U.S. patent application number 15/357010 was filed with the patent office on 2017-03-09 for fungal strains.
The applicant listed for this patent is Codexis, Inc.. Invention is credited to Dipnath Baidyaroy, Ish K. Dhawan.
Application Number | 20170067033 15/357010 |
Document ID | / |
Family ID | 45997182 |
Filed Date | 2017-03-09 |
United States Patent
Application |
20170067033 |
Kind Code |
A1 |
Baidyaroy; Dipnath ; et
al. |
March 9, 2017 |
FUNGAL STRAINS
Abstract
The present invention provides improved fungal strains. In some
embodiments, the improved fungal strains find use in hydrolyzing
cellulosic material to glucose.
Inventors: |
Baidyaroy; Dipnath;
(Fremont, CA) ; Dhawan; Ish K.; (Foster City,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Codexis, Inc. |
Redwood City |
CA |
US |
|
|
Family ID: |
45997182 |
Appl. No.: |
15/357010 |
Filed: |
November 21, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14717447 |
May 20, 2015 |
9528098 |
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15357010 |
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13286860 |
Nov 1, 2011 |
9068235 |
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14717447 |
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61409217 |
Nov 2, 2010 |
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61409472 |
Nov 2, 2010 |
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61409480 |
Nov 2, 2010 |
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61497661 |
Jun 16, 2011 |
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61409186 |
Nov 2, 2010 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12P 19/12 20130101;
C12Y 302/01021 20130101; C12P 2203/00 20130101; Y02P 20/52
20151101; C12N 9/2437 20130101; C12N 9/2445 20130101; C12P 7/10
20130101; C12P 19/02 20130101; Y02E 50/10 20130101; C12N 9/0006
20130101; C12P 19/14 20130101; C13K 1/02 20130101; C12Y 101/99018
20130101; Y02E 50/16 20130101 |
International
Class: |
C12N 9/04 20060101
C12N009/04; C12N 9/42 20060101 C12N009/42 |
Claims
1. An enzyme mixture comprising two or more cellulose hydrolyzing
enzymes, wherein at least one of the two or more cellulose
hydrolyzing enzymes is expressed by a fungal cell that has been
genetically modified to reduce the amount of endogenous glucose
and/or cellobiose oxidizing enzyme activity that is produced by the
fungal cell.
2. An enzyme mixture comprising two or more cellulose hydrolyzing
enzymes, wherein at least one of the two or more cellulose
hydrolyzing enzymes is expressed by a fungal cell that has been
genetically modified to reduce the amount of endogenous glucose
and/or cellobiose oxidizing enzyme activity that is secreted by the
fungal cell.
3. The enzyme mixture of claim 1, comprising two or more cellulose
hydrolyzing enzymes, wherein at least one of the two or more
cellulose hydrolyzing enzymes is expressed by a fungal cell that
has been genetically modified to reduce the amount of endogenous
glucose and/or cellobiose oxidizing enzyme activity that is
secreted by the fungal cell and further to increase the expression
of at least one saccharide hydrolyzing enzyme.
4. The enzyme mixture of claim 1, wherein the enzyme mixture is a
cell-free mixture.
5. The enzyme mixture of claim 1, wherein a substrate of the enzyme
mixture comprises pretreated lignocellulose.
6. The enzyme mixture of claim 1, wherein the enzyme mixture
comprises at least one cellulase enzyme selected from
endoglucanases (EGs), beta-glucosidases (BGLs), Type 1
cellobiohydrolases (CBH1s), Type 2 cellobiohydrolases (CBH2s),
and/or glycoside hydrolase 61s (GH61s), and/or variants of said
cellulase enzyme.
7. The enzyme mixture of claim 6, wherein the enzyme mixture
comprises at least one beta-glucosidase.
8. The enzyme mixture of claim 1, further comprising at least one
cellobiose dehydrogenase.
9. The enzyme mixture of claim 8, wherein said celliobiose
dehydrogenase is CDH1 and/or CDH2.
10. The enzyme mixture of claim 1, further comprising at least one
cellulase enzyme and/or at least one additional enzyme.
11. The enzyme mixture of claim 1, wherein the enzyme mixture has
been subjected to a purification process to selectively remove one
or more glucose and/or cellobiose oxidizing enzymes from the enzyme
mixture.
12. The enzyme mixture of claim 11, wherein the purification
process comprises selective precipitation to separate the glucose
and/or cellobiose oxidizing enzymes from other enzymes present in
the enzyme mixture.
13. The enzyme mixture of claim 1, wherein the enzyme mixture
comprises an inhibitor of one or more glucose and/or cellobiose
oxidizing enzymes.
Description
[0001] The present application claims priority to U.S. patent
application Ser. No. 14/717,447, filed May 20, 2015, which claims
priority to U.S. patent application Ser. No. 13/286,860, filed Nov.
1, 2011, now U.S. Pat. No. 9,068,235, which claims priority to U.S.
Prov. Patent Appln. Ser. Nos. 61/409,186, 61/409,217, 61/409,472,
and 61/409,480, all of which were filed on Nov. 2, 2010, and U.S.
Prov. Patent Appln. Ser. No. 61/497,661, filed on Jun. 16, 2011,
all of which are hereby incorporated by reference herein, in their
entireties and for all purposes.
REFERENCE TO A "SEQUENCE LISTING," A TABLE, OR A COMPUTER PROGRAM
LISTING APPENDIX SUBMITTED AS AN ASCII TEXT FILE
[0002] The Sequence Listing written in file CX3-082US1_ST25.TXT,
created on Dec. 7, 2011, 47,145 bytes, machine format IBM-PC,
MS-Windows operating system, is hereby incorporated by
reference.
FIELD OF THE INVENTION
[0003] The present invention provides improved fungal strains. In
some embodiments, the improved fungal strains find use in
hydrolyzing cellulosic material to glucose.
BACKGROUND OF THE INVENTION
[0004] Cellulose is a polymer of the simple sugar glucose linked by
beta-1,4 glycosidic bonds. Many microorganisms produce enzymes that
hydrolyze beta-linked glucans. These enzymes include
endoglucanases, cellobiohydrolases, and beta-glucosidases.
Endoglucanases digest the cellulose polymer at random locations,
opening it to attack by cellobiohydrolases. Cellobiohydrolases
sequentially release molecules of cellobiose from the ends of the
cellulose polymer. Cellobiose is a water-soluble beta-1,4-linked
dimer of glucose. Beta-glucosidases hydrolyze cellobiose to
glucose.
[0005] The conversion of lignocellulosic feedstocks into ethanol
has the advantages of the ready availability of large amounts of
feedstock, the desirability of avoiding burning or land-filling the
materials, and lower overall greenhouse gas production. Wood,
agricultural residues, herbaceous crops, and municipal solid wastes
have been considered as feedstocks for ethanol production. These
materials primarily consist of cellulose, hemicellulose, and
lignin. Once the cellulose is converted to glucose, the glucose is
easily fermented by yeast into ethanol.
SUMMARY OF THE INVENTION
[0006] The present invention provides improved fungal strains. In
some embodiments, the improved fungal strains find use in
hydrolyzing cellulosic material to glucose.
[0007] The present invention provides a fungal cell that has been
genetically modified to reduce the amount of endogenous cellobiose
dehydrogenase activity that is secreted by the cell, wherein the
fungal cell is from the family Chaetomiaceae, wherein said cell
comprises a deletion in the cellobiose dehydrogenase 1 (cdh1) gene.
In some embodiments, the fungal cell is a species of
Myceliophthora. In some further embodiments, the fungal cell is
Myceliophthora thermophila. In some embodiments, the fungal cell
has been genetically modified to disrupt the secretion signal
peptide of the cellobiose dehydrogenase. In some additional
embodiments, the fungal cell has been genetically modified to
reduce the amount of the endogenous cellobiose dehydrogenase
expressed by the cell. In some further embodiments, the fungal cell
has been genetically modified to disrupt a translation initiation
sequence in the transcript encoding the endogenous cellobiose
dehydrogenase. In some still additional embodiments, the fungal
cell has been genetically modified to introduce a frameshift
mutation in the transcript encoding the endogenous cellobiose
dehydrogenase. In some further embodiments, the fungal cell has
been genetically modified to reduce the transcription level of a
gene encoding the endogenous cellobiose dehydrogenase. In some
additional embodiments, the fungal cell has been genetically
modified to disrupt the promoter of a gene encoding the endogenous
cellobiose dehydrogenase. In some embodiments, the fungal cell has
been genetically modified to at least partially delete a gene
encoding the endogenous cellobiose dehydrogenase. In some further
embodiments, the fungal cell has been genetically modified to
reduce the catalytic efficiency of the endogenous cellobiose
dehydrogenase. In some additional embodiments, one or more residues
in an active site of the cellobiose dehydrogenase in the fungal
cell have been genetically modified. In some still further
embodiments, one or more residues in a heme binding domain of the
cellobiose dehydrogenase in the fungal cell have been genetically
modified.
[0008] In some embodiments, the present invention provides fungal
cells comprising cellobiose dehydrogenase. In some embodiments, the
cellobiose dehydrogenase comprises an amino acid sequence that is
at least about 85%, about 88%, about 90%, about 93%, about 95%,
about 97%, about 98%, or about 99% identical to SEQ ID NO:2. In
some additional embodiments, the fungal cell has been modified such
that the cell secretes a reduced amount of endogenous cellobiose
dehydrogenase 1 (cdhl), as compared to a fungal cell prior to or
without such modification.
[0009] The present invention also provides an enzyme mixture
comprising two or more cellulose hydrolyzing enzymes, wherein at
least one of the two or more cellulose hydrolyzing enzymes is
expressed by the fungal cell. In some embodiments, the enzyme
mixture is a cell-free mixture. In some additional embodiments,
pretreated lignocellulose comprises at least one substrate of the
enzyme mixture. In some further embodiments, the pretreated
lignocellulose comprises lignocellulose treated by at least one
treatment method selected from acid pretreatment, ammonia
pretreatment, steam explosion, and/or organic solvent
extraction.
[0010] The present invention also provides methods for generating
glucose comprising contacting at least one cellulose substrate with
an enzyme mixture comprising two or more cellulose hydrolyzing
enzymes, wherein at least one of the two or more cellulose
hydrolyzing enzymes is expressed by a fungal cell provided herein.
The present invention also provides methods for generating glucose,
comprising contacting at least one cellulose substrate with at
least one enzyme mixture provided herein. In some further
embodiments, the enzyme mixture is a cell-free mixture. In some
additional embodiments, the cellulose substrate is pretreated
lignocellulose. In still some further embodiments, the pretreated
lignocellulose comprises lignocellulose treated by at least one
treatment method selected from acid pretreatment, ammonia
pretreatment, steam explosion, and/or organic solvent extraction In
some additional embodiments, the methods of the present invention
further comprise fermenting the glucose to an end product. In some
further embodiments, the end product is a fuel alcohol or a
precursor industrial chemical. In some additional embodiments, the
fuel alcohol is ethanol or butanol. In still some additional
embodiments, the methods, enzyme mixtures, and/or fungal cells of
the present invention provide at least one cellulose degrading
enzyme that is homologous or heterologous to the fungal cell.
[0011] The present invention also provides fermentation media
comprising at least one fungal cell and/or at least one enzyme
mixture as provided herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1 provides the nucleotide and amino acid sequences of
M. thermophila CDH1 (SEQ ID NOS:1 and 2, respectively).
[0013] FIG. 2 provides a graph showing the relative
saccharification efficiency of CF-200 and CF-400, as measured in
glucose produced from 100 g/kg glucan (pre-treated corn stover).
Reactions were run at 24.6% solids, with 128 mM NaOAc, at pH 5,
55.degree. C., 3% enzyme, in 110 .mu.L volumes. Glucose was
measured using the GOPOD assay. Error bars represent .+-.1 SD,
n=4.
DESCRIPTION OF THE INVENTION
[0014] The present invention provides improved fungal strains. In
some embodiments, the improved fungal strains find use in
hydrolyzing cellulosic material to glucose. As indicated herein,
the present invention provides improved fungal strains for the
conversion of cellulose to glucose. In particular, the improved
fungal strains provided herein are genetically modified to reduce
the amount of endogenous cellobiose dehydrogenase activity secreted
by the cells. Prior to the present invention, it was generally
believed that cellobiose dehydrogenase enhances the rate of
cellulose hydrolysis by reducing the concentration of cellobiose,
which is a potent inhibitor of some cellulase components (See e.g.,
Mansfield et al., Appl. Environ. Microbiol., 63:3804-3809 [1997];
and Igarishi et al., Eur. J. Biochem., 253:101-106 [1998]).
Furthermore, cellobiose dehydrogenase has been reported as playing
a critical role in contributing to synergistic enhancement during
cellulose degradation by preventing hydrolysis product inhibition
(See e.g., Hai et al., J. Appl. Glycosci., 49:9-17 [2002]). It was
also generally believed that cellobiose dehydrogenase was useful in
delignifying lignocellulose, thereby enhancing cellulose
degradation. Recently, it has been reported that cellobiose
dehydrogenases can enhance the activity of cellulolytic enhancing
proteins from Glycosyl Hydrolase Family 61 (See e.g.,
WO2010/080532A1), and may find use in reactions for redox balance
purposes.
[0015] Contrary to general understanding in the art, the present
invention provides genetic modifications (e.g., deletion) of a
cellobiose dehydrogenase-encoding gene in cellulase-producing
fungal cells. This modification results in an improvement in the
yield of fermentable sugars from enzyme mixtures secreted by the
genetically modified cells. Thus, reduction of cellobiose
dehydrogenase secreted by a cellulase-producing organism provides a
mixture of cellulase enzymes that can improve yield of fermentable
sugars during enzymatic hydrolysis of cellulose-containing
substrates. In addition, deletion of the cdh gene provides
additional room in the fungal cell genome for introduction of other
sequences (e.g., heterologous sequences encoding proteins of
interest).
[0016] Accordingly, provided herein is a fungal cell that has been
genetically modified to reduce the amount of endogenous cellobiose
dehydrogenase activity that is secreted by the cell, wherein the
fungal cell is from the family Chaetomiaceae, and wherein the
fungal cell is capable of secreting a cellulose-containing enzyme
mixture. In some embodiments, the fungal cell is capable of
secreting an enzyme mixture comprising two or more cellulase
enzymes. In some embodiments, the fungal cell is a Chaetomiaceae
family member of the genus Achaetomium, Aporothielavia,
Chaetomidium, Chaetomium, Corylomyces, Corynascus, Farrowia,
Thielavia, Zopfiella, or Myceliophthora. In some embodiments, the
genetically modified fungal cell provided herein is a Chaetomiaceae
family member selected from the genera Myceliophthora, Thielavia,
Corynascus, or Chaetomium.
[0017] It is recognized that fungal taxonomy continues to undergo
reorganization. Thus, it is intended that all aspects of the
present invention encompass genera and species that have been
reclassified, including but not limited to such organisms as
Myceliophthora thermophila, which has also been given various other
names (e.g., Sporotrichum thermophile, Sporotrichum thermophilum,
Thelavia heterothallica, Corynascus heterothallica, Chrysoporium
thermophilum, and Myceliophthora indica). Indeed, it is intended
that the present invention encompass all teleomorphs, anamorphs,
and synonyms, basionyms, or taxonomic equivalents thereof.
[0018] In some embodiments, the fungal cell has been genetically
modified to reduce the amount of endogenous cellobiose
dehydrogenase activity that is secreted by the cell. In some
embodiments, the fungal cell is a species of Myceliophthora,
Thielavia, Sporotrichum, Corynascus, Acremonium, Chaetomium,
Ctenomyces, Scytalidium, Talaromyces, or Thermoascus. In some
embodiments, the fungal cell is Sporotrichum cellulophilum,
Thielavia terrestris, Corynascus heterothallicus, Thielavia
heterothallica, Chaetomium globosum, Talaromyces stipitatus, or
Myceliophthora thermophila. In some embodiments, the fungal cell is
an isolated fungal cell.
[0019] In some embodiments, the fungal cell has been genetically
modified to reduce the amount of endogenous cellobiose
dehydrogenase secreted by the cell. In some embodiments, the fungal
cell has been genetically modified to disrupt the secretion signal
peptide of cellobiose dehydrogenase. In some embodiments, the
fungal cell has been genetically modified to reduce the amount of
the endogenous cellobiose dehydrogenase expressed by the cell. For
example, in some embodiments, the fungal cell is genetically
modified to disrupt a translation initiation sequence, while in
some other embodiments, the fungal cell is genetically modified to
introduce a frameshift mutation in the transcript encoding the
endogenous cellobiose dehydrogenase. In some other embodiments, the
fungal cell has been genetically modified to reduce the
transcription level of a gene encoding the endogenous cellobiose
dehydrogenase. For example, in some embodiments, the fungal cell is
genetically modified to disrupt the promoter of a gene encoding the
endogenous cellobiose dehydrogenase. For example, in some
embodiments, the fungal cell is genetically modified to disrupt the
gene encoding the endogenous cellobiose dehydrogenase through use
of stop codons, terminator elimination, transposons, etc. In some
additional embodiments, the fungal cell has been genetically
modified to at least partially delete a gene encoding the
endogenous cellobiose dehydrogenase. In some other embodiments, the
fungal cell has been genetically modified to reduce the catalytic
efficiency of the endogenous cellobiose dehydrogenase. In some
embodiments, the fungal cell has been genetically modified, such
that one or more residues in an active site of the cellobiose
dehydrogenase have been mutated. In some embodiments, one or more
residues in a heme binding domain of the cellobiose dehydrogenase
of the fungal cell have been genetically modified. Indeed, it is
intended that any suitable means for modifying the fungal cell to
reduce the amount of cellobiose dehydrogenase expressed and/or
secreted by the cell will find use in the present invention.
[0020] In some embodiments, the cellobiose dehydrogenase is
encompassed within EC 1.1.99.18. In some embodiments, the
cellobiose dehydrogenase comprises an amino acid sequence that is
at least about 85%, about 86%, about 87%, about 88%, about 89%,
about 90%, about 91%, about 92%, about 93%, about 94%, about 95%,
about 96%, about 97%, about 98%, about 99%, or about 100% identical
to SEQ ID NO:2.
[0021] In some embodiments, the fungal cell further comprises at
least one gene encoding at least one cellulose degrading enzyme
that is heterologous to the fungal cell. For example, in some
embodiments, the fungal cell overexpresses a homologous or
heterologous gene encoding a cellulose degrading enzyme such as
beta-glucosidase. In some embodiments, the fungal cell
overexpresses beta-glucosidase and has been genetically modified to
reduce the amount of endogenous cellobiose dehydrogenase activity
secreted by the cell.
[0022] The present invention also provides enzyme mixtures
comprising two or more cellulose hydrolyzing enzymes, wherein at
least one of the two or more cellulose hydrolyzing enzymes is
expressed by a fungal cell as provided herein. For example, in some
embodiments, the fungal cell is a cell that has been genetically
modified to reduce the amount of endogenous cellobiose
dehydrogenase activity secreted by the cell, wherein the fungal
cell is a member of the genus Myceliophthora, Thielavia,
Sporotrichum, Corynascus, Acremonium, Chaetomium, Ctenomyces,
Scytalidium, Talaromyces, or Thermoascus. In some embodiments, the
enzyme mixture is a cell-free mixture. In some additional
embodiments, a substrate of the enzyme mixture comprises pretreated
lignocellulose. In some embodiments, the pretreated lignocellulose
comprises lignocellulose treated by acid pretreatment, ammonia
pretreatment, steam explosion, and/or organic solvent extraction.
In some embodiments, the enzyme mixture further comprises at least
one cellulose degrading enzyme that is heterologous to the fungal
cell. In some embodiments, at least one of the two or more
cellulose hydrolyzing enzymes is expressed by an isolated fungal
cell.
[0023] The present invention also provides methods for generating
glucose that comprise contacting cellulose with a mixture of at
least two enzymes. For example, in some embodiments, the methods
comprise contacting cellulose with an enzyme mixture comprising two
or more cellulose hydrolyzing enzymes, wherein at least one of the
two or more cellulose hydrolyzing enzymes is expressed by a fungal
cell as described herein. In some embodiments, the methods comprise
contacting cellulose with an enzyme mixture comprising two or more
cellulose hydrolyzing enzymes, wherein at least one of the two or
more cellulose hydrolyzing enzymes is expressed by a cell that has
been genetically modified to reduce the amount of endogenous
cellobiose dehydrogenase activity secreted by the cell, wherein the
fungal cell is Myceliophthora, Thielavia, Sporotrichum, Corynascus,
Acremonium, Chaetomium, Ctenomyces, Scytalidium, Talaromyces, or
Thermoascus. In some embodiments, the methods result in an
increased yield of glucose and/or cellobiose from the hydrolyzed
cellulose and decreased oxidation of the cellobiose to oxidized
sugar products, such as gluconolactone, gluconate, gluconic acid,
cellobionolactone, and/or cellobionic acid from the hydrolyzed
cellulose.
[0024] In some embodiments, the enzyme mixture is a cell-free
mixture. In some further embodiments, the cellulose substrate
comprises pretreated lignocellulose. In some additional
embodiments, the pretreated lignocellulose comprises lignocellulose
treated by at least one treatment method such as acid pretreatment,
ammonia pretreatment, steam explosion and/or organic solvent
extraction.
[0025] In some embodiments, the methods further comprise
fermentation of the glucose to an end product such as a fuel
alcohol or a precursor industrial chemical. In some embodiments,
the fuel alcohol is ethanol or butanol. In some embodiments, the
methods comprise contacting cellulose with an enzyme mixture that
further comprises a cellulose degrading enzyme that is heterologous
to the fungal cell.
[0026] Also provided herein are fermentation media comprising the
fungal cell of any of the above embodiments, and/or comprising the
enzyme mixture derived from the fungal cell of any of the above
embodiments.
Definitions
[0027] Unless otherwise indicated, the practice of the present
invention involves conventional techniques commonly used in
molecular biology, protein engineering, and microbiology, which are
within the skill of the art. Such techniques are well-known and
described in numerous texts and reference works well known to those
of skill in the art. All patents, patent applications, articles and
publications mentioned herein, both supra and infra, are hereby
expressly incorporated herein by reference.
[0028] Unless defined otherwise herein, all technical and
scientific terms used herein have the same meaning as commonly
understood by one of ordinary skill in the art to which this
invention pertains. Many technical dictionaries are known to those
of skill in the art. Although any suitable methods and materials
similar or equivalent to those described herein find use in the
practice of the present invention, some preferred methods and
materials are described herein. It is to be understood that this
invention is not limited to the particular methodology, protocols,
and reagents described, as these may vary, depending upon the
context they are used by those of skill in the art. Accordingly,
the terms defined immediately below are more fully described by
reference to the application as a whole.
[0029] Also, as used herein, the singular "a", "an," and "the"
include the plural references, unless the context clearly indicates
otherwise. Numeric ranges are inclusive of the numbers defining the
range. Thus, every numerical range disclosed herein is intended to
encompass every narrower numerical range that falls within such
broader numerical range, as if such narrower numerical ranges were
all expressly written herein. It is also intended that every
maximum (or minimum) numerical limitation disclosed herein includes
every lower (or higher) numerical limitation, as if such lower (or
higher) numerical limitations were expressly written herein.
Furthermore, the headings provided herein are not limitations of
the various aspects or embodiments of the invention which can be
had by reference to the application as a whole. Accordingly, the
terms defined immediately below are more fully defined by reference
to the application as a whole. Nonetheless, in order to facilitate
understanding of the invention, a number of terms are defined
below. Unless otherwise indicated, nucleic acids are written left
to right in 5' to 3' orientation; amino acid sequences are written
left to right in amino to carboxy orientation, respectively.
[0030] As used herein, the term "comprising" and its cognates are
used in their inclusive sense (i.e., equivalent to the term
"including" and its corresponding cognates).
[0031] As used herein, "substrate" refers to a substance or
compound that is converted or meant to be converted into another
compound by the action of an enzyme. The term includes not only a
single compound but also combinations of compounds, such as
solutions, mixtures and other materials which contain at least one
substrate.
[0032] As used herein, "conversion" refers to the enzymatic
transformation of a substrate to the corresponding product.
"Percent conversion" refers to the percent of the substrate that is
converted to the product within a period of time under specified
conditions. Thus, for example, the "enzymatic activity" or
"activity" of a cellobiose dehydrogenase ("CDH" or "cdh")
polypeptide can be expressed as "percent conversion" of the
substrate to the product.
[0033] As used herein, "secreted activity" refers to enzymatic
activity of cellobiose oxidizing enzymes produced by a fungal cell
that is present in an extracellular environment. An extracellular
environment can be, for example, an extracellular milieu such as a
culture medium. The secreted activity is influenced by the total
amount of cellobiose oxidizing enzyme secreted, and also is
influenced by the catalytic efficiency of the secreted cellobiose
oxidizing enzyme.
[0034] As used herein, a "reduction in catalytic efficiency" refers
to a reduction in the activity of the cellobiose oxidizing enzyme,
relative to unmodified cellobiose oxidizing enzyme, as measured
using standard techniques, as provided herein or otherwise known in
the art.
[0035] As used herein, the term "enzyme mixture" refers to a
combination of at least two enzymes. In some embodiments, at least
two enzymes are present in a composition. In some additional
embodiments, the enzyme mixtures are present within a cell (e.g., a
fungal cell). In some embodiments, each or some of the enzymes
present in an enzyme mixture are produced by different fungal cells
and/or different fungal cultures. In some further embodiments, all
of the enzymes present in an enzyme mixture are produced by the
same cell. In some embodiments, the enzyme mixtures comprise
cellulase enzymes, while in some additional embodiments, the enzyme
mixtures comprise enzymes other than cellulases. In some
embodiments, the enzyme mixtures comprise at least one cellulase
and at least one enzyme other than a cellulase. In some
embodiments, the enzyme mixtures comprise enzymes including, but
not limited to endoxylanases (EC 3.2.1.8), beta-xylosidases (EC
3.2.1.37), alpha-L-arabinofuranosidases (EC 3.2.1.55),
alpha-glucuronidases (EC 3.2.1.139), acetylxylanesterases (EC
3.1.1.72), feruloyl esterases (EC 3.1.1.73), coumaroyl esterases
(EC 3.1.1.73), alpha-galactosidases (EC 3.2.1.22),
beta-galactosidases (EC 3.2.1.23), beta-mannanases (EC 3.2.1.78),
beta-mannosidases (EC 3.2.1.25), endo-polygalacturonases (EC
3.2.1.15), pectin methyl esterases (EC 3.1.1.11), endo-galactanases
(EC 3.2.1.89), pectin acetyl esterases (EC 3.1.1.6), endo-pectin
lyases (EC 4.2.2.10), pectate lyases (EC 4.2.2.2), alpha
rhamnosidases (EC 3.2.1.40), exo-galacturonases (EC 3.2.1.82),
exo-galacturonases (EC 3.2.1.67), exopolygalacturonate lyases (EC
4.2.2.9), rhamnogalacturonan endolyases EC (4.2.2.B3),
rhamnogalacturonan acetylesterases (EC 3.2.1.B11),
rhamnogalacturonan galacturonohydrolases (EC 3.2.1.B11),
endo-arabinanases (EC 3.2.1.99), laccases (EC 1.10.3.2),
manganese-dependent peroxidases (EC 1.10.3.2), amylases (EC
3.2.1.1), glucoamylases (EC 3.2.1.3), lipases, lignin peroxidases
(EC 1.11.1.14), and/or proteases.
[0036] In some additional embodiments, the present invention
further provides enzyme mixtures comprising at least one expansin
and/or expansin-like protein, such as a swollenin (See e.g.,
Salheimo et al., Eur. J. Biochem., 269:4202-4211 [2002]) and/or a
swollenin-like protein. Expansins are implicated in loosening of
the cell wall structure during plant cell growth. Expansins have
been proposed to disrupt hydrogen bonding between cellulose and
other cell wall polysaccharides without having hydrolytic activity.
In this way, they are thought to allow the sliding of cellulose
fibers and enlargement of the cell wall. Swollenin, an
expansin-like protein contains an N-terminal Carbohydrate Binding
Module Family 1 domain (CBD) and a C-terminal expansin-like domain.
In some embodiments, an expansin-like protein and/or swollenin-like
protein comprises one or both of such domains and/or disrupts the
structure of cell walls (e.g., disrupting cellulose structure),
optionally without producing detectable amounts of reducing sugars.
In some additional embodiments, the enzyme mixtures comprise at
least one polypeptide product of a cellulose integrating protein,
scaffoldin and/or a scaffoldin-like protein (e.g., CipA or CipC
from Clostridium thermocellum or Clostridium cellulolyticum
respectively). In some additional embodiments, the enzyme mixtures
comprise at least one cellulose induced protein and/or modulating
protein (e.g., as encoded by cipl or cip2 gene and/or similar genes
from Trichoderma reesei; See e.g., Foreman et al., J. Biol. Chem.,
278:31988-31997 [2003]). In some additional embodiments, the enzyme
mixtures comprise at least one member of each of the classes of the
polypeptides described above, several members of one polypeptide
class, or any combination of these polypeptide classes to provide
enzyme mixtures suitable for various uses. Any combination of at
least one two, three, four, five, or more than five enzymes and/or
polypeptides find use in various enzyme mixtures provided herein.
Indeed, it is not intended that the enzyme mixtures of the present
invention be limited to any particular enzymes, polypeptides, nor
combinations, as any suitable enzyme mixture finds use in the
present invention.
[0037] As used herein, the term "saccharide" refers to any
carbohydrate comprising monosaccharides (e.g., glucose, ribose,
fructose, galactose, etc.), disaccharides (e.g., sucrose, lactose,
maltose, cellobiose, trehalose, melibiose, etc.), oligosaccharides
(e.g., raffinose, stachyose, amylose, etc.), and polysaccharides
(e.g., starch, glycogen, cellulose, chitin, xylan, arabinoxylan,
mannan, fucoidan, galactomannan, callose, laminarin,
chrysolaminarin, amylopectin, dextran, dextrins, maltodextrins,
inulin, oligofructose, polydextrose, etc.). The term encompasses
simple carbohydrates, as well as complex carbohydrates. Indeed, it
is not intended that the present invention be limited to any
particular saccharide, as various saccharides and forms of
saccharides find use in the present invention.
[0038] As used herein, the term "saccharide hydrolyzing enzyme"
refers to any enzyme that hydrolyzes at least one sachharide.
[0039] As used herein, the terms "cellobiose oxidizing enzyme"
refer to enzymes that oxidize cellobiose. In some embodiments,
cellobiose oxidizing enzymes include cellobiose dehydrogenase (EC
1.1.99.18).
[0040] As used herein, the terms "cellobiose dehydrogenase," "CDH,"
and "cdh" refer to a cellobiose: acceptor 1-oxidoreductase that
catalyzes the conversion of cellobiose in the presence of an
acceptor to cellobiono-1,5-lactone and a reduced acceptor. Examples
of cellobiose dehydrogenases fall into the enzyme classification
(E.C. 1.1.99.18). Typically 2,6-dichloroindophenol can act as
acceptor, as can iron, especially Fe(SCN).sub.3, molecular oxygen,
ubiquinone, or cytochrome C, and other polyphenolics, such as
lignin. Substrates of the enzyme include cellobiose,
cello-oligosaccharides, lactose, and
D-glucosyl-1,4-.beta.-D-mannose, glucose, maltose, mannobiose,
thiocellobiose, galactosyl-mannose, xylobiose, xylose. Electron
donors include beta-1-4 dihexoses with glucose or mannose at the
reducing end, though alpha-1-4 hexosides, hexoses, pentoses, and
beta-1-4 pentomers can act as substrates for at least some of these
enzymes (See e.g., Henriksson et al, Biochim. Biophys. Acta--Prot.
Struct. Mol. Enzymol., 1383: 48-54 [1998]; and Schou et al.,
Biochem. J., 330: 565-571 [1998]). In some embodiments, the
cellobiose dehydrogenase of interest in the present invention is
CDH1, which is encoded by the cdh1 gene.
[0041] As used herein, the terms "oxidation", "oxidize(d)" and the
like as used herein refer to the enzymatic formation of one or more
cellobiose oxidation products. When used in reference to a
percentage of oxidized cellobiose, those percentages reflect a
weight percent (w/w) relative to the initial amount of substrate.
For example, when the enzyme mixture is contacted with cellobiose,
the percentage of oxidized cellobiose reflects a weight percent
(w/w) relative to the initial amount of cellobiose present in
solution. Where the enzyme mixture is contacted with a cellulose
substrate, the percentage of oxidized cellobiose reflects a weight
percent (w/w) based on the maximum amount (wt %) of glucose that
could be produced from the total hydrolyzed cellulose (i.e.,
Gmax).
[0042] As used herein, "cellulose" refers to a polymer of the
simple sugar glucose linked by beta-1,4 glycosidic bonds.
[0043] As used herein, "cellobiose" refers to a water-soluble
beta-1,4-linked dimer of glucose.
[0044] As used herein, the term "cellodextrin" refers to a gluocose
polymer of varying length (i.e., comprising at least two glucose
monomers). Each glucose monomer is linked via a beta-1,4 glycosidic
bond. A cellodextrin is classified by its degree of polymerization
(DP), which indicates the number of glucose monomers the
cellodextrin contains. The most common cellodextrins are:
cellobiose (DP=2); cellotriose (DP=3); cellotetrose (DP=4);
cellopentose (DP=5); and cellohexose (DP=6). In some embodiments,
cellodextrins have a DP of 2-6 (i.e., cellobiose, cellotriose,
cellotetrose, cellopentose, and/or cellohexose). In some
embodiments, cellodextrins have a DP greater than 6. The degree of
polymerization of cellodextin molecules can be measured (e.g., by
mass spectrometry, including but not limited to matrix-assisted
laser desorption/ionization (MALDI) mass spectrometry and
electrospray ionization ion trap (ESI-IT) mass spectrometry).
Methods of measuring the degree of polymerization of cellodextrin
molecules are known in the art (See e.g., Melander et al.,
Biomacromol., 7:1410-1421 [2006]).
[0045] As used herein, the term "cellulase" refers to any enzyme
that is capable of degrading cellulose. Thus, the term encompasses
enzymes capable of hydrolyzing cellulose (.beta.-1,4-glucan or
.beta.-D-glucosidic linkages) to shorter cellulose chains,
oligosaccharides, cellobiose and/or glucose. "Cellulases" are
divided into three sub-categories of enzymes: 1,4-.beta.-D-glucan
glucanohydrolase ("endoglucanase" or "EG"); 1,4-.beta.-D-glucan
cellobiohydrolase ("exoglucanase," "cellobiohydrolase," or "CBH");
and .beta.-D-glucoside-glucohydrolase (".beta.-glucosidase,"
"cellobiase," "BG," or "BGL"). These enzymes act in concert to
catalyze the hydrolysis of cellulose-containing substrates.
Endoglucanases break internal bonds and disrupt the crystalline
structure of cellulose, exposing individual cellulose
polysaccharide chains ("glucans"). Cellobiohydrolases incrementally
shorten the glucan molecules, releasing mainly cellobiose units (a
water-soluble .beta.-1,4-linked dimer of glucose) as well as
glucose, cellotriose, and cellotetrose. Beta-glucosidases split the
cellobiose into monomers. Cellulases often comprise a mixture of
different types of cellulolytic enzymes (endoglucanases and
cellobiohydrolases) that act synergistically to break down the
cellulose to soluble di- or oligosaccharides such as cellobiose,
which are then further hydrolyzed to glucose by beta-glucosidase.
Cellulase enzymes are produced by a wide variety of microorganisms.
Cellulases (and hemicellulases) from filamentous fungi and some
bacteria are widely exploited for many industrial applications that
involve processing of natural fibers to sugars.
[0046] As used herein, a "cellulase-producing fungal cell" is a
fungal cell that produces at least one cellulase enzyme (i.e.,
"cellulose hydrolyzing enzyme"). In some embodiments, the
cellulase-producing fungal cells provided herein express and
secrete a mixture of cellulose hydrolyzing enzymes.
[0047] As used herein, the terms "cellulose hydrolyzing enzyme,"
"cellulolytic enzyme," and like terms refer to an enzyme that acts
in the process of breaking down cellulose to soluble di- or
oligosaccharides such as cellobiose, which are then further
hydrolyzed to glucose by beta-glucosidase. A mixture of cellulose
hydrolyzing enzymes is also referred to herein as "cellulases," a
"cellulase-containing mixture," and/or a "cellulase mixture."
[0048] As used herein, the terms "endoglucanase" and "EG" refer to
a category of cellulases (EC 3.2.1.4) that catalyze the hydrolysis
of internal .beta.-1,4 glucosidic bonds of cellulose. The term
"endoglucanase" is further defined herein as 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), lichenan, 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 based on a reduction in
substrate viscosity or increase in reducing ends determined by a
reducing sugar assay (See e.g., Zhang et al., Biotechnol. Adv.,
24:452-481 [2006]). For purposes of the present invention,
endoglucanase activity is determined using carboxymethyl cellulose
(CMC) hydrolysis (See e.g., Ghose, Pur. Appl. Chem., 59:257-268
[1987]).
[0049] As used herein, "EG1" refers to a carbohydrate active enzyme
expressed from a nucleic acid sequence coding for a glycohydrolase
(GH) Family 7 catalytic domain classified under EC 3.2.1.4 or any
protein, polypeptide or catalytically active fragment thereof. In
some embodiments, the EG1 is functionally linked to a carbohydrate
binding module (CBM), such as a Family 1 cellulose binding
domain
[0050] As used herein, the term "EG2" refers to a carbohydrate
active enzyme expressed from a nucleic acid sequence coding for a
glycohydrolase (GH) Family 5 catalytic domain classified under EC
3.2.1.4 or any protein, polypeptide or catalytically active
fragment thereof. In some embodiments, the EG2 is functionally
linked to a carbohydrate binding module (CBM), such as a Family 1
cellulose binding domain.
[0051] As used herein, the term "EG3" refers to a carbohydrate
active enzyme expressed from a nucleic acid sequence coding for a
glycohydrolase (GH) Family 12 catalytic domain classified under EC
3.2.1.4 or any protein, polypeptide or catalytically active
fragment thereof. In some embodiments, the EG3 is functionally
linked to a carbohydrate binding module (CBM), such as a Family 1
cellulose binding domain.
[0052] As used herein, the term "EG4" refers to a carbohydrate
active enzyme expressed from a nucleic acid sequence coding for a
glycohydrolase (GH) Family 61 catalytic domain classified under EC
3.2.1.4 or any protein, polypeptide or fragment thereof. In some
embodiments, the EG4 is functionally linked to a carbohydrate
binding module (CBM), such as a Family 1 cellulose binding
domain
[0053] As used herein, the term "EG5" refers to a carbohydrate
active enzyme expressed from a nucleic acid sequence coding for a
glycohydrolase (GH) Family 45 catalytic domain classified under EC
3.2.1.4 or any protein, polypeptide or fragment thereof. In some
embodiments, the EG5 is functionally linked to a carbohydrate
binding module (CBM), such as a Family 1 cellulose binding
domain
[0054] As used herein, the term "EG6" refers to a carbohydrate
active enzyme expressed from a nucleic acid sequence coding for a
glycohydrolase (GH) Family 6 catalytic domain classified under EC
3.2.1.4 or any protein, polypeptide or fragment thereof. In some
embodiments, the EG6 is functionally linked to a carbohydrate
binding module (CBM), such as a Family 1 cellulose binding
domain
[0055] As used herein, the terms "cellobiohydrolase" and "CBH"
refer to a category of cellulases (EC 3.2.1.91) that hydrolyze
glycosidic bonds in cellulose. The term "cellobiohydrolase" is
further defined herein as a 1,4-beta-D-glucan cellobiohydrolase
(E.C. 3.2.1.91), which catalyzes the hydrolysis of
1,4-beta-D-glucosidic linkages in cellulose, cellooligosaccharides,
or any beta-1,4-linked glucose containing polymer, releasing
cellobiose from the reducing or non-reducing ends of the chain (See
e.g., Teeri, Tr. Biotechnol., 15:160-167 [1997]; and Teeri et al.,
Biochem. Soc. Trans., 26:173-178 [1998]). In some embodiments,
cellobiohydrolase activity is determined using a fluorescent
disaccharide derivative 4-methylumbelliferyl-.beta.-D-lactoside
(See e.g., van Tilbeurgh et al., FEBS Lett., 149:152-156 [1982];
and van Tilbeurgh and Claeyssens, FEBS Lett., 187:283-288
[1985]).
[0056] As used herein, the terms "CBH1" and "type 1
cellobiohydrolase" refer to a carbohydrate active enzyme expressed
from a nucleic acid sequence coding for a glycohydrolase (GH)
Family 7 catalytic domain classified under EC 3.2.1.91 or any
protein, polypeptide or catalytically active fragment thereof. In
some embodiments, the CBH1 is functionally linked to a carbohydrate
binding module (CBM), such as a Family 1 cellulose binding
domain.
[0057] As used herein, the terms "CBH2" and "type 2
cellobiohydrolase" refer to a carbohydrate active enzyme expressed
from a nucleic sequence coding for a glycohydrolase (GH) Family 6
catalytic domain classified under EC 3.2.1.91 or any protein,
polypeptide or catalytically active fragment thereof. Type 2
cellobiohydrolases are also commonly referred to as "the Ce16
family." In some embodiments, the CBH2 is functionally linked to a
carbohydrate binding module (CBM), such as a Family 1 cellulose
binding domain
[0058] As used herein, the terms "beta-glucosidase," "cellobiase,"
and "BGL" refers to a category of cellulases (EC 3.2.1.21) that
catalyze the hydrolysis of cellobiose to glucose. The term
"beta-glucosidase" is further defined herein as a beta-D-glucoside
glucohydrolase (E.C. 3.2.1.21), which catalyzes the hydrolysis of
terminal non-reducing beta-D-glucose residues with the release of
beta-D-glucose. Beta-glucosidase activity can be determined using
any suitable method (See e.g., J. Basic Microbiol., 42: 55-66
[2002]). One unit of beta-glucosidase activity is defined as 1.0
pmole 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.
[0059] As used herein, the term "glycoside hydrolase 61" and "GH61"
refers to a category of cellulases that enhance cellulose
hydrolysis when used in conjunction with one or more additional
cellulases. The GH61 family of cellulases is described, for
example, in the Carbohydrate Active Enzymes (CAZY) database (See
e.g., Harris et al., Biochem., 49(15):3305-16 [2010]).
[0060] A "hemicellulase" as used herein, refers to a polypeptide
that can catalyze hydrolysis of hemicellulose into small
polysaccharides such as oligosaccharides, or monomeric saccharides.
Hemicellulloses include xylan, glucuonoxylan, arabinoxylan,
glucomannan and xyloglucan. Hemicellulases include, for example,
the following: endoxylanases, beta-xylosidases,
alpha-L-arabinofuranosidases, alpha -D-glucuronidases, feruloyl
esterases, coumaroyl esterases, alpha-galactosidases,
beta-galactosidases, beta-mannanases, and beta-mannosidases.
[0061] As used herein, the terms "xylan degrading activity" and
"xylanolytic activity" are defined herein as 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) (See e.g., Biely and Puchard, J.
Sci. Food Agr. 86:1636-1647 [2006]; Spanikova and Biely, FEBS
Lett., 580:4597-4601 [2006]; and Herrmann et al., Biochem. J.,
321:375-381 [1997]).
[0062] Total xylan degrading activity can be measured by
determining the reducing sugars formed from various types of xylan,
including oat spelt, beechwood, and larchwood xylans, or by
photometric determination of dyed xylan fragments released from
various covalently dyed xylans. A common total xylanolytic activity
assay is based on production of reducing sugars from polymeric
4-O-methyl glucuronoxylan (See e.g., Bailey et al., J. Biotechnol.,
23:257-270 [1992]). In some embodiments, 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 (See e.g., Lever, Anal. Biochem., 47:273-279
[1972]).
[0063] As used herein the term "xylanase activity" refers to a
1,4-beta-D-xylan-xylohydrolase activity (E.C. 3.2.1.8) that
catalyzes the endo-hydrolysis of 1,4-beta-D-xylosidic linkages in
xylans. In some embodiments, xylanase activity is determined using
birchwood xylan as substrate. One unit of xylanase activity is
defined as 1.0 .mu.mole of reducing sugar (measured in glucose
equivalents; See e.g., Lever, Anal. Biochem., 47:273-279 [1972])
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.
[0064] As used herein, the term "beta-xylosidase activity" refers
to 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 In some embodiments of the present invention, one unit of
beta-xylosidase activity 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.
[0065] As used herein, the term "acetylxylan esterase activity"
refers to a carboxylesterase activity (EC 3.1.1.72) that catalyses
the hydrolysis of acetyl groups from polymeric xylan, acetylated
xylose, acetylated glucose, alpha-napthyl acetate, and
p-nitrophenyl acetate. In some embodiments 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.RTM. 20. One unit of acetylxylan esterase
activity is defined as the amount of enzyme capable of releasing 1
pmole of p-nitrophenolate anion per minute at pH 5, 25.degree.
C.
[0066] As used herein, the term "feruloyl esterase activity" refers
to a 4-hydroxy-3-methoxycinnamoyl-sugar hydrolase activity (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.
In some embodiments 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 activity equals the amount of enzyme capable of releasing
1 .mu.mole of p-nitrophenolate anion per minute at pH 5, 25.degree.
C.
[0067] As used herein, the term "alpha-glucuronidase activity"
refers to an alpha-D-glucosiduronate glucuronohydrolase activity
(EC 3.2.1.139) that catalyzes the hydrolysis of an
alpha-D-glucuronoside to D-glucuronate and an alcohol. One unit of
alpha-glucuronidase activity equals the amount of enzyme capable of
releasing 1 pmole of glucuronic or 4-O-methylglucuronic acid per
minute at pH 5, 40.degree. C. (See e.g., de Vries, J. Bacteriol.,
180:243-249 [1998]).
[0068] As used herein the term "alpha-L-arabinofuranosidase
activity" refers to an alpha-L-arabinofuranoside
arabinofuranohydrolase activity (EC 3.2.1.55) that catalyzes the
hydrolysis of terminal non-reducing alpha-L-arabinofuranoside
residues in alpha-L-arabinosides. The enzyme activity 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, arabinofuranosidase, polysaccharide
alpha-L-arabinofuranosidase, alpha-L-arabinofuranoside hydrolase,
L-arabinosidase and 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).
[0069] Enzymatic lignin depolymerization can be accomplished by
lignin peroxidases, manganese peroxidases, laccases and cellobiose
dehydrogenases (CDH), often working in synergy. These extracellular
enzymes, essential for lignin degradation, are often referred to as
"lignin-modifying enzymes" or "LMEs." Three of these enzymes
comprise two glycosylated heme-containing peroxidases: lignin
peroxidase (LIP); Mn-dependent peroxidase (MNP); and, a
copper-containing phenoloxidase laccase (LCC). Although the details
of the reaction scheme of lignin biodegradation are not fully
understood to date, without being bound by theory, it is suggested
that these enzymes employ free radicals for depolymerization
reactions.
[0070] As used herein, the term "laccase" refers to the copper
containing oxidase enzymes that are found in many plants, fungi and
microorganisms. Laccases are enzymatically active on phenols and
similar molecules and perform a one electron oxidation. Laccases
can be polymeric and the enzymatically active form can be a dimer
or trimer.
[0071] As used herein, the term "Mn-dependent peroxidase" refers to
peroxidases that require Mn. The enzymatic activity of Mn-dependent
peroxidase (MnP) in is dependent on Mn.sup.2+. Without being bound
by theory, it has been suggested that the main role of this enzyme
is to oxidize Mn.sup.2+ to Mn.sup.3+ (See e.g., Glenn et al. Arch.
Biochem. Biophys., 251:688-696 [1986]). Subsequently, phenolic
substrates are oxidized by the Mn.sup.3+ generated.
[0072] As used herein, the term "lignin peroxidase" refers to an
extracellular heme that catalyses the oxidative depolymerization of
dilute solutions of polymeric lignin in vitro. Some of the
substrates of LiP, most notably 3,4-dimethoxybenzyl alcohol
(veratryl alcohol, VA), are active redox compounds that have been
shown to act as redox mediators. VA is a secondary metabolite
produced at the same time as LiP by ligninolytic cultures of P.
chrysosporium and without being bound by theory, has been proposed
to function as a physiological redox mediator in the LiP-catalysed
oxidation of lignin in vivo (See e.g., Harvey et al., FEBS Lett.
195:242-246 [1986]).
[0073] As used herein, the term "glucoamylase" (EC 3.2.1.3) refers
to enzymes that catalyze the release of D-glucose from non-reducing
ends of oligo- and poly-saccharide molecules. Glucoamylase is also
generally considered a type of amylase known as
amylo-gludosidase.
[0074] As used hererin, the term "amylase" (EC 3.2.1.1) refers to
starch cleaving enzymes that degrade starch and related compounds
by hydrolyzing the alpha-1,4 and/or alpha-1,6 glucosidic linkages
in an endo- or an exo-acting fashion. Amylases include
alpha-amylases (EC 3.2.1.1); beta-amylases (3.2.1.2),
amylo-amylases (EC 3.2.1.3), alpha-glucosidases (EC 3.2.1.20),
pullulanases (EC 3.2.1.41), and isoamylases (EC 3.2.1.68). In some
embodiments, the amylase is an alpha-amylase.
[0075] As used herein, the term "pectinase" refers to enzymes that
catalyze the hydrolysis of pectin into smaller units such as
oligosaccharide or monomeric saccharides. In some embodiments, the
enzyme mixtures comprise any pectinase, for example an
endo-polygalacturonase, a pectin methyl esterase, an
endo-galactanase, a pectin acetyl esterase, an endo-pectin lyase,
pectate lyase, alpha rhamnosidase, an exo-galacturonase, an
exo-polygalacturonate lyase, a rhamnogalacturonan hydrolase, a
rhamnogalacturonan lyase, a rhamnogalacturonan acetyl esterase, a
rhamnogalacturonan galacturonohydrolase and/or a
xylogalacturonase.
[0076] As used herein, the term "endo-polygalacturonase" (EC
3.2.1.15) refers to enzymes that catalyze the random hydrolysis of
1,4-alpha-D-galactosiduronic linkages in pectate and other
galacturonans. This enzyme may also be referred to as
"polygalacturonase pectin depolymerase," "pectinase,"
"endopolygalacturonase," "pectolase," "pectin hydrolase," "pectin
polygalacturonase," "poly-alpha-1,4-galacturonide
glycanohydrolase," "endogalacturonase," "endo-D-galacturonase," or"
poly(1,4-alpha-D-galacturonide) glycanohydrolase."
[0077] As used herein, the term "pectin methyl esterase" (EC
3.1.1.11) refers to enzymes that catalyze the reaction: pectin+n
H.sub.2O=n methanol+pectate. The enzyme may also been known as
"pectin esterase," "pectin demethoxylase," "pectin methoxylase,"
"pectin methylesterase," "pectase," "pectinoesterase," or" pectin
pectylhydrolase."
[0078] As used herein, the term "endo-galactanase" (EC 3.2.1.89)
refers to enzymes that catalyze the endohydrolysis of
1,4-beta-D-galactosidic linkages in arabinogalactans. The enzyme
may also be known as "arabinogalactan endo-1,4-beta-galactosidase,"
"endo-1,4-beta-galactanase," galactanase," "arabinogalactanase," or
"arabinogalactan 4-.beta.-D-galactanohydrolase."
[0079] As used herein, the term "pectin acetyl esterase" refers to
enzymes that catalyze the deacetylation of the acetyl groups at the
hydroxyl groups of GaIUA residues of pectin.
[0080] As used herein, the term "one endo-pectin lyase" (EC
4.2.2.10) refers to enzymes that catalyze the eliminative cleavage
of (1.fwdarw.4)-alpha-D-galacturonan methyl ester to give
oligosaccharides with
4-deoxy-6-O-methyl-alpha-D-galact-4-enuronosyl groups at their
non-reducing ends. The enzyme may also be known as "pectin lyase,"
"pectin trans-eliminase." "endo-pectin lyase,"
"polymethylgalacturonic transeliminase," "pectin
methyltranseliminase," "pectolyase," "PL," "PNL," " PMGL," or
"(1.fwdarw.4)-6-O-methyl-.alpha.-D-galacturonan lyase."
[0081] As used herein, the term "pectate lyase" (EC 4.2.2.2) refers
to enzymes that catalyze the eliminative cleavage of
(1.fwdarw.4)-alpha-D-galacturonan to give oligosaccharides with
4-deoxy-alpha-D-galact-4-enuronosyl groups at their non-reducing
ends. The enzyme may also be known as "polygalacturonic
transeliminase," "pectic acid transeliminase," "polygalacturonate
lyase," "endopectin methyltranseliminase," "pectate
transeliminase," "endogalacturonate transeliminase," "pectic acid
lyase," "pectic lyase," "alpha-1,4-D-endopolygalacturonic acid
lyase," "PGA lyase," "PPase-N," "endo-alpha-1,4-polygalacturonic
acid lyase," "polygalacturonic acid lyase," "pectin
trans-eliminase," "polygalacturonic acid trans-eliminase," or
"(1.fwdarw.4)-alpha-D-galacturonan lyase."
[0082] As used herein, the term "alpha-rhamnosidase" (EC 3.2.1.40)
refers to enzymes that catalyze the hydrolysis of terminal
non-reducing alpha-L-rhamnose residues in alpha-L-rhamnosides or
alternatively in rhamnogalacturonan. This enzyme may also be known
as "alpha-L-rhamnosidase T," "alpha-L-rhamnosidase N," or
"alpha-L-rhamnoside rhamnohydrolase."
[0083] As used herein, the term "exo-galacturonase" (EC 3.2.1.82)
refers to enzymes that hydrolyze pectic acid from the non-reducing
end, releasing digalacturonate. The enzyme may also be known as
"exo-poly-alpha-galacturonosidase," "exopolygalacturonosidase," or
"exopolygalacturanosidase."
[0084] As used herein, the term "exo-galacturan 1,4-alpha
galacturonidase" (EC 3.2.1.67) refers to enzymes that catalyze
reactions of the following types:
(1,4-alpha-D-galacturonide)n+H2O=(1,4-alpha-D-galacturonide)n-i+D--
galacturonate. The enzyme may also be known as "poly [1->4)
alpha-D-galacturonide] galacturonohydrolase,"
"exopolygalacturonase," "poly(galacturonate) hydrolase,"
"exo-D-galacturonase," "exo-D-galacturonanase,"
"exopoly-D-galacturonase," or "poly(1,4-alpha-D-galacturonide)
galacturonohydrolase."
[0085] As used herein, the term "exopolygalacturonate lyase" (EC
4.2.2.9) refers to enzymes that catalyze eliminative cleavage of
4-(4-deoxy-.alpha.-D-galact-4-enuronosyl)-D-galacturonate from the
reducing end of pectate (i.e. de-esterified pectin). This enzyme
may be known as "pectate disaccharide-lyase," "pectate exo-lyase,"
"exopectic acid transeliminase," "exopectate lyase,"
"exopolygalacturonic acid-trans-eliminase," "PATE," "exo-PATE,"
"exo-PGL," or "(1.fwdarw.4)-alpha-D-galacturonan
reducing-end-disaccharide-lyase."
[0086] As used herein, the term "rhamnogalacturonanase" refers to
enzymes that hydrolyze the linkage between galactosyluronic acid
and rhamnopyranosyl in an endo-fashion in strictly alternating
rhamnogalacturonan structures, consisting of the disaccharide
[(1,2-alpha-L-rhamnoyl-(1,4)-alpha-galactosyluronic acid].
[0087] As used herein, the term "rhamnogalacturonan lyase" refers
to enzymes that cleave alpha-L-Rhap-(1.fwdarw.4)-alpha-D-GalpA
linkages in an endo-fashion in rhamnogalacturonan by
beta-elimination.
[0088] As used herein, the term "rhamnogalacturonan acetyl
esterase" refers to enzymes that catalyze the deacetylation of the
backbone of alternating rhamnose and galacturonic acid residues in
rhamnogalacturonan.
[0089] As used herein, the term "rhamnogalacturonan
galacturonohydrolase" refers to enzymes that hydrolyze galacturonic
acid from the non-reducing end of strictly alternating
rhamnogalacturonan structures in an exo-fashion. This enzyme may
also be known as "xylogalacturonan hydrolase."
[0090] As used herein, the term "endo-arabinanase" (EC 3.2.1.99)
refers to enzymes tha catalyze endohydrolysis of
1,5-alpha-arabinofuranosidic linkages in 1,5-arabinans. The enzyme
may also be known as "endo-arabinase," "arabinan
endo-1,5-.alpha.-L-arabinosidase," "endo-1,5-alpha-L-arabinanase,"
"endo-alpha-1,5-arabanase," "endo-arabanase," or
"1,5-alpha-L-arabinan 1,5-alpha-L-arabinanohydrolase."
[0091] As used herein, "protease" includes enzymes that hydrolyze
peptide bonds (peptidases), as well as enzymes that hydrolyze bonds
between peptides and other moieties, such as sugars
(glycopeptidases). Many proteases are characterized under EC 3.4,
and are suitable for use in the present invention. Some specific
types of proteases include but are not limited to, cysteine
proteases including pepsin, papain and serine proteases including
chymotrypsins, carboxypeptidases and metalloendopeptidases.
[0092] As used herein, "lipase" includes enzymes that hydrolyze
lipids, fatty acids, and acylglycerides, including
phosphoglycerides, lipoproteins, diacylglycerols, and the like. In
plants, lipids are used as structural components to limit water
loss and pathogen infection. These lipids include waxes derived
from fatty acids, as well as cutin and suberin.
[0093] As used herein, the terms "isolated" and "purified" are used
to refer to a molecule (e.g., an isolated nucleic acid, polypeptide
[including, but not limited to enzymes], etc.) or other component
that is removed from at least one other component with which it is
naturally associated. Thus, the terms refer to a material that is
removed from its original environment (e.g., the natural
environment, if it is naturally occurring). It is intended that the
term encompass any suitable method for removing at least one
component with which the molecule is naturally associated. In some
embodiments, the terms also encompass cells that are separated from
other cells and/or media components. It is intended that any
suitable separation method finds use in the present invention. In
some embodiments, a material is said to be "purified" when it is
present in a particular composition in a higher or lower
concentration than exists in a naturally-occurring or wild-type
organism or in combination with components not normally present
upon expression from a naturally-occurring or wild-type organism.
For example, a naturally-occurring polynucleotide or polypeptide
present in a living animal is not isolated, but the same
polynucleotide or polypeptide, separated from some or all of the
coexisting materials in the natural system, is isolated. In some
embodiments, such polynucleotides are part of a vector, and/or such
polynucleotides or polypeptides are part of a composition, and
still considered to be isolated, in that such vector or composition
is not part of its natural environment. In some embodiments, a
nucleic acid or protein is said to be purified, for example, if it
gives rise to essentially one band in an electrophoretic gel or
blot.
[0094] The term "isolated," when used in reference to a DNA
sequence, refers to a DNA sequence that has been removed from its
natural genetic milieu and is thus free of other extraneous or
unwanted coding sequences, and is in a form suitable for use within
genetically engineered protein production systems. Such isolated
molecules are those that are separated from their natural
environment and include cDNA and genomic clones. Isolated DNA
molecules of the present invention are free of other genes with
which they are ordinarily associated, but may include naturally
occurring 5' and 3' untranslated regions (e.g., promoters and
terminators). The identification of associated regions will be
evident to one of ordinary skill in the art (See e.g., Dynan and
Tijan, Nature 316:774-78 [1985]). The term "an isolated DNA
sequence" is alternatively referred to as "a cloned DNA
sequence."
[0095] The term "isolated," when used in reference to a protein,
refers to a protein that is found in a condition other than its
native environment. In some embodiments, the isolated protein is
substantially free of other proteins, particularly other homologous
proteins. An isolated protein is more than about 10% pure,
preferably more than about 20% pure, and even more preferably more
than about 30% pure, as determined by SDS-PAGE. Further aspects of
the invention encompass the protein in a highly purified form
(i.e., more than about 40% pure, more than about 60% pure, more
than about 70% pure, more than about 80% pure, more than about 90%
pure, more than about 95% pure, more than about 97% pure, more than
about 98% pure, or even more than about 99% pure), as determined by
SDS-PAGE.
[0096] By "purification" or "isolation," when used in reference to
the cellobiose dehydrogenase, it is meant that the cellobiose
dehydrogenase is altered from its natural state by virtue of
separating the cellobiose dehydrogenase from some or all of the
naturally occurring constituents with which it is associated in
nature. This may be accomplished by any suitable method, including
art recognized separation techniques, including but not limited to
ion exchange chromatography, affinity chromatography, hydrophobic
separation, dialysis, protease treatment, ammonium sulphate
precipitation or other protein salt precipitation, centrifugation,
size exclusion chromatography, filtration, microfiltration, gel
electrophoresis, separation on a gradient or any other suitable
methods, to remove whole cells, cell debris, impurities, extraneous
proteins, or enzymes undesired in the final composition. It is
further possible to then add constituents to the cellobiose
dehydrogenase-containing composition which provide additional
benefits, for example, activating agents, anti-inhibition agents,
desirable ions, compounds to control pH, other enzymes, etc.
[0097] As used herein, the phrase "substantially pure polypeptide"
refers to a composition in which the polypeptide species is the
predominant species present (i.e., on a molar or weight basis, it
is more abundant than any other individual macromolecular species
in the composition), and is generally a substantially purified
composition when the object species comprises at least about 50
percent of the macromolecular species present by mole or % weight.
Generally, a substantially pure enzyme composition will comprise
about 60% or more, about 70% or more, about 80% or more, about 90%
or more, about 95% or more, or about 98% or more of all
macromolecular species by mole or % weight present in the
composition. Solvent species, small molecules (<500 Daltons),
and elemental ion species are not considered macromolecular
species.
[0098] As used herein, the term "purification process" used in
reference to an enzyme mixture encompasses any process that
physically removes an undesired component of the enzyme mixture.
Thus, in some embodiments, purification processes provided herein
include purification methodologies that physically remove
cellobiose oxidizing activity the enzyme mixture or vice versa. It
is contemplated that any suitable purification process known in the
art will find use in the present invention. Indeed, it is not
intended that the present invention be limited to any particular
purification process.
[0099] As used herein, the term "cell-free enzyme mixture"
comprises enzymes that have been separated from any cells,
including the cells that secreted the enzymes. Cell-free enzyme
mixtures can be prepared by any of a variety of methodologies that
are known in the art, such as filtration or centrifugation
methodologies. In some embodiments, the enzyme mixture can be, for
example, partially cell-free, substantially cell-free, or entirely
cell-free.
[0100] As used herein, "polynucleotide" refers to a polymer of
deoxyribonucleotides or ribonucleotides in either single- or
double-stranded form, and complements thereof.
[0101] A polynucleotide is said to "encode" an RNA or a polypeptide
if, in its native state or when manipulated by methods known to
those of skill in the art, it can be transcribed and/or translated
to produce the RNA, the polypeptide or a fragment thereof. The
anti-sense strand of such a nucleic acid is also said to encode the
sequences. As is known in the art, DNA can be transcribed by an RNA
polymerase to produce RNA, but RNA can be reverse transcribed by
reverse transcriptase to produce a DNA. Thus, a DNA molecule can
effectively encode an RNA molecule and vice versa.
[0102] The terms "protein" and "polypeptide" are used
interchangeably herein to refer to a polymer of amino acid
residues. In addition, the terms "amino acid" "polypeptide," and
"peptide" encompass naturally-occurring and synthetic amino acids,
as well as amino acid analogs. Naturally occurring amino acids are
those encoded by the genetic code, as well as those amino acids
that are later modified (e.g., hydroxyproline, y-carboxyglutamate,
and O-phosphoserine).
[0103] As used herein, "protein of interest" and "polypeptide of
interest" refer to a protein/polypeptide that is desired and/or
being assessed. In some embodiments, the protein of interest is
expressed intracellularly, while in other embodiments, it is a
secreted polypeptide. In some embodiments, the "protein of
interest" or "polypeptide of interest" includes the enzymes of the
present invention. In some embodiments, the protein of interest is
a secreted polypeptide which is fused to a signal peptide (i.e., an
amino-terminal extension on a protein to be secreted). Nearly all
secreted proteins use an amino-terminal protein extension which
plays a crucial role in the targeting to and translocation of
precursor proteins across the membrane. This extension is
proteolytically removed by a signal peptidase during or immediately
following membrane transfer.
[0104] As used herein, the term "amino acid analogs" refers to
compounds that have the same basic chemical structure as a
naturally occurring amino acid (i.e., an a-carbon that is bound to
a hydrogen, a carboxyl group, an amino group, and an R group,
including but not limited to homoserine, norleucine, methionine
sulfoxide, and methionine methyl sulfonium). In some embodiments,
these analogs have modified R groups (e.g., norleucine) and/or
modified peptide backbones, but retain the same basic chemical
structure as a naturally occurring amino acid.
[0105] Amino acids are referred to herein by either their commonly
known three letter symbols or by the one-letter symbols recommended
by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides,
likewise, may be referred to by their commonly accepted
single-letter codes.
[0106] An amino acid or nucleotide base "position" is denoted by a
number that sequentially identifies each amino acid (or nucleotide
base) in the reference sequence based on its position relative to
the N-terminus (or 5'-end). Due to deletions, insertions,
truncations, fusions, and the like that must be taken into account
when determining an optimal alignment, the amino acid residue
number in a test sequence determined by simply counting from the
N-terminus will not necessarily be the same as the number of its
corresponding position in the reference sequence. For example, in a
case where a test sequence has a deletion relative to an aligned
reference sequence, there will be no amino acid in the variant that
corresponds to a position in the reference sequence at the site of
deletion. Where there is an insertion in an aligned test sequence,
that insertion will not correspond to a numbered amino acid
position in the reference sequence. In the case of truncations or
fusions there can be stretches of amino acids in either the
reference or aligned sequence that do not correspond to any amino
acid in the corresponding sequence.
[0107] The terms "wild-type sequence" and "naturally-occurring
sequence" are used interchangeably herein, to refer to a
polypeptide or polynucleotide sequence that is native or naturally
occurring in a host cell. In some embodiments, the wild-type
sequence refers to a sequence of interest that is the starting
point of a protein engineering project. The wild-type sequence may
encode either a homologous or heterologous protein. A homologous
protein is one the host cell would produce without intervention. A
heterologous protein is one that the host cell would not produce
but for intervention.
[0108] As used herein, "naturally-occurring enzyme" refers to an
enzyme having the unmodified amino acid sequence identical to that
found in nature (i.e., "wild-type"). Naturally occurring enzymes
include native enzymes (i.e., those enzymes naturally expressed or
found in the particular microorganism).
[0109] As used herein, the term "reference enzyme" refers to an
enzyme to which another enzyme of the present invention (e.g., a
"test" enzyme) is compared in order to determine the presence of an
improved property in the other enzyme being evaluated. In some
embodiments, a reference enzyme is a wild-type enzyme. In some
embodiments, the reference enzyme is an enzyme to which a test
enzyme of the present invention is compared in order to determine
the presence of an improved property in the test enzyme being
evaluated, including but not limited to improved thermoactivity,
improved thermostability, and/or improved stability. In some
embodiments, a reference enzyme is a wild-type enzyme.
[0110] As used herein, the term "biologically active fragment,"
refers to a polypeptide that has an amino-terminal and/or
carboxy-terminal deletion(s) and/or internal deletion(s), but where
the remaining amino acid sequence is identical to the corresponding
positions in the sequence to which it is being compared and that
retains substantially all of the activity of the full-length
polypeptide.
[0111] As used herein, the term "recombinant" refers to a
polynucleotide or polypeptide that does not naturally occur in a
host cell. In some embodiments, recombinant molecules contain two
or more naturally-occurring sequences that are linked together in a
way that does not occur naturally. In some embodiments,
"recombinant cells" express genes that are not found in identical
form within the native (i.e., non-recombinant) form of the cell
and/or express native genes that are otherwise abnormally
over-expressed, under-expressed, and/or not expressed at all due to
deliberate human intervention. Recombinant cells contain at least
one recombinant polynucleotide or polypeptide. A nucleic acid
construct, nucleic acid (e.g., a polynucleotide), polypeptide, or
host cell is referred to herein as "recombinant" when it is
non-naturally occurring, artificial or engineered. "Recombination,"
"recombining" and generating a "recombined" nucleic acid generally
encompass the assembly of at least two nucleic acid fragments. The
present invention also provides a recombinant nucleic acid
construct comprising at least one CDH polynucleotide sequence that
hybridizes under stringent hybridization conditions to the
complement of a polynucleotide which encodes a polypeptide having
the amino acid sequence of SEQ ID NO:2.
[0112] Nucleic acids "hybridize" when they associate, typically in
solution. Nucleic acids hybridize due to a variety of
well-characterized physico-chemical forces, such as hydrogen
bonding, solvent exclusion, base stacking and the like. As used
herein, the term "stringent hybridization wash conditions" in the
context of nucleic acid hybridization experiments, such as Southern
and Northern hybridizations, are sequence dependent, and are
different under different environmental parameters. An extensive
guide to the hybridization of nucleic acids is found in Tijssen,
1993, "Laboratory Techniques in Biochemistry and Molecular
Biology-Hybridization with Nucleic Acid Probes," Part I, Chapter 2
(Elsevier, N.Y.), which is incorporated herein by reference. For
polynucleotides of at least 100 nucleotides in length, low to very
high stringency conditions are defined as follows: prehybridization
and hybridization at 42.degree. C. in 5.times.SSPE, 0.3% SDS, 200
.mu.g/ml sheared and denatured salmon sperm DNA, and either 25%
formamide for low stringencies, 35% formamide for medium and
medium-high stringencies, or 50% formamide for high and very high
stringencies, following standard Southern blotting procedures. For
polynucleotides of at least 100 nucleotides in length, the carrier
material is finally washed three times each for 15 minutes using
2.times.SSC, 0.2% SDS 50.degree. C. (low stringency), at 55.degree.
C. (medium stringency), at 60.degree. C. (medium-high stringency),
at 65.degree. C. (high stringency), or at 70.degree. C. (very high
stringency).
[0113] Moderately stringent conditions encompass those known in the
art and described in various standard texts and include the use of
washing solution and hybridization conditions (e.g., temperature,
ionic strength and % SDS). An example of moderately stringent
conditions involves overnight incubation at 37.degree. C. in a
solution comprising: 20% formamide, 5.times.SSC (150 mM NaCl, 15 mM
trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5.times.
Denhardt's solution, 10% dextran sulfate, and 20 mg/mL denatured
sheared salmon sperm DNA, followed by washing the filters in
1.times.SSC at about 37-50.degree. C. The skilled artisan will
recognize how to adjust the temperature, ionic strength, etc. as
necessary to accommodate factors such as probe length and the
like.
[0114] As used in some embodiments herein, stringent conditions or
high stringency conditions utilize: (1) low ionic strength and high
temperature for washing, for example 0.015 M sodium chloride/0.0015
M sodium citrate/0.1% sodium dodecyl sulfate at 50.degree. C.; (2)
during hybridization a denaturing agent, such as formamide, for
example, 50% (v/v) formamide with 0.1% bovine serum albumin/0 1%
Ficol1/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at
pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at
42.degree. C.; or (3) 50% formamide, 5.times.SSC (0.75 M NaCl,
0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1%
sodium pyrophosphate, 5.times. Denhardt's solution, sonicated
salmon sperm DNA (50 .mu.g/mL), 0.1% SDS, and 10% dextran sulfate
at 42.degree. C., with washes at 42.degree. C. in 0.2.times.SSC
(sodium chloride/sodium citrate) and 50% formamide at 55.degree.
C., followed by a high-stringency wash consisting of 0.1.times.SSC
containing EDTA at 55.degree. C.
[0115] As used herein, the terms "library of mutants" and "library
of variants" used in reference to cells, refer to a population of
cells which are identical in most of their genome but include
different homologues of one or more genes. Such libraries can be
used, for example, to identify genes or operons with improved
traits. When used in reference to polypeptides or nucleic acids,
"library" refers to a set (i.e., a plurality) of heterogeneous
polypeptides or nucleic acids. A library is composed of "members."
Sequence differences between library members are responsible for
the diversity present in the library. The library may take the form
of a simple mixture of polypeptides or nucleic acids, or may be in
the form of organisms or cells, for example bacteria, viruses,
animal or plant cells and the like, transformed with a library of
nucleic acids.
[0116] As used herein, the term "starting gene" refers to a gene of
interest that encodes a protein of interest that is to be improved,
deleted, mutated, and/or otherwise changed using the present
invention.
[0117] The term "property" and grammatical equivalents thereof in
the context of a nucleic acid, as used herein, refer to any
characteristic or attribute of a nucleic acid that can be selected
or detected. These properties include, but are not limited to, a
property affecting binding to a polypeptide, a property conferred
on a cell comprising a particular nucleic acid, a property
affecting gene transcription (e.g., promoter strength, promoter
recognition, promoter regulation, and/or enhancer function), a
property affecting RNA processing (e.g., RNA splicing, RNA
stability, RNA conformation, and/or post-transcriptional
modification), a property affecting translation (e.g., level,
regulation, binding of mRNA to ribosomal proteins, and/or
post-translational modification). For example, a binding site for a
transcription factor, polymerase, regulatory factor, etc., of a
nucleic acid may be altered to produce desired characteristics or
to identify undesirable characteristics.
[0118] The term "property" and grammatical equivalents thereof in
the context of a polypeptide (including proteins), as used herein,
refer to any characteristic or attribute of a polypeptide that can
be selected or detected. These properties include, but are not
limited to oxidative stability, substrate specificity, catalytic
activity, thermal stability, alkaline stability, pH activity
profile, resistance to proteolytic degradation, k.sub.m, k.sub.cat,
k.sub.cat/k.sub.m ratio, protein folding, inducing an immune
response, not inducing an immune response, ability to bind to a
ligand, ability to bind to a receptor, ability to be secreted,
ability to be displayed on the surface of a cell, ability to
oligomerize, ability to signal, ability to stimulate cell
proliferation, ability to inhibit cell proliferation, ability to
induce apoptosis, ability to be modified by phosphorylation or
glycosylation, and/or ability to treat disease, etc. Indeed, it is
not intended that the present invention be limited to any
particular property.
[0119] As used herein, "similarity" refers to an identical or
conservative amino acid substitution thereof as defined below.
Accordingly, a change to an identical or conservative substitution
for the purposes of similarity is viewed as not comprising a
change. A deletion of an amino acid or a non-conservative amino
acid substitution is viewed herein as comprising a change.
Calculation of percent similarity is performed in the same manner
as performed for percent identity.
[0120] As used herein, "conservative substitution," as used with
respect to amino acids, refers to the substitution of an amino acid
with a chemically similar amino acid Amino acid substitutions that
do not generally alter the specific activity are well known in the
art and are described in numerous textbooks. The most commonly
occurring exchanges are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser,
Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro,
Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly, as well
as these in reverse. As used herein, a conservative substitute for
a residue is another residue in the same group.
[0121] In some embodiments, conservative amino acid substitution
can be a substitution such as the conservative substitutions shown
in Table A. The substitutions shown are based on amino acid
physical-chemical properties, and as such, are independent of
organism. In some embodiments, the conservative amino acid
substitution is a substitution listed under the heading of
exemplary substitutions.
TABLE-US-00001 TABLE A Substitutions Original Residue Conservative
Substitutions Exemplary Substitutions Ala (A) val; leu; ile Val Arg
(R) lys; gln; asn Lys Asn (N) gln; his; lys; arg Gln Asp (D) Glu
Glu Cys (C) Ser Ser Gln (Q) Asn Asn Glu (E) Asp Asp Gly (G) pro;
ala Ala His (H) asn; gln; lys; arg Arg Ile (I) leu; val; met; ala;
phe Leu Leu (L) ile; val; met; ala; phe Ile Lys (K) arg; gln; asn
Arg Met (M) leu; phe; ile Leu Phe (F) leu; val; ile; ala; tyr Leu
Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Ser Ser Trp (W) tyr; phe
Tyr Tyr (Y) trp; phe; thr; ser Phe Val (V) ile; leu; met; phe; ala
Leu
[0122] As used herein, the terms "numbered with reference to" or
"corresponding to," when used in the context of the numbering of a
given amino acid or polynucleotide sequence, refers to the
numbering of the residues of a specified reference sequence when
the given amino acid or polynucleotide sequence is compared to the
reference sequence.
[0123] The following nomenclature may be used to describe
substitutions in a reference sequence relative to a reference
sequence or a variant polypeptide or nucleic acid sequence:
"R-#-V," where # refers to the position in the reference sequence,
R refers to the amino acid (or base) at that position in the
reference sequence, and V refers to the amino acid (or base) at
that position in the variant sequence.
[0124] The term "amino acid substitution set" or "substitution set"
refers to a group of amino acid substitutions. A substitution set
can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or
more amino acid substitutions.
[0125] As used herein, "identity" and "percent identity," in the
context of two or more polypeptide sequences, refers to two or more
sequences or subsequences that are the same or have a specified
percentage of amino acid residues that are the same (e.g., share at
least about 70%, at least about 75%, at least about 80%, at least
about 85%, at least about 88% identity, at least about 89%, at
least about 90%, at least about 91%, at least about 92%, at least
about 93%, at least about 94%, at least about 95%, at least about
96%, at least about 97%, at least about 98%, or at least about 99%
identity) over a specified region to a reference sequence, when
compared and aligned for maximum correspondence over a comparison
window, or designated region as measured using a sequence
comparison algorithms or by manual alignment and visual inspection.
In some embodiments, the terms "percent identity," "% identity",
"percent identical," and "% identical," are used interchangeably
herein to refer to the percent amino acid or polynucleotide
sequence identity that is obtained by ClustalW analysis (version W
1.8 available from European Bioinformatics Institute, Cambridge,
UK), counting the number of identical matches in the alignment and
dividing such number of identical matches by the length of the
reference sequence, and using the following ClustalW parameters to
achieve slow/more accurate pairwise optimal alignments--DNA/Protein
Gap Open Penalty:15/10; DNA/Protein Gap Extension Penalty:6.66/0.1;
Protein weight matrix: Gonnet series; DNA weight matrix:
Identity.
[0126] Two sequences are "aligned" when they are aligned for
similarity scoring using a defined amino acid substitution matrix
(e.g., BLOSUM62), gap existence penalty and gap extension penalty
so as to arrive at the highest score possible for that pair of
sequences Amino acid substitution matrices and their use in
quantifying the similarity between two sequences are well known in
the art (See, e.g., Dayhoff et al., in Dayhoff [ed.], Atlas of
Protein Sequence and Structure," Vol. 5, Suppl. 3, Natl. Biomed.
Res. Round., Washington D.C. [1978]; pp. 345-352; and Henikoff et
al., Proc. Natl. Acad. Sci. USA, 89:10915-10919 [1992], both of
which are incorporated herein by reference). The BLOSUM62 matrix is
often used as a default scoring substitution matrix in sequence
alignment protocols such as Gapped BLAST 2.0. The gap existence
penalty is imposed for the introduction of a single amino acid gap
in one of the aligned sequences, and the gap extension penalty is
imposed for each additional empty amino acid position inserted into
an already opened gap. The alignment is defined by the amino acid
position of each sequence at which the alignment begins and ends,
and optionally by the insertion of a gap or multiple gaps in one or
both sequences so as to arrive at the highest possible score. While
optimal alignment and scoring can be accomplished manually, the
process is facilitated by the use of a computer-implemented
alignment algorithm (e.g., gapped BLAST 2.0; See, Altschul et al.,
Nucleic Acids Res., 25:3389-3402 [1997], which is incorporated
herein by reference), and made available to the public at the
National Center for Biotechnology Information Website). Optimal
alignments, including multiple alignments can be prepared using
readily available programs such as PSI-BLAST (See e.g., Altschul et
al., supra).
[0127] The present invention also provides a recombinant nucleic
acid construct comprising a CDH polynucleotide sequence that
hybridizes under stringent hybridization conditions to the
complement of a polynucleotide which encodes a polypeptide having
the amino acid sequence of SEQ ID NO:2. Two nucleic acid or
polypeptide sequences that have 100% sequence identity are said to
be "identical." A nucleic acid or polypeptide sequence is said to
have "substantial sequence identity" to a reference sequence when
the sequences have at least about 70%, at least about 75%, at least
about 80%, at least about 85%, at least about 90%, at least about
91%, at least about 92%, at least about 93%, at least about 94%, at
least about 95%, at least about 96%, at least about 97%, at least
about 98%, or at least about 99%, or greater sequence identity as
determined using the methods described herein, such as BLAST using
standard parameters.
[0128] As used herein, a "secretion signal peptide" can be a
propeptide, a prepeptide or both. For example, the term
"propeptide" refers to a protein precursor that is cleaved to yield
a "mature protein." The signal peptide is cleaved from the
pre-protein by a signal peptidase prior to secretion to result in
the "mature" or "secreted" protein. The terms "prepeptide" ad
"pre-protein" refer to a polypeptide synthesized with an N-terminal
signal peptide that targets it for secretion. Accordingly, a
"pre-pro-peptide" is a polypeptide that contains a signal peptide
that targets the polypeptide for secretion and which is cleaved off
to yield a mature polypeptide. Signal peptides are found at the
N-terminus of the protein and are typically composed of between 6
to 136 basic and hydrophobic amino acids.
[0129] As used herein, "transcription" and like terms refer to the
conversion of the information encoded in a gene to an RNA
transcript. Accordingly, a reduction of the transcription level of
a cellobiose oxidizing enzyme is a reduction in the amount of RNA
transcript of an RNA coding for a cellobiose oxidizing enzyme.
[0130] As used herein, the terms "DNA construct" and "transforming
DNA" are used interchangeably to refer to DNA used to introduce
sequences into a host cell or organism. The DNA may be generated in
vitro by PCR or any other suitable technique(s) known to those in
the art. In some embodiments, the DNA construct comprises a
sequence of interest (e.g., as an "incoming sequence"). In some
embodiments, the sequence is operably linked to additional elements
such as control elements (e.g., promoters, etc.). In some
embodiments, the DNA construct further comprises at least one
selectable marker. In some further embodiments, the DNA construct
comprises an incoming sequence flanked by homology boxes. In some
further embodiments, the transforming DNA comprises other
non-homologous sequences, added to the ends (e.g., stuffer
sequences or flanks). In some embodiments, the ends of the incoming
sequence are closed such that the transforming DNA forms a closed
circle. The transforming sequences may be wild-type, mutant or
modified. In some embodiments, the DNA construct comprises
sequences homologous to the host cell chromosome. In some other
embodiments, the DNA construct comprises non-homologous sequences.
Once the DNA construct is assembled in vitro, it may be used to: 1)
insert heterologous sequences into a desired target sequence of a
host cell; 2) mutagenize a region of the host cell chromosome
(i.e., replace an endogenous sequence with a heterologous
sequence); 3) delete target genes; and/or 4) introduce a
replicating plasmid into the host. In some embodiments, the
incoming sequence comprises at least one selectable marker. This
sequence can code for one or more proteins of interest. It can have
other biological functions. In many cases the incoming sequence
comprises at least one selectable marker, such as a gene that
confers antimicrobial resistance.
[0131] As used herein, a "vector" is a polynucleotide construct for
introducing a polynucleotide sequence into a cell. In some
embodiments, the vector comprises a suitable control sequence
operably linked to and capable of effecting the expression of the
polypeptide encoded in the polynucleotide sequence in a suitable
host. An "expression vector" has a promoter sequence operably
linked to the polynucleotide sequence (e.g., transgene) to drive
expression in a host cell, and in some embodiments a transcription
terminator sequence. In some embodiments, the vectors are deletion
vectors. In some embodiments, vectors comprise polynucleotide
sequences that produce small interfering RNA or antisense RNA
transcripts that interfere with the translation of a target
polynucleotide sequence.
[0132] As used herein, a "deletion vector" comprises polynucleotide
sequences homologous to a polynucleotide sequences 5' and 3' to a
target sequence to be deleted from a host genome so as to direct
recombination and replacement of the target sequence with a
polynucleotiode between the 5' and 3' targeting sequences.
[0133] As used herein, the term "expression" includes any step
involved in the production of the polypeptide including, but not
limited to, transcription, post-transcriptional modification,
translation, and post-translational modification. In some
embodiments, the term also encompasses secretion of the polypeptide
from a cell. In general the term, "expression" refers to conversion
of the information encoded in a gene to the protein encoded by that
gene. Thus, a "reduction of the amount of an expressed cellobiose
oxidizing enzyme" is a reduction in the amount of the cellobiose
oxidizing enzyme that is eventually translated by the cell.
[0134] As used herein, the term "overexpress" is intended to
encompass increasing the expression of a protein to a level greater
than the cell normally produces. It is intended that the term
encompass overexpression of endogenous, as well as heterologous
proteins. In some embodiments, overexpression includes an elevated
transcription rate and/or level of the gene compared to the
endogenous transcription rate and/or level for that gene. For
example, in some embodiments, a heterologous gene is introduced
into a fungal cell to express a gene encoding a heterologous enzyme
such as a beta-glucosidase from another organism In some other
embodiments, a heterologous gene is introduced into a fungal cell
to overexpress a gene encoding a homologous enzyme such as a
beta-glucosidase.
[0135] In some embodiments, the heterologous gene is a gene that
has been modified to overexpress the gene product. In some
embodiments, "overexpression" refers to any state in which a gene
is caused to be expressed at an elevated rate or level as compared
to the endogenous expression rate or level for that gene. In some
embodiments, overexpression includes elevated translation rate
and/or level of the gene compared to the endogenous translation
rate and/or level for that gene.
[0136] As used herein, the term "produces" refers to the production
of proteins and/or other compounds by cells. It is intended that
the term encompass any step involved in the production of
polypeptides including, but not limited to, transcription,
post-transcriptional modification, translation, and
post-translational modification. In some embodiments, the term also
encompasses secretion of the polypeptide from a cell.
[0137] As used herein, a "cellobiose dehydrogenase that is secreted
by a cell" is a cellobiose dehydrogenase produced by the cell in a
manner such that the cellobiose dehydrogenase is exported across a
cell membrane and then subsequently released into the extracellular
milieu, such as into culture media.
[0138] As used herein, a "polynucleotide sequence that has been
adapted for expression" is a polynucleotide sequence that has been
inserted into an expression vector or otherwise modified to contain
regulatory elements necessary for expression of the polynucleotide
in the host cell, positioned in such a manner as to permit
expression of the polynucleotide in the host cell. Such regulatory
elements required for expression include promoter sequences,
transcription initiation sequences and, optionally, enhancer
sequences. For example, in some embodiments, a polynucleotide
sequence is inserted into a plasmid adapted for expression in the
fungal host cell.
[0139] As used herein, the term "plasmid" refers to a circular
double-stranded (ds) DNA construct used as a cloning vector. In
some embodiments, plasmids form an extrachromosomal
self-replicating genetic element in some eukaryotes and/or
prokaryotes, while in some other embodiments, plasmids integrate
into the host cell chromosome.
[0140] As used herein, a "control sequence" includes all
components, which are necessary or advantageous for the expression
of a polynucleotide of the present disclosure. Each control
sequence may be native or foreign to the polynucleotide of
interest. Such control sequences include, but are not limited to,
leaders, polyadenylation sequences, propeptide sequences,
promoters, signal peptide sequences, and transcription
terminators.
[0141] As used herein, "operably linked" refers to a configuration
in which a control sequence is appropriately placed (i.e., in a
functional relationship) at a position relative to a polynucleotide
of interest such that the control sequence directs or regulates the
expression of the polynucleotide and/or polypeptide of
interest.
[0142] As used herein, a nucleic acid is "operably linked" when it
is placed into a functional relationship with another nucleic acid
sequence. For example, DNA encoding a secretory leader (i.e., a
signal peptide), is operably linked to DNA for a polypeptide if it
is expressed as a preprotein that participates in the secretion of
the polypeptide; a promoter or enhancer is operably linked to a
coding sequence if it affects the transcription of the sequence; or
a ribosome binding site is operably linked to a coding sequence if
it is positioned so as to facilitate translation. Generally,
"operably linked" means that the DNA sequences being linked are
contiguous, and, in the case of a secretory leader, contiguous and
in reading phase. However, enhancers do not have to be contiguous
Linking is accomplished by ligation at convenient restriction
sites. If such sites do not exist, the synthetic oligonucleotide
adaptors or linkers are used in accordance with conventional
practice.
[0143] As used herein the term "gene" refers to a polynucleotide
(e.g., a DNA segment), that encodes a polypeptide and includes
regions preceding and following the coding regions as well as
intervening sequences (introns) between individual coding segments
(exons).
[0144] As used herein, an "endogenous" or "homologous" gene refers
to a gene (including, but not limited to wild-type) that is found
in a parental strain of a host cell (e.g., fungal or bacterial
cell). As used herein in making comparisons between nucleic acid
sequences, "homologous genes" (or "homologue" genes) refers to
genes from different, but usually related species, which correspond
to each other and which are identical or very similar to each
other. The term encompasses genes that are separated by speciation
(i.e., the development of new species) (e.g., orthologous genes),
as well as genes that have been separated by genetic duplication
(e.g., paralogous genes).
[0145] As used herein, an amino acid or nucleotide sequence (e.g.,
a promoter sequence, signal peptide, terminator sequence, etc.) is
"heterologous" to another sequence with which it is operably linked
if the two sequences are not associated in nature.
[0146] As used herein, a "heterologous enzyme" refers to an enzyme
that is encoded by a "heterologous gene." However, it is also
contemplated that a heterologous gene encodes an endogenous or
homologous enzyme, as described herein. In general, the term
"heterologous gene" refers to a gene that occurs in a form not
found in a parental strain of the host fungal cell (including but
not limited to wild-type). Thus, in some embodiments, a
heterologous gene is a gene that is derived from a species that is
different from the species of the fungal cell expressing the gene
and recognized anamorphs, teleomorphs or taxonomic equivalents of
the fungal cell expressing the gene. In some embodiments, a
heterologous gene is a modified version of a gene that is
endogenous to the host fungal cell, which endogenous gene has been
subjected to manipulation and then introduced or transformed into
the host cell. For example, in some embodiments, a heterologous
gene has an endogenous coding sequence, but has modifications to
the promoter sequence. Similarly, in some embodiments, a
heterologous gene encodes the same amino acid sequence as an
endogenous gene, but has modifications to the codon usage or to
noncoding regions such as introns, or a combination thereof. For
example, in some embodiments, a heterologous gene comprises
modifications to the coding sequence to encode a non-wild type
polypeptide. In some other embodiments, a heterologous gene has the
same promoter sequence, 5' and 3' untranslated regions and coding
regions as a parental strain, but be located in another region of
the same chromosome, or on an entirely different chromosome as
compared to a parental strain of the host cell.
[0147] As used herein, the term "introduced" used in the context of
inserting a nucleic acid sequence into a cell, means
transformation, transduction, conjugation, transfection, and/or any
other suitable method(s) known in the art for inserting nucleic
acid sequences into host cells. Any suitable means for the
introduction of nucleic acid into host cells find use in the
present invention.
[0148] As used herein, the terms "transformed" and "transformation"
used in reference to a cell refer to a cell that has a non-native
nucleic acid sequence integrated into its genome or has an episomal
plasmid that is maintained through multiple generations. In some
embodiments, the terms "transformed" and "stably transformed" refer
to a cell that has a non-native (i.e., heterologous) polynucleotide
sequence integrated into its genome or as an episomal plasmid that
is maintained for at least two generations.
[0149] As used herein, the terms "host cell" and "host strain"
refer to suitable hosts for expression vectors comprising
polynucleotide sequences (e.g., DNA) as provided herein. In some
embodiments, the host cells are prokaryotic or eukaryotic cells
that have been transformed or transfected with vectors constructed
using recombinant techniques as known in the art. Transformed hosts
are capable of either replicating vectors encoding at least one
protein of interest and/or expressing the desired protein of
interest. In addition, reference to a cell of a particular strain
refers to a parental cell of the strain as well as progeny and
genetically modified derivatives. Genetically modified derivatives
of a parental cell include progeny cells that contain a modified
genome or episomal plasmids that confer for example, antibiotic
resistance, improved fermentation, etc. In some embodiments, host
cells are genetically modified to have characteristics that improve
protein secretion, protein stability or other properties desirable
for expression and/or secretion of a protein. For example, knockout
of Alp1 function results in a cell that is protease deficient.
Knockout of pyr5 function results in a cell with a pyrimidine
deficient phenotype. In some embodiments, host cells are modified
to delete endogenous cellulase protein-encoding sequences or
otherwise eliminate expression of one or more endogenous
cellulases. In some embodiments, expression of one or more
endogenous cellulases is inhibited to increase production of
cellulases of interest. Genetic modification can be achieved by any
suitable genetic engineering techniques and/or classical
microbiological techniques (e.g., chemical or UV mutagenesis and
subsequent selection). Using recombinant technology, nucleic acid
molecules can be introduced, deleted, inhibited or modified, in a
manner that results in increased yields of enzyme within the
organism or in the culture. For example, knockout of Alp1 function
results in a cell that is protease deficient. Knockout of pyr5
function results in a cell with a pyrimidine deficient phenotype.
In some genetic engineering approaches, homologous recombination is
used to induce targeted gene modifications by specifically
targeting a gene in vivo to suppress expression of the encoded
protein. In an alternative approach, siRNA, antisense, and/or
ribozyme technology finds use in inhibiting gene expression.
[0150] The terms "modified sequence" and "modified genes" are used
interchangeably herein to refer to a sequence that includes a
deletion, insertion, substitution or any other interruption of a
naturally occurring nucleic acid sequence. In some embodiments, the
expression product of the modified sequence is a truncated protein
(e.g., if the modification is a deletion or interruption of the
sequence). In some embodiments, the truncated protein retains
biological activity. In some alternative embodiments, the
expression product of the modified sequence is an elongated protein
(e.g., modifications comprising an insertion into the nucleic acid
sequence). In some further embodiments, an insertion leads to a
truncated protein (e.g., when the insertion results in the
formation of a stop codon). Thus, an insertion may result in either
a truncated protein or an elongated protein as an expression
product.
[0151] As used herein, the terms "mutant nucleic acid sequence,"
"mutant nucleotide sequence," and "mutant gene" are used
interchangeably in reference to a nucleotide sequence that has an
alteration in at least one codon occurring in a host cell's
wild-type nucleotide sequence. The expression product of the mutant
sequence is a protein with an altered amino acid sequence relative
to the wild-type. In some embodiments, the expression product has
an altered functional capacity (e.g., enhanced enzymatic
activity).
[0152] As used herein, the term "targeted randomization" refers to
a process that produces a plurality of sequences where one or
several positions have been randomized In some embodiments,
randomization is complete (i.e., all four nucleotides, A, T, G, and
C can occur at a randomized position). In some alternative
embodiments, randomization of a nucleotide is limited to a subset
of the four nucleotides. Targeted randomization can be applied to
one or several codons of a sequence, coding for one or several
proteins of interest. When expressed, the resulting libraries
produce protein populations in which one or more amino acid
positions can contain a mixture of all 20 amino acids or a subset
of amino acids, as determined by the randomization scheme of the
randomized codon. In some embodiments, the individual members of a
population resulting from targeted randomization differ in the
number of amino acids, due to targeted or random insertion or
deletion of codons. In some further embodiments, synthetic amino
acids are included in the protein populations produced. In some
additional embodiments, the majority of members of a population
resulting from targeted randomization show greater sequence
homology to the consensus sequence than the starting gene. In some
embodiments, the sequence encodes one or more proteins of interest.
In some alternative embodiments, the proteins have differing
biological functions.
[0153] As used herein, "deletion" refers to modification of the
polypeptide by removal of one or more amino acids from the
reference polypeptide. Deletions can comprise removal of 1 or more
amino acids, 2 or more amino acids, 3 or more amino acids, 4 or
more amino acids, 5 or more amino acids, 6 or more amino acids, 7
or more amino acids, 8 or more amino acids, 9 or more amino acids,
10 or more amino acids, 15 or more amino acids, or 20 or more amino
acids, up to 10% of the total number of amino acids, or up to 20%
of the total number of amino acids making up the polypeptide while
retaining enzymatic activity and/or retaining the improved
properties of an engineered cellobiose dehydrogenase enzyme.
Deletions may be present in the internal portions and/or terminal
portions of the polypeptide. In some embodiments, the deletion
comprises a continuous segment, while in other embodiments, it is
discontinuous.
[0154] As used herein, a "gene deletion" or "deletion mutation" is
a mutation in which at least part part of a sequence of the DNA
making up the gene is missing. Thus, a "deletion" in reference to
nucleic acids is a loss or replacement of genetic material
resulting in a complete or partial disruption of the sequence of
the DNA making up the gene. In some embodiments, complete or
near-complete deletion of the gene sequence is contemplated.
However, a deletion mutation need not completely remove the entire
gene sequence for the cellobiose oxidizing enzyme in order to
reduce the endogenous cellobiose oxidizing enzyme activity secreted
by the fungal cell. For example, a partial deletion that removes
one or more nucleotides encoding an amino acid in a cellobiose
oxidizing enzyme active site, encoding a secretion signal, or
encoding another portion of the cellobiose oxidizing enzyme that
plays a role in endogenous cellobiose oxidizing enzyme activity
being secreted by the fungal cell. Any number of nucleotides can be
deleted, from a single base to an entire piece of a chromosome.
Thus, in some embodiments, the term "deletion" refers to the
removal of a gene necessary for encoding a specific protein (e.g.,
cdh1). In this case, the strain having this deletion can be
referred to as a "deletion strain."
[0155] As used herein, "fragment" refers to a polypeptide that has
an amino-terminal and/or carboxy-terminal and/or internal deletion,
as compared to a reference polypeptide, but where the remaining
amino acid sequence is identical to the corresponding positions in
the reference sequence. Fragments can typically have about 80%,
about 90%, about 95%, about 98%, or about 99% of the full-length
cellobiose dehydrogenase polypeptide, for example the polypeptide
of SEQ ID NO:2. In some instances, the sequences of the
non-naturally occurring and wild-type cellobiose dehydrogenase
polypeptides disclosed herein can include an initiating methionine
(M) residue (i.e., M at position 1). However, the skilled artisan
will recognize that this initiating methionine residue can be
removed during the course of biological processing of the enzyme,
such as in a host cell or in vitro translation system, to generate
a mature enzyme lacking the initiating methionine residue, but
otherwise retaining the enzyme's properties. Thus, for each of the
cellobiose dehydrogenase polypeptides disclosed herein having an
amino acid sequence comprising an initiating methionine, the
present disclosure also encompasses the polypeptide with the
initiating methionine residue deleted (i.e., a fragment of the
cellobiose dehydrogenase polypeptide lacking a methionine at
position 1).
[0156] As used herein, a "conditional mutation" is a mutation that
has wild-type phenotype under certain environmental conditions and
a mutant phenotype under certain other conditions.
[0157] As used herein, the term "screening" has its usual meaning
in the art and is, in general a multi-step process. In the first
step, a mutant nucleic acid or variant polypeptide therefrom is
provided. In the second step, a property of the mutant nucleic acid
or variant polypeptide is determined In the third step, the
determined property is compared to a property of the corresponding
precursor nucleic acid, to the property of the corresponding
naturally occurring polypeptide or to the property of the starting
material (e.g., the initial sequence) for the generation of the
mutant nucleic acid. It will be apparent to the skilled artisan
that the screening procedure for obtaining a nucleic acid or
protein with an altered property depends upon the property of the
starting material, and the modification of which the generation of
the mutant nucleic acid is intended to facilitate. The skilled
artisan will therefore appreciate that the invention is not limited
to any specific property to be screened for and that the following
description of properties lists illustrative examples only. Methods
for screening for any particular property are generally described
in the art. For example, one can measure binding, pH optima,
specificity, etc., before and after mutation, wherein a change
indicates an alteration. In some embodiments, the screens are
performed in a high-throughput manner, including multiple samples
being screened simultaneously, including, but not limited to assays
utilizing chips, phage display, multiple substrates and/or
indicators, and/or any other suitable method known in the art.
[0158] As used in some embodiments, screens encompass selection
steps in which variants of interest are enriched from a population
of variants. Examples of these embodiments include the selection of
variants that confer a growth advantage to the host organism, as
well as phage display or any other method of display, where
variants can be captured from a population of variants based on
their binding or catalytic properties. In some embodiments, a
library of variants is exposed to stress (e.g., exposure to heat,
protease, or denaturing conditions). Subsequently, variants that
are still intact are identified in a screen or enriched by
selection. It is intended that the term encompass any suitable
means for selection. Indeed, it is not intended that the present
invention be limited to any particular method of screening.
[0159] As used herein, a "genetically modified" and/or "genetically
engineered cell" (e.g., a "geneticially engineered fungal cell"
and/or a "genetically modified fungal cell") is a cell whose
genetic material has been altered using genetic engineering
techniques. A genetically modified cell also refers to a derivative
of or the progeny of a cell whose genetic material has been altered
using genetic engineering techniques. An example of a genetic
modification as a result of genetic engineering techniques includes
a modification to the genomic DNA; another example of a genetic
modification as a result of genetic engineering techniques includes
introduction of a stable heterologous nucleic acid into the cell.
For example, as provided herein, a genetically modified fungal cell
as provided herein is a fungal cell that whose genetic material has
been altered in such a way as to either reduce the amount of
secreted cellobiose oxidizing enzyme activity, or to reduce the
ability of the secreted enzyme to oxidize cellobiose.
[0160] In some embodiments, mutant DNA sequences are generated
using site saturation mutagenesis in at least one codon. In some
other embodiments, site saturation mutagenesis is performed for two
or more codons. In some further embodiments, mutant DNA sequences
have more than about 50%, more than about 55%, more than about 60%,
more than about 65%, more than about 70%, more than about 75%, more
than about 80%, more than about 85%, more than about 90%, more than
about 95%, or more than about 98% homology with the wild-type
sequence. In some alternative embodiments, mutant DNA is generated
in vivo using any suitable known mutagenic procedure including, but
not limited to the use of radiation, nitrosoguanidine, etc. The
desired DNA sequence is then isolated and used in the methods
provided herein.
[0161] As used herein, the terms "amplification" and "gene
amplification" refer to a method by which specific DNA sequences
are disproportionately replicated such that the amplified gene
becomes present in a higher copy number than was initially present
in the genome. In some embodiments, selection of cells by growth in
the presence of a drug (e.g., an inhibitor of an inhibitable
enzyme) results in the amplification of either the endogenous gene
encoding the gene product required for growth in the presence of
the drug or by amplification of exogenous (i.e., input) sequences
encoding this gene product, or both. "Amplification" is a special
case of nucleic acid replication involving template specificity. It
is to be contrasted with non-specific template replication (i.e.,
replication that is template-dependent but not dependent on a
specific template). Template specificity is here distinguished from
fidelity of replication (i.e., synthesis of the proper
polynucleotide sequence) and nucleotide (ribo- or deoxyribo-)
specificity. Template specificity is frequently described in terms
of "target" specificity. Target sequences are "targets" in the
sense that they are sought to be sorted out from other nucleic
acid. Amplification techniques have been designed primarily for
this sorting out.
[0162] As used herein, the term "primer" refers to an
oligonucleotide, whether occurring naturally as in a purified
restriction digest or produced synthetically, that is capable of
acting as a synthesis initiation point when placed under conditions
in which synthesis of a primer extension product which is
complementary to a nucleic acid strand is induced (i.e., in the
presence of nucleotides and an inducing agent such as DNA
polymerase and at a suitable temperature and pH). The primer is
preferably single stranded for maximum efficiency in amplification,
but may alternatively be double stranded. If double stranded, the
primer is first treated to separate its strands before being used
to prepare extension products. In some embodiments, the primer is
an oligodeoxyribonucleotide. The primer must be sufficiently long
to prime the synthesis of extension products in the presence of the
inducing agent. As known in the art, the exact lengths of the
primers will depend on many factors, including temperature, source
of primer and the use of the method.
[0163] The terms "mutagenic primer" and "mutagenic oligonucleotide"
(used interchangeably herein) refer to oligonucleotide compositions
which correspond to a portion of a template sequence and which are
capable of hybridizing thereto. With respect to mutagenic primers,
the primer will not precisely match the template nucleic acid, the
mismatch or mismatches in the primer being used to introduce the
desired mutation into the nucleic acid library.
[0164] As used herein, the terms "non-mutagenic primer" and
"non-mutagenic oligonucleotide" (used interchangeably herein) are
intended to refer to oligonucleotide compositions which will match
precisely to a template nucleic acid. In some embodiments of the
invention, only mutagenic primers are used. In some other
embodiments, the primers are designed so that for at least one
region at which a mutagenic primer has been included, there is also
non-mutagenic primer included in the oligonucleotide mixture. By
adding a mixture of mutagenic primers and non-mutagenic primers
corresponding to at least one of the mutagenic primers, it is
possible to produce a resulting nucleic acid library in which a
variety of combinatorial mutational patterns are presented. For
example, if it is desired that some of the members of the mutant
nucleic acid library retain their precursor sequence at certain
positions while other members are mutant at such sites, the
non-mutagenic primers provide the ability to obtain a specific
level of non-mutant members within the nucleic acid library for a
given residue. The methods of the invention employ mutagenic and
non-mutagenic oligonucleotides which are generally between about
10-50 bases in length, or more preferably, about 15-45 bases in
length. However, it may be necessary to use primers that are either
shorter than about 10 bases or longer than about 50 bases to obtain
the mutagenesis result desired. With respect to corresponding
mutagenic and non-mutagenic primers, it is not necessary that the
corresponding oligonucleotides be of identical length, but only
that there is overlap in the region corresponding to the mutation
to be added. Primers may be added in a pre-defined ratio according
to the present invention. For example, if it is desired that the
resulting library have a significant level of a certain specific
mutation and a lesser amount of a different mutation at the same or
different site, it is possible to produce the desired biased
library by adjusting the amount of primer added. Alternatively, by
adding lesser or greater amounts of non-mutagenic primers, it is
possible to adjust the frequency with which the corresponding
mutation(s) are produced in the mutant nucleic acid library.
[0165] As used herein, the phrase "contiguous mutations" refers to
mutations which are presented within the same oligonucleotide
primer. For example, contiguous mutations may be adjacent or nearby
each other, however, they will be introduced into the resulting
mutant template nucleic acids by the same primer.
[0166] As used herein, the phrase "discontiguous mutations" refers
to mutations which are presented in separate oligonucleotide
primers. For example, discontiguous mutations will be introduced
into the resulting mutant template nucleic acids by separately
prepared oligonucleotide primers.
[0167] As used herein, the term "degenerate codon" refers to a
codon used to represent a set of different codons (also referred to
as an "ambiguous codon"). For example, the degenerate codon "NNT"
represents a set of 16 codons having the base triplet sequence (A,
C, T, or G)/(A, C, T, or G)/T.
[0168] As used herein, "coding sequence" refers to that portion of
a polynucleotide that encodes an amino acid sequence of a protein
(e.g., a gene).
[0169] As used herein, the term "probe" refers to an
oligonucleotide (i.e., a sequence of nucleotides), whether
occurring naturally as in a purified restriction digest or produced
synthetically, recombinantly or by PCR amplification, that is
capable of hybridizing to another oligonucleotide of interest. A
probe may be single-stranded or double-stranded. Probes are useful
in the detection, identification and isolation of particular gene
sequences. It is contemplated that any probe used in the present
invention will be labeled with any "reporter molecule," so that is
detectable in any detection system, including, but not limited to
enzyme (e.g., ELISA, as well as enzyme-based histochemical assays),
fluorescent, radioactive, and luminescent systems. It is not
intended that the present invention be limited to any particular
detection system or label.
[0170] As used herein, the term "target," when used in reference to
the polymerase chain reaction, refers to the region of nucleic acid
bounded by the primers used for polymerase chain reaction. Thus,
the "target" is sought to be sorted out from other nucleic acid
sequences. A "segment" is defined as a region of nucleic acid
within the target sequence.
[0171] As used herein, the term "polymerase chain reaction" (PCR)
refers to the methods of U.S. Pat. Nos. 4,683,195 4,683,202, and
4,965,188, hereby incorporated by reference, which include methods
for increasing the concentration of a segment of a target sequence
in a mixture of genomic DNA without cloning or purification. This
method for amplifying the target sequence is well known in the
art.
[0172] As used herein, the term "amplification reagents" refers to
those reagents (deoxyribonucleotide triphosphates, buffer, etc.),
needed for amplification except for primers, nucleic acid template
and the amplification enzyme. Typically, amplification reagents
along with other reaction components are placed and contained in a
reaction vessel (test tube, microwell, etc.).
[0173] As used herein, the terms "restriction endonucleases" and
"restriction enzymes" refer to bacterial enzymes, each of which cut
double-stranded DNA at or near a specific nucleotide sequence.
[0174] A "restriction site" refers to a nucleotide sequence
recognized and cleaved by a given restriction endonuclease and is
frequently the site for insertion of DNA fragments. In some
embodiments of the invention, restriction sites are engineered into
the selective marker and into 5' and 3' ends of the DNA
construct.
[0175] As used herein, "homologous recombination" means the
exchange of DNA fragments between two DNA molecules or paired
chromosomes at the site of identical or nearly identical nucleotide
sequences. In some embodiments, chromosomal integration is
homologous recombination.
[0176] As used herein, the term "C1" refers to Myceliophthora
thermophilia, including the fungal strain described by Garg (See,
Garg, Mycopathol., 30: 3-4 [1966]).
[0177] As used herein, "Chrysosporium lucknowense" includes the
strains described in U.S. Pat. Nos. 6,015,707, 5,811,381 and
6,573,086; U.S. Pat. Pub. Nos. 2007/0238155, US 2008/0194005, U.S.
2009/0099079; International Pat. Pub. Nos., WO 2008/073914 and WO
98/15633, all of which are incorporated herein by reference, and
include, without limitation, Chrysosporium lucknowense Garg 27K,
VKM-F 3500 D (Accession No. VKM F-3500-D), C1 strain UV13-6
(Accession No. VKM F-3632 D), C1 strain NG7C-19 (Accession No. VKM
F-3633 D), and C1 strain UV18-25 (VKM F-3631 D), all of which have
been deposited at the All-Russian Collection of Microorganisms of
Russian Academy of Sciences (VKM), Bakhurhina St. 8, Moscow,
Russia, 113184, and any derivatives thereof. Although initially
described as Chrysosporium lucknowense, C1 may currently be
considered a strain of Myceliophthora thermophila. Other C1 strains
include cells deposited under accession numbers ATCC 44006, CBS
(Centraalbureau voor Schimmelcultures) 122188, CBS 251.72, CBS
143.77, CBS 272.77, CBS122190, CBS122189, and VKM F-3500D.
Exemplary C1 derivatives include modified organisms in which one or
more endogenous genes or sequences have been deleted or modified
and/or one or more heterologous genes or sequences have been
introduced. Derivatives include, but are not limited to UV18#100f
.DELTA.alpl, UV18#100f .DELTA.pyr5 .DELTA.alpl, UV18#100.f
.DELTA.alpl .DELTA.pep4 .DELTA.alp2, UV18#100.f .DELTA.pyr5
.DELTA.alpl .DELTA.pep4 .DELTA.alp2 and UV18#100.f .DELTA.pyr4
.DELTA.pyr5 .DELTA.aIp1 .DELTA.pep4 .DELTA.alp2, as described in WO
2008073914 and WO 2010107303, each of which is incorporated herein
by reference.
[0178] As used herein, the term "culturing" refers to growing a
population of microbial cells under suitable conditions in a liquid
or solid medium. It is contemplated that the culturing be carried
out in any suitable format, equipment (e.g., shake flasks,
fermentation tanks, bioreactors, etc.). It is also intended that
the culturing be conducted using any suitable process methods,
including but not limited to batch, fed-batch, and/or continuous
culturing. Indeed, it is contemplated that any combination of
suitable methods will find use.
[0179] In a "batch process," all the necessary materials, with the
exception of oxygen for aerobic processes, are placed in a reactor
at the start of the operation and the fermentation is allowed to
proceed until completion, at which point the product is harvested.
In some embodiments, batch processes for producing the fungal
cells, enzymes, and/or enzyme mixtures of the present invention are
carried out in a shake-flask or a bioreactor.
[0180] In a "fed-batch process," the culture is fed continuously or
sequentially with one or more media components without the removal
of the culture fluid.
[0181] In a "continuous process," fresh medium is supplied and
culture fluid is removed continuously at volumetrically equal rates
to maintain the culture at a steady growth rate. In reference to
continous processes, "steady state" refers to a state in which the
concentration of reactants does not vary appreciably, and
"quasi-steady state" refers to a state in which, subsequent to the
initiation of the reaction, the concentration of reactants
fluctuates within a range consistent with normal operation of the
continuous hydrolysis process.
[0182] As used herein, the term "saccharification" refers to the
process in which substrates (e.g., cellulosic biomass) are broken
down via the action of cellulases to produce fermentable sugars
(e.g. monosaccharides such as but not limited to glucose).
[0183] As used herein, the term "fermentable sugars" refers to
simple sugars (e.g., monosaccharides, disaccharides and short
oligosaccharides), including but not limited to glucose, xylose,
galactose, arabinose, mannose and sucrose. Indeed, a fermentable
sugar is any sugar that a microorganism can utilize or ferment.
[0184] As used herein the term "soluble sugars" refers to
water-soluble pentose and hexose monomers and oligomers of up to
about six monomer units. It is intended that the term encompass any
water soluble mono- and/or oligosaccharides.
[0185] As used herein, the term "fermentation" is used broadly to
refer to the process of obtaining energy from the oxidation of
organic compounds (e.g., carbohydrates). Indeed, "fermentation"
broadly refers to the chemical conversion of a sugar source to an
end product through the use of a fermenting organism. In some
embodiments, the term encompasses cultivation of a microorganism or
a culture of microorganisms that use sugars, such as fermentable
sugars, as an energy source to obtain a desired product.
[0186] As used herein, the term "fermenting organism" refers to any
organism, including prokaryotic, as well as eukaryotic organisms
(e.g., bacterial organisms, as well as fungal organisms such as
yeast and filamentous fungi), suitable for producing a desired end
product. Especially suitable fermenting organisms are able to
ferment (i.e., convert) sugars, including but not limited to
glucose, fructose, maltose, xylose, mannose and/or arabinose,
directly or indirectly into at least one desired end product. In
some embodiments, yeast that find use in the present invention
include, but are not limited to strains of the genus Saccharomyces
(e.g., strains of Saccharomyces cerevisiae and Saccharomyces
uvarum), strains of the genus Pichia (e.g., Pichia stipitis such as
Pichia stipitis CBS 5773 and Pichia pastoris), and strains of the
genus Candida (e.g., Candida utilis, Candida arabinofermentans,
Candida diddensii, Candida sonorensis, Candida shehatae, Candida
tropicalis, and Candida boidinii). Other fermenting organisms
include, but are not limited to strains of Zymomonas, Hansenula
(e.g., Hansenula polymorpha and Hansenula anomala), Kluyveromyces
(e.g., Kluyveromyces fragilis), and Schizosaccharomyces (e.g.,
Schizosaccharomyces pombe).
[0187] As used herein, the term "slurry" refers to an aqueous
solution in which are dispersed one or more solid components, such
as a cellulosic substrate. Thus, the term "slurry" refers to a
suspension of solids in a liquid. In some embodiments, the
cellulosic substrate is slurried in a liquid at a concentration
that is thick, but can still be pumped. For example, in some
embodiments, the liquid is water, a recycled process stream, and/or
a treated effluent. However, it is not intended that the present
invention be limited to any particular liquid and/or solid.
[0188] The terms "biomass," and "biomass substrate," encompass any
suitable materials for use in saccharification reactions. The terms
encompass, but are not limited to, materials that comprise
cellulose (i.e., "cellulosic biomass," "cellulosic feedstock," and
"cellulosic substrate"), as well as lignocellulosic biomass.
Indeed, the term "biomass" encompasses any living or dead
biological material that contains a polysaccharide substrate,
including but not limited to cellulose, starch, other forms of
long-chain carbohydrate polymers, and mixtures of such sources. In
some embodiments, it is assembled entirely or primarily from
glucose or xylose, and in some embodiments, optionally also
contains various other pentose and/or hexose monomers. Biomass can
be derived from plants, animals, or microorganisms, and includes,
but is not limited to agricultural, industrial, and forestry
residues, industrial and municipal wastes, and terrestrial and
aquatic crops grown for energy purposes. Examples of biomass
substrates include, but are not limited to, wood, wood pulp, paper
pulp, corn fiber, corn grain, corn cobs, crop residues such as corn
husks, corn stover, grasses, wheat, wheat straw, barley, barley
straw, hay, rice, rice straw, switchgrass, waste paper, paper and
pulp processing waste, woody or herbaceous plants, fruit or
vegetable pulp, distillers grain, grasses, rice hulls, cotton,
hemp, flax, sisal, sugar cane bagasse, sorghum, soy, switchgrass,
components obtained from milling of grains, trees, branches, roots,
leaves, wood chips, sawdust, shrubs and bushes, vegetables, fruits,
and flowers and any suitable mixtures thereof. In some embodiments,
the biomass comprises, but is not limited to cultivated crops
(e.g., grasses, including C4 grasses, such as switch grass, cord
grass, rye grass, miscanthus, reed canary grass, or any combination
thereof), sugar processing residues, for example, but not limited
to, bagasse (e.g., sugar cane bagasse, beet pulp [e.g., sugar
beet], or a combination thereof), agricultural residues (e.g.,
soybean stover, corn stover, corn fiber, rice straw, sugar cane
straw, rice, rice hulls, barley straw, corn cobs, wheat straw,
canola straw, oat straw, oat hulls, corn fiber, hemp, flax, sisal,
cotton, or any combination thereof), fruit pulp, vegetable pulp,
distillers' grains, forestry biomass (e.g., wood, wood pulp, paper
pulp, recycled wood pulp fiber, sawdust, hardwood, such as aspen
wood, softwood, or a combination thereof). Furthermore, in some
embodiments, the biomass comprises cellulosic waste material and/or
forestry waste materials, including but not limited to, paper and
pulp processing waste, municipal paper waste, newsprint, cardboard
and the like. In some embodiments, biomass comprises one species of
fiber, while in some alternative embodiments, the biomass comprises
a mixture of fibers that originate from different biomasses. In
some embodiments, the biomass also comprises transgenic plants that
express ligninase and/or cellulase enzymes (See e.g., US
2008/0104724 A1).
[0189] As used herein, "lignocellulose" refers to a matrix of
cellulose, hemicellulose and lignin. Economic production of
biofuels from lignocellulosic biomass typically involves conversion
of the cellulose and hemicellulose components to fermentable
sugars, typically monosaccharides such as glucose (from the
cellulose) and xylose and arabinose (from the hemicelluloses).
Nearly complete conversion can be achieved by a chemical
pretreatment of the lignocellulose followed by enzymatic hydrolysis
with cellulase enzymes. The chemical pretreatment step renders the
cellulose more susceptible to enzymatic hydrolysis and, in some
cases, also hydrolyzes the hemicellulose component. Numerous
chemical pretreatment processes are known in the art, and include,
but are not limited to, mild acid pretreatment at high temperatures
and dilute acid, ammonium pretreatment or organic solvent
extraction.
[0190] Lignin is a more complex and heterogeneous biopolymer than
either cellulose or hemicellulose and comprises a variety of
phenolic subunits. Enzymatic lignin depolymerization can be
accomplished by lignin peroxidases, manganese peroxidases, laccases
and cellobiose dehydrogenases (CDH), often working in synergy.
However, as the name suggests, CDH enzymes also oxidize cellobiose
to cellobionolactone. Several reports indicate that the oxidation
of cellobiose by CDH enhances the rate of cellulose hydrolysis by
cellulases by virtue of reducing the concentrations of cellobiose,
which is a potent inhibitor of some cellulase components (Mansfield
et al., Appl. Environ. Microbiol., 63: 3804-3809 [1997]; and
Igarishi et al., Eur. J. Biochem., 253: 101-106 [1998]). Recently,
it has been reported that CDHs can enhance the activity of
cellulolytic enhancing proteins from Glycosyl Hydrolase family 61
(See e.g., WO2010/080532A1).
[0191] As used herein, the term "lignocellulosic biomass" refers to
any plant biomass comprising cellulose and hemicellulose, bound to
lignin
[0192] In some embodiments, the biomass is optionally pretreated to
increase the susceptibility of cellulose to hydrolysis by chemical,
physical and biological pretreatments (such as steam explosion,
pulping, grinding, acid hydrolysis, solvent exposure, and the like,
as well as combinations thereof). Various lignocellulosic
feedstocks find use, including those that comprise fresh
lignocellulosic feedstock, partially dried lignocellulosic
feedstock, fully dried lignocellulosic feedstock, and/or any
combination thereof. In some embodiments, lignocellulosic
feedstocks comprise cellulose in an amount greater than about 20%,
more preferably greater than about 30%, more preferably greater
than about 40% (w/w). For example, in some embodiments, the
lignocellulosic material comprises from about 20% to about 90%
(w/w) cellulose, or any amount therebetween, although in some
embodiments, the lignocellulosic material comprises less than about
19%, less than about 18%, less than about 17%, less than about 16%,
less than about 15%, less than about 14%, less than about 13%, less
than about 12%, less than about 11%, less than about 10%, less than
about 9%, less than about 8%,less than about 7%, less than about
6%, or less than about 5% cellulose (w/w). Furthermore, in some
embodiments, the lignocellulosic feedstock comprises lignin in an
amount greater than about 10%, more typically in an amount greater
than about 15% (w/w). In some embodiments, the lignocellulosic
feedstock comprises small amounts of sucrose, fructose and/or
starch. The lignocellulosic feedstock is generally first subjected
to size reduction by methods including, but not limited to,
milling, grinding, agitation, shredding, compression/expansion, or
other types of mechanical action. Size reduction by mechanical
action can be performed by any type of equipment adapted for the
purpose, for example, but not limited to, hammer mills,
tub-grinders, roll presses, refiners and hydrapulpers. In some
embodiments, at least 90% by weight of the particles produced from
the size reduction have lengths less than between about 1/16 and
about 4 in (the measurement may be a volume or a weight average
length). In some embodiments, the equipment used to reduce the
particle size is a hammer mill or shredder. Subsequent to size
reduction, the feedstock is typically slurried in water, as this
facilitates pumping of the feedstock. In some embodiments,
lignocellulosic feedstocks of particle size less than about 6
inches do not require size reduction.
[0193] As used herein, the term "lignocellulosic feedstock" refers
to any type of lignocellulosic biomass that is suitable for use as
feedstock in saccharification reactions.
[0194] As used herein, the term "pretreated lignocellulosic
feedstock," refers to lignocellulosic feedstocks that have been
subjected to physical and/or chemical processes to make the fiber
more accessible and/or receptive to the actions of cellulolytic
enzymes, as described above.
[0195] As used herein, the terms "lignocellulose-competent,"
"lignocellulose-utilizing" and like terms refer to an organism that
secretes enzymes that participate in lignin breakdown and
hydrolysis. For example, in some embodiments,
lignocellulose-competent fungal cells secrete one or more lignin
peroxidases, manganese peroxidases, laccases and/or cellobiose
dehydrogenases (CDH). These extracellular enzymes, essential for
lignin degradation, are often referred to as "lignin-modifying
enzymes" or "LMEs."
[0196] A biomass substrate is said to be "pretreated" when it has
been processed by some physical and/or chemical means to facilitate
saccharification. As described further herein, in some embodiments,
the biomass substrate is "pretreated," or treated using methods
known in the art, such as chemical pretreatment (e g., ammonia
pretreatment, dilute acid pretreatment, dilute alkali pretreatment,
or solvent exposure), physical pretreatment (e.g., steam explosion
or irradiation), mechanical pretreatment (e.g., grinding or
milling) and biological pretreatment (e.g., application of
lignin-solubilizing microorganisms) and combinations thereof, to
increase the susceptibility of cellulose to hydrolysis.
[0197] In some embodiments, the substrate is slurried prior to
pretreatment. In some embodiments, the consistency of the slurry is
between about 2% and about 30% and more typically between about 4%
and about 15%. In some embodiments, the slurry is subjected to a
water and/or acid soaking operation prior to pretreatment. In some
embodiments, the slurry is dewatered using any suitable method to
reduce steam and chemical usage prior to pretreatment. Examples of
dewatering devices include, but are not limited to pressurized
screw presses (See e.g., WO 2010/022511, incorporated herein by
reference) pressurized filters and extruders.
[0198] In some embodiments, the pretreatment is carried out to
hydrolyze hemicellulose, and/or a portion thereof present in
lignocellulose to monomeric pentose and hexose sugars (e.g.,
xylose, arabinose, mannose, galactose, and/or any combination
thereof). In some embodiments, the pretreatment is carried out so
that nearly complete hydrolysis of the hemicellulose and a small
amount of conversion of cellulose to glucose occurs. In some
embodiments, an acid concentration in the aqueous slurry from about
0.02% (w/w) to about 2% (w/w), or any amount therebetween, is
typically used for the treatment of the cellulosic substrate. Any
suitable acid finds use in these methods, including but not limited
to, hydrochloric acid, nitric acid, and/or sulfuric acid. In some
embodiments, the acid used during pretreatment is sulfuric acid.
Steam explosion is one method of performing acid pretreatment of
biomass substrates (See e.g., U.S. Pat. No. 4,461,648). Another
method of pretreating the slurry involves continuous pretreatment
(i.e., the cellulosic biomass is pumped though a reactor
continuously). This methods are well-known to those skilled in the
art (See e.g., U.S. Pat. No. 7,754,457).
[0199] In some embodiments, alkali is used in the pretreatment. In
contrast to acid pretreatment, pretreatment with alkali may not
hydrolyze the hemicellulose component of the biomass. Rather, the
alkali reacts with acidic groups present on the hemicellulose to
open up the surface of the substrate. In some embodiments, the
addition of alkali alters the crystal structure of the cellulose so
that it is more amenable to hydrolysis. Examples of alkali that
find use in the pretreatment include, but are not limited to
ammonia, ammonium hydroxide, potassium hydroxide, and sodium
hydroxide. One method of alkali pretreatment is Ammonia Freeze
Explosion, Ammonia Fiber Explosion or Ammonia Fiber Expansion
("AFEX" process; See e.g., U.S. Pat. Nos. 5,171,592; 5,037,663;
4,600,590; 6,106,888; 4,356,196; 5,939,544; 6,176,176; 5,037,663;
and 5,171,592). During this process, the cellulosic substrate is
contacted with ammonia or ammonium hydroxide in a pressure vessel
for a sufficient time to enable the ammonia or ammonium hydroxide
to alter the crystal structure of the cellulose fibers. The
pressure is then rapidly reduced, which allows the ammonia to flash
or boil and explode the cellulose fiber structure. In some
embodiments, the flashed ammonia is then recovered using methods
known in the art. In some alternative methods, dilute ammonia
pretreatment is utilized. The dilute ammonia pretreatment method
utilizes more dilute solutions of ammonia or ammonium hydroxide
than AFEX (See e.g., WO 2009/045651 and US 2007/0031953). This
pretreatment process may or may not produce any
monosaccharides.
[0200] An additional pretreatment process for use in the present
invention includes chemical treatment of the cellulosic substrate
with organic solvents, in methods such as those utilizing organic
liquids in pretreatment systems (See e.g., U.S. Pat. No.
4,556,430). These methods have the advantage that the low boiling
point liquids easily can be recovered and reused. Other
pretreatments, such as the Organosolv.TM. process, also use organic
liquids (See e.g., U.S. Pat. No. 7,465,791). Subjecting the
substrate to pressurized water may also be a suitable pretreatment
method (See e.g., Weil et al., Appl. Biochem. Biotechnol., 68:
21-40 [1997]). In some embodiments, the pretreated cellulosic
biomass is processed after pretreatment by any of several steps,
such as dilution with water, washing with water, buffering,
filtration, or centrifugation, or any combination of these
processes, prior to enzymatic hydrolysis, as is familiar to those
skilled in the art.
[0201] The pretreatment produces a pretreated feedstock composition
(e.g., a "pretreated feedstock slurry") that contains a soluble
component including the sugars resulting from hydrolysis of the
hemicellulose, optionally acetic acid and other inhibitors, and
solids including unhydrolyzed feedstock and lignin. In some
embodiments, the soluble components of the pretreated feedstock
composition are separated from the solids to produce a soluble
fraction. In some embodiments, the soluble fraction, including the
sugars released during pretreatment and other soluble components
(e.g., inhibitors), is then sent to fermentation. However, in some
embodiments in which the hemicellulose is not effectively
hydrolyzed during the pretreatment one or more additional steps are
included (e.g., a further hydrolysis step(s) and/or enzymatic
treatment step(s) and/or further alkali and/or acid treatment) to
produce fermentable sugars. In some embodiments, the separation is
carried out by washing the pretreated feedstock composition with an
aqueous solution to produce a wash stream and a solids stream
comprising the unhydrolyzed, pretreated feedstock. Alternatively,
the soluble component is separated from the solids by subjecting
the pretreated feedstock composition to a solids-liquid separation,
using any suitable method (e.g., centrifugation, microfiltration,
plate and frame filtration, cross-flow filtration, pressure
filtration, vacuum filtration, etc.). Optionally, in some
embodiments, a washing step is incorporated into the solids-liquids
separation. In some embodiments, the separated solids containing
cellulose, then undergo enzymatic hydrolysis with cellulase enzymes
in order to convert the cellulose to glucose. In some embodiments,
the pretreated feedstock composition is fed into the fermentation
process without separation of the solids contained therein. In some
embodiments, the unhydrolyzed solids are subjected to enzymatic
hydrolysis with cellulase enzymes to convert the cellulose to
glucose after the fermentation process. In some embodiments, the
pretreated cellulosic feedstock is subjected to enzymatic
hydrolysis with cellulase enzymes.
[0202] As used herein, the term "chemical treatment" refers to any
chemical pretreatment that promotes the separation and/or release
of cellulose, hemicellulose, and/or lignin.
[0203] As used herein, the term "physical pretreatment" refers to
any pretreatment that promotes the separation and/or release of
cellulose, hemicellulose, and/or lignin from cellulosic
material.
[0204] As used herein, the term "mechanical pretreatment" refers to
any mechanical means for treating biomass, including but not
limited to various types of grinding or milling (e.g., dry milling,
wet milling, or vibratory ball milling)
[0205] As used herein, 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.
[0206] As used herein, the term "recovered" refers to the
harvesting, isolating, collecting, or recovering of protein from a
cell and/or culture medium. In the context of saccharification, it
is used in reference to the harvesting of fermentable sugars
produced during the saccharification reaction from the culture
medium and/or cells. In the context of fermentation, it is used in
reference to harvesting the fermentation product from the culture
medium and/or cells. Thus, a process can be said to comprise
"recovering" a product of a reaction (such as a soluble sugar
recovered from saccharification) if the process includes separating
the product from other components of a reaction mixture subsequent
to at least some of the product being generated in the
reaction.
[0207] As used herein, "increasing" the yield of a product (such as
a fermentable sugar) from a reaction occurs when a particular
component of interest is present during the reaction (e.g., enzyme)
causes more product to be produced, compared with a reaction
conducted under the same conditions with the same substrate and
other substituents, but in the absence of the component of interest
(e.g., without enzyme).
[0208] As used herein, a reaction is said to be "substantially
free" of a particular enzyme if the amount of that enzyme compared
with other enzymes that participate in catalyzing the reaction is
less than about 2%, about 1%, or about 0.1% (wt/wt).
[0209] As used herein, "fractionating" a liquid (e.g., a culture
broth) means applying a separation process (e.g., salt
precipitation, column chromatography, size exclusion, and
filtration) or a combination of such processes to provide a
solution in which a desired protein (e.g., a cellulase enzyme,
and/or a combination thereof) comprises a greater percentage of
total protein in the solution than in the initial liquid
product.
[0210] As used herein, the term "enzymatic hydrolysis," refers to
the hydrolysis of a substrate by an enzyme. In some embodiments,
the hydrolysis comprises methods in which at least one enzyme is
contacted with at least one substrate to produce an end product. In
some embodiments, the enzymatic hydrolysis methods comprise at
least one cellulase and at least one glycosidase enzyme and/or a
mixture glycosidases that act on polysaccharides, (e.g.,
cellulose), to convert all or a portion thereof to fermentable
sugars. "Hydrolyzing" and/or "hydrolysis" of cellulose or other
polysaccharide occurs when at least some of the glycosidic bonds
between two monosaccharides present in the substrate are
hydrolyzed, thereby detaching from each other the two monomers that
were previously bonded.
[0211] It is intended that the enzymatic hydrolysis be carried out
with any suitable type of enzyme(s) capable of hydrolyzing at least
one substrate to at least one end-product. In some embodiments, the
substrate is cellulose, while in some other embodiments, it is
lignocelluloses, and in still further embodiments, it is another
composition (e.g., starch). In some embodiments, the end-product
comprises at least one fermentable sugar. It is further intended
that the enzymatic hydrolysis encompass processes carried out with
any suitable type of cellulase enzymes capable of hydrolyzing the
cellulose to glucose, regardless of their source. It is intended
that any suitable source of enzyme will find use in the present
invention, including but not limited to enzymes obtained from
fungi, such as Trichoderma spp., Aspergillus spp., Hypocrea spp.,
Humicola spp., Neurospora spp., Orpinomyces spp., Gibberella spp.,
Emericella spp., Chaetomium spp., Chrysosporium spp., Fusarium
spp., Penicillium spp., Magnaporthe spp., Phanerochaete spp.,
Trametes spp., Lentinula edodes, Gleophyllum trabeiu, Ophiostoma
piliferum, Corpinus cinereus, Geomyces pannorum, Cryptococcus
laurentii, Aureobasidium pullulans, Amorphotheca resinae,
Leucosporidium scotti, Cunninghamella elegans, Thermomyces
lanuginosus, Myceliopthora thermophila, and Sporotrichum
thermophile, as well as those obtained from bacteria of the genera
Bacillus, Thermomyces, Clostridium, Streptomyces and
Thermobifida.
[0212] In some embodiments, the enzymatic hydrolysis is carried out
at a pH and temperature that is at or near the optimum for the
cellulase enzymes being used. For example, in some embodiments, the
enzymatic hydrolysis is carried out at about 30.degree. C. to about
75.degree. C., or any suitable temperature therebetween, for
example a temperature of about 30.degree. C., about 35.degree. C.,
about 40.degree. C., about 45.degree. C., about 50.degree. C.,
about 55.degree. C., about 60.degree. C., about 65.degree. C.,
about 70.degree. C., about 75.degree. C., or any temperature
therebetween, and a pH of about 3.5 to about 7.5, or any pH
therebetween (e.g., about 3.5, about 4.0, about 4.5, about 5.0,
about 5.5, about 6.0, about 6.5, about 7.0, about 7.5, or any
suitable pH therebetween). In some embodiments, the initial
concentration of cellulose, prior to the start of enzymatic
hydrolysis, is preferably about 0.1% (w/w) to about 20% (w/w), or
any suitable amount therebetween (e.g., about 0.1%, about 0.5%,
about 1%, about 2%, about 4%, about 6%, about 8%, about 10%, about
12%, about 14%, about 15%, about 18%, about 20%, or any suitable
amount therebetween). In some embodiments, the combined dosage of
all cellulase enzymes is about 0.001 to about 100 mg protein per
gram cellulose, or any suitable amount therebetween (e.g., about
0.001, about 0.01, about 0.1, about 1, about 5, about 10, about 15,
about 20, about 25, about 30, about 40, about 50, about 60, about
70, about 80, about 90, about 100 mg protein per gram cellulose or
any amount therebetween). The enzymatic hydrolysis is carried out
for any suitable time period. In some embodiments, the enzymatic
hydrolysis is carried out for a time period of about 0.5 hours to
about 200 hours, or any time therebetween (e.g., about 2 hours to
about 100 hours, or any suitable time therebetween). For example,
in some embodiments, it is carried out for about 0.5, about 1,
about 2, about 5, about 7, about 10, about 12, about 14, about 15,
about 20, about 25, about 30, about 35, about 40, about 45, about
50, about 55, about 60, about 65, about 70, about 75, about 80,
about 85, about 90, about 95, about 100, about 120, about 140,
about 160, about 180, about 200, or any suitable time
therebetween.
[0213] In some embodiments, the enzymatic hydrolysis is batch
hydrolysis, continuous hydrolysis, and/or a combination thereof. In
some embodiments, the hydrolysis is agitated, unmixed, or a
combination thereof. The enzymatic hydrolysis is typically carried
out in a hydrolysis reactor. The cellulase enzyme composition is
added to the pretreated lignocellulosic substrate prior to, during,
or after the addition of the substrate to the hydrolysis reactor.
Indeed it is not intended that reaction conditions be limited to
those provided herein, as modifications are well-within the
knowledge of those skilled in the art. In some embodiments,
following cellulose hydrolysis, any insoluble solids present in the
resulting lignocellulosic hydrolysate, including but not limited to
lignin, are removed using conventional solid-liquid separation
techniques prior to any further processing. In some embodiments,
these solids are burned to provide energy for the entire
process.
[0214] As used herein, the "total available cellulose" is the
amount (wt %) of cellulose that is accessible to enzymatic
hydrolysis. Total available cellulose is typically equal to, or
very close to being equal to, the amount of initial cellulose
present in a hydrolysis reaction.
[0215] As used herein, the "residual cellulose" is the portion (wt
%) of the total available cellulose in the hydrolysis mixture that
remains unhydrolyzed. Residual cellulose can be measured using any
suitable method known in the art. For example, it can be directly
measured using IR spectroscopy, or it can be measured by
determining the amount of glucose generated by concentrated acid
hydrolysis of the residual solids.
[0216] As used herein, the "total hydrolyzed cellulose" is the
portion of the total available cellulose that is hydrolyzed in the
hydrolysis mixture. For example, the total hydrolyzed cellulose can
be calculated as the difference between the "total available
cellulose" and the "residual cellulose."
[0217] As used herein, the "theoretical maximum glucose yield" is
the maximum amount (wt %) of glucose that could be produced under
given conditions from the total available cellulose.
[0218] As used herein, "Gmax" refers to the maximum amount (wt %)
of glucose that could be produced from the total hydrolyzed
cellulose. Gmax can be calculated, for example, by directly
measuring the amount of residual cellulose remaining at the end of
a reaction under a given reaction conditions, subtracting the
amount of residual cellulose from the total available cellulose to
determine the total hydrolyzed cellulose, and then calculating the
amount of glucose that could be produced from the total hydrolyzed
cellulose.
[0219] It will be appreciated by those skilled in the art that when
calculating theoretical values such as Gmax and theoretical maximum
glucose yield, the mass of two hydrogen atoms and one oxygen atom
that are added to the glucose molecule in the course of the
hydrolysis reaction are taken into account. For example, when a
polymer of "n" glucose units is hydrolyzed, (n-1) units of water
are added to the glucose molecules formed in the hydrolysis, so the
weight of the glucose produced is about 10% greater than the weight
of cellulose consumed in the hydrolysis (e.g., hydrolysis of 1 g
cellulose would produce about 1.1 g glucose).
[0220] Thus, as an example, where 5 g of total available cellulose
is present at the beginning of a hydrolysis reaction, and 2 g of
residual cellulose remains after the reaction, the total hydrolyzed
cellulose is 3 g cellulose. A theoretical maximum glucose yield of
100% (w/w) under the reaction conditions is about 5.5 g of glucose.
Gmax is calculated based on the 3 g of cellulose that was released
or converted in the reaction by hydrolysis. Thus, in this example,
a Gmax of 100% (w/w) is about 3.3 g of glucose. Cellulose levels,
either the total available amount present in the substrate or the
amount of unhydrolyzed or residual cellulose, can be quantified by
any of a variety of methods known in the art, such as by IR
spectroscopy or by measuring the amount of glucose generated by
concentrated acid hydrolysis of the cellulose (See e.g., U.S. Pat.
Nos. 6,090,595 and 7,419,809).
[0221] As used herein, the term "undissolved solids" refers to
solid material which is suspended, but not dissolved, in a liquid.
As is well known in the art, the concentration of suspended or
undissolved solids can be determined by any suitable method (e.g.,
by filtering a sample of the slurry using glass microfiber filter
paper, washing the filter cake with water, and drying the cake
overnight at about 105.degree. C.).
[0222] As used herein, the terms "unhydrolyzed solids,"
"unconverted solids," and the like refer to cellulose that is not
digested by the cellulase enzyme(s), as well as non-cellulosic, or
other, materials that are inert to the cellulase enzyme(s), present
in the feedstock.
[0223] As used herein, the term "by-product" refers to an organic
molecule that is an undesired product of a particular process
(e.g., saccharification).
[0224] As used herein, the term "antibodies" refers to
immunoglobulins. Antibodies include but are not limited to
immunoglobulins obtained directly from any species from which it is
desirable to obtain antibodies. In addition, the present invention
encompasses modified antibodies. The term also refers to antibody
fragments that retain the ability to bind to the epitope that the
intact antibody binds and includes polyclonal antibodies,
monoclonal antibodies, chimeric antibodies, anti-idiotype (anti-ID)
antibodies. Antibody fragments include, but are not limited to the
complementarity-determining regions (CDRs), single-chain fragment
variable regions (scFv), heavy chain variable region (VH), and
light chain variable region (VL) fragments.
[0225] As used herein, the terms "thermally stable" and
"thermostable" refer to enzymes of the present invention that
retain a specified amount of enzymatic activity after exposure to
identified temperatures over a given period of time under
conditions prevailing during the use of the enzyme, for example,
when exposed to altered temperatures. "Altered temperatures"
include increased or decreased temperatures. In some embodiments,
the enzymes retain at least about 50%, about 60%, about 70%, about
75%, about 80%, about 85%, about 90%, about 92%, about 95%, about
96%, about 97%, about 98%, or about 99% enzymatic activity after
exposure to altered temperatures over a given time period, for
example, at least about 60 minutes, about 120 minutes, about 180
minutes, about 240 minutes, about 300 minutes, etc.
[0226] As used herein, the term "thermophilic fungus" refers to any
fungus which exhibits optimum growth at a temperature of at least
about 37.degree. C., and generally below about 100.degree. C., such
as for example between about 37.degree. C. to about 80.degree. C.,
between about 37.degree. C. to about 75.degree. C., between about
40.degree. C. to about 65.degree. C., or between about 40.degree.
C. to about 60.degree. C. Typically, the optimum growth is
exhibited at a temperature of at least about 40.degree. to about
60.degree. C.
[0227] As used herein, "solvent stable" refers to a polypeptide
that maintains similar activity (more than for example, about 60%
to about 80%) after exposure to varying concentrations (e.g., about
5 to about 99%) of a non-aqueous solvent (e.g., isopropyl alcohol,
tetrahydrofuran, 2-methyltetrahydrofuran, acetone, toluene,
butylacetate, methyl tert-butylether, etc.) for a period of time
(e.g., about 0.5 to about 24 hrs) compared to a reference
polypeptide.
[0228] As used herein, the term "oxidation stable" refers to
enzymes of the present invention that retain a specified amount of
enzymatic activity over a given period of time under conditions
prevailing during the use of the invention, for example while
exposed to or contacted with oxidizing agents. In some embodiments,
the enzymes retain at least about 50%, about 60%, about 70%, about
75%, about 80%, about 85%, about 90%, about 92%, about 95%, about
96%, about 97%, about 98%, or about 99% enzymatic activity after
contact with an oxidizing agent over a given time period, for
example, at least about 1 minute, about 3 minutes, about 5 minutes,
about 8 minutes, about 12 minutes, about 16 minutes, about 20
minutes, etc.
[0229] As used herein, "pH stable" refers to a polypeptide that
maintains similar activity (more than for example, about 60% to
about 80%) after exposure to low or high pH (e.g., about 4.5 to
about 6 or about 8 to about 12) for a period of time (e.g., 0.5-24
hrs) compared to a reference polypeptide.
[0230] As used herein, the term "enhanced stability" in the context
of an oxidation, chelator, thermal and/or pH stable enzyme refers
to a higher retained enzymatic activity over time as compared to
other enzymes and/or wild-type enzymes.
[0231] As used herein, the term "diminished stability" in the
context of an oxidation, chelator, thermal and/or pH stable enzyme
refers to a lower retained enzymatic activity over time as compared
to other enzymes and/or wild-type enzymes.
DETAILED DESCRIPTION OF THE INVENTION
[0232] The present invention provides improved fungal strains. In
some embodiments, the improved fungal strain finds use in
hydrolyzing cellulosic material to glucose. As indicated herein,
the present invention provides fungal strains that have reduced
secreted activity of an endogenous cellobiose dehydrogenase. In
some embodiments, the fungal strains secrete enzyme mixtures that
improve the yield of fermentable sugars from cellulose. Previous
reports have indicated that the oxidation of cellobiose by
cellobiose dehydrogenase enhances the rate of cellulose hydrolysis
by cellulases. In contrast to the traditional thinking in the art,
the present invention surprisingly provides genomic modifications
that reduce cellobiose dehydrogenase activity and result in
improvement in the yield of fermentable sugars from cellulose.
Advantageously, the genetically modified cellulase-producing fungal
cells provided herein secrete enzyme mixtures that result in
improved yields of fermentable sugars such as glucose from
cellulose.
[0233] Lignocellulose (also "lignocellulosic biomass") comprises a
matrix of cellulose, hemicellulose and lignin. Economic production
of biofuels from lignocellulosic biomass typically involves
conversion of the cellulose and hemicellulose components to
fermentable sugars, typically monosaccharides such as glucose (from
the cellulose) and xylose and arabinose (from the hemicelluloses).
Nearly complete conversion can be achieved by a chemical
pretreatment of the lignocellulose followed by enzymatic hydrolysis
with cellulase enzymes. The chemical pretreatment step renders the
cellulose more susceptible to enzymatic hydrolysis and in some
cases, also hydrolyzes the hemicellulose component. Numerous
chemical pretreatment processes known in the art find use in the
present invention, and include, but are not limited to, mild acid
pretreatment at high temperatures and dilute acid, ammonium
pretreatment and/or organic solvent extraction.
[0234] Cellulase is typically a mixture of different types of
cellulolytic enzymes (e.g., endoglucanases and cellobiohydrolases,
the latter are also referred to as "exoglucanases") that act
synergistically to break down the cellulose to soluble di- or
oligosaccharides such as cellobiose, which are then further
hydrolyzed to glucose by beta-glucosidase. Cellulase enzymes are
produced by a wide variety of microorganisms. Cellulases, as well
as hemicellulases from filamentous fungi and some bacteria are
widely exploited for many industrial applications that involve
processing of natural fibers to sugars.
[0235] Lignin is a more complex and heterogeneous biopolymer than
either cellulose or hemicellulose and comprises a variety of
phenolic subunits. Enzymatic lignin depolymerization can be
accomplished by lignin peroxidases, manganese peroxidases,
laccases, and/or cellobiose dehydrogenases (CDH), often working in
synergy. However, as the name suggests, CDH enzymes also oxidize
cellobiose to cellobionolactone. Several reports indicate that the
oxidation of cellobiose by CDH enhances the rate of cellulose
hydrolysis by cellulases by virtue of reducing the concentrations
of cellobiose, which is a potent inhibitor of some cellulase
components (See e.g., Mansfield et al., Appl. Environ. Microbiol.,
63: 3804-3809 [1997]; and Igarishi et al., Eur. J. Biochem.,
253:101-106 [1998]). Recently, it has been reported that CDHs can
enhance the activity of cellulolytic enhancing proteins from
Glycosyl Hydrolase family 61 (See e.g., WO2010/080532A1).
[0236] Among the cellulase-producing filamentous fungi, there are
those that also produce a variety of enzymes involved in lignin
degradation. For example, organisms of such genera as
Myceliophthora, Chrysosporium, Sporotrichum, Thielavia,
Phanerochaete and Trametes produce and secrete a mixture of
cellulases, hemicellulases and lignin degrading enzymes. These
types of organisms are commonly called "white rot fungi" by virtue
of their ability to digest lignin and to distinguish them from the
"brown rot" fungi (such as Trichoderma) which typically cannot
digest lignin.
Genetically Modified Fungal Cells
[0237] The genetically modified fungal cells provided herein permit
a reduction in the amount of endogenous cellobiose dehydrogenase
activity that is secreted by the cell. In some genetically modified
fungal cells provided herein, the cellobiose dehydrogenase activity
secreted by the cell is reduced by at least about 5%, about 10%,
about 15%, about 20%, about 25%, about 30%, about 35%, about 40%,
about 45%, about 50%, about 55%, about 60%, about 65%, about 70%,
about 75%, about 80%, about 85%, about 90%, about 91%, about 92%,
about 93%, about 94%, about 95%, about 96%, about 97%, about 98%,
about 99%, or more, relative to the level of cellobiose
dehydrogenase activity secreted by the unmodified parental fungal
cell grown or cultured under essentially the same culture
conditions. In some embodiments, a genetically modified fungal cell
provides at least about 5%, about 10%, about 15%, about 20%, about
25%, about 30%, about 35%, about 40%, about 45%, about 50%, about
55%, about 60%, about 65%, about 70%, about 75%, about 80%, about
85%, about 90%, about 91%, about 92%, about 93%, about 94%, about
95%, about 96%, about 97%, about 98%, about 99% or more, relative
to the level of cellobiose dehydrogenase activity secreted by the
unmodified parental fungal cell grown or cultured under essentially
the same conditions.
[0238] It will be readily appreciated that any suitable genetic
modification known in the art can be employed to reduce the
secreted activity of the endogenous cellobiose dehydrogenase. For
example, as described below, modifications contemplated herein
include modifications that reduce the amount of cellobiose
dehydrogenase secreted by the cell. Modifications that reduce the
amount of cellobiose dehydrogenase expressed by the cell are also
contemplated. Additional embodiments include modifications that
reduce the transcription level of cellobiose dehydrogenase. Still
further embodiments include the complete or partial deletion of a
gene encoding cellobiose dehydrogenase. Other embodiments include
modifications that reduce the catalytic efficiency of cellobiose
dehydrogenase.
Secreted Enzymes
[0239] In some embodiments, the fungal cells of the present
invention have been genetically modified to reduce the amount of
the endogenous cellobiose dehydrogenase secreted by the cell. A
reduction in the amount of secreted cellobiose dehydrogenase can be
a complete or partial reduction of the cellobiose dehydrogenase
secreted to the extracellular milieu Reduction in the amount of
secreted cellobiose dehydrogenase can be accomplished by reducing
the amount of cellobiose dehydrogenase produced by the cell and/or
by reducing the ability of the cell to secrete the cellobiose
dehydrogenase produced by the cell. Methods for reducing the
ability of the cell to secrete a polypeptide can be performed
according to any of a variety of suitable methods known in the art
(See e.g., Fass and Engels J. Biol. Chem., 271:15244-15252 [1996],
which is incorporated by reference herein in its entirety). For
example, the gene encoding a secreted polypeptide can be modified
to delete or inactivate a secretion signal peptide. In some
embodiments, the fungal cells have been genetically modified to
disrupt the N-terminal secretion signal peptide of the cellobiose
dehydrogenase. In some embodiments, the amount of cellobiose
dehydrogenase secreted by the cell is reduced by at least about 5%,
about 10%, about 15%, about 20%, about 25%, about 30%, about 35%,
about 40%, about 45%, about 50%, about 55%, about 60%, about 65%,
about 70%, about 75%, about 80%, about 85%, about 90%, about 91%,
about 92%, about 93%, about 94%, about 95%, about 96%, about 97%,
about 98%, about 99%, or more, relative to the secretion of
cellobiose dehydrogenase in an unmodified organism grown or
cultured under essentially the same culture conditions.
[0240] Furthermore, in some embodiments, the total amount of
cellobiose dehydrogenase activity is reduced by at least about 5%,
about 10%, about 15%, about 20%, about 25%, about 30%, about 35%,
about 40%, about 45%, about 50%, about 55%, about 60%, about 65%,
about 70%, about 75%, about 80%, about 85%, about 90%, about 91%,
about 92%, about 93%, about 94%, about 95%, about 96%, about 97%,
about 98%, about 99%, or more, relative to the total amount of
cellobiose dehydrogenase secreted in an unmodified organism grown
or cultured under essentially the same culture conditions.
[0241] Decreased secretion of a cellobiose dehydrogenase can be
determined by any of a variety of suitable methods known in the art
for detection of protein or enzyme levels. For example, the levels
of cellobiose dehydrogenase in the supernatant of a fungal culture
can be detected using Western blotting techniques or any other
suitable protein detection techniques that use an antibody specific
to cellobiose dehydrogenase. Similarly, secreted cellobiose
dehydrogenase activity in the supernatant of a fungal culture can
be measured using assays for cellobiose dehydrogenase activity as
described in greater detail herein.
Expression Level
[0242] In some embodiments, the fungal cells have been genetically
modified to reduce the amount of the endogenous cellobiose
dehydrogenase expressed by the cell. As used herein, expression
refers to conversion of the information encoded in a gene to the
protein encoded by that gene. Thus, a reduction of the amount of an
expressed cellobiose dehydrogenase represents a reduction in the
amount of the cellobiose dehydrogenase that is eventually
translated by the cell. In some such embodiments, the reduction in
the expression is accomplished by reducing the amount of mRNA that
is transcribed from a gene encoding cellobiose dehydrogenase. In
some other embodiments, the reduction in the expression is
accomplished by reducing the amount of protein that is translated
from a mRNA encoding cellobiose dehydrogenase.
[0243] The amount of cellobiose dehydrogenase expressed by the cell
can be reduced by at least about 5%, about 10%, about 15%, about
20%, about 25%, about 30%, about 35%, about 40%, about 45%, about
50%, about 55%, about 60%, about 65%, about 70%, about 75%, about
80%, about 85%, about 90%, about 91%, about 92%, about 93%, about
94%, about 95%, about 96%, about 97%, about 98%, about 99%, or
more, relative to the expression of cellobiose dehydrogenase in an
unmodified fungal cell. In some such embodiments, the reduction in
the expression is accomplished by reducing the amount of mRNA that
is transcribed from a gene encoding cellobiose dehydrogenase in an
unmodified organism grown or cultured under essentially the same
culture conditions.
[0244] Furthermore, in some embodiments, a reduction in the
expression level of a cellobiose dehydrogenase results in at least
about 5%, about 10%, about 15%, about 20%, about 25%, about 30%,
about 35%, about 40%, about 45%, about 50%, about 55%, about 60%,
about 65%, about 70%, about 75%, about 80%, 85% about, 90%, about
91%, about 92%, about 93%, about 94%, about 95%, about 96%, about
97%, about 98%, or about a 99% reduction in the total expression
level of cellobiose dehydrogenase activity by the fungal cell
relative to an unmodified fungal cell grown or cultured under
essentially the same culture conditions.
[0245] Decreased expression of a cellobiose dehydrogenase can be
determined by any of a variety of methods known in the art for
detection of protein or enzyme levels. For example, the levels of
cellobiose dehydrogenase in the supernatant of a fungal culture can
be detected using Western blotting techniques or any other suitable
protein detection techniques that use an antibody specific to
cellobiose dehydrogenase.
[0246] Methods for reducing expression of a polypeptide are well
known and can be performed using any of a variety of suitable
methods known in the art. For example, the gene encoding a secreted
polypeptide can be modified to disrupt a translation initiation
sequence such as a Shine-Delgarno sequence or a Kozak consensus
sequence. Furthermore, the gene encoding a secreted polypeptide can
be modified to introduce a frameshift mutation in the transcript
encoding the endogenous cellobiose dehydrogenase. It will also be
recognized that usage of uncommon codons can result in reduced
expression of a polypeptide. It will be appreciated that in some
embodiments, the gene encoding the cellobiose dehydrogenase has at
least one nonsense mutation that results in the translation of a
truncated protein.
[0247] Other methods of reducing the amount of expressed
polypeptide include post-transcriptional RNA silencing
methodologies such as antisense RNA and RNA interference. Antisense
techniques are well-established, and include using a nucleotide
sequence complementary to the nucleic acid sequence of the gene.
More specifically, expression of the gene by a fungal cell may be
reduced or eliminated by introducing a nucleotide sequence
complementary to the nucleic acid sequence, which may be
transcribed in the cell and is capable of hybridizing to the mRNA
produced in the cell. Under conditions allowing the complementary
anti-sense nucleotide sequence to hybridize to the mRNA, the amount
of protein translated is thus reduced or eliminated. Methods for
expressing antisense RNA are known in the art (See e.g., Ngiam et
al., Appl Environ Microbiol., 66(2):775-82 [2000]; and Zrenner et
al., Planta., 190(2):247-52 [1993]), both of which are hereby
incorporated by reference herein in their entirety).
[0248] Furthermore, modification, downregulation or inactivation of
the gene may be obtained via RNA interference (RNAi) techniques
(See e.g., Kadotani et al. Mol. Plant Microbe Interact., 16:769-76
[2003], which is incorporated by reference herein in its entirety).
RNA interference methodologies include double stranded RNA (dsRNA),
short hairpin RNAs (shRNAs) and small interfering RNAs (siRNAs).
Potent silencing using dsRNA may be obtained using any suitable
technique (See e.g., Fire et al., Nature 391:806-11 [1998]).
Silencing using shRNAs is also well-established (See e.g., Paddison
et al., Genes Dev., 16:948-958 [2002]). Silencing using siRNA
techniques are also known (See e.g., Miyagishi et al., Nat.
Biotechnol., 20:497-500 [2002]). The content of each of the
above-cited references is incorporated by reference herein in its
entirety.
Transcription Level
[0249] In some embodiments, the fungal cells of the present
invention have been genetically modified to reduce the
transcription level of a gene encoding the endogenous cellobiose
dehydrogenase. As used herein, transcription and similar terms
refer to the conversion of the information encoded in a gene to an
RNA transcript. Accordingly, a reduction of the transcription level
of a cellobiose dehydrogenase is a reduction in the amount of RNA
transcript of an RNA coding for a cellobiose dehydrogenase. In some
embodiments, the transcription level is reduced by at least about
5%, about 10%, about 15%, about 20%, about 25%, about 30%, about
35%, about 40%, about 45%, about 50%, about 55%, about 60%, about
65%, about 70%, about 75%, about 80%, about 85%, about 90%, about
91%, about 92%, about 93%, about 94%, about 95%, about 96%, about
97%, about 98%, about 99%, or more, relative to the transcription
level of a cellobiose dehydrogenase in an unmodified organism grown
or cultured under essentially the same culture conditions.
[0250] Furthermore, in some embodiments, a reduction in the
transcription level of a cellobiose dehydrogenase results in at
least about 5%, about 10%, about 15%, about 20%, about 25%, about
30%, about 35%, about 40%, about 45%, about 50%, about 55%, about
60%, about 65%, about 70%, about 75%, about 80%, about 85%, about
90%, about 91%, about 92%, about 93%, about 94%, about 95%, about
96%, about 97%, about 98%, or about a 99% reduction in the total
cellobiose dehydrogenase secreted by the fungal cell relative to an
unmodified organism grown or cultured under essentially the same
culture conditions. Decreased transcription can be determined by
any of a variety of methods known in the art for detection of
transcription levels. For example, the levels of transcription of a
particular mRNA in a fungal cell can be detected using quantitative
RT-PCR techniques or other RNA detection techniques that
specifically detect a particular mRNA. Methods for reducing
transcription level of a gene can be performed according to any
suitable method known in the art, and include partial or complete
deletion of the gene, and disruption or replacement of the promoter
of the gene such that transcription of the gene is greatly reduced
or even inhibited. For example, the promoter of the gene can be
replaced with a weak promoter (See e.g., U.S. Pat. No. 6,933,133,
which is incorporated by reference herein in its entirety). Thus,
where the weak promoter is operably linked with the coding sequence
of an endogenous polypeptide, transcription of that gene is greatly
reduced or inhibited.
Gene Deletion
[0251] In some embodiments, the fungal cells of the present
invention have been genetically modified to at least partially
delete a gene encoding the endogenous cellobiose dehydrogenase.
Typically, this deletion reduces or eliminates the total amount of
endogenous cellobiose dehydrogenase secreted by the fungal cell. In
some embodiments, complete or near-complete deletion of the gene
sequence is contemplated. However, a deletion mutation need not
completely remove the entire gene sequence encoding cellobiose
dehydrogenase, in order to reduce the amount of endogenous
cellobiose dehydrogenase secreted by the fungal cell. For example,
in some embodiments, there is a partial deletion that removes one
or more nucleotides encoding an amino acid in a cellobiose
dehydrogenase active site, encoding a secretion signal, or encoding
another portion of the cellobiose dehydrogenase that plays a role
in endogenous cellobiose dehydrogenase activity being secreted by
the fungal cell.
[0252] A deletion in a gene encoding cellobiose dehydrogenase in
accordance with the embodiments provided herein include a deletion
of one or more nucleotides in the gene encoding the cellobiose
dehydrogenase. In some embodiments, there is a deletion of at least
about 5%, about 10%, about 15%, about 20%, about 25%, about 30%,
about 35%, about 40%, about 45%, about 50%, about 55%, about 60%,
about 65%, about 70%, about 75%, about 80%, about 85%, about 90%,
about 91%, about 92%, about 93%, about 94%, about 95%, about 96%,
about 97%, about 98%, about 99%, or about 100%, of the gene
encoding the cellobiose dehydrogenase, wherein the amount of
cellobiose dehydrogenase secreted by the cell is reduced.
[0253] Thus, in some embodiments, the deletion results in at least
about 5%, about 10%, about 15%, about 20%, about 25%, about 30%,
about 35%, about 40%, about 45%, about 50%, about 55%, about 60%,
about 65%, about 70%, about 75%, about 80%, about 85%, about 90%,
about 91%, about 92%, about 93%, about 94%, about 95%, about 96%,
about 97%, about 98%, or about a 99% reduction in the activity of
the cellobiose dehydrogenase secreted by the fungal cell, relative
to the activity of cellobiose dehydrogenase secreted by an
unmodified organism grown or cultured under essentially the same
culture conditions.
[0254] Furthermore, in some embodiments, the deletion results in at
least about 5%, about 10%, about 15%, about 20%, about 25%, about
30%, about 35%, about 40%, about 45%, about 50%, about 55%, about
60%, about 65%, about 70%, about 75%, about 80%, about 85%, about
90%, about 91%, about 92%, about 93%, about 94%, about 95%, about
96%, about 97%, about 98%, or about a 99% reduction in the total
cellobiose dehydrogenase secreted by the fungal cell relative to an
unmodified fungal cell grown or cultured under essentially the same
culture conditions.
[0255] Deletion of a cellobiose dehydrogenase gene can be detected
and confirmed by any of a variety of methods known in the art for
detection of gene deletions, including the methods provide herein
and in the Examples. For example, as exemplified herein, gene
deletion can be confirmed using PCR amplification of the modified
genomic region. It will be appreciated that additional suitable
techniques for confirming deletion can be used and are well known,
including Southern blot techniques, DNA sequencing of the modified
genomic region, and screening for positive or negative markers
incorporated during recombination events.
[0256] Methods for complete and/or partial deletion of a gene are
well-known and the genetically modified fungal cells described
herein can be generated using any of a variety of deletion methods
known in the art that result in a reduction in the amount of
endogenous cellobiose dehydrogenase secreted by the cells. Such
methods may advantageously include standard gene disruption using
homologous flanking markers (See e.g., Rothstein, Meth. Enzymol.,
101:202-211 [1983], incorporated herein by reference in its
entirety). Additional techniques for gene deletion include
PCR-based methods for standard deletion (See e.g., Davidson et al.,
Microbiol., 148:2607-2615 [2002], incorporated herein by reference
in its entirety).
[0257] Additional gene deletion techniques include
"positive-negative" cassettes; cre/lox based deletion, biolistic
transformation to increase homologous recombination, and
Agrobacterium-mediated gene disruption. The "positive-negative"
method employs cassettes which consist of one marker gene for
positive screening and another marker gene for negative screening
(See e.g., Chang et al., Proc. Natl. Acad. Sci. USA 84:4959-4963
[1987]). Cre/lox based methodologies employ elimination of marker
genes using expression of Cre recombinase (See e.g., Florea et al.,
Fung. Genet. Biol., 46:721-730 [2009]).
[0258] Methods to introduce DNA or RNA into fungal cells are known
to those of skill in the art and include, but are not limited to
PEG-mediated transformation of protoplasts, electroporation,
biolistic transformation, and Agrobacterium-mediated
transformation. Biolistic transformation employs a process in which
DNA or RNA is introduced into cells on micron-sized particles, thus
increasing delivery of a deletion construct to the fungal cell (See
e.g., Davidson et al., Fung. Genet. Biol., 29:38-48 [2000]).
Similarly, Agrobacterium-mediated transformation in conjunction
with linear or split-marker deletion cassettes can facilitate
delivery of deletion constructs to the target cell (See e.g., Wang
et al., Curr. Genet., 56:297-307 [2010]).
[0259] Further methods for complete or partial gene deletion
include disruption of the gene. Such gene disruption techniques are
known to those of skill in the art, including, but not limited to
insertional mutagenesis, the use of transposons, and marked
integration. However, it will be appreciated that any suitable
technique that provides for disruption of the coding sequence or
any other functional aspect of a gene finds use in generating the
genetically modified fungal cells provided herein. Methods of
insertional mutagenesis can be performed according to any suitable
method known in the art (See e.g., Combier et al., FEMS Microbiol
Lett., 220:141-8 [2003], which is incorporated by reference herein
in its entirety). In addition, Agrobacterium-mediated insertional
mutagenesis can be used to insert a sequence that disrupts the
function of the encoded gene, such as disruption of the coding
sequence or any other functional aspect of the gene.
[0260] Transposon mutagenesis methodologies provide another means
for gene disruption. Transposon mutagenesis is well known in the
art, and can be performed using in vivo techniques (See e.g., Firon
et al., Eukaryot. Cell 2:247-55 [2003]); or by the use of in vitro
techniques (See e.g., Adachi et al., Curr. Genet., 42:123-7 [2002])
; both of these references are incorporated by reference in their
entireties. Thus, targeted gene disruption using transposon
mutagenesis can be used to insert a sequence that disrupts the
function of the encoded gene, such as disruption of the coding
sequence or any other functional aspect of the gene.
[0261] Restriction enzyme-mediated integration (REMI) is another
methodology for gene disruption, and is well known in the art (See
e.g., Thon et al., Mol. Plant Microbe Interact., 13:1356-65 [2000],
which is incorporated by reference herein in its entirety). REMI
generates insertions into genomic restriction sites in an
apparently random manner, some of which cause mutations. Thus,
insertional mutants that demonstrate a disruption in the gene
encoding the endogenous cellobiose dehydrogenase can be selected
and utilized as provided herein.
Catalytic Disruption
[0262] In some other embodiments, the fungal cell has been
genetically modified to reduce the catalytic efficiency of the
cellobiose dehydrogenase. A reduction in catalytic efficiency
refers to a reduction in the activity of cellobiose dehydrogenase,
relative to unmodified cellobiose dehydrogenase, as measured using
standard techniques, as provided herein or otherwise known in the
art. Thus, a genetic modification that reduces catalytic efficiency
can result in, for example, a translated protein product that has a
reduction in enzymatic activity.
[0263] A reduction in catalytic efficiency is a reduction of
cellobiose dehydrogenase activity of about 5%, about 10%, about
15%, about 20%, about 25%, about 30%, about 35%, about 40%, about
45%, about 50%, about 55%, about 60%, about 65%, about 70%, about
75%, about 80%, about 85%, about 90%, about 91%, about 92%, about
93%, about 94%, about 95%, about 96%, about 97%, about 98%, about
99%, or more, relative to unmodified cellobiose dehydrogenase, as
measured using standard techniques. In some further embodiments,
the genetic modification results in a reduction of cellobiose
dehydrogenase activity of at least about 5%, about 10%, about 15%,
about 20%, about 25%, about 30%, about 35%, about 40%, about 45%,
about 50%, about 55%, about 60%, about 65%, about 70%, about 75%,
about 80%, about 85%, about 90%, about 91%, about 92%, about 93%,
about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%
in the total cellobiose dehydrogenase activity secreted by the
fungal cell, as compared to unmodified cellobiose dehydrogenase, as
measured using standard techniques.
[0264] Methods for reducing catalytic efficiency of dehydrogenases
are well known, and as such, any of a variety of suitable methods
known in the art for reducing catalytic efficiency find use in
genetically modifying the fungal cells provided herein. Thus, for
example, the fungal cell can be genetically modified to inactivate
one or more residues in an active site of the cellobiose
dehydrogenase (See e.g., Frederik et at., Biochem., 42:4049-4056
[2003], incorporated by reference herein in its entirety). For
example, one or more residues can be modified to decrease substrate
binding, and/or one or more residues can be modified to decrease
the catalytic activity of the cellobiose dehydrogenase.
Accordingly, one or more residues in the electron acceptor (e.g.,
flavin) binding domain, or any substrate binding domain of
cellobiose dehydrogenase can be performed to reduce or inactivate
the catalytic efficiency of the cellobiose dehydrogenase.
Similarly, it will be apparent that mutation of residues outside an
active site can result in allosteric change in the shape or
activity of the cellobiose dehydrogenase, such that the catalytic
efficient of the enzyme is reduced.
[0265] In some embodiments, other domains are targeted for at least
one mutation which results in reducing catalytic efficiency of the
endogenous cellobiose dehydrogenase. For example, in some
embodiments, a mutation to one or more residues in a heme-binding
domain of cellobiose dehydrogenase can result in reduced catalytic
efficiency (See e.g., Rotsaert et al., Arch. Biochem. Biophys.,
390:206-14 [2001], which is incorporated by reference herein in its
entirety).
Fungal Cells
[0266] As indicated herein, the present invention provides fungal
cells from the family Chaetomiaceae that have been genetically
modified to reduce the amount of endogenous cellobiose
dehydrogenase activity that is secreted by the cell, where the
fungal cell is capable of secreting a cellulase-containing enzyme
mixture. The Chaetomiaceae are a family of fungi in the Ascomycota,
class Sordariomycetes. The family Chaetomiaceae includes the genera
Achaetomium, Aporothielavia, Chaetomidium, Chaetomium, Corylomyces,
Corynascus, Farrowia, Thielavia, Zopfiella, and Myceliophthora. In
some embodiments, the genetically modified fungal cell provided
herein is a Chaetomiaceae family member selected from
Myceliophthora, Thielavia, Corynascus, and Chaetomium.
[0267] In some embodiments, the genetically modified fungal cell is
an anamorph or teleomorph of a Chaetomiaceae family member selected
from Myceliophthora, Thielavia, Corynascus, and Chaetomium. In some
embodiments, the genetically modified fungal cell is selected from
Sporotrichum, Chrysosporium, Paecilomyces, Talaromyces and
Acremonium. It is also contemplated that the genetically modified
fungal cell can also be selected from the genera Ctenomyces,
Thermoascus, and Scytalidium, including anamorphs and teleomorphs
of fungal cells of these genera. In some embodiments, the
genetically modified fungal cell is selected from the strains of
Sporotrichum cellulophilum, Thielavia heterothallica, Corynascus
heterothallicus, Thielavia terrestris, and Myceliophthora
thermophila, including anamorphs and teleomorphs thereof. It is not
intended that the present invention be limited to any particular
genus within the Chaetomiaceae family In some further embodiments,
the genetically modified fungal cell is a thermophilic species of
Acremonium, Arthroderma, Corynascus, Thielavia, Myceliophthora,
Thermoascus, Chromocleista, Byssochlamys, Sporotrichum, Chaetomium,
Chrysosporium, Scytalidium, Ctenomyces, Paecilomyces, or
Talaromyces. It will be understood that for all of the
aforementioned species, the genetically modified fungal cell
presented herein encompasses both the perfect and imperfect states,
and other taxonomic equivalents (e.g., anamorphs), regardless of
the species name by which they are known (See e.g., Cannon,
Mycopathol., 111:75-83 [1990]; Moustafa et al., Persoonia
14:173-175 [1990]; Upadhyay et al., Mycopathol., 87:71-80 [1984];
Guarro et al., Mycotaxon 23: 419-427 [1985]; Awao et al., Mycotaxon
16:436-440 [1983]; and von Klopotek, Arch. Microbiol., 98:365-369
[1974]). Those skilled in the art will readily recognize the
identity of appropriate equivalents. Accordingly, it will be
understood that, unless otherwise stated, the use of a particular
species designation in the present disclosure also refers to
species that are related by anamorphic or teleomorphic
relationship. For example, the following species are anamorphs or
teleomorphs and may therefore be considered as synonymous:
Myceliophthora thermophila, Sporotrichum thermophile, Sporotrichum
thermophilum, Sporotrichum cellulophilum, Chrysosporium
thermophile, Corynascus heterothallicus, and Thielavia
heterothallica.
[0268] In some embodiments, the genetically modified fungal cells
provided herein are cellulase-producing fungal cells. In some
embodiments, the cellulase-producing fungal cells express and
secrete a mixture of cellulose hydrolyzing enzymes. In some
embodiments, the genetically modified fungal cells provided herein
are fungal cells from the family Chaetomiaceae that secrete two or
more cellulose hydrolyzing enzymes (e.g., endoglucanase,
cellobiohydrolase, and/or beta-glucosidase). In some additional
embodiments, the cellulase-producing fungal cells produce two or
more of these enzymes, in any combination.
[0269] Additionally, in some embodiments, the genetically modified
fungal cell is derived from a lignocellulose-competent parental
fungal cell. In some embodiments, lignocellulose-competent fungal
cells secrete one or more lignin peroxidases, manganese
peroxidases, laccases and/or cellobiose dehydrogenases (CDH).
[0270] The present invention also provides a fungal culture in a
vessel comprising a genetically modified fungal cell as described
hereinabove. In some embodiments, the vessel comprises a liquid
medium, such as fermentation medium. For example, the vessel can be
a flask, bioprocess reactor, or any suitable container. In some
embodiments, the vessel comprises a solid growth medium. For
example, the solid medium can be an agar medium such as potato
dextrose agar, carboxymethylcellulose, cornmeal agar, and any other
suitable medium. In some embodiments, the fungal cell described
hereinabove is an isolated fungal cell.
Cellobiose Dehydrogenase
[0271] As indicated herein, the terms "cellobiose dehydrogenase"
and "CDH" refer to a cellobiose: acceptor 1-oxidoreductase that
catalyzes the conversion of cellobiose in the presence of an
acceptor to cellobiono-1,5-lactone and a reduced acceptor. Examples
of cellobiose dehydrogenases fall into the enzyme classification
(E.C. 1.1.99.18). Typically 2,6-dichloroindophenol can act as
acceptor, as can iron, especially Fe(SCN).sub.3, molecular oxygen,
ubiquinone, or cytochrome C, and other polyphenolics, such as
lignin. Substrates of the enzyme include cellobiose,
cello-oligosaccharides, lactose, and
D-glucosyl-1,4-.beta.-D-mannose, glucose, maltose, mannobiose,
thiocellobiose, galactosyl-mannose, xylobiose, xylose. Electron
donors include beta-1-4 dihexoses with glucose or mannose at the
reducing end, though alpha-1-4 hexosides, hexoses, pentoses, and
beta-1-4 pentomers can act as substrates for at least some of these
enzymes (See e..g, Henriksson et al., Biochim. Biophys. Acta Prot.
Struct. Mol. Enzymol., 1383: 48-54 [1998]; and Schou et al.,
Biochem. J., 330: 565-571 [1998]).
[0272] In some embodiments, a CDH enzyme contains both the
conserved glucose-methanol-choline (GMC) oxido-reductase N and the
GMC oxido-reductase C domains. In some other embodiments, a CDH
contains the GMC oxido-reductase N domain alone. The GMC
oxidoreductases are FAD flavoprotein oxidoreductases (See e.g.,
Cavener, J. Mol. Biol., 223:811-814 [1992]; and Vrielink and Blow,
Biochem., 32:11507-15 [1993]). The GMC oxidoreductases include a
variety of proteins, such as choline dehydrogenase, methanol
oxidase, and cellobiose dehydrogenase (CDH), which share a number
of regions with sequence similarities. One of these regions,
located in the N-terminal section, corresponds to the FAD
ADP-binding domain, as further defined by the Pfam database under
the entry GMC_oxred_N (PF00732). Similarly, the C-terminal
conserved domain (GMC oxido-reductase C domain) is defined as set
forth in the Pfam database under the entry GMC_oxred_C
(PF05199).
[0273] Cellobiose dehydrogenases can be categorized into two
families The first family contains a catalytic portion and the
second family contains a catalytic portion and a cellulose binding
motif (CBM). The 3-dimensional structure of an exemplary cellobiose
dehydrogenase features two globular domains, each containing one of
two cofactors, namely a heme or a flavin. The active site lies at a
cleft between the two domains. Oxidation of cellobiose typically
occurs via 2-electron transfer from cellobiose to the flavin,
generating cellobiono-1,5-lactone and reduced flavin. The active
FAD is regenerated by electron transfer to the heme group, leaving
a reduced heme. The native state heme is regenerated by reaction
with the oxidizing substrate at the second active site. In some
embodiments, the acceptor is preferentially iron ferricyanide,
cytochrome C, or an oxidized phenolic compound such as
dichloroindophenol (DCIP), an acceptor commonly used for
colorimetric assays. Metal ions and 0.sub.2 are also acceptors, but
for most cellobiose dehydrogenases the reaction rate of cellobiose
oxidase for these acceptors is several orders of magnitude lower
than that observed for iron or organic oxidants. Following
cellobionolactone release, the product may undergo spontaneous
ring-opening to generate cellobionic acid (See e.g., Hallberg et
al., J. Biol. Chem., 278:7160-7166 [2003]). Those of skill in the
art will appreciate that cellobiose dehydrogenase enzyme activity
typically employs the presence of oxygen or an equivalent redox
acceptor, which may be, for example, lignin, molecular oxygen,
cytochrome c, redox dyes, benzoquinones and/or Fe.sup.2+
complexes.
[0274] Cellobiose dehydrogenase activity can be measured using any
of a variety of suitable methods known in the art (See e.g., Schou
et al., Biochem J., 220:565-71 [1998], which is incorporated by
reference in its entirety). For example, DCPIP
(2,6-dichlorophenolindophenol) reduction by CDH activity in the
presence of cellobiose can be monitored by absorbance at 530 nm
[0275] As provided herein, a fungal cell that has been genetically
modified to reduce the secreted activity of a cellobiose
dehydrogenase typically has reduced secreted activity of an
endogenous cellobiose dehydrogenase. Accordingly, one or more
cellobiose dehydrogenase enzymes from each of the fungal species
described herein can be targeted for genetic modification. In some
embodiments, the cellobiose dehydrogenase is from a fungal species
in the family Chaetomiaceae. Some examples of cellobiose
dehydrogenase enzymes identified from Chaetomiaceae family members
are set forth in Table 1, below. In some embodiments, the
cellobiose dehydrogenase is from a fungal species selected from
Sporotrichum cellulophilum, Thielavia heterothallica, Corynascus
heterothallicus, Thielavia terrestris, Chaetomium globosum and
Myceliophthora thermophila. Some cellobiose dehydrogenase enzymes
identified from these species are set forth in the table below. The
proteins listed in the table below are examples of cellobiose
dehydrogenase that are known in the art, or identified herein as
being a cellobiose dehydrogenase.
TABLE-US-00002 TABLE 1 Cellobiose Dehydrogenase Sequences GMC oxred
N GMC oxred C Accession Number Organism Domain Domain AAC26221
Myceliophthora thermophila 251-554 645-781 XP_001229896.1
Chaetomium globosum CBS 226-529 620-757 148.51 JGIThite5441
Thielavia terrestris 253-555 647-783 JGIThite4524 Thielavia
terrestris 36-337 NA XP_001225932.1 Chaetomium globosum CBS 36-338
NA 148.51 JGIThite6738 Thielavia terrestris 249-550 642-779 CDH2
derived from a C1 strain Myceliophthora thermophila 249-550 NA
XP_001226549.1 Chaetomium globosum CBS 249-521 549-667 148.51
*Accession numbers for Thielavia terrestris refer to the U.S.
Department of Energy (DOE) Joint Genome Institute (JGI) genome
sequence
[0276] Certain amino acid sequences encoding cellobiose
dehydrogenase are provided herein. For example, the nucleotide
sequence encoding Myceliophthora thermophila CDH1 is set forth
herein as SEQ ID NO:1, and the encoded amino acid sequence of
Myceliophthora thermophila CDH1 is set forth as SEQ ID NO:2.
[0277] In some embodiments, the cellobiose dehydrogenase is
cellobiose dehydrogenase EC 1.1.99.18. In some embodiments, the
cellobiose dehydrogenase is a cellobiose dehydrogenase with the
amino acid sequence of Myceliophthora thermophila CDH1 as set forth
in SEQ ID NO:2. In some other embodiments, the cellobiose
dehydrogenase comprises an amino acid sequence provided in the
GenBank entry of any one of the accession numbers set forth in
Table 1. In some embodiments, the cellobiose dehydrogenase is
encoded by a nucleic acid sequence that is at least about 60%,
about 61%, about 62%, about 63%, about 64%, about 65%, about 66%,
about 67%, about 68%, about 69%, about 70%, about 71%, about 72%,
about 73%, about 74%, about 75%, about 76%, about 77%, about 78%,
about 79%, about 80%, about 81%, about 82%, about 83%, about 84%,
about 85%, about 86%, about 87%, about 88%, about 89%, about 90%,
about 91%, about 92%, about 93%, about 94%, about 95%, about 96%,
about 97%, about 98%, about 99%, or about 100% identical to SEQ ID
NO:1. In some embodiments, the cellobiose dehydrogenase is encoded
by a nucleic acid sequence that is at least about 60%, about 61%,
about 62%, about 63%, about 64%, about 65%, about 66%, about 67%,
about 68%, about 69%, about 70%, about 71%, about 72%, about 73%,
about 74%, about 75%, about 76%, about 77%, about 78%, about 79%,
about 80%, about 81%, about 82%, about 83%, about 84%, about 85%,
about 86%, about 87%, about 88%, about 89%, about 90%, about 91%,
about 92%, about 93%, about 94%, about 95%, about 96%, about 97%,
about 98%, about 99%, or about 100% identical to a nucleic acid
sequence encoding the amino acid sequence set forth as SEQ ID NO:2,
or an amino acid sequence provided in the GenBank entry of any one
of the accession numbers set forth in Table 1. In some embodiments,
the cellobiose dehydrogenase is encoded by a nucleic acid sequence
that can selectively hybridize to SEQ ID NO:1, under moderately
stringent or stringent conditions, as described hereinabove. In
some embodiments, the cellobiose dehydrogenase is encoded by a
nucleic acid sequence that can selectively hybridize under
moderately stringent or stringent conditions to a nucleic acid
sequence that encodes SEQ ID NO:2, or an amino acid sequence
provided in the GenBank entry of any one of the accession numbers
set forth in Table 1.
[0278] In some embodiments, the cellobiose dehydrogenase comprises
an amino acid sequence with at least about 50%, about 51%, about
52%, about 53%, about 54%, about 55%, about 56%, about 57%, about
58%, about 59%, about 60%, about 61%, about 62%, about 63%, about
64%, about 65%, about 66%, about 67%, about 68%, about 69%, about
70%, about 71%, about 72%, about 73%, about 74%, about 75%, about
76%, about 77%, about 78%, about 79%, about 80%, about 81%, about
82%, about 83%, about 84%, about 85% about 86%, about 87%, about
88%, about 89%, about 90%, about 91%, about 92%, about 93%, about
94%, about 95%, about 96%, about 97%, about 98%, about 99%, or
about 100% similarity to the amino acid sequence set forth as SEQ
ID NO:2, or an amino acid sequence provided in the GenBank entry of
any one of the accession numbers set forth in Table 1. Cellobiose
dehydrogenase sequences can be identified by any of a variety of
methods known in the art. For example, a sequence alignment can be
conducted against a database, for example against the NCBI
database, and sequences with the lowest HMM E-value can be
selected.
[0279] In some embodiments, the fungal cells of the present
invention have been genetically modified to reduce the amount of
cellobiose dehydrogenase activity from two or more endogenous
cellobiose dehydrogenase enzymes secreted by the cell. In some
embodiments, a first of the two or more cellobiose dehydrogenases
comprises an amino acid sequence that is at least about 60%, about
61%, about 62%, about 63%, about 64%, about 65%, about 66%, about
67%, about 68%, about 69%, about 70%, about 71%, about 72%, about
73%, about 74%, about 75%, about 76%, about 77%, about 78%, about
79%, about 80%, about 81%, about 82%, about 83%, about 84%, about
85%, about 86%, about 87%, about 88%, about 89%, about 90%, about
91%, about 92%, about 93%, about 94%, about 95%, about 96%, about
97%, about 98%, or about 99% identical to SEQ ID NO:2, and a second
of the two or more cellobiose dehydrogenase enzymes comprises an
amino acid sequence that is at least about 60%, about 61%, about
62%, about 63%, about 64%, about 65%, about 66%, about 67%, about
68%, about 69%, about 70%, about 71%, about 72%, about 73%, about
74%, about 75%, about 76%, about 77%, about 78%, about 79%, about
80%, about 81%, about 82%, about 83%, about 84%, about 85%, about
86%, about 87%, about 88%, about 89%, about 90%, about 91%, about
92%, about 93%, about 94%, about 95%, about 96%, about 97%, about
98%, or about 99% identical to SEQ ID NO: 2.
Enzyme Mixtures
[0280] Also provided herein are enzyme mixtures that comprise at
least one or more cellulose hydrolyzing enzymes expressed by a
fungal cell that has been genetically modified to reduce the amount
of endogenous cellobiose dehydrogenase activity secreted by the
cell, as described herein. Cellulase enzymes are produced by a wide
variety of microorganisms. Cellulases (and hemicellulases) from
filamentous fungi and some bacteria are widely exploited for many
industrial applications that involve processing of natural fibers
to sugars. It is contemplated that mixtures of any enzymes set
forth herein will find use in the present invention.
[0281] In some embodiments, the enzyme mixture comprises at least
one or more cellulose hydrolyzing enzymes expressed by a fungal
cell that has been genetically modified to reduce the amount of
endogenous cellobiose dehydrogenase activity that is secreted by
the cell, as described herein. In some embodiments, the fungal cell
is a lignocellulose-utilizing cell from the family Chaetomiaceae.
In some embodiments, the genetically modified fungal cell provided
herein is a Chaetomiaceae family member selected from
Myceliophthora, Thielavia, Corynascus, or Chaetomium. In some other
embodiments, the genetically modified fungal cell can also be an
anamorph or teleomorph of a Chaetomiaceae family member selected
from Myceliophthora, Thielavia, Corynascus, or Chaetomium. In
addition, the genetically modified fungal cell can also be selected
from Sporotrichum or Acremonium or Talaromyces. It is also
contemplated that the genetically modified fungal cell be selected
from Ctenomyces, Thermoascus, and Scytalidium, including anamorphs
and teleomorphs of fungal cells from those genera. In some
embodiments, the fungal cell is a species selected from
Sporotrichum cellulophilum, Thielavia heterothallica, Corynascus
heterothallicus, Thielavia terrestris, Chaetomium globosum
Talaromyces stipitatus and Myceliophthora thermophila, including
anamorphs and teleomorphs thereof.
[0282] In addition to the enzymes described above, other enzymes
such as laccases find use in the mixtures of the present invention.
Laccases are copper containing oxidase enzymes that are found in
many plants, fungi and microorganisms. Laccases are enzymatically
active on phenols and similar molecules and perform a one electron
oxidation. Laccases can be polymeric and the enzymatically active
form can be a dimer or trimer.
[0283] Mn-dependent peroxidases also find use in the mixtures of
the present invention. The enzymatic activity of Mn-dependent
peroxidase (MnP) in is dependent on Mn.sup.2+. Without being bound
by theory, it has been suggested that the main role of this enzyme
is to oxidize Mn.sup.2+ to Mn.sup.3+ (See e.g., Glenn et al. Arch.
Biochem. Biophys., 251:688-696 [1986]). Subsequently, phenolic
substrates are oxidized by the Mn.sup.3+ generated.
[0284] Lignin peroxidases also find use in the mixtures of the
present invention. Lignin peroxidase is an extracellular heme that
catalyzes the oxidative depolymerization of dilute solutions of
polymeric lignin in vitro. Some of the substrates of LiP, most
notably 3,4-dimethoxybenzyl alcohol (veratryl alcohol, VA), are
active redox compounds that have been shown to act as redox
mediators. VA is a secondary metabolite produced at the same time
as LiP by ligninolytic cultures of P. chrysosporium and without
being bound by theory, has been proposed to function as a
physiological redox mediator in the LiP-catalysed oxidation of
lignin in vivo (See e.g., Harvey et al., FEBS Lett., 195:242-246
[1986]).
[0285] In some embodiments, it may be advantageous to utilize an
enzyme mixture that is cell-free. A cell-free enzyme mixture
typically comprises enzymes that have been separated from any
cells, including the cells that secreted the enzymes. Cell-free
enzyme mixtures can be prepared using any of a variety of suitable
methodologies that are known in the art (e.g., filtration or
centrifugation). In some embodiments, the enzyme mixture is
partially cell-free, substantially cell-free, or entirely
cell-free.
[0286] In some embodiments, two or more cellulases and any
additional enzymes present in the cellulase enzyme mixture are
secreted from a single genetically modified fungal cell or by
different microbes in combined or separate fermentations.
Similarly, two or more cellulases and any additional enzymes
present in the cellulase enzyme mixture may be expressed
individually or in sub-groups from different strains of different
organisms and the enzymes combined in vitro to make the cellulase
enzyme mixture. It is also contemplated that the cellulases and any
additional enzymes in the enzyme mixture are expressed individually
or in sub-groups from different strains of a single organism, and
the enzymes combined to make the cellulase enzyme mixture.
[0287] In some embodiments, the enzyme mixture comprises at least
one or more cellulose hydrolyzing enzymes expressed by a fungal
cell that has been genetically modified to reduce the amount of
endogenous cellobiose dehydrogenase activity that is secreted by
the cell, as described herein. In some embodiments, the fungal cell
is a lignocellulose-utilizing cell from the family Chaetomiaceae.
In some embodiments, the genetically modified fungal cell provided
herein is a Chaetomiaceae family member selected from
Myceliophthora, Thielavia, Corynascus, and Chaetomium. The
genetically modified fungal cell can also be an anamorph or
teleomorph of a Chaetomiaceae family member selected from
Myceliophthora, Thielavia, Corynascus, and Chaetomium. In addition,
the genetically modified fungal cell can also be selected from
Sporotrichum, Acremonium, Ctenomyces, Scytalidium and Thermoascus,
including anamorphs and teleomorphs of fungal cells from these
genera. In some embodiments, the fungal cell is a species selected
from Sporotrichum cellulophilum, Thielavia heterothallica,
Corynascus heterothallicus, Thielavia terrestris, Chaetomium
globosum, Talaromyces stipitatus, and Myceliophthora thermophila,
including anamorphs and teleomorphs thereof.
[0288] In some embodiments, the cellulase enzyme mixture of the
present invention is produced in a fermentation process in which
the fungal cells described herein are grown in submerged liquid
culture fermentation. In some embodiments, submerged liquid
fermentations of fungal cells are incubated using batch, fed-batch
or continuous processing. In a batch process, all the necessary
materials, with the exception of oxygen for aerobic processes, are
placed in a reactor at the start of the operation and the
fermentation is allowed to proceed until completion, at which point
the product is harvested. In some embodiments, batch processes for
producing the enzyme mixture of the present invention are carried
out in a shake-flask or a bioreactor. In some embodiments in which
a fed-batch process is used, the culture is fed continuously or
sequentially with one or more media components without the removal
of the culture fluid. In continuous processes, fresh medium is
supplied and culture fluid is removed continuously at
volumetrically equal rates to maintain the culture at a steady
growth rate. Those of skill in the art will appreciate that
fermentation medium is typically liquid, and comprises a carbon
source, a nitrogen source as well as other nutrients, vitamins and
minerals which can be added to the fermentation media to improve
growth and enzyme production of the fungal cells. These other media
components may be added prior to, simultaneously with or after
inoculation of the culture with the fungal cells.
[0289] In some embodiments of the process for producing the enzyme
mixture of the present invention, the carbon source comprises a
carbohydrate that will induce the expression of the cellulase
enzymes from the fungal cell. For example, in some embodiments, the
carbon source comprises one or more of cellulose, cellobiose,
sophorose, xylan, xylose, xylobiose, and/or related oligo- or
poly-saccharides known to induce expression of cellulases and
beta-glucosidase in such fungal cells. In some embodiments
utilizing batch fermentation, the carbon source is added to the
fermentation medium prior to or simultaneously with inoculation. In
some embodiments utilizing fed-batch or continuous operations, the
carbon source is supplied continuously or intermittently during the
fermentation process. For example, in some embodiments, the carbon
source is supplied at a carbon feed rate of between about 0.2 and
about 2.5 g carbon/L of culture/h, or any suitable amount
therebetween.
[0290] The methods for producing the enzyme mixture of the present
invention may be carried at any suitable temperature, typically
from about 20.degree. C. to about 100.degree. C., or any suitable
temperature therebetween, for example from about 20 .degree. C. to
about 80.degree. C. , 25.degree. C. to about 65.degree. C., or any
suitable temperature therebetween, or from about 20.degree. C.,
about 22.degree. C., about 25.degree. C., about 26.degree. C.,
about 27.degree. C., about 28.degree. C., about 29.degree. C.,
about 30.degree. C., about 32.degree. C., about 35.degree. C.,
about 37.degree. C., about 40.degree. C., about 45.degree. C.,
about 50.degree. C., about 55.degree. C., about 60.degree. C.,
about 65.degree. C., about 70.degree. C., about 75.degree. C.,
about 80.degree. C,about 85.degree. C. C, about 90.degree. C.,
about 95.degree. C., and/or any suitable temperature
therebetween.
[0291] The methods for producing enzyme mixture of the present
invention may be carried out at any suitable pH, typically from
about 3.0 to 8.0, or any suitable pH therebetween, for example from
about pH 3.5 to pH 6.8, or any suitable pH therebetween, for
example from about pH 3.0, about 3.2, about 3.4, about 3.5, about
3.7, about 3.8, about 4.0, about 4.1, about 4.2, about 4.3, about
4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about
5.0, about 5.2, about 5.4, about 5.5, about 5.7, about 5.8, about
6.0, about 6.2, about 6.5, about 6.8, about 7.0, about 7.2, about
7.5, about 8.0, or any suitable pH therebetween.
[0292] In some embodiments, the enzyme mixture is contained in a
vessel comprising a genetically modified fungal cell as described
herein. In some embodiments, the vessel comprises a liquid medium.
In some embodiments, the vessel is a flask, bioprocess reactor, or
any other suitable container. In some embodiments, the enzyme
mixture is in a liquid volume. In some embodiments, the liquid
volume can be greater than about 0.01 mL, about 0.1 mL, about 1 mL,
about 10 mL, about 100 mL, about 1000 mL, or greater than about 10
L, about 50 L, about 100 L, about 200 L, about 300 L, about 400 L,
about 500 L, about 600 L, about 700 L, about 800 L, about 900 L,
about 1000 L, about 10,000 L, about 50,000 L, about 100,000 L,
about 250,000 L, about 500,000 L or greater than about 1,000,000
L.
[0293] In some embodiments, following fermentation, the
fermentation medium containing the fungal cells is used, or the
fermentation medium containing the fungal cells and the enzyme
mixture is used, or the enzyme mixture is separated from the fungal
cells, for example by filtration or centrifugation, and the enzyme
mixture in the fermentation medium is used. In some embodiments,
low molecular solutes such as unconsumed components of the
fermentation medium are removed by ultrafiltration. In some
embodiments, the enzyme mixture is concentrated by evaporation,
precipitation, sedimentation, filtration, or any suitable means. In
some embodiments, chemicals such as glycerol, sucrose, sorbitol,
etc., are added to stabilize the enzyme mixture. In some
embodiments, other chemicals, such as sodium benzoate or potassium
sorbate, are added to the enzyme mixture to prevent growth of
microbial contaminants.
Methods for Generating Glucose
[0294] The present invention also provides processes for generating
glucose, comprising contacting cellulose with the enzyme mixture
described herein. For example, in some embodiments, the process
comprises contacting cellulose with an enzyme mixture comprising
two or more cellulose hydrolyzing enzymes, wherein at least one of
the two or more cellulose hydrolyzing enzymes is expressed by a
fungal cell as described herein. In some embodiments, the method
for generating glucose from cellulose using the enzyme mixture is
batch hydrolysis, continuous hydrolysis, or a combination thereof.
In some embodiments, the hydrolysis is agitated, unmixed, or a
combination thereof.
[0295] The methods for generating glucose from cellulose may be
carried out at any suitable temperature, including between about
30.degree. C. and about 80.degree. C., or any suitable temperature
therebetween, for example a temperature of about 30.degree. C.,
about 35.degree. C., about 40.degree. C., about 45.degree. C.,
about 50.degree. C., about 55.degree. C., about 60.degree. C.,
about 65.degree. C., about 70.degree. C., about 75.degree. C.,
about 80.degree. C. or any suitable temperature therebetween, and a
pH of about 3.0 to about 8.0, or any suitable pH therebetween, for
example at a pH of about 3.0, about 3.5, about 4.0, about 4.5,
about 5.0, about 5.5, about 6.0, about 6.5, about 7.0, about 7.5,
about 8.0, or any sutiable pH therebetween. The initial
concentration of cellulose in the hydrolysis reactor, prior to the
start of hydrolysis, is preferably about 0.1% (w/w) to about 15%
(w/w), or any suitable amount therebetween, for example about 2,
about 4, about 6, about 8, about 10, about 12, about 14, about 15%,
or any suitable amount therebetween.
[0296] The dosage of the cellulase enzyme mixture may be about 0.1
to about 100 mg protein per gram cellulose, or any suitable amount
therebetween, for example about 0.1, about 0.5, about 1, about 5,
about 10, about 15, about 20, about 25, about 30, about 40, about
50, about 60, about 70, about 80, about 90, about 100 mg protein
per gram cellulose or any suitable amount therebetween. The
hydrolysis may be carried out for a time period of about 0.5 hours
to about 200 hours, or any suitable time therebetwee. For example,
in some embodiments, the hydrolysis is carried out for a period of
about 15 hours to about 100 hours, or any time therebetween, or it
may be carried out for about 0.5 hour, about 1 hour, about 2 hours,
about 4 hours, about 8 hours, about 12 hours, about 15 hours, about
20 hours, about 25 hours, about 30 hours, about 35 hours, about 40
hours, about 45 hours, about 50 hours, about 55 hours, about 60
hours, about 65 hours, about 70 hours, about 75 hours, about 80
hours, about 85 hours, about 90 hours, about 95 hours, about 100
hours, about 120 hours, about 140 hours, about 160 hours, about 180
hours, about 200 hours, or any suitable time therebetween. It
should be appreciated that the reaction conditions are not meant to
limit the invention in any manner and may be adjusted as desired by
those of skill in the art.
[0297] In some embodiments, the enzymatic hydrolysis is typically
carried out in a hydrolysis reactor. The enzyme mixture is added to
the pretreated lignocellulosic feedstock (also referred to as the
"substrate") prior to, during, or after the addition of the
substrate to the hydrolysis reactor.
[0298] In methods of contacting cellulosic material with an enzyme
mixture, various environmental conditions may be adjusted according
to any variety of methods known in the art in order to maximize the
formation of a hydrolysis product such as glucose. For example,
temperature, pH, % dissolved oxygen, and stirring speed can each be
independently adjusted. In some embodiments, the enzyme mixture is
a cell-free mixture, as described herein.
[0299] The methods for generating glucose, as described herein,
using the enzyme mixture with reduced cellobiose dehydrogenase
activity result in a higher yield of glucose from the enzymatically
hydrolyzed cellulose than a corresponding process using an enzyme
mixture with its full complement of cellobiose dehydrogenase
activity. Further, such methods result in decreased conversion of
the cellobiose products in the enzymatic hydrolysate to oxidized
products.
[0300] In some embodiments of the methods using the genetically
modified cells and/or enzyme mixtures provided herein, improved
glucose yield can be measured and quantified. As described herein,
glucose yield can be described in terms of the amount of generated
glucose per theoretical maximum glucose yield, or in terms of Gmax.
It will be appreciated by those skilled in the art that when
calculating theoretical values such as Gmax and theoretical maximum
glucose yield, the mass of two hydrogen atoms and one oxygen atom
that are added to the glucose molecule in the course of the
hydrolysis reaction is taken into account. For example, when a
polymer of "n" glucose units is hydrolyzed, (n-1) units of water
are added to the glucose molecules formed in the hydrolysis, so the
weight of the glucose produced is about 10% greater than the weight
of cellulose consumed in the hydrolysis (e.g., hydrolysis of 1 g
cellulose produces about 1.1 g glucose). Thus, as an example, where
5 g of total available cellulose is present at the beginning of a
hydrolysis reaction, and 2 g of residual cellulose remains after
the reaction, the total hydrolyzed cellulose is 3 g cellulose. A
theoretical maximum glucose yield of 100% (w/w) under the reaction
conditions is about 5.5 g of glucose. Gmax is calculated based on
the 3 g of cellulose that was released or converted in the reaction
by hydrolysis. Thus, in this example, a Gmax of 100% (w/w) is about
3.3 g of glucose. Cellulose levels, either the total available
amount present in the substrate or the amount of unhydrolyzed or
residual cellulose, can be quantified by any of a variety of
suitable methods known in the art, such as by IR spectroscopy or by
measuring the amount of glucose generated by concentrated acid
hydrolysis of the cellulose (See e.g., U.S. Pat. Nos. 6,090,595 and
7,419,809, both of which are incorporated by reference herein in
their entireties).
[0301] For example, in some embodiments, the cellulose content is
determined by acid hydrolysis of the cellulose, followed by glucose
concentration determination, taking into account the water
necessary to hydrolyze the cellulose (See e.g., U.S. Pat. Nos.
6,090,595 and 7,419,809). In one example, a slurry of feedstock is
centrifuged, washed with water, and suspended in sulfuric acid at a
net sulfuric acid concentration of 70%. The slurry is incubated at
40.degree. C. for 30 minutes, followed by dilution in deionized
water to 2% sulfuric acid. At this time point, the samples are
steam-autoclaved at 121.degree. C. for 1 hour, to convert the
oligomers to monomeric glucose. The glucose concentration is
measured by HPLC or any suitable enzymatic assay. In some
alternative embodiments, the cellulose content is analyzed by
infrared spectroscopy as described in Example 1. For example,
solids can be washed and placed on the detection crystal of an
infrared spectrometer and the absorbance measured between 500-4000
cm.sup.-1.
[0302] Glucose levels can be quantified by any of a variety of
suitable methods known in the art (See e.g., U.S. Pat. Nos.
6,090,595 and 7,419,809). For example, glucose concentrations can
be determined using a coupled enzymatic assay based on glucose
oxidase and horseradish peroxidase (See e.g., Trinder, Ann. Clin.
Biochem., 6:24-27 [1969], which is incorporated herein by reference
in its entirety). Additional methods of glucose quantification
include chromatographic methods (See e.g., U.S. Pat. Nos. 6,090,595
and 7,419,809). Cellobiose levels can be measured by any number of
suitable HPLC methods known to those of skill in the art (See e.g.,
Kotiranta et al., Appl. Biochem. Biotechnol., 81:81-90 [1999]),
which is incorporated herein by reference in its entirety).
[0303] Similarly, decreased conversion of cellobiose and glucose
products to oxidized products such as cellobionolactone and
gluconolactone can be quantified by any of a variety of suitable
methods known in the art. For example, products of glucose or
cellobiose oxidation can be detected and quantified using infrared
spectroscopy, or by chromatographic methodologies such as HPLC (See
e.g., Rakotomanga et al., J. Chromatog. B., 4:277-284 [1991]; and
Mansfield et al., Appl. Environ. Microbiol., 64:3804-3809 [1997],
both of which are incorporated herein by reference in their
entireties). Accordingly, total oxidation of glucose or cellobiose
can be determined, for example, as a function of total oxidation
products per theoretical maximum glucose yield, or as a function of
Gmax.
Cellulosic Material
[0304] Any material containing cellulose finds use in the present
invention. 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.
[0305] Cellulose is generally found, for example, in the stems,
leaves, hulls, husks, and cobs of plants or leaves, branches, and
wood of trees. 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 e.g., Wiselogel et al., in Charles E. Wyman, (ed.), Handbook
on Bioethanol, Taylor & Francis, Washington D.C. [1995], at pp.
105-118; Wyman, Biores. Technol., 50:3-16 [1994]; Lynd, Appl.
Biochem. Biotechnol., 24/25: 695-719 [1990]; and Mosier et al.,
Adv. Biochem. Eng. Biotechnol., 65:23-40 [1999]). It is understood
that in some embodiments, the cellulose is in the form of
lignocellulose, a plant cell wall material containing lignin,
cellulose, and hemicellulose in a mixed matrix. In some
embodiments, the cellulosic material is lignocellulose.
[0306] A pretreated lignocellulosic feedstock is a material of
plant origin that, prior to pretreatment, contains at least 10%
cellulose (dry weight), more preferably greater than about 30%
cellulose, even more preferably greater than 40% cellulose, for
example about 10%, about 11%, about 12%, about 13%, about 14%,
about 15%, about 16%, about 17%, about 18%, about 19%, about 20%,
about 21%, about 22%, about 23%, about 24%, about 25%, about 26%,
about 27%, about 28%, about 29%, about 30%, about 31%, about 32%,
about 33%, about 34%, about 35%, about 36%, about 37%, about 38%,
about 39%, about 40%, about 41%. about 42%, about 43%, about 44%,
about 45%, about 46%, about 47%, about 48%, about 49%, about 50%,
about 55%, about 60%, about 65%, about 70%, about 75%, about 80%,
about 85%, about 90%, about 95%, or any suitable percent
therebetween, and at least about 10% lignin (dry weight), or at
least about 12% (dry weight) and that has been subjected to
physical and/or chemical processes to make the fiber more
accessible and/or receptive to the actions of cellulolytic enzymes.
In some embodiments, the lignocellulosic feedstock may contain
higher levels of cellulose after pretreatment. For example, if acid
pretreatment is employed, the hemicellulose component is
hydrolyzed, which increases the relative level of cellulose. In
this case, the pretreated feedstock may contain greater than about
20% cellulose and greater than about 12% lignin.
[0307] Lignocellulosic feedstocks that find use in the invention
include, but are not limited to, agricultural residues such as corn
stover, wheat straw, barley straw, rice straw, oat straw, canola
straw, sugarcane straw and soybean stover; fiber process residues
such as corn fiber, sugar beet pulp, pulp mill fines and rejects or
sugar cane bagasse; forestry residues such as aspen wood, other
hardwoods, softwood, and sawdust; or grasses such as switch grass,
miscanthus, cord grass, and reed canary grass. In some embodiments,
the lignocellulosic feedstock is first subjected to size reduction
by any of a variety of methods including, but not limited to,
milling, grinding, agitation, shredding, compression/expansion,
and/or other types of mechanical action. Size reduction by
mechanical action can be performed by any type of equipment adapted
for the purpose, for example, but not limited to, a hammer mill
Pretreatment
[0308] In some embodiments, a substrate of the enzyme mixture
comprises pretreated cellulosic material. Thus, for example, in
some methods described herein, any pretreatment process known in
the art can be used to disrupt plant cell wall components of
cellulosic material (See e.g., Chandra et al., Adv. Biochem. Engin.
Biotechnol., 108: 67-93 [2007]; Galbe and Zacchi, Adv. Biochem.
Engin. Biotechnol., 108: 41-65 [2007]; Hendriks and Zeeman, Biores.
Technol., 100: 10-18 [2009]; Mosier et al., Biores. Technol., 96:
673-686 [2005]; Taherzadeh and Karimi, Int. J. Mol. Sci.,
9:1621-1651 [2008]; and Yang and Wyman, Biofuels Bioprod. Bioref.
Biofpr. 2: 26-40 [2008]; all of which are hereby incorporated by
reference in their entireties).
[0309] In some embodiments, the cellulosic material is subjected to
particle size reduction, pre-soaking, wetting, washing, or
conditioning prior to pretreatment using any of a variety of
suitable methods known in the art. Conventional pretreatments
include, but are not limited to, steam pretreatment (with or
without explosion), dilute acid pretreatment, hot water
pretreatment, alkaline pretreatment, lime pretreatment, wet
oxidation, wet explosion, ammonia fiber expansion, dilute ammonia
pretreatment, 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. In some
embodiments, the cellulosic material is pretreated before
hydrolysis and/or fermentation. In some embodiments, pretreatment
is preferably performed prior to the hydrolysis. In some
alternative embodiments, pretreatment is carried out simultaneously
with enzyme hydrolysis to release fermentable sugars, such as
glucose, xylose, and/or cellobiose. In some embodiments, the
pretreatment step itself results in some conversion of biomass to
fermentable sugars, even in absence of enzymes.
[0310] 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., hemicelluloses), 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. In some embodiments, steam pretreatment is
preferably done at about 140.degree. C. to about 230.degree. C.,
while in other embodiments it is done at about 160.degree. C. to
about 200.degree. C., and in additional embodiments, it is done at
about 170.degree. C. to about 190.degree. C., where the optimal
temperature range depends on any addition of a chemical catalyst.
In some embodiments, residence time for the steam pretreatment is
about 1 to about 15 minutes, while in other embodiments it is about
3 to about 12 minutes, and in still other embodiments, it is about
4 to about 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. 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 (See e.g., U.S. Pat. No.
4,451,648; Duff and Murray, Biores. Technol., 855:1-33 [1996];
Galbe and Zacchi, Appl. Microbiol. Biotechnol., 59: 618-628 [2002];
and U.S. Patent Appln. Publ. No. 2002/0164730, all of which are
incorporated herein by reference in their entireties). 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. A catalyst such as
H.sub.2SO.sub.4 or SO.sub.2 (typically about 0.3 to about 3% w/w)
is often added prior to steam pretreatment, which decreases the
time and temperature, increases the recovery, and improves
enzymatic hydrolysis (See e.g., Ballesteros et al., Appl. Biochem.
Biotechnol., 129-132: 496-508 [2006]; Varga et al., Appl. Biochem.
Biotechnol., 113-116: 509-523 [2004]; and Sassner et al., Enz.
Microb. Technol., 39: 756-762 [2006]).
[0311] 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, but are not
limited to, dilute acid pretreatment, dilute alkali pretreatment
(See e.g., U.S. Pat. Appln. Pub. Nos. 2007/0031918 and
2007/0037259), lime pretreatment, wet oxidation, ammonia
fiber/freeze explosion or expansion (AFEX), ammonia percolation
(APR), dilute ammonia pretreatment, and organosolv pretreatments
(See e.g., WO 2006/110891, WO 2006/11899, WO 2006/11900, and WO
2006/110901).
[0312] 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) (See e.g., Duff and Murray,
supra; Schell et al., Biores. Technol., 91: 179-188 [2004]; and Lee
et al., Adv. Biochem. Eng. Biotechnol., 65: 93-115 [1999]).
[0313] In some embodiments, lime pretreatment is performed with
calcium carbonate, sodium hydroxide, or ammonia at low temperatures
of about 85.degree. C. to about 150.degree. C. and residence times
from about 1 hour to several days (Wyman et al., Biores. Technol.,
96: 1959-1966 [2005]; and Mosier et al., Biores. Technol. 96:
673-686 [2005]).
[0314] Wet oxidation is a thermal pretreatment performed typically
at about 180.degree. C. to about 200.degree. C. for about 5 to
about 15 minutes with addition of an oxidative agent such as
hydrogen peroxide or over-pressure of oxygen (See e.g., Schmidt and
Thomsen, Biores. Technol., 64:139-151 [1998]; Palonen et al., Appl.
Biochem. Biotechnol., 117: 1-17 [2004]; Varga et al., Biotechnol.
Bioeng., 88: 567-574 [2004]; Martin et al., J. Chem. Technol.
Biotechnol., 81: 1669-1677 [2006]). The pretreatment is performed
at preferably about 1% to about 40% dry matter, about 2 to about
30% dry matter, or about 5 to about 20% dry matter, and often the
initial pH is increased by the addition of an alkali such as sodium
carbonate. In some embodiments, a modification of the wet oxidation
pretreatment method, known as wet explosion (combination of wet
oxidation and steam explosion), finds use. This method can handle
dry matter up to about 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
(See e.g., WO 2006/032282).
[0315] In some embodiments, ammonia fiber expansion (AFEX) finds
use. This method involves treating cellulosic material with liquid
or gaseous ammonia at moderate temperatures such as about 90 to
about 100.degree. C. and high pressure such as about 17 to about 20
bar for about 5 to about 10 minutes, where the dry matter content
can be as high as about 60% (See e.g., Gollapalli et al., Appl.
Biochem. Biotechnol., 98: 23-35 [2002]; Chundawat et al.,
Biotechnol. Bioeng., 96:219-231 [2007]; Alizadeh et al., Appl.
Biochem. Biotechnol., 121: 1133-1141 [2005]; and Teymouri et al.,
Biores. Technol., 96: 2014-2018 [2005]). AFEX pretreatment results
in the depolymerization of cellulose and partial hydrolysis of
hemicellulose. Lignin-carbohydrate complexes are cleaved. Dilute
ammonia pretreatment utilizes more dilute solutions of ammonia than
AFEX and may be conducted at a temperature of about 100.degree. C.
to about 150.degree. C., or any suitable temperature therebetween
(See e.g., U.S. Pat. Appln. Pub. Nos. 2007/0031918 and
2007/0037259, herein incorporated by reference in their
entireties). In some embodiments, the duration of the dilute
ammonia pretreatment is about 1 to about 20 minutes, or any
suitable duration therebetween.
[0316] In some embodiments, organosolv pretreatment finds use. This
method delignifies cellulosic material by extraction using aqueous
ethanol (about 40% to about 60% ethanol) at about 160.degree. C. to
about 200.degree. C. for about 30 to about 60 minutes (See e.g.,
Pan et al., Biotechnol. Bioeng., 90: 473-481 [2005]; Pan et al.,
Biotechnol. Bioeng., 94: 851-861 [2006]; and Kurabi et al., Appl.
Biochem. Biotechnol., 121: 219-230 [2005]). Sulfuric acid is
usually added as a catalyst. In organosolv pretreatment, the
majority of hemicellulose is removed.
[0317] Other examples of suitable pretreatment methods are known in
the art (See e.g., Schell et al., Appl. Biochem. Biotechnol.,
105:69-85 [2003]; Mosier et al., Biores. Technol., 96: 673-686
[2005]; and U.S. Pat. Appln. Publ. No. 2002/0164730).
[0318] In some embodiments, 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 nitric
acid, phosphoric acid, hydrogen chloride or mixtures thereof. Mild
acid treatment is conducted in the pH range of about 1 to about 5,
or about 1 to about 4, or about 1 to about 3. In some embodiments,
the acid concentration is in the range of from about 0.01 to about
20 wt % acid, while in other embodiments, it is in the range of
from about 0.05 to about 10 wt % acid, in other embodiments, it is
in the range of from about 0.1 to about 5 wt % acid, and in still
other embodiments, it is in the range of from about 0.2 to about
2.0 wt % acid. The acid is contacted with cellulosic material and
held at a temperature in the range of preferably about 160.degree.
C. to about 220.degree. C., and more preferably about 165.degree.
C. to about 195.degree. C., for periods ranging from seconds to
minutes to (e.g., about 1 second to about 60 minutes).
[0319] In some embodiments, pretreatment takes place in an aqueous
slurry. In some embodiments, cellulosic material is present during
pretreatment in amounts preferably between about 10 to about 80 wt
%, or about 20 to about 70 wt %, or about 30 to about 60 wt %, or
about 50 wt %. The pretreated cellulosic material can be unwashed
or washed using any suitable method known in the art (e.g., washed
with water).
[0320] Physical Pretreatment. Physical pretreatment can involve
high pressure and/or high temperature (steam explosion). In some
embodiments, high pressure physical pretreatment involves pressure
in the range of about 300 to about 600 psi, or about 350 to about
550 psi, or about 400 to about 500 psi, or about 450 psi. In some
other embodiments, high temperature pretreatment involves the use
of treatment temperatures in the range of about 100.degree. C. to
about 300.degree. C., or about 140.degree. C. to about 235.degree.
C. In some embodiments, mechanical pretreatment is performed in a
batch-process, steam gun hydrolyzer system that uses high pressure
and high temperature as defined above (e.g., Sunds Hydrolyzer;
Sunds Defibrator AB, Sweden).
[0321] Combined Physical and Chemical Pretreatment. In some
embodiments, combined physical and chemical pretreatments find use.
Indeed, cellulosic material can be pretreated both physically and
chemically. For example, in some embodiments, the pretreatment step
involves 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. In some
additional embodiments, mechanical pretreatment is also used in
conjunction with the physical and chemical pretreatments. Thus, in
some embodiments, 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.
[0322] Biological Pretreatment. In some embodiments, biological
pretreatment techniques find use. In some embodiments, these
methods involve applying lignin-solubilizing microorganisms (See
e.g., Hsu, in Wyman (ed.), Handbook on Bioethanol: Production and
Utilization, Taylor & Francis, Washington, D.C., at pp. 179-212
[1996]; Ghosh and Singh, Adv. Appl. Microbiol., 39:295-333 [1993];
McMillan, in Baker and Overend (eds.), Enzymatic Conversion of
Biomass for Fuels Production, ACS Symposium Series 566, American
Chemical Society, Washington, D.C., chapter 15 [1994]; Gong et al.,
Adv. Biochem. Engineer. Biotechnol., 65: 207-241 [1999]; Olsson and
Hahn-Hagerdal, Enz. Microb. Tech., 18:312-331 [1996]; and Vallander
and Eriksson, Adv. Biochem. Eng. Biotechnol. 42: 63-95 [1990]).
[0323] In some embodiments, the soluble compounds derived from
pretreatment process are subsequently separated from the solids.
For example, in some embodiments, the separation step comprises one
or more of standard mechanical means (e.g., screening, sieving,
centrifugation or filtration) to achieve the separation. In some
other embodiments, the soluble compounds are not separated from the
solids following pretreatment. Those of skill in the art appreciate
that pretreatment may be conducted as a batch, fed-batch or
continuous process. It will also be appreciated that pretreatment
may be conducted at low, medium or high solids consistency as
desired (See e.g., WO2010/022511, which is incorporated herein by
reference in its entirety).
Fermentation
[0324] In some embodiments, methods for generating sugar(s)
described herein further comprise fermentation of the resultant
sugar(s) to an end product. Fermentation involves the conversion of
a sugar source to an end product through the use of a fermenting
organism. Any suitable organism finds use in the present invention,
including bacterial and fungal organisms (e.g., yeast and
filamentous fungi), suitable for producing a desired end product.
Especially suitable fermenting organisms are able to ferment (i.e.,
convert), sugars, such as glucose, fructose, maltose, xylose,
mannose and/or arabinose, directly or indirectly into a desired end
product. Examples of fermenting organisms include fungal organisms
such as yeast. In some embodiments, yeast strains, including but
not limited to the following genera find use: the genus
Saccharomyces (e.g., S. cerevisiae and S. uvarum); Pichia (e.g.,P.
stipitis and P. pastoris); Candida (e.g.,C. utilis, C.
arabinofermentans, C. diddensii, C. sonorensis, C. shehatae, C.
tropicalis, and C. boidinii). Other fermenting organisms include,
but are not limited to strains of Zymomonas, Hansenula (e.g., H.
polymorpha and H. anomala), Kluyveromyces (e.g., K fragilis), and
Schizosaccharomyces (e.g., S. pombe).
[0325] In some embodiments, the fermenting organisms are strains of
Escherichia (e.g., E. coli), Zymomonas (e.g., Z. mobilis),
Zymobacter (e.g., Z. palmae), Klebsiella (e.g., K. oxytoca),
Leuconostoc (e.g., L. mesenteroides), Clostridium (e.g., C.
butyricum), Enterobacter (e.g., E. aerogenes) and
Thermoanaerobacter (e.g., Thermoanaerobacter BG1L1 [See e.g.,
Georgieva and Ahring, Appl. Microbiol, Biotech., 77: 61-86] T.
ethanolicus, T. thermosaccharolyticum, or T. mathranii),
Lactobacillus, Corynebacterium glutamicum R, Bacillus
thermoglucosidaisus, and Geobacillus thermoglucosidasius. It is not
intended that the fermenting organism be limited to these
particular strains, as any suitable organism finds use in the
present invention.
[0326] The fermentation conditions depend on the desired
fermentation product and can easily be determined by one of
ordinary skill in the art. In some embodiments involving ethanol
fermentation by yeast, fermentation is typically ongoing for
between about 1 hour to about 120 hours, or about 12 to about 96
hours. In some embodiments, the fermentation is carried out at a
temperature between about 20.degree. C. to about 40.degree. C., or
between about 26.degree. C. and about 34.degree. C., or about
32.degree. C. In some embodiments, the fermentation pH is from
about pH 3 to about pH 7, while in some other embodiments, the pH
is about 4 to about 6.
[0327] In some embodiments, enzymatic hydrolysis and fermentation
are conducted in separate vessels, so that each biological reaction
can occur under its respective optimal conditions (e.g.,
temperature). In some other embodiments, the methods for producing
glucose from cellulose are conducted simultaneously with
fermentation in a simultaneous saccharification and fermentation
(i.e., "SSF") reaction. In some embodiments, SSF is typically
carried out at temperatures of about 28.degree. C. to about
50.degree. C., or about 30.degree. C. to about 40.degree. C., or
about 35.degree. C. to about 38.degree. C., which is a compromise
between the about 50.degree. C. optimum for most cellulase enzyme
mixtures and the about 28.degree. C. to about 30.degree. C. optimum
for most yeast. However, it is not intended that the present
invention be limited to any particular temperature, as any suitable
temperature finds use in the present invention.
[0328] In some embodiments, the methods for generating glucose
further comprise fermentation of the glucose to a desired end
product. It is not intended that the methods provided herein be
limited to the production of any specific end product. In some
embodiments, end products include fuel alcohols or precursor
industrial chemicals. For example, in some embodiments,
fermentation products include precursor industrial chemicals such
as alcohols (e.g., ethanol, methanol and/or butanol); organic acids
(e.g., butyric acid, citric acid, acetic acid, itaconic acid,
lactic acid, and/or gluconic acid); ketones (e.g., acetone); amino
acids (e.g., glutamic acid); gases (e.g., H.sub.2 and/or CO.sub.2);
antimicrobials (e.g., penicillin and/or tetracycline); enzymes;
vitamins (e.g., riboflavin, B.sub.12, and/or beta-carotene); and/or
hormones. In some embodiments, the end product is a fuel alcohol.
Suitable fuel alcohols are known in the art and include, but are
not limited to lower alcohols such as methanol, ethanol, butanol
and propyl alcohols.
Increased Expression of Saccharide Hydrolyzing Enzymes
[0329] In some embodiments provided herein, the fungal cell is
further genetically modified to increase its production of one or
more saccharide hydrolyzing enzymes. For example, in some
embodiments, the fungal cell overexpresses a homologous or
heterologous gene encoding a saccharide hydrolysis enzyme such as
beta-glucosidase. In some embodiments, the one or more saccharide
hydrolysis enzyme is a cellulase enzyme described herein. For
example, in some embodiments, the enzyme is any one of a variety of
endoglucanases, cellobiohydrolases, beta-glucosidases,
endoxylanases, beta-xylosidases, arabinofuranosidases,
alpha-glucuronidases, acetylxylan esterases, feruloyl esterases,
and alpha-glucuronyl esterases, and/or any other enzyme involved in
saccharide hydrolysis. In some embodiments, the fungal cell is
genetically modified to increase expression of beta-glucosidase.
Thus, in some embodiments, the fungal cell comprises a
polynucleotide sequence for increased expression of
beta-glucosidase-encoding polynucleotide. In some embodiments, the
fungal cell is further genetically modified to delete
polynucleotides encoding one or more endogenous cellobiose
dehydrogenase enzymes.
[0330] In some embodiments, the saccharide hydrolyzing enzyme is
endogenous to the fungal cell, while in other embodiments, the
saccharide hydrolyzing enzyme is exogenous to the fungal cell. In
some additional embodiments, the enzyme mixture further comprises a
saccharide hydrolyzing enzyme that is heterologous to the fungal
cell. Still further, in some embodiments, the methods for
generating glucose comprise contacting cellulose with an enzyme
mixture that comprises a saccharide hydrolyzing enzyme that is
heterologous to the fungal cell.
[0331] In some embodiments, a fungal cell is genetically modified
to increase the expression of a saccharide hydrolysis enzyme using
any of a variety of suitable methods known to those of skill in the
art. In some embodiments, the hydrolyzing enzyme-encoding
polynucleotide sequence is adapted for increased expression in a
host fungal cell. As used herein, a polynucleotide sequence that
has been adapted for expression is a polynucleotide sequence that
has been inserted into an expression vector or otherwise modified
to contain regulatory elements necessary for expression of the
polynucleotide in the host cell, positioned in such a manner as to
permit expression of the polynucleotide in the host cell. Such
regulatory elements required for expression include promoter
sequences, transcription initiation sequences and, optionally,
enhancer sequences. For example, in some embodiments, a
polynucleotide sequence is inserted into a plasmid vector adapted
for expression in the fungal host cell.
EXPERIMENTAL
[0332] The present invention is described in further detail in the
following Examples, which are not in any way intended to limit the
scope of the invention as claimed.
[0333] In the experimental disclosure below, the following
abbreviations apply: ppm (parts per million); M (molar); mM
(millimolar), uM and .mu.M (micromolar); nM (nanomolar); mol
(moles); gm and g (gram); mg (milligrams); ug and .mu.g
(micrograms); L and 1 (liter); ml and mL (milliliter); cm
(centimeters); mm (millimeters); um and .mu.m (micrometers); sec.
(seconds); min(s) (minute(s)); h(s) (hour(s)); U (units); MW
(molecular weight); rpm (rotations per minute); .degree. C.
(degrees Centigrade); wt % (weight percent); w.r.t. (with regard
to); DNA (deoxyribonucleic acid); RNA (ribonucleic acid); HPLC
(high pressure liquid chromatography); MS (mass spectroscopy); LC
(liquid chromatography); LC/MS (liquid chromatography/mass
spectroscopy); LC/MS/MS (liquid chromatography/multi-stage mass
spectroscopy); HMF (hydroxymethylfurfural); YPD (Yeast extract 10
g/L; Peptone 20 g/L; Dextrose 20 g/L); DCPIP
(2,6-dichlorophenolindophenol); CV (column volume); NREL (National
Renewable Energy Laboratory, Golden, Colo.); ARS (ARS Culture
Collection or NRRL Culture Collection, Peoria, Ill.); Lallemand
(Lallemand Ethanol Technology, Milwaukee, Wis.); Cayla
(Cayla-InvivoGen, Toulouse, France); Agilent New Brunswick (New
Brunswick Scientific Co., Edison, N.J.); Sigma (Sigma Aldrich, St.
Louis, Mo.); Eppendorf (Eppendorf AG, Hamburg, Germany); GE
Healthcare (GE Healthcare, Waukesha, Wis.); Bruker Optics (Bruker
Optics, Inc., Billerica, Mass.); Specac (Specac, Inc., Cranston,
R.I.); Invitrogen (Invitrogen, Corp., Carlsbad, Calif.); Alphalyse
(Alphalyse, Inc., Palo Alto, Calif.); Promega (Promega, Corp.,
Madison, Wis.); Sartorius (Sartorius-Stedim Biotech, SA, Aubagne,
France); Finnzymes (Finnzymes Oy, Espoo, FI [part of Thermo Fisher
Scientific]), CalBiochem (CalBiochem, EMD Chemicals, Inc.,
Gibbstown, N.J.); and Bio-Rad (Bio-Rad Laboratories, Hercules,
Calif.).
[0334] The following CDH sequences from M. thermophila (C1) find
use in the present invention. SEQ ID NOS:1 and 2 provide CDH1
nucleic acid and amino acid sequences, respectively. SEQ ID NO:3 is
the amino acid sequence of CDH2, while SEQ ID NO:4 is the amino
acid sequence of CDH3, SEQ ID NO:5 is the amino acid sequence of
CDH4, SEQ ID NO:6 is the amino acid sequence of CDH5, SEQ ID NO:7
is the amino acid sequence of CDH6, and SEQ ID NO:8 is the amino
acid sequence of CDH7.
TABLE-US-00003 CDH1: (SEQ ID NO: 1)
atgaggacctcctctcgtttaatcggtgcccttgcggcggcactcttgc
cgtctgcccttgcgcagaacaacgcgccggtaaccttcaccgacccgga
ctcgggcattaccttcaacacgtggggtctcgccgaggattctccccag
actaagggcggtttcacttttggtgttgctctgccctctgatgccctca
cgacagacgccaaggagttcatcggttacttgaaatgcgcgaggaacga
tgagagcggttggtgcggtgtctccctgggcggccccatgaccaactcg
ctcctcatcgcggcctggccccacgaggacaccgtctacacctctctcc
gcttcgccaccggctatgccatgccggatgtctaccagggggacgccga
gatcacccaggtctcctcctctgtcaactcgacgcacttcagcctcatc
ttcaggtgcgagaactgcctgcaatggagtcaaagcggcgccaccggcg
gtgcctccacctcgaacggcgtgttggtcctcggctgggtccaggcatt
cgccgaccccggcaacccgacctgccccgaccagatcaccctcgagcag
cacgacaacggcatgggtatctggggtgcccagctcaactccgacgccg
ccagcccgtcctacaccgagtgggccgcccaggccaccaagaccgtcac
gggtgactgcggcggtcccaccgagacctctgtcgtcggtgtccccgtt
ccgacgggcgtctcgttcgattacatcgtcgtgggcggcggtgccggtg
gcatccccgccgccgacaagctcagcgaggccggcaagagtgtgctgct
catcgagaagggctttgcctcgaccgccaacaccggaggcactctcggc
cccgagtggctcgagggccacgaccttacccgctttgacgtgccgggtc
tgtgcaaccagatctgggttgactccaaggggatcgcttgcgaggatac
cgaccagatggctggctgtgtcctcggcggcggtaccgccgtgaatgcc
ggcctgtggttcaagccctactcgctcgactgggactacctcttcccta
gtggttggaagtacaaagacgtccagccggccatcaaccgcgccctctc
gcgcatcccgggcaccgatgctccctcgaccgacggcaagcgctactac
caacagggcttcgacgtcctctccaagggcctggccggcggcggctgga
cctcggtcacggccaataacgcgccagacaagaagaaccgcaccttctc
ccatgcccccttcatgttcgccggcggcgagcgcaacggcccgctgggc
acctacttccagaccgccaagaagcgcagcaacttcaagctctggctca
acacgtcggtcaagcgcgtcatccgccagggcggccacatcaccggcgt
cgaggtcgagccgttccgcgacggcggttaccaaggcatcgtccccgtc
accaaggttacgggccgcgtcatcctctctgccggtacctttggcagtg
caaagatcctgctgaggagcggtatcggtccgaacgatcagctgcaggt
tgtcgcggcctcggagaaggatggccctaccatgatcagcaactcgtcc
tggatcaacctgcctgtcggctacaacctggatgaccacctcaacaccg
acactgtcatctcccaccccgacgtcgtgttctacgacttctacgaggc
gtgggacaatcccatccagtctgacaaggacagctacctcaactcgcgc
acgggcatcctcgcccaagccgctcccaacattgggcctatgttctggg
aagagatcaagggtgcggacggcattgttcgccagctccagtggactgc
ccgtgtcgagggcagcctgggtgcccccaacggcaagaccatgaccatg
tcgcagtacctcggtcgtggtgccacctcgcgcggccgcatgaccatca
ccccgtccctgacaactgtcgtctcggacgtgccctacctcaaggaccc
caacgacaaggaggccgtcatccagggcatcatcaacctgcagaacgcc
ctcaagaacgtcgccaacctgacctggctcttccccaactcgaccatca
cgccgcgccaatacgttgacagcatggtcgtctccccgagcaaccggcg
ctccaaccactggatgggcaccaacaagatcggcaccgacgacgggcgc
aagggcggctccgccgtcgtcgacctcaacaccaaggtctacggcaccg
acaacctcttcgtcatcgacgcctccatcttccccggcgtgcccaccac
caaccccacctcgtacatcgtgacggcgtcggagcacgcctcggcccgc
atcctcgccctgcccgacctcacgcccgtccccaagtacgggcagtgcg
gcggccgcgaatggagcggcagcttcgtctgcgccgacggctccacgtg
ccagatgcagaacgagtggtactcgcagtgcttgtga (SEQ ID NO: 2)
MRTSSRLIGALAAALLPSALAQNNAPVTFTDPDSGITFNTWGLAEDSPQ
TKGGFTFGVALPSDALTTDAKEFIGYLKCARNDESGWCGVSLGGPMTNS
LLIAAWPHEDTVYTSLRFATGYAMPDVYQGDAEITQVSSSVNSTHFSLI
FRCENCLQWSQSGATGGASTSNGVLVLGWVQAFADPGNPTCPDQITLEQ
HDNGMGIWGAQLNSDAASPSYTEWAAQATKTVTGDCGGPTETSVVGVPV
PTGVSFDYIVVGGGAGGIPAADKLSEAGKSVLLIEKGFASTANTGGTLG
PEWLEGHDLTRFDVPGLCNQIWVDSKGIACEDTDQMAGCVLGGGTAVNA
GLWFKPYSLDWDYLFPSGWKYKDVQPAINRALSRIPGTDAPSTDGKRYY
QQGFDVLSKGLAGGGWTSVTANNAPDKKNRTFSHAPFMFAGGERNGPLG
TYFQTAKKRSNFKLWLNTSVKRVIRQGGHITGVEVEPFRDGGYQGIVPV
TKVTGRVILSAGTFGSAKILLRSGIGPNDQLQVVAASEKDGPTMISNSS
WINLPVGYNLDDHLNTDTVISHPDVVFYDFYEAWDNPIQSDKDSYLNSR
TGILAQAAPNIGPMFWEEIKGADGIVRQLQWTARVEGSLGAPNGKTMTM
SQYLGRGATRGRMTITPSLTTVVSDVPYLKDPNDKEAVIQGIINLQNAL
KNVANLTWLFPNSTITPRQYVDSMVVSPSNRRSNHWMGTNKIGTDDGRK
GGSAVVDLNTKVYGTDNLFVIDASIFPGVPTTNPTSYIVTASEHASARI
LALPDLTPVPKYGQCGGREWSGSFVCADGSTCQMQNEWYSQCL CDH2: (SEQ ID NO: 3)
MKLLSRVGATALAATLSLQQCAAQMTEGTYTDEATGIQFKTWTASEGAP
FTFGLTLPADALEKDATEYIGLLRCQITDPASPSWCGISHGQSGQMTQA
LLLVAWASEDTVYTSFRYATGYTLPGLYTGDAKLTQISSSVSEDSFEVL
FRCENCFSWDQDGTKGNVSTSNGNLVLGRAAAKDGVTGPTCPDTAEFGF
HDNGFGQWGAVLEGATSDSYEEWAKLATTTPETTCDGTGPGDKECVPAP
EDTYDYIVVGAGAGGITVADKLSEAGHKVLLIEKGPPSTGLWNGTMKPE
WLESTDLTRFDVPGLCNQIWVDSAGIACTDTDQMAGCVLGGGTAVNAGL
WWKPHPADWDENFPEGWKSSDLADATERVFKRIPGTSHPSQDGKLYRQE
GFEVISKGLANAGWKEISANEAPSEKNHTYAHTEFMFSGGERGGPLATY
LASAAERSNFNLWLNTAVRRAVRSGSKVTGVELECLTDGGFSGTVNLNE
GGGVIFSAGAFGSAKLLLRSGIGPEDQLEIVASSKDGETFTPKDEWINL
PVGHNLIDHLNTDLIITHPDVVFYDFYAAWDEPITEDKEAYLNSRSGIL
AQAAPNIGPMMWDQVTPSDGITRQFQWTCRVEGDSSKTNSTHAMTLSQY
LGRGVVSRGRMGITSGLSTTVAEHPYLHNNGDLEAVIQGIQNVVDALSQ
VADLEWVLPPPDGTVADYVNSLIVSPANRRANHWMGTAKLGTDDGRSGG
TSVVDLDTKVYGTDNLFVVDASVFPGMSTGNPSAMIVIVAEQAAQRILA LRS CDH3: (SEQ ID
NO: 4) MKFLRKSDRGSVLGSTLFSLAFLFYSPPTAAQSPPPDGAVYDYIVIGSGP
GGGVVGANLAKAGYSVLLLEAGDDSPGAGFGVYTPTVTWDFYVKHYPEGD
PRDNQYSHLTWLTPDGRYWVGQSGAPEGSRLLGVYYPRGATLGGSSMINA
MVVWLPNDSDWDYHAEVTGDDSWRAENMHKIFQKIEKNNYLPRGTANHGF
DGWFQTQMGTMVQTNRTGPLQGNGVMTTYAQDWNLTIPMSDLLIRDPNEI
GPDRDQTSSIYGQVSHQFANGNRYSSRHYVQDAVSSGANLTVSLTSLATR
ILFDTVTEPDSPRATGVEYLFGKSLYRGDRRRADGAIGVNRTAVARREVI
VSGGAFNSPQLLLLSGIGNATELEALGIPVIRDLPGVGRNLMDNQEMPIV
GTGSPGGGPGAVAGVAMYKTRHPAHGERDMFLFGGPGFLFRGFWPNEAVH
LPDEPAQPVYGVSMVKGSSVNNGGWVKLRSRDPTDTPEINFNHYAVGAEY
DLEAVKDTVAWIRSVYRRVGIATVEPPCARGPDENGYCGEEDEAWIHKQT
FGHHPTSTNKIGADDDPTAVLDSKFRVRGVRALRVVDASAFARIPGVFPV
VSTFMISQKASDDILAELEAESR CDH4: (SEQ ID NO: 5)
MGFLAATLVSCAALASAASIPRPHAKRQVSQLRDDYDFVIVGGGTSGLT
VADRLTEAFPAKNVLVIEYGDVHYAPGTFDPPTDWITPQPDAPPSWSFN
SLPNPDMANTTAFVLAGQVVGGSSAVNGMFFDRASRHDYDAWTAVGGSG
FEQSSHKWDWEGLFPFFQKSVTFTEPPADIVQKYHYTWDLSAYGNGSTP
IYSSYPVFQWADQPLLNQAWQEMGINPVTECAGGDKEGVCWVPASQHPV
TARRSHAGLGHYADVLPRANYDLLVQHQVVRVVFPNGPSHGPPLVEARS
LADNHLFNVTVKGEVIISAGALHTPTVLQRSGIGPASFLDDAGIPVTLD
LPGVGANLQDHCGPPVTWNYTEPYTGFFPLPSEMVNNATFKAEAITGFD
EVPARGPYTLAGGNNAIFVSLPHLTADYGAITANIRAMVADGTAASYLA
ADVRTIPGMVAGYEAQLLVLADLLDNPEAPSLETPWATSEAPQTSSVLA
FLLHPLSRGSVRLNLSDPLAQPVLDYRSGSNPVDIDLHLAHVRFLRGLL
DTPTMQARGALETAPGSAVADSDEALGEYVRSHSTLSFMHPCCTAAMLP
EDRGGVVGPDLKVHGAEGLRVVDMSVMPLLPGAHLSATAYAVGEKAADI IIQEWMDKEQ CDH5:
(SEQ ID NO: 6) MELLRVSLAAVALSPLILFGVAAAHPTARSIARSTILDGADGLLPEYDY
IIIGGGTSGLTVADRLTENRKRKFSRSPLPTSPARSSPAWCYSVLVLER
GIFQNSSSVTTISGGSRGLFDPSLTFNINSVPQAGLDNRSIAVIGGLIL
GGSSGVNGLQVLRGQREDYDRWGSYFGPNSDWSWKGLLPYFKKAWNFHP
PRPELVSQFDIKYDPSYWGNTSDVHASFPTTFWPVLKLEMAAFGDIPGV
EYPPDSASGETGAYWHPASVDPATVLRSFARPAHWDNIEAARPNYHTLT
GQRVLKVAFDGNRATSVVFVPANATDHSTARSVKAKKEIVLAAGAIHTP
QILQASGVGPKQVLKEAGVPLVVDAPGVGSNFQDQPYVVAPTFNFTKFP
FHPDFYDMILNQTFIAEAQAQFEKDRTGPHTIASGYCGSWLPLQIIAPN
SWKDIARRYESQDPAAYLPAGTDETVIEGYRAQQKALARSMRSKQSAMY
NFFLRGGYEEGSVVYLHPTSRGTVRINRSDPFFSPPEVDYRALSNPTDL
EVLLEFTPFTRRYFLETRLKSLDPVELSPGANVTAPADIEAWLRSVMIP
SSFHPIGTAAMLPRHLGGVVDENLLVYGVEGLSVVDASVMPDLPGSYTQ
QTVYAIAEKAADLIKSRA CDH6: (SEQ ID NO: 7)
MQVASKLVAVTGGALALWLHPVAAQEGCTNISSTETYDYIVVGSGAGGI
PVADRLSEAGHKVLLIEKGPPSTGRWGGIMKPEWLIGTNLTRFDVPGLC
NQIWADPTGAICTDVDQMAGCMLGGGTAVNAGLWWKPHPADWDVNFPEG
WHSEDMAEATERVFERIPGTITPSMDGKRYLSQGFDMLGGSLEAAGWEY
LVPNEHPDRKNRTYGHSTFMYSGGERGGPLATYLVSAVQREGFTLWMNT
TVTRIIREGGHATGVEVQCSNSEAGQAGIVPLTPKTGRVIVSAGAFGSA
KLLFRSGIGPKDQLNIVKNSTDGPSMISEDQWIELPVGYNLNDHVGTDI
EIAHPDVVFYDYYGAWDEPIVEDTERYVANRTGPLAQAAPNIGPIFWET
IKGSDGVSRHLQWQARVEGKLNTSMTITQYLGTGSRSRGRMTITRRLNT
VVSTPPYLRDEYDREAVIQGIANLRESLKGVANLTWITPPSNVTVEDFV
DSIPATPARRCSNHWIGTAKIGLDDGREGGTSVVDLNTKVYGTDNIFVV
DASIFPGHITGNPSAAIVIAAEYAAAKILALPAPEDAAS CDH7: (SEQ ID NO: 8)
MASVDLDQPFDYIVVGGGTAGLVVANRLSEDSNVRVLVVEAGADRNADP
LVLTPGLVAGLYGKDEYDWNFSSPPQPTLNNRRINQARGKMLGGTSGLN
FMMLLYPSKGNIDSWAALGNPSWNYDALAPYLRKFATVHPSPQSARDLL
GLTYIDESLAAGDGPIQVSHTDGHNVTNKAWLETFASLGLEVSTDPRDG
KALGAFQNHASIDPATHTRSFAGPAYYTPDVAKRPNLVVLTETLVARVL
FDTAGGEGDAVATGVEIITKDGQKKQVSACGEVILAAGALQSPQILELS
GVGGRELLEKHNIPVVVDNPNVGEHVQDHPIVCQSFEVADGVPSGDVLR
DPNVLQAVVGMYQSGGGAGPLGQSVISVAYTPLVDGSGVVSAEAKAELL
ARHESSFSTAEGKVLRDLVESPSEATFEFLLFPSQVDIPENPTSMAQYI
TPVLPENYISVMTFIHQPFSRGKVHITSPDIRAAPLWDPRYNSDPLDLE
LLARGVQFVERIVDSATPFGRVLKQGGKRQPPLRADDLETAREIVRQRQ
ISVFHVSGSCTMRPRDQGGVVDERLRVYGTRGLRVVDASVFPIEPVGNI
QSVVYAVAERAADLIKEDRAKA
EXAMPLE 1
Fungal Strains and Methods
[0335] This Example describes the production of variants of fungal
strain C1.
Strain Nomenclature
[0336] Strain CF-200 (UV18#100f.DELTA.alp1) is a derivative C1
strain. Strain CF-400 is a derivative of C1 strain
("UV18#100f.DELTA.alp1.DELTA.pyr5"), further modified by deletion
of cdh1, wherein cdh1 comprises the polynucleotide sequence of SEQ
ID NO:1. Cellulolytic enzymes from these strains were produced by
submerged liquid culture fermentation using methods and a suitable
fungal growth medium, as well-known in the art.
GOPOD Assay
[0337] The GOPOD assay kits (Sigma-Aldrich) used in these
experiments to measure the amount of glucose produced. In these
experiments, 10 ul of test sample was added to 190 ul of the GOPOD
assay mix provided in the kit. The reaction was allowed to shake
for 30 min at 50.degree. C. Absorbance of the solution was measured
at 510 nm to determine the amount of glucose produced. The glucose
concentration of the samples was calculated in comparison with the
glucose standards (0-150 g/L).
EXAMPLE 2
Purification of C1 CDH1
[0338] In this Example, 400 mL of C1 supernatant produced using the
methods of Example 1 were first concentrated to 140 mL using a
rotary evaporator. Then, 63 mL of the concentrate was
buffer-exchanged into 20 mM MOPS buffer, pH 7.0, using 4 in-line
Hi-Prep 26/10 desalting columns (GE Healthcare, 17-5087-02). The
resulting buffer-exchanged supernatant (.about.150g/L total
protein) was loaded onto a column containing 500 mL DEAE Fast Flow
resin (GE Healthcare, 17-0709-01) pre-equilibrated with 20 mM pH7.0
MOPS buffer. The column was rinsed with 1 column volume (CV) of 20
mM MOPS (pH7.0) and then a 0-300 mM sodium chloride gradient was
run over 12 column volumes. Fractions were collected and analyzed
by NuPage.RTM. Novex.RTM. Bis-tris SDS-PAGE gels (Invitrogen,
NP0322BOX). The SDS-PAGE bands corresponding to the apparent
molecular weight of CDH1 were analyzed by MS (performed by
Alphalyse). The mass-mapping analysis confirmed the presence of
CDH1 in late-eluting fractions. Fractions containing CDH1, as
demonstrated by SDS-PAGE gel, and confirmed by MS were pooled and
concentrated by ultrafiltration using Sartorius centrifugal 10 kDa
filter (Sartorius-Stedim, VS2002). Then, 10 mL 500 mM piperazine
(pH 5.6) and 45 mL saturated ammonium sulfate were added to 45 mL
of the CDH1-containing pool and the resulting mixture was loaded
onto a Phenyl FF (high sub) 16/10 column (GE Healthcare,
28-9365-45) pre-equilibrated with 1.6M ammonium sulfate in 50 mM
piperazine, pH 5.6. A gradient of 1.6 M to 0 M ammonium sulfate in
50 mM piperazine, pH 5.6, was run over 30 CV. Fractions were
collected and SDS-PAGE gel analysis was performed on the selected
fractions as described above, revealing that CDH1 eluted in the
final rinse step with approximately 80-90% purity.
[0339] CDH1 activity was measured using a DCPIP reduction assay
similar to that described by Schou et. al. (See, Schou et al.,
Biochem J., 330:565-71 [1998]). In a UV-transparent flat-bottom
96-well plate, 50 .mu.L CDH1-containing fractions were added to 150
.mu.L of a solution of 1.0 g/L cellobiose and 100 .mu.M DCPIP in
100 mM sodium acetate, pH 5.0. Samples were agitated briefly at
room temperature and then the absorbance at 530 nm (A.sub.530) was
measured for 10 minutes. C1 CDH1-containing fractions displayed a
rapid drop in absorbance at 530 nm DCPIP assays were performed
using varying amounts of glucose or cellobiose with purified CDH1.
Serial dilutions of cellobiose (1.0 g/L to 7.8 mg/L) and glucose
(10 g/L to 78 mg/L) were prepared in a 96-well shallow-well plate.
Then, 20 .mu.L glucose and cellobiose standards were added to 160
.mu.L/well 200 mM DCPIP (in 100 mM pH 5.0 sodium acetate).
Reactions were initiated by addition of 20 .mu.L CDH1 solution.
Absorbance at 530 nm was monitored for 30 minutes. Comparisons of
the rates of decrease in absorbance at 530 nm indicate that C1 CDH1
is approximately 10-fold more active on cellobiose than
glucose.
EXAMPLE 3
Making of CDH1 Split Marker Deletion Constructs
[0340] Genomic DNA was isolated from the C1 strain using standard
procedures. Briefly, hyphal inoculum was seeded into a growth
medium and allowed to grow for 72 hours at 35.degree. C. The
mycelial mat was collected by centrifugation, washed, and 50 .mu.L
DNA extraction buffer (200 mM TRIS, pH 8.0; 250 mM NaCl; 125 mM
EDTA; 0.5% SDS) was added. The mycelia were ground with a conical
grinder, re-extracted with 250 .mu.L extraction buffer, and the
suspension was centrifuged. The supernatant was transferred to a
new tube containing 300 .mu.L isopropanol. DNA was collected by
centrifugation, washed twice with 70% ethanol, and diluted in 100
.mu.L of water.
[0341] Genomic DNA fragments flanking the cdh1 gene were cloned
using primers cf09067 and cf09068 (cdh1 upstream homology) and
primers cf09069 and cf09070 (cdh1 downstream homology). PCR
reactions were performed by using the GoTaq.RTM. polymerase
(Promega) following the manufacturer's instructions using 0.2 uM of
each primer. The amplification conditions were 95.degree. C. for 2
minutes, 35 cycles of 95.degree. C. for 30 seconds, 55.degree. C.
for 30 seconds (for upstream homology) or 53.degree. C. for 30
seconds (for downstream homology), 72.degree. C. for 1 minute and
final extension at 72.degree. C. for 5 minutes. The pyr5 gene was
PCR amplified as a split marker from a vector using primers cf09024
and cf09025 (for the 5' portion of the gene) and cf09026 and
cf09027 (for the 3' portion of the gene). PCR reactions were
performed using the GoTaq.RTM. polymerase (Promega) following the
manufacturer's instructions using 0.2 uM of each primer. The
amplification conditions were 95.degree. C. for 2 minutes, 35
cycles of 95.degree. C. for 30 seconds, 53.degree. C. for 30
seconds, 72.degree. C. for 1 minute and final extension at
72.degree. C. for 5 minutes. The primers used are shown in Table
3-1. In separate strand overlap extension reactions (See, Horton et
al., Meth. Enzymol., 217:270-279 [1993]), the PCR products
resulting from primers cf09067 and cf09068 and primers cf09026 and
cf09027 were fused, as were the PCR products resulting from primers
cf09069 and cf09070, and primers cf09024 and cf09025. PCR reactions
were performed by using Finnzymes' Phusion.RTM. DNA polymerase
following the manufacturer's instructions including 3% DMSO and
using 0.2 uM of each primer. The amplification conditions were
98.degree. C. for 1 minute, 35 cycles of 98.degree. C. for 10
seconds, 62.degree. C. for 20 seconds, 72.degree. C. for 2 minutes
and final extension at 72.degree. C. for 5 minutes. The strand
overlap extension products were used for cdh1 deletion.
TABLE-US-00004 TABLE 3-1 Primer Names and Sequences Primer SEQ ID
Name Sequence (5'-3') NO: cf09067 CACGCGGGGTTCTTTCTCCATCTC 9
cf09068 TGAGGAAAACGCCGAGACTGAGCTCGACTCTG 10 CCGGCCTACCTACGA cf09069
ATCAGTTGGGTGCACGAGTGGGTTTTGATGGG 11 GAGTTGAGTTTGTGAA cf09070
GGATGGATGAGGTTGTTTTTGAGC 12 cf09024 AACCCACTCGTGCACCCAACTGAT 13
cf09025 GACCACGATGCCGGCTACGATACC 14 cf09026
ACATGGCCCCACTCGCTTCTTACA 15
EXAMPLE 4
Transformation Method
[0342] C1 cells and derivative strains were inoculated into 100 mL
growth medium in a 500 mL Erlenmeyer flask using 10.sup.6
spores/mL. The culture was incubated for 48 hours at 35.degree. C.,
250 rpm. To harvest the mycelia, the culture was filtered over a
sterile Myracloth filter (CalBiochem) and washed with 100 mL 1700
mosmol NaCl/CaCl.sub.2 solution (0.6 M NaCl, 0.27 M
CaCl.sub.2*H.sub.2O). The washed mycelia were transferred into a 50
mL tube and weighed. Caylase (20 mg/gram mycelia; Cayla) was
dissolved in 1700 mosmol NaCl/CaCl.sub.2 and UV-sterilized for 90
sec. Then, 3 mL of sterile Caylase solution was added into the tube
containing washed mycelia and mixed. Then, 15 mL of 1700 mosmol
NaCl/CaCl.sub.2 solution was added into the tube and mixed. The
mycelium/Caylase suspension was incubated at 30.degree. C., 70 rpm
for 2 hours. Protoplasts were harvested by filtering through a
sterile Myracloth filter into a sterile 50 mL tube. 25 mL cold STC
(1.2 M sorbitol, 50 mM CaCl.sub.2*H.sub.2O, 35 mM NaCl, 10 mM
Tris-HCl) was added to the flow through and spun down at 2720 rpm
for 10 min at 4.degree. C. The pellet was resuspended in 50 mL STC
and centrifuged again. After the washing steps, the pellet was
resuspended in 1 mL STC.
[0343] Then, 2 .mu.g DNA of each strand overlap extension product
was pipetted into the bottom of a 15 mL sterile tube and 1 .mu.L
aurintricarboxylic acid and 100 .mu.L of the protoplast suspension
were added. The contents were mixed and the protoplasts were
incubated with the DNA at room temperature for 25 min Then, 1.7 mL
PEG4000 solution (60% PEG4000; polyethylene glycol, average
molecular weight 4000 daltons), 50 mM CaCl.sub.2.H.sub.2O, 35 mM
NaCl, 10 mM Tris-HCl) was added and mixed thoroughly. The solution
was kept at room temperature for 20 min The tube was filled with
STC, mixed and centrifuged at 2500 rpm for 10 min at 4.degree. C.
The STC was poured off and the pellet was resuspended in the
remaining STC and plated on minimal selective media plates. The
plates were incubated for 5 days at 35.degree. C. Colonies were
restreaked and checked for the deletion of cdh1; colonies with this
deletion were designated as strain "CF-400".
EXAMPLE 5
Confirmation of CDH1 Deletion
[0344] Genomic DNA was prepared as described in Example 3. PCR
reactions were performed by using the GoTaq.RTM. polymerase
(Promega) following the manufacturer's instructions using 0.2 uM of
each primer (primers cf09112 and cf09113). The amplification
conditions were 95.degree. C. for 2 minutes, 35 cycles of
95.degree. C. for 30 seconds, 54.degree. C. for 30 seconds,
72.degree. C. for 30 seconds and final extension at 72.degree. C.
for 5 minutes. PCR was also conducted using primers cf09110 and
cf09111 and GoTaq.RTM. polymerase (Promega) following the
manufacturer's instructions using 0.2 uM of each primer. The
amplification conditions were 95.degree. C. for 2 minutes, 35
cycles of 95.degree. C. for 30 seconds, 55.4.degree. C. for 30
seconds, 72.degree. C. for 30 seconds and final extension at
72.degree. C. for 5 minutes). These primers were used in separate
PCR reactions to confirm absence of the cdh1 gene. Primers cf09181
and cf09091 were used in PCR to confirm proper junction structure
and targeting of the pyr5 marker construct (See, Table 5-1). The
PCR reaction was performed by using the GoTaq.RTM. polymerase
(Promega) following the manufacturer's instructions using 0.2 uM of
each primer. The amplification conditions were 95.degree. C. for 2
minutes, 35 cycles of 95.degree. C. for 30 seconds, 54.4.degree. C.
for 30 seconds, 72.degree. C. for 3 minutes 30 seconds, and final
extension at 72.degree. C. for 5 minutes. PCR products were run on
an agarose gel to confirm a banding pattern indicative of cdh1
deletion.
TABLE-US-00005 TABLE 5-1 Primer Names and Sequences Primer name
Sequence (5'-3') SEQ ID NO: cf09110 AAGCGTGCCGATTTTCCTGATTTC 16
cf09111 GCATTTCTGGGGCGGTTAGCA 17 cf09112 TCATCGACGCCTCCATCTTCC 18
cf09113 TTTCGGTTGTCGTGTTTCCATTAT 19 cf09181 GGAGATCCTGGAGGATTTCC 20
cf09091 CAGGCGGTGTGCGTTATCAAAA 21
[0345] A colorimetric dichlorophenolindophenol (DCPIP) assay was
used to test for deletion of cdh1 in CF-400. Deletion of cdh1 was
determined by observing a decreased ability to reduce the DCPIP
substrate compared to a parent strain. Cells of the parental C1
strain and putative cdh1 delete strain were grown and the
supernatants tested for DCPIP activity. In these tests, 160 .mu.L
of freshly made DCPIP reagent solution (0.2 mM DCPIP in 100 mM
sodium acetate, pH 5.0), 20 .mu.L cellobiose solution (1 g/L
cellobiose in deionized water), and 20 mLs of undiluted cell
supernatant were combined in microtiter plates. The absorbance of
the solution was immediately measured over time at 530 nm in
kinetic mode for 30 minutes to track loss of absorbance as a result
of DCPIP reduction. Supernatant from strains displaying decreased
ability to reduce the DCPIP substrate were run on SDS-PAGE to
confirm the absence of CDH1. Proteins from culture supernatants of
submerged liquid culture fermentations of CF-400 and the
untransformed parent were separated by SDS-PAGE using standard
protocols. The proteins were visualized by staining with Simply
Blue Safe Stain (Invitrogen), as per manufacturer's instructions.
The Cdhl protein was observed as a .about.90 kD band in the
untransformed parent but was absent in CF-400.
EXAMPLE 6
Hydrolysis of Corn Stover
[0346] In these experiments, acid pretreated corn stover (NREL) was
pH adjusted to 5.0 with aqueous ammonium hydroxide. The material
was 41.3% solids, with a moisture content of 58.7%. The glucan
content in the solids was 40.7%. The acid pretreated corn stover
was loaded into a 96-well plate and diluted with sodium acetate
buffer to an average volume of 110 .mu.L per well with 128 mM
sodium acetate, at pH 5. The total solids loading were 24.7% in all
experiments, and the concentration of glucan was 100 g glucan/kg
reaction. CF-200 and CF-400 enzyme supernatants were used at 3 g
cellulase/kg reaction. A set of wells in the 96-well plate was also
run wherein water was used in place of enzyme to serve as a
control, due to the presence of free glucose in the substrate. The
level of this control was subtracted from the final measured
glucose concentration. The plate was sealed once all reaction
components were added and placed in a shaker at 55.degree. C.
rotating at 950 rpm for 73 hours. At the end of reaction, the plate
was allowed to cool. Samples were withdrawn, diluted and
subsequently analyzed by GO assay kit (Sigma) to determine glucose
production. The results are provided in FIG. 2. As indicated, the
CF-200 supernatant generated 52.1 g/L glucose, while CF-400
supernatant generated 69.4 g/L glucose. CF-400 supernatant
exhibited higher saccharification performance, indicating that
deletion of cdh1 gene reduces formation of the gluconate from
glucose during the saccharification reaction.
[0347] While particular embodiments of the present invention have
been illustrated and described, it will be apparent to those
skilled in the art that various other changes and modifications can
be made without departing from the spirit and scope of the present
invention. Therefore, it is intended that the present invention
encompass all such changes and modifications with the scope of the
present invention.
[0348] The present invention has been described broadly and
generically herein. Each of the narrower species and subgeneric
groupings falling within the generic disclosure also form part(s)
of the invention. The invention described herein suitably may be
practiced in the absence of any element or elements, limitation or
limitations which is/are not specifically disclosed herein. The
terms and expressions which have been employed are used as terms of
description and not of limitation. There is no intention that in
the use of such terms and expressions, of excluding any equivalents
of the features described and/or shown or portions thereof, but it
is recognized that various modifications are possible within the
scope of the claimed invention. Thus, it should be understood that
although the present invention has been specifically disclosed by
some preferred embodiments and optional features, modification and
variation of the concepts herein disclosed may be utilized by those
skilled in the art, and that such modifications and variations are
considered to be within the scope of this invention.
Sequence CWU 1
1
2112487DNAMyceliophthora thermophila 1atgaggacct cctctcgttt
aatcggtgcc cttgcggcgg cactcttgcc gtctgccctt 60gcgcagaaca acgcgccggt
aaccttcacc gacccggact cgggcattac cttcaacacg 120tggggtctcg
ccgaggattc tccccagact aagggcggtt tcacttttgg tgttgctctg
180ccctctgatg ccctcacgac agacgccaag gagttcatcg gttacttgaa
atgcgcgagg 240aacgatgaga gcggttggtg cggtgtctcc ctgggcggcc
ccatgaccaa ctcgctcctc 300atcgcggcct ggccccacga ggacaccgtc
tacacctctc tccgcttcgc caccggctat 360gccatgccgg atgtctacca
gggggacgcc gagatcaccc aggtctcctc ctctgtcaac 420tcgacgcact
tcagcctcat cttcaggtgc gagaactgcc tgcaatggag tcaaagcggc
480gccaccggcg gtgcctccac ctcgaacggc gtgttggtcc tcggctgggt
ccaggcattc 540gccgaccccg gcaacccgac ctgccccgac cagatcaccc
tcgagcagca cgacaacggc 600atgggtatct ggggtgccca gctcaactcc
gacgccgcca gcccgtccta caccgagtgg 660gccgcccagg ccaccaagac
cgtcacgggt gactgcggcg gtcccaccga gacctctgtc 720gtcggtgtcc
ccgttccgac gggcgtctcg ttcgattaca tcgtcgtggg cggcggtgcc
780ggtggcatcc ccgccgccga caagctcagc gaggccggca agagtgtgct
gctcatcgag 840aagggctttg cctcgaccgc caacaccgga ggcactctcg
gccccgagtg gctcgagggc 900cacgacctta cccgctttga cgtgccgggt
ctgtgcaacc agatctgggt tgactccaag 960gggatcgctt gcgaggatac
cgaccagatg gctggctgtg tcctcggcgg cggtaccgcc 1020gtgaatgccg
gcctgtggtt caagccctac tcgctcgact gggactacct cttccctagt
1080ggttggaagt acaaagacgt ccagccggcc atcaaccgcg ccctctcgcg
catcccgggc 1140accgatgctc cctcgaccga cggcaagcgc tactaccaac
agggcttcga cgtcctctcc 1200aagggcctgg ccggcggcgg ctggacctcg
gtcacggcca ataacgcgcc agacaagaag 1260aaccgcacct tctcccatgc
ccccttcatg ttcgccggcg gcgagcgcaa cggcccgctg 1320ggcacctact
tccagaccgc caagaagcgc agcaacttca agctctggct caacacgtcg
1380gtcaagcgcg tcatccgcca gggcggccac atcaccggcg tcgaggtcga
gccgttccgc 1440gacggcggtt accaaggcat cgtccccgtc accaaggtta
cgggccgcgt catcctctct 1500gccggtacct ttggcagtgc aaagatcctg
ctgaggagcg gtatcggtcc gaacgatcag 1560ctgcaggttg tcgcggcctc
ggagaaggat ggccctacca tgatcagcaa ctcgtcctgg 1620atcaacctgc
ctgtcggcta caacctggat gaccacctca acaccgacac tgtcatctcc
1680caccccgacg tcgtgttcta cgacttctac gaggcgtggg acaatcccat
ccagtctgac 1740aaggacagct acctcaactc gcgcacgggc atcctcgccc
aagccgctcc caacattggg 1800cctatgttct gggaagagat caagggtgcg
gacggcattg ttcgccagct ccagtggact 1860gcccgtgtcg agggcagcct
gggtgccccc aacggcaaga ccatgaccat gtcgcagtac 1920ctcggtcgtg
gtgccacctc gcgcggccgc atgaccatca ccccgtccct gacaactgtc
1980gtctcggacg tgccctacct caaggacccc aacgacaagg aggccgtcat
ccagggcatc 2040atcaacctgc agaacgccct caagaacgtc gccaacctga
cctggctctt ccccaactcg 2100accatcacgc cgcgccaata cgttgacagc
atggtcgtct ccccgagcaa ccggcgctcc 2160aaccactgga tgggcaccaa
caagatcggc accgacgacg ggcgcaaggg cggctccgcc 2220gtcgtcgacc
tcaacaccaa ggtctacggc accgacaacc tcttcgtcat cgacgcctcc
2280atcttccccg gcgtgcccac caccaacccc acctcgtaca tcgtgacggc
gtcggagcac 2340gcctcggccc gcatcctcgc cctgcccgac ctcacgcccg
tccccaagta cgggcagtgc 2400ggcggccgcg aatggagcgg cagcttcgtc
tgcgccgacg gctccacgtg ccagatgcag 2460aacgagtggt actcgcagtg cttgtga
24872828PRTMyceliophthora thermophila 2Met Arg Thr Ser Ser Arg Leu
Ile Gly Ala Leu Ala Ala Ala Leu Leu 1 5 10 15 Pro Ser Ala Leu Ala
Gln Asn Asn Ala Pro Val Thr Phe Thr Asp Pro 20 25 30 Asp Ser Gly
Ile Thr Phe Asn Thr Trp Gly Leu Ala Glu Asp Ser Pro 35 40 45 Gln
Thr Lys Gly Gly Phe Thr Phe Gly Val Ala Leu Pro Ser Asp Ala 50 55
60 Leu Thr Thr Asp Ala Lys Glu Phe Ile Gly Tyr Leu Lys Cys Ala Arg
65 70 75 80 Asn Asp Glu Ser Gly Trp Cys Gly Val Ser Leu Gly Gly Pro
Met Thr 85 90 95 Asn Ser Leu Leu Ile Ala Ala Trp Pro His Glu Asp
Thr Val Tyr Thr 100 105 110 Ser Leu Arg Phe Ala Thr Gly Tyr Ala Met
Pro Asp Val Tyr Gln Gly 115 120 125 Asp Ala Glu Ile Thr Gln Val Ser
Ser Ser Val Asn Ser Thr His Phe 130 135 140 Ser Leu Ile Phe Arg Cys
Glu Asn Cys Leu Gln Trp Ser Gln Ser Gly 145 150 155 160 Ala Thr Gly
Gly Ala Ser Thr Ser Asn Gly Val Leu Val Leu Gly Trp 165 170 175 Val
Gln Ala Phe Ala Asp Pro Gly Asn Pro Thr Cys Pro Asp Gln Ile 180 185
190 Thr Leu Glu Gln His Asp Asn Gly Met Gly Ile Trp Gly Ala Gln Leu
195 200 205 Asn Ser Asp Ala Ala Ser Pro Ser Tyr Thr Glu Trp Ala Ala
Gln Ala 210 215 220 Thr Lys Thr Val Thr Gly Asp Cys Gly Gly Pro Thr
Glu Thr Ser Val 225 230 235 240 Val Gly Val Pro Val Pro Thr Gly Val
Ser Phe Asp Tyr Ile Val Val 245 250 255 Gly Gly Gly Ala Gly Gly Ile
Pro Ala Ala Asp Lys Leu Ser Glu Ala 260 265 270 Gly Lys Ser Val Leu
Leu Ile Glu Lys Gly Phe Ala Ser Thr Ala Asn 275 280 285 Thr Gly Gly
Thr Leu Gly Pro Glu Trp Leu Glu Gly His Asp Leu Thr 290 295 300 Arg
Phe Asp Val Pro Gly Leu Cys Asn Gln Ile Trp Val Asp Ser Lys 305 310
315 320 Gly Ile Ala Cys Glu Asp Thr Asp Gln Met Ala Gly Cys Val Leu
Gly 325 330 335 Gly Gly Thr Ala Val Asn Ala Gly Leu Trp Phe Lys Pro
Tyr Ser Leu 340 345 350 Asp Trp Asp Tyr Leu Phe Pro Ser Gly Trp Lys
Tyr Lys Asp Val Gln 355 360 365 Pro Ala Ile Asn Arg Ala Leu Ser Arg
Ile Pro Gly Thr Asp Ala Pro 370 375 380 Ser Thr Asp Gly Lys Arg Tyr
Tyr Gln Gln Gly Phe Asp Val Leu Ser 385 390 395 400 Lys Gly Leu Ala
Gly Gly Gly Trp Thr Ser Val Thr Ala Asn Asn Ala 405 410 415 Pro Asp
Lys Lys Asn Arg Thr Phe Ser His Ala Pro Phe Met Phe Ala 420 425 430
Gly Gly Glu Arg Asn Gly Pro Leu Gly Thr Tyr Phe Gln Thr Ala Lys 435
440 445 Lys Arg Ser Asn Phe Lys Leu Trp Leu Asn Thr Ser Val Lys Arg
Val 450 455 460 Ile Arg Gln Gly Gly His Ile Thr Gly Val Glu Val Glu
Pro Phe Arg 465 470 475 480 Asp Gly Gly Tyr Gln Gly Ile Val Pro Val
Thr Lys Val Thr Gly Arg 485 490 495 Val Ile Leu Ser Ala Gly Thr Phe
Gly Ser Ala Lys Ile Leu Leu Arg 500 505 510 Ser Gly Ile Gly Pro Asn
Asp Gln Leu Gln Val Val Ala Ala Ser Glu 515 520 525 Lys Asp Gly Pro
Thr Met Ile Ser Asn Ser Ser Trp Ile Asn Leu Pro 530 535 540 Val Gly
Tyr Asn Leu Asp Asp His Leu Asn Thr Asp Thr Val Ile Ser 545 550 555
560 His Pro Asp Val Val Phe Tyr Asp Phe Tyr Glu Ala Trp Asp Asn Pro
565 570 575 Ile Gln Ser Asp Lys Asp Ser Tyr Leu Asn Ser Arg Thr Gly
Ile Leu 580 585 590 Ala Gln Ala Ala Pro Asn Ile Gly Pro Met Phe Trp
Glu Glu Ile Lys 595 600 605 Gly Ala Asp Gly Ile Val Arg Gln Leu Gln
Trp Thr Ala Arg Val Glu 610 615 620 Gly Ser Leu Gly Ala Pro Asn Gly
Lys Thr Met Thr Met Ser Gln Tyr 625 630 635 640 Leu Gly Arg Gly Ala
Thr Ser Arg Gly Arg Met Thr Ile Thr Pro Ser 645 650 655 Leu Thr Thr
Val Val Ser Asp Val Pro Tyr Leu Lys Asp Pro Asn Asp 660 665 670 Lys
Glu Ala Val Ile Gln Gly Ile Ile Asn Leu Gln Asn Ala Leu Lys 675 680
685 Asn Val Ala Asn Leu Thr Trp Leu Phe Pro Asn Ser Thr Ile Thr Pro
690 695 700 Arg Gln Tyr Val Asp Ser Met Val Val Ser Pro Ser Asn Arg
Arg Ser 705 710 715 720 Asn His Trp Met Gly Thr Asn Lys Ile Gly Thr
Asp Asp Gly Arg Lys 725 730 735 Gly Gly Ser Ala Val Val Asp Leu Asn
Thr Lys Val Tyr Gly Thr Asp 740 745 750 Asn Leu Phe Val Ile Asp Ala
Ser Ile Phe Pro Gly Val Pro Thr Thr 755 760 765 Asn Pro Thr Ser Tyr
Ile Val Thr Ala Ser Glu His Ala Ser Ala Arg 770 775 780 Ile Leu Ala
Leu Pro Asp Leu Thr Pro Val Pro Lys Tyr Gly Gln Cys 785 790 795 800
Gly Gly Arg Glu Trp Ser Gly Ser Phe Val Cys Ala Asp Gly Ser Thr 805
810 815 Cys Gln Met Gln Asn Glu Trp Tyr Ser Gln Cys Leu 820 825
3787PRTMyceliophthora thermophila 3Met Lys Leu Leu Ser Arg Val Gly
Ala Thr Ala Leu Ala Ala Thr Leu 1 5 10 15 Ser Leu Gln Gln Cys Ala
Ala Gln Met Thr Glu Gly Thr Tyr Thr Asp 20 25 30 Glu Ala Thr Gly
Ile Gln Phe Lys Thr Trp Thr Ala Ser Glu Gly Ala 35 40 45 Pro Phe
Thr Phe Gly Leu Thr Leu Pro Ala Asp Ala Leu Glu Lys Asp 50 55 60
Ala Thr Glu Tyr Ile Gly Leu Leu Arg Cys Gln Ile Thr Asp Pro Ala 65
70 75 80 Ser Pro Ser Trp Cys Gly Ile Ser His Gly Gln Ser Gly Gln
Met Thr 85 90 95 Gln Ala Leu Leu Leu Val Ala Trp Ala Ser Glu Asp
Thr Val Tyr Thr 100 105 110 Ser Phe Arg Tyr Ala Thr Gly Tyr Thr Leu
Pro Gly Leu Tyr Thr Gly 115 120 125 Asp Ala Lys Leu Thr Gln Ile Ser
Ser Ser Val Ser Glu Asp Ser Phe 130 135 140 Glu Val Leu Phe Arg Cys
Glu Asn Cys Phe Ser Trp Asp Gln Asp Gly 145 150 155 160 Thr Lys Gly
Asn Val Ser Thr Ser Asn Gly Asn Leu Val Leu Gly Arg 165 170 175 Ala
Ala Ala Lys Asp Gly Val Thr Gly Pro Thr Cys Pro Asp Thr Ala 180 185
190 Glu Phe Gly Phe His Asp Asn Gly Phe Gly Gln Trp Gly Ala Val Leu
195 200 205 Glu Gly Ala Thr Ser Asp Ser Tyr Glu Glu Trp Ala Lys Leu
Ala Thr 210 215 220 Thr Thr Pro Glu Thr Thr Cys Asp Gly Thr Gly Pro
Gly Asp Lys Glu 225 230 235 240 Cys Val Pro Ala Pro Glu Asp Thr Tyr
Asp Tyr Ile Val Val Gly Ala 245 250 255 Gly Ala Gly Gly Ile Thr Val
Ala Asp Lys Leu Ser Glu Ala Gly His 260 265 270 Lys Val Leu Leu Ile
Glu Lys Gly Pro Pro Ser Thr Gly Leu Trp Asn 275 280 285 Gly Thr Met
Lys Pro Glu Trp Leu Glu Ser Thr Asp Leu Thr Arg Phe 290 295 300 Asp
Val Pro Gly Leu Cys Asn Gln Ile Trp Val Asp Ser Ala Gly Ile 305 310
315 320 Ala Cys Thr Asp Thr Asp Gln Met Ala Gly Cys Val Leu Gly Gly
Gly 325 330 335 Thr Ala Val Asn Ala Gly Leu Trp Trp Lys Pro His Pro
Ala Asp Trp 340 345 350 Asp Glu Asn Phe Pro Glu Gly Trp Lys Ser Ser
Asp Leu Ala Asp Ala 355 360 365 Thr Glu Arg Val Phe Lys Arg Ile Pro
Gly Thr Ser His Pro Ser Gln 370 375 380 Asp Gly Lys Leu Tyr Arg Gln
Glu Gly Phe Glu Val Ile Ser Lys Gly 385 390 395 400 Leu Ala Asn Ala
Gly Trp Lys Glu Ile Ser Ala Asn Glu Ala Pro Ser 405 410 415 Glu Lys
Asn His Thr Tyr Ala His Thr Glu Phe Met Phe Ser Gly Gly 420 425 430
Glu Arg Gly Gly Pro Leu Ala Thr Tyr Leu Ala Ser Ala Ala Glu Arg 435
440 445 Ser Asn Phe Asn Leu Trp Leu Asn Thr Ala Val Arg Arg Ala Val
Arg 450 455 460 Ser Gly Ser Lys Val Thr Gly Val Glu Leu Glu Cys Leu
Thr Asp Gly 465 470 475 480 Gly Phe Ser Gly Thr Val Asn Leu Asn Glu
Gly Gly Gly Val Ile Phe 485 490 495 Ser Ala Gly Ala Phe Gly Ser Ala
Lys Leu Leu Leu Arg Ser Gly Ile 500 505 510 Gly Pro Glu Asp Gln Leu
Glu Ile Val Ala Ser Ser Lys Asp Gly Glu 515 520 525 Thr Phe Thr Pro
Lys Asp Glu Trp Ile Asn Leu Pro Val Gly His Asn 530 535 540 Leu Ile
Asp His Leu Asn Thr Asp Leu Ile Ile Thr His Pro Asp Val 545 550 555
560 Val Phe Tyr Asp Phe Tyr Ala Ala Trp Asp Glu Pro Ile Thr Glu Asp
565 570 575 Lys Glu Ala Tyr Leu Asn Ser Arg Ser Gly Ile Leu Ala Gln
Ala Ala 580 585 590 Pro Asn Ile Gly Pro Met Met Trp Asp Gln Val Thr
Pro Ser Asp Gly 595 600 605 Ile Thr Arg Gln Phe Gln Trp Thr Cys Arg
Val Glu Gly Asp Ser Ser 610 615 620 Lys Thr Asn Ser Thr His Ala Met
Thr Leu Ser Gln Tyr Leu Gly Arg 625 630 635 640 Gly Val Val Ser Arg
Gly Arg Met Gly Ile Thr Ser Gly Leu Ser Thr 645 650 655 Thr Val Ala
Glu His Pro Tyr Leu His Asn Asn Gly Asp Leu Glu Ala 660 665 670 Val
Ile Gln Gly Ile Gln Asn Val Val Asp Ala Leu Ser Gln Val Ala 675 680
685 Asp Leu Glu Trp Val Leu Pro Pro Pro Asp Gly Thr Val Ala Asp Tyr
690 695 700 Val Asn Ser Leu Ile Val Ser Pro Ala Asn Arg Arg Ala Asn
His Trp 705 710 715 720 Met Gly Thr Ala Lys Leu Gly Thr Asp Asp Gly
Arg Ser Gly Gly Thr 725 730 735 Ser Val Val Asp Leu Asp Thr Lys Val
Tyr Gly Thr Asp Asn Leu Phe 740 745 750 Val Val Asp Ala Ser Val Phe
Pro Gly Met Ser Thr Gly Asn Pro Ser 755 760 765 Ala Met Ile Val Ile
Val Ala Glu Gln Ala Ala Gln Arg Ile Leu Ala 770 775 780 Leu Arg Ser
785 4623PRTMyceliophthora thermophila 4Met Lys Phe Leu Arg Lys Ser
Asp Arg Gly Ser Val Leu Gly Ser Thr 1 5 10 15 Leu Phe Ser Leu Ala
Phe Leu Phe Tyr Ser Pro Pro Thr Ala Ala Gln 20 25 30 Ser Pro Pro
Pro Asp Gly Ala Val Tyr Asp Tyr Ile Val Ile Gly Ser 35 40 45 Gly
Pro Gly Gly Gly Val Val Gly Ala Asn Leu Ala Lys Ala Gly Tyr 50 55
60 Ser Val Leu Leu Leu Glu Ala Gly Asp Asp Ser Pro Gly Ala Gly Phe
65 70 75 80 Gly Val Tyr Thr Pro Thr Val Thr Trp Asp Phe Tyr Val Lys
His Tyr 85 90 95 Pro Glu Gly Asp Pro Arg Asp Asn Gln Tyr Ser His
Leu Thr Trp Leu 100 105 110 Thr Pro Asp Gly Arg Tyr Trp Val Gly Gln
Ser Gly Ala Pro Glu Gly 115 120 125 Ser Arg Leu Leu Gly Val Tyr Tyr
Pro Arg Gly Ala Thr Leu Gly Gly 130 135 140 Ser Ser Met Ile Asn Ala
Met Val Val Trp Leu Pro Asn Asp Ser Asp 145 150 155 160 Trp Asp Tyr
His Ala Glu Val Thr Gly Asp Asp Ser Trp Arg Ala Glu 165 170 175 Asn
Met His Lys Ile Phe Gln Lys Ile Glu Lys Asn Asn Tyr Leu Pro 180 185
190 Arg Gly Thr Ala Asn His Gly Phe Asp Gly Trp Phe Gln Thr Gln Met
195 200 205 Gly Thr Met Val Gln Thr Asn Arg Thr Gly Pro Leu Gln Gly
Asn Gly 210 215 220 Val Met Thr Thr Tyr Ala Gln Asp Trp Asn Leu Thr
Ile Pro Met Ser 225 230 235 240 Asp Leu Leu Ile Arg Asp Pro Asn Glu
Ile Gly Pro Asp Arg Asp Gln 245 250 255 Thr Ser Ser Ile Tyr Gly Gln
Val Ser His Gln Phe Ala Asn Gly Asn 260 265 270 Arg Tyr Ser Ser Arg
His Tyr Val Gln Asp Ala Val Ser Ser Gly Ala 275 280 285 Asn
Leu Thr Val Ser Leu Thr Ser Leu Ala Thr Arg Ile Leu Phe Asp 290 295
300 Thr Val Thr Glu Pro Asp Ser Pro Arg Ala Thr Gly Val Glu Tyr Leu
305 310 315 320 Phe Gly Lys Ser Leu Tyr Arg Gly Asp Arg Arg Arg Ala
Asp Gly Ala 325 330 335 Ile Gly Val Asn Arg Thr Ala Val Ala Arg Arg
Glu Val Ile Val Ser 340 345 350 Gly Gly Ala Phe Asn Ser Pro Gln Leu
Leu Leu Leu Ser Gly Ile Gly 355 360 365 Asn Ala Thr Glu Leu Glu Ala
Leu Gly Ile Pro Val Ile Arg Asp Leu 370 375 380 Pro Gly Val Gly Arg
Asn Leu Met Asp Asn Gln Glu Met Pro Ile Val 385 390 395 400 Gly Thr
Gly Ser Pro Gly Gly Gly Pro Gly Ala Val Ala Gly Val Ala 405 410 415
Met Tyr Lys Thr Arg His Pro Ala His Gly Glu Arg Asp Met Phe Leu 420
425 430 Phe Gly Gly Pro Gly Phe Leu Phe Arg Gly Phe Trp Pro Asn Glu
Ala 435 440 445 Val His Leu Pro Asp Glu Pro Ala Gln Pro Val Tyr Gly
Val Ser Met 450 455 460 Val Lys Gly Ser Ser Val Asn Asn Gly Gly Trp
Val Lys Leu Arg Ser 465 470 475 480 Arg Asp Pro Thr Asp Thr Pro Glu
Ile Asn Phe Asn His Tyr Ala Val 485 490 495 Gly Ala Glu Tyr Asp Leu
Glu Ala Val Lys Asp Thr Val Ala Trp Ile 500 505 510 Arg Ser Val Tyr
Arg Arg Val Gly Ile Ala Thr Val Glu Pro Pro Cys 515 520 525 Ala Arg
Gly Pro Asp Glu Asn Gly Tyr Cys Gly Glu Glu Asp Glu Ala 530 535 540
Trp Ile His Lys Gln Thr Phe Gly His His Pro Thr Ser Thr Asn Lys 545
550 555 560 Ile Gly Ala Asp Asp Asp Pro Thr Ala Val Leu Asp Ser Lys
Phe Arg 565 570 575 Val Arg Gly Val Arg Ala Leu Arg Val Val Asp Ala
Ser Ala Phe Ala 580 585 590 Arg Ile Pro Gly Val Phe Pro Val Val Ser
Thr Phe Met Ile Ser Gln 595 600 605 Lys Ala Ser Asp Asp Ile Leu Ala
Glu Leu Glu Ala Glu Ser Arg 610 615 620 5647PRTMyceliophthora
thermophila 5Met Gly Phe Leu Ala Ala Thr Leu Val Ser Cys Ala Ala
Leu Ala Ser 1 5 10 15 Ala Ala Ser Ile Pro Arg Pro His Ala Lys Arg
Gln Val Ser Gln Leu 20 25 30 Arg Asp Asp Tyr Asp Phe Val Ile Val
Gly Gly Gly Thr Ser Gly Leu 35 40 45 Thr Val Ala Asp Arg Leu Thr
Glu Ala Phe Pro Ala Lys Asn Val Leu 50 55 60 Val Ile Glu Tyr Gly
Asp Val His Tyr Ala Pro Gly Thr Phe Asp Pro 65 70 75 80 Pro Thr Asp
Trp Ile Thr Pro Gln Pro Asp Ala Pro Pro Ser Trp Ser 85 90 95 Phe
Asn Ser Leu Pro Asn Pro Asp Met Ala Asn Thr Thr Ala Phe Val 100 105
110 Leu Ala Gly Gln Val Val Gly Gly Ser Ser Ala Val Asn Gly Met Phe
115 120 125 Phe Asp Arg Ala Ser Arg His Asp Tyr Asp Ala Trp Thr Ala
Val Gly 130 135 140 Gly Ser Gly Phe Glu Gln Ser Ser His Lys Trp Asp
Trp Glu Gly Leu 145 150 155 160 Phe Pro Phe Phe Gln Lys Ser Val Thr
Phe Thr Glu Pro Pro Ala Asp 165 170 175 Ile Val Gln Lys Tyr His Tyr
Thr Trp Asp Leu Ser Ala Tyr Gly Asn 180 185 190 Gly Ser Thr Pro Ile
Tyr Ser Ser Tyr Pro Val Phe Gln Trp Ala Asp 195 200 205 Gln Pro Leu
Leu Asn Gln Ala Trp Gln Glu Met Gly Ile Asn Pro Val 210 215 220 Thr
Glu Cys Ala Gly Gly Asp Lys Glu Gly Val Cys Trp Val Pro Ala 225 230
235 240 Ser Gln His Pro Val Thr Ala Arg Arg Ser His Ala Gly Leu Gly
His 245 250 255 Tyr Ala Asp Val Leu Pro Arg Ala Asn Tyr Asp Leu Leu
Val Gln His 260 265 270 Gln Val Val Arg Val Val Phe Pro Asn Gly Pro
Ser His Gly Pro Pro 275 280 285 Leu Val Glu Ala Arg Ser Leu Ala Asp
Asn His Leu Phe Asn Val Thr 290 295 300 Val Lys Gly Glu Val Ile Ile
Ser Ala Gly Ala Leu His Thr Pro Thr 305 310 315 320 Val Leu Gln Arg
Ser Gly Ile Gly Pro Ala Ser Phe Leu Asp Asp Ala 325 330 335 Gly Ile
Pro Val Thr Leu Asp Leu Pro Gly Val Gly Ala Asn Leu Gln 340 345 350
Asp His Cys Gly Pro Pro Val Thr Trp Asn Tyr Thr Glu Pro Tyr Thr 355
360 365 Gly Phe Phe Pro Leu Pro Ser Glu Met Val Asn Asn Ala Thr Phe
Lys 370 375 380 Ala Glu Ala Ile Thr Gly Phe Asp Glu Val Pro Ala Arg
Gly Pro Tyr 385 390 395 400 Thr Leu Ala Gly Gly Asn Asn Ala Ile Phe
Val Ser Leu Pro His Leu 405 410 415 Thr Ala Asp Tyr Gly Ala Ile Thr
Ala Asn Ile Arg Ala Met Val Ala 420 425 430 Asp Gly Thr Ala Ala Ser
Tyr Leu Ala Ala Asp Val Arg Thr Ile Pro 435 440 445 Gly Met Val Ala
Gly Tyr Glu Ala Gln Leu Leu Val Leu Ala Asp Leu 450 455 460 Leu Asp
Asn Pro Glu Ala Pro Ser Leu Glu Thr Pro Trp Ala Thr Ser 465 470 475
480 Glu Ala Pro Gln Thr Ser Ser Val Leu Ala Phe Leu Leu His Pro Leu
485 490 495 Ser Arg Gly Ser Val Arg Leu Asn Leu Ser Asp Pro Leu Ala
Gln Pro 500 505 510 Val Leu Asp Tyr Arg Ser Gly Ser Asn Pro Val Asp
Ile Asp Leu His 515 520 525 Leu Ala His Val Arg Phe Leu Arg Gly Leu
Leu Asp Thr Pro Thr Met 530 535 540 Gln Ala Arg Gly Ala Leu Glu Thr
Ala Pro Gly Ser Ala Val Ala Asp 545 550 555 560 Ser Asp Glu Ala Leu
Gly Glu Tyr Val Arg Ser His Ser Thr Leu Ser 565 570 575 Phe Met His
Pro Cys Cys Thr Ala Ala Met Leu Pro Glu Asp Arg Gly 580 585 590 Gly
Val Val Gly Pro Asp Leu Lys Val His Gly Ala Glu Gly Leu Arg 595 600
605 Val Val Asp Met Ser Val Met Pro Leu Leu Pro Gly Ala His Leu Ser
610 615 620 Ala Thr Ala Tyr Ala Val Gly Glu Lys Ala Ala Asp Ile Ile
Ile Gln 625 630 635 640 Glu Trp Met Asp Lys Glu Gln 645
6655PRTMyceliophthora thermophila 6Met Glu Leu Leu Arg Val Ser Leu
Ala Ala Val Ala Leu Ser Pro Leu 1 5 10 15 Ile Leu Phe Gly Val Ala
Ala Ala His Pro Thr Ala Arg Ser Ile Ala 20 25 30 Arg Ser Thr Ile
Leu Asp Gly Ala Asp Gly Leu Leu Pro Glu Tyr Asp 35 40 45 Tyr Ile
Ile Ile Gly Gly Gly Thr Ser Gly Leu Thr Val Ala Asp Arg 50 55 60
Leu Thr Glu Asn Arg Lys Arg Lys Phe Ser Arg Ser Pro Leu Pro Thr 65
70 75 80 Ser Pro Ala Arg Ser Ser Pro Ala Trp Cys Tyr Ser Val Leu
Val Leu 85 90 95 Glu Arg Gly Ile Phe Gln Asn Ser Ser Ser Val Thr
Thr Ile Ser Gly 100 105 110 Gly Ser Arg Gly Leu Phe Asp Pro Ser Leu
Thr Phe Asn Ile Asn Ser 115 120 125 Val Pro Gln Ala Gly Leu Asp Asn
Arg Ser Ile Ala Val Ile Gly Gly 130 135 140 Leu Ile Leu Gly Gly Ser
Ser Gly Val Asn Gly Leu Gln Val Leu Arg 145 150 155 160 Gly Gln Arg
Glu Asp Tyr Asp Arg Trp Gly Ser Tyr Phe Gly Pro Asn 165 170 175 Ser
Asp Trp Ser Trp Lys Gly Leu Leu Pro Tyr Phe Lys Lys Ala Trp 180 185
190 Asn Phe His Pro Pro Arg Pro Glu Leu Val Ser Gln Phe Asp Ile Lys
195 200 205 Tyr Asp Pro Ser Tyr Trp Gly Asn Thr Ser Asp Val His Ala
Ser Phe 210 215 220 Pro Thr Thr Phe Trp Pro Val Leu Lys Leu Glu Met
Ala Ala Phe Gly 225 230 235 240 Asp Ile Pro Gly Val Glu Tyr Pro Pro
Asp Ser Ala Ser Gly Glu Thr 245 250 255 Gly Ala Tyr Trp His Pro Ala
Ser Val Asp Pro Ala Thr Val Leu Arg 260 265 270 Ser Phe Ala Arg Pro
Ala His Trp Asp Asn Ile Glu Ala Ala Arg Pro 275 280 285 Asn Tyr His
Thr Leu Thr Gly Gln Arg Val Leu Lys Val Ala Phe Asp 290 295 300 Gly
Asn Arg Ala Thr Ser Val Val Phe Val Pro Ala Asn Ala Thr Asp 305 310
315 320 His Ser Thr Ala Arg Ser Val Lys Ala Lys Lys Glu Ile Val Leu
Ala 325 330 335 Ala Gly Ala Ile His Thr Pro Gln Ile Leu Gln Ala Ser
Gly Val Gly 340 345 350 Pro Lys Gln Val Leu Lys Glu Ala Gly Val Pro
Leu Val Val Asp Ala 355 360 365 Pro Gly Val Gly Ser Asn Phe Gln Asp
Gln Pro Tyr Val Val Ala Pro 370 375 380 Thr Phe Asn Phe Thr Lys Phe
Pro Phe His Pro Asp Phe Tyr Asp Met 385 390 395 400 Ile Leu Asn Gln
Thr Phe Ile Ala Glu Ala Gln Ala Gln Phe Glu Lys 405 410 415 Asp Arg
Thr Gly Pro His Thr Ile Ala Ser Gly Tyr Cys Gly Ser Trp 420 425 430
Leu Pro Leu Gln Ile Ile Ala Pro Asn Ser Trp Lys Asp Ile Ala Arg 435
440 445 Arg Tyr Glu Ser Gln Asp Pro Ala Ala Tyr Leu Pro Ala Gly Thr
Asp 450 455 460 Glu Thr Val Ile Glu Gly Tyr Arg Ala Gln Gln Lys Ala
Leu Ala Arg 465 470 475 480 Ser Met Arg Ser Lys Gln Ser Ala Met Tyr
Asn Phe Phe Leu Arg Gly 485 490 495 Gly Tyr Glu Glu Gly Ser Val Val
Tyr Leu His Pro Thr Ser Arg Gly 500 505 510 Thr Val Arg Ile Asn Arg
Ser Asp Pro Phe Phe Ser Pro Pro Glu Val 515 520 525 Asp Tyr Arg Ala
Leu Ser Asn Pro Thr Asp Leu Glu Val Leu Leu Glu 530 535 540 Phe Thr
Pro Phe Thr Arg Arg Tyr Phe Leu Glu Thr Arg Leu Lys Ser 545 550 555
560 Leu Asp Pro Val Glu Leu Ser Pro Gly Ala Asn Val Thr Ala Pro Ala
565 570 575 Asp Ile Glu Ala Trp Leu Arg Ser Val Met Ile Pro Ser Ser
Phe His 580 585 590 Pro Ile Gly Thr Ala Ala Met Leu Pro Arg His Leu
Gly Gly Val Val 595 600 605 Asp Glu Asn Leu Leu Val Tyr Gly Val Glu
Gly Leu Ser Val Val Asp 610 615 620 Ala Ser Val Met Pro Asp Leu Pro
Gly Ser Tyr Thr Gln Gln Thr Val 625 630 635 640 Tyr Ala Ile Ala Glu
Lys Ala Ala Asp Leu Ile Lys Ser Arg Ala 645 650 655
7578PRTMyceliophthora thermophila 7Met Gln Val Ala Ser Lys Leu Val
Ala Val Thr Gly Gly Ala Leu Ala 1 5 10 15 Leu Trp Leu His Pro Val
Ala Ala Gln Glu Gly Cys Thr Asn Ile Ser 20 25 30 Ser Thr Glu Thr
Tyr Asp Tyr Ile Val Val Gly Ser Gly Ala Gly Gly 35 40 45 Ile Pro
Val Ala Asp Arg Leu Ser Glu Ala Gly His Lys Val Leu Leu 50 55 60
Ile Glu Lys Gly Pro Pro Ser Thr Gly Arg Trp Gly Gly Ile Met Lys 65
70 75 80 Pro Glu Trp Leu Ile Gly Thr Asn Leu Thr Arg Phe Asp Val
Pro Gly 85 90 95 Leu Cys Asn Gln Ile Trp Ala Asp Pro Thr Gly Ala
Ile Cys Thr Asp 100 105 110 Val Asp Gln Met Ala Gly Cys Met Leu Gly
Gly Gly Thr Ala Val Asn 115 120 125 Ala Gly Leu Trp Trp Lys Pro His
Pro Ala Asp Trp Asp Val Asn Phe 130 135 140 Pro Glu Gly Trp His Ser
Glu Asp Met Ala Glu Ala Thr Glu Arg Val 145 150 155 160 Phe Glu Arg
Ile Pro Gly Thr Ile Thr Pro Ser Met Asp Gly Lys Arg 165 170 175 Tyr
Leu Ser Gln Gly Phe Asp Met Leu Gly Gly Ser Leu Glu Ala Ala 180 185
190 Gly Trp Glu Tyr Leu Val Pro Asn Glu His Pro Asp Arg Lys Asn Arg
195 200 205 Thr Tyr Gly His Ser Thr Phe Met Tyr Ser Gly Gly Glu Arg
Gly Gly 210 215 220 Pro Leu Ala Thr Tyr Leu Val Ser Ala Val Gln Arg
Glu Gly Phe Thr 225 230 235 240 Leu Trp Met Asn Thr Thr Val Thr Arg
Ile Ile Arg Glu Gly Gly His 245 250 255 Ala Thr Gly Val Glu Val Gln
Cys Ser Asn Ser Glu Ala Gly Gln Ala 260 265 270 Gly Ile Val Pro Leu
Thr Pro Lys Thr Gly Arg Val Ile Val Ser Ala 275 280 285 Gly Ala Phe
Gly Ser Ala Lys Leu Leu Phe Arg Ser Gly Ile Gly Pro 290 295 300 Lys
Asp Gln Leu Asn Ile Val Lys Asn Ser Thr Asp Gly Pro Ser Met 305 310
315 320 Ile Ser Glu Asp Gln Trp Ile Glu Leu Pro Val Gly Tyr Asn Leu
Asn 325 330 335 Asp His Val Gly Thr Asp Ile Glu Ile Ala His Pro Asp
Val Val Phe 340 345 350 Tyr Asp Tyr Tyr Gly Ala Trp Asp Glu Pro Ile
Val Glu Asp Thr Glu 355 360 365 Arg Tyr Val Ala Asn Arg Thr Gly Pro
Leu Ala Gln Ala Ala Pro Asn 370 375 380 Ile Gly Pro Ile Phe Trp Glu
Thr Ile Lys Gly Ser Asp Gly Val Ser 385 390 395 400 Arg His Leu Gln
Trp Gln Ala Arg Val Glu Gly Lys Leu Asn Thr Ser 405 410 415 Met Thr
Ile Thr Gln Tyr Leu Gly Thr Gly Ser Arg Ser Arg Gly Arg 420 425 430
Met Thr Ile Thr Arg Arg Leu Asn Thr Val Val Ser Thr Pro Pro Tyr 435
440 445 Leu Arg Asp Glu Tyr Asp Arg Glu Ala Val Ile Gln Gly Ile Ala
Asn 450 455 460 Leu Arg Glu Ser Leu Lys Gly Val Ala Asn Leu Thr Trp
Ile Thr Pro 465 470 475 480 Pro Ser Asn Val Thr Val Glu Asp Phe Val
Asp Ser Ile Pro Ala Thr 485 490 495 Pro Ala Arg Arg Cys Ser Asn His
Trp Ile Gly Thr Ala Lys Ile Gly 500 505 510 Leu Asp Asp Gly Arg Glu
Gly Gly Thr Ser Val Val Asp Leu Asn Thr 515 520 525 Lys Val Tyr Gly
Thr Asp Asn Ile Phe Val Val Asp Ala Ser Ile Phe 530 535 540 Pro Gly
His Ile Thr Gly Asn Pro Ser Ala Ala Ile Val Ile Ala Ala 545 550 555
560 Glu Tyr Ala Ala Ala Lys Ile Leu Ala Leu Pro Ala Pro Glu Asp Ala
565 570 575 Ala Ser 8610PRTMyceliophthora thermophila 8Met Ala Ser
Val Asp Leu Asp Gln Pro Phe Asp Tyr Ile Val Val Gly 1 5 10 15 Gly
Gly Thr Ala Gly Leu Val Val Ala Asn Arg Leu Ser Glu Asp Ser 20 25
30 Asn Val Arg Val Leu Val Val Glu Ala Gly Ala Asp Arg Asn Ala Asp
35 40 45 Pro Leu Val Leu Thr Pro Gly Leu Val Ala Gly Leu Tyr Gly
Lys Asp 50 55 60 Glu Tyr Asp Trp Asn Phe Ser Ser Pro Pro Gln Pro
Thr Leu Asn Asn 65 70
75 80 Arg Arg Ile Asn Gln Ala Arg Gly Lys Met Leu Gly Gly Thr Ser
Gly 85 90 95 Leu Asn Phe Met Met Leu Leu Tyr Pro Ser Lys Gly Asn
Ile Asp Ser 100 105 110 Trp Ala Ala Leu Gly Asn Pro Ser Trp Asn Tyr
Asp Ala Leu Ala Pro 115 120 125 Tyr Leu Arg Lys Phe Ala Thr Val His
Pro Ser Pro Gln Ser Ala Arg 130 135 140 Asp Leu Leu Gly Leu Thr Tyr
Ile Asp Glu Ser Leu Ala Ala Gly Asp 145 150 155 160 Gly Pro Ile Gln
Val Ser His Thr Asp Gly His Asn Val Thr Asn Lys 165 170 175 Ala Trp
Leu Glu Thr Phe Ala Ser Leu Gly Leu Glu Val Ser Thr Asp 180 185 190
Pro Arg Asp Gly Lys Ala Leu Gly Ala Phe Gln Asn His Ala Ser Ile 195
200 205 Asp Pro Ala Thr His Thr Arg Ser Phe Ala Gly Pro Ala Tyr Tyr
Thr 210 215 220 Pro Asp Val Ala Lys Arg Pro Asn Leu Val Val Leu Thr
Glu Thr Leu 225 230 235 240 Val Ala Arg Val Leu Phe Asp Thr Ala Gly
Gly Glu Gly Asp Ala Val 245 250 255 Ala Thr Gly Val Glu Ile Ile Thr
Lys Asp Gly Gln Lys Lys Gln Val 260 265 270 Ser Ala Cys Gly Glu Val
Ile Leu Ala Ala Gly Ala Leu Gln Ser Pro 275 280 285 Gln Ile Leu Glu
Leu Ser Gly Val Gly Gly Arg Glu Leu Leu Glu Lys 290 295 300 His Asn
Ile Pro Val Val Val Asp Asn Pro Asn Val Gly Glu His Val 305 310 315
320 Gln Asp His Pro Ile Val Cys Gln Ser Phe Glu Val Ala Asp Gly Val
325 330 335 Pro Ser Gly Asp Val Leu Arg Asp Pro Asn Val Leu Gln Ala
Val Val 340 345 350 Gly Met Tyr Gln Ser Gly Gly Gly Ala Gly Pro Leu
Gly Gln Ser Val 355 360 365 Ile Ser Val Ala Tyr Thr Pro Leu Val Asp
Gly Ser Gly Val Val Ser 370 375 380 Ala Glu Ala Lys Ala Glu Leu Leu
Ala Arg His Glu Ser Ser Phe Ser 385 390 395 400 Thr Ala Glu Gly Lys
Val Leu Arg Asp Leu Val Glu Ser Pro Ser Glu 405 410 415 Ala Thr Phe
Glu Phe Leu Leu Phe Pro Ser Gln Val Asp Ile Pro Glu 420 425 430 Asn
Pro Thr Ser Met Ala Gln Tyr Ile Thr Pro Val Leu Pro Glu Asn 435 440
445 Tyr Ile Ser Val Met Thr Phe Ile His Gln Pro Phe Ser Arg Gly Lys
450 455 460 Val His Ile Thr Ser Pro Asp Ile Arg Ala Ala Pro Leu Trp
Asp Pro 465 470 475 480 Arg Tyr Asn Ser Asp Pro Leu Asp Leu Glu Leu
Leu Ala Arg Gly Val 485 490 495 Gln Phe Val Glu Arg Ile Val Asp Ser
Ala Thr Pro Phe Gly Arg Val 500 505 510 Leu Lys Gln Gly Gly Lys Arg
Gln Pro Pro Leu Arg Ala Asp Asp Leu 515 520 525 Glu Thr Ala Arg Glu
Ile Val Arg Gln Arg Gln Ile Ser Val Phe His 530 535 540 Val Ser Gly
Ser Cys Thr Met Arg Pro Arg Asp Gln Gly Gly Val Val 545 550 555 560
Asp Glu Arg Leu Arg Val Tyr Gly Thr Arg Gly Leu Arg Val Val Asp 565
570 575 Ala Ser Val Phe Pro Ile Glu Pro Val Gly Asn Ile Gln Ser Val
Val 580 585 590 Tyr Ala Val Ala Glu Arg Ala Ala Asp Leu Ile Lys Glu
Asp Arg Ala 595 600 605 Lys Ala 610 924DNAArtificial
SequenceSynthetic DNA primer 9cacgcggggt tctttctcca tctc
241047DNAArtificial SequenceSynthetic DNA primer 10tgaggaaaac
gccgagactg agctcgactc tgccggccta cctacga 471148DNAArtificial
SequenceSynthetic DNA primer 11atcagttggg tgcacgagtg ggttttgatg
gggagttgag tttgtgaa 481224DNAArtificial SequenceSynthetic DNA
primer 12ggatggatga ggttgttttt gagc 241324DNAArtificial
SequenceSynthetic DNA primer 13aacccactcg tgcacccaac tgat
241424DNAArtificial SequenceSynthetic DNA primer 14gaccacgatg
ccggctacga tacc 241524DNAArtificial SequenceSynthetic DNA primer
15acatggcccc actcgcttct taca 241624DNAArtificial SequenceSynthetic
DNA primer 16aagcgtgccg attttcctga tttc 241721DNAArtificial
SequenceSynthetic DNA primer 17gcatttctgg ggcggttagc a
211821DNAArtificial SequenceSynthetic DNA primer 18tcatcgacgc
ctccatcttc c 211924DNAArtificial SequenceSynthetic DNA primer
19tttcggttgt cgtgtttcca ttat 242020DNAArtificial SequenceSynthetic
DNA primer 20ggagatcctg gaggatttcc 202122DNAArtificial
SequenceSynthetic DNA primer 21caggcggtgt gcgttatcaa aa 22
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