U.S. patent application number 13/945557 was filed with the patent office on 2014-01-09 for methods for producing secreted polypeptides.
This patent application is currently assigned to NOVOZYMES, INC.. The applicant listed for this patent is Howard Brody, Ana Fidantsef, Suchindra Maiyuran. Invention is credited to Howard Brody, Ana Fidantsef, Suchindra Maiyuran.
Application Number | 20140011262 13/945557 |
Document ID | / |
Family ID | 33551405 |
Filed Date | 2014-01-09 |
United States Patent
Application |
20140011262 |
Kind Code |
A1 |
Maiyuran; Suchindra ; et
al. |
January 9, 2014 |
Methods For Producing Secreted Polypeptides
Abstract
The present invention relates to methods for producing a
polypeptide, comprising: (a) cultivating a fungal host cell in a
medium conducive for the production of the polypeptide, wherein the
fungal host cell comprises a nucleic acid construct comprising a
first nucleotide sequence encoding a signal peptide operably linked
to a second nucleotide sequence encoding the polypeptide, wherein
the first nucleotide sequence is foreign to the second nucleotide
sequence and the 3' end of the first nucleotide sequence is
immediately upstream of the initiator codon of the second
nucleotide sequence. The present invention also relates to the
isolated signal peptide sequences and to constructs, vectors, and
fungal host cells comprising the signal peptide sequences operably
linked to nucleotide sequences encoding polypeptides.
Inventors: |
Maiyuran; Suchindra; (Gold
River, CA) ; Fidantsef; Ana; (Davis, CA) ;
Brody; Howard; (Davis, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Maiyuran; Suchindra
Fidantsef; Ana
Brody; Howard |
Gold River
Davis
Davis |
CA
CA
CA |
US
US
US |
|
|
Assignee: |
NOVOZYMES, INC.
Davis
CA
|
Family ID: |
33551405 |
Appl. No.: |
13/945557 |
Filed: |
July 18, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13350384 |
Jan 13, 2012 |
8497115 |
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13945557 |
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12135611 |
Jun 9, 2008 |
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13350384 |
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10837318 |
Apr 30, 2004 |
7393664 |
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12135611 |
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60467766 |
May 2, 2003 |
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Current U.S.
Class: |
435/254.21 ;
435/254.11; 435/254.2; 435/254.22; 435/254.23; 435/254.3;
435/254.4; 435/254.5; 435/254.6; 435/254.7; 435/254.8; 435/320.1;
536/23.74 |
Current CPC
Class: |
C12N 15/81 20130101;
C12P 21/02 20130101; C07K 2319/02 20130101; C12N 15/80
20130101 |
Class at
Publication: |
435/254.21 ;
536/23.74; 435/320.1; 435/254.11; 435/254.2; 435/254.22;
435/254.23; 435/254.3; 435/254.7; 435/254.8; 435/254.4; 435/254.5;
435/254.6 |
International
Class: |
C12N 15/81 20060101
C12N015/81; C12N 15/80 20060101 C12N015/80 |
Goverment Interests
STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED
RESEARCH AND DEVELOPMENT
[0002] This invention was made with Government support under NREL
Subcontract No. ZCO-30017-02, Prime Contract DE-AC36-98G010337
awarded by the Department of Energy. The government has certain
rights in this invention.
Claims
1. A nucleic acid construct comprising a first polynucleotide
comprising a nucleotide sequence encoding a signal peptide operably
linked to a second polynucleotide comprising a nucleotide sequence
encoding a polypeptide, wherein the first polynucleotide encoding
the signal peptide is foreign to the second polynucleotide encoding
the polypeptide, and the 3' end of the first polynucleotide
encoding the signal peptide is immediately upstream of the
initiator codon of the second polynucleotide encoding the
polypeptide; wherein the nucleotide sequence encoding the signal
peptide is: (a) a nucleotide sequence encoding a signal peptide
comprising an amino acid sequence having at least 90% sequence
identity to SEQ ID NO: 37; or (b) a nucleotide sequence encoding a
signal peptide comprising a sequence having at least 90% sequence
identity to SEQ ID NO: 36.
2. The nucleic acid construct of claim 1, wherein the nucleotide
sequence encoding a signal peptide comprises an amino acid sequence
having at least 95% sequence identity to SEQ ID NO: 37.
3. The nucleic acid construct of claim 2, wherein the nucleotide
sequence encoding a signal peptide comprises an amino acid sequence
having at least 97% sequence identity to SEQ ID NO: 37.
4. The nucleic acid construct of claim 1, wherein the nucleotide
sequence encoding a signal peptide comprises a sequence having at
least 95% sequence identity to SEQ ID NO: 36.
5. The nucleic acid construct of claim 4, wherein the nucleotide
sequence encoding a signal peptide comprises a sequence having at
least 97% sequence identity to SEQ ID NO: 36.
6. A recombinant expression vector comprising the nucleic acid
construct of claim 1.
7. A recombinant host cell comprising the nucleic acid construct of
claim 1.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a divisional of U.S. application Ser.
No. 13/350,384, filed Jan. 13, 2012, which is a divisional of U.S.
application Ser. No. 12/135,611, filed Jun. 9, 2008, now abandoned,
which is a divisional of U.S. application Ser. No. 10/837,318,
filed Apr. 30, 2004, now U.S. Pat. No. 7,393,664, which claims the
benefit of U.S. Provisional Application No. 60/467766, filed May 2,
2003, which applications are incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] The present invention relates to methods for producing
secreted polypeptides. The present invention also relates to
isolated nucleotide sequences encoding signal peptides and nucleic
acid constructs, vectors, and host cells comprising the signal
peptide sequences operably linked to nucleotide sequences encoding
polypeptides.
[0005] 2. Description of the Related Art
[0006] The recombinant production of a heterologous protein in a
fungal host cell, particularly a filamentous fungal cell such as
Aspergillus or a yeast cell such Saccharomyces, may provide for a
more desirable vehicle for producing the protein in commercially
relevant quantities.
[0007] Recombinant production of a heterologous protein is
generally accomplished by constructing an expression cassette in
which the DNA coding for the protein is placed under the expression
control of a promoter, excised from a regulated gene, suitable for
the host cell. The expression cassette is introduced into the host
cell, usually by plasmid-mediated transformation. Production of the
heterologous protein is then achieved by culturing the transformed
host cell under inducing conditions necessary for the proper
functioning of the promoter contained on the expression
cassette.
[0008] Improvement of the recombinant production of proteins
generally requires the availability of new regulatory sequences
which are suitable for controlling the expression of the proteins
in a host cell.
[0009] U.S. Pat. No. 6,015,703 discloses genetic constructs
comprising a promoter, a xylanase secretion signal, and a mature
beta-glucosidase coding region. The disclosed constructs, when
expressed in recombinant microbes, dramatically increase the amount
of beta-glucosidase produced relative to untransformed
microbes.
[0010] WO 91/17243 discloses an endoglucanase V and the gene
thereof from Humicola insolens DSM 1800.
[0011] It is an object of the present invention to provide improved
methods for producing a polypeptide in a fungal host cell using
signal peptide sequences.
SUMMARY OF THE INVENTION
[0012] The present invention relates to methods for producing a
secreted polypeptide, comprising:
[0013] (a) cultivating a fungal host cell in a medium conducive for
the production of the polypeptide, wherein the fungal host cell
comprises a nucleic acid construct comprising a first nucleotide
sequence encoding a signal peptide operably linked to a second
nucleotide sequence encoding the polypeptide, wherein the first
nucleotide sequence is foreign to the second nucleotide sequence,
the 3' end of the first nucleotide sequence is immediately upstream
of the initiator codon of the second nucleotide sequence, and the
first nucleotide sequence is selected from the group consisting of:
[0014] (i) a nucleotide sequence encoding a signal peptide having
an amino acid sequence which has at least 70% identity with SEQ ID
NO: 37; [0015] (ii) a nucleotide sequence having at least 70%
homology with SEQ ID NO: 36; and [0016] (iii) a nucleotide sequence
which hybridizes under stringency conditions with the nucleotides
of SEQ ID NO: 36, or its complementary strand, wherein the
stringency conditions are defined as prehybridization,
hybridization, and washing post-hybridization at 5.degree. C. to
10.degree. C. below the calculated T.sub.m in 0.9 M NaCl, 0.09 M
Tris-HCl pH 7.6, 6 mM EDTA, 0.5% NP-40, 1.times. Denhardt's
solution, 1 mM sodium pyrophosphate, 1 mM sodium monobasic
phosphate, 0.1 mM ATP, and 0.2 mg of yeast RNA per ml, and washing
once in 6.times.SCC plus 0.1% SDS for 15 minutes and twice each for
15 minutes using 6.times.SSC at 5.degree. C. to 10.degree. C. below
the calculated T.sub.m; and
[0017] (b) isolating the secreted polypeptide from the cultivation
medium.
[0018] The present invention also relates to isolated signal
peptide sequences and to constructs, vectors, and fungal host cells
comprising the signal peptide sequences operably linked to
nucleotide sequences encoding polypeptides.
BRIEF DESCRIPTION OF THE FIGURES
[0019] FIG. 1 shows a restriction map of pAILo1.
[0020] FIG. 2 shows a restriction map of pMJ04.
[0021] FIG. 3 shows a restriction map of pCaHj527.
[0022] FIG. 4 shows a restriction map of pMT2188.
[0023] FIG. 5 shows a restriction map of pCaHj568.
[0024] FIG. 6 shows a restriction map of pMJ05.
[0025] FIG. 7 shows a restriction map of pSMai130.
[0026] FIG. 8 shows the DNA sequence (SEQ ID NO: 34) and deduced
amino acid sequence (SEQ ID NO: 35) of the secretion signal
sequence of an Aspergillus oryzae beta-glucosidase.
[0027] FIG. 9 shows the DNA sequence (SEQ ID NO: 36) and deduced
amino acid sequence (SEQ ID NO: 37) of the secretion signal
sequence of a Humicola insolens endoglucanase V.
[0028] FIG. 10 shows a restriction map of pSMai135.
[0029] FIG. 11 shows a restriction map of pSATe101.
[0030] FIG. 12 shows a restriction map of pSATe111.
[0031] FIG. 13 shows a restriction map of pALFd1.
[0032] FIG. 14 shows a restriction map of pAILo2.
[0033] FIG. 15 shows a restriction map of pEJG97.
[0034] FIGS. 16A and 16B show the genomic DNA sequence and the
deduced amino acid sequence of an Aspergillus fumigatus
beta-glucosidase (SEQ ID NOS: 46 and 47, respectively). The
predicted signal peptide is underlined and predicted introns are
italicized.
[0035] FIG. 17 shows a restriction map of
pCR4Blunt-TOPOAfcDNA5'.
[0036] FIG. 18 shows a restriction map of
pCR4Blunt-TOPOAfcDNA3'.
[0037] FIG. 19 shows a restriction map of pCR4Blunt-TOPOAfcDNA.
[0038] FIG. 20 shows a restriction map of pALFd7.
[0039] FIG. 21 shows a restriction map of pALFd6.
DETAILED DESCRIPTION OF THE INVENTION
[0040] The present invention relates to methods for producing a
polypeptide, comprising: (a) cultivating a fungal host cell in a
medium conducive for the production of the polypeptide, wherein the
fungal host cell comprises a nucleic acid construct comprising a
first nucleotide sequence encoding a signal peptide operably linked
to a second nucleotide sequence encoding the polypeptide, wherein
the first nucleotide sequence is foreign to the second nucleotide
sequence and the 3' end of the first nucleotide sequence is
immediately upstream of the initiator codon of the second
nucleotide sequence. The first nucleotide sequence is selected from
the group consisting of: (i) a nucleotide sequence encoding a
signal peptide having an amino acid sequence which has at least 70%
identity with SEQ ID NO: 37; (ii) a nucleotide sequence having at
least 70% homology with SEQ ID NO: 36; and (iii) a nucleotide
sequence which hybridizes under stringency conditions with the
nucleotides of SEQ ID NO: 36, or its complementary strand, wherein
the stringency conditions are defined as prehybridization,
hybridization, and washing post-hybridization at 5.degree. C. to
10.degree. C. below the calculated T.sub.m in 0.9 M NaCl, 0.09 M
Tris-HCl pH 7.6, 6 mM EDTA, 0.5% NP-40, 1.times. Denhardt's
solution, 1 mM sodium pyrophosphate, 1 mM sodium monobasic
phosphate, 0.1 mM ATP, and 0.2 mg of yeast RNA per ml, and washing
once in 6.times.SCC plus 0.1% SDS for 15 minutes and twice each for
15 minutes using 6.times.SSC at 5.degree. C. to 10.degree. C. below
the calculated T.sub.m; and (b) isolating the secreted polypeptide
from the cultivation medium.
[0041] In the production methods of the present invention, the
fungal host cells are cultivated in a nutrient medium suitable for
production of the polypeptide using methods known in the art. For
example, the cell may be cultivated by shake flask cultivation, or
small-scale or large-scale fermentation (including continuous,
batch, fed-batch, or solid state fermentations) in laboratory or
industrial fermentors performed in a suitable medium and under
conditions allowing the polypeptide to be expressed and/or
isolated. The cultivation takes place in a suitable nutrient medium
comprising carbon and nitrogen sources and inorganic salts, using
procedures known in the art. Suitable media are available from
commercial suppliers or may be prepared according to published
compositions (e.g., in catalogues of the American Type Culture
Collection).
[0042] The polypeptides may be detected using methods known in the
art that are specific for the polypeptides. These detection methods
may include use of specific antibodies, formation of an enzyme
product, or disappearance of an enzyme substrate.
[0043] In the methods of the present invention, the fungal cell
preferably produces at least about 25% more, more preferably at
least about 50% more, more preferably at least about 75% more, more
preferably at least about 100% more, even more preferably at least
about 200% more, most preferably at least about 300% more, and even
most preferably at least about 400% more polypeptide relative to a
fungal cell containing a native signal peptide sequence operably
linked to a nucleotide sequence encoding the polypeptide when
cultured under identical production conditions.
[0044] The resulting secreted polypeptide can be recovered directly
from the medium by methods known in the art. For example, the
polypeptide may be recovered from the nutrient medium by
conventional procedures including, but not limited to,
centrifugation, filtration, extraction, spray-drying, evaporation,
or precipitation.
[0045] The polypeptides may be purified by a variety of procedures
known in the art including, but not limited to, chromatography
(e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and
size exclusion), electrophoretic procedures (e.g., preparative
isoelectric focusing), differential solubility (e.g., ammonium
sulfate precipitation), SDS-PAGE, or extraction (see, e.g., Protein
Purification, J.-C. Janson and Lars Ryden, editors, VCH Publishers,
New York, 1989).
Signal Peptide Sequences
[0046] The term "signal peptide sequence" is defined herein as a
peptide coding region that codes for an amino acid sequence linked
to the amino terminus of a polypeptide and directs the encoded
polypeptide into the cell's secretory pathway.
[0047] The term "operably linked" is defined herein as a
configuration in which a control sequence, e.g., a signal peptide
sequence, is appropriately placed at a position relative to a
coding sequence such that the control sequence directs the
production of a polypeptide encoded by the coding sequence.
[0048] The term "coding sequence" is defined herein as a nucleotide
sequence that is transcribed into mRNA which is translated into a
polypeptide when placed under the control of the appropriate
control sequences. The boundaries of the coding sequence are
generally determined by the start codon located at the beginning of
the open reading frame of the 5' end of the mRNA and a stop codon
located at the 3' end of the open reading frame of the mRNA. A
coding sequence can include, but is not limited to, genomic DNA,
cDNA, semisynthetic, synthetic, and recombinant nucleotide
sequences.
[0049] The 5' end of the polypeptide coding sequence may contain a
native signal peptide coding region naturally linked in translation
reading frame with the segment of the coding region which encodes
the polypeptide, wherein the signal peptide coding region of the
present invention may simply replace the natural signal peptide
coding region in order to enhance secretion of the polypeptide.
Alternatively, the 5' end of the polypeptide coding sequence may
lack a native signal peptide coding region.
[0050] In the methods of the present invention, the signal peptide
sequence is foreign to the nucleotide sequence encoding a
polypeptide of interest, but the signal peptide sequence or
nucleotide sequence may be native to the fungal host cell.
[0051] In a first aspect, the isolated nucleotide sequences
encoding a signal peptide have a degree of identity to SEQ ID NO:
37 of at least about 70%, preferably at least about 75%, more
preferably at least about 80%, more preferably at least about 85%,
even more preferably at least about 90%, most preferably at least
about 95%, and even most preferably at least about 97%, which have
the ability to direct a polypeptide into a cell's secretory pathway
(hereinafter "homologous signal peptides"). In a preferred aspect,
the homologous signal peptides have an amino acid sequence which
differs by five amino acids, preferably by four amino acids, more
preferably by three amino acids, even more preferably by two amino
acids, and most preferably by one amino acid from SEQ ID NO: 37.
For purposes of the present invention, the degree of identity
between two amino acid sequences is determined by the Clustal
method (Higgins, 1989, CABIOS 5: 151-153) using the LASERGENE.TM.
MEGALIGN.TM. software (DNASTAR, Inc., Madison, Wis.) with an
identity table and the following multiple alignment parameters: Gap
penalty of 10 and gap length penalty of 10. Pairwise alignment
parameters are Ktuple=1, gap penalty=3, windows=5, and
diagonals=5.
[0052] Preferably, the nucleotide sequences encode signal peptides
that comprise the amino acid sequence of SEQ ID NO: 37, or allelic
variants thereof; or fragments thereof that have the ability to
direct the polypeptide into a cell's secretory pathway. In a more
preferred aspect, a nucleotide sequence of the present invention
encodes a signal peptide that comprises the amino acid sequence of
SEQ ID NO: 37. In another preferred aspect, the nucleotide sequence
encodes a signal peptide that consists of the amino acid sequence
of SEQ ID NO: 37, or a fragment thereof, wherein the signal peptide
fragment has the ability to direct a polypeptide into a cell's
secretory pathway. In another more preferred aspect, the nucleotide
sequence of the present invention encodes a signal peptide that
consists of the amino acid sequence of SEQ ID NO: 37.
[0053] The present invention also encompasses nucleotide sequences
which encode a signal peptide having the amino acid sequence of SEQ
ID NO: 37, which differ from SEQ ID NO: 36 by virtue of the
degeneracy of the genetic code. The present invention also relates
to subsequences of SEQ ID NO: 36 which encode fragments of SEQ ID
NO: 37 which have the ability to direct a polypeptide into a cell's
secretory pathway.
[0054] A subsequence of SEQ ID NO: 36 is a nucleic acid sequence
encompassed by SEQ ID NO: 36 except that one or more nucleotides
from the 5' and/or 3' end have been deleted. Preferably, a
subsequence contains at least 45 nucleotides, more preferably at
least 51 nucleotides, and most preferably at least 57 nucleotides.
A fragment of SEQ ID NO: 37 is a polypeptide having one or more
amino acids deleted from the amino and/or carboxy terminus of this
amino acid sequence. Preferably, a fragment contains at least 15
amino acid residues, more preferably at least 17 amino acid
residues, and most preferably at least 19 amino acid residues.
[0055] An allelic variant denotes any of two or more alternative
forms of a gene occupying the same chomosomal locus. Allelic
variation arises naturally through mutation, and may result in
polymorphism within populations. Gene mutations can be silent (no
change in the encoded signal peptide) or may encode signal peptides
having altered amino acid sequences. The allelic variant of a
signal peptide is a peptide encoded by an allelic variant of a
gene.
[0056] In a preferred aspect, the first nucleotide sequence is the
signal peptide coding sequence of the endoglucanase V gene
contained in Humicola insolens DSM 1800.
[0057] In a second aspect, the isolated nucleic acid sequences
encoding a signal peptide have a degree of homology to SEQ ID NO:
36 of at least about 70%, preferably at least about 75%, more
preferably at least about 80%, more preferably at least about 85%,
even more preferably at least about 90% homology, most preferably
at least about 95% homology, and even most preferably at least
about 97% homology, which encode a signal peptide; or allelic
variants and subsequences of SEQ ID NO: 36 which encode signal
peptide fragments which have the ability to direct a polypeptide
into a cell's secretory pathway. For purposes of the present
invention, the degree of homology between two nucleic acid
sequences is determined by the Wilbur-Lipman method (Wilbur and
Lipman, 1983, Proceedings of the National Academy of Science USA
80: 726-730) using the LASERGENE.TM. MEGALIGN.TM. software
(DNASTAR, Inc., Madison, Wis.) with an identity table and the
following multiple alignment parameters: Gap penalty of 10 and gap
length penalty of 10. Pairwise alignment parameters are Ktuple=3,
gap penalty=3, and windows=20.
[0058] In a third aspect, the isolated nucleotide sequences encode
signal peptides, wherein the nucleotide sequences hybridize under
stringency conditions with the nucleotides of SEQ ID NO: 36, or its
complementary strand (J. Sambrook, E. F. Fritsch, and T. Maniatus,
1989, Molecular Cloning, A Laboratory Manual, 2d edition, Cold
Spring Harbor, N.Y.).
[0059] The nucleotide sequence of SEQ ID NO: 36 or a subsequence
thereof, as well as the amino acid sequence of SEQ ID NO: 37 or a
fragment thereof, may be used to design a nucleic acid probe to
identify and clone DNA encoding signal peptides from strains of
different genera or species according to methods well known in the
art. In particular, such probes can be used for hybridization with
the genomic or cDNA of the genus or species of interest, following
standard Southern blotting procedures, in order to identify and
isolate the corresponding gene therein. Such probes can be
considerably shorter than the entire sequence, but should be at
least 15, preferably at least 25, and more preferably at least 35
nucleotides in length. Both DNA and RNA probes can be used. The
probes are typically labeled for detecting the corresponding gene
(for example, with .sup.32P, .sup.3H, .sup.35S, biotin, or avidin).
Such probes are encompassed by the present invention.
[0060] Thus, a genomic DNA or cDNA library prepared from such other
organisms may be screened for DNA which hybridizes with the probes
described above and which encodes a signal peptide. Genomic or
other DNA from such other organisms may be separated by agarose or
polyacrylamide gel electrophoresis, or other separation techniques.
DNA from the libraries or the separated DNA may be transferred to
and immobilized on nitrocellulose or other suitable carrier
material. In order to identify a clone or DNA which is homologous
with SEQ ID NO: 36 or a subsequence thereof, the carrier material
is used in a Southern blot. For purposes of the present invention,
hybridization indicates that the nucleic acid sequence hybridizes
to a labeled nucleic acid probe corresponding to the nucleic acid
sequence shown in SEQ ID NO: 36, its complementary strand, or a
subsequence thereof, under stringency conditions defined herein.
Molecules to which the nucleic acid probe hybridizes under these
conditions can be detected using X-ray film.
[0061] In a preferred aspect, the nucleic acid probe is a
nucleotide sequence which encodes the signal peptide of SEQ ID NO:
37, or a subsequence thereof. In another preferred aspect, the
nucleic acid probe is SEQ ID NO: 36. In another preferred aspect,
the nucleic acid probe is the signal peptide coding sequence of the
endoglucanase V gene contained in Humicola insolens DSM 1800.
[0062] For short probes which are about 15 nucleotides to about 60
nucleotides in length, stringency conditions are defined as
prehybridization, hybridization, and washing post-hybridization at
5.degree. C. to 10.degree. C. below the calculated T.sub.m using
the calculation according to Bolton and McCarthy (1962, Proceedings
of the National Academy of Sciences USA 48: 1390) in 0.9 M NaCl,
0.09 M Tris-HCl pH 7.6, 6 mM EDTA, 0.5% NP-40, 1.times. Denhardt's
solution, 1 mM sodium pyrophosphate, 1 mM sodium monobasic
phosphate, 0.1 mM ATP, and 0.2 mg of yeast RNA per ml following
standard Southern blotting procedures.
[0063] For short probes which are about 15 nucleotides to about 60
nucleotides in length, the carrier material is washed once in
6.times. SCC plus 0.1% SDS for 15 minutes and twice each for 15
minutes using 6.times. SSC at 5.degree. C. to 10.degree. C. below
the calculated T.sub.m.
[0064] In a fourth aspect, the isolated nucleic acid sequences
encode variants of the signal peptide having an amino acid sequence
of SEQ ID NO: 37 comprising a substitution, deletion, and/or
insertion of one or more amino acids.
[0065] The amino acid sequences of the variant signal peptides may
differ from the amino acid sequence of SEQ ID NO: 37 by an
insertion or deletion of one or more amino acid residues and/or the
substitution of one or more amino acid residues by different amino
acid residues. Preferably, amino acid changes are of a minor
nature, such as conservative amino acid substitutions that do not
significantly affect the activity of the signal peptide; or small
deletions, typically of one to about 5 amino acids.
[0066] Examples of conservative substitutions are within the group
of basic amino acids (arginine, lysine and histidine), acidic amino
acids (glutamic acid and aspartic acid), polar amino acids
(glutamine and asparagine), hydrophobic amino acids (leucine,
isoleucine and valine), aromatic amino acids (phenylalanine,
tryptophan and tyrosine), and small amino acids (glycine, alanine,
serine, threonine and methionine). Amino acid substitutions that do
not generally alter the specific activity are known in the art and
are described, for example, by H. Neurath and R. L. Hill, 1979, In,
The Proteins, Academic Press, New York. The most commonly occurring
exchanges are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr,
Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn,
Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly as well as these in
reverse.
[0067] The present invention also relates to the isolated signal
peptide sequences disclosed supra.
Polypeptide Encoding Nucleotide Sequences
[0068] The polypeptide encoded by the second nucleotide sequence
may be native or heterologous to the fungal host cell of
interest.
[0069] The term "polypeptide" is not meant herein to refer to a
specific length of the encoded product and, therefore, encompasses
peptides, oligopeptides, and proteins. The term "heterologous
polypeptide" is defined herein as a polypeptide which is not native
to the fungal cell, a native polypeptide in which modifications
have been made to alter the native sequence, or a native
polypeptide whose expression is quantitatively altered as a result
of a manipulation of the gene encoding the polypeptide by
recombinant DNA techniques. The fungal cell may contain one or more
copies of the nucleotide sequence encoding the polypeptide.
[0070] Preferably, the polypeptide is a hormone or variant thereof,
enzyme, receptor or portion thereof, antibody or portion thereof,
or reporter. In a preferred aspect, the polypeptide is an
oxidoreductase, transferase, hydrolase, lyase, isomerase, or
ligase. In a more preferred aspect, the polypeptide is an
aminopeptidase, amylase, carbohydrase, carboxypeptidase, catalase,
cellulase, cellobiohydrolase, chitinase, cutinase, cyclodextrin
glycosyltransferase, deoxyribonuclease, endoglucanase, esterase,
alpha-galactosidase, beta-galactosidase, glucoamylase,
alpha-glucosidase, beta-glucosidase, invertase, laccase, lipase,
mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase,
phospholipase, phytase, polyphenoloxidase, proteolytic enzyme,
ribonuclease, transglutaminase, xylanase, or beta-xylosidase. In a
most preferred aspect, the polypeptide is an endoglucanase,
cellobiohydrolase, and/or beta-glucosidase useful in converting
cellulose to glucose including, but not limited to, endoglucanase
I, endoglucanase II, endoglucanse III, endoglucanase IV,
endoglucanase V, cellobiohydrolase I, cellobiohydrolase II, and
beta-glucosidase. Endoglucanase and cellobiohydrolase enzymes are
collectively referred to as "cellulases."
[0071] The nucleotide sequence encoding a polypeptide of interest
may be obtained from any prokaryotic, eukaryotic, or other source.
For purposes of the present invention, the term "obtained from" as
used herein in connection with a given source shall mean that the
polypeptide is produced by the source or by a cell in which a gene
from the source has been inserted.
[0072] The techniques used to isolate or clone a nucleotide
sequence encoding a polypeptide of interest are known in the art
and include isolation from genomic DNA, preparation from cDNA, or a
combination thereof. The cloning of the nucleotide sequence from
such genomic DNA can be effected, e.g., by using the well known
polymerase chain reaction (PCR). See, for example, Innis et al.,
1990, PCR Protocols: A Guide to Methods and Application, Academic
Press, New York. The cloning procedures may involve excision and
isolation of a desired nucleotide fragment comprising the
nucleotide sequence encoding the polypeptide, insertion of the
fragment into a vector molecule, and incorporation of the
recombinant vector into the mutant fungal cell where multiple
copies or clones of the nucleotide sequence will be replicated. The
nucleotide sequence may be of genomic, cDNA, RNA, semisynthetic,
synthetic origin, or any combinations thereof.
[0073] In the methods of the present invention, the polypeptide may
also include a fused or hybrid polypeptide in which another
polypeptide is fused at the N-terminus or the C-terminus of the
polypeptide or fragment thereof. A fused polypeptide is produced by
fusing a nucleotide sequence (or a portion thereof) encoding one
polypeptide to a nucleotide sequence (or a portion thereof)
encoding another polypeptide. Techniques for producing fusion
polypeptides are known in the art, and include, ligating the coding
sequences encoding the polypeptides so that they are in frame and
expression of the fused polypeptide is under control of the same
promoter(s) and terminator. The hybrid polypeptide may comprise a
combination of partial or complete polypeptide sequences obtained
from at least two different polypeptides wherein one or more may be
heterologous to the mutant fungal cell.
Nucleic Acid Constructs
[0074] The present invention also relates to nucleic acid
constructs comprising a nucleotide sequence encoding a polypeptide
operably linked to a signal peptide sequence of the present
invention and one or more control sequences which direct the
expression of the coding sequence in a suitable host cell under
conditions compatible with the control sequences. Expression will
be understood to include any step involved in the production of the
polypeptide including, but not limited to, transcription,
post-transcriptional modification, translation, post-translational
modification, and secretion.
[0075] "Nucleic acid construct" is defined herein as a nucleotide
molecule, either single- or double-stranded, which is isolated from
a naturally occurring gene or which has been modified to contain
segments of nucleic acids combined and juxtaposed in a manner that
would not otherwise exist in nature. The term nucleic acid
construct is synonymous with the term expression cassette when the
nucleic acid construct contains a coding sequence and all the
control sequences required for expression of the coding
sequence.
[0076] An isolated nucleotide sequence encoding a polypeptide may
be further manipulated in a variety of ways to provide for
expression of the polypeptide. Manipulation of the nucleotide
sequence prior to its insertion into a vector may be desirable or
necessary depending on the expression vector. The techniques for
modifying nucleotide sequences utilizing recombinant DNA methods
are well known in the art.
[0077] In the methods of the present invention, the nucleotide
sequence may comprise one or more native control sequences or one
or more of the native control sequences may be replaced with one or
more control sequences foreign to the nucleotide sequence for
improving expression of the coding sequence in a host cell.
[0078] The term "control sequences" is defined herein to include
all components which are necessary or advantageous for the
expression of a polypeptide of interest. Each control sequence may
be native or foreign to the nucleotide sequence encoding the
polypeptide. Such control sequences include, but are not limited
to, a leader, polyadenylation sequence, propeptide sequence, signal
peptide sequence of the present invention, and transcription
terminator. At a minimum, the control sequences include a signal
peptide sequence of the present invention, and transcriptional and
translational stop signals. The control sequences may be provided
with linkers for the purpose of introducing specific restriction
sites facilitating ligation of the control sequences with the
coding region of the nucleotide sequence encoding a
polypeptide.
[0079] The control sequence may be an appropriate promoter
sequence, which is recognized by a host cell for expression of the
nucleotide sequence. The promoter sequence contains transcriptional
control sequences which mediate the expression of the polypeptide.
The promoter may be any sequence which shows transcriptional
activity in the host cell of choice including mutant, truncated,
and hybrid promoters, and may be obtained from genes encoding
extracellular or intracellular polypeptides either homologous or
heterologous to the host cell.
[0080] Examples of suitable promoters for directing the
transcription of the nucleic acid constructs of the present
invention in a filamentous fungal host cell are promoters obtained
from the genes for Aspergillus oryzae TAKA amylase, Rhizomucor
miehei aspartic proteinase, Aspergillus niger neutral
alpha-amylase, Aspergillus niger acid stable alpha-amylase,
Aspergillus niger or Aspergillus awamori glucoamylase (glaA),
Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease,
Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans
acetamidase, Fusarium venenatum amyloglucosidase, Fusarium
oxysporum trypsin-like protease (WO 96/00787), Trichoderma reesei
cellobiohydrolase I, Trichoderma reesei cellobiohydrolase II,
Trichoderma reesei endoglucanase I, Trichoderma reesei
endoglucanase II, Trichoderma reesei endoglucanase III, Trichoderma
reesei endoglucanase IV, Trichoderma reesei endoglucanase V,
Trichoderma reesei xylanase I, Trichoderma reesei xylanase II,
Trichoderma reesei beta-xylosidase, as well as the NA2-tpi promoter
(a hybrid of the promoters from the genes for Aspergillus niger
neutral alpha-amylase and Aspergillus oryzae triose phosphate
isomerase); and mutant, truncated, and hybrid promoters
thereof.
[0081] In a yeast host, useful promoters are obtained from the
genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces
cerevisiae galactokinase (GAL1), Saccharomyces cerevisiae alcohol
dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase
(ADH1,ADH2/GAP), Saccharomyces cerevisiae triose phosphate
isomerase (TPI), Saccharomyces cerevisiae metallothionine (CUP1),
and Saccharomyces cerevisiae 3-phosphoglycerate kinase. Other
useful promoters for yeast host cells are described by Romanos et
al., 1992, Yeast 8: 423-488.
[0082] The control sequence may be a suitable transcription
terminator sequence, which is recognized by a host cell to
terminate transcription. The terminator sequence is operably linked
to the 3' terminus of the nucleotide sequence encoding the
polypeptide. Any terminator which is functional in the host cell of
choice may be used in the present invention.
[0083] Preferred terminators for filamentous fungal host cells are
obtained from the genes for Aspergillus oryzae TAKA amylase,
Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate
synthase, Aspergillus niger alpha-glucosidase, and Fusarium
oxysporum trypsin-like protease.
[0084] Preferred terminators for yeast host cells are obtained from
the genes for Saccharomyces cerevisiae enolase, Saccharomyces
cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae
glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators
for yeast host cells are described by Romanos et al., 1992,
supra.
[0085] The control sequence may also be a suitable leader sequence,
a nontranslated region of an mRNA which is important for
translation by the host cell. The leader sequence is operably
linked to the 5' terminus of the nucleotide sequence encoding the
polypeptide. Any leader sequence that is functional in the host
cell of choice may be used in the present invention.
[0086] Preferred leaders for filamentous fungal host cells are
obtained from the genes for Aspergillus oryzae TAKA amylase and
Aspergillus nidulans triose phosphate isomerase.
[0087] Suitable leaders for yeast host cells are obtained from the
genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces
cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae
alpha-factor, and Saccharomyces cerevisiae alcohol
dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase
(ADH2/GAP).
[0088] The control sequence may also be a polyadenylation sequence,
which is operably linked to the 3' terminus of the nucleotide
sequence and which, when transcribed, is recognized by the host
cell as a signal to add polyadenosine residues to transcribed mRNA.
Any polyadenylation sequence which is functional in the host cell
of choice may be used in the present invention.
[0089] Preferred polyadenylation sequences for filamentous fungal
host cells are obtained from the genes for Aspergillus oryzae TAKA
amylase, Aspergillus niger glucoamylase, Aspergillus nidulans
anthranilate synthase, Fusarium oxysporum trypsin-like protease,
and Aspergillus niger alpha-glucosidase.
[0090] Useful polyadenylation sequences for yeast host cells are
described by Guo and Sherman, 1995, Molecular Cellular Biology 15:
5983-5990.
[0091] The control sequence may also be a propeptide coding region
that codes for an amino acid sequence positioned at the amino
terminus of a polypeptide. The resultant polypeptide is known as a
proenzyme or propolypeptide (or a zymogen in some cases). A
propolypeptide is generally inactive and can be converted to a
mature active polypeptide by catalytic or autocatalytic cleavage of
the propeptide from the propolypeptide. The propeptide coding
region may be obtained from the genes for Saccharomyces cerevisiae
alpha-factor, Rhizomucor miehei aspartic proteinase, and
Myceliophthora thermophila laccase (WO 95/33836).
[0092] Where both signal peptide and propeptide regions are present
at the amino terminus of a polypeptide, the propeptide region is
positioned next to the amino terminus of a polypeptide and the
signal peptide region is positioned next to the amino terminus of
the propeptide region.
[0093] It may also be desirable to add regulatory sequences which
allow the regulation of the expression of the polypeptide relative
to the growth of the host cell. Examples of regulatory systems are
those which cause the expression of the gene to be turned on or off
in response to a chemical or physical stimulus, including the
presence of a regulatory compound. In yeast, the ADH2 system or
GAL1 system may be used. In filamentous fungi, the TAKA
alpha-amylase promoter, Aspergillus niger glucoamylase promoter,
and Aspergillus oryzae glucoamylase promoter may be used as
regulatory sequences. Other examples of regulatory sequences are
those which allow for gene amplification. In eukaryotic systems,
these include the dihydrofolate reductase gene which is amplified
in the presence of methotrexate, and the metallothionein genes
which are amplified with heavy metals. In these cases, the
nucleotide sequence encoding the polypeptide would be operably
linked with the regulatory sequence.
Expression Vectors
[0094] The present invention also relates to recombinant expression
vectors comprising a signal peptide sequence of the present
invention, a nucleotide sequence encoding a polypeptide of
interest, and transcriptional and translational stop signals. The
various nucleotide and control sequences described above may be
joined together to produce a recombinant expression vector which
may include one or more convenient restriction sites to allow for
insertion or substitution of the promoter and/or nucleotide
sequence encoding the polypeptide at such sites. Alternatively, the
nucleotide sequence may be expressed by inserting the nucleotide
sequence or a nucleic acid construct comprising the signal peptide
sequence and/or nucleotide sequence encoding the polypeptide into
an appropriate vector for expression. In creating the expression
vector, the coding sequence is located in the vector so that the
coding sequence is operably linked with a signal peptide sequence
of the present invention and one or more appropriate control
sequences for expression.
[0095] The recombinant expression vector may be any vector (e.g., a
plasmid or virus) which can be conveniently subjected to
recombinant DNA procedures and can bring about the expression of
the nucleotide sequence. The choice of the vector will typically
depend on the compatibility of the vector with the host cell into
which the vector is to be introduced. The vectors may be linear or
closed circular plasmids.
[0096] The vectors of the present invention preferably contain one
or more selectable markers which permit easy selection of
transformed cells. A selectable marker is a gene the product of
which provides for biocide or viral resistance, resistance to heavy
metals, prototrophy to auxotrophs, and the like. Suitable markers
for yeast host cells include, but are not limited to, ADE2, HIS3,
LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in a
filamentous fungal host cell include, but are not limited to, amdS
(acetamidase), argB (ornithine carbamoyltransferase), bar
(phosphinothricin acetyltransferase), hygB (hygromycin
phosphotransferase), niaD (nitrate reductase), pyrG
(orotidine-5'-phosphate decarboxylase), sC (sulfate
adenyltransferase), trpC (anthranilate synthase), as well as
equivalents thereof. Preferred for use in an Aspergillus cell are
the amdS and pyrG genes of Aspergillus nidulans or Aspergillus
oryzae and the bar gene of Streptomyces hygroscopicus.
[0097] The vector may be an autonomously replicating vector, i.e.,
a vector which exists as an extrachromosomal entity, the
replication of which is independent of chromosomal replication,
e.g., a plasmid, an extrachromosomal element, a minichromosome, or
an artificial chromosome. The vector may contain any means for
assuring self-replication. Alternatively, the vector may be one
which, when introduced into the host cell, is integrated into the
genome and replicated together with the chromosome(s) into which it
has been integrated. Furthermore, a single vector or plasmid or two
or more vectors or plasmids which together contain the total DNA to
be introduced into the genome of the host cell, or a transposon may
be used.
[0098] The vectors of the present invention preferably contain an
element(s) that permits stable integration of the vector into the
host cell's genome or autonomous replication of the vector in the
cell independent of the genome.
[0099] For integration into the host cell genome, the vector may
rely on the nucleotide sequence encoding the polypeptide or any
other element of the vector for stable integration of the vector
into the genome by homologous or nonhomologous recombination.
Alternatively, the vector may contain additional nucleotide
sequences for directing integration by homologous recombination
into the genome of the host cell. The additional nucleotide
sequences enable the vector to be integrated into the host cell
genome at a precise location(s) in the chromosome(s). To increase
the likelihood of integration at a precise location, the
integrational elements should preferably contain a sufficient
number of nucleotides, such as 100 to 1,500 base pairs, preferably
400 to 1,500 base pairs, and most preferably 800 to 1,500 base
pairs, which are highly homologous with the corresponding target
sequence to enhance the probability of homologous recombination.
The integrational elements may be any sequence that is homologous
with the target sequence in the genome of the host cell.
Furthermore, the integrational elements may be non-encoding or
encoding nucleotide sequences. On the other hand, the vector may be
integrated into the genome of the host cell by non-homologous
recombination.
[0100] For autonomous replication, the vector may further comprise
an origin of replication enabling the vector to replicate
autonomously in the host cell in question. The origin of
replication may be any plasmid replicator mediating autonomous
replication which functions in a cell. The term "origin of
replication" or "plasmid replicator" is defined herein as a
nucleotide sequence that enables a plasmid or vector to replicate
in vivo.
[0101] Examples of origins of replication for use in a yeast host
cell are the 2 micron origin of replication, ARS1, ARS4, the
combination of ARS1 and CEN3, and the combination of ARS4 and CEN6.
The origin of replication may be one having a mutation which makes
its functioning temperature-sensitive in the host cell (see, e.g.,
Ehrlich, 1978, Proceedings of the National Academy of Sciences USA
75: 1433).
[0102] Examples of origins of replication useful in a filamentous
fungal cell are AMA1 and ANS1 (Gems et al., 1991, Gene 98:61-67;
Cullen et al., 1987, Nucleic Acids Research 15: 9163-9175; WO
00/24883). Isolation of the AMA1 gene and construction of plasmids
or vectors comprising the gene can be accomplished according to the
methods disclosed in WO 00/24883.
[0103] More than one copy of a nucleotide sequence encoding a
polypeptide may be inserted into the host cell to increase
production of the gene product. An increase in the copy number of
the nucleotide sequence can be obtained by integrating at least one
additional copy of the sequence into the host cell genome or by
including an amplifiable selectable marker gene with the nucleotide
sequence where cells containing amplified copies of the selectable
marker gene, and thereby additional copies of the nucleotide
sequence, can be selected for by cultivating the cells in the
presence of the appropriate selectable agent.
[0104] The procedures used to ligate the elements described above
to construct the recombinant expression vectors of the present
invention are well known to one skilled in the art (see, e.g.,
Sambrook et al., 1989, supra).
Host Cells
[0105] The present invention also relates to recombinant host
cells, comprising a signal peptide sequence of the present
invention operably linked to a nucleotide sequence encoding a
polypeptide, which are advantageously used in the recombinant
production of the polypeptides. A vector comprising a signal
peptide sequence of the present invention operably linked to a
nucleotide sequence encoding a polypeptide is introduced into a
host cell so that the vector is maintained as a chromosomal
integrant or as a self-replicating extra-chromosomal vector as
described earlier. The term "host cell" encompasses any progeny of
a parent cell that is not identical to the parent cell due to
mutations that occur during replication. The choice of a host cell
will to a large extent depend upon the gene encoding the
polypeptide and its source.
[0106] The host cell may be any fungal cell useful in the methods
of the present invention. "Fungi" as used herein includes the phyla
Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota (as
defined by Hawksworth et al., In, Ainsworth and Bisby's Dictionary
of The Fungi, 8th edition, 1995, CAB International, University
Press, Cambridge, UK) as well as the Oomycota (as cited in
Hawksworth et al., 1995, supra, page 171) and all mitosporic fungi
(Hawksworth et al., 1995, supra).
[0107] In a preferred aspect, the fungal host cell is a yeast cell.
"Yeast" as used herein includes ascosporogenous yeast
(Endomycetales), basidiosporogenous yeast, and yeast belonging to
the Fungi Imperfecti (Blastomycetes). Since the classification of
yeast may change in the future, for the purposes of this invention,
yeast shall be defined as described in Biology and Activities of
Yeast (Skinner, F. A., Passmore, S. M., and Davenport, R. R., eds,
Soc. App. Bacteriol. Symposium Series No. 9, 1980).
[0108] In a more preferred aspect, the yeast host cell is a
Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces,
Schizosaccharomyces, or Yarrowia cell. In a most preferred aspect,
the yeast host cell is a Saccharomyces carlsbergensis,
Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces
douglasii, Saccharomyces kluyveri, Saccharomyces norbensis or
Saccharomyces oviformis cell. In another most preferred aspect, the
yeast host cell is a Kluyveromyces lactis cell. In another most
preferred aspect, the yeast host cell is a Yarrowia lipolytica
cell.
[0109] In another preferred aspect, the fungal host cell is a
filamentous fungal cell. "Filamentous fungi" include all
filamentous forms of the subdivision Eumycota and Oomycota (as
defined by Hawksworth et al., 1995, supra). The filamentous fungi
are characterized by a mycelial wall composed of chitin, cellulose,
glucan, chitosan, mannan, and other complex polysaccharides.
Vegetative growth is by hyphal elongation and carbon catabolism is
obligately aerobic. In contrast, vegetative growth by yeasts such
as Saccharomyces cerevisiae is by budding of a unicellular thallus
and carbon catabolism may be fermentative.
[0110] In a more preferred aspect, the filamentous fungal host cell
is a cell of a species of, but not limited to, Acremonium,
Aspergillus, Fusarium, Humicola, Mucor, Myceliophthora, Neurospora,
Penicillium, Thielavia, Tolypocladium, or Trichoderma.
[0111] In an even more preferred aspect, the filamentous fungal
host cell is an Aspergillus awamori, Aspergillus foetidus,
Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger or
Aspergillus oryzae cell. In another even more preferred aspect, the
filamentous fungal host cell is a Fusarium bactridioides, Fusarium
cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium
graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium
negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum,
Fusarium sambucinum, Fusarium sarcochroum, Fusarium
sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium
trichothecioides, or Fusarium venenatum cell. In another even more
preferred aspect, the filamentous fungal host cell is a Humicola
insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora
thermophila, Neurospora crassa, Penicillium purpurogenum, Thielavia
terrestris, Trichoderma harzianum, Trichoderma koningii,
Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma
viride cell.
[0112] In a most preferred aspect, the Fusarium venenatum cell is
Fusarium venenatum A3/5, which was originally deposited as Fusarium
graminearum ATCC 20334 and recently reclassified as Fusarium
venenatum by Yoder and Christianson, 1998, Fungal Genetics and
Biology 23: 62-80 and O'Donnell et al., 1998, Fungal Genetics and
Biology 23: 57-67; as well as taxonomic equivalents of Fusarium
venenatum regardless of the species name by which they are
currently known. In another preferred aspect, the Fusarium
venenatum cell is a morphological mutant of Fusarium venenatum A3/5
or Fusarium venenatum ATCC 20334, as disclosed in WO 97/26330.
[0113] In another most preferred aspect, the Trichoderma cell is
Trichoderma reesei ATCC 56765, Trichoderma reesei ATCC 13631,
Trichoderma reesei CBS 526.94, Trichoderma reesei CBS 529.94,
Trichoderma longibrachiatum CBS 528.94, Trichoderma longibrachiatum
ATCC 2106, Trichoderma longibrachiatum CBS 592.94, Trichoderma
viride NRRL 3652, Trichoderma viride CBS 517.94, and Trichoderma
viride NIBH FERM/BP 447.
[0114] Fungal cells may be transformed by a process involving
protoplast formation, transformation of the protoplasts, and
regeneration of the cell wall in a manner known per se. Suitable
procedures for transformation of Aspergillus host cells are
described in EP 238 023 and Yelton et al., 1984, Proceedings of the
National Academy of Sciences USA 81: 1470-1474. Suitable procedures
for transformation of Trichoderma reesei host cells is described in
Penttila et al, 1987, Gene 61: 155-164, and Gruber et al., 1990,
Curr Genet. 18(1):71-6. Suitable methods for transforming Fusarium
species are described by Malardier et al., 1989, Gene 78: 147-156
and WO 96/00787. Yeast may be transformed using the procedures
described by Becker and Guarente, In Abelson, J. N. and Simon, M.
I., editors, Guide to Yeast Genetics and Molecular Biology, Methods
in Enzymology, Volume 194, pp 182-187, Academic Press, Inc., New
York; Ito et al., 1983, Journal of Bacteriology 153: 163; and
Hinnen et al., 1978, Proceedings of the National Academy of
Sciences USA 75: 1920.
Degradation of Biomass
[0115] The present invention also relates to methods for degrading
or converting a cellulose-containing and/or
hemicellulose-containing biomass, comprising treating the biomass
with an effective amount of one or more polypeptides obtained by
the methods of the present invention, wherein the one or more
polypeptides have enzyme activity against the cellulose-containing
and/or hemicellulose-containing biomass. For example, the methods
of the present invention may be used to produce enzymes and host
cells for use in the production of ethanol from biomass. Ethanol
can be produced by enzymatic degradation of biomass and conversion
of the released polysaccharides to ethanol. This kind of ethanol is
often referred to as bioethanol or biofuel. It can be used as a
fuel additive or extender in blends of from less than 1% and up to
100% (a fuel substitute).
[0116] The methods of the present invention may also be used to
produce enzymes and host cells for use in the production of
monosaccharides, disaccharides, and polysaccharides as chemical or
fermentation feedstocks from biomass for the production of ethanol,
plastics, or other products or intermediates. The enzymes may be in
the form of a crude fermentation broth with or without the cells
removed or in the form of a semi-purified or purified enzyme
preparation. Alternatively, a host cell of the present invention
may be used as a source of one or more enzymes in a fermentation
process with the biomass.
[0117] Biomass can include, but is not limited to, wood resources,
municipal solid waste, wastepaper, and crop residues (see, for
example, Wiselogel et al., 1995, in Handbook on Bioethanol (Charles
E. Wyman, editor), pp. 105-118, Taylor & Francis, Washington
D.C.; Wyman, 1994, Bioresource Technology 50: 3-16; Lynd, 1990,
Applied Biochemistry and Biotechnology 24/25: 695-719; Mosier et
al., 1999, Recent Progress in Bioconversion of Lignocellulosics, in
Advances in Biochemical Engineering/Biotechnology, T. Scheper,
managing editor, Volume 65, pp. 23-40, Springer-Verlag, New
York).
[0118] The predominant polysaccharide in the primary cell wall of
biomass is cellulose, the second most abundant is hemi-cellulose,
and the third is pectin. The secondary cell wall, produced after
the cell has stopped growing, also contains polysaccharides and is
strengthened through 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 helps
stabilize the cell wall matrix.
[0119] Three major classes of glycohydrolases are used to breakdown
cellulosic biomass:
[0120] (1) The "endo-1,4-beta-glucanases" or
1,4-beta-D-glucan-4-glucanohydrolases (EC 3.2.1.4), which act
randomly on soluble and insoluble 1,4-beta-glucan substrates.
[0121] (2) The "exo-1,4-beta-D-glucanases" including both the
1,4-beta-D-glucan glucohydrolases (EC 3.2.1.74), which liberate
D-glucose from 1,4-beta-D-glucans and hydrolyze D-cellobiose
slowly, and cellobiohydrolases (1,4-beta-D-glucan
cellobiohydrolases, EC 3.2.1.91), which liberate D-cellobiose from
1,4-beta-glucans.
[0122] (3) The "beta-D-glucosidases" or beta-D-glucoside
glucohydrolases (EC 3.2.1.21), which act to release D-glucose units
from cellobiose and soluble cellodextrins, as well as an array of
glycosides.
[0123] These three classes of enzymes work together synergistically
resulting in efficient decrystallization and hydrolysis of native
cellulose from biomass to yield reducing sugars.
[0124] The methods of the present invention may also be used to
produce other enzymes in conjunction with the above-noted enzymes
to further degrade the hemicellulose component of the biomass
substrate, (see, for example, Brigham et al., 1995, in Handbook on
Bioethanol (Charles E. Wyman, editor), pp. 119-141, Taylor &
Francis, Washington D.C.; Lee, 1997, Journal of Biotechnology 56:
1-24). Such enzymes include, but are not limited to, enzymes that
degrade beta-1,3-1,4-glucan such as endo-beta-1,3(4)-glucanase,
endoglucanase (beta-glucanase, cellulase), and beta-glucosidase;
degrade xyloglucans such as xyloglucanase, endoglucanase, and
cellulase; degrade xylan such as xylanase, xylosidase,
alpha-arabinofuranosidase, alpha-glucuronidase, and acetyl xylan
esterase; degrade mannan such as mannanase, mannosidase,
alpha-galactosidase, and mannan acetyl esterase; degrade galactan
such as galactanase; degrade arabinan such as arabinanase; degrade
homogalacturonan such as pectate lyase, pectin lyase, pectate
lyase, polygalacturonase, pectin acetyl esterase, and pectin methyl
esterase; degrade rhamnogalacturonan such as
alpha-arabinofuranosidase, beta-galactosidase, galactanase,
arabinanase, alpha-arabinofuranosidase, rhamnogalacturonase,
rhamnogalacturonan lyase, and rhamnogalacturonan acetyl esterase;
degrade xylogalacturonan such as xylogalacturonosidase,
xylogalacturonase, and rhamnogalacturonan lyase; and degrade lignin
such as lignin peroxidases, manganese-dependent peroxidases, hybrid
peroxidases, with combined properties of lignin peroxidases and
manganese-dependent peroxidases, and laccases. Other enzymes
include esterases, lipases, oxidases, phospholipases, phytases,
proteases, and peroxidases.
[0125] The present invention is further described by the following
examples which should not be construed as limiting the scope of the
invention.
EXAMPLES
Strains
[0126] Trichoderma reesei RutC30 (ATCC 56765; Montenecourt and
Eveleigh, 1979, Adv. Chem. Ser. 181: 289-301) was derived from
Trichoderma reesei Qm6A (ATCC 13631; Mandels and Reese, 1957, J.
Bacteriol. 73: 269-278). Trichoderma reesei RutC30 and
Saccharomyces cerevisiae YNG318 (MAT.alpha., ura3-52,
leu-2.DELTA.2, pep4A1, his4-539) (WO97/07205) were used as hosts
for expression of Aspergillus oryzae beta-glucosidase. Aspergillus
fumigatus PaHa34 was used as the source of the Family GH3A
beta-glucosidase.
Media and Buffer Solutions
[0127] YP medium was composed per liter of 10 g of yeast extract
and 20 g of bactopeptone.
[0128] Yeast selection medium was composed per liter of 6.7 g of
yeast nitrogen base, 0.8 g of complete supplement mixture (CSM,
Qbiogene, Inc., Carlsbad, Calif.; missing uracil and containing 40
mg/ml of adenine), 5 g of casamino acids (without amino acids), 100
ml of 0.5 M succinate pH 5.0, 40 ml of 50% glucose, 1 ml of 100 mM
CuSO.sub.4, 50 mg of ampicillin, and 25 mg of chloramphenicol.
[0129] Yeast selection plate medium was composed per liter of yeast
selection medium supplemented with 20 g of bacto agar and 150 mg of
5-bromo-4-chloro-3-indolyl-beta-D-glucopyranoside (X-Glc, INALCO
SPA, Milano, Italy) but lacking both ampicillin and
chloramphenicol.
[0130] COVE selection plates were composed per liter of 342.3 g of
sucrose, 20 ml of COVE salt solution, 10 mM acetamide, 15 mM
CsCl.sub.2, and 25 g of Noble agar.
[0131] COVE2 plates were composed per liter of 30 g of sucrose, 20
ml COVE salt solution, 10 mM acetamide, and 25 g of Noble agar.
[0132] COVE salt solution was composed per liter of 26 g of KCl, 26
g of MgSO.sub.4.7H.sub.2O, 76 g of KH.sub.2PO.sub.4, and 50 ml of
COVE trace metals.
[0133] COVE trace metals solution was composed per liter of 0.04 g
of NaB.sub.4O.sub.7.10H.sub.2O, 0.4 g of CuSO.sub.4.5H.sub.2O, 1.2
g of FeSO.sub.4.7H.sub.2O, 0.7 g of MnSO.sub.4.H.sub.2O, 0.8 g of
Na.sub.2MoO.sub.2.2H.sub.2O, and 10 g of ZnSO.sub.4.7H.sub.2O.
[0134] Cellulase-inducing media was composed per liter of 20 g of
Arbocel B800-natural cellulose fibers (J. Rettenmaier USA LP,
Schoolcraft, Mich.), 10 g of corn steep solids (Sigma Chemical Co.,
St. Louis, Mo.), 1.45 g of (NH.sub.4).sub.2SO.sub.4, 2.08 g of
KH.sub.2PO.sub.4, 0.28 g of CaCl.sub.2, 0.42 g of
MgSO.sub.4.7H.sub.2O, 0.42 ml Trichoderma reesei Trace Metals, and
2 drops of pluronic acid; pH to 6.0 with 10 N NaoH.
[0135] Trichoderma reesei trace metals solution was composed per
liter of 216 g of FeCl.sub.3.6H.sub.2O, 58 g of
ZnSO.sub.4.7H.sub.2O, 27 g of MnSO.sub.4.H.sub.2O, 10 g of
CuSO.sub.4.5H.sub.2O, 2.4 g of H.sub.3BO.sub.3, and 336 g of citric
acid.
[0136] PEG Buffer was composed per liter of 500 g of PEG 4000 (BDH,
Poole, England), 10 mM CaCl.sub.2, and 10 mM Tris-HCl pH 7.5
(filter sterilize).
[0137] STC was composed per liter of 1 M sorbitol, 10 mM
CaCl.sub.2, and 10 mM Tris-HCl pH 7.5 (filter sterilize).
[0138] Inoculum Medium was composed per liter of 20 g of glucose,
10 g of corn steep solids (Sigma Chemical Co., St. Louis, Mo.),
1.45 g of (NH.sub.4).sub.2SO.sub.4, 2.08 g of KH.sub.2PO.sub.4,
0.28 g of CaCl.sub.2, 0.42 g of MgSO.sub.4.7H.sub.2O, 0.42 ml of
Trichoderma reesei trace metals solution, and 2 drops of pluronic
acid; final pH 5.0.
[0139] Fermentation Medium was composed per liter of 4 g of
glucose, 10 g of corn steep solids, 30 g of Arbocel B800-natural
cellulose fibers (J. Rettenmaier USA LP, Schoolcraft, Mich.), 3.8 g
of (NH.sub.4).sub.2SO.sub.4, 2.8 g of KH.sub.2PO.sub.4, 2.08 g of
CaCl.sub.2, 1.63 g of MgSO.sub.4.7H.sub.2O, 0.75 ml of Trichoderma
reesei trace metals solution, and 1.8 ml of pluronic acid.
[0140] Feed Medium was composed per liter of 600 g of glucose, 20 g
of Cellulose B800, 35.5 g of H.sub.3PO4, and 5 ml of pluronic
acid.
Beta-glucosidase Activity Assay
[0141] For Trichoderma reesei samples, beta-glucosidase activity
was determined at ambient temperature using 25 .mu.l aliquots of
culture supernatants, diluted 1:10 in 50 mM succinate pH 5.0, using
200 .mu.l of 0.5 mg/ml p-nitrophenyl-beta-D-glucopyranoside as
substrate in 50 mM succinate pH 5.0. After 15 minutes incubation
the reaction was stopped by adding 100 .mu.l of 1 M Tris-HCl pH 8.0
and the absorbance was read spectrophotometrically at 405 nm.
[0142] For Saccharomyces cerevisiae samples, culture supernatant
samples were diluted 0.6-fold with 0.1 M succinate pH 5.0 in
96-wells microtiter plates. Twenty five .mu.l of the diluted
samples were taken from each well and added to a new 96-well plate,
containing 200 .mu.l of 1 mg/ml
p-nitrophenyl-beta-D-glucopyranoside substrate. The plates were
incubated at ambient temperature for 1.5 hours and the reaction
stopped by adding 2 M Tris-HCl pH 9. The plates were then read
spectrophotometrically at 405 nm.
[0143] One unit of beta-glucosidase activity corresponded to
production of 1 .mu.mol of p-nitrophenyl per minute per liter at pH
5.0, ambient temperature. Aspergillus niger beta-glucosidase
(Novozyme 188, Novozymes A/S, Bagsv.ae butted.rd, Denmark) was used
as an enzyme standard.
DNA Sequencing
[0144] DNA sequencing was performed on an ABI3700 (Applied
Biosystems, Foster City, Calif.) using dye terminator chemistry
(Giesecke et al., 1992, Journal of Virol. Methods 38: 47-60).
Sequences were assembled using phred/phrap/consed (University of
Washington, Seattle Wash.) with sequence specific primers.
Example 1
Construction of pAILo1 Expression Vector
[0145] Expression vector pAILo1 was constructed by modifying pBANe6
(U.S. Pat. No. 6,461,837), which comprises the NA2-tpi promoter,
Aspergillus niger amyloglucosidase terminator sequence (AMG
terminator), and Aspergillus nidulans acetamidase gene (amdS).
Modification of pBANe6 was performed by first eliminating three Nco
I restriction sites at positions 2051, 2722, and 3397 bp from the
amdS selection marker by site directed mutagenesis. All changes
were designed to be "silent" leaving the actual protein sequence of
the amdS gene product unchanged. Removal of these three sites was
performed simultaneously with a GeneEditor Site-Directed
Mutagenesis Kit (Promega, Madison, Wis.) according to the
manufacturer's instructions using the following primers (underlined
nucleotide represents the changed base):
TABLE-US-00001 AMDS3NcoMut (2050): 5'-GTGCCCCATGATACGCCTCCGG-3'
(SEQ ID NO: 1) AMDS2NcoMut (2721): 5'-GAGTCGTATTTCCAAGGCTCCTGACC-3'
(SEQ ID NO: 2) AMDS1NcoMut (3396): 5'-GGAGGCCATGAAGTGGACCAACGG-3'
(SEQ ID NO: 3)
[0146] A plasmid comprising all three expected sequence changes was
then submitted to site-directed mutagenesis, using a QuickChange
Mutagenesis Kit (Stratagene, La Jolla, Calif.), to eliminate the
Nco I restriction site at the end of the AMG terminator at position
1643. The following primers (underlined nucleotide represents the
changed base) were used for mutagenesis: [0147] Upper Primer to
mutagenize the Aspergillus niger amyloglucosidase (AMG) terminator
sequence:
TABLE-US-00002 [0147] (SEQ ID NO: 4)
5'-CACCGTGAAAGCCATGCTCTTTCCTTCGTGTAGAAGACCAGACAG-3'
[0148] Lower Primer to mutagenize the Aspergillus niger
amyloglucosidase (AMG) terminator sequence:
TABLE-US-00003 [0148] (SEQ ID NO: 5)
5'-CTGGTCTTCTACACGAAGGAAAGAGCATGGCTTTCACGGTGTCTG-3'
[0149] The last step in the modification of pBANe6 was the addition
of a new Nco I restriction site at the beginning of the polylinker
using a QuickChange Mutagenesis Kit and the following primers
(underlined nucleotides represent the changed bases) to yield
pAILo1 (FIG. 1). [0150] Upper Primer to mutagenize the Aspergillus
niger amylase promoter (NA2-tpi):
TABLE-US-00004 [0150] (SEQ ID NO: 6)
5'-CTATATACACAACTGGATTTACCATGGGCCCGCGGCCGCAGATC-3'
[0151] Lower Primer to mutagenize the Aspergillus niger amylase
promoter (NA2-tpi):
TABLE-US-00005 [0151] (SEQ ID NO: 7)
5'-GATCTGCGGCCGCGGGCCCATGGTAAATCCAGTTGTGTATATAG-3'
[0152] The amdS gene of pAILo1 was swapped with the Aspergillus
nidulans pyrG gene. Plasmid pBANe10 (FIG. 14) was used as a source
for the pyrG gene as a selection marker. Analysis of the sequence
of pBANe10 showed that the pyrG marker was contained within an Nsi
I restriction fragment and does not contain either Nco I or Pac I
restriction sites. Since the amdS is also flanked by Nsi I
restriction sites the strategy to switch the selection marker was a
simple swap of Nsi I restriction fragments. Plasmid DNA from pAILo1
and pBANe10 were digested with the restriction enzyme Nsi I and the
products purified by agarose gel electrophoresis. The Nsi I
fragment from pBANe10 containing the pyrG gene was ligated to the
backbone of pAILo1 to replace the original Nsi I DNA fragment
containing the amdS gene. Recombinant clones were analyzed by
restriction digest to determine that they had the correct insert
and also its orientation. A clone with the pyrG gene transcribed in
the counterclockwise direction was selected. The new plasmid has
been designated pAILo2 (FIG. 15).
Example 2
Construction of pMJ04 Expression Vector
[0153] Expression vector pMJ04 was constructed by PCR amplifying
the Trichoderma reesei exocellobiohydrolase 1 gene (cbh1)
terminator from Trichoderma reesei RutC30 genomic DNA using primers
993429 (antisense) and 993428 (sense) shown below. The antisense
primer was engineered to have a Pac I site at the 5'-end and a Spe
I site at the 3'-end of the sense primer.
TABLE-US-00006 Primer 993429 (antisense):
5'-AACGTTAATTAAGGAATCGTTTTGTGTTT-3' (SEQ ID NO: 8) Primer 993428
(sense): 5'-AGTACTAGTAGCTCCGTGGCGAAAGCCTG-3' (SEQ ID NO: 9)
[0154] Trichoderma reesei RutC30 genomic DNA was isolated using a
DNeasy Plant Maxi Kit (Qiagen, Chatsworth, Calif.).
[0155] The amplification reactions (50 .mu.l) were composed of
1.times. ThermoPol Reaction Buffer (New England Biolabs, Beverly,
Mass.), 0.3 mM dNTPs, 100 ng of Trichoderma reesei RutC30 genomic
DNA, 0.3 .mu.M primer 993429, 0.3 .mu.M primer 993428, and 2 units
of Vent polymerase (New England Biolabs, Beverly, Mass.). The
reactions were incubated in an Eppendorf Mastercycler 5333
(Eppendorf Scientific, Inc., Westbury, N.Y.) programmed as follows:
5 cycles each for 30 seconds at 94.degree. C., 30 seconds at
50.degree. C., and 60 seconds at 72.degree. C., followed by 25
cycles each for 30 seconds at 94.degree. C., 30 seconds at
65.degree. C., and 120 seconds at 72.degree. C. (5 minute final
extension). The reaction products were isolated on a 1.0% agarose
gel using 40 mM Tris base-20 mM sodium acetate-1 mM disodium EDTA
(TAE) buffer where a 229 bp product band was excised from the gel
and purified using a QIAquick Gel Extraction Kit (QIAGEN,
Chatsworth, Calif.) according to the manufacturer's
instructions.
[0156] The resulting PCR fragment was digested with Pac I and Spe I
and ligated into pAILo01 digested with the same restriction enzymes
using a Rapid Ligation Kit (Roche, Indianapolis, Ind.), to generate
pMJ04 (FIG. 2).
Example 3
Construction of pCaHj568 Expression Vector
[0157] Expression plasmid pCaHj568 was constructed from pCaHj170
(U.S. Pat. No. 5,763,254) and pMT2188. Plasmid pCaHj170 comprises
the Humicola insolens endoglucanase V (EGV) coding region. Plasmid
pMT2188 was constructed as follows: The pUC19 origin of replication
was PCR amplified from pCaHj483 (WO 98/00529) with primers 142779
and 142780 shown below. Primer 142780 introduces a BbuI site in the
PCR fragment.
TABLE-US-00007 (SEQ ID NO: 10) 142779:
5'-TTGAATTGAAAATAGATTGATTTAAAACTTC-3' (SEQ ID NO: 11) 142780:
5'-TTGCATGCGTAATCATGGTCATAGC-3'
[0158] The Expand PCR system (Roche Molecular Biochemicals, Basel,
Switserland) was used for the amplification following the
manufacturer's instructions for this and the subsequent PCR
amplifications. PCR products were separated on a 1% agarose gel
using TAE buffer and an 1160 bp fragment was isolated and purified
using a Jetquick Gel Extraction Spin Kit (Genomed, Wielandstr,
Germany).
[0159] The URA3 gene was amplified from the Saccharomyces cerevisae
cloning vector pYES2 (Invitrogen, Carlsbad, Calif.) using primers
140288 and 142778 below. Primer 140288 introduces an Eco RI site in
the PCR fragment.
TABLE-US-00008 (SEQ ID NO: 12) 140288:
5'-TTGAATTCATGGGTAATAACTGATAT-3' (SEQ ID NO: 13) 142778:
5'-AAATCAATCTATTTTCAATTCAATTCATCATT-3'
[0160] PCR products were separated on a 1% agarose gel using TAE
buffer and an 1126 bp fragment was isolated and purified using a
Jetquick Gel Extraction Spin Kit.
[0161] The two PCR fragments were fused by mixing and amplification
using primers 142780 and 140288 shown above by overlap method
splicing (Horton et al., 1989, Gene 77: 61-68). PCR products were
separated on 1% agarose gel using TAE buffer and a 2263 bp fragment
was isolated and purified using a Jetquick Gel Extraction Spin
Kit.
[0162] The resulting fragment was digested with Eco RI and Bbu I
and ligated to the largest fragment of pCaHj483 digested with the
same enzymes. The ligation mixture was used to transform
pyrF-negative E. coli strain DB6507 (ATCC 35673) made competent by
the method of Mandel and Higa, 1970, J. Mol. Biol. 45: 154.
Transformants were selected on solid M9 medium (Sambrook et al.,
1989, Molecular Cloning, A Laboratory Manual, 2nd edition, Cold
Spring Harbor Laboratory Press) supplemented per liter with 1 g of
casaminoacids, 500 .mu.g of thiamine, and 10 mg of kanamycin. A
plasmid from one transformant was isolated and designated pCaHj527
(FIG. 3).
[0163] The NA2/tpi promoter present on pCaHj527 was subjected to
site directed mutagenesis by a simple PCR approach. Nucleotides
134-144 were converted from GTACTAAAACC to CCGTTAAATTT using
mutagenic primer 141223:
TABLE-US-00009 Primer 141223: (SEQ ID NO: 14)
5'-GGATGCTGTTGACTCCGGAAATTTAACGGTTTGGTCTTGCATCCC-3'
Nucleotides 423-436 were converted from ATGCAATTTAAACT to
CGGCAATTTAACGG using mutagenic primer 141222:
TABLE-US-00010 Primer 141222: (SEQ ID NO: 15)
5'-GGTATTGTCCTGCAGACGGCAATTTAACGGCTTCTGCGAATCGC-3'
[0164] The resulting plasmid was designated pMT2188 (FIG. 4).
[0165] The Humicola insolens endoglucanase V coding region was
transferred from pCaHj170 as a Bam HI-Sal I fragment into pMT2188
digested with Bam HI and Xho I to generate pCaHj568 (FIG. 5).
Example 4
Construction of pMJ05 Expression Vector
[0166] Expression vector pMJ05 was constructed by PCR amplifying
the 915 bp Humicola insolens endoglucanase V coding region from
pCaHj568 using primers HiEGV-F and HiEGV-R shown below.
TABLE-US-00011 HiEGV-F (sense): (SEQ ID NO: 16)
5'-AAGCTTAAGCATGCGTTCCTCCCCCCTCC-3' HiEGV-R (antisense): (SEQ ID
NO: 17) 5'-CTGCAGAATTCTACAGGCACTGATGGTACCAG-3'
[0167] The amplification reactions (50 .mu.l) were composed of
1.times. ThermoPol Reaction Buffer, 0.3 mM dNTPs, 10 ng/.mu.l
pCaHj568 plasmid, 0.3 .mu.M HiEGV-F primer, 0.3 .mu.M HiEGV-R
primer, and 2 units of Vent polymerase. The reactions were
incubated in an Eppendorf Mastercycler 5333 programmed as follows:
5 cycles each for 30 seconds at 94.degree. C., 30 seconds at
50.degree. C., and 60 seconds at 72.degree. C., followed by 25
cycles each for 30 seconds at 94.degree. C., 30 seconds at
65.degree. C., and 120 seconds at 72.degree. C. (5 minute final
extension). The reaction products were isolated on a 1.0% agarose
gel using TAE buffer where a 937 bp product band was excised from
the gel and purified using a QIAquick Gel Extraction Kit according
to the manufacturer's instructions.
[0168] This 937 bp purified fragment was used as template DNA for
subsequent amplifications using the following primers:
TABLE-US-00012 HiEGV-R (antisense): (SEQ ID NO: 18)
5'-CTGCAGAATTCTACAGGCACTGATGGTACCAG-3' HiEGV-F-overlap (sense):
(SEQ ID NO: 19) 5'-ACCGCGGACTGCGCATCATGCGTTCCTCCCCCCTCC-3'
Primer sequences in italics are homologous to 17 bp of the
Trichoderma reesei cbh1 promoter and underlined primer sequences
are homologous to 29 bp of the Humicola insolens endoglucanase V
coding region. The 36 bp overlap between the promoter and the
coding sequence allowed precise fusion of the 994 bp fragment
comprising the Trichoderma reesei cbh1 promoter to the 918 bp
fragment comprising the Humicola insolens endoglucanase V open
reading frame.
[0169] The amplification reactions (50 .mu.l) were composed of
1.times. ThermoPol Reaction Buffer, 0.3 mM dNTPs, 1 ul of 937 bp
purified PCR fragment, 0.3 .mu.M HiEGV-F-overlap primer, 0.3 .mu.M
HiEGV-R primer, and 2 units of Vent polymerase. The reactions were
incubated in an Eppendorf Mastercycler 5333 programmed as follows:
5 cycles each for 30 seconds at 94.degree. C., 30 seconds at
50.degree. C., and 60 seconds at 72.degree. C., followed by 25
cycles each for 30 seconds at 94.degree. C., 30 seconds at
65.degree. C., and 120 seconds at 72.degree. C. (5 minute final
extension). The reaction products were isolated on a 1.0% agarose
gel using TAE buffer where a 945 bp product band was excised from
the gel and purified using a QIAquick Gel Extraction Kit according
to the manufacturer's instructions.
[0170] A separate PCR was performed to amplify the Trichoderma
reesei cbh1 promoter sequence extending from 994 bp upstream of the
ATG start codon of the gene from Trichoderma reesei RutC30 genomic
DNA using the following primers (sense primer was engineered to
have a Sal I restriction site at the 5'-end):
TABLE-US-00013 TrCBHIpro-F (sense):
5'-AAACGTCGACCGAATGTAGGATTGTTATC-3' (SEQ ID NO: 20) TrCBHIpro-R
(antisense): 5'-GATGCGCAGTCCGCGGT-3' (SEQ ID NO: 21)
[0171] The amplification reactions (50 .mu.l) were composed of
1.times. ThermoPol Reaction Buffer, 0.3 mM dNTPs, 100 ng of
Trichoderma reesei RutC30 genomic DNA, 0.3 .mu.M TrCBHIpro-F
primer, 0.3 .mu.M TrCBHIpro-R primer, and 2 units of Vent
polymerase. The reactions were incubated in an Eppendorf
Mastercycler 5333 programmed as follows: 30 cycles each for 30
seconds at 94.degree. C., 30 seconds at 55.degree. C., and 120
seconds at 72.degree. C. (5 minute final extension). The reaction
products were isolated on a 1.0% agarose gel using TAE buffer where
a 998 bp product band was excised from the gel and purified using a
QIAquick Gel Extraction Kit according to the manufacturer's
instructions.
[0172] The 998 bp purified PCR fragment was used to as template DNA
for subsequent amplifications using the following primers:
TABLE-US-00014 TrCBHIpro-F: (SEQ ID NO: 22)
5'-AAACGTCGACCGAATGTAGGATTGTTATC-3' TrCBHIpro-R-overlap: (SEQ ID
NO: 23) 5'-GGAGGGGGGAGGAACGCATGATGCGCAGTCCGCGGT-3'
[0173] Sequences in italics are homologous to 17 bp of the
Trichoderma reesei cbh1 promoter and underlined sequences are
homologous to 29 bp of the Humicola insolens endoglucanase V coding
region. The 36 bp overlap between the promoter and the coding
sequence allowed precise fusion of the 994 bp fragment comprising
the Trichoderma reesei cbh1 promoter to the 918 bp fragment
comprising the Humicola insolens endoglucanase V open reading
frame.
[0174] The amplification reactions (50 .mu.l) were composed of
1.times. ThermoPol Reaction Buffer, 0.3 mM dNTPs, 1 .mu.l of 998 bp
purified PCR fragment, 0.3 .mu.M TrCBH1pro-F primer, 0.3 .mu.M
TrCBH1pro-R-overlap primer, and 2 units of Vent polymerase. The
reactions were incubated in an Eppendorf Mastercycler 5333
programmed as follows: 5 cycles each for 30 seconds at 94.degree.
C., 30 seconds at 50.degree. C., and 60 seconds at 72.degree. C.,
followed by 25 cycles each for 30 seconds at 94.degree. C., 30
seconds at 65.degree. C., and 120 seconds at 72.degree. C. (5
minute final extension). The reaction products were isolated on a
1.0% agarose gel using TAE buffer where a 1017 bp product band was
excised from the gel and purified using a QIAquick Gel Extraction
Kit according to the manufacturer's instructions.
[0175] The 1017 bp Trichoderma reesei cbh1 promoter PCR fragment
and the 945 bp Humicola insolens endoglucanase V PCR fragments were
used as template DNA for subsequent amplification using the
following primers to precisely fuse the 994 bp Trichoderma reesei
cbh1 promoter to the 918 bp Humicola insolens endoglucanase V
coding region using overlapping PCR.
TABLE-US-00015 TrCBHIpro-F: (SEQ ID NO: 24)
5'-AAACGTCGACCGAATGTAGGATTGTTATC-3' HiEGV-R: (SEQ ID NO: 25)
5'-CTGCAGAATTCTACAGGCACTGATGGTACCAG-3'
[0176] The amplification reactions (50 .mu.l) were composed of
1.times. ThermoPol Reaction Buffer, 0.3 mM dNTPs, 0.3 .mu.M
TrCBH1pro-F primer, 0.3 .mu.M HiEGV-R primer, and 2 U of Vent
polymerase.
[0177] The reactions were incubated in an Eppendorf Mastercycler
5333 programmed as follows: 5 cycles each for 30 seconds at
94.degree. C., 30 seconds at 50.degree. C., and 60 seconds at
72.degree. C., followed by 25 cycles each for 30 seconds at
94.degree. C., 30 seconds at 65.degree. C., and 120 seconds at
72.degree. C. (5 minute final extension). The reaction products
were isolated on a 1.0% agarose gel using TAE buffer where a 1926
bp product band was excised from the gel and purified using a
QIAquick Gel Extraction Kit according to the manufacturer's
instructions.
[0178] The resulting 1926 bp fragment was cloned into
pCR-Blunt-II-TOPO vector using a Zero Blunt.TM. TOPO PCR Cloning
Kit (Invitrogen, Carlsbad, Calif.) following the manufacturer's
protocol. The resulting plasmid was digested with Not I and Sal I
and the 1926 bp fragment purified and ligated into pMJ04 expression
vector which was also digested with the same two restriction
enzymes, to generate pMJ05 (FIG. 6).
Example 5
Construction of pSMai130 Expression Vector
[0179] A 2586 bp DNA fragment spanning from the ATG start codon to
the TAA stop codon of the Aspergillus oryzae beta-glucosidase
coding sequence (SEQ ID NO: 42 for cDNA sequence and SEQ ID NO: 43
for the deduced amino acid sequence; E. coli DSM 14240) was
amplified by PCR from pJaL660 (WO 2002/095014) as template with
primers 993467 (sense) and 993456 (antisense) shown below. A Spe I
site was engineered at the 5' end of the antisense primer to
facilitate ligation. Primer sequences in italics are homologous to
24 bp of the Trichoderma reesei cbh1 promoter and underlined
sequences are homologous to 22 bp of the Aspergillus oryzae
beta-glucosidase coding region.
TABLE-US-00016 Primer 993467: (SEQ ID NO: 26)
5'-ATAGTCAACCGCGGACTGCGCATCATGAAGCTTGGTTGGATCGAGG- 3' Primer
993456: (SEQ ID NO: 27) 5'-ACTAGTTTACTGGGCCTTAGGCAGCG-3'
[0180] The amplification reactions (50 .mu.l) were composed of Pfx
Amplification Buffer (Invitrogen, Carlsbad, Calif.), 0.25 mM dNTPs,
10 ng of pJaL660 plasmid, 6.4 .mu.M primer 993467, 3.2 .mu.M primer
993456, 1 mM MgCl.sub.2, and 2.5 units of Pfx DNA polymerase
(Invitrogen, Carlsbad, Calif.). The reactions were incubated in an
Eppendorf Mastercycler 5333 programmed as follows: 30 cycles each
for 60 seconds at 94.degree. C., 60 seconds at 55.degree. C., and
180 seconds at 72.degree. C. (15 minute final extension). The
reaction products were isolated on a 1.0% agarose gel using TAE
buffer where a 2586 bp product band was excised from the gel and
purified using a QIAquick Gel Extraction Kit according to the
manufacturer's instructions.
[0181] A separate PCR was performed to amplify the Trichoderma
reesei cbh1 promoter sequence extending from 1000 bp upstream of
the ATG start codon of the gene, using primer 993453 (sense) and
primer 993463 (antisense) shown below to generate a 1000 bp PCR
fragment. Primer sequences in italics are homologous to the 24 bp
of the Trichoderma reesei cbh1 promoter and underlined primer
sequences are homologous to the 22 bp of the Aspergillus oryzae
beta-glucosidase coding region. The 46 bp overlap between the
promoter and the coding sequence allows precise fusion of the 1000
bp fragment comprising the Trichoderma reesei cbh1 promoter to the
2586 bp fragment comprising the Aspergillus oryzae beta-glucosidase
open reading frame.
TABLE-US-00017 Primer 993453: (SEQ ID NO: 28)
5'-GTCGACTCGAAGCCCGAATGTAGGAT-3' Primer 993463: (SEQ ID NO: 29)
5'-CCTCGATCCAACCAAGCTTCATGATGCGCAGTCCGCGGTTGACTA-3'
[0182] The amplification reactions (50 .mu.l) were composed of Pfx
Amplification Buffer, 0.25 mM dNTPs, 100 ng of Trichoderma reesei
RutC30 genomic DNA, 6.4 .mu.M primer 993453, 3.2 .mu.M primer
993463, 1 mM MgCl.sub.2, and 2.5 units of Pfx DNA polymerase. The
reactions were incubated in an Eppendorf Mastercycler 5333
programmed as follows: 30 cycles each for 60 seconds at 94.degree.
C., 60 seconds at 55.degree. C., and 180 seconds at 72.degree. C.
(15 minute final extension). The reaction products were isolated on
a 1.0% agarose gel using TAE buffer where a 1000 bp product band
was excised from the gel and purified using a QIAquick Gel
Extraction Kit according to the manufacturer's instructions.
[0183] The purified fragments were used as template DNA for
subsequent amplification using primer 993453 (sense) and primer
993456 (antisense) shown above to precisely fuse the 1000 bp
fragment comprising the Trichoderma reesei cbh1 promoter to the
2586 bp fragment comprising the Aspergillus oryzae beta-glucosidase
open reading frame by overlapping PCR.
[0184] The amplification reactions (50 .mu.l) were composed of Pfx
Amplification Buffer, 0.25 mM dNTPs, 6.4 .mu.M primer 99353, 3.2
.mu.M primer 993456, 1 mM MgCl.sub.2, and 2.5 units of Pfx DNA
polymerase. The reactions were incubated in an Eppendorf
Mastercycler 5333 programmed as follows: 30 cycles each for 60
seconds at 94.degree. C., 60 seconds at 60.degree. C., and 240
seconds at 72.degree. C. (15 minute final extension).
[0185] The resulting 3586 bp fragment was digested with Sal I and
Spe I and ligated into pMJ04, digested with the same two
restriction enzymes, to generate pSMai130 (FIG. 7).
Example 6
Construction of pSMai135
[0186] The Aspergillus oryzae beta-glucosidase coding region (minus
the native signal sequence, see FIG. 8) from Lys-20 to the TM stop
codon was PCR amplified from pJaL660 as template with primer 993728
(sense) and primer 993727 (antisense) shown below. Sequences in
italics are homologous to 20 bp of the Humicola insolens
endoglucanase V signal sequence and sequences underlined are
homologous to 22 bp of the Aspergillus oryzae beta-glucosidase
coding region. A Spe I site was engineered into the 5' end of the
antisense primer.
TABLE-US-00018 Primer 993728: (SEQ ID NO: 30)
5'-TGCCGGTGTTGGCCCTTGCCAAGGATGATCTCGCGTACTCCC-3' Primer 993727:
(SEQ ID NO: 31) 5'-GACTAGTCTTACTGGGCCTTAGGCAGCG-3'
[0187] The amplification reactions (50 .mu.l) were composed of Pfx
Amplification Buffer, 0.25 mM dNTPs, 10 ng/.mu.l pJal660, 6.4 .mu.M
primer 993728, 3.2 .mu.M primer 993727, 1 mM MgCl.sub.2, and 2.5
units of Pfx DNA polymerase. The reactions were incubated in an
Eppendorf Mastercycler 5333 programmed as follows: 30 cycles each
for 60 seconds at 94.degree. C., 60 seconds at 55.degree. C., and
180 seconds at 72.degree. C. (15 minute final extension). The
reaction products were isolated on a 1.0% agarose gel using TAE
buffer where a 2523 bp product band was excised from the gel and
purified using a QIAquick Gel Extraction Kit according to the
manufacturer's instructions.
[0188] A separate PCR amplification was performed to amplify 1000
bp of the Trichoderma reesei cbh1 promoter and 63 bp of the
putative Humicola insolens endoglucanase V signal sequence (ATG
start codon to Ala-21, FIG. 9, SEQ ID NO: 36), using primer 993724
(sense) and primer 993729 (antisense) shown below. Primer sequences
in italics are homologous to 20 by of the Humicola insolens
endoglucanase V signal sequence and underlined primer sequences are
homologous to the 22 bp of the Aspergillus oryzae beta-glucosidase
coding region. Plasmid pMJ05, which comprises the Humicola insolens
endoglucanase V coding region under the control of the cbh1
promoter, was used as a template to generate a 1063 bp fragment
comprising the Trichoderma reesei cbh1 promoter/Humicola insolens
endoglucanase V signal sequence fragment. A 42 bp of overlap was
shared between the Trichoderma reesei cbh1 promoter/Humicola
insolens endoglucanase V signal sequence and the Aspergillus oryzae
coding sequence to provide a perfect linkage between the promoter
and the ATG start codon of the 2523 bp Aspergillus oryzae
beta-glucosidase.
TABLE-US-00019 Primer 993724: (SEQ ID NO: 32)
5'-ACGCGTCGACCGAATGTAGGATTGTTATCC-3' Primer 993729: (SEQ ID NO: 33)
5'-GGGAGTACGCGAGATCATCCTTGGCAAGGGCCAACACCGGCA-3'
[0189] The amplification reactions (50 .mu.l) were composed of Pfx
Amplification Buffer, 0.25 mM dNTPs, 10 ng/.mu.l pMJ05, 6.4 .mu.M
primer 993728, 3.2 .mu.M primer 993727, 1 mM MgCl.sub.2, and 2.5
units of Pfx DNA polymerase. The reactions were incubated in an
Eppendorf Mastercycler 5333 programmed as follows: 30 cycles each
for 60 seconds at 94.degree. C., 60 seconds at 60.degree. C., and
240 seconds at 72.degree. C. (15 minute final extension). The
reaction products were isolated on a 1.0% agarose gel using TAE
buffer where a 1063 bp product band was excised from the gel and
purified using a QIAquick Gel Extraction Kit according to the
manufacturer's instructions.
[0190] The purified overlapping fragments were used as a template
for amplification using primer 993724 (sense) and primer 993727
(antisense) described above to precisely fuse the 1063 bp fragment
comprising the Trichoderma reesei cbh1 promoter/Humicola insolens
endoglucanase V signal sequence to the 2523 bp fragment comprising
the Aspergillus oryzae beta-glucosidase open reading frame by
overlapping PCR.
[0191] The amplification reactions (50 .mu.l) were composed of Pfx
Amplification Buffer, 0.25 mM dNTPs, 6.4 .mu.M primer 993724, 3.2
.mu.M primer 993727, 1 mM MgCl.sub.2, and 2.5 units of Pfx DNA
polymerase. The reactions were incubated in an Eppendorf
Mastercycler 5333 programmed as follows: 30 cycles each for 60
seconds at 94.degree. C., 60 seconds at 60.degree. C., and 240
seconds at 72.degree. C. (15 minute final extension). The reaction
products were isolated on a 1.0% agarose gel using TAE buffer where
a 3591 bp product band was excised from the gel and purified using
a QIAquick Gel Extraction Kit according to the manufacturer's
instructions.
[0192] The resulting 3591 bp fragment was digested with Sal I and
Spe I and ligated into pMJ04 digested with the same restriction
enzymes to generate pSMai135 (FIG. 10).
Example 7
Expression of Aspergillus oryzae Beta-Glucosidase Comparing Native
and Heterologous Humicola insolens Endoglucanase V Secretion Signal
in Trichoderma reesei
[0193] Plasmid pSMai130, in which the Aspergillus oryzae
beta-glucosidase is expressed from the cbh1 promoter and native
secretion signal (FIG. 8, SEQ ID NOs: 34 (DNA sequence) and 35
(deduced amino acid sequence)), or pSMai135 encoding the mature
Aspergillus oryzae beta-glucosidase enzyme linked to the Humicola
insolens endoglucanase V secretion signal (FIG. 9, SEQ ID NOs: 36
(DNA sequence) and 37 (deduced amino acid sequence)), was
introduced into Trichoderma reesei RutC30 by PEG-mediated
transformation (Penttila et al., 1987, supra). Both plasmids
contain the Aspergillus nidulans amdS gene to enable transformants
to grow on acetamide as the sole nitrogen source.
[0194] Trichoderma reesei RutC30 was cultivated at 27.degree. C.
and 90 rpm in 25 ml of YP medium supplemented with 2% (w/v) glucose
and 10 mM uridine for 17 hours. Mycelia was collected by filtration
using Millipore's Vacuum Driven Disposable Filtration System
(Millipore, Bedford, Mass.) and washed twice with deionized water
and twice with 1.2 M sorbitol. Protoplasts were generated by
suspending the washed mycelia in 20 ml of 1.2 M sorbitol containing
15 mg of Glucanex (Novozymes A/S, Bagsvrd, Denmark) per ml and 0.36
units of chitinase (Sigma Chemical Co., St. Louis, Mo.) per ml and
incubating for 15-25 minutes at 34.degree. C. with gentle shaking
at 90 rpm. Protoplasts were collected by centrifuging for 7 minutes
at 400.times.g and washed twice with cold 1.2 M sorbitol. The
protoplasts were counted using a haemacytometer and re-suspended in
STC to a final concentration of 1.times.10.sup.8 protoplasts per
ml. Excess protoplasts were stored in a Cryo 1.degree. C. Freezing
Container (Nalgene, Rochester, N.Y.) at -80.degree. C.
[0195] Approximately 7 .mu.g of Pme I digested expression plasmid
(pSMai130 or pSMai135) was added to 100 .mu.l of protoplast
solution and mixed gently, followed by 260 .mu.l of PEG buffer,
mixed, and incubated at room temperature for 30 minutes. STC (3 ml)
was then added, mixed and the transformation solution was plated
onto COVE plates using Aspergillus nidulans amdS selection. The
plates were incubated at 28.degree. C. for 5-7 days. Transformants
were sub-cultured onto COVE2 plates and grown at 28.degree. C.
[0196] One hundred and ten amdS positive transformants were
obtained with pSMai130 and 65 transformants with pSMai135. Twenty
transformants designated SMA130 obtained with pSMai130 (native
secretion signal) and 67 transformants designated SMA135 obtained
with pSMai135 (heterologous secretion signal) twere subcultured
onto fresh plates containing acetamide and allowed to sporulate for
7 days at 28.degree. C.
[0197] The 20 SMA130 and 67 SMA135 Trichoderma reesei transformants
were cultivated in 125 ml baffled shake flasks containing 25 ml of
cellulase-inducing media at pH 6.0 inoculated with spores of the
transformants and incubated at 28.degree. C. and 200 rpm for 7
days. Trichoderma reesei RutC30 was run as a control. Culture broth
samples were removed at day 7. One ml of each culture broth was
centrifuged at 15,700.times.g for 5 minutes in a micro-centrifuge
and the supernatants transferred to new tubes. Samples were stored
at 4.degree. C. until enzyme assay. The supernatants were assayed
for beta-glucosidase activity using
p-nitrophenyl-beta-D-glucopyranoside as substrate, as described
above.
[0198] All 20 SMA130 transformants exhibited equivalent
beta-glucosidase activity to that of the host strain, Trichoderma
reesei RutC30. In contrast, a number of SMA135 transformants showed
beta-glucosidase activities several-fold more than that of
Trichoderma reesei RutC30. Transformant SMA135-04 produced the
highest beta-glucosidase activity having 7 times more
beta-glucosidase activity than produced by Trichoderma reesei
RutC30 as a control.
[0199] SDS-PAGE was carried out using Criterion Tris-HCl (5%
resolving) gels (BioRad, Hercules, Calif.) with The Criterion
System (BioRad, Hercules, Calif.). Five .mu.l of day 7 supernatants
(see above) were suspended in 2.times. concentration of Laemmli
Sample Buffer (BioRad, Hercules, Calif.) and boiled in the presence
of 5% beta-mercaptoethanol for 3 minutes. The supernatant samples
were loaded onto a polyacrylamide gel and subjected to
electrophoresis with 1.times. Tris/Glycine/SDS as running buffer
(BioRad, Hercules, Calif.). The resulting gel was stained with
BioRad's Bio-Safe Coomassie Stain.
[0200] No beta-glucosidase protein was visible by SDS-PAGE for the
Trichoderma reesei SMA130 transformant culture broth supernatants.
In contrast, 26 of the 38 Trichoderma reesei SMA135 transformants
produced a protein of approximately 110 kDa that was not visible in
Trichoderma reesei RutC30 as control. Transformant Trichoderma
reesei SMA135-04 produced the highest level of
beta-glucosidase.
Example 8
Fermentation of Aspergillus oryzae SMA135-04
[0201] Fermentation was performed on Aspergillus oryzae SMA135-04
to determine the production level of beta-glucosidase activity.
Trichoderma reesei RutC30 (host strain) was run as a control.
Spores of Trichoderma reesei SMA135-04 were inoculated into 500 ml
shake flasks, containing 100 ml of Inoculum Medium. The flasks were
placed into an orbital shaker at 28.degree. C. for approximately 48
hours at which time 50 ml of the culture was inoculated into 1.8
liters of Fermentation Medium (see above) in a 2 liter fermentation
vessel. The fermentations were run at a pH of 5.0, 28.degree. C.,
with minimum dissolved oxygen at a 25% at a 1.0 VVM air flow and an
agitation of 1100. Feed Medium was administrated into the
fermentation vessel at 18 hours with a feed rate of 3.6 g/hour for
33 hours and then 7.2 g/hour. The fermentations ran for 165 hours
at which time the final fermentation broths were centrifuged and
the supernatants stored at -20.degree. C. until beta-glucosidase
activity assay using the procedure described earlier.
[0202] Beta-glucosidase activity on the Trichoderma reesei
SMA135-04 fermentation sample was determined to be approximately 8
times more active than that of Trichoderma reesei RutC30.
Example 9
Construction of pSATe111 and pALFd1 Saccharomyces cerevisiae
Expression Vectors
[0203] A 2,605 bp DNA fragment comprising the region from the ATG
start codon to the TM stop codon of the Aspergillus oryzae
beta-glucosidase coding sequence (SEQ ID NO: 42 for cDNA sequence
and SEQ ID NO: 43 for the deduced amino acid sequence) was
amplified by PCR from pJaL660 (WO 2002/095014) as template with
primers 992127 (sense) and 992328 (antisense) shown below:
TABLE-US-00020 992127: (SEQ ID NO: 38)
5'-GCAGATCTACCATGAAGCTTGGTTGGATCGAG-3' 992328: (SEQ ID NO: 39)
5'-GCCTCAGATTACTGGGCCTTAGGCAGCGAG-3'
[0204] Primer 992127 has an upstream Bgl II site and the primer
992328 has a downstream Xho I site.
[0205] The amplification reactions (50 .mu.l) were composed of
1.times. PCR buffer containing MgCl.sub.2 (Roche Applied Science,
Manheim, Germany), 0.25 mM dNTPs, 50 .mu.M primer 992127, 50 .mu.M
primer 992328, 80 ng of pJaL660, and 2.5 units of Pwo DNA
Polymerase (Roche Applied Science, Manheim, Germany). The reactions
were incubated in an Eppendorf Mastercycler 5333 programmed for 1
cycle at 94.degree. C. for 5 minutes followed by 25 cycles each at
94.degree. C. for 60 seconds, 55.degree. C. for 60 seconds, and
72.degree. C. for 120 seconds (10 minute final extension). The PCR
product was then subcloned into the pCR-Blunt II-TOPO vector using
the ZeroBlunt.TM. TOPO PCR Cloning Kit (Invitrogen, Carlsbad,
Calif.) following the manufacturer's instructions to generate
plasmid pSATe101 (FIG. 11). Plasmid pSATe101 was digested with Bgl
II and Xho I to liberate the beta-glucosidase gene. The reaction
products were isolated on a 1.0% agarose gel using TAE buffer where
a 2.6 kb product band was excised from the gel and purified using a
QIAquick Gel Extraction Kit according to the manufacturer's
instructions.
[0206] The 2.6 kb PCR product was digested and cloned into Bam HI
and Xho I sites of the copper inducible 2 .mu.m yeast expression
vector pCu426 (Labbe and Thiele, 1999, Methods Enzymol. 306:
145-53), to generate pSATe111 (FIG. 12).
[0207] Plasmid pALFd1 was constructed to determine if enhanced
Aspergillus oryzae beta-glucosidase production and secretion could
also be achieved in Saccharomyces cerevisiae by swapping the native
Aspergillus oryzae beta-glucosidase secretion signal with the
Humicola insolens endoglucanase V signal peptide.
[0208] Plasmid pSATe111 was digested with Xho I and Spe I to
release 2.6 kb (Aspergillus oryzae beta-glucosidase) and 6 kb (rest
of the vector) fragments. The 6 kb fragment was isolated and
ligated to the 2.6 kb PCR fragment, containing the Aspergillus
oryzae beta-glucosidase coding region (minus the secretion signal
sequence) and the Humicola insolens endoglucanase V signal
sequence, which was amplified from pSMai135 using primers 993950
and 993951 shown below. The primers contain the Xho I and Spe I
restriction sites at their ends for subsequent subcloning into the
Xho I and Spe I restriction sites of pSATe111.
TABLE-US-00021 Primer 993950: (SEQ ID NO: 40)
5'-AATCCGACTAGTGGATCTACCATGCGTTCCTCCCCCCTCC-3' Primer 993951: (SEQ
ID NO: 41) 5'-GCGGGCCTCGAGTTACTGGGCCTTAGGCAGCG-3'
[0209] The amplification reactions (100 .mu.l) were composed of PCR
Thermo Pol Buffer, 0.20 mM dNTPs, 0.14 .mu.g of pSMai135 plasmid
DNA, 50 .mu.M primer 993950, 50 .mu.M primer 993951, and 2 units of
Vent DNA polymerase. The reactions were incubated in a RoboCycler
Gradient 40 Thermal Cycler (Stratagene, La Jolla, Calif.)
programmed as follows: one cycle of 1 minute at 95.degree. C., 25
cycles each for 1 minute at 95.degree. C., 1 minute at 60 or
64.degree. C., and 3 minutes at 72.degree. C. (10 minute final
extension). The reaction products were visualized on a 0.7% agarose
gel using TAE buffer. The resulting 2.6 kb fragment bands were
purified using a PCR MinElute PCR Purification (QIAGEN, Chatsworth,
Calif.) according to the manufacturer's instructions. The purified
fragments were combined and digested with Xho I and Spe I and
ligated into pSATe111 digested with the same two restriction
enzymes to generate pALFd1 (FIG. 13).
Example 10
Expression of Aspergillus oryzae BbetaGlucosidaseComparingNative
and Heterologous Secretion Signal in Saccharomyces cerevisiae
[0210] Plasmid pALFd1 (approximately 600 ng) was transformed into
freshly made Saccharomyces cerevisiae YNG 318 competent cells
according to the YEASTMAKER Yeast Transformation Protocol, CLONTECH
Laboratories, Inc., Palo Alto, Calif. Transformed cells were plated
onto yeast selection plates containing 0.15 mg of the chromogenic
substrate 5-bromo-4-chloro-3-indolyl-beta-D-glucopyranoside per ml,
which yield blue colonies when beta-glucosidase is present. The
plates were incubated at 30.degree. C. for 4 days.
[0211] Colonies harboring the expression vector with the Humicola
insolens endoglucanase V secretion signal were generally darker
blue in color than the colonies that had the native Aspergillus
oryzae beta-glucosidase signal sequence, indicating that more
Aspergillus oryzae beta-glucosidase was secreted using the Humicola
insolens endoglucanase V secretion signal. Approximately, 242 blue
colonies from both constructs were picked using an automated colony
picker (QPix, Genetix USA, Inc., Boston, Mass.). The 242
transformants were inoculated into yeast selection medium (which
contains copper) to induce expression and secretion of Aspergillus
oryzae beta-glucosidase. Broth from day 7 96-well culture was taken
from each of the 245 colonies and assayed for beta-glucosidase
activity using p-nitrophenyl-beta-D-glucopyranoside as substrate as
described above. The results showed that colonies expressing
beta-glucosidase with the heterologous signal sequence were 6.6
times more active than the colonies that were transformed with the
Aspergillus oryzae beta-glucosidase with the native secretion
signal.
Example 11
Identification of a Glycosyl Hydrolase Family GH3A Gene in the
Genomic Sequence of Aspergillus fumigatus
[0212] A tblastn search (Altschul et al., 1997, Nucleic Acids Res.
25: 3389-3402) of the Aspergillus fumigatus partial genome sequence
(The Institute for Genomic Research, Rockville, Md.) was carried
out using as query a beta-glucosidase protein sequence from
Aspergillus aculeates (Accession No. P48825). Several genes were
identified as putative Family GH3A homologs based upon a high
degree of similarity to the query sequence at the amino acid level.
One genomic region of approximately 3000 bp with greater than 70%
identity to the query sequence at the amino acid level was chosen
for further study.
Example 12
Aspergillus fumigatus Genomic DNA Extraction
[0213] Aspergillus fumigatus PaHa34 was grown in 250 ml of potato
dextrose medium in a baffled shake flask at 37.degree. C. and 240
rpm. Mycelia were harvested by filtration, washed twice in TE
buffer (10 mM Tris-1 mM EDTA), and frozen under liquid nitrogen.
Frozen mycelia were ground by mortar and pestle to a fine powder,
which was resuspended in pH 8.0 buffer containing 10 mM Tris, 100
mM EDTA, 1% Triton X-100, 0.5 M guanidine-HCl, and 200 mM NaCl.
DNase-free RNase A was added at a concentration of 20 .mu.g/ml and
the lysate was incubated at 37.degree. C. for 30 minutes. Cellular
debris was removed by centrifugation, and DNA was isolated by using
a Qiagen Maxi 500 column (QIAGEN Inc., Chatsworth, Calif.). The
columns were equilibrated in 10 ml of QBT washed with 30 ml of QC,
and eluted with 15 ml of QF (all buffers from QIAGEN Inc.,
Chatsworth, Calif.). DNA was precipitated in isopropanol, washed in
70% ethanol, and recovered by centrifugation. The DNA was
resuspended in TE buffer.
Example 13
Cloning of the Family GH3A Beta-Glucosidase Gene and Construction
of an Aspergillus oryzae Expression Vector
[0214] Two synthetic oligonucleotide primers shown below were
designed to PCR amplify an Aspergillus fumigatus PaHa34 gene
encoding a Family GH3A beta-glucosidase from the genomic DNA
prepared in Example 14. An InFusion Cloning Kit (BD Biosciences,
Palo Alto, Calif.) was used to clone the fragment directly into the
expression vector, pAILo2 (FIG. 14), without the need for
restriction digests and ligation.
TABLE-US-00022 Forward primer: (SEQ ID NO: 44)
5'-ACTGGATTTACCATGAGATTCGGTTGGCTCG-3' Reverse primer: (SEQ ID NO:
45) 5'-AGTCACCTCTAGTTACTAGTAGACACGGGGC-3'
Bold letters represent coding sequence. The remaining sequence is
homologous to the insertion sites of pAILo2, described in Example
7.
[0215] Fifty picomoles of each of the primers above were used in a
PCR reaction containing 100 ng of Aspergillus fumigatus genomic
DNA, 1.times. Pfx Amplification Buffer, 1.5 .mu.l of 10 mM blend of
dATP, dTTP, dGTP, and dCTP, 2.5 units of Pfx DNA Polymerase, 1
.mu.l of 50 mM MgSO.sub.4 and 2.5 .mu.l of 10.times. pCRx Enhancer
solution (Invitrogen, Carlsbad, Calif.) in a final volume of 50
.mu.l. The reactions were incubated in an Eppendorf Mastercycler
5333 programmed as follows: one cycle at 94.degree. C. for 2
minutes; and 30 cycles each at 94.degree. C. for 15 seconds,
55.degree. C. for 30 seconds, and 68.degree. C. for 3 minutes. The
heat block then went to a 4.degree. C. soak cycle.
[0216] The reaction products were isolated on a 1.0% agarose gel
using TAE buffer where a 3 kb product band was excised from the gel
and purified using a QIAquick Gel Extraction Kit according to the
manufacturer's instructions.
[0217] The fragment was then cloned into the pAILo2 expression
vector using an Infusion Cloning Kit. The vector was digested with
Nco I and Pac I. The fragment was purified by gel electrophoresis
and Qiaquick gel purification. The gene fragment and digested
vector were ligated together in a reaction resulting in the
expression plasmid pEJG97 (FIG. 15) in which transcription of the
Family GH3A beta-glucosidase gene was under the control of the
NA2-tpi promoter. The ligation reaction (50 .mu.l) was composed of
1.times. InFusion Buffer (BD Biosciences, Palo Alto, Calif.),
1.times. BSA (BD Biosciences, Palo Alto, Calif.), 1 .mu.l of
Infusion enzyme (diluted 1:10) (BD Biosciences, Palo Alto, Calif.),
150 ng of pAILo2 digested with Nco I and Pac I, and 50 ng of the
Aspergillus fumigatus beta-glucosidase purified PCR product. The
reaction was incubated at room temperature for 30 minutes. One
.mu.l of the reaction was used to transform E. coli XL10 Solopac
Gold cells (Stratagene, La Jolla, Calif.). An E. coli transformant
containing the pEJG97 plasmid was detected by restriction digestion
of the plasmid DNA.
Example 14
Characterization of the Aspergillus fumigatus Genomic Sequence
Encoding a Family GH3A Beta-Glucosidase
[0218] DNA sequencing of the Aspergillus fumigatus beta-glucosidase
gene from pEJG97 was performed as described previously using a
primer walking strategy. A gene model for the Aspergillus fumigatus
sequence was constructed based on similarity to homologous genes
from Aspergillus aculeatus, Aspergillus niger, and Aspergillus
kawachii. The nucleotide sequence (SEQ ID NO: 46) and deduced amino
acid sequence (SEQ ID NO: 47) are shown in FIGS. 16A and 16B. The
genomic fragment encodes a polypeptide of 863 amino acids,
interrupted by 8 introns of 62, 55, 58, 63, 58, 58, 63 and 51 bp.
The %G+C content of the gene is 54.3%. Using the SignalP software
program (Nielsen et al., 1997, Protein Engineering 10: 1-6), a
signal peptide of 19 residues was predicted. The predicted mature
protein contains 844 amino acids with a molecular mass of 91.7
kDa.
[0219] A comparative alignment of beta-glucosidase sequences was
determined using the Clustal W method (Higgins, 1989, CABIOS 5:
151-153) using the LASERGENE.TM. MEGALIGN.TM. software (DNASTAR,
Inc., Madison, Wis.) with an identity table and the following
multiple alignment parameters: Gap penalty of 10 and gap length
penalty of 10. Pairwise alignment parameters were Ktuple=1, gap
penalty=3, windows=5, and diagonals=5. The alignment showed that
the deduced amino acid sequence of the Aspergillus fumigatus
beta-glucosidase gene shares 78%, 76%, and 76% identity to the
deduced amino acid sequences of the Aspergillus aculeatus
(accession number P48825), Aspergillus niger (accession number
000089), and Aspergillus kawachii (accession number P87076)
beta-glucosidases.
Example 15
Expression of the Aspergillus fumigatus Family GH3A
Beta-Glucosidase Gene in Aspergillus oryzae JAL250
[0220] Aspergillus oryzae JaL250 protoplasts were prepared
according to the method of Christensen et al., 1988, Bio/Technology
6: 1419-1422. Five .mu.g of pEJG97 (as well as pAILo2 as a vector
control) was used to transform Aspergillus oryzae JAL250.
[0221] The transformation of Aspergillus oryzae Jal250 with pEJG97
yielded about 100 transformants. Ten transformants were isolated to
individual PDA plates.
[0222] Confluent PDA plates of five of the ten transformants were
washed with 5 ml of 0.01% Tween 20 and inoculated separately into
25 ml of MDU2BP medium in 125 ml glass shake flasks and incubated
at 34.degree. C., 250 rpm. Five days after incubation, 0.5 .mu.l of
supernatant from each culture was analyzed using 8-16% Tris-Glycine
SDS-PAGE gels (Invitrogen, Carlsbad, Calif.) according to the
manufacturer's instructions. SDS-PAGE profiles of the cultures
showed that one of the transformants (designated transformant 1)
had a major band of approximately 130 kDa.
Example 16
Extraction of Total RNA from Aspergillus oryzae
[0223] The Aspergillus oryzae transformant described in Example 13
was frozen in liquid nitrogen and stored at -80.degree. C.
Subsequently, the frozen tissue was ground in an electric coffee
grinder with a few chips of dry ice added to keep the powdered
mycelia frozen. Then, the ground material was transferred with a
spatula to a 50 ml sterile conical tube which had been previously
filled with 20 ml of Fenozol (Active Motif, Inc., Carlsbad,
Calif.). The mixture was mixed rapidly to dissolve the frozen
material to a thick solution, and placed in a 50.degree. C. water
bath for 15 minutes. Five ml of RNase-free chloroform was added to
the mixture and vortexed vigorously. Then, the mixture was allowed
to stand at room temperature for 10 minutes. Next the mixture was
centrifuged at 1300.times.g in a Sorvall RT7 centrifuge (Sorvall,
Inc, Newtown, Conn.) at room temperature for 20 minutes. The top
phase was transferred to a new conical tube and an equal volume of
phenol-chloroform-isoamylalcohol (25:24:1) was added. The mixture
was vortexed and centrifuged for 10 minutes. This procedure was
repeated twice so that three phenol-chloroform isoamylalcohol
extractions were done. Then, the top phase was transferred to a new
tube and an equal volume of chloroform:isoamylalcohol (24:1) was
added. The mixture was vortexed once again and centrifuged for 10
minutes. After centrifugation, the aqueous phase (approximately 5
ml) was transferred to a new Oak Ridge tube and 0.5 ml of 3 M
sodium acetate pH 5.2 and 6.25 ml of isopropanol were added. The
mixture was mixed and incubated at room temperature for 15 minutes.
Subsequently, the mixture was centrifuged at 12,000.times.g for 30
minutes, at 4.degree. C. in a Sorvall RCSB (Sorvall, Inc, Newtown,
Conn.). Following centrifugation, the supernatant was removed and
18 ml of 70% ethanol was carefully added to the pellet. Another
centrifugation step was done for 10 minutes at 4.degree. C. at
12,000.times.g. The supernatant was carefully removed and the
pellet was air dried. The RNA pellet was resuspended in 500 .mu.l
of diethyl pyrocarbonate (DEPC)-treated water. Heating at
65.degree. C. for 10 minutes aided in resuspension. The total RNA
was stored at -80.degree. C. Quantitation and assessing RNA quality
was done on an Agilent Bioanalyzer 2100 (Englewood, Colo.) using
RNA chips. All the materials and reagents used in this protocol
were RNAse-free.
Example 17
Cloning of the Aspergillus fumigatus Beta-Glucosidase cDNA
Sequence
[0224] The total RNA described in Example 16 was used to clone the
Aspergillus fumigatus beta-glucosidase cDNA sequence (SEQ ID NO: 48
for cDNA sequence and SEQ ID NO: 49 for the deduced amino acid
sequence). The mRNA from the total RNA was purified using a
Poly(A)Purist Mag Kit (Ambion, Inc., Austin, Tex.) following the
manufacturer's instructions. The Aspergillus fumigatus
beta-glucosidase cDNA sequence, was then amplified in two
fragments: a 1,337 bp DNA fragment spanning from the ATG start
codon to the 1,332 position (labeled as 5' fragment) and a second
1,300 bp DNA fragment (labeled 3' fragment) spanning from the 1,303
position until the stop codon using the ProStar UltraHF RT-PCR
System (Stratagene, La Jolla, Calif.), following the manufacturer's
protocol for a 50 .mu.l reaction using 200 ng of poly-A mRNA with
primes Afuma (sense) and Afumc (antisense) for the 5' fragment and
primers Afumd (sense) and Afumb (antisense) for the 3' fragment as
shown below:
TABLE-US-00023 (SEQ ID NO: 50) Afuma:
5'-GGCTCATGAGATTCGGTTGGCTCGAGGTC-3' (SEQ ID NO: 51) Afumc:
5'-GCCGTTATCACAGCCGCGGTCGGGGCAGCC-3' (SEQ ID NO: 52) Afumd:
5'-GGCTGCCCCGACCGCGGCTGTGATAACGGC-3' (SEQ ID NO: 53) Afumb:
5'-GCTTAATTAATCTAGTAGACACGGGGCAGAGGCGC-3'
Primer Afuma has an upstream Bsp HI site and primer Afumb has a
downstream Pac I site. Twenty nine nucleotides at the 3'-end of the
1,337 fragment overlapped with the 5'-end of the 1,303 fragment. In
the overlap region there was a unique Sac II site.
[0225] Both fragments were subcloned individually into the
pCR-BluntII-TOPO vector using a Zero Blunt.TM. TOPO PCR Cloning Kit
for sequencing, following the manufacturer's protocol, generating
plasmids pCR4Blunt-TOPOAfcDNA5' (FIG. 17) and
pCR4Blunt-TOPOAfcDNA3' (FIG. 18), containing the 5' and 3'
fragments, respectively.
[0226] The entire coding region of both Aspergillus fumigatus
beta-glucosidase fragments was confirmed by sequencing using 0.5
.mu.l of each plasmid DNA and 3.2 pmol of the following
primers:
TABLE-US-00024 (SEQ ID NO: 54) BGLU1.for: 5'-ACACTGGCGGAGAAGG-3'
(SEQ ID NO: 55) BGLU2.for: 5'-GCCCAGGGATATGGTTAC-3' (SEQ ID NO: 56)
BGLU3.for: 5'-CGACTCTGGAGAGGGTTTC-3' (SEQ ID NO: 57) BGLU4.rev:
5'-GGACTGGGTCATCACAAAG-3' (SEQ ID NO: 58) BGLU5.rev:
5'-GCGAGAGGTCATCAGCA-3' (SEQ ID NO: 59) M13 forward:
5'-GTAAAACGACGGCCAGT-3' (SEQ ID NO: 60) M13 reverse:
5'-CAGGAAACAGCTATGA-3'
[0227] Sequencing results indicated the presence of several
nucleotide changes when comparing the Aspergillus fumigatus
beta-glucosidase cDNA sequence obtained to the Aspergillus
fumigatus beta-glucosidase cDNA sequence deduced from genome data
of The Institute for Genomic Research (Rockville, Md.). At position
500, T was replaced by C, so that the coding sequence GTT was
changed to GCT, so that valine was replaced by alanine. At position
903, T was replaced by C, so that the coding sequence CCC was
changed to CCT, however, this change was silent. At position 2,191,
G was replaced by C, so that the coding sequence CAG was changed to
GAG, so that glutamic acid was replaced by glutamine. Finally, at
position 2,368, C was replaced by T, so that the coding sequence
CTG was changed to TTG, however, this change was also silent.
[0228] Once the two fragments had been sequenced, both clones
containing each fragment were digested using approximately 9 .mu.g
of each plasmid DNA with Sac II and Pme I. Digestion of the
pCR4Blunt-TOPOAfcDNA5' vector with the above enzymes generated a
fragment of 3,956 bp (containing most of the vector) and a second
fragment of and 1,339 bp (containing the Aspergillus fumigatus
beta-glucosidase cDNA 5' fragment). Digestion of the
pCR4Blunt-TOPOAfcDNA3' vector with these same enzymes generated a
5,227 bp fragment (containing most of the pCR4Blunt-TOPO vector and
the Aspergillus fumigatus beta-glucosidase cDNA 3' fragment) and a
second fragment of 31 bp.
[0229] Digested pCR4Blunt-TOPOAfcDNA3' was treated with shrimp
alkaline phosphatase for dephosphorylation of the digested DNA
products by adding 1.times. SAP buffer and 1 .mu.l of shrimp
alkaline phosphatase (Roche Applied Science, Manheim, Germany) and
incubating the reaction for 10 minutes at 37.degree. C. followed by
incubation at 85.degree. C. for 10 minutes for enzyme inactivation.
Both digestions were run on 0.7% agarose gel with TAE buffer and
purified using a QIAGEN Gel Purification Kit according to the
manufacturer's instructions. The 1,339 bp band generated from the
pCR4Blunt-TOPOAfcDNA5' digestion and the 5,527 bp fragment
generated from the pCR4Blunt-TOPOAfcDNA3' digestion were ligated by
using the Rapid DNA Ligation Kit (Roche Applied Science, Manheim,
Germany) following the manufacturer's instructions. The ligation
reaction was transformed into XL1-Blue E. coli
subcloning-competetent cells according to the manufacturer's
instructions (Stratagene, La Jolla, Calif.). Upon transformation,
plasmid DNA from an isolated colony was sequenced to confirm that
both the 5' and 3' fragments of the Aspergillus fumigatus
beta-glucosidase cDNA were subcloned in tandem generating a 6,566
bp pCR4Blunt-TOPOAfcDNA vector (FIG. 19).
Example 18
Construction of the pALFd6 and pALFd7 Sacharomyces cerevisiae
Expression Vectors
[0230] The Aspergillus fumigatus beta-glucosidase full length cDNA
was amplified by PCR using the following primers that have homology
to pCu426 and the 5' and 3' sequences of the Aspergillus fumigatus
beta-glucosidase cDNA:
AfumigatusBGUpper:
TABLE-US-00025 [0231] (SEQ ID NO: 61)
5'-CTTCTTGTTAGTGCAATATCATATAGAAGTCATCGACTAGTGGATCTA
CCATGAGATTCGGTTGGCTCG-3'
ATGAGATTCGGTTGGCTCG has homology to the 5' end of the Aspergillus
fumigatus cDNA
AfumigatusBGLower:
TABLE-US-00026 [0232] (SEQ ID NO: 62)
5'-GCGTGAATGTAAGCGTGACATAACTAATTACATGACTCGAGCTAGTAG
ACACGGGGCAGAG-3'
CTAGTAGACACGGGGCAGAG has homology to the 3' end of the Aspergillus
fumigatus cDNA
[0233] The amplification reaction (100 .mu.l) was composed of 0.5
.mu.l of the pCR4Blunt-TOPOAfcDNA plasmid containing the
Aspergillus fumigatus cDNA sequence, 1.times. Pfx Amplification
Buffer, 50 .mu.M each of dATP, dCTP, dGTP, and dTTP, 50 pmole of
each above primer, 1.5 mM MgSO.sub.4, and 2.5 units of Platinum Pfx
DNA polymerase. The reactions were incubated in an RoboCycler
Gradient 40 programmed for 1 cycle at 95.degree. C. for 5 minutes;
25 cycles each at 95.degree. C. for 1 minute, 50.degree. C. for 1
minute; and 72.degree. C. for 3 minutes; and a final extension
cycle at 72.degree. C. for 10 minutes. The PCR reaction was
purified using a QIAquick PCR Purification Kit (QIAGEN Inc.,
Valencia, Calif.). DNA was eluted into 30 .mu.l of EB buffer
(QIAGEN Inc., Valencia, Calif.). The PCR product comprised 37 bp of
homologous DNA sequence which was mixed with 1 .mu.l of pCU426
gapped with Spe I and Xho I for cotransformation into Saccharomyces
cerevisiae YNG318 competent cells as described in Example 10. These
colonies did not turn blue, suggesting some sequencing error in the
Aspergillus fumigatus beta-glucosidase cDNA sequence. Further
sequencing of the Aspergillus fumigatus cDNA sequence indicated an
insertion of an extra nucleotide in the cDNA sequence, which
disrupted the open-reading frame of the enzyme. Therefore, this
construct had to be fixed.
[0234] Simultaneously to expressing the Aspergillus fumigatus
beta-glucosidase cDNA in Saccharomyces cerevisiae, the Humicola
insolens endoglucanase V signal sequence was swapped with the
native signal sequence of the Aspergillus fumigatus cDNA sequence
also for expression in Saccharomyces cerevisiae to compare the
expression of the enzymes with both signal sequences. The
Aspergillus fumigatus cDNA sequence was amplified by PCR with a
primer that has homology to the Humicola insolens endoglucanase V
signal sequence in pALFd1 as well as homology to the 5'-end of the
mature Aspergillus fumigatus beta-glucosidase cDNA sequence. The
primers used for amplification of the Aspergillus fumigatus
beta-glucosidase cDNA sequence are the AfumigatusBGLower primer
described before and the HiEGVAfumigatus primer described
below:
HiEGVAfumigatus:
TABLE-US-00027 [0235] (SEQ ID NO: 63)
5'-CCGCTCCGCCGTTGTGGCCGCCCTGCCGGTGTTGGCCCTTGCCGAATT
GGCTTTCTCTCC-3'
GAATTGGCTTTCTCTCC has homology to the 5' end of the Aspergillus
fumigatus mature sequence.
[0236] The amplification reaction (100 .mu.l) was composed of 0.5
.mu.l of the pCR4Blunt-TOPOAfcDNA plasmid containing the
Aspergillus fumigatus cDNA sequence, 1.times. Pfx Amplification
Buffer, 50 .mu.M each of dATP, dCTP, dGTP, and dTTP, 50 pmole of
each above primer, 1.5 mM MgSO.sub.4, and 2.5 units of Platinum Pfx
DNA polymerase. The reactions were incubated in an RoboCycler
Gradient 40 programmed for 1 cycle at 95.degree. C. for 5 minutes;
25 cycles each at 95.degree. C. for 1 minute, 50.degree. C. for 1
minute; and 72.degree. C. for 3 minutes; and a final extension
cycle at 72.degree. C. for 10 minutes. The PCR reaction was
purified using a QIAquick PCR Purification Kit. DNA was eluted into
10 .mu.l of EB buffer. Three ul of the clean-up PCR product was
mixed with 1.8 .mu.l of pALFd1 gapped with Eco NI and Xho I for
cotransformation into Saccharomyces cerevisiae YNG318 competent
cells as described in Example 10. These colonies turned light blue.
However, one colony stood out by being very blue. DNA rescue from
this colony was done according to the protocol described by Kaiser
and Auer, 1993, BioTechniques 14: 552, except 20 .mu.l of yeast
lysis buffer (1% SDS, 10 mM Tris-HCl, 1 mM EDTA pH 8) was used, and
the plasmid was transformed into E. coli SURE
electroporation-competent cells (Stratagene, La Jolla, Calif.) for
sequencing. Full-length sequencing indicated the Aspergillus
fumigatus beta-glucosidase cDNA sequence was correct. This plasmid
was designated pALFd7 (FIG. 20), which comprised the Aspergillus
fumigatus beta-glucosidase cDNA sequence with the Humicola insolens
endoglucanase V signal sequence for yeast expression.
[0237] To produce a yeast expression vector containing the correct
Aspergillus fumigatus cDNA sequence with its native signal
sequence, the region containing the correct nucleotide sequence
from the yeast expression vector containing the Aspergillus
fumigatus cDNA sequence with the Humicola insolens endoglucanase V
signal sequence (pALFd7) was amplified by PCR using the above
BGLU.5rev primer and the following primer:
TABLE-US-00028 BGL.7for: 5'-CTGGCGTTGGCGCTGTC-3' (SEQ ID NO:
64)
[0238] The amplification reaction (100 .mu.l) was composed of 0.5
.mu.l of pALFd7, 1.times. Pfx Amplification Buffer, 50 .mu.M each
of dATP, dCTP, dGTP, and dTTP, 50 pmole of each above primer, 1.5
mM MgSO.sub.4, and 2.5 units of Platinum Pfx DNA polymerase. The
reactions were incubated in an RoboCycler Gradient 40 programmed
for 1 cycle at 95.degree. C. for 5 minutes; 25 cycles each at
95.degree. C. for 1 minute, 50.degree. C. for 1 minute; and
72.degree. C. for 1 minutes; and a final extension cycle at
72.degree. C. for 10 minutes.
[0239] The 701 bp PCR fragment was purified using a QIAquick PCR
Purification Kit. DNA was eluted into 10 .mu.l of EB buffer. Three
ul of the clean-up PCR product was mixed with 3 .mu.l of the yeast
expression vector containing the Aspergillus fumigatus cDNA
sequence with the native signal sequence and the extra nucleotide
gapped with the Sac II and Xma I vector for cotransformation into
Saccharomyces cerevisiae YNG318 competent cells as described as
described in Example 10. These colonies turned blue. DNA rescue
from one randomly picked blue colony was done as above, the plasmid
was transformed into E. coli SURE electroporation-competent cells
(Stratagene, La Jolla, Calif.) for sequencing. Full-length
sequencing indicated the Aspergillus fumigatus beta-glucosidase
cDNA sequence was correct. This yeast expression vector was
designated pALFd6 (FIG. 21), which comprised the Aspergillus
fumigatus cDNA sequence with its native signal sequence.
Example 19
Expression of Aspergillus fumigatus Beta-Glucosidase Comparing
Native and Heterologous Secretion Signal in Saccharomyces
cerevisiae
[0240] Plasmids pALFd6 (containing the Aspergillus fumigatus with
its native signal sequence) and pALFd7 (containing the Aspergillus
fumigatus with the heterologous signal sequence), approximately 1
.mu.g, were individually transformed into freshly made
Saccharomyces cerevisiae YNG318 competent cells, plated onto yeast
selection plates, and were incubated at 30.degree. C. for 4 days as
described in Example 10.
[0241] Two blue colonies from both constructs were picked manually
and inoculated into yeast selection medium (which contains copper)
to induce expression and secretion of Aspergillus oryzae
beta-glucosidase. Broth from day 5 was then assayed in duplicate
for beta-glucosidase activity using
p-nitrophenyl-beta-D-glucopyranoside as substrate as described
above. Cultures expressing beta-glucosidase with the heterologous
signal sequence produced 2.5-fold more beta-glucosidase than
cultures expressing beta-glucosidase with its native signal
sequence.
Deposit of Biological Material
[0242] The following biological material has been deposited under
the terms of the Budapest Treaty with the Agricultural Research
Service Patent Culture Collection, Northern Regional Research
Center, 1815 University Street, Peoria, Ill., 61604, and given the
following accession number:
TABLE-US-00029 Deposit Accession Number Date of Deposit E. coli
TOP10 (pEJG113) NRRL B-30695 Oct. 17, 2003
[0243] The strain has been deposited under conditions that assure
that access to the culture will be available during the pendency of
this patent application to one determined by the Commissioner of
Patents and Trademarks to be entitled thereto under 37 C.F.R.
.sctn.1.14 and 35 U.S.C. .sctn.122. The deposit represents a
substantially pure culture of the deposited strain. The deposit is
available as required by foreign patent laws in countries wherein
counterparts of the subject application, or its progeny are filed.
However, it should be understood that the availability of a deposit
does not constitute a license to practice the subject invention in
derogation of patent rights granted by governmental action.
[0244] The invention described and claimed herein is not to be
limited in scope by the specific aspects herein disclosed, since
these aspects are intended as illustrations of several aspects of
the invention. Any equivalent aspects are intended to be within the
scope of this invention. Indeed, various modifications of the
invention in addition to those shown and described herein will
become apparent to those skilled in the art from the foregoing
description. Such modifications are also intended to fall within
the scope of the appended claims. In the case of conflict, the
present disclosure including definitions will control.
[0245] Various references are cited herein, the disclosures of
which are incorporated by reference in their entireties.
Sequence CWU 1
1
64122DNAAspergillus oryzae 1gtgccccatg atacgcctcc gg
22226DNAAspergillus oryzae 2gagtcgtatt tccaaggctc ctgacc
26324DNAAspergillus oryzae 3ggaggccatg aagtggacca acgg
24445DNAAspergillus niger 4caccgtgaaa gccatgctct ttccttcgtg
tagaagacca gacag 45545DNAAspergillus niger 5ctggtcttct acacgaagga
aagagcatgg ctttcacggt gtctg 45644DNAAspergillus oryzae 6ctatatacac
aactggattt accatgggcc cgcggccgca gatc 44744DNAAspergillus oryzae
7gatctgcggc cgcgggccca tggtaaatcc agttgtgtat atag
44829DNATrichoderma reesei 8aacgttaatt aaggaatcgt tttgtgttt
29929DNATrichoderma reesei 9agtactagta gctccgtggc gaaagcctg
291031DNAHumicola insolens 10ttgaattgaa aatagattga tttaaaactt c
311125DNAHumicola insolens 11ttgcatgcgt aatcatggtc atagc
251226DNASaccharomyces cerevisiae 12ttgaattcat gggtaataac tgatat
261332DNASaccharomyces cerevisiae 13aaatcaatct attttcaatt
caattcatca tt 321445DNAAspergillus oryzae 14ggatgctgtt gactccggaa
atttaacggt ttggtcttgc atccc 451544DNAAspergillus oryzae
15ggtattgtcc tgcagacggc aatttaacgg cttctgcgaa tcgc
441629DNAHumicola insolens 16aagcttaagc atgcgttcct cccccctcc
291732DNAHumicola insolens 17ctgcagaatt ctacaggcac tgatggtacc ag
321832DNAHumicola insolens 18ctgcagaatt ctacaggcac tgatggtacc ag
321936DNAHumicola insolens 19accgcggact gcgcatcatg cgttcctccc
ccctcc 362029DNATrichoderma reesei 20aaacgtcgac cgaatgtagg
attgttatc 292117DNATrichoderma reesei 21gatgcgcagt ccgcggt
172229DNATrichoderma reesei 22aaacgtcgac cgaatgtagg attgttatc
292336DNATrichoderma reesei 23ggagggggga ggaacgcatg atgcgcagtc
cgcggt 362429DNATrichoderma reesei 24aaacgtcgac cgaatgtagg
attgttatc 292532DNATrichoderma reesei 25ctgcagaatt ctacaggcac
tgatggtacc ag 322646DNATrichoderma reesei 26atagtcaacc gcggactgcg
catcatgaag cttggttgga tcgagg 462726DNATrichoderma reesei
27actagtttac tgggccttag gcagcg 262826DNATrichoderma reesei
28gtcgactcga agcccgaatg taggat 262945DNATrichoderma reesei
29cctcgatcca accaagcttc atgatgcgca gtccgcggtt gacta
453042DNAAspergillus oryzae 30tgccggtgtt ggcccttgcc aaggatgatc
tcgcgtactc cc 423128DNAAspergillus oryzae 31gactagtctt actgggcctt
aggcagcg 283230DNAAspergillus oryzae 32acgcgtcgac cgaatgtagg
attgttatcc 303342DNAAspergillus oryzae 33gggagtacgc gagatcatcc
ttggcaaggg ccaacaccgg ca 423457DNAAspergillus oryzae 34atgaagcttg
gttggatcga ggtggccgca ttggcggctg cctcagtagt cagtgcc
573519PRTAspergillus oryzae 35Met Lys Leu Gly Trp Ile Glu Val Ala
Ala Leu Ala Ala Ala Ser Val 1 5 10 15 Val Ser Ala 3663DNAHumicola
insolens 36atgcgttcct cccccctcct ccgctccgcc gttgtggccg ccctgccggt
gttggccctt 60gcc 633721PRTHumicola insolens 37Met Arg Ser Ser Pro
Leu Leu Arg Ser Ala Val Val Ala Ala Leu Pro 1 5 10 15 Val Leu Ala
Leu Ala 20 3832DNAAspergillus oryzae 38gcagatctac catgaagctt
ggttggatcg ag 323930DNAAspergillus oryzae 39gcctcagatt actgggcctt
aggcagcgag 304040DNAHumicola insolens 40aatccgacta gtggatctac
catgcgttcc tcccccctcc 404132DNAHumicola insolens 41gcgggcctcg
agttactggg ccttaggcag cg 32422586DNAAspergillus
oryzaeCDS(1)..(2583) 42atg aag ctt ggt tgg atc gag gtg gcc gca ttg
gcg gct gcc tca gta 48Met Lys Leu Gly Trp Ile Glu Val Ala Ala Leu
Ala Ala Ala Ser Val 1 5 10 15 gtc agt gcc aag gat gat ctc gcg tac
tcc cct cct ttc tac cct tcc 96Val Ser Ala Lys Asp Asp Leu Ala Tyr
Ser Pro Pro Phe Tyr Pro Ser 20 25 30 cca tgg gca gat ggt cag ggt
gaa tgg gcg gaa gta tac aaa cgc gct 144Pro Trp Ala Asp Gly Gln Gly
Glu Trp Ala Glu Val Tyr Lys Arg Ala 35 40 45 gta gac ata gtt tcc
cag atg acg ttg aca gag aaa gtc aac tta acg 192Val Asp Ile Val Ser
Gln Met Thr Leu Thr Glu Lys Val Asn Leu Thr 50 55 60 act gga aca
gga tgg caa cta gag agg tgt gtt gga caa act ggc agt 240Thr Gly Thr
Gly Trp Gln Leu Glu Arg Cys Val Gly Gln Thr Gly Ser 65 70 75 80 gtt
ccc aga ctc aac atc ccc agc ttg tgt ttg cag gat agt cct ctt 288Val
Pro Arg Leu Asn Ile Pro Ser Leu Cys Leu Gln Asp Ser Pro Leu 85 90
95 ggt att cgt ttc tcg gac tac aat tca gct ttc cct gcg ggt gtt aat
336Gly Ile Arg Phe Ser Asp Tyr Asn Ser Ala Phe Pro Ala Gly Val Asn
100 105 110 gtc gct gcc acc tgg gac aag acg ctc gcc tac ctt cgt ggt
cag gca 384Val Ala Ala Thr Trp Asp Lys Thr Leu Ala Tyr Leu Arg Gly
Gln Ala 115 120 125 atg ggt gag gag ttc agt gat aag ggt att gac gtt
cag ctg ggt cct 432Met Gly Glu Glu Phe Ser Asp Lys Gly Ile Asp Val
Gln Leu Gly Pro 130 135 140 gct gct ggc cct ctc ggt gct cat ccg gat
ggc ggt aga aac tgg gaa 480Ala Ala Gly Pro Leu Gly Ala His Pro Asp
Gly Gly Arg Asn Trp Glu 145 150 155 160 ggt ttc tca cca gat cca gcc
ctc acc ggt gta ctt ttt gcg gag acg 528Gly Phe Ser Pro Asp Pro Ala
Leu Thr Gly Val Leu Phe Ala Glu Thr 165 170 175 att aag ggt att caa
gat gct ggt gtc att gcg aca gct aag cat tat 576Ile Lys Gly Ile Gln
Asp Ala Gly Val Ile Ala Thr Ala Lys His Tyr 180 185 190 atc atg aac
gaa caa gag cat ttc cgc caa caa ccc gag gct gcg ggt 624Ile Met Asn
Glu Gln Glu His Phe Arg Gln Gln Pro Glu Ala Ala Gly 195 200 205 tac
gga ttc aac gta agc gac agt ttg agt tcc aac gtt gat gac aag 672Tyr
Gly Phe Asn Val Ser Asp Ser Leu Ser Ser Asn Val Asp Asp Lys 210 215
220 act atg cat gaa ttg tac ctc tgg ccc ttc gcg gat gca gta cgc gct
720Thr Met His Glu Leu Tyr Leu Trp Pro Phe Ala Asp Ala Val Arg Ala
225 230 235 240 gga gtc ggt gct gtc atg tgc tct tac aac caa atc aac
aac agc tac 768Gly Val Gly Ala Val Met Cys Ser Tyr Asn Gln Ile Asn
Asn Ser Tyr 245 250 255 ggt tgc gag aat agc gaa act ctg aac aag ctt
ttg aag gcg gag ctt 816Gly Cys Glu Asn Ser Glu Thr Leu Asn Lys Leu
Leu Lys Ala Glu Leu 260 265 270 ggt ttc caa ggc ttc gtc atg agt gat
tgg acc gct cat cac agc ggc 864Gly Phe Gln Gly Phe Val Met Ser Asp
Trp Thr Ala His His Ser Gly 275 280 285 gta ggc gct gct tta gca ggt
ctg gat atg tcg atg ccc ggt gat gtt 912Val Gly Ala Ala Leu Ala Gly
Leu Asp Met Ser Met Pro Gly Asp Val 290 295 300 acc ttc gat agt ggt
acg tct ttc tgg ggt gca aac ttg acg gtc ggt 960Thr Phe Asp Ser Gly
Thr Ser Phe Trp Gly Ala Asn Leu Thr Val Gly 305 310 315 320 gtc ctt
aac ggt aca atc ccc caa tgg cgt gtt gat gac atg gct gtc 1008Val Leu
Asn Gly Thr Ile Pro Gln Trp Arg Val Asp Asp Met Ala Val 325 330 335
cgt atc atg gcc gct tat tac aag gtt ggc cgc gac acc aaa tac acc
1056Arg Ile Met Ala Ala Tyr Tyr Lys Val Gly Arg Asp Thr Lys Tyr Thr
340 345 350 cct ccc aac ttc agc tcg tgg acc agg gac gaa tat ggt ttc
gcg cat 1104Pro Pro Asn Phe Ser Ser Trp Thr Arg Asp Glu Tyr Gly Phe
Ala His 355 360 365 aac cat gtt tcg gaa ggt gct tac gag agg gtc aac
gaa ttc gtg gac 1152Asn His Val Ser Glu Gly Ala Tyr Glu Arg Val Asn
Glu Phe Val Asp 370 375 380 gtg caa cgc gat cat gcc gac cta atc cgt
cgc atc ggc gcg cag agc 1200Val Gln Arg Asp His Ala Asp Leu Ile Arg
Arg Ile Gly Ala Gln Ser 385 390 395 400 act gtt ctg ctg aag aac aag
ggt gcc ttg ccc ttg agc cgc aag gaa 1248Thr Val Leu Leu Lys Asn Lys
Gly Ala Leu Pro Leu Ser Arg Lys Glu 405 410 415 aag ctg gtc gcc ctt
ctg gga gag gat gcg ggt tcc aac tcg tgg ggc 1296Lys Leu Val Ala Leu
Leu Gly Glu Asp Ala Gly Ser Asn Ser Trp Gly 420 425 430 gct aac ggc
tgt gat gac cgt ggt tgc gat aac ggt acc ctt gcc atg 1344Ala Asn Gly
Cys Asp Asp Arg Gly Cys Asp Asn Gly Thr Leu Ala Met 435 440 445 gcc
tgg ggt agc ggt act gcg aat ttc cca tac ctc gtg aca cca gag 1392Ala
Trp Gly Ser Gly Thr Ala Asn Phe Pro Tyr Leu Val Thr Pro Glu 450 455
460 cag gcg att cag aac gaa gtt ctt cag ggc cgt ggt aat gtc ttc gcc
1440Gln Ala Ile Gln Asn Glu Val Leu Gln Gly Arg Gly Asn Val Phe Ala
465 470 475 480 gtg acc gac agt tgg gcg ctc gac aag atc gct gcg gct
gcc cgc cag 1488Val Thr Asp Ser Trp Ala Leu Asp Lys Ile Ala Ala Ala
Ala Arg Gln 485 490 495 gcc agc gta tct ctc gtg ttc gtc aac tcc gac
tca gga gaa ggc tat 1536Ala Ser Val Ser Leu Val Phe Val Asn Ser Asp
Ser Gly Glu Gly Tyr 500 505 510 ctt agt gtg gat gga aat gag ggc gat
cgt aac aac atc act ctg tgg 1584Leu Ser Val Asp Gly Asn Glu Gly Asp
Arg Asn Asn Ile Thr Leu Trp 515 520 525 aag aac ggc gac aat gtg gtc
aag acc gca gcg aat aac tgt aac aac 1632Lys Asn Gly Asp Asn Val Val
Lys Thr Ala Ala Asn Asn Cys Asn Asn 530 535 540 acc gtt gtc atc atc
cac tcc gtc gga cca gtt ttg atc gat gaa tgg 1680Thr Val Val Ile Ile
His Ser Val Gly Pro Val Leu Ile Asp Glu Trp 545 550 555 560 tat gac
cac ccc aat gtc act ggt att ctc tgg gct ggt ctg cca ggc 1728Tyr Asp
His Pro Asn Val Thr Gly Ile Leu Trp Ala Gly Leu Pro Gly 565 570 575
cag gag tct ggt aac tcc att gcc gat gtg ctg tac ggt cgt gtc aac
1776Gln Glu Ser Gly Asn Ser Ile Ala Asp Val Leu Tyr Gly Arg Val Asn
580 585 590 cct ggc gcc aag tct cct ttc act tgg ggc aag acc cgg gag
tcg tat 1824Pro Gly Ala Lys Ser Pro Phe Thr Trp Gly Lys Thr Arg Glu
Ser Tyr 595 600 605 ggt tct ccc ttg gtc aag gat gcc aac aat ggc aac
gga gcg ccc cag 1872Gly Ser Pro Leu Val Lys Asp Ala Asn Asn Gly Asn
Gly Ala Pro Gln 610 615 620 tct gat ttc acc cag ggt gtt ttc atc gat
tac cgc cat ttc gat aag 1920Ser Asp Phe Thr Gln Gly Val Phe Ile Asp
Tyr Arg His Phe Asp Lys 625 630 635 640 ttc aat gag acc cct atc tac
gag ttt ggc tac ggc ttg agc tac acc 1968Phe Asn Glu Thr Pro Ile Tyr
Glu Phe Gly Tyr Gly Leu Ser Tyr Thr 645 650 655 acc ttc gag ctc tcc
gac ctc cat gtt cag ccc ctg aac gcg tcc cga 2016Thr Phe Glu Leu Ser
Asp Leu His Val Gln Pro Leu Asn Ala Ser Arg 660 665 670 tac act ccc
acc agt ggc atg act gaa gct gca aag aac ttt ggt gaa 2064Tyr Thr Pro
Thr Ser Gly Met Thr Glu Ala Ala Lys Asn Phe Gly Glu 675 680 685 att
ggc gat gcg tcg gag tac gtg tat ccg gag ggg ctg gaa agg atc 2112Ile
Gly Asp Ala Ser Glu Tyr Val Tyr Pro Glu Gly Leu Glu Arg Ile 690 695
700 cat gag ttt atc tat ccc tgg atc aac tct acc gac ctg aag gca tcg
2160His Glu Phe Ile Tyr Pro Trp Ile Asn Ser Thr Asp Leu Lys Ala Ser
705 710 715 720 tct gac gat tct aac tac ggc tgg gaa gac tcc aag tat
att ccc gaa 2208Ser Asp Asp Ser Asn Tyr Gly Trp Glu Asp Ser Lys Tyr
Ile Pro Glu 725 730 735 ggc gcc acg gat ggg tct gcc cag ccc cgt ttg
ccc gct agt ggt ggt 2256Gly Ala Thr Asp Gly Ser Ala Gln Pro Arg Leu
Pro Ala Ser Gly Gly 740 745 750 gcc gga gga aac ccc ggt ctg tac gag
gat ctt ttc cgc gtc tct gtg 2304Ala Gly Gly Asn Pro Gly Leu Tyr Glu
Asp Leu Phe Arg Val Ser Val 755 760 765 aag gtc aag aac acg ggc aat
gtc gcc ggt gat gaa gtt cct cag ctg 2352Lys Val Lys Asn Thr Gly Asn
Val Ala Gly Asp Glu Val Pro Gln Leu 770 775 780 tac gtt tcc cta ggc
ggc ccg aat gag ccc aag gtg gta ctg cgc aag 2400Tyr Val Ser Leu Gly
Gly Pro Asn Glu Pro Lys Val Val Leu Arg Lys 785 790 795 800 ttt gag
cgt att cac ttg gcc cct tcg cag gag gcc gtg tgg aca acg 2448Phe Glu
Arg Ile His Leu Ala Pro Ser Gln Glu Ala Val Trp Thr Thr 805 810 815
acc ctt acc cgt cgt gac ctt gca aac tgg gac gtt tcg gct cag gac
2496Thr Leu Thr Arg Arg Asp Leu Ala Asn Trp Asp Val Ser Ala Gln Asp
820 825 830 tgg acc gtc act cct tac ccc aag acg atc tac gtt gga aac
tcc tca 2544Trp Thr Val Thr Pro Tyr Pro Lys Thr Ile Tyr Val Gly Asn
Ser Ser 835 840 845 cgg aaa ctg ccg ctc cag gcc tcg ctg cct aag gcc
cag taa 2586Arg Lys Leu Pro Leu Gln Ala Ser Leu Pro Lys Ala Gln 850
855 860 43861PRTAspergillus oryzae 43Met Lys Leu Gly Trp Ile Glu
Val Ala Ala Leu Ala Ala Ala Ser Val 1 5 10 15 Val Ser Ala Lys Asp
Asp Leu Ala Tyr Ser Pro Pro Phe Tyr Pro Ser 20 25 30 Pro Trp Ala
Asp Gly Gln Gly Glu Trp Ala Glu Val Tyr Lys Arg Ala 35 40 45 Val
Asp Ile Val Ser Gln Met Thr Leu Thr Glu Lys Val Asn Leu Thr 50 55
60 Thr Gly Thr Gly Trp Gln Leu Glu Arg Cys Val Gly Gln Thr Gly Ser
65 70 75 80 Val Pro Arg Leu Asn Ile Pro Ser Leu Cys Leu Gln Asp Ser
Pro Leu 85 90 95 Gly Ile Arg Phe Ser Asp Tyr Asn Ser Ala Phe Pro
Ala Gly Val Asn 100 105 110 Val Ala Ala Thr Trp Asp Lys Thr Leu Ala
Tyr Leu Arg Gly Gln Ala 115 120 125 Met Gly Glu Glu Phe Ser Asp Lys
Gly Ile Asp Val Gln Leu Gly Pro 130 135 140 Ala Ala Gly Pro Leu Gly
Ala His Pro Asp Gly Gly Arg Asn Trp Glu 145 150 155 160 Gly Phe Ser
Pro Asp Pro Ala Leu Thr Gly Val Leu Phe Ala Glu Thr 165 170 175 Ile
Lys Gly Ile Gln Asp Ala Gly Val Ile Ala Thr Ala Lys His Tyr 180 185
190 Ile Met Asn Glu Gln Glu His Phe Arg Gln Gln Pro Glu Ala Ala Gly
195 200 205 Tyr Gly Phe Asn Val Ser Asp Ser Leu Ser Ser Asn Val Asp
Asp Lys 210 215 220 Thr Met His Glu Leu Tyr Leu Trp Pro Phe Ala
Asp
Ala Val Arg Ala 225 230 235 240 Gly Val Gly Ala Val Met Cys Ser Tyr
Asn Gln Ile Asn Asn Ser Tyr 245 250 255 Gly Cys Glu Asn Ser Glu Thr
Leu Asn Lys Leu Leu Lys Ala Glu Leu 260 265 270 Gly Phe Gln Gly Phe
Val Met Ser Asp Trp Thr Ala His His Ser Gly 275 280 285 Val Gly Ala
Ala Leu Ala Gly Leu Asp Met Ser Met Pro Gly Asp Val 290 295 300 Thr
Phe Asp Ser Gly Thr Ser Phe Trp Gly Ala Asn Leu Thr Val Gly 305 310
315 320 Val Leu Asn Gly Thr Ile Pro Gln Trp Arg Val Asp Asp Met Ala
Val 325 330 335 Arg Ile Met Ala Ala Tyr Tyr Lys Val Gly Arg Asp Thr
Lys Tyr Thr 340 345 350 Pro Pro Asn Phe Ser Ser Trp Thr Arg Asp Glu
Tyr Gly Phe Ala His 355 360 365 Asn His Val Ser Glu Gly Ala Tyr Glu
Arg Val Asn Glu Phe Val Asp 370 375 380 Val Gln Arg Asp His Ala Asp
Leu Ile Arg Arg Ile Gly Ala Gln Ser 385 390 395 400 Thr Val Leu Leu
Lys Asn Lys Gly Ala Leu Pro Leu Ser Arg Lys Glu 405 410 415 Lys Leu
Val Ala Leu Leu Gly Glu Asp Ala Gly Ser Asn Ser Trp Gly 420 425 430
Ala Asn Gly Cys Asp Asp Arg Gly Cys Asp Asn Gly Thr Leu Ala Met 435
440 445 Ala Trp Gly Ser Gly Thr Ala Asn Phe Pro Tyr Leu Val Thr Pro
Glu 450 455 460 Gln Ala Ile Gln Asn Glu Val Leu Gln Gly Arg Gly Asn
Val Phe Ala 465 470 475 480 Val Thr Asp Ser Trp Ala Leu Asp Lys Ile
Ala Ala Ala Ala Arg Gln 485 490 495 Ala Ser Val Ser Leu Val Phe Val
Asn Ser Asp Ser Gly Glu Gly Tyr 500 505 510 Leu Ser Val Asp Gly Asn
Glu Gly Asp Arg Asn Asn Ile Thr Leu Trp 515 520 525 Lys Asn Gly Asp
Asn Val Val Lys Thr Ala Ala Asn Asn Cys Asn Asn 530 535 540 Thr Val
Val Ile Ile His Ser Val Gly Pro Val Leu Ile Asp Glu Trp 545 550 555
560 Tyr Asp His Pro Asn Val Thr Gly Ile Leu Trp Ala Gly Leu Pro Gly
565 570 575 Gln Glu Ser Gly Asn Ser Ile Ala Asp Val Leu Tyr Gly Arg
Val Asn 580 585 590 Pro Gly Ala Lys Ser Pro Phe Thr Trp Gly Lys Thr
Arg Glu Ser Tyr 595 600 605 Gly Ser Pro Leu Val Lys Asp Ala Asn Asn
Gly Asn Gly Ala Pro Gln 610 615 620 Ser Asp Phe Thr Gln Gly Val Phe
Ile Asp Tyr Arg His Phe Asp Lys 625 630 635 640 Phe Asn Glu Thr Pro
Ile Tyr Glu Phe Gly Tyr Gly Leu Ser Tyr Thr 645 650 655 Thr Phe Glu
Leu Ser Asp Leu His Val Gln Pro Leu Asn Ala Ser Arg 660 665 670 Tyr
Thr Pro Thr Ser Gly Met Thr Glu Ala Ala Lys Asn Phe Gly Glu 675 680
685 Ile Gly Asp Ala Ser Glu Tyr Val Tyr Pro Glu Gly Leu Glu Arg Ile
690 695 700 His Glu Phe Ile Tyr Pro Trp Ile Asn Ser Thr Asp Leu Lys
Ala Ser 705 710 715 720 Ser Asp Asp Ser Asn Tyr Gly Trp Glu Asp Ser
Lys Tyr Ile Pro Glu 725 730 735 Gly Ala Thr Asp Gly Ser Ala Gln Pro
Arg Leu Pro Ala Ser Gly Gly 740 745 750 Ala Gly Gly Asn Pro Gly Leu
Tyr Glu Asp Leu Phe Arg Val Ser Val 755 760 765 Lys Val Lys Asn Thr
Gly Asn Val Ala Gly Asp Glu Val Pro Gln Leu 770 775 780 Tyr Val Ser
Leu Gly Gly Pro Asn Glu Pro Lys Val Val Leu Arg Lys 785 790 795 800
Phe Glu Arg Ile His Leu Ala Pro Ser Gln Glu Ala Val Trp Thr Thr 805
810 815 Thr Leu Thr Arg Arg Asp Leu Ala Asn Trp Asp Val Ser Ala Gln
Asp 820 825 830 Trp Thr Val Thr Pro Tyr Pro Lys Thr Ile Tyr Val Gly
Asn Ser Ser 835 840 845 Arg Lys Leu Pro Leu Gln Ala Ser Leu Pro Lys
Ala Gln 850 855 860 4431DNAAspergillus fumigatus 44actggattta
ccatgagatt cggttggctc g 314531DNAAspergillus fumigatus 45agtcacctct
agttactagt agacacgggg c 31463060DNAAspergillus fumigatus
46atgagattcg gttggctcga ggtggccgct ctgacggccg cttctgtagc caatgcccag
60gtttgtgatg ctttcccgtc attgtttcgg atatagttga caatagtcat ggaaataatc
120aggaattggc tttctctcca ccattctacc cttcgccttg ggctgatggc
cagggagagt 180gggcagatgc ccatcgacgc gccgtcgaga tcgtttctca
gatgacactg gcggagaagg 240ttaaccttac aacgggtact gggtgggttg
cgactttttt gttgacagtg agctttcttc 300actgaccatc tacacagatg
ggaaatggac cgatgcgtcg gtcaaaccgg cagcgttccc 360aggtaagctt
gcaattctgc aacaacgtgc aagtgtagtt gctaaaacgc ggtggtgcag
420acttggtatc aactggggtc tttgtggcca ggattcccct ttgggtatcc
gtttctgtga 480gctatacccg cggagtcttt cagtccttgt attatgtgct
gatgattgtc tctgtatagc 540tgacctcaac tccgccttcc ctgctggtac
taatgtcgcc gcgacatggg acaagacact 600cgcctacctt cgtggcaagg
ccatgggtga ggaattcaac gacaagggcg tggacatttt 660gctggggcct
gctgctggtc ctctcggcaa atacccggac ggcggcagaa tctgggaagg
720cttctctcct gatccggttc tcactggtgt acttttcgcc gaaactatca
agggtatcca 780agacgcgggt gtgattgcta ctgccaagca ttacattctg
aatgaacagg agcatttccg 840acaggttggc gaggcccagg gatatggtta
caacatcacg gagacgatca gctccaacgt 900ggatgacaag accatgcacg
agttgtacct ttggtgagta gttgacactg caaatgagga 960ccttgattga
tttgactgac ctggaatgca ggccctttgc agatgctgtg cgcggtaaga
1020ttttccgtag acttgacctc gcgacgaaga aatcgctgac gaaccatcgt
agctggcgtt 1080ggcgctgtca tgtgttccta caatcaaatc aacaacagct
acggttgtca aaacagtcaa 1140actctcaaca agctcctcaa ggctgagctg
ggcttccaag gcttcgtcat gagtgactgg 1200agcgctcacc acagcggtgt
cggcgctgcc ctcgctgggt tggatatgtc gatgcctgga 1260gacatttcct
tcgacgacgg actctccttc tggggcacga acctaactgt cagtgttctt
1320aacggcaccg ttccagcctg gcgtgtcgat gacatggctg ttcgtatcat
gaccgcgtac 1380tacaaggttg gtcgtgaccg tcttcgtatt ccccctaact
tcagctcctg gacccgggat 1440gagtacggct gggagcattc tgctgtctcc
gagggagcct ggaccaaggt gaacgacttc 1500gtcaatgtgc agcgcagtca
ctctcagatc atccgtgaga ttggtgccgc tagtacagtg 1560ctcttgaaga
acacgggtgc tcttcctttg accggcaagg aggttaaagt gggtgttctc
1620ggtgaagacg ctggttccaa cccgtggggt gctaacggct gccccgaccg
cggctgtgat 1680aacggcactc ttgctatggc ctggggtagt ggtactgcca
acttccctta ccttgtcacc 1740cccgagcagg ctatccagcg agaggtcatc
agcaacggcg gcaatgtctt tgctgtgact 1800gataacgggg ctctcagcca
gatggcagat gttgcatctc aatccaggtg agtgcgggct 1860cttagaaaaa
gaacgttctc tgaatgaagt tttttaacca ttgcgaacag cgtgtctttg
1920gtgtttgtca acgccgactc tggagagggt ttcatcagtg tcgacggcaa
cgagggtgac 1980cgcaaaaatc tcactctgtg gaagaacggc gaggccgtca
ttgacactgt tgtcagccac 2040tgcaacaaca cgattgtggt tattcacagt
gttgggcccg tcttgatcga ccggtggtat 2100gataacccca acgtcactgc
catcatctgg gccggcttgc ccggtcagga gagtggcaac 2160tccctggtcg
acgtgctcta tggccgcgtc aaccccagcg ccaagacccc gttcacctgg
2220ggcaagactc gggagtctta cggggctccc ttgctcaccg agcctaacaa
tggcaatggt 2280gctccccagg atgatttcaa cgagggcgtc ttcattgact
accgtcactt tgacaagcgc 2340aatgagaccc ccatttatga gtttggccat
ggcttgagct acaccacctt tggttactct 2400caccttcggg ttcaggccct
caatagttcg agttcggcat atgtcccgac tagcggagag 2460accaagcctg
cgccaaccta tggtgagatc ggtagtgccg ccgactacct gtatcccgag
2520ggtctcaaaa gaattaccaa gtttatttac ccttggctca actcgaccga
cctcgaggat 2580tcttctgacg acccgaacta cggctgggag gactcggagt
acattcccga aggcgctagg 2640gatgggtctc ctcaacccct cctgaaggct
ggcggcgctc ctggtggtaa ccctaccctt 2700tatcaggatc ttgttagggt
gtcggccacc ataaccaaca ctggtaacgt cgccggttat 2760gaagtccctc
aattggtgag tgacccgcat gttccttgcg ttgcaatttg gctaactcgc
2820ttctagtatg tttcactggg cggaccgaac gagcctcggg tcgttctgcg
caagttcgac 2880cgaatcttcc tggctcctgg ggagcaaaag gtttggacca
cgactcttaa ccgtcgtgat 2940ctcgccaatt gggatgtgga ggctcaggac
tgggtcatca caaagtaccc caagaaagtg 3000cacgtcggca gctcctcgcg
taagctgcct ctgagagcgc ctctgccccg tgtctactag 306047863PRTAspergillus
fumigatus 47Met Arg Phe Gly Trp Leu Glu Val Ala Ala Leu Thr Ala Ala
Ser Val 1 5 10 15 Ala Asn Ala Gln Glu Leu Ala Phe Ser Pro Pro Phe
Tyr Pro Ser Pro 20 25 30 Trp Ala Asp Gly Gln Gly Glu Trp Ala Asp
Ala His Arg Arg Ala Val 35 40 45 Glu Ile Val Ser Gln Met Thr Leu
Ala Glu Lys Val Asn Leu Thr Thr 50 55 60 Gly Thr Gly Trp Glu Met
Asp Arg Cys Val Gly Gln Thr Gly Ser Val 65 70 75 80 Pro Arg Leu Gly
Ile Asn Trp Gly Leu Cys Gly Gln Asp Ser Pro Leu 85 90 95 Gly Ile
Arg Phe Ser Asp Leu Asn Ser Ala Phe Pro Ala Gly Thr Asn 100 105 110
Val Ala Ala Thr Trp Asp Lys Thr Leu Ala Tyr Leu Arg Gly Lys Ala 115
120 125 Met Gly Glu Glu Phe Asn Asp Lys Gly Val Asp Ile Leu Leu Gly
Pro 130 135 140 Ala Ala Gly Pro Leu Gly Lys Tyr Pro Asp Gly Gly Arg
Ile Trp Glu 145 150 155 160 Gly Phe Ser Pro Asp Pro Val Leu Thr Gly
Val Leu Phe Ala Glu Thr 165 170 175 Ile Lys Gly Ile Gln Asp Ala Gly
Val Ile Ala Thr Ala Lys His Tyr 180 185 190 Ile Leu Asn Glu Gln Glu
His Phe Arg Gln Val Gly Glu Ala Gln Gly 195 200 205 Tyr Gly Tyr Asn
Ile Thr Glu Thr Ile Ser Ser Asn Val Asp Asp Lys 210 215 220 Thr Met
His Glu Leu Tyr Leu Trp Pro Phe Ala Asp Ala Val Arg Ala 225 230 235
240 Gly Val Gly Ala Val Met Cys Ser Tyr Asn Gln Ile Asn Asn Ser Tyr
245 250 255 Gly Cys Gln Asn Ser Gln Thr Leu Asn Lys Leu Leu Lys Ala
Glu Leu 260 265 270 Gly Phe Gln Gly Phe Val Met Ser Asp Trp Ser Ala
His His Ser Gly 275 280 285 Val Gly Ala Ala Leu Ala Gly Leu Asp Met
Ser Met Pro Gly Asp Ile 290 295 300 Ser Phe Asp Asp Gly Leu Ser Phe
Trp Gly Thr Asn Leu Thr Val Ser 305 310 315 320 Val Leu Asn Gly Thr
Val Pro Ala Trp Arg Val Asp Asp Met Ala Val 325 330 335 Arg Ile Met
Thr Ala Tyr Tyr Lys Val Gly Arg Asp Arg Leu Arg Ile 340 345 350 Pro
Pro Asn Phe Ser Ser Trp Thr Arg Asp Glu Tyr Gly Trp Glu His 355 360
365 Ser Ala Val Ser Glu Gly Ala Trp Thr Lys Val Asn Asp Phe Val Asn
370 375 380 Val Gln Arg Ser His Ser Gln Ile Ile Arg Glu Ile Gly Ala
Ala Ser 385 390 395 400 Thr Val Leu Leu Lys Asn Thr Gly Ala Leu Pro
Leu Thr Gly Lys Glu 405 410 415 Val Lys Val Gly Val Leu Gly Glu Asp
Ala Gly Ser Asn Pro Trp Gly 420 425 430 Ala Asn Gly Cys Pro Asp Arg
Gly Cys Asp Asn Gly Thr Leu Ala Met 435 440 445 Ala Trp Gly Ser Gly
Thr Ala Asn Phe Pro Tyr Leu Val Thr Pro Glu 450 455 460 Gln Ala Ile
Gln Arg Glu Val Ile Ser Asn Gly Gly Asn Val Phe Ala 465 470 475 480
Val Thr Asp Asn Gly Ala Leu Ser Gln Met Ala Asp Val Ala Ser Gln 485
490 495 Ser Ser Val Ser Leu Val Phe Val Asn Ala Asp Ser Gly Glu Gly
Phe 500 505 510 Ile Ser Val Asp Gly Asn Glu Gly Asp Arg Lys Asn Leu
Thr Leu Trp 515 520 525 Lys Asn Gly Glu Ala Val Ile Asp Thr Val Val
Ser His Cys Asn Asn 530 535 540 Thr Ile Val Val Ile His Ser Val Gly
Pro Val Leu Ile Asp Arg Trp 545 550 555 560 Tyr Asp Asn Pro Asn Val
Thr Ala Ile Ile Trp Ala Gly Leu Pro Gly 565 570 575 Gln Glu Ser Gly
Asn Ser Leu Val Asp Val Leu Tyr Gly Arg Val Asn 580 585 590 Pro Ser
Ala Lys Thr Pro Phe Thr Trp Gly Lys Thr Arg Glu Ser Tyr 595 600 605
Gly Ala Pro Leu Leu Thr Glu Pro Asn Asn Gly Asn Gly Ala Pro Gln 610
615 620 Asp Asp Phe Asn Glu Gly Val Phe Ile Asp Tyr Arg His Phe Asp
Lys 625 630 635 640 Arg Asn Glu Thr Pro Ile Tyr Glu Phe Gly His Gly
Leu Ser Tyr Thr 645 650 655 Thr Phe Gly Tyr Ser His Leu Arg Val Gln
Ala Leu Asn Ser Ser Ser 660 665 670 Ser Ala Tyr Val Pro Thr Ser Gly
Glu Thr Lys Pro Ala Pro Thr Tyr 675 680 685 Gly Glu Ile Gly Ser Ala
Ala Asp Tyr Leu Tyr Pro Glu Gly Leu Lys 690 695 700 Arg Ile Thr Lys
Phe Ile Tyr Pro Trp Leu Asn Ser Thr Asp Leu Glu 705 710 715 720 Asp
Ser Ser Asp Asp Pro Asn Tyr Gly Trp Glu Asp Ser Glu Tyr Ile 725 730
735 Pro Glu Gly Ala Arg Asp Gly Ser Pro Gln Pro Leu Leu Lys Ala Gly
740 745 750 Gly Ala Pro Gly Gly Asn Pro Thr Leu Tyr Gln Asp Leu Val
Arg Val 755 760 765 Ser Ala Thr Ile Thr Asn Thr Gly Asn Val Ala Gly
Tyr Glu Val Pro 770 775 780 Gln Leu Tyr Val Ser Leu Gly Gly Pro Asn
Glu Pro Arg Val Val Leu 785 790 795 800 Arg Lys Phe Asp Arg Ile Phe
Leu Ala Pro Gly Glu Gln Lys Val Trp 805 810 815 Thr Thr Thr Leu Asn
Arg Arg Asp Leu Ala Asn Trp Asp Val Glu Ala 820 825 830 Gln Asp Trp
Val Ile Thr Lys Tyr Pro Lys Lys Val His Val Gly Ser 835 840 845 Ser
Ser Arg Lys Leu Pro Leu Arg Ala Pro Leu Pro Arg Val Tyr 850 855 860
482592DNAAspergillus fumigatus 48atgagattcg gttggctcga ggtggccgct
ctgacggccg cttctgtagc caatgcccag 60gaattggctt tctctccacc attctaccct
tcgccttggg ctgatggcca gggagagtgg 120gcagatgccc atcgacgcgc
cgtcgagatc gtttctcaga tgacactggc ggagaaggtt 180aaccttacaa
cgggtactgg atgggaaatg gaccgatgcg tcggtcaaac cggcagcgtt
240cccagacttg gtatcaactg gggtctttgt ggccaggatt cccctttggg
tatccgtttc 300tctgacctca actccgcctt ccctgctggt actaatgtcg
ccgcgacatg ggacaagaca 360ctcgcctacc ttcgtggcaa ggccatgggt
gaggaattca acgacaaggg cgtggacatt 420ttgctggggc ctgctgctgg
tcctctcggc aaatacccgg acggcggcag aatctgggaa 480ggcttctctc
ctgatccggt tctcactggt gtacttttcg ccgaaactat caagggtatc
540caagacgcgg gtgtgattgc tactgccaag cattacattc tgaatgaaca
ggagcatttc 600cgacaggttg gcgaggccca gggatatggt tacaacatca
cggagacgat cagctccaac 660gtggatgaca agaccatgca cgagttgtac
ctttggccct ttgcagatgc tgtgcgcgct 720ggcgttggcg ctgtcatgtg
ttcctacaat caaatcaaca acagctacgg ttgtcaaaac 780agtcaaactc
tcaacaagct cctcaaggct gagctgggct tccaaggctt cgtcatgagt
840gactggagcg ctcaccacag cggtgtcggc gctgccctcg ctgggttgga
tatgtcgatg 900cctggagaca tttccttcga cgacggactc tccttctggg
gcacgaacct aactgtcagt 960gttcttaacg gcaccgttcc agcctggcgt
gtcgatgaca tggctgttcg tatcatgacc 1020gcgtactaca aggttggtcg
tgaccgtctt cgtattcccc ctaacttcag ctcctggacc 1080cgggatgagt
acggctggga gcattctgct gtctccgagg gagcctggac caaggtgaac
1140gacttcgtca atgtgcagcg cagtcactct cagatcatcc gtgagattgg
tgccgctagt 1200acagtgctct tgaagaacac gggtgctctt cctttgaccg
gcaaggaggt taaagtgggt 1260gttctcggtg aagacgctgg ttccaacccg
tggggtgcta acggctgccc cgaccgcggc 1320tgtgataacg gcactcttgc
tatggcctgg ggtagtggta ctgccaactt cccttacctt 1380gtcacccccg
agcaggctat ccagcgagag gtcatcagca acggcggcaa tgtctttgct
1440gtgactgata acggggctct cagccagatg gcagatgttg catctcaatc
cagcgtgtct 1500ttggtgtttg tcaacgccga ctctggagag ggtttcatca
gtgtcgacgg caacgagggt 1560gaccgcaaaa atctcactct gtggaagaac
ggcgaggccg tcattgacac tgttgtcagc 1620cactgcaaca acacgattgt
ggttattcac agtgttgggc ccgtcttgat cgaccggtgg 1680tatgataacc
ccaacgtcac tgccatcatc tgggccggct tgcccggtca ggagagtggc
1740aactccctgg tcgacgtgct ctatggccgc gtcaacccca gcgccaagac
cccgttcacc 1800tggggcaaga ctcgggagtc ttacggggct cccttgctca
ccgagcctaa caatggcaat 1860ggtgctcccc aggatgattt caacgagggc
gtcttcattg actaccgtca ctttgacaag 1920cgcaatgaga cccccattta
tgagtttggc catggcttga gctacaccac ctttggttac 1980tctcaccttc
gggttcaggc cctcaatagt tcgagttcgg catatgtccc gactagcgga
2040gagaccaagc ctgcgccaac ctatggtgag atcggtagtg ccgccgacta
cctgtatccc 2100gagggtctca aaagaattac caagtttatt tacccttggc
tcaactcgac cgacctcgag 2160gattcttctg acgacccgaa ctacggctgg
gaggactcgg agtacattcc cgaaggcgct 2220agggatgggt ctcctcaacc
cctcctgaag gctggcggcg ctcctggtgg taaccctacc 2280ctttatcagg
atcttgttag ggtgtcggcc accataacca acactggtaa cgtcgccggt
2340tatgaagtcc ctcaattgta tgtttcactg ggcggaccga acgagcctcg
ggtcgttctg 2400cgcaagttcg accgaatctt cctggctcct ggggagcaaa
aggtttggac cacgactctt 2460aaccgtcgtg atctcgccaa ttgggatgtg
gaggctcagg actgggtcat cacaaagtac 2520cccaagaaag tgcacgtcgg
cagctcctcg cgtaagctgc ctctgagagc gcctctgccc 2580cgtgtctact ag
259249863PRTAspergillus fumigatus 49Met Arg Phe Gly Trp Leu Glu Val
Ala Ala Leu Thr Ala Ala Ser Val 1 5 10 15 Ala Asn Ala Gln Glu Leu
Ala Phe Ser Pro Pro Phe Tyr Pro Ser Pro 20 25 30 Trp Ala Asp Gly
Gln Gly Glu Trp Ala Asp Ala His Arg Arg Ala Val 35 40 45 Glu Ile
Val Ser Gln Met Thr Leu Ala Glu Lys Val Asn Leu Thr Thr 50 55 60
Gly Thr Gly Trp Glu Met Asp Arg Cys Val Gly Gln Thr Gly Ser Val 65
70 75 80 Pro Arg Leu Gly Ile Asn Trp Gly Leu Cys Gly Gln Asp Ser
Pro Leu 85 90 95 Gly Ile Arg Phe Ser Asp Leu Asn Ser Ala Phe Pro
Ala Gly Thr Asn 100 105 110 Val Ala Ala Thr Trp Asp Lys Thr Leu Ala
Tyr Leu Arg Gly Lys Ala 115 120 125 Met Gly Glu Glu Phe Asn Asp Lys
Gly Val Asp Ile Leu Leu Gly Pro 130 135 140 Ala Ala Gly Pro Leu Gly
Lys Tyr Pro Asp Gly Gly Arg Ile Trp Glu 145 150 155 160 Gly Phe Ser
Pro Asp Pro Val Leu Thr Gly Val Leu Phe Ala Glu Thr 165 170 175 Ile
Lys Gly Ile Gln Asp Ala Gly Val Ile Ala Thr Ala Lys His Tyr 180 185
190 Ile Leu Asn Glu Gln Glu His Phe Arg Gln Val Gly Glu Ala Gln Gly
195 200 205 Tyr Gly Tyr Asn Ile Thr Glu Thr Ile Ser Ser Asn Val Asp
Asp Lys 210 215 220 Thr Met His Glu Leu Tyr Leu Trp Pro Phe Ala Asp
Ala Val Arg Ala 225 230 235 240 Gly Val Gly Ala Val Met Cys Ser Tyr
Asn Gln Ile Asn Asn Ser Tyr 245 250 255 Gly Cys Gln Asn Ser Gln Thr
Leu Asn Lys Leu Leu Lys Ala Glu Leu 260 265 270 Gly Phe Gln Gly Phe
Val Met Ser Asp Trp Ser Ala His His Ser Gly 275 280 285 Val Gly Ala
Ala Leu Ala Gly Leu Asp Met Ser Met Pro Gly Asp Ile 290 295 300 Ser
Phe Asp Asp Gly Leu Ser Phe Trp Gly Thr Asn Leu Thr Val Ser 305 310
315 320 Val Leu Asn Gly Thr Val Pro Ala Trp Arg Val Asp Asp Met Ala
Val 325 330 335 Arg Ile Met Thr Ala Tyr Tyr Lys Val Gly Arg Asp Arg
Leu Arg Ile 340 345 350 Pro Pro Asn Phe Ser Ser Trp Thr Arg Asp Glu
Tyr Gly Trp Glu His 355 360 365 Ser Ala Val Ser Glu Gly Ala Trp Thr
Lys Val Asn Asp Phe Val Asn 370 375 380 Val Gln Arg Ser His Ser Gln
Ile Ile Arg Glu Ile Gly Ala Ala Ser 385 390 395 400 Thr Val Leu Leu
Lys Asn Thr Gly Ala Leu Pro Leu Thr Gly Lys Glu 405 410 415 Val Lys
Val Gly Val Leu Gly Glu Asp Ala Gly Ser Asn Pro Trp Gly 420 425 430
Ala Asn Gly Cys Pro Asp Arg Gly Cys Asp Asn Gly Thr Leu Ala Met 435
440 445 Ala Trp Gly Ser Gly Thr Ala Asn Phe Pro Tyr Leu Val Thr Pro
Glu 450 455 460 Gln Ala Ile Gln Arg Glu Val Ile Ser Asn Gly Gly Asn
Val Phe Ala 465 470 475 480 Val Thr Asp Asn Gly Ala Leu Ser Gln Met
Ala Asp Val Ala Ser Gln 485 490 495 Ser Ser Val Ser Leu Val Phe Val
Asn Ala Asp Ser Gly Glu Gly Phe 500 505 510 Ile Ser Val Asp Gly Asn
Glu Gly Asp Arg Lys Asn Leu Thr Leu Trp 515 520 525 Lys Asn Gly Glu
Ala Val Ile Asp Thr Val Val Ser His Cys Asn Asn 530 535 540 Thr Ile
Val Val Ile His Ser Val Gly Pro Val Leu Ile Asp Arg Trp 545 550 555
560 Tyr Asp Asn Pro Asn Val Thr Ala Ile Ile Trp Ala Gly Leu Pro Gly
565 570 575 Gln Glu Ser Gly Asn Ser Leu Val Asp Val Leu Tyr Gly Arg
Val Asn 580 585 590 Pro Ser Ala Lys Thr Pro Phe Thr Trp Gly Lys Thr
Arg Glu Ser Tyr 595 600 605 Gly Ala Pro Leu Leu Thr Glu Pro Asn Asn
Gly Asn Gly Ala Pro Gln 610 615 620 Asp Asp Phe Asn Glu Gly Val Phe
Ile Asp Tyr Arg His Phe Asp Lys 625 630 635 640 Arg Asn Glu Thr Pro
Ile Tyr Glu Phe Gly His Gly Leu Ser Tyr Thr 645 650 655 Thr Phe Gly
Tyr Ser His Leu Arg Val Gln Ala Leu Asn Ser Ser Ser 660 665 670 Ser
Ala Tyr Val Pro Thr Ser Gly Glu Thr Lys Pro Ala Pro Thr Tyr 675 680
685 Gly Glu Ile Gly Ser Ala Ala Asp Tyr Leu Tyr Pro Glu Gly Leu Lys
690 695 700 Arg Ile Thr Lys Phe Ile Tyr Pro Trp Leu Asn Ser Thr Asp
Leu Glu 705 710 715 720 Asp Ser Ser Asp Asp Pro Asn Tyr Gly Trp Glu
Asp Ser Glu Tyr Ile 725 730 735 Pro Glu Gly Ala Arg Asp Gly Ser Pro
Gln Pro Leu Leu Lys Ala Gly 740 745 750 Gly Ala Pro Gly Gly Asn Pro
Thr Leu Tyr Gln Asp Leu Val Arg Val 755 760 765 Ser Ala Thr Ile Thr
Asn Thr Gly Asn Val Ala Gly Tyr Glu Val Pro 770 775 780 Gln Leu Tyr
Val Ser Leu Gly Gly Pro Asn Glu Pro Arg Val Val Leu 785 790 795 800
Arg Lys Phe Asp Arg Ile Phe Leu Ala Pro Gly Glu Gln Lys Val Trp 805
810 815 Thr Thr Thr Leu Asn Arg Arg Asp Leu Ala Asn Trp Asp Val Glu
Ala 820 825 830 Gln Asp Trp Val Ile Thr Lys Tyr Pro Lys Lys Val His
Val Gly Ser 835 840 845 Ser Ser Arg Lys Leu Pro Leu Arg Ala Pro Leu
Pro Arg Val Tyr 850 855 860 5029DNAAspergillus fumigatus
50ggctcatgag attcggttgg ctcgaggtc 295130DNAAspergillus fumigatus
51gccgttatca cagccgcggt cggggcagcc 305230DNAAspergillus fumigatus
52ggctgccccg accgcggctg tgataacggc 305335DNAAspergillus fumigatus
53gcttaattaa tctagtagac acggggcaga ggcgc 355416DNAAspergillus
fumigatus 54acactggcgg agaagg 165518DNAAspergillus fumigatus
55gcccagggat atggttac 185619DNAAspergillus fumigatus 56cgactctgga
gagggtttc 195719DNAAspergillus fumigatus 57ggactgggtc atcacaaag
195817DNAAspergillus fumigatus 58gcgagaggtc atcagca
175917DNAAspergillus fumigatus 59gtaaaacgac ggccagt
176016DNAAspergillus fumigatus 60caggaaacag ctatga
166169DNAAspergillus fumigatus 61cttcttgtta gtgcaatatc atatagaagt
catcgactag tggatctacc atgagattcg 60gttggctcg 696261DNAAspergillus
fumigatus 62gcgtgaatgt aagcgtgaca taactaatta catgactcga gctagtagac
acggggcaga 60g 616360DNAAspergillus fumigatus 63ccgctccgcc
gttgtggccg ccctgccggt gttggccctt gccgaattgg ctttctctcc
606417DNAAspergillus fumigatus 64ctggcgttgg cgctgtc 17
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