulate, a liquid, a stabilized
liquid, or a stabilized protected enzyme. Liquid enzyme
preparations may, for instance, be stabilized by adding stabilizers
such as a sugar, a sugar alcohol or another polyol, and/or lactic
acid or another organic acid according to established
processes.
[0318] The optimum amounts of the enzymes and polypeptides having
glucuronyl esterase activity depend on several factors including,
but not limited to, the mixture of component cellulolytic enzymes,
the cellulosic substrate, the concentration of cellulosic
substrate, the pretreatment(s) of the cellulosic substrate,
temperature, time, pH, and inclusion of fermenting organism (e.g.,
yeast for Simultaneous Saccharification and Fermentation).
[0319] In a preferred aspect, an effective amount of cellulolytic
enzyme(s) to cellulosic material is about 0.5 to about 50 mg,
preferably at about 0.5 to about 40 mg, more preferably at about
0.5 to about 25 mg, more preferably at about 0.75 to about 20 mg,
more preferably at about 0.75 to about 15 mg, even more preferably
at about 0.5 to about 10 mg, and most preferably at about 2.5 to
about 10 mg per g of cellulosic material.
[0320] In another preferred aspect, an effective amount of
polypeptide(s) having glucuronyl esterase activity to cellulosic
material is about 0.01 to about 50.0 mg, preferably about 0.01 to
about 40 mg, more preferably about 0.01 to about 30 mg, more
preferably about 0.01 to about 20 mg, more preferably about 0.01 to
about 10 mg, more preferably about 0.01 to about 5 mg, more
preferably at about 0.025 to about 1.5 mg, more preferably at about
0.05 to about 1.25 mg, more preferably at about 0.075 to about 1.25
mg, more preferably at about 0.1 to about 1.25 mg, even more
preferably at about 0.15 to about 1.25 mg, and most preferably at
about 0.25 to about 1.0 mg per g of cellulosic material.
[0321] In another preferred aspect, an effective amount of
polypeptide(s) having glucuronyl esterase activity to cellulolytic
enzyme(s) is about 0.005 to about 1.0 g, preferably at about 0.01
to about 1.0 g, more preferably at about 0.15 to about 0.75 g, more
preferably at about 0.15 to about 0.5 g, more preferably at about
0.1 to about 0.5 g, even more preferably at about 0.1 to about 0.5
g, and most preferably at about 0.05 to about 0.2 g per g of
cellulolytic enzyme(s).
[0322] The enzymes can be derived or obtained from any suitable
origin, including, bacterial, fungal, yeast, plant, or mammalian
origin. The term "obtained" means herein that the enzyme may have
been isolated from an organism that naturally produces the enzyme
as a native enzyme. The term "obtained" also means herein that the
enzyme may have been produced recombinantly in a host organism
employing methods described herein, wherein the recombinantly
produced enzyme is either native or foreign to the host organism or
has a modified amino acid sequence, e.g., having one or more
(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.
[0323] A polypeptide having cellulolytic enzyme activity or xylan
degrading 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,
or Oceanobacillus polypeptide having cellulolytic enzyme activity
or xylan degrading activity, or a Gram negative bacterial
polypeptide such as an E. coli, Pseudomonas, Salmonella,
Campylobacter, Helicobacter, Flavobacterium, Fusobacterium,
Ilyobacter, Neisseria, or Ureaplasma polypeptide having
cellulolytic enzyme activity or xylan degrading activity.
[0324] In a preferred aspect, the polypeptide is a Bacillus
alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus
circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus,
Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus
megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus
subtilis, or Bacillus thuringiensis polypeptide having cellulolytic
enzyme activity or xylan degrading activity.
[0325] In another preferred aspect, the polypeptide is a
Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus
uberis, or Streptococcus equi subsp. Zooepidemicus polypeptide
having cellulolytic enzyme activity or xylan degrading
activity.
[0326] In another preferred aspect, the polypeptide is a
Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces
coelicolor, Streptomyces griseus, or Streptomyces lividans
polypeptide having cellulolytic enzyme activity or xylan degrading
activity.
[0327] The polypeptide having cellulolytic enzyme activity or xylan
degrading activity may also be a fungal polypeptide, and more
preferably a yeast polypeptide such as a Candida, Kluyveromyces,
Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia polypeptide
having cellulolytic enzyme activity or xylan degrading activity; or
more preferably a filamentous fungal polypeptide such as an
Acremonium, Agaricus, Alternaria, Aspergillus, Aureobasidium,
Botryosphaeria, Ceriporiopsis, Chaetomidium, Chrysosporium,
Claviceps, Cochliobolus, Coprinopsis, Coptotermes, Corynascus,
Cryphonectria, Cryptococcus, Diplodia, Exidia, Filibasidium,
Fusarium, Gibberella, Holomastigotoides, Humicola, Irpex,
Lentinula, Leptospaeria, Magnaporthe, Melanocarpus, Meripilus,
Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces,
Penicillium, Phanerochaete, Piromyces, Poitrasia, Pseudoplectania,
Pseudotrichonympha, Rhizomucor, Schizophyllum, Scytalidium,
Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trichoderma,
Trichophaea, Verticillium, Volvariella, or Xylaria polypeptide
having cellulolytic enzyme activity or xylan degrading
activity.
[0328] In a preferred aspect, the polypeptide is a Saccharomyces
carlsbergensis, Saccharomyces cerevisiae, Saccharomyces
diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri,
Saccharomyces norbensis, or Saccharomyces oviformis polypeptide
having cellulolytic enzyme activity or xylan degrading
activity.
[0329] In another preferred aspect, the polypeptide is an
Acremonium cellulolyticus, Aspergillus aculeatus, Aspergillus
awamori, Aspergillus fumigatus, Aspergillus foetidus, Aspergillus
japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus
oryzae, Chrysosporium keratinophilum, Chrysosporium lucknowense,
Chrysosporium tropicum, Chrysosporium merdarium, Chrysosporium
inops, Chrysosporium pannicola, Chrysosporium queenslandicum,
Chrysosporium zonatum, Fusarium bactridioides, Fusarium cerealis,
Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum,
Fusarium graminum, Fusarium heterosporum, Fusarium negundi,
Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium
sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides,
Fusarium suiphureum, Fusarium torulosum, Fusarium trichothecioides,
Fusarium venenatum, Humicola grisea, Humicola insolens, Humicola
lanuginosa, Irpex lacteus, Mucor miehei, Myceliophthora
thermophila, Neurospora crassa, Penicillium funiculosum,
Penicillium purpurogenum, Phanerochaete chrysosporium, Thielavia
achromatica, Thielavia albomyces, Thielavia albopilosa, Thielavia
australeinsis, Thielavia fimeti, Thielavia microspora, Thielavia
ovispora, Thielavia peruviana, Thielavia spededonium, Thielavia
setosa, Thielavia subthermophila, Thielavia terrestris, Trichoderma
harzianum, Trichoderma koningii, Trichoderma longibrachiatum,
Trichoderma reesei, Trichoderma viride, or Trichophaea saccata
polypeptide having cellulolytic enzyme activity or xylan degrading
activity.
[0330] Chemically modified or protein engineered mutants of
polypeptides having cellulolytic enzyme activity or xylan degrading
activity may also be used.
[0331] One or more (several) components of the enzyme composition
may be a recombinant component, i.e., produced by cloning of a DNA
sequence encoding the single component and subsequent cell
transformed with the DNA sequence and expressed in a host (see, for
example, WO 91/17243 and WO 91/17244). The host is preferably a
heterologous host (enzyme is foreign to host), but the host may
under certain conditions also be a homologous host (enzyme is
native to host). Monocomponent cellulolytic proteins may also be
prepared by purifying such a protein from a fermentation broth.
[0332] Examples of commercial cellulolytic protein preparations
suitable for use in the present invention include, for example,
CELLIC.TM. Ctec (Novozymes A/S), CELLUCLAST.TM. (Novozymes A/S),
NOVOZYM.TM. 188 (Novozymes A/S), CELLUZYME.TM. (Novozymes A/S),
CEREFLO.TM. (Novozymes A/S), and ULTRAFLO.TM. (Novozymes A/S),
ACCELERASE.TM. (Genencor Int.), LAMINEX.TM. (Genencor Int.),
SPEZYME.TM. CP (Genencor Int.), ROHAMENT.TM. 7069 W (Rohm GmbH),
FIBREZYME.RTM. LDI (Dyadic International, Inc.), FIBREZYME.RTM. LBR
(Dyadic International, Inc.), or VISCOSTAR.RTM. 150 L (Dyadic
International, Inc.). The cellulase enzymes are added in amounts
effective from about 0.001 to about 5.0 wt % of solids, more
preferably from about 0.025 to about 4.0 wt % of solids, and most
preferably from about 0.005 to about 2.0 wt % of solids. The
cellulase enzymes are added in amounts effective from about 0.001
to about 5.0 wt % of solids, more preferably from about 0.025 to
about 4.0 wt % of solids, and most preferably from about 0.005 to
about 2.0 wt % of solids.
[0333] Examples of bacterial endoglucanases that can be used in the
methods of the present invention, include, but are not limited to,
an Acidothermus cellulolyticus endoglucanase (WO 91/05039; WO
93/15186; U.S. Pat. No. 5,275,944; WO 96/02551; U.S. Pat. No.
5,536,655, WO 00/70031, WO 05/093050); Thermobifida fusca
endoglucanase III (WO 05/093050); and Thermobifida fusca
endoglucanase V (WO 05/093050).
[0334] Examples of fungal endoglucanases that can be used in the
methods of the present invention, include, but are not limited to,
a Trichoderma reesei endoglucanase I (Penttila et al., 1986, Gene
45: 253-263; GENBANK.TM. accession no. M15665); Trichoderma reesei
endoglucanase II (Saloheimo, et al., 1988, Gene 63:11-22;
GENBANK.TM. accession no. M19373); Trichoderma reesei endoglucanase
III (Okada et al., 1988, Appl. Environ. Microbiol. 64: 555-563;
GENBANK.TM. accession no. AB003694); and Trichoderma reesei
endoglucanase V (Saloheimo et al., 1994, Molecular Microbiology 13:
219-228; GENBANK.TM. accession no. Z33381); Aspergillus aculeatus
endoglucanase (Ooi et al., 1990, Nucleic Acids Research 18: 5884);
Aspergillus kawachii endoglucanase (Sakamoto et al., 1995, Current
Genetics 27: 435-439); Erwinia carotovara endoglucanase (Saarilahti
et al., 1990, Gene 90: 9-14); Fusarium oxysporum endoglucanase
(GENBANK.TM. accession no. L29381); Humicola grisea var. thermoidea
endoglucanase (GENBANK.TM. accession no. AB003107); Melanocarpus
albomyces endoglucanase (GENBANK.TM. accession no. MAL515703);
Neurospora crassa endoglucanase (GENBANK.TM. accession no.
XM_324477); Humicola insolens endoglucanase V; Myceliophthora
thermophila CBS 117.65 endoglucanase; basidiomycete CBS 495.95
endoglucanase; basidiomycete CBS 494.95 endoglucanase; Thielavia
terrestris NRRL 8126 CEL6B endoglucanase; Thielavia terrestris NRRL
8126 CEL6C endoglucanase); Thielavia terrestris NRRL 8126 CEL7C
endoglucanase; Thielavia terrestris NRRL 8126 CEL7E endoglucanase;
Thielavia terrestris NRRL 8126 CEL7F endoglucanase; Cladorrhinum
foecundissimum ATCC 62373 CEL7A endoglucanase; and Trichoderma
reesei strain No. VTT-D-80133 endoglucanase (GENBANK.TM. accession
no. M15665).
[0335] Examples of cellobiohydrolases useful in the methods of the
present invention include, but are not limited to, Trichoderma
reesei cellobiohydrolase I; Trichoderma reesei cellobiohydrolase
II; Humicola insolens cellobiohydrolase I, Myceliophthora
thermophila cellobiohydrolase II, Thielavia terrestris
cellobiohydrolase II (CEL6A), Chaetomium thermophilum
cellobiohydrolase I, and Chaetomium thermophilum cellobiohydrolase
II.
[0336] Examples of beta-glucosidases useful in the methods of the
present invention include, but are not limited to, Aspergillus
oryzae beta-glucosidase; Aspergillus fumigatus beta-glucosidase;
Penicillium brasilianum IBT 20888 beta-glucosidase; Aspergillus
niger beta-glucosidase; and Aspergillus aculeatus
beta-glucosidase.
[0337] The Aspergillus oryzae polypeptide having beta-glucosidase
activity can be obtained according to WO 2002/095014. The
Aspergillus fumigatus polypeptide having beta-glucosidase activity
can be obtained according to WO 2005/047499. The Penicillium
brasilianum polypeptide having beta-glucosidase activity can be
obtained according to WO 2007/019442. The Aspergillus niger
polypeptide having beta-glucosidase activity can be obtained
according to Dan et al., 2000, J. Biol. Chem. 275: 4973-4980. The
Aspergillus aculeatus polypeptide having beta-glucosidase activity
can be obtained according to Kawaguchi et al., 1996, Gene 173:
287-288.
[0338] The beta-glucosidase may be a fusion protein. In one aspect,
the beta-glucosidase is the Aspergillus oryzae beta-glucosidase
variant BG fusion protein or the Aspergillus oryzae
beta-glucosidase fusion protein obtained according to WO
2008/057637.
[0339] Other 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.
[0340] Other cellulolytic enzymes that may be used in the present
invention are described in EP 495 257, EP 531 315, EP 531 372, WO
1989/09259, WO 1994/07998, WO 1995/24471, WO 1996/11262, WO
1996/29397, WO 1996/034108, WO 1997/14804, WO 1998/08940, WO
1998/012307, WO 1998/13465, WO 1998/015619, WO 1998/015633, WO
1998/028411, WO 1999/06574, WO 1999/10481, WO 1999/025846, WO
1999/025847, WO 1999/031255, WO 2000/009707, WO 2002/050245, WO
2002/0076792, WO 2002/101078, WO 2003/027306, WO 2003/052054, WO
2003/052055, WO 2003/052056, WO 2003/052057, WO 2003/052118, WO
2004/016760, WO 2004/043980, WO 2004/048592, WO 2005/001065, WO
2005/028636, WO 2005/093050, WO 2005/093073, WO 2006/074005, WO
2006/117432, WO 2007/071818, WO 2007/071820, WO 2008/008070, WO
2008/008793, U.S. Pat. No. 4,435,307, U.S. Pat. No. 5,457,046, U.S.
Pat. No. 5,648,263, U.S. Pat. No. 5,686,593, U.S. Pat. No.
5,691,178, U.S. Pat. No. 5,763,254, and U.S. Pat. No.
5,776,757.
[0341] In the methods of the present invention, any GH61
polypeptide having cellulolytic enhancing activity can be used.
[0342] In a first aspect, the polypeptide having cellulolytic
enhancing activity comprises the following motifs:
TABLE-US-00001 (SEQ ID NO: 7 or SEQ ID NO: 8)
[ILMV]-P-X(4,5)-G-X-Y-[ILMV]-X-R-X-[EQ]-X(4)-[HNQ] and (SEQ ID NO:
9) [FW]-[TF]-K-[AIV],
[0343] 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.
[0344] The polypeptide comprising the above-noted motifs may
further comprise:
TABLE-US-00002 (SEQ ID NO: 10 or SEQ ID NO: 11)
H-X(1,2)-G-P-X(3)-[YW]-[AILMV], (SEQ ID NO: 12)
[EQ]-X-Y-X(2)-C-X-[EHQN]-[FILV]-X-[ILV], or (SEQ ID NO: 10 or SEQ
ID NO: 11) H-X(1,2)-G-P-X(3)-[YW]-[AILMV] and (SEQ ID NO: 12)
[EQ]-X-Y-X(2)-C-X-[EHQN]-[FILV]-X-[ILV],
[0345] 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.
[0346] In a preferred aspect, the polypeptide having cellulolytic
enhancing activity further comprises H-X(1,2)-G-P-X(3)-[YW]-[AILMV]
(SEQ ID NO: 10 or SEQ ID NO: 11). In another preferred aspect, the
isolated polypeptide having cellulolytic enhancing activity further
comprises [EQ]-X-Y-X(2)-C-X-[EHQN]-[FILV]-X-[ILV] (SEQ ID NO: 12).
In another preferred aspect, the polypeptide having cellulolytic
enhancing activity further comprises H-X(1,2)-G-P-X(3)-[YW]-[AILMV]
(SEQ ID NO: 10 or SEQ ID NO: 11) and
[EQ]-X-Y-X(2)-C-X-[EHQN]-[FILV]-X-[ILV] (SEQ ID NO: 12).
[0347] In a second aspect, the polypeptide having cellulolytic
enhancing activity comprises the following motif:
TABLE-US-00003 (SEQ ID NO: 13 or SEQ ID NO: 14)
[ILMV]-P-x(4,5)-G-x-Y-[ILMV]-x-R-x-[EQ]-x(3)-A- [HNQ],
wherein x is any amino acid, x(4,5) is any amino acid at 4 or 5
contiguous positions, and x(3) is any amino acid at 3 contiguous
positions. In the above motif, the accepted IUPAC single letter
amino acid abbreviation is employed.
[0348] Examples of polypeptides having cellulolytic enhancing
activity useful in the methods of the present invention include,
but are not limited to, polypeptides having cellulolytic enhancing
activity from Thielavia terrestris (WO 2005/074647); polypeptides
having cellulolytic enhancing activity from Thermoascus aurantiacus
(WO 2005/074656); polypeptides having cellulolytic enhancing
activity from Trichoderma reesei (WO 2007/089290); and polypeptides
having cellulolytic enhancing activity from Myceliophthora
thermophila (WO 2009/085935; WO 2009/085859; WO 2009/085864; WO
2009/085868).
[0349] Examples of commercial xylan degrading enzyme preparations
suitable for use in the present invention include, for example,
SHEARZYME.TM. (Novozymes A/S), CELLIC.TM. Htec (Novozymes A/S),
VISCOZYME.RTM. (Novozymes A/S), ULTRAFLO.RTM. (Novozymes A/S),
PULPZYME.RTM. HC (Novozymes A/S), MULTIFECT.RTM. Xylanase
(Genencor), ECOPULP.RTM. TX-200A (AB Enzymes), HSP 6000 Xylanase
(DSM), DEPOL.TM. 333P (Biocatalysts Limit, Wales, UK), DEPOL.TM.
740 L. (Biocatalysts Limit, Wales, UK), and DEPOL.TM. 762P
(Biocatalysts Limit, Wales, UK).
[0350] Examples of xylanases useful in the methods of the present
invention include, but are not limited to, Aspergillus aculeatus
xylanase (GeneSeqP:AAR63790; WO 94/21785), Aspergillus fumigatus
xylanases (WO 2006/078256), and Thielavia terrestris NRRL 8126
xylanases (WO 2009/079210).
[0351] Examples of beta-xylosidases useful in the methods of the
present invention include, but are not limited to, Trichoderma
reesei beta-xylosidase (UniProtKB/TrEMBL accession number Q92458),
Talaromyces emersonii (SwissProt accession number Q8X212), and
Neurospora crassa (SwissProt accession number Q7SOW4).
[0352] Examples of acetylxylan esterases useful in the methods of
the present invention include, but are not limited to, Hypocrea
jecorina acetylxylan esterase (WO 2005/001036), Neurospora crassa
acetylxylan esterase (UniProt accession number q7s259), Thielavia
terrestris NRRL 8126 acetylxylan esterase (WO 2009/042846),
Chaetomium globosum acetylxylan esterase (Uniprot accession number
Q2GWX4), Chaetomium gracile acetylxylan esterase (GeneSeqP
accession number AAB82124), Phaeosphaeria nodorum acetylxylan
esterase (Uniprot accession number Q0UHJ1), and Humicola insolens
DSM 1800 acetylxylan esterase (WO 2009/073709).
[0353] Examples of ferulic acid esterases useful in the methods of
the present invention include, but are not limited to, Humicola
insolens DSM 1800 feruloyl esterase (WO 2009/076122), Neurospora
crassa feruloyl esterase (UniProt accession number Q9HGR3), and
Neosartorya fischeri feruloyl esterase (UniProt Accession number
A1D9T4).
[0354] Examples of arabinofuranosidases useful in the methods of
the present invention include, but are not limited to, Humicola
insolens DSM 1800 arabinofuranosidase (WO 2009/073383) and
Aspergillus niger arabinofuranosidase (GeneSeqP accession number
AAR94170).
[0355] Examples of alpha-glucuronidases useful in the methods of
the present invention include, but are not limited to, Aspergillus
clavatus alpha-glucuronidase (UniProt accession number alcc12),
Trichoderma reesei alpha-glucuronidase (Uniprot accession number
Q99024), Talaromyces emersonii alpha-glucuronidase (UniProt
accession number Q8X211), Aspergillus niger alpha-glucuronidase
(Uniprot accession number Q96WX9), Aspergillus terreus
alpha-glucuronidase (SwissProt accession number Q0CJP9), and
Aspergillus fumigatus alpha-glucuronidase (SwissProt accession
number Q4WW45).
[0356] The enzymes and proteins used in the methods of the present
invention may be produced by fermentation of the above-noted
microbial strains on a nutrient medium containing suitable carbon
and nitrogen sources and inorganic salts, using procedures known in
the art (see, e.g., Bennett, J. W. and LaSure, L. (eds.), More Gene
Manipulations in Fungi, Academic Press, C A, 1991). Suitable media
are available from commercial suppliers or may be prepared
according to published compositions (e.g., in catalogues of the
American Type Culture Collection). Temperature ranges and other
conditions suitable for growth and enzyme production are known in
the art (see, e.g., Bailey, J. E., and Ollis, D. F., Biochemical
Engineering Fundamentals, McGraw-Hill Book Company, N Y, 1986).
[0357] The fermentation can be any method of cultivation of a cell
resulting in the expression or isolation of an enzyme. Fermentation
may, therefore, be understood as comprising shake flask
cultivation, or small- or large-scale fermentation (including
continuous, batch, fed-batch, or solid state fermentations) in
laboratory or industrial fermentors performed in a suitable medium
and under conditions allowing the enzyme to be expressed or
isolated. The resulting enzymes produced by the methods described
above may be recovered from the fermentation medium and purified by
conventional procedures.
[0358] Fermentation.
[0359] The fermentable sugars obtained from the hydrolyzed
cellulosic material can be fermented by one or more (several)
fermenting microorganisms capable of fermenting the sugars directly
or indirectly into a desired fermentation product. "Fermentation"
or "fermentation process" refers to any fermentation process or any
process comprising a fermentation step. Fermentation processes also
include fermentation processes used in the consumable alcohol
industry (e.g., beer and wine), dairy industry (e.g., fermented
dairy products), leather industry, and tobacco industry. The
fermentation conditions depend on the desired fermentation product
and fermenting organism and can easily be determined by one skilled
in the art.
[0360] In the fermentation step, sugars, released from 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.
[0361] 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.
[0362] 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).
[0363] "Fermenting microorganism" refers to any microorganism,
including bacterial and fungal organisms, suitable for use in a
desired fermentation process to produce a fermentation product. The
fermenting organism can be C.sub.6 and/or C.sub.5 fermenting
organisms, or a combination thereof. Both C.sub.6 and C.sub.5
fermenting organisms are well known in the art. Suitable fermenting
microorganisms are able to ferment, i.e., convert, sugars, such as
glucose, xylose, xylulose, arabinose, maltose, mannose, galactose,
or oligosaccharides, directly or indirectly into the desired
fermentation product.
[0364] Examples of bacterial and fungal fermenting organisms
producing ethanol are described by Lin et al., 2006, Appl.
Microbiol. Biotechnol. 69: 627-642.
[0365] Examples of fermenting microorganisms that can ferment
C.sub.6 sugars include bacterial and fungal organisms, such as
yeast. Preferred yeast includes strains of the Saccharomyces spp.,
preferably Saccharomyces cerevisiae.
[0366] Examples of fermenting organisms that can ferment C.sub.5
sugars include bacterial and fungal organisms, such as yeast.
Preferred C.sub.5 fermenting yeast include strains of Pichia,
preferably Pichia stipitis, such as Pichia stipitis CBS 5773;
strains of Candida, preferably Candida boidinii, Candida brassicae,
Candida sheatae, Candida diddensii, Candida pseudotropicalis, or
Candida utilis.
[0367] Other fermenting organisms include strains of Zymomonas,
such as Zymomonas mobilis; Hansenula, such as Hansenula anomala;
Kluyveromyces, such as K. fragilis; Schizosaccharomyces, such as S.
pombe; and E. coli, especially E. coli strains that have been
genetically modified to improve the yield of ethanol.
[0368] In a preferred aspect, the yeast is a Saccharomyces spp. In
a more preferred aspect, the yeast is Saccharomyces cerevisiae. In
another more preferred aspect, the yeast is Saccharomyces
distaticus. In another more preferred aspect, the yeast is
Saccharomyces uvarum. In another preferred aspect, the yeast is a
Kluyveromyces. In another more preferred aspect, the yeast is
Kluyveromyces marxianus. In another more preferred aspect, the
yeast is Kluyveromyces fragilis. In another preferred aspect, the
yeast is a Candida. In another more preferred aspect, the yeast is
Candida boidinii. In another more preferred aspect, the yeast is
Candida brassicae. In another more preferred aspect, the yeast is
Candida diddensii. In another more preferred aspect, the yeast is
Candida pseudotropicalis. In another more preferred aspect, the
yeast is Candida utilis. In another preferred aspect, the yeast is
a Clavispora. In another more preferred aspect, the yeast is
Clavispora lusitaniae. In another more preferred aspect, the yeast
is Clavispora opuntiae. In another preferred aspect, the yeast is a
Pachysolen. In another more preferred aspect, the yeast is
Pachysolen tannophilus. In another preferred aspect, the yeast is a
Pichia. In another more preferred aspect, the yeast is a Pichia
stipitis. In another preferred aspect, the yeast is a
Bretannomyces. In another more preferred aspect, the yeast is
Bretannomyces clausenii (Philippidis, G. P., 1996, Cellulose
bioconversion technology, in Handbook on Bioethanol: Production and
Utilization, Wyman, C. E., ed., Taylor & Francis, Washington,
DC, 179-212).
[0369] Bacteria that can efficiently ferment hexose and pentose to
ethanol include, for example, Zymomonas mobilis and Clostridium
thermocellum (Philippidis, 1996, supra).
[0370] In a preferred aspect, the bacterium is a Zymomonas. In a
more preferred aspect, the bacterium is Zymomonas mobilis. In
another preferred aspect, the bacterium is a Clostridium. In
another more preferred aspect, the bacterium is Clostridium
thermocellum.
[0371] Commercially available yeast suitable for ethanol production
includes, e.g., ETHANOL RED.TM. yeast (available from
Fermentis/Lesaffre, USA), FALI.TM. (available from Fleischmann's
Yeast, USA), SUPERSTART.TM. and THERMOSACC.TM. fresh yeast
(available from Ethanol Technology, WI, USA), BIOFERM.TM. AFT and
XR (available from NABC--North American Bioproducts Corporation,
GA, USA), GERT STRAND.TM. (available from Gert Strand AB, Sweden),
and FERMIOL.TM. (available from DSM Specialties).
[0372] 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.
[0373] The cloning of heterologous genes into various fermenting
microorganisms has led to the construction of organisms capable of
converting hexoses and pentoses to ethanol (cofermentation) (Chen
and Ho, 1993, Cloning and improving the expression of Pichia
stipitis xylose reductase gene in Saccharomyces cerevisiae, Appl.
Biochem. Biotechnol. 39-40: 135-147; Ho et al., 1998, Genetically
engineered Saccharomyces yeast capable of effectively cofermenting
glucose and xylose, Appl. Environ. Microbiol. 64: 1852-1859; Kotter
and Ciriacy, 1993, Xylose fermentation by Saccharomyces cerevisiae,
Appl. Microbiol. Biotechnol. 38: 776-783; Walfridsson et al., 1995,
Xylose-metabolizing Saccharomyces cerevisiae strains overexpressing
the TKL1 and TALI genes encoding the pentose phosphate pathway
enzymes transketolase and transaldolase, Appl. Environ. Microbiol.
61: 4184-4190; Kuyper et al., 2004, Minimal metabolic engineering
of Saccharomyces cerevisiae for efficient anaerobic xylose
fermentation: a proof of principle, FEMS Yeast Research 4: 655-664;
Beall et al., 1991, Parametric studies of ethanol production from
xylose and other sugars by recombinant Escherichia coli, Biotech.
Bioeng. 38: 296-303; Ingram et al., 1998, Metabolic engineering of
bacteria for ethanol production, Biotechnol. Bioeng. 58: 204-214;
Zhang et al., 1995, Metabolic engineering of a pentose metabolism
pathway in ethanologenic Zymomonas mobilis, Science 267: 240-243;
Deanda et al., 1996, Development of an arabinose-fermenting
Zymomonas mobilis strain by metabolic pathway engineering, Appl.
Environ. Microbiol. 62: 4465-4470; WO 2003/062430, xylose
isomerase).
[0374] In a preferred aspect, the genetically modified fermenting
microorganism is Saccharomyces cerevisiae. In another preferred
aspect, the genetically modified fermenting microorganism is
Zymomonas mobilis. In another preferred aspect, the genetically
modified fermenting microorganism is Escherichia coli. In another
preferred aspect, the genetically modified fermenting microorganism
is Klebsiella oxytoca. In another preferred aspect, the genetically
modified fermenting microorganism is Kluyveromyces sp.
[0375] It is well known in the art that the organisms described
above can also be used to produce other substances, as described
herein.
[0376] The fermenting microorganism is typically added to the
degraded lignocellulose or hydrolysate and the fermentation is
performed for about 8 to about 96 hours, such as about 24 to about
60 hours. The temperature is typically between about 26.degree. C.
to about 60.degree. C., in particular about 32.degree. C. or
50.degree. C., and at about pH 3 to about pH 8, such as around pH
4-5, 6, or 7.
[0377] In a preferred aspect, the yeast and/or another
microorganism is 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 a preferred aspect, the temperature is
preferably between about 20.degree. C. to about 60.degree. C., more
preferably about 25.degree. C. to about 50.degree. C., and most
preferably about 32.degree. C. to about 50.degree. C., in
particular about 32.degree. C. or 50.degree. C., and the pH is
generally from about pH 3 to about pH 7, preferably around pH 4-7.
However, some fermenting organisms, e.g., bacteria, have higher
fermentation temperature optima. Yeast or another microorganism is
preferably applied in amounts of approximately 10.sup.5 to
10.sup.12, preferably from approximately 10.sup.7 to 10.sup.10,
especially approximately 2.times.10.sup.8 viable cell count per ml
of fermentation broth. Further guidance in respect of using yeast
for fermentation can be found in, e.g., "The Alcohol Textbook"
(Editors K. Jacques, T. P. Lyons and D. R. Kelsall, Nottingham
University Press, United Kingdom 1999), which is hereby
incorporated by reference.
[0378] For ethanol production, following the fermentation the
fermented slurry is distilled to extract the ethanol. The ethanol
obtained according to the methods of the invention can be used as,
e.g., fuel ethanol, drinking ethanol, i.e., potable neutral
spirits, or industrial ethanol.
[0379] A fermentation stimulator can be used in combination with
any of the processes described herein to further improve the
fermentation process, and in particular, the performance of the
fermenting microorganism, such as, rate enhancement and ethanol
yield. A "fermentation stimulator" refers to stimulators for growth
of the fermenting microorganisms, in particular, yeast. Preferred
fermentation stimulators for growth include vitamins and minerals.
Examples of vitamins include multivitamins, biotin, pantothenate,
nicotinic acid, meso-inositol, thiamine, pyridoxine,
para-aminobenzoic acid, folic acid, riboflavin, and Vitamins A, B,
C, D, and E. See, for example, Alfenore et al., Improving ethanol
production and viability of Saccharomyces cerevisiae by a vitamin
feeding strategy during fed-batch process, Springer-Verlag (2002),
which is hereby incorporated by reference. Examples of minerals
include minerals and mineral salts that can supply nutrients
comprising P, K, Mg, S, Ca, Fe, Zn, Mn, and Cu.
[0380] Fermentation Products:
[0381] A fermentation product can be any substance derived from the
fermentation. The fermentation product can be, without limitation,
an alcohol (e.g., arabinitol, butanol, ethanol, glycerol, methanol,
1,3-propanediol, sorbitol, and xylitol); an organic acid (e.g.,
acetic acid, acetonic acid, adipic acid, ascorbic acid, citric
acid, 2,5-diketo-D-gluconic acid, formic acid, fumaric acid,
glucaric acid, gluconic acid, glucuronic acid, glutaric acid,
3-hydroxypropionic acid, itaconic acid, lactic acid, malic acid,
malonic acid, oxalic acid, oxaloacetic acid, propionic acid,
succinic acid, and xylonic acid); a ketone (e.g., acetone); an
amino acid (e.g., aspartic acid, glutamic acid, glycine, lysine,
serine, and threonine); and a gas (e.g., methane, hydrogen
(H.sub.2), carbon dioxide (CO.sub.2), and carbon monoxide (CO)).
The fermentation product can also be protein as a high value
product.
[0382] In a preferred aspect, the fermentation product is an
alcohol. It will be understood that the term "alcohol" encompasses
a substance that contains one or more hydroxyl moieties. In a more
preferred aspect, the alcohol is arabinitol. In another more
preferred aspect, the alcohol is butanol. In another more preferred
aspect, the alcohol is ethanol. In another more preferred aspect,
the alcohol is glycerol. In another more preferred aspect, the
alcohol is methanol. In another more preferred aspect, the alcohol
is 1,3-propanediol. In another more preferred aspect, the alcohol
is sorbitol. In another more preferred aspect, the alcohol is
xylitol. See, for example, Gong, C. S., Cao, N. J., Du, J., and
Tsao, G. T., 1999, Ethanol production from renewable resources, in
Advances in Biochemical Engineering/Biotechnology, Scheper, T.,
ed., Springer-Verlag Berlin Heidelberg, Germany, 65: 207-241;
Silveira, M. M., and Jonas, R., 2002, The biotechnological
production of sorbitol, Appl. Microbiol. Biotechnol. 59: 400-408;
Nigam and Singh, 1995, Processes for fermentative production of
xylitol--a sugar substitute, Process Biochemistry 30(2): 117-124;
Ezeji, Qureshi, and Blaschek, 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.
[0383] In another preferred aspect, the fermentation product is an
organic acid. In another more preferred aspect, the organic acid is
acetic acid. In another more preferred aspect, the organic acid is
acetonic acid. In another more preferred aspect, the organic acid
is adipic acid. In another more preferred aspect, the organic acid
is ascorbic acid. In another more preferred aspect, the organic
acid is citric acid. In another more preferred aspect, the organic
acid is 2,5-diketo-D-gluconic acid. In another more preferred
aspect, the organic acid is formic acid. In another more preferred
aspect, the organic acid is fumaric acid. In another more preferred
aspect, the organic acid is glucaric acid. In another more
preferred aspect, the organic acid is gluconic acid. In another
more preferred aspect, the organic acid is glucuronic acid. In
another more preferred aspect, the organic acid is glutaric acid.
In another preferred aspect, the organic acid is 3-hydroxypropionic
acid. In another more preferred aspect, the organic acid is
itaconic acid. In another more preferred aspect, the organic acid
is lactic acid. In another more preferred aspect, the organic acid
is malic acid. In another more preferred aspect, the organic acid
is malonic acid. In another more preferred aspect, the organic acid
is oxalic acid. In another more preferred aspect, the organic acid
is propionic acid. In another more preferred aspect, the organic
acid is succinic acid. In another more preferred aspect, the
organic acid is xylonic acid. See, for example, Chen and Lee, 1997,
Membrane-mediated extractive fermentation for lactic acid
production from cellulosic biomass, Appl. Biochem. Biotechnol.
63-65: 435-448.
[0384] 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.
[0385] In another preferred aspect, the fermentation product is an
amino acid. In another more preferred aspect, the organic acid is
aspartic acid. In another more preferred aspect, the amino acid is
glutamic acid. In another more preferred aspect, the amino acid is
glycine. In another more preferred aspect, the amino acid is
lysine. In another more preferred aspect, the amino acid is serine.
In another more preferred aspect, the amino acid is threonine. See,
for example, Richard and Margaritis, 2004, Empirical modeling of
batch fermentation kinetics for poly(glutamic acid) production and
other microbial biopolymers, Biotechnology and Bioengineering
87(4): 501-515.
[0386] In another preferred aspect, the fermentation product is a
gas. In another more preferred aspect, the gas is methane. In
another more preferred aspect, the gas is H.sub.2. In another more
preferred aspect, the gas is CO.sub.2. In another more preferred
aspect, the gas is CO. See, for example, Kataoka, N., A. Miya, and
K. Kiriyama, 1997, Studies on hydrogen production by continuous
culture system of hydrogen-producing anaerobic bacteria, Water
Science and Technology 36 (6-7): 41-47; and Gunaseelan V. N. in
Biomass and Bioenergy, Vol. 13 (1-2), pp. 83-114, 1997, Anaerobic
digestion of biomass for methane production: A review.
[0387] Recovery.
[0388] The fermentation product(s) can be optionally recovered from
the fermentation medium using any method known in the art
including, but not limited to, chromatography, electrophoretic
procedures, differential solubility, distillation, or extraction.
For example, alcohol is separated from the fermented cellulosic
material and purified by conventional methods of distillation.
Ethanol with a purity of up to about 96 vol. % can be obtained,
which can be used as, for example, fuel ethanol, drinking ethanol,
i.e., potable neutral spirits, or industrial ethanol.
Signal Peptide
[0389] The present invention also relates to an isolated
polynucleotide encoding a signal peptide comprising or consisting
of amino acids 1 to 17 of SEQ ID NO:2, amino acids 1 to 17 of SEQ
ID NO:4 or amino acids 1 to 26 of SEQ ID NO:6. The polynucleotides
may further comprise a gene encoding a protein, which is operably
linked to the signal peptide and/or propeptide. The protein is
preferably foreign to the signal peptide. In one aspect, the
polynucleotide encoding the signal peptide is nucleotides 33 to 83
of SEQ ID NO:1, amino acids 81 to 131 of SEQ ID NO:3, amino acids
235 to 312 of SEQ ID NO:5.
[0390] The present invention also relates to nucleic acid
constructs, expression vectors and recombinant host cells
comprising such polynucleotides.
[0391] The present invention also relates to methods of producing a
protein, comprising: (a) cultivating a recombinant host cell
comprising such polynucleotide; and (b) recovering the protein.
[0392] 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.
[0393] Preferably, the protein is a hormone or variant thereof,
enzyme, receptor or portion thereof, antibody or portion thereof,
or reporter. For example, the protein may be a hydrolase,
isomerase, ligase, lyase, oxidoreductase, or transferase, e.g., an
aminopeptidase, amylase, carbohydrase, carboxypeptidase, catalase,
cellobiohydrolase, cellulase, chitinase, cutinase, cyclodextrin
glycosyltransferase, deoxyribonuclease, endoglucanase, esterase,
alpha-galactosidase, beta-galactosidase, glucoamylase,
alpha-glucosidase, beta-glucosidase, invertase, laccase, lipase,
mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase,
phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease,
transglutaminase, xylanase, or beta-xylosidase.
[0394] The gene may be obtained from any prokaryotic, eukaryotic,
or other source.
[0395] The present invention is further described by the following
examples that should not be construed as limiting the scope of the
invention.
EXAMPLES
Strains
[0396] The enzymes included herein are isolated from a diverse
range of microorganisms including Cerrena unicolor (SEQ ID NO:1+2),
Trichoderma reesei (SEQ ID NO:3+4) and Chaetomium globosum (SEQ ID
NO:5+6).
Media and Solutions
[0397] The reaction conditions, media and solutions provided herein
are included for inspiration and may be replaced by alternative
methods, reaction conditions and media where the skilled person
finds it applicable.
Hydrolysis Conditions
TABLE-US-00004 [0398] Conditions Total reaction volume 2 ml
Hydrolysis time 24 or 48 h Beta-glucanase composition 5 mg
protein/g DM (H. insolens) Beta-xylosidase 1 mg protein/g DM
(Trichoderma reesei) .alpha.-glucuronidase (if added) 1 mg
protein/g DM Substrate Pretreated corn fiber (140.degree. C., 150
min) Substrate loading 2.5% Buffer 50 mM Succinic acid pH 5.0
Instruments Thermomixer at 50.degree. C. and 1300 rpm
[0399] The 0.05 g pretreated corn fiber was transferred to plastic
vials. Enzymes and buffer was added and the plastic vials
containing a total reaction volume of 2 ml was placed on a
thermomixer at 50.degree. C. and 1300 rprm for 24 or 48 hours.
Determination of Arabinose and Xylose
[0400] Arabinose and xylose were determined by carbohydrate
hydrolysis using dilute hydrochloric acid. The pretreated corn
fiber was transferred to 125 ml conical flasks and diluted to
contain approximately 10% dry matter. The corn fiber sample was
preheated at 100.degree. C. in an oil bath. Hydrolysis was started
by adding 5 ml of 2 M hydrochloric acid for 2 hours at 100.degree.
C. After incubation the flasks were cooled on ice and neutralized
with 4 M sodium hydroxide. Samples were filtered with a
MINISART.RTM. 0.2 micron syringe filter (Sartorius AG, Goettingen,
Germany) and analyzed for arabinose and xylose on a DIONEX
BIOLC.RTM. System (Dionex Corporation, Sunnyvale, Calif., USA).
Determination of Glucose
[0401] Glucose concentration was determined with a DIONEX.RTM.
BIOLC.RTM. System according to the following method. Samples (10
.mu.l) were loaded onto a DIONEX BIOLC.RTM. System equipped with a
DIONEX.RTM. CARBOPAC.TM. PA1 analytical column (4.times.250 mm)
(Dionex Corporation, Sunnyvale, Calif., USA) combined with a
CARBOPAC.TM. PA1 guard column (4.times.50 mm) (Dionex Corporation,
Sunnyvale, Calif., USA). The monosaccharides were separated
isocratically with 10 mM potassium hydroxide at a flow rate of 1 ml
per minute and detected by a pulsed electrochemical detector in the
pulsed amperiometric detection mode. The potential of the electrode
was programmed for +0.1 volt (t=0-0.4 second) to -2.0 volt
(t=0.41-0.42 second) to 0.6 volt (t=0.43 second) and finally -0.1
volt (t=0.44-0.50 second), while integrating the resulting signal
from t=0.2-0.4 second.
Determination of Glucuronic Acid
[0402] Glucuronic acid concentration was determined with a
DIONEX.RTM. ICS3000.RTM. System according to the following method.
Samples (10 .mu.l) were loaded onto a DIONEX ICS3000.RTM. System
equipped with a DIONEX.RTM. CARBOPAC.TM. PA1 analytical column
(4.times.250 mm) (Dionex Corporation, Sunnyvale, Calif., USA)
combined with a CARBOPAC.TM. PA1 guard column (4.times.50 mm)
(Dionex Corporation, Sunnyvale, Calif., USA). Glucuronic acid was
separated isocratically with 101 mM sodium hydroxide and 160 mM
sodium acetate at a flow rate of 1 ml per minute and detected by a
pulsed electrochemical detector in the pulsed amperiometric
detection mode. The potential of the electrode was programmed for
+0.1 volt (t=0-0.4 second) to -2.0 volt (t=0.41-0.42 second) to 0.6
volt (t=0.43 second) and finally -0.1 volt (t=0.44-0.50 second),
while integrating the resulting signal from t=0.2-0.4 second. Pure
glucuronic acid dissolved in deionised water was used as a
standard. Standards of the following concentration were used: 5,
10, 25, 50, 100, 250 and 500 .mu.g/ml were used to determine the
concentration of glucuronic acid in the hydrolysed samples.
Example 1
Effect of Glucuronyl Esterase on Hydrolysis of Pretreated Corn
Fiber
[0403] FIG. 1 shows the conversion of pretreated corn fiber after
hydrolysis for 48 hours with and without addition of glucuronyl
esterase.
[0404] As apparent from FIG. 1, addition of glucuronyl esterase to
hydrolysis mixtures comprising .beta.-glucanase and
.beta.-xylosidase enhances the total hydrolysis.
[0405] The effect of glucuronyl esterase on hydrolysis of
pretreated corn fiber was evaluated. Corn fiber is a fraction from
the wet milling of corn kernels. Corn fiber is the seed coat and
residual endosperm left after starch is removed and further
processed. Corn fiber was pretreated by autoclaving at 140.degree.
C. for 150 minutes. The amount of theoretical arabinose, glucose
and xylose in the substrate was determined to be 114, 302, and 204
g per kg dry matter using the following methods.
[0406] Arabinose and xylose were determined by carbohydrate
hydrolysis using dilute hydrochloric acid. The pretreated corn
fiber was transferred to 125 ml conical flasks and diluted to
contain approximately 10% dry matter. The corn fiber sample was
preheated at 100.degree. C. in an oil bath. Hydrolysis was started
by adding 5 ml of 2 M hydrochloric acid for 2 hours at 100.degree.
C. After incubation the flasks were cooled on ice and neutralized
with 4 M sodium hydroxide. Samples were filtered with a
MINISART.RTM. 0.2 micron syringe filter (Sartorius AG, Goettingen,
Germany) and analyzed for arabinose and xylose on a DIONEX
BIOLC.RTM. System (Dionex Corporation, Sunnyvale, Calif., USA).
[0407] The hydrolysis of the pretreated corn fiber was conducted
with a H. insolens beta-glucanase composition and a Trichoderma
reesei beta-xylosidase. The Trichoderma reesei beta-xylosidase was
obtained recombinantly by expression in Aspergillus oryzae as
described in Rasmussen et al., 2006, Biotechnology and
Bioengineering 94: 869-876 using standard cultivation methods for
Aspergillus oryzae.
[0408] The hydrolysis of the pretreated corn fiber was performed in
2 ml EPPENDORF.RTM. tubes (Eppendorf AG, Germany) at a temperature
of 50.degree. C. and a pH of 5.0 in 50 mM succinic acid. Samples
were incubated in a THERMOMIXER.RTM. Comfort (Eppendorf AG,
Germany) that subjected each sample with constant heating and
mixing. The substrate amount used was 2.5 w/w % in a total sample
volume of 2 ml. The Chaetomium globosum glucuronyl esterase was
added at an enzyme loading of 1 mg enzyme per g of dry matter on
top of both the H. insolens beta-glucanase composition and the
Trichoderma reesei beta-xylosidase. H. insolens beta-glucanase
composition was added at a loading of 5 mg enzyme per g of dry
matter and the Trichoderma reesei beta-xylosidase at a loading of 1
mg enzyme per g of dry matter. Hydrolysis was terminated after 48
hours by heating the samples for 10 minutes at 100.degree. C. in a
heat block (Techne Inc., Burlington N.J., USA).
[0409] Conversion was calculated by determining the amount of
sugars released from the substrate as a percentage of what was
added from the start using the formula below but not including
initial monomeric sugars. T-tests were performed with a two tailed
distribution and equal variance of sample data.
Conversion (%)=(Sugar amount in hydrolysate/Sugar amount in added
substrate).times.100
[0410] Comparing the conversion of pretreated corn fiber when
adding the Chaetomium globosum glucuronyl esterase at an enzyme
loading of 1 mg of enzyme per gram dry matter together with 1 mg
enzyme per g of dry matter of Trichoderma reesei beta-xylosidase
and 5 mg enzyme per g of dry matter of H. insolens beta-glucanase
composition to just adding 1 mg enzyme per g of dry matter of
beta-xylosidase from Trichoderma reesei and 5 mg enzyme per g of
dry matter of H. insolens beta-glucanase composition demonstrated a
significant (P 0.0323) increase in conversion from 44.1 to 46.1
(Table 1).
TABLE-US-00005 TABLE 1 Standard Samples Mean Conversion deviation
T-test H. insolens beta-glucanase 44.1 1.0 0.0323 composition and
Trichoderma reesei beta-xylosidase H. insolens beta-glucanase 46.1
0.3 composition, Trichoderma reesei beta-xylosidase, and Chaetomium
globosum glucuronyl esterase
Example 2
[0411] In a further aspect, the invention relates to the enhanced
release of glucuronic acid of pretreated corn fiber after
hydrolysis with addition of glucuronyl esterase. As shown in FIG.
2, addition of glucuronyl esterase stimulates the release of
glucuronic acid during hydrolysis of pretreated corn fiber.
[0412] Comparing the release of glucuronic acid from pretreated
corn fiber when adding the Chaetomium globosum glucuronyl esterase
at an enzyme loading of 1 mg of enzyme per gram dry matter together
with 1 mg enzyme per g of dry matter of Trichoderma reesei
beta-xylosidase and 5 mg enzyme per g of dry matter of H. insolens
beta-glucanase composition to just adding 1 mg enzyme per g of dry
matter of beta-xylosidase from Trichoderma reesei and 5 mg enzyme
per g of dry matter of H. insolens beta-glucanase composition
demonstrated a significant (P 0.0449) increase in glucuronic acid
release from 4.0 to 5.3 g/kg DM (Table 2).
TABLE-US-00006 TABLE 2 Mean release Standard Samples (g/kg DM)
deviation T-test H. insolens beta-glucanase 4.0 0.2 0.0449
composition and Trichoderma reesei beta-xylosidase H. insolens
beta-glucanase 5.3 0.7 composition, Trichoderma reesei
beta-xylosidase, and Chaetomium globosum glucuronyl esterase
Aspects
[0413] Further, the present invention relates to the following
aspects:
Aspect 1. An isolated polypeptide having glucuronyl esterase
activity, selected from the group consisting of:
[0414] (a) a polypeptide having at least 80%, at least 85%, at
least 90%, at least 91%, at least 92%, at least 93%, at least 94%,
at least 95%, at least 96%, at least 97%, at least 98%, at least
99%, or 100% sequence identity to the mature polypeptide of SEQ ID
NO:2; or
at least 95%, at least 96%, at least 97%, at least 98%, at least
99%, or 100% sequence identity to the mature polypeptide of SEQ ID
NO:4 or 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;
[0415] (b) a polypeptide encoded by a polynucleotide that
hybridizes under high stringency conditions, or very high
stringency conditions with (i) the mature polypeptide coding
sequence of SEQ ID NO:1, (ii) the cDNA sequence thereof, or (iii)
the full-length complement of (i) or (ii); or
[0416] or under very high stringency conditions with (iv) the
mature polypeptide coding sequence of SEQ ID NO:3, (v) the cDNA
sequence thereof, or (vi) the full-length complement of (iv) or
(v);
[0417] or under very high stringency conditions with (vii) the
mature polypeptide coding sequence of SEQ ID NO:5, (viii) the cDNA
sequence thereof, or (ix) the full-length complement of (vii) or
(viii);
[0418] or under medium stringency conditions, medium-high
stringency conditions, high stringency conditions, or very high
stringency conditions with (x) the mature polypeptide coding
sequence of SEQ ID NO:7, (xi) the cDNA sequence thereof, or (xii)
the full-length complement of (x) or (xi);
[0419] (c) a polypeptide encoded by a polynucleotide having at
least 80%, at least 85%, at least 85%, at least 90%, at least 91%,
at least 92%, at least 93%, at least 94%, at least 95%, at least
96%, at least 97%, at least 98%, at least 99%, or 100% sequence
identity to the mature polypeptide coding sequence of SEQ ID NO:1
or the cDNA sequence thereof or
[0420] having 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 or
[0421] having at least 85%, at least 90%, at least 91%, at least
92%, at least 93%, at least 94%, at least 95%, at least 96%, at
least 97%, at least 98%, at least 99%, or 100% sequence identity to
the mature polypeptide coding sequence of SEQ ID NO:5 or the cDNA
sequence thereof or
[0422] having at least 80%, at least 85%, at least 85%, at least
90%, at least 91%, at least 92%, at least 93%, at least 94%, at
least 95%, at least 96%, at least 97%, at least 98%, at least 99%,
or 100% sequence identity to the mature polypeptide coding sequence
of SEQ ID NO:7 or the cDNA sequence thereof.
[0423] (d) a variant of the mature polypeptide of SEQ ID NO:2, SEQ
ID NO:4 or SEQ ID NO:6 comprising a substitution, deletion, and/or
insertion at one or more positions; and
[0424] (e) a fragment of the polypeptide of (a), (b), (c) or (d)
that has glucuronyl esterase activity.
Aspect 2. The polypeptide of aspect 1, comprising or consisting one
of the sequences SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6. Aspect 3.
The polypeptide of any of aspects 1-2, comprising or consisting of
the mature polypeptide of one of the sequences SEQ ID NO:2, SEQ ID
NO:4 or SEQ ID NO:6. Aspect 4. The polypeptide of aspect 3, wherein
the mature polypeptide is amino acids 101 to 474 of SEQ ID NO:2, 94
to 460 of SEQ ID NO:4 or 21 to 392 of SEQ ID NO:6. Aspect 5. The
polypeptide of any of aspects 1-4, which is a fragment of SEQ ID
NO:2, SEQ ID NO:4 or SEQ ID NO:6 wherein the fragment has
glucuronyl esterase activity. Aspect 6. A composition comprising
the polypeptide of any of aspects 1-5. Aspect 7. An isolated
polynucleotide encoding the polypeptide of any of aspects 1-5.
Aspect 8. A nucleic acid construct or expression vector comprising
the polynucleotide of aspect 7 operably linked to one or more
control sequences that direct the production of the polypeptide in
an expression host. Aspect 9. A recombinant host cell comprising
the polynucleotide of aspect 7 operably linked to one or more
control sequences that direct the production of the polypeptide.
Aspect 10. A method of producing the polypeptide of any of aspects
1-5, comprising:
[0425] (a) cultivating a cell, which in its wild-type form produces
the polypeptide, under conditions conducive for production of the
polypeptide; and
[0426] (b) recovering the polypeptide.
Aspect 11. A method of producing a polypeptide having glucuronyl
esterase activity, comprising:
[0427] (a) cultivating the host cell of aspect 9 under conditions
conducive for production of the polypeptide; and
[0428] (b) recovering the polypeptide.
Aspect 12. A transgenic plant, plant part or plant cell transformed
with a polynucleotide encoding the polypeptide of any of aspects
1-5. Aspect 13. A method of producing a polypeptide having
glucuronyl esterase activity, comprising:
[0429] (a) cultivating the transgenic plant or plant cell of aspect
12 under conditions conducive for production of the polypeptide;
and
[0430] (b) recovering the polypeptide.
Aspect 14. A method of producing a mutant of a parent cell,
comprising inactivating a polynucleotide encoding the polypeptide
of any of aspects 1-5, which results in the mutant producing less
of the polypeptide than the parent cell. Aspect 15. A mutant cell
produced by the method of aspect 14. Aspect 16. The mutant cell of
aspect 15, further comprising a gene encoding a native or
heterologous protein. Aspect 17. A method of producing a protein,
comprising:
[0431] (a) cultivating the mutant cell of aspect 15 or 16 under
conditions conducive for production of the protein; and
[0432] (b) recovering the protein.
Aspect 18. A double-stranded inhibitory RNA (dsRNA) molecule
comprising a subsequence of the polynucleotide of aspect 7, wherein
optionally the dsRNA is an siRNA or an miRNA molecule. Aspect 19.
The double-stranded inhibitory RNA (dsRNA) molecule of aspect 18,
which is about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more
duplex nucleotides in length. Aspect 20. A method of inhibiting the
expression of a polypeptide having glucuronyl esterase activity in
a cell, comprising administering to the cell or expressing in the
cell the double-stranded inhibitory RNA (dsRNA) molecule of aspect
18 or 19. Aspect 21. The method of aspect 20, wherein the dsRNA is
about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more duplex
nucleotides in length. Aspect 22. A cell produced by the method of
aspect 20 or 21. Aspect 23. The cell of aspect 22, further
comprising a gene encoding a native or heterologous protein. Aspect
24. A method of producing a protein, comprising:
[0433] (a) cultivating the cell of aspect 22 or 23 under conditions
conducive for production of the protein; and
[0434] (b) recovering the protein.
Aspect 25. An isolated polynucleotide encoding a signal peptide
comprising or consisting of amino acids 1 to 17 of SEQ ID NO:2 or
amino acids 1 to 17 of SEQ ID NO:4 or amino acids 1 to 20 of SEQ ID
NO:6. Aspect 26. A nucleic acid construct or expression vector
comprising a gene encoding a protein operably linked to the
polynucleotide of aspect 25, wherein the gene is foreign to the
polynucleotide encoding the signal peptide. Aspect 27. A
recombinant host cell comprising a gene encoding a protein operably
linked to the polynucleotide of aspect 25, wherein the gene is
foreign to the polynucleotide encoding the signal peptide. Aspect
28. A method of producing a protein, comprising:
[0435] (a) cultivating a recombinant host cell comprising a gene
encoding a protein operably linked to the polynucleotide of aspect
24 or 25, wherein the gene is foreign to the polynucleotide
encoding the signal peptide, under conditions conducive for
production of the protein; and
[0436] (b) recovering the protein.
Aspect 29. A method for degrading or converting a cellulosic
material, comprising: treating the cellulosic material with an
enzyme composition in the presence of the polypeptide having
glucuronyl esterase activity of any of aspects 1-5. Aspect 30. The
method of aspect 29, wherein the cellulosic material is pretreated.
Aspect 31. The method of aspect 29 or 30, wherein the enzyme
composition comprises one or more enzymes selected from the group
consisting of a cellulase, a polypeptide having cellulolytic
enhancing activity, a hemicellulase, an esterase, a protease, a
laccase, or a peroxidase. Aspect 32. The method of aspect 31,
wherein the cellulase is one or more enzymes selected from the
group consisting of an endoglucanase, a cellobiohydrolase, and a
beta-glucosidase. Aspect 33. The method of aspect 31, wherein the
hemicellulase is one or more enzymes selected from the group
consisting of a xylanase, an acetyxylan esterase, a feruloyl
esterase, an arabinofuranosidase, a xylosidase, and a
glucuronidase. Aspect 34. The method of any of aspects 29-33,
further comprising recovering the degraded cellulosic material.
Aspect 35. The method of aspect 34, wherein the degraded cellulosic
material is a sugar. Aspect 36. The method of aspect 35, wherein
the sugar is selected from the group consisting of glucose, xylose,
mannose, galactose, and arabinose. Aspect 37. A method for
producing a fermentation product, comprising:
[0437] (a) saccharifying a cellulosic material with an enzyme
composition in the presence of the polypeptide having glucuronyl
esterase activity of any of aspects 1-5;
[0438] (b) fermenting the saccharified cellulosic material with one
or more fermenting microorganisms to produce the fermentation
product; and
[0439] (c) recovering the fermentation product from the
fermentation.
Aspect 38. The method of aspect 37, wherein the cellulosic material
is pretreated. Aspect 39. The method of aspect 37 or 38, wherein
the enzyme composition comprises one or more enzymes selected from
the group consisting of a cellulase, a polypeptide having
cellulolytic enhancing activity, a hemicellulase, an esterase, a
protease, a laccase, or a peroxidase. Aspect 40. The method of
aspect 39, wherein the cellulase is one or more enzymes selected
from the group consisting of an endoglucanase, a cellobiohydrolase,
and a beta-glucosidase. Aspect 41. The method of aspect 39, wherein
the hemicellulase is one or more enzymes selected from the group
consisting of a xylanase, an acetyxylan esterase, a feruloyl
esterase, an arabinofuranosidase, a xylosidase, and a
glucuronidase. Aspect 42. The method of any of aspects 37-41,
wherein steps (a) and (b) are performed simultaneously in a
simultaneous saccharification and fermentation. Aspect 43. The
method of any of aspects 37-42, wherein the fermentation product is
an alcohol, an organic acid, a ketone, an amino acid, or a gas.
Aspect 44. A method of fermenting a cellulosic material,
comprising: fermenting the cellulosic material with one or more
fermenting microorganisms, wherein the cellulosic material is
saccharified with an enzyme composition in the presence of a
polypeptide having glucuronyl esterase activity of any of aspects
1-5. Aspect 45. The method of aspect 44, wherein the fermenting of
the cellulosic material produces a fermentation product. Aspect 46.
The method of aspect 45, further comprising recovering the
fermentation product from the fermentation. Aspect 47. The method
of any of aspects 44-46, wherein the cellulosic material is
pretreated before saccharification. Aspect 48. The method of any of
aspects 44-47, wherein the enzyme composition comprises one or more
enzymes selected from the group consisting of a cellulase, a
polypeptide having cellulolytic enhancing activity, a
hemicellulase, an esterase, a protease, a laccase, or a peroxidase.
Aspect 49. The method of aspect 48, wherein the cellulase is one or
more enzymes selected from the group consisting of an
endoglucanase, a cellobiohydrolase, and a beta-glucosidase. Aspect
50. The method of aspect 48, wherein the hemicellulase is one or
more enzymes selected from the group consisting of a xylanase, an
acetyxylan esterase, a feruloyl esterase, an arabinofuranosidase, a
xylosidase, and a glucuronidase. Aspect 51. The method of any of
aspects 45-50, wherein the fermentation product is an alcohol, an
organic acid, a ketone, an amino acid, or a gas.
Sequence CWU 1
1
1411580DNACerrena unicolor 1cttctttgta ggctaaccgt cagatcaaca
aaatgttcaa gccatctttc gtagctctcg 60cgctcgtctc gtacgcgact gctcaggctt
ctgctcctca atggggtcag tgtggtggca 120taggatggac cggacctact
gcatgtccat caggctgggc atgtcagcaa cttaacgcgt 180actactcgca
gtgtctccag ggagccgcac ctgcacctgc acgtaccaca gctgcccctc
240ctccccctcc tgctactact gccgcgcccc ctccacccac cacatccgcg
ccgaccggta 300gttctcccgt agctggagca tgcggtgcca ttgcttccac
cgtccccaat tacaacaacg 360cgaagttgcc cgatccattc acttttgcca
acggtactgc acttcgcaca aaggctgact 420ggtcatgtcg tcgtgcagag
atcagtgctt tgatccagaa ctacgaagct ggaactctcc 480ctcccaagcc
gcctgtcgtc actgcttcgt tctcgaagtc tggcaacaca ggtactttgg
540ccattactgc tggtcttagc aacagccaga ctatcaaatt ctctccgaca
atttcatacc 600ctagcggtac tcctccggcc aatggctggc cactgatcat
tgcgtacgag ggtggtagca 660ttcccattcc cgccggggtc gcgacattga
cctatagcaa cagcgacatg gctcaacaaa 720acagcgcctc cagcagaggc
cagggtctct tctaccagct ctacggtagc acgcacagtg 780ctagtgccat
gactgcctgg gtgtggggtg tcagccgtat catcgacgct ttggagatga
840caccgactgc acaaatcaac acccagcgga tcggcgttac gggttgctcc
cgtgacggca 900agggtgctct tatggccggt gcctttgagg agcgtatcgc
tttgaccatc cctcaagagt 960ccggctccgg aggtgatgct tgctggaggt
tgtcgaagta tgagatcgat aacggcaacc 1020aagtgcagga cgcagtcgag
atcgtcggcg aaaacgtttg gttctcgacc aatttcaaca 1080actacgttca
gaaactcccc actgtgcccg aagaccacca tctcctcgct gccatggtcg
1140caccccgggc gatgatctca ttcgagaaca ccgattactt gtggttgagc
cccatgagca 1200gcttcgggtg catgactgcc gcacataccg tctggcaggg
tctcggcatt gccgactcgc 1260acggtttcgc ccaagtcggt ggtcacgctc
actgtgcatg gccgtccagc cttactcctc 1320aactcaatgc tttcatcaac
cgattcttac tcgatcaaag tgcgactaca aacgtcttca 1380caaccaacaa
ccagtttggt aaggttcagt ggaacgctgc gaactggatc acctggacca
1440ctcccacttt gacctgattg aggccccggg tggtgtgtgg ctagtagtgg
gaactaatag 1500ttgacattgt atttaccatt ccattccata cttgcgtact
agttgaagca cgcgtattct 1560tcatatggcg ttggtctgat 15802474PRTCerrena
unicolor 2Met Phe Lys Pro Ser Phe Val Ala Leu Ala Leu Val Ser Tyr
Ala Thr 1 5 10 15 Ala Gln Ala Ser Ala Pro Gln Trp Gly Gln Cys Gly
Gly Ile Gly Trp 20 25 30 Thr Gly Pro Thr Ala Cys Pro Ser Gly Trp
Ala Cys Gln Gln Leu Asn 35 40 45 Ala Tyr Tyr Ser Gln Cys Leu Gln
Gly Ala Ala Pro Ala Pro Ala Arg 50 55 60 Thr Thr Ala Ala Pro Pro
Pro Pro Pro Ala Thr Thr Ala Ala Pro Pro 65 70 75 80 Pro Pro Thr Thr
Ser Ala Pro Thr Gly Ser Ser Pro Val Ala Gly Ala 85 90 95 Cys Gly
Ala Ile Ala Ser Thr Val Pro Asn Tyr Asn Asn Ala Lys Leu 100 105 110
Pro Asp Pro Phe Thr Phe Ala Asn Gly Thr Ala Leu Arg Thr Lys Ala 115
120 125 Asp Trp Ser Cys Arg Arg Ala Glu Ile Ser Ala Leu Ile Gln Asn
Tyr 130 135 140 Glu Ala Gly Thr Leu Pro Pro Lys Pro Pro Val Val Thr
Ala Ser Phe 145 150 155 160 Ser Lys Ser Gly Asn Thr Gly Thr Leu Ala
Ile Thr Ala Gly Leu Ser 165 170 175 Asn Ser Gln Thr Ile Lys Phe Ser
Pro Thr Ile Ser Tyr Pro Ser Gly 180 185 190 Thr Pro Pro Ala Asn Gly
Trp Pro Leu Ile Ile Ala Tyr Glu Gly Gly 195 200 205 Ser Ile Pro Ile
Pro Ala Gly Val Ala Thr Leu Thr Tyr Ser Asn Ser 210 215 220 Asp Met
Ala Gln Gln Asn Ser Ala Ser Ser Arg Gly Gln Gly Leu Phe 225 230 235
240 Tyr Gln Leu Tyr Gly Ser Thr His Ser Ala Ser Ala Met Thr Ala Trp
245 250 255 Val Trp Gly Val Ser Arg Ile Ile Asp Ala Leu Glu Met Thr
Pro Thr 260 265 270 Ala Gln Ile Asn Thr Gln Arg Ile Gly Val Thr Gly
Cys Ser Arg Asp 275 280 285 Gly Lys Gly Ala Leu Met Ala Gly Ala Phe
Glu Glu Arg Ile Ala Leu 290 295 300 Thr Ile Pro Gln Glu Ser Gly Ser
Gly Gly Asp Ala Cys Trp Arg Leu 305 310 315 320 Ser Lys Tyr Glu Ile
Asp Asn Gly Asn Gln Val Gln Asp Ala Val Glu 325 330 335 Ile Val Gly
Glu Asn Val Trp Phe Ser Thr Asn Phe Asn Asn Tyr Val 340 345 350 Gln
Lys Leu Pro Thr Val Pro Glu Asp His His Leu Leu Ala Ala Met 355 360
365 Val Ala Pro Arg Ala Met Ile Ser Phe Glu Asn Thr Asp Tyr Leu Trp
370 375 380 Leu Ser Pro Met Ser Ser Phe Gly Cys Met Thr Ala Ala His
Thr Val 385 390 395 400 Trp Gln Gly Leu Gly Ile Ala Asp Ser His Gly
Phe Ala Gln Val Gly 405 410 415 Gly His Ala His Cys Ala Trp Pro Ser
Ser Leu Thr Pro Gln Leu Asn 420 425 430 Ala Phe Ile Asn Arg Phe Leu
Leu Asp Gln Ser Ala Thr Thr Asn Val 435 440 445 Phe Thr Thr Asn Asn
Gln Phe Gly Lys Val Gln Trp Asn Ala Ala Asn 450 455 460 Trp Ile Thr
Trp Thr Thr Pro Thr Leu Thr 465 470 31563DNAHypocrea jecorina
3atagagcagc gctcatagaa actagatgct agcaccttag caaagccgaa gtcgatattt
60cgttgttcag aagtaacaag atggcttccc gcttctttgc tcttctcctt ttagcgatcc
120caatccaggc ccaatctcca gtctggggac aatgtggtgg aattggttgg
tctggcccaa 180caacttgtgt tggaggtgcg acttgtgtat catataaccc
ttattactcg caatgtattc 240ccagtacaca ggcttcatcg agcatagcct
ctacaacgct ggtcacatca tttacgacca 300ccactgctac gaggacttcg
gcatcaacgc ctccagcgag cagtacaggt gcaggcggcg 360caacatgctc
agcactgccg ggctccatta ccctgagatc caacgcaaag ctcaacgatc
420tgtttacaat gttcaatgga gataaggtca ccacgaaaga caaattctcg
tgccgccagg 480cagagatgtc ggagctaata caacgatatg agctcggcac
cctgcccgga cgaccaagca 540ctctcacagc ctcattctcg ggcaatacgt
tgaccatcaa ttgcggagag gccggaaagt 600caatttcatt cacagtcacg
atcacttatc catcttccgg aacagcacca taccctgcga 660ttatcggcta
tggaggcggc agtcttccag ctcccgccgg ggttgccatg atcaacttta
720acaatgacaa catagcagcc caagttaata caggcagccg cggacagggc
aagttctacg 780atctctacgg gagctcgcac tccgcgggcg ccatgaccgc
atgggcctgg ggagtaagcc 840gagtcattga tgctcttgag cttgtaccag
gcgcaagaat agacaccacc aagattggcg 900tgacggggtg ttcacgaaat
ggcaaaggcg caatggtcgc aggtgctttc gagaaacgaa 960tcgttctgac
acttccccag gagtcgggcg ccggtggctc tgcgtgctgg aggatttcag
1020actacttaaa gtcccaagga gccaatatcc agaccgcgtc tgagatcatt
ggcgaagacc 1080cctggttctc gactactttc aacagctacg tcaaccaagt
gccggtgttg ccgtttgacc 1140accattcgct tgctgccttg atagccccga
gaggattatt cgtcatcgac aacaatattg 1200actggctcgg cccacaaagc
tgctttggct gtatgacagc tgctcacatg gcatggcaag 1260ctttgggtgt
ctcggaccac atgggctatt cgcagattgg agctcacgca cactgcgcgt
1320tcccatcaaa ccagcaatcg caacttactg cctttgttca gaaattcttg
ctgggccagt 1380ccacaaatac ggcgattttc caaagcgact tttcggccaa
tcaaagccaa tggatcgact 1440ggacaacccc aacgctgagt tgagtcttac
ggccagggaa acgcgcatat ttggcgattg 1500gcggttcctg tattatgact
tggtaaccca agccatacca agcttagcag agggtgttga 1560aag
15634460PRTHypocrea jecorina 4Met Ala Ser Arg Phe Phe Ala Leu Leu
Leu Leu Ala Ile Pro Ile Gln 1 5 10 15 Ala Gln Ser Pro Val Trp Gly
Gln Cys Gly Gly Ile Gly Trp Ser Gly 20 25 30 Pro Thr Thr Cys Val
Gly Gly Ala Thr Cys Val Ser Tyr Asn Pro Tyr 35 40 45 Tyr Ser Gln
Cys Ile Pro Ser Thr Gln Ala Ser Ser Ser Ile Ala Ser 50 55 60 Thr
Thr Leu Val Thr Ser Phe Thr Thr Thr Thr Ala Thr Arg Thr Ser 65 70
75 80 Ala Ser Thr Pro Pro Ala Ser Ser Thr Gly Ala Gly Gly Ala Thr
Cys 85 90 95 Ser Ala Leu Pro Gly Ser Ile Thr Leu Arg Ser Asn Ala
Lys Leu Asn 100 105 110 Asp Leu Phe Thr Met Phe Asn Gly Asp Lys Val
Thr Thr Lys Asp Lys 115 120 125 Phe Ser Cys Arg Gln Ala Glu Met Ser
Glu Leu Ile Gln Arg Tyr Glu 130 135 140 Leu Gly Thr Leu Pro Gly Arg
Pro Ser Thr Leu Thr Ala Ser Phe Ser 145 150 155 160 Gly Asn Thr Leu
Thr Ile Asn Cys Gly Glu Ala Gly Lys Ser Ile Ser 165 170 175 Phe Thr
Val Thr Ile Thr Tyr Pro Ser Ser Gly Thr Ala Pro Tyr Pro 180 185 190
Ala Ile Ile Gly Tyr Gly Gly Gly Ser Leu Pro Ala Pro Ala Gly Val 195
200 205 Ala Met Ile Asn Phe Asn Asn Asp Asn Ile Ala Ala Gln Val Asn
Thr 210 215 220 Gly Ser Arg Gly Gln Gly Lys Phe Tyr Asp Leu Tyr Gly
Ser Ser His 225 230 235 240 Ser Ala Gly Ala Met Thr Ala Trp Ala Trp
Gly Val Ser Arg Val Ile 245 250 255 Asp Ala Leu Glu Leu Val Pro Gly
Ala Arg Ile Asp Thr Thr Lys Ile 260 265 270 Gly Val Thr Gly Cys Ser
Arg Asn Gly Lys Gly Ala Met Val Ala Gly 275 280 285 Ala Phe Glu Lys
Arg Ile Val Leu Thr Leu Pro Gln Glu Ser Gly Ala 290 295 300 Gly Gly
Ser Ala Cys Trp Arg Ile Ser Asp Tyr Leu Lys Ser Gln Gly 305 310 315
320 Ala Asn Ile Gln Thr Ala Ser Glu Ile Ile Gly Glu Asp Pro Trp Phe
325 330 335 Ser Thr Thr Phe Asn Ser Tyr Val Asn Gln Val Pro Val Leu
Pro Phe 340 345 350 Asp His His Ser Leu Ala Ala Leu Ile Ala Pro Arg
Gly Leu Phe Val 355 360 365 Ile Asp Asn Asn Ile Asp Trp Leu Gly Pro
Gln Ser Cys Phe Gly Cys 370 375 380 Met Thr Ala Ala His Met Ala Trp
Gln Ala Leu Gly Val Ser Asp His 385 390 395 400 Met Gly Tyr Ser Gln
Ile Gly Ala His Ala His Cys Ala Phe Pro Ser 405 410 415 Asn Gln Gln
Ser Gln Leu Thr Ala Phe Val Gln Lys Phe Leu Leu Gly 420 425 430 Gln
Ser Thr Asn Thr Ala Ile Phe Gln Ser Asp Phe Ser Ala Asn Gln 435 440
445 Ser Gln Trp Ile Asp Trp Thr Thr Pro Thr Leu Ser 450 455 460
51491DNAChaetomium globosum 5atgaccgaag gagctaccct catttacact
tccaatccct tcgtaggcgg ttgtccgaca 60gaaacactgc ccgggcgacg ggatgcgaac
atctcggggg ttattctagc gattgacggc 120ctcactcggg cgaatggccc
ccgggcctcg gtggtgatat accccgacag gccctcgccg 180tggttctttg
ccttgagcaa acaaagccgc ctagacagat cgtcgacgga aacaatgcgt
240tcccttctac acacgctcgc cgcggcagcg atcggcagcg ccggcgccga
cgcccacccc 300ctgatccccc ggcagggcgg cggcaacaac acaatccaat
gcccccccac cccctcgccg 360ttcccgacct ggcagcagct cccgctgcag
tcgtctctgc ccgatccttt cctgccactg 420caatacacca cgcccggcga
tgcggcggac gtggtggcgg gccgcggcga gggccgggtg 480aagacgcccg
aggagtggta ccagtgccgg cagcccgaga tcctgcacat gctgcaggag
540taccagtacg gctactaccc ggaccacggc caggagacgg tgcaggccac
gcgcagcggc 600aacacgctga gcatcaccgt ggcggccggc ggcaagacgg
gccggttcag cgcgaccgtc 660acgctgccgt cgggggcgtc cgcgtctaag
cccgcccccg tggtcatcaa catcggtggc 720atgcagaacc aggcttatct
gagtgcgggc attgccgtcg cgcagtttga ttacacctcg 780gtggcgcccg
atagcaatgc gaagacgggg gcgttctgga gcatctacaa cgggagagac
840atcggtgtgt tgacggcctg ggcgtggggc ttccaccgca cgctcgacgc
tattaacatg 900acggtgctcg agatcgacgc cgggcgggtg ggcgtgacgg
ggtgttccag gctaggaaaa 960gcggcgctcg cggcggggct cttcgacacc
cgcatcacgc tcacgatgcc catgtcgtcg 1020ggggtgcagg gcatgggccc
gtaccggtac tacagcatga gcgggcaggg cgagaacctc 1080gagaacagca
agcagggggc cgggtggtgg accagcagca agctaggggc gtttatcaac
1140cactccgaga acctgccgta cgacgcgcac accatcgcgg cggccatcgc
gccgagggcg 1200ctagtcattg accaagggac gggtgaccag tttgtcaaca
gcaagggcac cgccgtcgtc 1260atctacccgg cggcgaaagt ggtgtacgac
tggctgggtg cgggtgacaa gatcgccatc 1320agcgtgcgtg ggggcgggca
ttgtgatatg agcggattca catccatcct gccgtatgtg 1380caaaagatct
tctttggtac accgacgagc aaggactata acaatttggg atcctacggg
1440tcgcctgtga cgaccgcctt cccatggggg acggctgttc ccaaggcatg a
14916496PRTChaetomium globosum 6Met Thr Glu Gly Ala Thr Leu Ile Tyr
Thr Ser Asn Pro Phe Val Gly 1 5 10 15 Gly Cys Pro Thr Glu Thr Leu
Pro Gly Arg Arg Asp Ala Asn Ile Ser 20 25 30 Gly Val Ile Leu Ala
Ile Asp Gly Leu Thr Arg Ala Asn Gly Pro Arg 35 40 45 Ala Ser Val
Val Ile Tyr Pro Asp Arg Pro Ser Pro Trp Phe Phe Ala 50 55 60 Leu
Ser Lys Gln Ser Arg Leu Asp Arg Ser Ser Thr Glu Thr Met Arg 65 70
75 80 Ser Leu Leu His Thr Leu Ala Ala Ala Ala Ile Gly Ser Ala Gly
Ala 85 90 95 Asp Ala His Pro Leu Ile Pro Arg Gln Gly Gly Gly Asn
Asn Thr Ile 100 105 110 Gln Cys Pro Pro Thr Pro Ser Pro Phe Pro Thr
Trp Gln Gln Leu Pro 115 120 125 Leu Gln Ser Ser Leu Pro Asp Pro Phe
Leu Pro Leu Gln Tyr Thr Thr 130 135 140 Pro Gly Asp Ala Ala Asp Val
Val Ala Gly Arg Gly Glu Gly Arg Val 145 150 155 160 Lys Thr Pro Glu
Glu Trp Tyr Gln Cys Arg Gln Pro Glu Ile Leu His 165 170 175 Met Leu
Gln Glu Tyr Gln Tyr Gly Tyr Tyr Pro Asp His Gly Gln Glu 180 185 190
Thr Val Gln Ala Thr Arg Ser Gly Asn Thr Leu Ser Ile Thr Val Ala 195
200 205 Ala Gly Gly Lys Thr Gly Arg Phe Ser Ala Thr Val Thr Leu Pro
Ser 210 215 220 Gly Ala Ser Ala Ser Lys Pro Ala Pro Val Val Ile Asn
Ile Gly Gly 225 230 235 240 Met Gln Asn Gln Ala Tyr Leu Ser Ala Gly
Ile Ala Val Ala Gln Phe 245 250 255 Asp Tyr Thr Ser Val Ala Pro Asp
Ser Asn Ala Lys Thr Gly Ala Phe 260 265 270 Trp Ser Ile Tyr Asn Gly
Arg Asp Ile Gly Val Leu Thr Ala Trp Ala 275 280 285 Trp Gly Phe His
Arg Thr Leu Asp Ala Ile Asn Met Thr Val Leu Glu 290 295 300 Ile Asp
Ala Gly Arg Val Gly Val Thr Gly Cys Ser Arg Leu Gly Lys 305 310 315
320 Ala Ala Leu Ala Ala Gly Leu Phe Asp Thr Arg Ile Thr Leu Thr Met
325 330 335 Pro Met Ser Ser Gly Val Gln Gly Met Gly Pro Tyr Arg Tyr
Tyr Ser 340 345 350 Met Ser Gly Gln Gly Glu Asn Leu Glu Asn Ser Lys
Gln Gly Ala Gly 355 360 365 Trp Trp Thr Ser Ser Lys Leu Gly Ala Phe
Ile Asn His Ser Glu Asn 370 375 380 Leu Pro Tyr Asp Ala His Thr Ile
Ala Ala Ala Ile Ala Pro Arg Ala 385 390 395 400 Leu Val Ile Asp Gln
Gly Thr Gly Asp Gln Phe Val Asn Ser Lys Gly 405 410 415 Thr Ala Val
Val Ile Tyr Pro Ala Ala Lys Val Val Tyr Asp Trp Leu 420 425 430 Gly
Ala Gly Asp Lys Ile Ala Ile Ser Val Arg Gly Gly Gly His Cys 435 440
445 Asp Met Ser Gly Phe Thr Ser Ile Leu Pro Tyr Val Gln Lys Ile Phe
450 455 460 Phe Gly Thr Pro Thr Ser Lys Asp Tyr Asn Asn Leu Gly Ser
Tyr Gly 465 470 475 480 Ser Pro Val Thr Thr Ala Phe Pro Trp Gly Thr
Ala Val Pro Lys Ala 485 490 495 719PRTThielavia
terrestrismisc_feature(1)..(1)X = I, L, M, or V 7Xaa Pro Xaa Xaa
Xaa Xaa Gly Xaa Tyr Xaa Xaa Arg Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa
Xaa 820PRTThielavia terrestrismisc_feature(1)..(1)X=I,L,M, or V
8Xaa Pro Xaa Xaa Xaa Xaa Xaa Gly Xaa Tyr Xaa Xaa Arg Xaa Xaa Xaa 1
5 10 15 Xaa Xaa Xaa Xaa 20 94PRTArtificial sequenceSynthetic
construct 9Xaa Xaa Lys Xaa 1 109PRTThielavia
terrestrismisc_feature(2)..(2)Xaa can be any naturally occurring
amino acid 10His Xaa Gly Pro Xaa Xaa Xaa Xaa Xaa 1 5
1110PRTThielavia terrestrismisc_feature(2)..(3)Xaa can be any
naturally occurring amino acid 11His Xaa Xaa Gly Pro Xaa Xaa Xaa
Xaa Xaa 1 5 10 1211PRTThielavia terrestrismisc_feature(1)..(1)X= E
or Q 12Xaa Xaa Tyr Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa 1 5 10
1319PRTThielavia
terrestrismisc_feature(1)..(1)X= I, L, M or V 13Xaa Pro Xaa Xaa Xaa
Xaa Gly Xaa Tyr Xaa Xaa Arg Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Ala Xaa
1420PRTThielavia terrestrismisc_feature(1)..(1)X= I, L, M or V
14Xaa Pro Xaa Xaa Xaa Xaa Xaa Gly Xaa Tyr Xaa Xaa Arg Xaa Xaa Xaa 1
5 10 15 Xaa Xaa Ala Xaa 20
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