U.S. patent application number 15/051169 was filed with the patent office on 2016-06-16 for polypeptides having lipase activity and polynucleotides encoding same.
This patent application is currently assigned to Novozymes Inc.. The applicant listed for this patent is Novozymes A/S, Novozymes Inc.. Invention is credited to Christopher Amolo, Kim Borch, Barbara Cherry, Haiyan Ge, Michael Lamsa, Janine Lin, Suzanne Otani, Shamkant Anant Patkar, Debbie Yaver.
Application Number | 20160168547 15/051169 |
Document ID | / |
Family ID | 35789088 |
Filed Date | 2016-06-16 |
United States Patent
Application |
20160168547 |
Kind Code |
A1 |
Otani; Suzanne ; et
al. |
June 16, 2016 |
Polypeptides Having Lipase Activity and Polynucleotides Encoding
Same
Abstract
The present invention relates to isolated polypeptides having
lipase activity and isolated polynucleotides encoding the
polypeptides. The invention also relates to nucleic acid
constructs, vectors, and host cells comprising the polynucleotides
as well as methods for producing and using the polypeptides.
Inventors: |
Otani; Suzanne; (Elk Grove,
CA) ; Yaver; Debbie; (Davis, CA) ; Ge;
Haiyan; (Davis, CA) ; Lin; Janine; (Davis,
CA) ; Amolo; Christopher; (West Sacramento, CA)
; Borch; Kim; (Birkerod, DK) ; Patkar; Shamkant
Anant; (Lyngby, DK) ; Lamsa; Michael;
(Woodland, CA) ; Cherry; Barbara; (Winters,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Novozymes Inc.
Novozymes A/S |
Davis
Bagsvaerd |
CA |
US
DK |
|
|
Assignee: |
Novozymes Inc.
Davis
CA
Novozymes A/S
Bagsvaerd
|
Family ID: |
35789088 |
Appl. No.: |
15/051169 |
Filed: |
February 23, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13747184 |
Jan 22, 2013 |
9303248 |
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15051169 |
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12696676 |
Jan 29, 2010 |
8377675 |
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13747184 |
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11241677 |
Sep 30, 2005 |
7666630 |
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12696676 |
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60614508 |
Sep 30, 2004 |
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60621304 |
Oct 21, 2004 |
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60621222 |
Oct 21, 2004 |
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Current U.S.
Class: |
426/18 ;
435/198 |
Current CPC
Class: |
C12Y 301/01003 20130101;
C12N 9/20 20130101 |
International
Class: |
C12N 9/20 20060101
C12N009/20 |
Claims
1. An isolated polypeptide having lipase activity, selected from
the group consisting of: (a) a polypeptide having an amino acid
sequence which has at least 60% identity with the mature
polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO:
8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, or SEQ ID NO: 16;
(b) a polypeptide which is encoded by a polynucleotide which
hybridizes under at least medium stringency conditions with (i) the
mature polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3,
SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO:
13, or SEQ ID NO: 15, (ii) the cDNA sequence contained in the
mature polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3,
SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO:
13, or SEQ ID NO: 15, or (iii) a complementary strand of (i) or
(ii); and (c) a variant comprising a conservative substitution,
deletion, and/or insertion of one or more amino acids of the mature
polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO:
8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, or SEQ ID NO:
16.
2. The polypeptide of claim 1, having an amino acid sequence which
has at least 60% identity with the mature polypeptide of SEQ ID NO:
2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID
NO: 12, SEQ ID NO: 14, or SEQ ID NO: 16.
3. The polypeptide of claim 2, having an amino acid sequence which
has at least 65% identity with the mature polypeptide of SEQ ID NO:
2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID
NO: 12, SEQ ID NO: 14, or SEQ ID NO: 16.
4. The polypeptide of claim 3, having an amino acid sequence which
has at least 70% identity with the mature polypeptide of SEQ ID NO:
2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID
NO: 12, SEQ ID NO: 14, or SEQ ID NO: 16.
5. The polypeptide of claim 4, having an amino acid sequence which
has at least 75% identity with the mature polypeptide of SEQ ID NO:
2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID
NO: 12, SEQ ID NO: 14, or SEQ ID NO: 16.
6. The polypeptide of claim 5, having an amino acid sequence which
has at least 80% identity with the mature polypeptide of SEQ ID NO:
2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID
NO: 12, SEQ ID NO: 14, or SEQ ID NO: 16.
7. The polypeptide of claim 6, having an amino acid sequence which
has at least 85% identity with the mature polypeptide of SEQ ID NO:
2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID
NO: 12, SEQ ID NO: 14, or SEQ ID NO: 16.
8. The polypeptide of claim 7, having an amino acid sequence which
has at least 90% identity with the mature polypeptide of SEQ ID NO:
2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID
NO: 12, SEQ ID NO: 14, or SEQ ID NO: 16.
9. The polypeptide of claim 8, having an amino acid sequence which
has at least 95% identity with the mature polypeptide of SEQ ID NO:
2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID
NO: 12, SEQ ID NO: 14, or SEQ ID NO: 16.
10. The polypeptide of claim 1, which comprises the amino acid
sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8,
SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, or SEQ ID NO: 16; or a
fragment thereof having lipase activity.
11. The polypeptide of claim 10, which comprises the amino acid
sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8,
SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, or SEQ ID NO: 16.
12. The polypeptide of claim 11, which comprises the mature
polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO:
8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, or SEQ ID NO:
16.
13. The polypeptide of claim 1, which consists of SEQ ID NO: 2, SEQ
ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12,
SEQ ID NO: 14, or SEQ ID NO: 16; or a fragment thereof having
lipase activity.
14. The polypeptide of claim 13, which consists of SEQ ID NO: 2,
SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO:
12, SEQ ID NO: 14, or SEQ ID NO: 16.
15. The polypeptide of claim 13, which consists of the mature
polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO:
8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, or SEQ ID NO:
16.
16-48. (canceled)
49. A method for preparing a dough or a baked product comprising
incorporating into a dough a polypeptide of claim 1.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application of U.S. patent
application Ser. No. 13/747,184 filed on Jan. 22, 2013 which is a
divisional of U.S. application Ser. No. 12/696,676, filed Jan. 29,
2010, which is a divisional application of U.S. patent application
Ser. No. 11/241,677, filed Sep. 30, 2005, now U.S. Pat. No.
7,666,630, which claims the benefit of U.S. Provisional Application
No. 60/614,508, filed Sep. 30, 2004, U.S. Provisional Application
No. 60/621,304, filed Oct. 21, 2004, and U.S. Provisional
Application No. 60/621,222, filed Oct. 21, 2004, which applications
are incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates to isolated polypeptides
having lipase activity and isolated polynucleotides encoding the
polypeptides. The invention also relates to nucleic acid
constructs, vectors, and host cells comprising the polynucleotides
as well as methods for producing and using the polypeptides.
[0004] 2. Description of the Related Art
[0005] Triacylglycerol hydrolyzing enzymes are enzymes that
catalyze the hydrolysis or formation of triglycerides.
Triacylglycerol hydrolyzing enzymes are a versatile group of
enzymes and often have more than one activity such as lipase,
phospholipase, lysophospholipase, cholesterol esterase, cutinase,
amidase, galactolipase, and other esterase type of activities.
Which activity is the predominant activity will depend on the
application of the enzyme and the conditions.
[0006] Triacylglycerol hydrolyzing enzymes belong to the IUBMB
Enzyme Nomenclature 3.1.1. EC 3 refers to hydrolases, EC 3.1 refers
to acting on ester bonds, and EC 3.1.1 refers to carboxylic ester
hydrolases. Related enzymes are classified in EC 3.1.4, which
refers to phosphoric diester hydrolases.
[0007] Lipases (EC 3.1.1.3) are enzymes that catalyze the
hydrolysis of a wide range of carboxy esters, e.g., triglycerides
to release fatty acid. Esterases (EC 3.1.1.1) are enzymes that
catalyze the hydrolysis of water-soluble carboxylic esters,
including short-chain fatty acid triglycerides, to produce an
alcohol and a carboxylic acid anion.
[0008] Some lipases also have phospholipase activity and/or
galactolipase activity (see, for example, U.S. Pat. No. 6,103,505
and U.S. Pat. No. 6,852,346).
[0009] Phospholipases are enzymes that catalyze the hydrolysis of
phospholipids which consist of a glycerol backbone with two fatty
acids in the sn1 and sn2 positions, which is esterified with a
phosphoric acid in the sn3 position. The phosphoric acid may, in
turn, be esterified to an amino alcohol.
[0010] There are several types of phospholipases which catalyze the
hydrolysis of the fatty acyl moieties. These phospholipases include
phospholipase A.sub.1 (EC 3.1.1.32), phospholipase A.sub.2 (EC
3.1.1.4), and lysophospholipase (EC 3.1.1.5). Phospholipase C (EC
3.1.4.3) and phospholipase D (EC 3.1.4.4) hydrolyze the phosphoric
acid group from a phospholipid, but do not hydrolyze fatty acids
like phospholipase A.sub.1, phospholipase A.sub.2 and phospholipase
B.
[0011] Phospholipase A.sub.1 (EC 3.1.1.32) catalyzes the
deacylation of one fatty acyl group in the sn1 position from a
diacylglycerophospholipid to produce lysophospholipid and fatty
acid. Phospholipase A.sub.2 (EC 3.1.1.4) catalyzes the deacylation
of one fatty acyl group in the sn2 position from a
diacylglycerophospholipid to produce lysophospholipid and fatty
acid. Lysophospholipase (EC 3.1.1.5), also known as phospholipase
B, catalyzes the hydrolysis of the fatty acyl group in a
lysophospholipid. Phospholipase C (EC 3.1.4.3) catalyzes the
hydrolysis of phosphatidylcholine to 1,2-diacylglycerol and choline
phosphate. Phospholipase D (EC 3.1.4.4) catalyzes the hydrolysis of
the terminal phosphate diester bond of phosphatidylcholine to
produce choline and phosphatidic acid.
[0012] Galactolipases (EC 3.1.1.26) catalyze the hydrolysis of
galactolipids by removing one or two fatty acids.
[0013] Detergents formulated with lipolytic enzymes are known to
have improved properties for removing fatty stains. For example,
LIPOLASE.TM. (Novozymes A/S, Bagsv.ae butted.rd, Denmark), a
microbial lipase obtained from the fungus Thermomyces lanuginosus
(also called Humicola lanuginosa), has been introduced into many
commercial brands of detergent. Lipases have also been used in
degumming processes and baking.
[0014] El-Shahed et al., 1988, Egypt. J. Microbiol. 23: 357-372 and
Mohawed et al., 1988, Egypt. J. Microbiol 23: 537-547 disclose two
Aspergillus fumigatus lipases.
[0015] WO 03/12071 discloses a gene encoding a lipase from
Aspergillus fumigatus.
[0016] Mayordomo et al., 2000, J. Agric. Chem. 48: 105-109 disclose
the isolation, purification, and characterization of a cold-active
lipase from Aspergillus nidulans.
[0017] Lipases have many commercial uses but very few lipases that
work under application conditions and can be produced with high
yields by microbial fermentation have been identified. There is a
need in the art for alternative lipases with improved
properties.
[0018] It is an object of the present invention to provide
polypeptides having lipase activity and polynucleotides encoding
the polypeptides.
SUMMARY OF THE INVENTION
[0019] The present invention relates to isolated polypeptides
having lipase activity selected from the group consisting of:
[0020] (a) a polypeptide having an amino acid sequence which has at
least 60% identity with the mature polypeptide of SEQ ID NO: 2, SEQ
ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12,
SEQ ID NO: 14, or SEQ ID NO: 16;
[0021] (b) a polypeptide which is encoded by a nucleotide sequence
which hybridizes under at least medium stringency conditions with
(i) the mature polypeptide coding sequence of SEQ ID NO: 1, SEQ ID
NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ
ID NO: 13, or SEQ ID NO: 15, (ii) the cDNA sequence contained in
the mature polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO:
3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID
NO: 13, or SEQ ID NO: 15, or (iii) a complementary strand of (i) or
(ii); and
[0022] (c) a variant comprising a conservative substitution,
deletion, and/or insertion of one or more amino acids of the mature
polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO:
8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, or SEQ ID NO:
16.
[0023] The present invention also relates to isolated
polynucleotides encoding polypeptides having lipase activity,
selected from the group consisting of:
[0024] (a) a polynucleotide encoding a polypeptide having an amino
acid sequence which has at least 60% identity with the mature
polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO:
8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, or SEQ ID NO:
16;
[0025] (b) a polynucleotide having at least 60% identity with the
mature polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3,
SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO:
13, or SEQ ID NO: 15; and
[0026] (c) a polynucleotide which hybridizes under at least medium
stringency conditions with (i) the mature polypeptide coding
sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7,
SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, or SEQ ID NO: 15, (ii)
the cDNA sequence contained in the mature polypeptide coding
sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7,
SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, or SEQ ID NO: 15, or
(iii) a complementary strand of (i) or (ii).
[0027] In a preferred aspect, the mature polypeptide is amino acids
25 to 396 of SEQ ID NO: 2, amino acids 25 to 283 of SEQ ID NO: 4,
amino acids 20 to 318 of SEQ ID NO: 6, amino acids 19 to 348 of SEQ
ID NO: 8, amino acids 25 to 393 of SEQ ID NO: 10, amino acids 20 to
294 of SEQ ID NO: 12, amino acids 25 to 308 of SEQ ID NO: 14, or
amino acids 26 to 404 of SEQ ID NO: 16. In another preferred
aspect, the mature polypeptide coding sequence is nucleotides 73 to
1256 of SEQ ID NO: 1, nucleotides 73 to 944 of SEQ ID NO: 3,
nucleotides 58 to 1085 of SEQ ID NO: 5, nucleotides 55 to 1044 of
SEQ ID NO: 7, nucleotides 73 to 1179 of SEQ ID NO: 9, nucleotides
58 to 1038 of SEQ ID NO: 11, nucleotides 73 to 1119 of SEQ ID NO:
13, or nucleotides 76 to 1280 of SEQ ID NO: 15.
[0028] The present invention also relates to nucleic acid
constructs, recombinant expression vectors, and recombinant host
cells comprising the polynucleotides.
[0029] The present invention also relates to methods for producing
such a polypeptide having lipase activity comprising: (a)
cultivating a recombinant host cell comprising a nucleic acid
construct comprising a polynucleotide encoding the polypeptide
under conditions conducive for production of the polypeptide; and
(b) recovering the polypeptide.
[0030] The present invention also relates to methods of using the
polypeptides having lipase activity in detergents, degumming, and
baking.
[0031] The present invention further relates to nucleic acid
constructs comprising a gene encoding a protein, wherein the gene
is operably linked to a nucleotide sequence encoding a signal
peptide comprising or consisting of nucleotides 1 to 72 of SEQ ID
NO: 1, nucleotides 1 to 72 of SEQ ID NO: 3, nucleotides 1 to 57 of
SEQ ID NO: 5, nucleotides 1 to 54 of SEQ ID NO: 7, nucleotides 1 to
72 of SEQ ID NO: 9, nucleotides 1 to 57 of SEQ ID NO: 11,
nucleotides 1 to 72 of SEQ ID NO: 13, or nucleotides 1 to 75 of SEQ
ID NO: 15, wherein the gene is foreign to the nucleotide
sequence.
BRIEF DESCRIPTION OF THE FIGURES
[0032] FIG. 1 shows a restriction map of pSMO224.
[0033] FIG. 2 shows the genomic DNA sequence and the deduced amino
acid sequence of an Aspergillus fumigatus lipase (Aspergillus
fumigatus lip1, SEQ ID NOs: 1 and 2, respectively).
[0034] FIG. 3 shows a restriction map of pAlLo1.
[0035] FIG. 4 shows a restriction map of pBM121b.
[0036] FIG. 5 shows a restriction map of pBM120a.
[0037] FIG. 6 shows a restriction map of pSMO218.
[0038] FIG. 7 shows a restriction map of pSMO223.
[0039] FIG. 8 shows the genomic DNA sequence and the deduced amino
acid sequence of an Aspergillus fumigatus lipase (Aspergillus
fumigatus lip2, SEQ ID NOs: 3 and 4, respectively).
[0040] FIG. 9 shows a restriction map of pHyGe026.
[0041] FIGS. 10A and 10B show the genomic DNA sequence and the
deduced amino acid sequence of a Magnaporthe grisea lipase
(Magnaporthe grisea lip1, SEQ ID NOs: 5 and 6, respectively).
[0042] FIG. 11 shows a restriction map of pHyGe010.
[0043] FIG. 12 shows a restriction map of pCrAm138.
[0044] FIG. 13 shows the genomic DNA sequence and the deduced amino
acid sequence of a Magnaporthe grisea lipase (Magnaporthe grisea
lip2, SEQ ID NOs: 7 and 8, respectively).
[0045] FIG. 14 shows a restriction map of pBM135g.
[0046] FIG. 15 shows the genomic DNA sequence and the deduced amino
acid sequence of a Magnaporthe grisea lipase (Magnaporthe grisea
lip3, SEQ ID NOs: 9 and 10, respectively).
[0047] FIG. 16 shows a restriction map of pJLin171.
[0048] FIG. 17 shows the genomic DNA sequence and the deduced amino
acid sequence of an Aspergillus nidulans lipase (Aspergillus
nidulans lip1, SEQ ID NOs: 11 and 12, respectively).
[0049] FIG. 18 shows a restriction map of pJLin167.
[0050] FIG. 19 shows a restriction map of pJLin170.
[0051] FIG. 20 shows the genomic DNA sequence and the deduced amino
acid sequence of an Aspergillus nidulans lipase (Aspergillus
nidulans lip2, SEQ ID NOs: 13 and 14, respectively).
[0052] FIG. 21 shows a restriction map of pJSF8c.
[0053] FIG. 22 shows a restriction map of pBM141.
[0054] FIG. 23 shows the genomic DNA sequence and the deduced amino
acid sequence of an Aspergillus nidulans lipase (Aspergillus
nidulans lip3, SEQ ID NOs: 15 and 16, respectively).
DEFINITIONS
[0055] Lipase activity: The term "lipase activity" is defined
herein as a triacylglycerol acylhydrolase activity (E.C. 3.1.1.3)
which catalyzes the hydrolysis of a triacylglycerol to fatty
acid(s). The substrate spectrum of lipases includes triglycerides,
diglycerides, and monoglycerides, but for the purpose of the
present invention, lipase activity is determined using
p-nitrophenyl butyrate as substrate. One unit of lipase activity
equals the amount of enzyme capable of releasing 1 .mu.mole of
butyric acid per minute at pH 7.5, 25.degree. C. Encompassed within
the term "lipase activity" are polypeptides that also have
phospholipase activity and/or galactolipase activity, as defined
herein.
[0056] The polypeptides of the present invention have at least 20%,
preferably at least 40%, more preferably at least 50%, more
preferably at least 60%, more preferably at least 70%, more
preferably at least 80%, even more preferably at least 90%, most
preferably at least 95%, and even most preferably at least 100% of
the lipase activity of the mature polypeptide of SEQ ID NO: 2, SEQ
ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12,
SEQ ID NO: 14, or SEQ ID NO: 16.
[0057] Phospholipase Activity: The term "phospholipase activity" is
defined herein as a phosphatidyl acylhydrolase (EC 3.1.1.4, EC
3.1.1.5, and EC 3.1.1.32) which catalyzes the hydrolysis of a fatty
acid from a phospholipid. Phospholipids consist of a glycerol
backbone with two fatty acids in the sn1 and sn2 positions, which
is esterified with a phosphoric acid in the sn3 position. The
phosphoric acid may, in turn, be esterified with an amino
alcohol.
[0058] Phospholipase A.sub.1 (EC 3.1.1.32) catalyzes the
deacylation of one fatty acyl group in the sn1 position from a
diacylglycerophospholipid to produce lysophospholipid and fatty
acid.
[0059] Phospholipase A.sub.2 (EC 3.1.1.4) catalyzes the deacylation
of one fatty acyl group in the sn2 position from a
diacylglycerophospholipid to produce lysophospholipid and fatty
acid.
[0060] Lysophospholipase (EC 3.1.1.5), also known as phospholipase
B, catalyzes the hydrolysis of the fatty acyl group in a
lysophospholipid.
[0061] For purposes of the present invention, phospholipase
A.sub.1, phospholipase A.sub.2, and lysophospholipase activity is
determined according to WO 2005/040410 using phosphatidylcholines
derived from soy (Avanti Polar Lipids Inc., AL, USA) as
substrate.
[0062] Galactolipase Activity: The term "galactolipase activity" is
defined herein as a 1,2-diacyl-3-beta-D-galactosyl-sn-glycerol
acylhydrolase (EC 3.1.1.26) which catalyzes the hydrolysis of
galactolipids by removing one or two fatty acids. Galactolipase
activity is determined according to WO 2005/040410 using
digalactosyldiglyceride (DGDG) or monogalactosyldiglyceride (MGDG)
extracted from wheat flour as substrate. DGDG is a galactolipid
that consists of two fatty acids and a digalactose. MGDG is a
galactolipid that consists of two fatty acids and a galactose.
[0063] Isolated Polypeptide: The term "isolated polypeptide" as
used herein refers to a polypeptide which is at least 20% pure,
preferably at least 40% pure, more preferably at least 60% pure,
even more preferably at least 80% pure, most preferably at least
90% pure, and even most preferably at least 95% pure, as determined
by SDS-PAGE.
[0064] Substantially Pure Polypeptide: The term "substantially pure
polypeptide" denotes herein a polypeptide preparation which
contains at most 10%, preferably at most 8%, more preferably at
most 6%, more preferably at most 5%, more preferably at most 4%,
more preferably at most 3%, even more preferably at most 2%, most
preferably at most 1%, and even most preferably at most 0.5% by
weight of other polypeptide material with which it is natively
associated. It is, therefore, preferred that the substantially pure
polypeptide is at least 92% pure, preferably at least 94% pure,
more preferably at least 95% pure, more preferably at least 96%
pure, more preferably at least 96% pure, more preferably at least
97% pure, more preferably at least 98% pure, even more preferably
at least 99%, most preferably at least 99.5% pure, and even most
preferably 100% pure by weight of the total polypeptide material
present in the preparation.
[0065] The polypeptides of the present invention are preferably in
a substantially pure form. In particular, it is preferred that the
polypeptides are in "essentially pure form", i.e., that the
polypeptide preparation is essentially free of other polypeptide
material with which it is natively associated. This can be
accomplished, for example, by preparing the polypeptide by means of
well-known recombinant methods or by classical purification
methods.
[0066] Herein, the term "substantially pure polypeptide" is
synonymous with the terms "isolated polypeptide" and "polypeptide
in isolated form."
[0067] Mature Polypeptide: The term "mature polypeptide" is defined
herein as a polypeptide having lipase activity that is in its final
form following translation and any post-translational
modifications, such as N-terminal processing, C-terminal
truncation, glycosylation, etc.
[0068] Identity: The relatedness between two amino acid sequences
or between two nucleotide sequences is described by the parameter
"identity".
[0069] 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.
[0070] For purposes of the present invention, the degree of
identity between two nucleotide 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.
[0071] Homologous Sequence: The term "homologous sequence" is
defined herein as a predicted protein which gives an E value (or
expectancy score) of less than 0.001 in a tfasty search (Pearson,
W. R., 1999, in Bioinformatics Methods and Protocols, S. Misener
and S. A. Krawetz, ed., pp. 185-219) with the Thermomyces
lanuginosus lipase (Accession No. O59952).
[0072] Polypeptide Fragment: The term "polypeptide fragment" is
defined herein as a polypeptide having one or more amino acids
deleted from the amino and/or carboxyl terminus of the mature
polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO:
8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, or SEQ ID NO: 16;
or a homologous sequence thereof; wherein the fragment has lipase
activity. In a preferred aspect, a fragment contains at least 325
amino acid residues, more preferably at least 340 amino acid
residues, and most preferably at least 355 amino acid residues of
the mature polypeptide of SEQ ID NO: 2 or a homologous sequence
thereof. In another preferred aspect, a fragment contains at least
215 amino acid residues, more preferably at least 230 amino acid
residues, and most preferably at least 245 amino acid residues of
the mature polypeptide of SEQ ID NO: 4 or a homologous sequence
thereof. In another preferred aspect, a fragment contains at least
255 amino acid residues, more preferably at least 270 amino acid
residues, and most preferably at least 285 amino acid residues of
the mature polypeptide of SEQ ID NO: 6 or a homologous sequence
thereof. In another preferred aspect, a fragment contains at least
285 amino acid residues, more preferably at least 300 amino acid
residues, and most preferably at least 315 amino acid residues of
the mature polypeptide of SEQ ID NO: 8 or a homologous sequence
thereof. In another preferred aspect, a fragment contains at least
320 amino acid residues, more preferably at least 335 amino acid
residues, and most preferably at least 350 amino acid residues of
the mature polypeptide of SEQ ID NO: 10 or a homologous sequence
thereof. In another preferred aspect, a fragment contains at least
230 amino acid residues, more preferably at least 245 amino acid
residues, and most preferably at least 260 amino acid residues of
the mature polypeptide of SEQ ID NO: 12 or a homologous sequence
thereof. In another preferred aspect, a fragment contains at least
240 amino acid residues, more preferably at least 255 amino acid
residues, and most preferably at least 270 amino acid residues of
the mature polypeptide of SEQ ID NO: 14 or a homologous sequence
thereof. In another preferred aspect, a fragment contains at least
320 amino acid residues, more preferably at least 340 amino acid
residues, and most preferably at least 360 amino acid residues of
the mature polypeptide of SEQ ID NO: 16 or a homologous sequence
thereof.
[0073] Subsequence: The term "subsequence" is defined herein as a
nucleotide sequence having one or more nucleotides deleted from the
5' and/or 3' end of the mature polypeptide coding sequence of SEQ
ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9,
SEQ ID NO: 11, SEQ ID NO: 13, or SEQ ID NO: 15; or a homologous
sequence thereof; wherein the subsequence encodes a polypeptide
fragment having lipase activity. In a preferred aspect, a
subsequence contains at least 975 nucleotides, more preferably at
least 1020 nucleotides, and most preferably at least 1060
nucleotides of the mature polypeptide coding sequence of SEQ ID NO:
1 or a homologous sequence thereof. In another preferred aspect, a
subsequence contains at least 645 nucleotides, more preferably at
least 690 nucleotides, and most preferably at least 735 nucleotides
of the mature polypeptide coding sequence of SEQ ID NO: 3 or a
homologous sequence thereof. In a preferred aspect, a subsequence
contains at least 765 nucleotides, more preferably at least 810
nucleotides, and most preferably at least 855 nucleotides of the
mature polypeptide coding sequence of SEQ ID NO: 5 or a homologous
sequence thereof. In another preferred aspect, a subsequence
contains at least 855 nucleotides, more preferably at least 900
nucleotides, and most preferably at least 945 nucleotides of the
mature polypeptide coding sequence of SEQ ID NO: 7 or a homologous
sequence thereof. In another preferred aspect, a subsequence
contains at least 960 nucleotides, more preferably at least 1005
nucleotides, and most preferably at least 1050 nucleotides of the
mature polypeptide coding sequence of SEQ ID NO: 9 or a homologous
sequence thereof. In a preferred aspect, a subsequence contains at
least 690 nucleotides, more preferably at least 735 nucleotides,
and most preferably at least 780 nucleotides of the mature
polypeptide coding sequence of SEQ ID NO: 11 or a homologous
sequence thereof. In another preferred aspect, a subsequence
contains at least 720 nucleotides, more preferably at least 765
nucleotides, and most preferably at least 810 nucleotides of the
mature polypeptide coding sequence of SEQ ID NO: 13 or a homologous
sequence thereof. In another preferred aspect, a subsequence
contains at least 1020 nucleotides, more preferably at least 1020
nucleotides, and most preferably at least 1080 nucleotides of the
mature polypeptide coding sequence of SEQ ID NO: 15 or a homologous
sequence thereof.
[0074] Allelic Variant: The term "allelic variant" denotes herein
any of two or more alternative forms of a gene occupying the same
chromosomal locus. Allelic variation arises naturally through
mutation, and may result in polymorphism within populations. Gene
mutations can be silent (no change in the encoded polypeptide) or
may encode polypeptides having altered amino acid sequences. An
allelic variant of a polypeptide is a polypeptide encoded by an
allelic variant of a gene.
[0075] Isolated Polynucleotide: The term "isolated polynucleotide"
as used herein refers to a polynucleotide which is at least 20%
pure, preferably at least 40% pure, more preferably at least 60%
pure, even more preferably at least 80% pure, most preferably at
least 90% pure, and even most preferably at least 95% pure, as
determined by agarose electrophoresis.
[0076] Substantially Pure Polynucleotide: The term "substantially
pure polynucleotide" as used herein refers to a polynucleotide
preparation free of other extraneous or unwanted nucleotides and in
a form suitable for use within genetically engineered protein
production systems. Thus, a substantially pure polynucleotide
contains at most 10%, preferably at most 8%, more preferably at
most 6%, more preferably at most 5%, more preferably at most 4%,
more preferably at most 3%, even more preferably at most 2%, most
preferably at most 1%, and even most preferably at most 0.5% by
weight of other polynucleotide material with which it is natively
associated. A substantially pure polynucleotide may, however,
include naturally occurring 5' and 3' untranslated regions, such as
promoters and terminators. It is preferred that the substantially
pure polynucleotide is at least 90% pure, preferably at least 92%
pure, more preferably at least 94% pure, more preferably at least
95% pure, more preferably at least 96% pure, more preferably at
least 97% pure, even more preferably at least 98% pure, most
preferably at least 99%, and even most preferably at least 99.5%
pure by weight. The polynucleotides of the present invention are
preferably in a substantially pure form. In particular, it is
preferred that the polynucleotides disclosed herein are in
"essentially pure form", i.e., that the polynucleotide preparation
is essentially free of other polynucleotide material with which it
is natively associated. Herein, the term "substantially pure
polynucleotide" is synonymous with the terms "isolated
polynucleotide" and "polynucleotide in isolated form." The
polynucleotides may be of genomic, cDNA, RNA, semisynthetic,
synthetic origin, or any combinations thereof.
[0077] Mature Polypeptide Coding Sequence: The term "mature
polypeptide coding sequence" is defined herein as a nucleotide
sequence that encodes a mature polypeptide having lipase
activity.
[0078] cDNA: The term "cDNA" is defined herein as a DNA molecule
which can be prepared by reverse transcription from a mature,
spliced, mRNA molecule obtained from a eukaryotic cell. cDNA lacks
intron sequences that are usually present in the corresponding
genomic DNA. The initial, primary RNA transcript is a precursor to
mRNA which is processed through a series of steps before appearing
as mature spliced mRNA. These steps include the removal of intron
sequences by a process called splicing. cDNA derived from mRNA
lacks, therefore, any intron sequences.
[0079] Nucleic Acid Construct: The term "nucleic acid construct" as
used herein refers to a nucleic acid molecule, either single- or
double-stranded, which is isolated from a naturally occurring gene
or which is modified to contain segments of nucleic acids in a
manner that would not otherwise exist in nature. The term nucleic
acid construct is synonymous with the term "expression cassette"
when the nucleic acid construct contains the control sequences
required for expression of a coding sequence of the present
invention.
[0080] Control Sequence: The term "control sequences" is defined
herein to include all components, which are necessary or
advantageous for the expression of a polynucleotide encoding a
polypeptide of the present invention. Each control sequence may be
native or foreign to the nucleotide sequence encoding the
polypeptide or native or foreign to each other. Such control
sequences include, but are not limited to, a leader,
polyadenylation sequence, propeptide sequence, promoter, signal
peptide sequence, and transcription terminator. At a minimum, the
control sequences include a promoter, and transcriptional and
translational stop signals. The control sequences may be provided
with linkers for the purpose of introducing specific restriction
sites facilitating ligation of the control sequences with the
coding region of the nucleotide sequence encoding a
polypeptide.
[0081] Operably Linked: The term "operably linked" denotes herein a
configuration in which a control sequence is placed at an
appropriate position relative to the coding sequence of the
polynucleotide sequence such that the control sequence directs the
expression of the coding sequence of a polypeptide.
[0082] Coding Sequence: When used herein the term "coding sequence"
means a nucleotide sequence, which directly specifies the amino
acid sequence of its protein product. The boundaries of the coding
sequence are generally determined by an open reading frame, which
usually begins with the ATG start codon or alternative start codons
such as GTG and TTG and ends with a stop codon such as TAA, TAG and
TGA. The coding sequence may be a DNA, cDNA, or recombinant
nucleotide sequence.
[0083] Expression: The term "expression" includes any step involved
in the production of the polypeptide including, but not limited to,
transcription, post-transcriptional modification, translation,
post-translational modification, and secretion.
[0084] Expression Vector: The term "expression vector" is defined
herein as a linear or circular DNA molecule that comprises a
polynucleotide encoding a polypeptide of the invention, and which
is operably linked to additional nucleotides that provide for its
expression.
[0085] Host Cell: The term "host cell", as used herein, includes
any cell type which is susceptible to transformation, transfection,
transduction, and the like with a nucleic acid construct or
expression vector comprising a polynucleotide of the present
invention.
[0086] Modification: The term "modification" means herein any
chemical modification of the polypeptide consisting of the mature
polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO:
8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, or SEQ ID NO: 16;
or a homologous sequence thereof; as well as genetic manipulation
of the DNA encoding such a polypeptide. The modification can be
substitutions, deletions and/or insertions of one or more amino
acids as well as replacements of one or more amino acid side
chains.
[0087] Artificial Variant: When used herein, the term "artificial
variant" means a polypeptide having lipase activity produced by an
organism expressing a modified nucleotide sequence of the mature
polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID
NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, or
SEQ ID NO: 15; or a homologous sequence thereof. The modified
nucleotide sequence is obtained through human intervention by
modification of the nucleotide sequence disclosed in SEQ ID NO: 1,
SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO:
11, SEQ ID NO: 13, or SEQ ID NO: 15; or a homologous sequence
thereof.
DETAILED DESCRIPTION OF THE INVENTION
Polypeptides Having Lipase Activity
[0088] In a first aspect, the present invention relates to isolated
polypeptides having an amino acid sequence which has a degree of
identity to the mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4,
SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID
NO: 14, or SEQ ID NO: 16, of at least 60%, preferably at least 65%,
more preferably at least 70%, more preferably at least 75%, more
preferably at least 80%, more preferably at least 85%, even more
preferably at least 90%, most preferably at least 95%, and even
most preferably at least 97%, 98%, or 99%, which have lipase
activity (hereinafter "homologous polypeptides"). In a preferred
aspect, the homologous polypeptides have an amino acid sequence
which differs by ten amino acids, preferably by five amino acids,
more preferably by four amino acids, even more preferably by three
amino acids, most preferably by two amino acids, and even most
preferably by one amino acid from the mature polypeptide of SEQ ID
NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ
ID NO: 12, SEQ ID NO: 14, or SEQ ID NO: 16.
[0089] A polypeptide of the present invention preferably comprises
the amino acid sequence of SEQ ID NO: 2 or an allelic variant
thereof; or a fragment thereof that has lipase activity. In a
preferred aspect, a polypeptide comprises the amino acid sequence
of SEQ ID NO: 2. In another preferred aspect, a polypeptide
comprises the mature polypeptide of SEQ ID NO: 2. In another
preferred aspect, a polypeptide comprises amino acids 25 to 396 of
SEQ ID NO: 2, or an allelic variant thereof; or a fragment thereof
that has lipase activity. In another preferred aspect, a
polypeptide comprises amino acids 25 to 396 of SEQ ID NO: 2. In
another preferred aspect, a polypeptide consists of the amino acid
sequence of SEQ ID NO: 2 or an allelic variant thereof; or a
fragment thereof that has lipase activity. In another preferred
aspect, a polypeptide consists of the amino acid sequence of SEQ ID
NO: 2. In another preferred aspect, a polypeptide consists of the
mature polypeptide of SEQ ID NO: 2. In another preferred aspect, a
polypeptide consists of amino acids 25 to 396 of SEQ ID NO: 2 or an
allelic variant thereof; or a fragment thereof that has lipase
activity. In another preferred aspect, a polypeptide consists of
amino acids 25 to 396 of SEQ ID NO: 2.
[0090] A polypeptide of the present invention preferably comprises
the amino acid sequence of SEQ ID NO: 4, or an allelic variant
thereof; or a fragment thereof that has lipase activity. In a
preferred aspect, a polypeptide comprises the amino acid sequence
of SEQ ID NO: 4. In another preferred aspect, a polypeptide
comprises the mature polypeptide of SEQ ID NO: 4. In another
preferred aspect, a polypeptide comprises amino acids 25 to 283 of
SEQ ID NO: 4, or an allelic variant thereof; or a fragment thereof
that has lipase activity. In another preferred aspect, a
polypeptide comprises amino acids 25 to 283 of SEQ ID NO: 4. In
another preferred aspect, a polypeptide consists of the amino acid
sequence of SEQ ID NO: 4 or an allelic variant thereof; or a
fragment thereof that has lipase activity. In another preferred
aspect, a polypeptide consists of the amino acid sequence of SEQ ID
NO: 4. In another preferred aspect, a polypeptide consists of amino
acids 25 to 283 of SEQ ID NO: 4 or an allelic variant thereof; or a
fragment thereof that has lipase activity. In another preferred
aspect, a polypeptide consists of amino acids 25 to 283 of SEQ ID
NO: 4.
[0091] A polypeptide of the present invention preferably comprises
the amino acid sequence of SEQ ID NO: 6, or an allelic variant
thereof; or a fragment thereof that has lipase activity. In a
preferred aspect, a polypeptide comprises the amino acid sequence
of SEQ ID NO: 6. In another preferred aspect, a polypeptide
comprises the mature polypeptide of SEQ ID NO: 6. In another
preferred aspect, a polypeptide comprises amino acids 20 to 318 of
SEQ ID NO: 6, or an allelic variant thereof; or a fragment thereof
that has lipase activity. In another preferred aspect, a
polypeptide comprises amino acids 20 to 318 of SEQ ID NO: 6. In
another preferred aspect, a polypeptide consists of the amino acid
sequence of SEQ ID NO: 6 or an allelic variant thereof; or a
fragment thereof that has lipase activity. In another preferred
aspect, a polypeptide consists of the amino acid sequence of SEQ ID
NO: 6. In another preferred aspect, a polypeptide consists of amino
acids 20 to 318 of SEQ ID NO: 6 or an allelic variant thereof; or a
fragment thereof that has lipase activity. In another preferred
aspect, a polypeptide consists of amino acids 20 to 318 of SEQ ID
NO: 6.
[0092] A polypeptide of the present invention preferably comprises
the amino acid sequence of SEQ ID NO: 8, or an allelic variant
thereof; or a fragment thereof that has lipase activity. In a
preferred aspect, a polypeptide comprises the amino acid sequence
of SEQ ID NO: 8. In another preferred aspect, a polypeptide
comprises the mature polypeptide of SEQ ID NO: 8. In another
preferred aspect, a polypeptide comprises amino acids 19 to 348 of
SEQ ID NO: 8, or an allelic variant thereof; or a fragment thereof
that has lipase activity. In another preferred aspect, a
polypeptide comprises amino acids 19 to 348 of SEQ ID NO: 8. In
another preferred aspect, a polypeptide consists of the amino acid
sequence of SEQ ID NO: 8 or an allelic variant thereof; or a
fragment thereof that has lipase activity. In another preferred
aspect, a polypeptide consists of the amino acid sequence of SEQ ID
NO: 8. In another preferred aspect, a polypeptide consists of amino
acids 19 to 348 of SEQ ID NO: 8 or an allelic variant thereof; or a
fragment thereof that has lipase activity. In another preferred
aspect, a polypeptide consists of amino acids 19 to 348 of SEQ ID
NO: 8.
[0093] A polypeptide of the present invention preferably comprises
the amino acid sequence of SEQ ID NO: 10, or an allelic variant
thereof; or a fragment thereof that has lipase activity. In a
preferred aspect, a polypeptide comprises the amino acid sequence
of SEQ ID NO: 10. In another preferred aspect, a polypeptide
comprises the mature polypeptide of SEQ ID NO: 10. In another
preferred aspect, a polypeptide comprises amino acids 25 to 393 of
SEQ ID NO: 10, or an allelic variant thereof; or a fragment thereof
that has lipase activity. In another preferred aspect, a
polypeptide comprises amino acids 25 to 393 of SEQ ID NO: 10. In
another preferred aspect, a polypeptide consists of the amino acid
sequence of SEQ ID NO: 10 or an allelic variant thereof; or a
fragment thereof that has lipase activity. In another preferred
aspect, a polypeptide consists of the amino acid sequence of SEQ ID
NO: 10. In another preferred aspect, a polypeptide consists of
amino acids 25 to 393 of SEQ ID NO: 10 or an allelic variant
thereof; or a fragment thereof that has lipase activity. In another
preferred aspect, a polypeptide consists of amino acids 25 to 393
of SEQ ID NO: 10.
[0094] A polypeptide of the present invention preferably comprises
the amino acid sequence of SEQ ID NO: 12, or an allelic variant
thereof; or a fragment thereof that has lipase activity. In a
preferred aspect, a polypeptide comprises the amino acid sequence
of SEQ ID NO: 12. In another preferred aspect, a polypeptide
comprises the mature polypeptide of SEQ ID NO: 12. In another
preferred aspect, a polypeptide comprises amino acids 20 to 294 of
SEQ ID NO: 12, or an allelic variant thereof; or a fragment thereof
that has lipase activity. In another preferred aspect, a
polypeptide comprises amino acids 20 to 294 of SEQ ID NO: 12. In
another preferred aspect, a polypeptide consists of the amino acid
sequence of SEQ ID NO: 12 or an allelic variant thereof; or a
fragment thereof that has lipase activity. In another preferred
aspect, a polypeptide consists of the amino acid sequence of SEQ ID
NO: 12. In another preferred aspect, a polypeptide consists of
amino acids 20 to 294 of SEQ ID NO: 12 or an allelic variant
thereof; or a fragment thereof that has lipase activity. In another
preferred aspect, a polypeptide consists of amino acids 20 to 294
of SEQ ID NO: 12.
[0095] A polypeptide of the present invention preferably comprises
the amino acid sequence of SEQ ID NO: 14, or an allelic variant
thereof; or a fragment thereof that has lipase activity. In a
preferred aspect, a polypeptide comprises the amino acid sequence
of SEQ ID NO: 14. In another preferred aspect, a polypeptide
comprises the mature polypeptide of SEQ ID NO: 14. In another
preferred aspect, a polypeptide comprises amino acids 25 to 308 of
SEQ ID NO: 14, or an allelic variant thereof; or a fragment thereof
that has lipase activity. In another preferred aspect, a
polypeptide comprises amino acids 25 to 308 of SEQ ID NO: 14. In
another preferred aspect, a polypeptide consists of the amino acid
sequence of SEQ ID NO: 14 or an allelic variant thereof; or a
fragment thereof that has lipase activity. In another preferred
aspect, a polypeptide consists of the amino acid sequence of SEQ ID
NO: 14. In another preferred aspect, a polypeptide consists of
amino acids 25 to 308 of SEQ ID NO: 14 or an allelic variant
thereof; or a fragment thereof that has lipase activity. In another
preferred aspect, a polypeptide consists of amino acids 25 to 308
of SEQ ID NO: 14.
[0096] A polypeptide of the present invention preferably comprises
the amino acid sequence of SEQ ID NO: 16, or an allelic variant
thereof; or a fragment thereof that has lipase activity. In a
preferred aspect, a polypeptide comprises the amino acid sequence
of SEQ ID NO: 16. In another preferred aspect, a polypeptide
comprises the mature polypeptide of SEQ ID NO: 16. In another
preferred aspect, a polypeptide comprises amino acids 26 to 404 of
SEQ ID NO: 16, or an allelic variant thereof; or a fragment thereof
that has lipase activity. In another preferred aspect, a
polypeptide comprises amino acids 26 to 404 of SEQ ID NO: 16. In
another preferred aspect, a polypeptide consists of the amino acid
sequence of SEQ ID NO: 16 or an allelic variant thereof; or a
fragment thereof that has lipase activity. In another preferred
aspect, a polypeptide consists of the amino acid sequence of SEQ ID
NO: 16. In another preferred aspect, a polypeptide consists of
amino acids 26 to 404 of SEQ ID NO: 16 or an allelic variant
thereof; or a fragment thereof that has lipase activity. In another
preferred aspect, a polypeptide consists of amino acids 26 to 404
of SEQ ID NO: 16.
[0097] In a second aspect, the present invention relates to
isolated polypeptides having lipase activity which are encoded by
polynucleotides which hybridize under very low stringency
conditions, preferably low stringency conditions, more preferably
medium stringency conditions, more preferably medium-high
stringency conditions, even more preferably high stringency
conditions, and most preferably very high stringency conditions
with (i) the mature polypeptide coding sequence of SEQ ID NO: 1,
SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO:
11, SEQ ID NO: 13, or SEQ ID NO: 15, (ii) the cDNA sequence
contained in the mature polypeptide coding sequence of SEQ ID NO:
1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID
NO: 11, SEQ ID NO: 13, or SEQ ID NO: 15, (iii) a subsequence of (i)
or (ii), or (iv) a complementary strand of (i), (ii), or (iii) (J.
Sambrook, E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning,
A Laboratory Manual, 2d edition, Cold Spring Harbor, N.Y.). A
subsequence of the mature polypeptide coding sequence of SEQ ID NO:
1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID
NO: 11, SEQ ID NO: 13, or SEQ ID NO: 15 contains at least 100
contiguous nucleotides or preferably at least 200 contiguous
nucleotides. Moreover, the subsequence may encode a polypeptide
fragment which has lipase activity. In a preferred aspect, the
mature polypeptide coding sequence is nucleotides 73 to 1256 of SEQ
ID NO: 1, nucleotides 73 to 944 of SEQ ID NO: 3, nucleotides 58 to
1085 of SEQ ID NO: 5, nucleotides 55 to 1044 of SEQ ID NO: 7,
nucleotides 73 to 1179 of SEQ ID NO: 9, nucleotides 58 to 1038 of
SEQ ID NO: 11, nucleotides 73 to 1116 of SEQ ID NO: 13, or
nucleotides 76 to 1280 of SEQ ID NO: 15.
[0098] The nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ
ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13,
or SEQ ID NO: 15; or a subsequence thereof; as well as the amino
acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID
NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, or SEQ ID NO:
16; or a fragment thereof; may be used to design a nucleic acid
probe to identify and clone DNA encoding polypeptides having lipase
activity from strains of different genera or species according to
methods well known in the art. In particular, such probes can be
used for hybridization with the genomic or cDNA of the genus or
species of interest, following standard Southern blotting
procedures, in order to identify and isolate the corresponding gene
therein. Such probes can be considerably shorter than the entire
sequence, but should be at least 14, preferably at least 25, more
preferably at least 35, and most preferably at least 70 nucleotides
in length. It is, however, preferred that the nucleic acid probe is
at least 100 nucleotides in length. For example, the nucleic acid
probe may be at least 200 nucleotides, preferably at least 300
nucleotides, more preferably at least 400 nucleotides, or most
preferably at least 500 nucleotides in length. Even longer probes
may be used, e.g., nucleic acid probes which are at least 600
nucleotides, at least preferably at least 700 nucleotides, more
preferably at least 800 nucleotides, or most preferably at least
900 nucleotides in length. Both DNA and RNA probes can be used. The
probes are typically labeled for detecting the corresponding gene
(for example, with .sup.32P, .sup.3H, .sup.35S, biotin, or avidin).
Such probes are encompassed by the present invention.
[0099] A genomic DNA or cDNA library prepared from such other
organisms may, therefore, be screened for DNA which hybridizes with
the probes described above and which encodes a polypeptide having
lipase activity. 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: 1, SEQ
ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11,
SEQ ID NO: 13, or SEQ ID NO: 15; or a subsequence thereof; the
carrier material is used in a Southern blot.
[0100] For purposes of the present invention, hybridization
indicates that the nucleotide sequence hybridizes to a labeled
nucleic acid probe corresponding to the mature polypeptide coding
sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7,
SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, or SEQ ID NO: 15, the
cDNA sequence contained in the mature polypeptide coding sequence
of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID
NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, or SEQ ID NO: 15; its
complementary strand; or a subsequence thereof; under very low to
very high stringency conditions. Molecules to which the nucleic
acid probe hybridizes under these conditions can be detected using
X-ray film.
[0101] In a preferred aspect, the nucleic acid probe is the mature
polypeptide coding sequence of SEQ ID NO: 1. In another preferred
aspect, the nucleic acid probe is nucleotides 73 to 1256 of SEQ ID
NO: 1. In another preferred aspect, the nucleic acid probe is a
polynucleotide sequence which encodes the polypeptide of SEQ ID NO:
2, or a subsequence thereof. In another preferred aspect, the
nucleic acid probe is SEQ ID NO: 1. In another preferred aspect,
the nucleic acid probe is the polynucleotide sequence contained in
plasmid pSMO224 which is contained in E. coli NRRL B-30774, wherein
the polynucleotide sequence thereof encodes a polypeptide having
lipase activity. In another preferred aspect, the nucleic acid
probe is the mature polypeptide coding region contained in plasmid
pSMO224 which is contained in E. coli NRRL B-30774.
[0102] In another preferred aspect, the nucleic acid probe is the
mature polypeptide coding sequence of SEQ ID NO: 3. In another
preferred aspect, the nucleic acid probe is nucleotides 73 to 944
of SEQ ID NO: 3. In another preferred aspect, the nucleic acid
probe is a polynucleotide sequence which encodes the polypeptide of
SEQ ID NO: 4, or a subsequence thereof. In another preferred
aspect, the nucleic acid probe is SEQ ID NO: 3. In another
preferred aspect, the nucleic acid probe is the polynucleotide
sequence contained in plasmid pSMO223 which is contained in E. coli
NRRL B-30773, wherein the polynucleotide sequence thereof encodes a
polypeptide having lipase activity. In another preferred aspect,
the nucleic acid probe is the mature polypeptide coding region
contained in plasmid pSMO223 which is contained in E. coli NRRL
B-30773.
[0103] In another preferred aspect, the nucleic acid probe is the
mature polypeptide coding sequence of SEQ ID NO: 5. In another
preferred aspect, the nucleic acid probe is nucleotides 58 to 1085
of SEQ ID NO: 5. In another preferred aspect, the nucleic acid
probe is a polynucleotide sequence which encodes the polypeptide of
SEQ ID NO: 6, or a subsequence thereof. In another preferred
aspect, the nucleic acid probe is SEQ ID NO: 5. In another
preferred aspect, the nucleic acid probe is the polynucleotide
sequence contained in plasmid pHyGe026 which is contained in E.
coli NRRL B-30772, wherein the polynucleotide sequence thereof
encodes a polypeptide having lipase activity. In another preferred
aspect, the nucleic acid probe is the mature polypeptide coding
region contained in plasmid pHyGe026 which is contained in E. coli
NRRL B-30772.
[0104] In another preferred aspect, the nucleic acid probe is the
mature polypeptide coding sequence of SEQ ID NO: 7. In another
preferred aspect, the nucleic acid probe is nucleotides 55 to 1044
of SEQ ID NO: 7. In another preferred aspect, the nucleic acid
probe is a polynucleotide sequence which encodes the polypeptide of
SEQ ID NO: 8, or a subsequence thereof. In another preferred
aspect, the nucleic acid probe is SEQ ID NO: 7. In another
preferred aspect, the nucleic acid probe is the polynucleotide
sequence contained in plasmid pCrAm138 which is contained in E.
coli NRRL B-30781, wherein the polynucleotide sequence thereof
encodes a polypeptide having lipase activity. In another preferred
aspect, the nucleic acid probe is the mature polypeptide coding
region contained in plasmid pCrAm138 which is contained in E. coli
NRRL B-30781.
[0105] In another preferred aspect, the nucleic acid probe is the
mature polypeptide coding sequence of SEQ ID NO: 9. In another
preferred aspect, the nucleic acid probe is nucleotides 73 to 1179
of SEQ ID NO: 9. In another preferred aspect, the nucleic acid
probe is a polynucleotide sequence which encodes the polypeptide of
SEQ ID NO: 10, or a subsequence thereof. In another preferred
aspect, the nucleic acid probe is SEQ ID NO: 9. In another
preferred aspect, the nucleic acid probe is the polynucleotide
sequence contained in plasmid pBM135g which is contained in E. coli
NRRL B-30779, wherein the polynucleotide sequence thereof encodes a
polypeptide having lipase activity. In another preferred aspect,
the nucleic acid probe is the mature polypeptide coding region
contained in plasmid pBM135g which is contained in E. coli NRRL
B-30779.
[0106] In another preferred aspect, the nucleic acid probe is the
mature polypeptide coding sequence of SEQ ID NO: 11. In another
preferred aspect, the nucleic acid probe is nucleotides 58 to 1038
of SEQ ID NO: 11. In another preferred aspect, the nucleic acid
probe is a polynucleotide sequence which encodes the polypeptide of
SEQ ID NO: 12, or a subsequence thereof. In another preferred
aspect, the nucleic acid probe is SEQ ID NO: 11. In another
preferred aspect, the nucleic acid probe is the polynucleotide
sequence contained in plasmid pJLin171 which is contained in E.
coli NRRL B-30755, wherein the polynucleotide sequence thereof
encodes a polypeptide having lipase activity. In another preferred
aspect, the nucleic acid probe is the mature polypeptide coding
region contained in plasmid pJLin171 which is contained in E. coli
NRRL B-30755.
[0107] In another preferred aspect, the nucleic acid probe is the
mature polypeptide coding sequence of SEQ ID NO: 13. In another
preferred aspect, the nucleic acid probe is nucleotides 73 to 1116
of SEQ ID NO: 13. In another preferred aspect, the nucleic acid
probe is a polynucleotide sequence which encodes the polypeptide of
SEQ ID NO: 18, or a subsequence thereof. In another preferred
aspect, the nucleic acid probe is SEQ ID NO: 13. In another
preferred aspect, the nucleic acid probe is the polynucleotide
sequence contained in plasmid pJLin170 which is contained in E.
coli NRRL B-30754, wherein the polynucleotide sequence thereof
encodes a polypeptide having lipase activity. In another preferred
aspect, the nucleic acid probe is the mature polypeptide coding
region contained in plasmid pJLin170 which is contained in E. coli
NRRL B-30754.
[0108] In another preferred aspect, the nucleic acid probe is the
mature polypeptide coding sequence of SEQ ID NO: 15. In another
preferred aspect, the nucleic acid probe is nucleotides 76 to 1280
of SEQ ID NO: 15. In another preferred aspect, the nucleic acid
probe is a polynucleotide sequence which encodes the polypeptide of
SEQ ID NO: 16, or a subsequence thereof. In another preferred
aspect, the nucleic acid probe is SEQ ID NO: 15. In another
preferred aspect, the nucleic acid probe is the polynucleotide
sequence contained in plasmid pBM141 which is contained in E. coli
NRRL B-30780, wherein the polynucleotide sequence thereof encodes a
polypeptide having lipase activity. In another preferred aspect,
the nucleic acid probe is the mature polypeptide coding region
contained in plasmid pBM141 which is contained in E. coli NRRL
B-30780.
[0109] For long probes of at least 100 nucleotides in length, very
low to very high stringency conditions are defined as
prehybridization and hybridization at 42.degree. C. in
5.times.SSPE, 0.3% SDS, 200 .mu.g/ml sheared and denatured salmon
sperm DNA, and either 25% formamide for very low and low
stringencies, 35% formamide for medium and medium-high
stringencies, or 50% formamide for high and very high stringencies,
following standard Southern blotting procedures for 12 to 24 hours
optimally.
[0110] For long probes of at least 100 nucleotides in length, the
carrier material is finally washed three times each for 15 minutes
using 2.times.SSC, 0.2% SDS preferably at least at 45.degree. C.
(very low stringency), more preferably at least at 50.degree. C.
(low stringency), more preferably at least at 55.degree. C. (medium
stringency), more preferably at least at 60.degree. C. (medium-high
stringency), even more preferably at least at 65.degree. C. (high
stringency), and most preferably at least at 70.degree. C. (very
high stringency).
[0111] For short probes which are about 15 nucleotides to about 70
nucleotides in length, stringency conditions are defined as
prehybridization, hybridization, and washing post-hybridization at
about 5.degree. C. to about 10.degree. C. below the calculated
T.sub.m using the calculation according to Bolton and McCarthy
(1962, Proceedings of the National Academy of Sciences USA 48:1390)
in 0.9 M NaCl, 0.09 M Tris-HCl pH 7.6, 6 mM EDTA, 0.5% NP-40,
1.times.Denhardt's solution, 1 mM sodium pyrophosphate, 1 mM sodium
monobasic phosphate, 0.1 mM ATP, and 0.2 mg of yeast RNA per ml
following standard Southern blotting procedures for 12 to 24 hours
optimally.
[0112] For short probes which are about 15 nucleotides to about 70
nucleotides in length, the carrier material is washed once in
6.times.SCC plus 0.1% SDS for 15 minutes and twice each for 15
minutes using 6.times.SSC at 5.degree. C. to 10.degree. C. below
the calculated T.sub.m.
[0113] In a third aspect, the present invention relates to
artificial variants comprising a conservative substitution,
deletion, and/or insertion of one or more amino acids of the mature
polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO:
8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, or SEQ ID NO: 16;
or a homologous sequence thereof. Preferably, amino acid changes
are of a minor nature, that is conservative amino acid
substitutions or insertions that do not significantly affect the
folding and/or activity of the protein; small deletions, typically
of one to about 30 amino acids; small amino- or carboxyl-terminal
extensions, such as an amino-terminal methionine residue; a small
linker peptide of up to about 20-25 residues; or a small extension
that facilitates purification by changing net charge or another
function, such as a poly-histidine tract, an antigenic epitope or a
binding domain.
[0114] Examples of conservative substitutions are within the group
of basic amino acids (arginine, lysine and histidine), acidic amino
acids (glutamic acid and aspartic acid), polar amino acids
(glutamine and asparagine), hydrophobic amino acids (leucine,
isoleucine and valine), aromatic amino acids (phenylalanine,
tryptophan and tyrosine), and small amino acids (glycine, alanine,
serine, threonine and methionine). Amino acid substitutions which
do not generally alter specific activity are known in the art and
are described, for example, by H. Neurath and R. L. Hill, 1979, In,
The Proteins, Academic Press, New York. The most commonly occurring
exchanges are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr,
Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn,
Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly.
[0115] In addition to the 20 standard amino acids, non-standard
amino acids (such as 4-hydroxyproline, 6-N-methyl lysine,
2-aminoisobutyric acid, isovaline, and alpha-methyl serine) may be
substituted for amino acid residues of a wild-type polypeptide. A
limited number of non-conservative amino acids, amino acids that
are not encoded by the genetic code, and unnatural amino acids may
be substituted for amino acid residues. "Unnatural amino acids"
have been modified after protein synthesis, and/or have a chemical
structure in their side chain(s) different from that of the
standard amino acids. Unnatural amino acids can be chemically
synthesized, and preferably, are commercially available, and
include pipecolic acid, thiazolidine carboxylic acid,
dehydroproline, 3- and 4-methylproline, and
3,3-dimethylproline.
[0116] Alternatively, the amino acid changes are of such a nature
that the physico-chemical properties of the polypeptides are
altered. For example, amino acid changes may improve the thermal
stability of the polypeptide, alter the substrate specificity,
change the pH optimum, and the like.
[0117] Essential amino acids in the parent polypeptide can be
identified according to procedures known in the art, such as
site-directed mutagenesis or alanine-scanning mutagenesis
(Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter
technique, single alanine mutations are introduced at every residue
in the molecule, and the resultant mutant molecules are tested for
biological activity (i.e., lipase activity) to identify amino acid
residues that are critical to the activity of the molecule. See
also, Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708. The
active site of the enzyme or other biological interaction can also
be determined by physical analysis of structure, as determined by
such techniques as nuclear magnetic resonance, crystallography,
electron diffraction, or photoaffinity labeling, in conjunction
with mutation of putative contact site amino acids. See, for
example, de Vos et al., 1992, Science 255: 306-312; Smith et al.,
1992, J. Mol. Biol. 224: 899-904; Wlodaver et al., 1992, FEBS Lett.
309: 59-64. The identities of essential amino acids can also be
inferred from analysis of identities with polypeptides which are
related to a polypeptide according to the invention.
[0118] Single or multiple amino acid substitutions can be made and
tested using known methods of mutagenesis, recombination, and/or
shuffling, followed by a relevant screening procedure, such as
those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241:
53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86:
2152-2156; WO 95/17413; or WO 95/22625. Other methods that can be
used include error-prone PCR, phage display (e.g., Lowman et al.,
1991, Biochem. 30: 10832-10837; U.S. Pat. No. 5,223,409; WO
92/06204), and region-directed mutagenesis (Derbyshire et al.,
1986, Gene 46: 145; Ner et al., 1988, DNA 7: 127).
[0119] Mutagenesis/shuffling methods can be combined with
high-throughput, automated screening methods to detect activity of
cloned, mutagenized polypeptides expressed by host cells (Ness et
al., 1999, Nature Biotechnology 17: 893-896). Mutagenized DNA
molecules that encode active polypeptides can be recovered from the
host cells and rapidly sequenced using standard methods in the art.
These methods allow the rapid determination of the importance of
individual amino acid residues in a polypeptide of interest, and
can be applied to polypeptides of unknown structure.
[0120] The total number of amino acid substitutions, deletions
and/or insertions of the mature polypeptide of SEQ ID NO: 2, such
as amino acids 25 to 396 of SEQ ID NO: 2, is 10, preferably 9, more
preferably 8, more preferably 7, more preferably at most 6, more
preferably 5, more preferably 4, even more preferably 3, most
preferably 2, and even most preferably 1.
[0121] The total number of amino acid substitutions, deletions
and/or insertions of the mature polypeptide of SEQ ID NO: 4, such
as amino acids 25 to 283 of SEQ ID NO: 4, is 10, preferably 9, more
preferably 8, more preferably 7, more preferably at most 6, more
preferably 5, more preferably 4, even more preferably 3, most
preferably 2, and even most preferably 1.
[0122] The total number of amino acid substitutions, deletions
and/or insertions of the mature polypeptide of SEQ ID NO: 6, such
as amino acids 20 to 318 of SEQ ID NO: 6, is 10, preferably 9, more
preferably 8, more preferably 7, more preferably at most 6, more
preferably 5, more preferably 4, even more preferably 3, most
preferably 2, and even most preferably 1.
[0123] The total number of amino acid substitutions, deletions
and/or insertions of the mature polypeptide of SEQ ID NO: 8, such
as amino acids 19 to 348 of SEQ ID NO: 8, is 10, preferably 9, more
preferably 8, more preferably 7, more preferably at most 6, more
preferably 5, more preferably 4, even more preferably 3, most
preferably 2, and even most preferably 1.
[0124] The total number of amino acid substitutions, deletions
and/or insertions of the mature polypeptide of SEQ ID NO: 10, such
as amino acids 25 to 393 of SEQ ID NO: 10, is 10, preferably 9,
more preferably 8, more preferably 7, more preferably at most 6,
more preferably 5, more preferably 4, even more preferably 3, most
preferably 2, and even most preferably 1.
[0125] The total number of amino acid substitutions, deletions
and/or insertions of the mature polypeptide of SEQ ID NO: 12, such
as amino acids 20 to 294 of SEQ ID NO: 12, is 10, preferably 9,
more preferably 8, more preferably 7, more preferably at most 6,
more preferably 5, more preferably 4, even more preferably 3, most
preferably 2, and even most preferably 1.
[0126] The total number of amino acid substitutions, deletions
and/or insertions of the mature polypeptide of SEQ ID NO: 14, such
as amino acids 25 to 308 of SEQ ID NO: 14, is 10, preferably 9,
more preferably 8, more preferably 7, more preferably at most 6,
more preferably 5, more preferably 4, even more preferably 3, most
preferably 2, and even most preferably 1.
[0127] The total number of amino acid substitutions, deletions
and/or insertions of the mature polypeptide of SEQ ID NO: 16, such
as amino acids 26 to 404 of SEQ ID NO: 16, is 10, preferably 9,
more preferably 8, more preferably 7, more preferably at most 6,
more preferably 5, more preferably 4, even more preferably 3, most
preferably 2, and even most preferably 1.
Sources of Polypeptides Having Lipase Activity
[0128] A polypeptide of the present invention may be obtained from
microorganisms of any genus. For purposes of the present invention,
the term "obtained from" as used herein in connection with a given
source shall mean that the polypeptide encoded by a nucleotide
sequence is produced by the source or by a strain in which the
nucleotide sequence from the source has been inserted. In a
preferred aspect, the polypeptide obtained from a given source is
secreted extracellularly.
[0129] A polypeptide of the present invention may be a bacterial
polypeptide. For example, the polypeptide may be a gram positive
bacterial polypeptide such as a Bacillus polypeptide having lipase
activity, e.g., a Bacillus alkalophilus, Bacillus
amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus
coagulans, Bacillus lautus, Bacillus lentus, Bacillus
licheniformis, Bacillus megaterium, Bacillus stearothermophilus,
Bacillus subtilis, or Bacillus thuringiensis polypeptide having
lipase activity; or a Streptomyces polypeptide having lipase
activity, e.g., a Streptomyces lividans or Streptomyces murinus
polypeptide having lipase activity; or a gram negative bacterial
polypeptide having lipase activity, e.g., an E. coli or a
Pseudomonas sp. polypeptide having lipase activity.
[0130] A polypeptide of the present invention may also be a fungal
polypeptide, and more preferably a yeast polypeptide such as a
Candida, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces,
or Yarrowia polypeptide; or more preferably a filamentous fungal
polypeptide such as an Acremonium, Aspergillus, Aureobasidium,
Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor,
Myceliophthora, Neocallimastix, Neurospora, Paecilomyces,
Penicillium, Piromyces, Schizophyllum, Talaromyces, Thermoascus,
Thielavia, Tolypocladium, or Trichoderma polypeptide having lipase
activity.
[0131] In a preferred aspect, the polypeptide is a Saccharomyces
carlsbergensis, Saccharomyces cerevisiae, Saccharomyces
diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri,
Saccharomyces norbensis, or Saccharomyces oviformis polypeptide
having lipase activity.
[0132] In another preferred aspect, the polypeptide is an
Aspergillus aculeatus, Aspergillus awamori, Aspergillus fumigatus,
Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans,
Aspergillus niger, Aspergillus oryzae, Fusarium bactridioides,
Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum,
Fusarium graminearum, Fusarium graminum, Fusarium heterosporum,
Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum,
Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum,
Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum,
Fusarium trichothecioides, Fusarium venenatum, Humicola insolens,
Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila,
Neurospora crassa, Penicillium purpurogenum, Trichoderma harzianum,
Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma
reesei, or Trichoderma viride polypeptide having lipase
activity.
[0133] In another preferred aspect, the polypeptide is a Thielavia
achromatica, Thielavia albomyces, Thielavia albopilosa, Thielavia
australeinsis, Thielavia fimeti, Thielavia microspora, Thielavia
ovispora, Thielavia peruviana, Thielavia spededonium, Thielavia
setosa, Thielavia subthermophila, Thielavia terrestris, Thielavia
terricola, Thielavia thermophila, Thielavia variospora, or
Thielavia wareingii polypeptide having lipase activity.
[0134] In a more preferred aspect, the polypeptide is an
Aspergillus fumigatus polypeptide, e.g., the polypeptide of SEQ ID
NO: 2 or SEQ ID NO: 4, or the mature polypeptide thereof.
[0135] In another more preferred aspect, the polypeptide is a
Magnaporthe grisea polypeptide, and most preferably a Magnaporthe
grisea FGSC 8958 (Fungal Genetics Stock Center, Kansas City, Mo.)
polypeptide, e.g., the polypeptide of SEQ ID NO: 6, SEQ ID NO: 8,
or SEQ ID NO: 10, or the mature polypeptide thereof.
[0136] In another more preferred aspect, the polypeptide is an
Aspergillus nidulans polypeptide, and most preferably an
Aspergillus nidulans A1000 (Fungal Genetics Stock Center, Kansas
City, Mo.) polypeptide, e.g., the polypeptide of SEQ ID NO: 12, SEQ
ID NO: 14, or SEQ ID NO: 16, or the mature polypeptide thereof.
[0137] It will be understood that for the aforementioned species
the invention encompasses both the perfect and imperfect states,
and other taxonomic equivalents, e.g., anamorphs, regardless of the
species name by which they are known. Those skilled in the art will
readily recognize the identity of appropriate equivalents.
[0138] Strains of these species are readily accessible to the
public in a number of culture collections, such as the American
Type Culture Collection (ATCC), Deutsche Sammlung von
Mikroorganismen und Zellkulturen GmbH (DSM), Centraalbureau Voor
Schimmelcultures (CBS), and Agricultural Research Service Patent
Culture Collection, Northern Regional Research Center (NRRL).
[0139] Furthermore, such polypeptides may be identified and
obtained from other sources including microorganisms isolated from
nature (e.g., soil, composts, water, etc.) using the
above-mentioned probes. Techniques for isolating microorganisms
from natural habitats are well known in the art. The polynucleotide
may then be obtained by similarly screening a genomic or cDNA
library of such a microorganism. Once a polynucleotide sequence
encoding a polypeptide has been detected with the probe(s), the
polynucleotide can be isolated or cloned by utilizing techniques
which are well known to those of ordinary skill in the art (see,
e.g., Sambrook et al., 1989, supra).
[0140] Polypeptides of the present invention also include fused
polypeptides or cleavable fusion polypeptides in which another
polypeptide is fused at the N-terminus or the C-terminus of the
polypeptide or fragment thereof. A fused polypeptide is produced by
fusing a nucleotide sequence (or a portion thereof) encoding
another polypeptide to a nucleotide sequence (or a portion thereof)
of the present invention. Techniques for producing fusion
polypeptides are known in the art, and include ligating the coding
sequences encoding the polypeptides so that they are in frame and
that expression of the fused polypeptide is under control of the
same promoter(s) and terminator.
Polynucleotides
[0141] The present invention also relates to isolated
polynucleotides having a nucleotide sequence which encode a
polypeptide of the present invention.
[0142] In a preferred aspect, the nucleotide sequence is set forth
in SEQ ID NO: 1. In another more preferred aspect, the nucleotide
sequence is the sequence contained in plasmid pSMO224 which is
contained in E. coli NRRL B-30774. In another preferred aspect, the
nucleotide sequence is the mature polypeptide coding region of SEQ
ID NO: 1. In another preferred aspect, the nucleotide sequence is
nucleotides 73 to 1256 of SEQ ID NO: 1. In another more preferred
aspect, the nucleotide sequence is the mature polypeptide coding
region contained in plasmid pSMO224 which is contained in E. coli
NRRL B-30774. The present invention also encompasses nucleotide
sequences which encode a polypeptide having the amino acid sequence
of SEQ ID NO: 2 or the mature polypeptide thereof, which differ
from SEQ ID NO: 1 or the mature polypeptide coding sequence thereof
by virtue of the degeneracy of the genetic code. The present
invention also relates to subsequences of SEQ ID NO: 1 which encode
fragments of SEQ ID NO: 2 that have lipase activity.
[0143] In another preferred aspect, the nucleotide sequence is set
forth in SEQ ID NO: 3. In another more preferred aspect, the
nucleotide sequence is the sequence contained in plasmid pSMO223
which is contained in E. coli NRRL B-30773. In another preferred
aspect, the nucleotide sequence is the mature polypeptide coding
region of SEQ ID NO: 3. In another preferred aspect, the nucleotide
sequence is nucleotides 73 to 944 of SEQ ID NO: 3. In another more
preferred aspect, the nucleotide sequence is the mature polypeptide
coding region contained in plasmid pSMO223 which is contained in E.
coli NRRL B-30773. The present invention also encompasses
nucleotide sequences which encode a polypeptide having the amino
acid sequence of SEQ ID NO: 4 or the mature polypeptide thereof,
which differ from SEQ ID NO: 3 or the mature polypeptide coding
sequence thereof by virtue of the degeneracy of the genetic code.
The present invention also relates to subsequences of SEQ ID NO: 3
which encode fragments of SEQ ID NO: 4 that have lipase
activity.
[0144] In another preferred aspect, the nucleotide sequence is set
forth in SEQ ID NO: 5. In another more preferred aspect, the
nucleotide sequence is the sequence contained in plasmid pHyGe026
which is contained in E. coli NRRL B-30772. In another preferred
aspect, the nucleotide sequence is the mature polypeptide coding
region of SEQ ID NO: 5. In another preferred aspect, the nucleotide
sequence is nucleotides 58 to 1085 of SEQ ID NO: 5. In another more
preferred aspect, the nucleotide sequence is the mature polypeptide
coding region contained in plasmid pHyGe026 which is contained in
E. coli NRRL B-30772. The present invention also encompasses
nucleotide sequences which encode a polypeptide having the amino
acid sequence of SEQ ID NO: 6 or the mature polypeptide thereof,
which differ from SEQ ID NO: 5 or the mature polypeptide coding
sequence thereof by virtue of the degeneracy of the genetic code.
The present invention also relates to subsequences of SEQ ID NO: 5
which encode fragments of SEQ ID NO: 6 that have lipase
activity.
[0145] In another preferred aspect, the nucleotide sequence is set
forth in SEQ ID NO: 7. In another more preferred aspect, the
nucleotide sequence is the sequence contained in plasmid pCrAm138
which is contained in E. coli NRRL B-30781. In another preferred
aspect, the nucleotide sequence is the mature polypeptide coding
region of SEQ ID NO: 7. In another preferred aspect, the nucleotide
sequence is nucleotides 55 to 1044 of SEQ ID NO: 7. In another more
preferred aspect, the nucleotide sequence is the mature polypeptide
coding region contained in plasmid pCrAm138 which is contained in
E. coli NRRL B-30781. The present invention also encompasses
nucleotide sequences which encode a polypeptide having the amino
acid sequence of SEQ ID NO: 8 or the mature polypeptide thereof,
which differ from SEQ ID NO: 7 or the mature polypeptide coding
sequence thereof by virtue of the degeneracy of the genetic code.
The present invention also relates to subsequences of SEQ ID NO: 7
which encode fragments of SEQ ID NO: 8 that have lipase
activity.
[0146] In another preferred aspect, the nucleotide sequence is set
forth in SEQ ID NO: 9. In another more preferred aspect, the
nucleotide sequence is the sequence contained in plasmid pBM135g
which is contained in E. coli NRRL B-30779. In another preferred
aspect, the nucleotide sequence is the mature polypeptide coding
region of SEQ ID NO: 9. In another preferred aspect, the nucleotide
sequence is nucleotides 73 to 1179 of SEQ ID NO: 9. In another more
preferred aspect, the nucleotide sequence is the mature polypeptide
coding region contained in plasmid pBM135g which is contained in E.
coli NRRL B-30779. The present invention also encompasses
nucleotide sequences which encode a polypeptide having the amino
acid sequence of SEQ ID NO: 10 or the mature polypeptide thereof,
which differ from SEQ ID NO: 9 or the mature polypeptide coding
sequence thereof by virtue of the degeneracy of the genetic code.
The present invention also relates to subsequences of SEQ ID NO: 9
which encode fragments of SEQ ID NO: 10 that have lipase
activity.
[0147] In another preferred aspect, the nucleotide sequence is set
forth in SEQ ID NO: 11. In another more preferred aspect, the
nucleotide sequence is the sequence contained in plasmid pJLin171
which is contained in E. coli NRRL B-30755. In another preferred
aspect, the nucleotide sequence is the mature polypeptide coding
region of SEQ ID NO: 11. In another preferred aspect, the
nucleotide sequence is nucleotides 58 to 1038 of SEQ ID NO: 11. In
another more preferred aspect, the nucleotide sequence is the
mature polypeptide coding region contained in plasmid pJLin171
which is contained in E. coli NRRL B-30755. In another more
preferred aspect, the nucleotide sequence is the mature polypeptide
coding region contained in plasmid pJLin171 which is contained in
E. coli NRRL B-30755. The present invention also encompasses
nucleotide sequences which encode a polypeptide having the amino
acid sequence of SEQ ID NO: 12 or the mature polypeptide thereof,
which differ from SEQ ID NO: 11 or the mature polypeptide coding
sequence thereof by virtue of the degeneracy of the genetic code.
The present invention also relates to subsequences of SEQ ID NO: 11
which encode fragments of SEQ ID NO: 12 that have lipase
activity.
[0148] In another preferred aspect, the nucleotide sequence is set
forth in SEQ ID NO: 13. In another more preferred aspect, the
nucleotide sequence is the sequence contained in plasmid pJLin170
which is contained in E. coli NRRL B-30754. In another preferred
aspect, the nucleotide sequence is the mature polypeptide coding
region of SEQ ID NO: 13. In another preferred aspect, the
nucleotide sequence is nucleotides 73 to 1116 of SEQ ID NO: 13. In
another more preferred aspect, the nucleotide sequence is the
mature polypeptide coding region contained in plasmid pJLin170
which is contained in E. coli NRRL B-30754. In another more
preferred aspect, the nucleotide sequence is the mature polypeptide
coding region contained in plasmid pJLin170 which is contained in
E. coli NRRL B-30754. The present invention also encompasses
nucleotide sequences which encode a polypeptide having the amino
acid sequence of SEQ ID NO: 14 or the mature polypeptide thereof,
which differ from SEQ ID NO: 13 or the mature polypeptide coding
sequence thereof by virtue of the degeneracy of the genetic code.
The present invention also relates to subsequences of SEQ ID NO: 13
which encode fragments of SEQ ID NO: 14 that have lipase
activity.
[0149] In another preferred aspect, the nucleotide sequence is set
forth in SEQ ID NO: 15. In another more preferred aspect, the
nucleotide sequence is the sequence contained in plasmid pBM141
which is contained in E. coli NRRL B-30780. In another preferred
aspect, the nucleotide sequence is the mature polypeptide coding
region of SEQ ID NO: 15. In another preferred aspect, the
nucleotide sequence is nucleotides 76 to 1280 of SEQ ID NO: 15. In
another more preferred aspect, the nucleotide sequence is the
mature polypeptide coding region contained in plasmid pBM141 which
is contained in E. coli NRRL B-30780. In another more preferred
aspect, the nucleotide sequence is the mature polypeptide coding
region contained in plasmid pBM141 which is contained in E. coli
NRRL B-30780. The present invention also encompasses nucleotide
sequences which encode a polypeptide having the amino acid sequence
of SEQ ID NO: 16 or the mature polypeptide thereof, which differ
from SEQ ID NO: 15 or the mature polypeptide coding sequence
thereof by virtue of the degeneracy of the genetic code. The
present invention also relates to subsequences of SEQ ID NO: 15
which encode fragments of SEQ ID NO: 16 that have lipase
activity.
[0150] The present invention also relates to mutant polynucleotides
comprising at least one mutation in the mature polypeptide coding
sequence of SEQ ID NO: 1, in which the mutant nucleotide sequence
encodes the mature polypeptide of SEQ ID NO: 2. In a preferred
aspect, the mature polypeptide is amino acids 25 to 396 of SEQ ID
NO: 2.
[0151] The present invention also relates to mutant polynucleotides
comprising at least one mutation in the mature polypeptide coding
sequence of SEQ ID NO: 3, in which the mutant nucleotide sequence
encodes the mature polypeptide of SEQ ID NO: 4. In a preferred
aspect, the mature polypeptide is amino acids 25 to 283 of SEQ ID
NO: 4.
[0152] The present invention also relates to mutant polynucleotides
comprising at least one mutation in the mature polypeptide coding
sequence of SEQ ID NO: 5, in which the mutant nucleotide sequence
encodes the mature polypeptide of SEQ ID NO: 6. In a preferred
aspect, the mature polypeptide is amino acids 20 to 318 of SEQ ID
NO: 6.
[0153] The present invention also relates to mutant polynucleotides
comprising at least one mutation in the mature polypeptide coding
sequence of SEQ ID NO: 7, in which the mutant nucleotide sequence
encodes the mature polypeptide of SEQ ID NO: 8. In a preferred
aspect, the mature polypeptide is amino acids 19 to 348 of SEQ ID
NO: 8.
[0154] The present invention also relates to mutant polynucleotides
comprising at least one mutation in the mature polypeptide coding
sequence of SEQ ID NO: 9, in which the mutant nucleotide sequence
encodes the mature polypeptide of SEQ ID NO: 10. In a preferred
aspect, the mature polypeptide is amino acids 25 to 393 of SEQ ID
NO: 10.
[0155] The present invention also relates to mutant polynucleotides
comprising at least one mutation in the mature polypeptide coding
sequence of SEQ ID NO: 11, in which the mutant nucleotide sequence
encodes the mature polypeptide of SEQ ID NO: 12. In a preferred
aspect, the mature polypeptide is amino acids 20 to 294 of SEQ ID
NO: 12.
[0156] The present invention also relates to mutant polynucleotides
comprising at least one mutation in the mature polypeptide coding
sequence of SEQ ID NO: 13, in which the mutant nucleotide sequence
encodes the mature polypeptide of SEQ ID NO: 14. In a preferred
aspect, the mature polypeptide is amino acids 25 to 308 of SEQ ID
NO: 14.
[0157] The present invention also relates to mutant polynucleotides
comprising at least one mutation in the mature polypeptide coding
sequence of SEQ ID NO: 15, in which the mutant nucleotide sequence
encodes the mature polypeptide of SEQ ID NO: 16. In a preferred
aspect, the mature polypeptide is amino acids 26 to 404 of SEQ ID
NO: 16.
[0158] The techniques used to isolate or clone a polynucleotide
encoding a polypeptide are known in the art and include isolation
from genomic DNA, preparation from cDNA, or a combination thereof.
The cloning of the polynucleotides of the present invention from
such genomic DNA can be effected, e.g., by using the well known
polymerase chain reaction (PCR) or antibody screening of expression
libraries to detect cloned DNA fragments with shared structural
features. See, e.g., Innis et al., 1990, PCR: A Guide to Methods
and Application, Academic Press, New York. Other nucleic acid
amplification procedures such as ligase chain reaction (LCR),
ligated activated transcription (LAT) and nucleotide sequence-based
amplification (NASBA) may be used. The polynucleotides may be
cloned from a strain of Thielavia, or another or related organism
and thus, for example, may be an allelic or species variant of the
polypeptide encoding region of the nucleotide sequence.
[0159] The present invention also relates to polynucleotides having
nucleotide sequences which have a degree of identity to the mature
polypeptide coding sequence of SEQ ID NO: 1 of at least 60%,
preferably at least 65%, more preferably at least 70%, more
preferably at least 75%, more preferably at least 80%, more
preferably at least 85%, more preferably at least 90%, even more
preferably at least 95%, and most preferably at least 97% identity,
which encode an active polypeptide. In a preferred aspect, the
mature polypeptide coding sequence is nucleotides 73 to 1256 of SEQ
ID NO: 1.
[0160] The present invention also relates to polynucleotides having
nucleotide sequences which have a degree of identity to the mature
polypeptide coding sequence of SEQ ID NO: 3 of at least 70%,
preferably at least 75%, more preferably at least 80%, more
preferably at least 85%, more preferably at least 90%, even more
preferably at least 95%, and most preferably at least 97% identity,
which encode an active polypeptide. In a preferred aspect, the
mature polypeptide coding sequence is nucleotides 73 to 944 of SEQ
ID NO: 3.
[0161] The present invention also relates to polynucleotides having
nucleotide sequences which have a degree of identity to the mature
polypeptide coding sequence of SEQ ID NO: 5 of at least 70%,
preferably at least 75%, more preferably at least 80%, more
preferably at least 85%, more preferably at least 90%, even more
preferably at least 95%, and most preferably at least 97% identity,
which encode an active polypeptide. In a preferred aspect, the
mature polypeptide coding sequence is nucleotides 58 to 1085 of SEQ
ID NO: 5.
[0162] The present invention also relates to polynucleotides having
nucleotide sequences which have a degree of identity to the mature
polypeptide coding sequence of SEQ ID NO: 7 of at least 70%,
preferably at least 75%, more preferably at least 80%, more
preferably at least 85%, more preferably at least 90%, even more
preferably at least 95%, and most preferably at least 97% identity,
which encode an active polypeptide. In a preferred aspect, the
mature polypeptide coding sequence is nucleotides 55 to 1044 of SEQ
ID NO: 7.
[0163] The present invention also relates to polynucleotides having
nucleotide sequences which have a degree of identity to the mature
polypeptide coding sequence of SEQ ID NO: 9 of at least 70%,
preferably at least 75%, more preferably at least 80%, more
preferably at least 85%, more preferably at least 90%, even more
preferably at least 95%, and most preferably at least 97% identity,
which encode an active polypeptide. In a preferred aspect, the
mature polypeptide coding sequence is nucleotides 73 to 1179 of SEQ
ID NO: 9.
[0164] The present invention also relates to polynucleotides having
nucleotide sequences which have a degree of identity to the mature
polypeptide coding sequence of SEQ ID NO: 11 of at least 70%,
preferably at least 75%, more preferably at least 75%, more
preferably at least 80%, more preferably at least 85%, more
preferably at least 90%, even more preferably at least 95%, and
most preferably at least 97% identity, which encode an active
polypeptide. In a preferred aspect, the mature polypeptide coding
sequence is nucleotides 58 to 1038 of SEQ ID NO: 11.
[0165] The present invention also relates to polynucleotides having
nucleotide sequences which have a degree of identity to the mature
polypeptide coding sequence of SEQ ID NO: 13 of at least 70%,
preferably at least 75%, more preferably at least 75%, more
preferably at least 80%, more preferably at least 85%, more
preferably at least 90%, even more preferably at least 95%, and
most preferably at least 97% identity, which encode an active
polypeptide. In a preferred aspect, the mature polypeptide coding
sequence is nucleotides 73 to 1116 of SEQ ID NO: 13.
[0166] The present invention also relates to polynucleotides having
nucleotide sequences which have a degree of identity to the mature
polypeptide coding sequence of SEQ ID NO: 15 of at least 70%,
preferably at least 75%, more preferably at least 75%, more
preferably at least 80%, more preferably at least 85%, more
preferably at least 90%, even more preferably at least 95%, and
most preferably at least 97% identity, which encode an active
polypeptide. In a preferred aspect, the mature polypeptide coding
sequence is nucleotides 76 to 1280 of SEQ ID NO: 15.
[0167] Modification of a nucleotide sequence encoding a polypeptide
of the present invention may be necessary for the synthesis of
polypeptides substantially similar to the polypeptide. The term
"substantially similar" to the polypeptide refers to non-naturally
occurring forms of the polypeptide. These polypeptides may differ
in some engineered way from the polypeptide isolated from its
native source, e.g., artificial variants that differ in specific
activity, thermostability, pH optimum, or the like. The variant
sequence may be constructed on the basis of the nucleotide sequence
presented as the polypeptide encoding region of SEQ ID NO: 1, SEQ
ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11,
SEQ ID NO: 13, or SEQ ID NO: 15, e.g., a subsequence thereof,
and/or by introduction of nucleotide substitutions which do not
give rise to another amino acid sequence of the polypeptide encoded
by the nucleotide sequence, but which correspond to the codon usage
of the host organism intended for production of the enzyme, or by
introduction of nucleotide substitutions which may give rise to a
different amino acid sequence. For a general description of
nucleotide substitution, see, e.g., Ford et al., 1991, Protein
Expression and Purification 2: 95-107.
[0168] It will be apparent to those skilled in the art that such
substitutions can be made outside the regions critical to the
function of the molecule and still result in an active polypeptide.
Amino acid residues essential to the activity of the polypeptide
encoded by an isolated polynucleotide of the invention, and
therefore preferably not subject to substitution, may be identified
according to procedures known in the art, such as site-directed
mutagenesis or alanine-scanning mutagenesis (see, e.g., Cunningham
and Wells, 1989, Science 244: 1081-1085). In the latter technique,
mutations are introduced at every positively charged residue in the
molecule, and the resultant mutant molecules are tested for lipase
activity to identify amino acid residues that are critical to the
activity of the molecule. Sites of substrate-enzyme interaction can
also be determined by analysis of the three-dimensional structure
as determined by such techniques as nuclear magnetic resonance
analysis, crystallography or photoaffinity labelling (see, e.g., de
Vos et al., 1992, Science 255: 306-312; Smith et al., 1992, Journal
of Molecular Biology 224: 899-904; Wlodaver et al., 1992, FEBS
Letters 309: 59-64).
[0169] The present invention also relates to isolated
polynucleotides encoding a polypeptide of the present invention,
which hybridize under very low stringency conditions, preferably
low stringency conditions, more preferably medium stringency
conditions, more preferably medium-high stringency conditions, even
more preferably high stringency conditions, and most preferably
very high stringency conditions with (i) the mature polypeptide
coding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID
NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, or SEQ ID NO:
15, (ii) the cDNA sequence contained in the mature polypeptide
coding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID
NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, or SEQ ID NO:
15, or (iii) a complementary strand of (i) or (ii); or allelic
variants and subsequences thereof (Sambrook et al., 1989, supra),
as defined herein. In a preferred aspect, the mature polypeptide
coding sequence of SEQ ID NO: 1 is nucleotides 73 to 1259. In
another preferred aspect, the mature polypeptide coding sequence of
SEQ ID NO: 3 is nucleotides 73 to 947. In another preferred aspect,
the mature polypeptide coding sequence of SEQ ID NO: 5 is
nucleotides 58 to 1088. In another preferred aspect, the mature
polypeptide coding sequence of SEQ ID NO: 7 is nucleotides 55 to
1044. In another preferred aspect, the mature polypeptide coding
sequence of SEQ ID NO: 9 is nucleotides 73 to 1179. In another
preferred aspect, the mature polypeptide coding sequence of SEQ ID
NO: 11 is nucleotides 58 to 1041. In another preferred aspect, the
mature polypeptide coding sequence of SEQ ID NO: 13 is nucleotides
73 to 1119. In another preferred aspect, the mature polypeptide
coding sequence of SEQ ID NO: 15 is nucleotides 76 to 1280.
[0170] The present invention also relates to isolated
polynucleotides obtained by (a) hybridizing a population of DNA
under very low, low, medium, medium-high, high, or very high
stringency conditions with (i) the mature polypeptide coding
sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7,
SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, or SEQ ID NO: 15, (ii)
the cDNA sequence contained in the mature polypeptide coding
sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7,
SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, or SEQ ID NO: 15, or
(iii) a complementary strand of (i) or (ii); and (b) isolating the
hybridizing polynucleotide, which encodes a polypeptide having
lipase activity. In a preferred aspect, the mature polypeptide
coding sequence of SEQ ID NO: 1 is nucleotides 73 to 1259. In
another preferred aspect, the mature polypeptide coding sequence of
SEQ ID NO: 3 is nucleotides 73 to 947. In another preferred aspect,
the mature polypeptide coding sequence of SEQ ID NO: 5 is
nucleotides 58 to 1088. In another preferred aspect, the mature
polypeptide coding sequence of SEQ ID NO: 7 is nucleotides 55 to
1044. In another preferred aspect, the mature polypeptide coding
sequence of SEQ ID NO: 9 is nucleotides 73 to 1179. In another
preferred aspect, the mature polypeptide coding sequence of SEQ ID
NO: 11 is nucleotides 58 to 1041. In another preferred aspect, the
mature polypeptide coding sequence of SEQ ID NO: 13 is nucleotides
73 to 1119. In another preferred aspect, the mature polypeptide
coding sequence of SEQ ID NO: 15 is nucleotides 76 to 1280.
Nucleic Acid Constructs
[0171] The present invention also relates to nucleic acid
constructs comprising an isolated polynucleotide of the present
invention operably linked to one or more control sequences that
direct the expression of the coding sequence in a suitable host
cell under conditions compatible with the control sequences.
[0172] An isolated polynucleotide encoding a polypeptide of the
present invention may be manipulated in a variety of ways to
provide for expression of the polypeptide. Manipulation of the
polynucleotide's sequence prior to its insertion into a vector may
be desirable or necessary depending on the expression vector. The
techniques for modifying polynucleotide sequences utilizing
recombinant DNA methods are well known in the art.
[0173] The control sequence may be an appropriate promoter
sequence, a nucleotide sequence which is recognized by a host cell
for expression of a polynucleotide encoding a polypeptide of the
present invention. The promoter sequence contains transcriptional
control sequences which mediate the expression of the polypeptide.
The promoter may be any nucleotide sequence which shows
transcriptional activity in the host cell of choice including
mutant, truncated, and hybrid promoters, and may be obtained from
genes encoding extracellular or intracellular polypeptides either
homologous or heterologous to the host cell.
[0174] Examples of suitable promoters for directing the
transcription of the nucleic acid constructs of the present
invention, especially in a bacterial host cell, are the promoters
obtained from the E. coli lac operon, Streptomyces coelicolor
agarase gene (dagA), Bacillus subtilis levansucrase gene (sacB),
Bacillus licheniformis alpha-amylase gene (amyL), Bacillus
stearothermophilus maltogenic amylase gene (amyM), Bacillus
amyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformis
penicillinase gene (penP), Bacillus subtilis xylA and xylB genes,
and prokaryotic beta-lactamase gene (Villa-Kamaroff et al., 1978,
Proceedings of the National Academy of Sciences USA 75: 3727-3731),
as well as the tac promoter (DeBoer et al., 1983, Proceedings of
the National Academy of Sciences USA 80: 21-25). Further promoters
are described in "Useful proteins from recombinant bacteria" in
Scientific American, 1980, 242: 74-94; and in Sambrook et al.,
1989, supra.
[0175] Examples of suitable promoters for directing the
transcription of the nucleic acid constructs of the present
invention in a filamentous fungal host cell are promoters obtained
from the genes for Aspergillus oryzae TAKA amylase, Rhizomucor
miehei aspartic proteinase, Aspergillus niger neutral
alpha-amylase, Aspergillus niger acid stable alpha-amylase,
Aspergillus niger or Aspergillus awamori glucoamylase (glaA),
Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease,
Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans
acetamidase, Fusarium venenatum amyloglucosidase (WO 00/56900),
Fusarium venenatum Daria (WO 00/56900), Fusarium venenatum Quinn
(WO 00/56900), Fusarium oxysporum trypsin-like protease (WO
96/00787), Trichoderma reesei beta-glucosidase, Trichoderma reesei
cellobiohydrolase I, Trichoderma reesei cellobiohydrolase II,
Trichoderma reesei endoglucanase I, Trichoderma reesei
endoglucanase II, Trichoderma reesei endoglucanase III, Trichoderma
reesei endoglucanase IV, Trichoderma reesei endoglucanase V,
Trichoderma reesei xylanase I, Trichoderma reesei xylanase II,
Trichoderma reesei beta-xylosidase, as well as the NA2-tpi promoter
(a hybrid of the promoters from the genes for Aspergillus niger
neutral alpha-amylase and Aspergillus oryzae triose phosphate
isomerase); and mutant, truncated, and hybrid promoters
thereof.
[0176] 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.
[0177] The control sequence may also be a suitable transcription
terminator sequence, a sequence recognized by a host cell to
terminate transcription. The terminator sequence is operably linked
to the 3' terminus of the nucleotide sequence encoding the
polypeptide. Any terminator which is functional in the host cell of
choice may be used in the present invention.
[0178] 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.
[0179] 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.
[0180] 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.
[0181] Preferred leaders for filamentous fungal host cells are
obtained from the genes for Aspergillus oryzae TAKA amylase and
Aspergillus nidulans triose phosphate isomerase.
[0182] 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).
[0183] The control sequence may also be a polyadenylation sequence,
a sequence operably linked to the 3' terminus of the nucleotide
sequence and which, when transcribed, is recognized by the host
cell as a signal to add polyadenosine residues to transcribed mRNA.
Any polyadenylation sequence which is functional in the host cell
of choice may be used in the present invention.
[0184] 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.
[0185] Useful polyadenylation sequences for yeast host cells are
described by Guo and Sherman, 1995, Molecular Cellular Biology 15:
5983-5990.
[0186] The control sequence may also be a signal peptide coding
region that codes for an amino acid sequence linked to the amino
terminus of a polypeptide and directs the encoded polypeptide into
the cell's secretory pathway. The 5' end of the coding sequence of
the nucleotide sequence may inherently contain a signal peptide
coding region naturally linked in translation reading frame with
the segment of the coding region which encodes the secreted
polypeptide. Alternatively, the 5' end of the coding sequence may
contain a signal peptide coding region which is foreign to the
coding sequence. The foreign signal peptide coding region may be
required where the coding sequence does not naturally contain a
signal peptide coding region. Alternatively, the foreign signal
peptide coding region may simply replace the natural signal peptide
coding region in order to enhance secretion of the polypeptide.
However, any signal peptide coding region which directs the
expressed polypeptide into the secretory pathway of a host cell of
choice, i.e., secreted into a culture medium, may be used in the
present invention.
[0187] Effective signal peptide coding regions for bacterial host
cells are the signal peptide coding regions obtained from the genes
for Bacillus NCIB 11837 maltogenic amylase, Bacillus
stearothermophilus alpha-amylase, Bacillus licheniformis
subtilisin, Bacillus licheniformis beta-lactamase, Bacillus
stearothermophilus neutral proteases (nprT, nprS, nprM), and
Bacillus subtilis prsA. Further signal peptides are described by
Simonen and Palva, 1993, Microbiological Reviews 57: 109-137.
[0188] Effective signal peptide coding regions for filamentous
fungal host cells are the signal peptide coding regions obtained
from the genes for Aspergillus oryzae TAKA amylase, Aspergillus
niger neutral amylase, Aspergillus niger glucoamylase, Rhizomucor
miehei aspartic proteinase, Humicola insolens cellulase, Humicola
insolens endoglucanase V, and Humicola lanuginosa lipase.
[0189] In a preferred aspect, the signal peptide coding region is
nucleotides 1 to 72 of SEQ ID NO: 1 which encode amino acids 1 to
24 of SEQ ID NO: 2.
[0190] In another preferred aspect, the signal peptide coding
region is nucleotides 1 to 72 of SEQ ID NO: 3 which encode amino
acids 1 to 24 of SEQ ID NO: 4.
[0191] In another preferred aspect, the signal peptide coding
region is nucleotides 1 to 42 of SEQ ID NO: 5 which encode amino
acids 1 to 19 of SEQ ID NO: 6.
[0192] In another preferred aspect, the signal peptide coding
region is nucleotides 1 to 54 of SEQ ID NO: 7 which encode amino
acids 1 to 18 of SEQ ID NO: 8.
[0193] In another preferred aspect, the signal peptide coding
region is nucleotides 1 to 72 of SEQ ID NO: 9 which encode amino
acids 1 to 24 of SEQ ID NO: 10.
[0194] In another preferred aspect, the signal peptide coding
region is nucleotides 1 to 57 of SEQ ID NO: 11 which encode amino
acids 1 to 19 of SEQ ID NO: 12.
[0195] In another preferred aspect, the signal peptide coding
region is nucleotides 1 to 72 of SEQ ID NO: 13 which encode amino
acids 1 to 24 of SEQ ID NO: 14.
[0196] In another preferred aspect, the signal peptide coding
region is nucleotides 1 to 75 of SEQ ID NO: 15 which encode amino
acids 1 to 25 of SEQ ID NO: 16.
[0197] Useful signal peptides for yeast host cells are obtained
from the genes for Saccharomyces cerevisiae alpha-factor and
Saccharomyces cerevisiae invertase. Other useful signal peptide
coding regions are described by Romanos et al., 1992, supra.
[0198] 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 Bacillus subtilis
alkaline protease (aprE), Bacillus subtilis neutral protease
(nprT), Saccharomyces cerevisiae alpha-factor, Rhizomucor miehei
aspartic proteinase, and Myceliophthora thermophila laccase (WO
95/33836).
[0199] 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.
[0200] It may also be desirable to add regulatory sequences which
allow the regulation of the expression of the polypeptide relative
to the growth of the host cell. Examples of regulatory systems are
those which cause the expression of the gene to be turned on or off
in response to a chemical or physical stimulus, including the
presence of a regulatory compound. Regulatory systems in
prokaryotic systems include the lac, tac, and trp operator systems.
In yeast, the ADH2 system or GAL1 system may be used. In
filamentous fungi, the TAKA alpha-amylase promoter, Aspergillus
niger glucoamylase promoter, and Aspergillus oryzae glucoamylase
promoter may be used as regulatory sequences. Other examples of
regulatory sequences are those which allow for gene amplification.
In eukaryotic systems, these include the dihydrofolate reductase
gene which is amplified in the presence of methotrexate, and the
metallothionein genes which are amplified with heavy metals. In
these cases, the nucleotide sequence encoding the polypeptide would
be operably linked with the regulatory sequence.
Expression Vectors
[0201] The present invention also relates to recombinant expression
vectors comprising a polynucleotide of the present invention, a
promoter, and transcriptional and translational stop signals. The
various nucleic acids and control sequences described herein may be
joined together to produce a recombinant expression vector which
may include one or more convenient restriction sites to allow for
insertion or substitution of the nucleotide sequence encoding the
polypeptide at such sites. Alternatively, a nucleotide sequence of
the present invention may be expressed by inserting the nucleotide
sequence or a nucleic acid construct comprising the sequence into
an appropriate vector for expression. In creating the expression
vector, the coding sequence is located in the vector so that the
coding sequence is operably linked with the appropriate control
sequences for expression.
[0202] The recombinant expression vector may be any vector (e.g., a
plasmid or virus) which can be conveniently subjected to
recombinant DNA procedures and can bring about expression of the
nucleotide sequence. The choice of the vector will typically depend
on the compatibility of the vector with the host cell into which
the vector is to be introduced. The vectors may be linear or closed
circular plasmids.
[0203] 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.
[0204] The vectors of the present invention preferably contain one
or more selectable markers which permit easy selection of
transformed, transfected, transduced, or the like cells. A
selectable marker is a gene the product of which provides for
biocide or viral resistance, resistance to heavy metals,
prototrophy to auxotrophs, and the like.
[0205] Examples of bacterial selectable markers are the dal genes
from Bacillus subtilis or Bacillus licheniformis, or markers which
confer antibiotic resistance such as ampicillin, kanamycin,
chloramphenicol, or tetracycline resistance. Suitable markers for
yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3.
Selectable markers for use in a filamentous fungal host cell
include, but are not limited to, amdS (acetamidase), argB
(ornithine carbamoyltransferase), bar (phosphinothricin
acetyltransferase), hph (hygromycin phosphotransferase), niaD
(nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase),
sC (sulfate adenyltransferase), and trpC (anthranilate synthase),
as well as equivalents thereof. Preferred for use in an Aspergillus
cell are the amdS and pyrG genes of Aspergillus nidulans or
Aspergillus oryzae and the bar gene of Streptomyces
hygroscopicus.
[0206] The vectors of the present invention preferably contain an
element(s) that permits integration of the vector into the host
cell's genome or autonomous replication of the vector in the cell
independent of the genome.
[0207] For integration into the host cell genome, the vector may
rely on the polynucleotide's sequence encoding the polypeptide or
any other element of the vector for integration into the genome by
homologous or nonhomologous recombination. Alternatively, the
vector may contain additional nucleotide sequences for directing
integration by homologous recombination into the genome of the host
cell at a precise location(s) in the chromosome(s). To increase the
likelihood of integration at a precise location, the integrational
elements should preferably contain a sufficient number of nucleic
acids, such as 100 to 10,000 base pairs, preferably 400 to 10,000
base pairs, and most preferably 800 to 10,000 base pairs, which
have a high degree of identity with the corresponding target
sequence to enhance the probability of homologous recombination.
The integrational elements may be any sequence that is homologous
with the target sequence in the genome of the host cell.
Furthermore, the integrational elements may be non-encoding or
encoding nucleotide sequences. On the other hand, the vector may be
integrated into the genome of the host cell by non-homologous
recombination.
[0208] 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.
[0209] Examples of bacterial origins of replication are the origins
of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184
permitting replication in E. coli, and pUB110, pE194, pTA1060, and
pAMB1 permitting replication in Bacillus.
[0210] 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.
[0211] 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.
[0212] More than one copy of a polynucleotide of the present
invention may be inserted into the host cell to increase production
of the gene product. An increase in the copy number of the
polynucleotide can be obtained by integrating at least one
additional copy of the sequence into the host cell genome or by
including an amplifiable selectable marker gene with the
polynucleotide where cells containing amplified copies of the
selectable marker gene, and thereby additional copies of the
polynucleotide, can be selected for by cultivating the cells in the
presence of the appropriate selectable agent.
[0213] 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
[0214] The present invention also relates to recombinant host
cells, comprising a polynucleotide of the present invention, which
are advantageously used in the recombinant production of the
polypeptides. A vector comprising a polynucleotide of the present
invention 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.
[0215] The host cell may be a unicellular microorganism, e.g., a
prokaryote, or a non-unicellular microorganism, e.g., a
eukaryote.
[0216] Useful unicellular microorganisms are bacterial cells such
as gram positive bacteria including, but not limited to, a Bacillus
cell, e.g., Bacillus alkalophilus, Bacillus amyloliquefaciens,
Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus
coagulans, Bacillus lautus, Bacillus lentus, Bacillus
licheniformis, Bacillus megaterium, Bacillus stearothermophilus,
Bacillus subtilis, and Bacillus thuringiensis, or a Streptomyces
cell, e.g., Streptomyces lividans and Streptomyces murinus, or gram
negative bacteria such as E. coli and Pseudomonas sp. In a
preferred aspect, the bacterial host cell is a Bacillus lentus,
Bacillus licheniformis, Bacillus stearothermophilus, or Bacillus
subtilis cell. In another preferred aspect, the Bacillus cell is an
alkalophilic Bacillus.
[0217] The introduction of a vector into a bacterial host cell may,
for instance, be effected by protoplast transformation (see, e.g.,
Chang and Cohen, 1979, Molecular General Genetics 168: 111-115),
using competent cells (see, e.g., Young and Spizizen, 1961, Journal
of Bacteriology 81: 823-829, or Dubnau and Davidoff-Abelson, 1971,
Journal of Molecular Biology 56: 209-221), electroporation (see,
e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), or
conjugation (see, e.g., Koehler and Thorne, 1987, Journal of
Bacteriology 169: 5771-5278).
[0218] The host cell may also be a eukaryote, such as a mammalian,
insect, plant, or fungal cell.
[0219] In a preferred aspect, the host cell is a fungal cell.
"Fungi" as used herein includes the phyla Ascomycota,
Basidiomycota, Chytridiomycota, and Zygomycota (as defined by
Hawksworth et al., In, Ainsworth and Bisby's Dictionary of The
Fungi, 8th edition, 1995, CAB International, University Press,
Cambridge, UK) as well as the Oomycota (as cited in Hawksworth et
al., 1995, supra, page 171) and all mitosporic fungi (Hawksworth et
al., 1995, supra).
[0220] In a more preferred aspect, the fungal host cell is a yeast
cell. "Yeast" as used herein includes ascosporogenous yeast
(Endomycetales), basidiosporogenous yeast, and yeast belonging to
the Fungi Imperfecti (Blastomycetes). Since the classification of
yeast may change in the future, for the purposes of this invention,
yeast shall be defined as described in Biology and Activities of
Yeast (Skinner, F. A., Passmore, S. M., and Davenport, R. R., eds,
Soc. App. Bacteriol. Symposium Series No. 9, 1980).
[0221] In an even more preferred aspect, the yeast host cell is a
Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces,
Schizosaccharomyces, or Yarrowia cell.
[0222] 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.
[0223] In another more preferred aspect, the fungal host cell is a
filamentous fungal cell. "Filamentous fungi" include all
filamentous forms of the subdivision Eumycota and Oomycota (as
defined by Hawksworth et al., 1995, supra). The filamentous fungi
are generally characterized by a mycelial wall composed of chitin,
cellulose, glucan, chitosan, mannan, and other complex
polysaccharides. Vegetative growth is by hyphal elongation and
carbon catabolism is obligately aerobic. In contrast, vegetative
growth by yeasts such as Saccharomyces cerevisiae is by budding of
a unicellular thallus and carbon catabolism may be
fermentative.
[0224] In an even more preferred aspect, the filamentous fungal
host cell is an Acremonium, Aspergillus, Aureobasidium,
Bjerkandera, Ceriporiopsis, Coprinus, Coriolus, Cryptococcus,
Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor,
Myceliophthora, Neocallimastix, Neurospora, Paecilomyces,
Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus,
Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium,
Trametes, or Trichoderma cell.
[0225] In a most preferred aspect, the filamentous fungal host cell
is an Aspergillus awamori, Aspergillus fumigatus, Aspergillus
foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus
niger or Aspergillus oryzae cell. In another most preferred aspect,
the filamentous fungal host cell is a Fusarium bactridioides,
Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum,
Fusarium graminearum, Fusarium graminum, Fusarium heterosporum,
Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum,
Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum,
Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum,
Fusarium trichothecioides, or Fusarium venenatum cell. In another
most preferred aspect, the filamentous fungal host cell is a
Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis aneirina,
Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis
pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa,
Ceriporiopsis subvermispora, Coprinus cinereus, Coriolus hirsutus,
Humicola insolens, Humicola lanuginosa, Mucor miehei,
Myceliophthora thermophila, Neurospora crassa, Penicillium
purpurogenum, Phanerochaete chrysosporium, Phlebia radiata,
Pleurotus eryngii, Thielavia terrestris, Trametes villosa, Trametes
versicolor, Trichoderma harzianum, Trichoderma koningii,
Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma
viride cell.
[0226] Fungal cells may be transformed by a process involving
protoplast formation, transformation of the protoplasts, and
regeneration of the cell wall in a manner known per se. Suitable
procedures for transformation of Aspergillus and Trichoderma host
cells are described in EP 238 023 and Yelton et al., 1984,
Proceedings of the National Academy of Sciences USA 81: 1470-1474.
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.
Methods of Production
[0227] The present invention also relates to methods for producing
a polypeptide of the present invention, comprising: (a) cultivating
a cell, which in its wild-type form is capable of producing the
polypeptide, under conditions conducive for production of the
polypeptide; and (b) recovering the polypeptide. In a preferred
aspect, the cell is of the genus Aspergillus. In a more preferred
aspect, the cell is Aspergillus fumigatus. In another more
preferred aspect, the cell is Aspergillus nidulans. In another
preferred aspect, the cell is of the genus Magnaporthe. In another
more preferred aspect, the cell is Magnaporthe grisea.
[0228] The present invention also relates to methods for producing
a polypeptide of the present invention, comprising: (a) cultivating
a host cell under conditions conducive for production of the
polypeptide; and (b) recovering the polypeptide.
[0229] The present invention also relates to methods for producing
a polypeptide of the present invention, comprising: (a) cultivating
a host cell under conditions conducive for production of the
polypeptide, wherein the host cell comprises a mutant nucleotide
sequence having at least one mutation in the mature polypeptide
coding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID
NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, or SEQ ID NO:
15, wherein the mutant nucleotide sequence encodes a polypeptide
which comprises or consists of the mature polypeptide of SEQ ID NO:
2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID
NO: 12, SEQ ID NO: 14, or SEQ ID NO: 16, and (b) recovering the
polypeptide.
[0230] In a preferred aspect, the mature polypeptide of SEQ ID NO:
2 is amino acids 25 to 396. In another preferred aspect, the mature
polypeptide of SEQ ID NO: 4 is amino acids 25 to 283. In another
preferred aspect, the mature polypeptide of SEQ ID NO: 6 is amino
acids 20 to 318. In another preferred aspect, the mature
polypeptide of SEQ ID NO: 8 is amino acids 19 to 348. In another
preferred aspect, the mature polypeptide of SEQ ID NO: 10 is amino
acids 25 to 393. In another preferred aspect, the mature
polypeptide of SEQ ID NO: 12 is amino acids 20 to 294. In another
preferred aspect, the mature polypeptide of SEQ ID NO: 14 is amino
acids 25 to 308. In another preferred aspect, the mature
polypeptide of SEQ ID NO: 16 is amino acids 26 to 404.
[0231] In the production methods of the present invention, the
cells are cultivated in a nutrient medium suitable for production
of the polypeptide using methods well known in the art. For
example, the cell may be cultivated by shake flask cultivation, and
small-scale or large-scale fermentation (including continuous,
batch, fed-batch, or solid state fermentations) in laboratory or
industrial fermentors performed in a suitable medium and under
conditions allowing the polypeptide to be expressed and/or
isolated. The cultivation takes place in a suitable nutrient medium
comprising carbon and nitrogen sources and inorganic salts, using
procedures known in the art. Suitable media are available from
commercial suppliers or may be prepared according to published
compositions (e.g., in catalogues of the American Type Culture
Collection). If the polypeptide is secreted into the nutrient
medium, the polypeptide can be recovered directly from the medium.
If the polypeptide is not secreted into the medium, it can be
recovered from cell lysates.
[0232] The polypeptides may be detected using methods known in the
art that are specific for the polypeptides. These detection methods
may include use of specific antibodies, formation of an enzyme
product, or disappearance of an enzyme substrate. For example, an
enzyme assay may be used to determine the activity of the
polypeptide as described herein.
[0233] The resulting polypeptide may be recovered using methods
known in the art. For example, the polypeptide may be recovered
from the nutrient medium by conventional procedures including, but
not limited to, centrifugation, filtration, extraction,
spray-drying, evaporation, or precipitation.
[0234] The polypeptides of the present invention may be purified by
a variety of procedures known in the art including, but not limited
to, chromatography (e.g., ion exchange, affinity, hydrophobic,
chromatofocusing, and size exclusion), electrophoretic procedures
(e.g., preparative isoelectric focusing), differential solubility
(e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction
(see, e.g., Protein Purification, J.-C. Janson and Lars Ryden,
editors, VCH Publishers, New York, 1989) to obtain substantially
pure polypeptides.
Plants
[0235] The present invention also relates to a transgenic plant,
plant part, or plant cell which has been transformed with a
nucleotide sequence encoding a polypeptide having lipase activity
of the present invention so as to express and produce the
polypeptide in recoverable quantities. The polypeptide may be
recovered from the plant or plant part. Alternatively, the plant or
plant part containing the recombinant polypeptide may be used as
such for improving the quality of a food or feed, e.g., improving
nutritional value, palatability, and rheological properties, or to
destroy an antinutritive factor.
[0236] The transgenic plant can be dicotyledonous (a dicot) or
monocotyledonous (a monocot). Examples of monocot plants are
grasses, such as meadow grass (blue grass, Poa), forage grass such
as Festuca, Lolium, temperate grass, such as Agrostis, and cereals,
e.g., wheat, oats, rye, barley, rice, sorghum, and maize
(corn).
[0237] Examples of dicot plants are tobacco, legumes, such as
lupins, potato, sugar beet, pea, bean and soybean, and cruciferous
plants (family Brassicaceae), such as cauliflower, rape seed, and
the closely related model organism Arabidopsis thaliana.
[0238] Examples of plant parts are stem, callus, leaves, root,
fruits, seeds, and tubers as well as the individual tissues
comprising these parts, e.g., epidermis, mesophyll, parenchyme,
vascular tissues, meristems. Specific plant cell compartments, such
as chloroplasts, apoplasts, mitochondria, vacuoles, peroxisomes and
cytoplasm are also considered to be a plant part. Furthermore, any
plant cell, whatever the tissue origin, is considered to be a plant
part. Likewise, plant parts such as specific tissues and cells
isolated to facilitate the utilisation of the invention are also
considered plant parts, e.g., embryos, endosperms, aleurone and
seeds coats.
[0239] Also included within the scope of the present invention are
the progeny of such plants, plant parts, and plant cells.
[0240] The transgenic plant or plant cell expressing a polypeptide
of the present invention may be constructed in accordance with
methods known in the art. In short, the plant or plant cell is
constructed by incorporating one or more expression constructs
encoding a polypeptide of the present invention into the plant host
genome or chloroplast genome and propagating the resulting modified
plant or plant cell into a transgenic plant or plant cell.
[0241] The expression construct is conveniently a nucleic acid
construct which comprises a polynucleotide encoding a polypeptide
of the present invention operably linked with appropriate
regulatory sequences required for expression of the nucleotide
sequence in the plant or plant part of choice. Furthermore, the
expression construct may comprise a selectable marker useful for
identifying host cells into which the expression construct has been
integrated and DNA sequences necessary for introduction of the
construct into the plant in question (the latter depends on the DNA
introduction method to be used).
[0242] The choice of regulatory sequences, such as promoter and
terminator sequences and optionally signal or transit sequences is
determined, for example, on the basis of when, where, and how the
polypeptide is desired to be expressed. For instance, the
expression of the gene encoding a polypeptide of the present
invention may be constitutive or inducible, or may be
developmental, stage or tissue specific, and the gene product may
be targeted to a specific tissue or plant part such as seeds or
leaves. Regulatory sequences are, for example, described by Tague
et al., 1988, Plant Physiology 86: 506.
[0243] For constitutive expression, the 35S-CaMV, the maize
ubiquitin 1, and the rice actin 1 promoter may be used (Franck et
al., 1980, Cell 21: 285-294, Christensen et al., 1992, Plant Mo.
Biol. 18: 675-689; Zhang et al, 1991, Plant Cell 3: 1155-1165).
Organ-specific promoters may be, for example, a promoter from
storage sink tissues such as seeds, potato tubers, and fruits
(Edwards & Coruzzi, 1990, Ann. Rev. Genet. 24: 275-303), or
from metabolic sink tissues such as meristems (Ito et al., 1994,
Plant Mol. Biol. 24: 863-878), a seed specific promoter such as the
glutelin, prolamin, globulin, or albumin promoter from rice (Wu et
al., 1998, Plant and Cell Physiology 39: 885-889), a Vicia faba
promoter from the legumin B4 and the unknown seed protein gene from
Vicia faba (Conrad et al., 1998, Journal of Plant Physiology 152:
708-711), a promoter from a seed oil body protein (Chen et al.,
1998, Plant and Cell Physiology 39: 935-941), the storage protein
napA promoter from Brassica napus, or any other seed specific
promoter known in the art, e.g., as described in WO 91/14772.
Furthermore, the promoter may be a leaf specific promoter such as
the rbcs promoter from rice or tomato (Kyozuka et al., 1993, Plant
Physiology 102: 991-1000, the chlorella virus adenine
methyltransferase gene promoter (Mitra and Higgins, 1994, Plant
Molecular Biology 26: 85-93), or the aldP gene promoter from rice
(Kagaya et al., 1995, Molecular and General Genetics 248: 668-674),
or a wound inducible promoter such as the potato pin2 promoter (Xu
et al., 1993, Plant Molecular Biology 22: 573-588). Likewise, the
promoter may inducible by abiotic treatments such as temperature,
drought, or alterations in salinity or induced by exogenously
applied substances that activate the promoter, e.g., ethanol,
oestrogens, plant hormones such as ethylene, abscisic acid, and
gibberellic acid, and heavy metals.
[0244] A promoter enhancer element may also be used to achieve
higher expression of a polypeptide of the present invention in the
plant. For instance, the promoter enhancer element may be an intron
which is placed between the promoter and the nucleotide sequence
encoding a polypeptide of the present invention. For instance, Xu
et al., 1993, supra, disclose the use of the first intron of the
rice actin 1 gene to enhance expression.
[0245] The selectable marker gene and any other parts of the
expression construct may be chosen from those available in the
art.
[0246] The nucleic acid construct is incorporated into the plant
genome according to conventional techniques known in the art,
including Agrobacterium-mediated transformation, virus-mediated
transformation, microinjection, particle bombardment, biolistic
transformation, and electroporation (Gasser et al., 1990, Science
244: 1293; Potrykus, 1990, Bio/Technology 8: 535; Shimamoto et al.,
1989, Nature 338: 274).
[0247] Presently, Agrobacterium tumefaciens-mediated gene transfer
is the method of choice for generating transgenic dicots (for a
review, see Hooykas and Schilperoort, 1992, Plant Molecular Biology
19: 15-38) and can also be used for transforming monocots, although
other transformation methods are often used for these plants.
Presently, the method of choice for generating transgenic monocots
is particle bombardment (microscopic gold or tungsten particles
coated with the transforming DNA) of embryonic calli or developing
embryos (Christou, 1992, Plant Journal 2: 275-281; Shimamoto, 1994,
Current Opinion Biotechnology 5: 158-162; Vasil et al., 1992,
Bio/Technology 10: 667-674). An alternative method for
transformation of monocots is based on protoplast transformation as
described by Omirulleh et al., 1993, Plant Molecular Biology 21:
415-428.
[0248] Following transformation, the transformants having
incorporated the expression construct are selected and regenerated
into whole plants according to methods well-known in the art. Often
the transformation procedure is designed for the selective
elimination of selection genes either during regeneration or in the
following generations by using, for example, co-transformation with
two separate T-DNA constructs or site specific excision of the
selection gene by a specific recombinase.
[0249] The present invention also relates to methods for producing
a polypeptide of the present invention comprising: (a) cultivating
a transgenic plant or a plant cell comprising a polynucleotide
encoding a polypeptide having lipase activity of the present
invention under conditions conducive for production of the
polypeptide; and (b) recovering the polypeptide.
Removal or Reduction of Lipase Activity
[0250] The present invention also relates to methods for producing
a mutant of a parent cell, which comprises disrupting or deleting a
polynucleotide sequence, or a portion thereof, encoding a
polypeptide of the present invention, which results in the mutant
cell producing less of the polypeptide than the parent cell when
cultivated under the same conditions.
[0251] The mutant cell may be constructed by reducing or
eliminating expression of a nucleotide sequence encoding a
polypeptide of the present invention using methods well known in
the art, for example, insertions, disruptions, replacements, or
deletions. In a preferred aspect, the nucleotide sequence is
inactivated. The nucleotide sequence to be modified or inactivated
may be, for example, the coding region or a part thereof essential
for activity, or a regulatory element required for the expression
of the coding region. An example of such a regulatory or control
sequence may be a promoter sequence or a functional part thereof,
i.e., a part that is sufficient for affecting expression of the
nucleotide sequence. Other control sequences for possible
modification include, but are not limited to, a leader,
polyadenylation sequence, propeptide sequence, signal peptide
sequence, transcription terminator, and transcriptional
activator.
[0252] Modification or inactivation of the nucleotide sequence may
be performed by subjecting the parent cell to mutagenesis and
selecting for mutant cells in which expression of the nucleotide
sequence has been reduced or eliminated. The mutagenesis, which may
be specific or random, may be performed, for example, by use of a
suitable physical or chemical mutagenizing agent, by use of a
suitable oligonucleotide, or by subjecting the DNA sequence to PCR
generated mutagenesis. Furthermore, the mutagenesis may be
performed by use of any combination of these mutagenizing
agents.
[0253] Examples of a physical or chemical mutagenizing agent
suitable for the present purpose include ultraviolet (UV)
irradiation, hydroxylamine, N-methyl-N'-nitro-N-nitrosoguanidine
(MNNG), O-methyl hydroxylamine, nitrous acid, ethyl methane
sulphonate (EMS), sodium bisulphite, formic acid, and nucleotide
analogues.
[0254] When such agents are used, the mutagenesis is typically
performed by incubating the parent cell to be mutagenized in the
presence of the mutagenizing agent of choice under suitable
conditions, and screening and/or selecting for mutant cells
exhibiting reduced or no expression of the gene.
[0255] Modification or inactivation of the nucleotide sequence may
be accomplished by introduction, substitution, or removal of one or
more nucleotides in the gene or a regulatory element required for
the transcription or translation thereof. For example, nucleotides
may be inserted or removed so as to result in the introduction of a
stop codon, the removal of the start codon, or a change in the open
reading frame. Such modification or inactivation may be
accomplished by site-directed mutagenesis or PCR generated
mutagenesis in accordance with methods known in the art. Although,
in principle, the modification may be performed in vivo, i.e.,
directly on the cell expressing the nucleotide sequence to be
modified, it is preferred that the modification be performed in
vitro as exemplified below.
[0256] An example of a convenient way to eliminate or reduce
expression of a nucleotide sequence by a cell is based on
techniques of gene replacement, gene deletion, or gene disruption.
For example, in the gene disruption method, a nucleic acid sequence
corresponding to the endogenous nucleotide sequence is mutagenized
in vitro to produce a defective nucleic acid sequence which is then
transformed into the parent cell to produce a defective gene. By
homologous recombination, the defective nucleic acid sequence
replaces the endogenous nucleotide sequence. It may be desirable
that the defective nucleotide sequence also encodes a marker that
may be used for selection of transformants in which the nucleotide
sequence has been modified or destroyed. In a particularly
preferred aspect, the nucleotide sequence is disrupted with a
selectable marker such as those described herein.
[0257] Alternatively, modification or inactivation of the
nucleotide sequence may be performed by established anti-sense or
RNAi techniques using a sequence complementary to the nucleotide
sequence. More specifically, expression of the nucleotide sequence
by a cell may be reduced or eliminated by introducing a sequence
complementary to the nucleotide sequence of the gene that may be
transcribed in the cell and is capable of hybridizing to the mRNA
produced in the cell. Under conditions allowing the complementary
anti-sense nucleotide sequence to hybridize to the mRNA, the amount
of protein translated is thus reduced or eliminated.
[0258] The present invention further relates to a mutant cell of a
parent cell which comprises a disruption or deletion of a
nucleotide sequence encoding the polypeptide or a control sequence
thereof, which results in the mutant cell producing less of the
polypeptide or no polypeptide compared to the parent cell.
[0259] The polypeptide-deficient mutant cells so created are
particularly useful as host cells for the expression of homologous
and/or heterologous polypeptides. Therefore, the present invention
further relates to methods for producing a homologous or
heterologous polypeptide comprising: (a) cultivating the mutant
cell under conditions conducive for production of the polypeptide;
and (b) recovering the polypeptide. The term "heterologous
polypeptides" is defined herein as polypeptides which are not
native to the host cell, a native protein in which modifications
have been made to alter the native sequence, or a native protein
whose expression is quantitatively altered as a result of a
manipulation of the host cell by recombinant DNA techniques.
[0260] In a further aspect, the present invention relates to a
method for producing a protein product essentially free of lipase
activity by fermentation of a cell which produces both a
polypeptide of the present invention as well as the protein product
of interest by adding an effective amount of an agent capable of
inhibiting lipase activity to the fermentation broth before,
during, or after the fermentation has been completed, recovering
the product of interest from the fermentation broth, and optionally
subjecting the recovered product to further purification.
[0261] In a further aspect, the present invention relates to a
method for producing a protein product essentially free of lipase
activity by cultivating the cell under conditions permitting the
expression of the product, subjecting the resultant culture broth
to a combined pH and temperature treatment so as to reduce the
lipase activity substantially, and recovering the product from the
culture broth. Alternatively, the combined pH and temperature
treatment may be performed on an enzyme preparation recovered from
the culture broth. The combined pH and temperature treatment may
optionally be used in combination with a treatment with an lipase
inhibitor.
[0262] In accordance with this aspect of the invention, it is
possible to remove at least 60%, preferably at least 75%, more
preferably at least 85%, still more preferably at least 95%, and
most preferably at least 99% of the lipase activity. Complete
removal of lipase activity may be obtained by use of this
method.
[0263] The combined pH and temperature treatment is preferably
carried out at a pH in the range of 2-4 or 9-11 and a temperature
in the range of at least 60-70.degree. C. for a sufficient period
of time to attain the desired effect, where typically, 30 to 60
minutes is sufficient.
[0264] The methods used for cultivation and purification of the
product of interest may be performed by methods known in the
art.
[0265] The methods of the present invention for producing an
essentially lipase-free product is of particular interest in the
production of eukaryotic polypeptides, in particular fungal
proteins such as enzymes. The enzyme may be selected from, e.g., an
amylolytic enzyme, lipolytic enzyme, proteolytic enzyme, cellulytic
enzyme, oxidoreductase, or plant cell-wall degrading enzyme.
Examples of such enzymes include an aminopeptidase, amylase,
amyloglucosidase, carbohydrase, carboxypeptidase, catalase,
cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase,
deoxyribonuclease, esterase, galactosidase, beta-galactosidase,
glucoamylase, glucose oxidase, glucosidase, haloperoxidase,
hemicellulase, invertase, isomerase, laccase, ligase, lipase,
lyase, mannosidase, oxidase, pectinolytic enzyme, peroxidase,
phytase, phenoloxidase, polyphenoloxidase, proteolytic enzyme,
ribonuclease, transferase, transglutaminase, or xylanase. The
lipase-deficient cells may also be used to express heterologous
proteins of pharmaceutical interest such as hormones, growth
factors, receptors, and the like.
[0266] It will be understood that the term "eukaryotic
polypeptides" includes not only native polypeptides, but also those
polypeptides, e.g., enzymes, which have been modified by amino acid
substitutions, deletions or additions, or other such modifications
to enhance activity, thermostability, pH tolerance and the
like.
[0267] In a further aspect, the present invention relates to a
protein product essentially free from lipase activity which is
produced by a method of the present invention.
Compositions
[0268] The present invention also relates to compositions
comprising a polypeptide of the present invention. Preferably, the
compositions are enriched in such a polypeptide. The term
"enriched" indicates that the lipase activity of the composition
has been increased, e.g., with an enrichment factor of at least
1.1.
[0269] The composition may comprise a polypeptide of the present
invention as the major enzymatic component, e.g., a mono-component
composition. Alternatively, the composition may comprise multiple
enzymatic activities, such as an aminopeptidase, amylase,
carbohydrase, carboxypeptidase, catalase, cellulase, chitinase,
cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease,
esterase, alpha-galactosidase, beta-galactosidase, glucoamylase,
alpha-glucosidase, beta-glucosidase, haloperoxidase, invertase,
laccase, lipase, mannosidase, oxidase, pectinolytic enzyme,
peptidoglutaminase, peroxidase, phytase, polyphenoloxidase,
proteolytic enzyme, ribonuclease, transglutaminase, or xylanase.
The additional enzyme(s) may be produced, for example, by a
microorganism belonging to the genus Aspergillus, preferably
Aspergillus aculeatus, Aspergillus awamori, Aspergillus fumigatus,
Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans,
Aspergillus niger, or Aspergillus oryzae; Fusarium, preferably
Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense,
Fusarium culmorum, Fusarium graminearum, Fusarium graminum,
Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum,
Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum,
Fusarium sarcochroum, Fusarium sulphureum, Fusarium toruloseum,
Fusarium trichothecioides, or Fusarium venenatum; Humicola,
preferably Humicola insolens or Humicola lanuginosa; or
Trichoderma, preferably Trichoderma harzianum, Trichoderma
koningii, Trichoderma longibrachiatum, Trichoderma reesei, or
Trichoderma viride.
[0270] The polypeptide compositions may be prepared in accordance
with methods known in the art and may be in the form of a liquid or
a dry composition. For instance, the polypeptide composition may be
in the form of a granulate or a microgranulate. The polypeptide to
be included in the composition may be stabilized in accordance with
methods known in the art.
[0271] Examples are given below of preferred uses of the
polypeptide compositions of the invention. The dosage of the
polypeptide composition of the invention and other conditions under
which the composition is used may be determined on the basis of
methods known in the art.
Uses
[0272] The present invention is also directed to methods for using
the polypeptides having lipase activity, or compositions
thereof.
[0273] Use in Degumming.
[0274] A polypeptide of the present invention may be used for
degumming an aqueous carbohydrate solution or slurry to improve its
filterability, particularly, a starch hydrolysate, especially a
wheat starch hydrolysate which is difficult to filter and yields
cloudy filtrates. The treatment may be performed using methods well
known in the art. See, for example, EP 219,269, EP 808,903, and
U.S. Pat. No. 6,103,505.
[0275] Use in Baking.
[0276] A polypeptide of the present invention may be used in baking
according to U.S. Pat. No. 6,558,715.
[0277] Use in Detergent.
[0278] The polypeptides of the present invention may be added to
and thus become a component of a detergent composition.
[0279] The detergent composition of the present invention may be
formulated, for example, as a hand or machine laundry detergent
composition including a laundry additive composition suitable for
pre-treatment of stained fabrics and a rinse added fabric softener
composition, or be formulated as a detergent composition for use in
general household hard surface cleaning operations, or be
formulated for hand or machine dishwashing operations.
[0280] In a specific aspect, the present invention provides a
detergent additive comprising a polypeptide of the present
invention as described herein. The detergent additive as well as
the detergent composition may comprise one or more enzymes such as
a protease, lipase, cutinase, an amylase, carbohydrase, cellulase,
pectinase, mannanase, arabinase, galactanase, xylanase, oxidase,
e.g., a laccase, and/or peroxidase.
[0281] In general the properties of the selected enzyme(s) should
be compatible with the selected detergent, (i.e., pH-optimum,
compatibility with other enzymatic and non-enzymatic ingredients,
etc.), and the enzyme(s) should be present in effective
amounts.
[0282] Cellulases:
[0283] Suitable cellulases include those of bacterial or fungal
origin. Chemically modified or protein engineered mutants are
included. Suitable cellulases include cellulases from the genera
Bacillus, Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium,
e.g., the fungal cellulases produced from Humicola insolens,
Myceliophthora thermophila and Fusarium oxysporum disclosed in U.S.
Pat. No. 4,435,307, U.S. Pat. No. 5,648,263, U.S. Pat. No.
5,691,178, U.S. Pat. No. 5,776,757 and WO 89/09259.
[0284] Especially suitable cellulases are the alkaline or neutral
cellulases having color care benefits. Examples of such cellulases
are cellulases described in EP 0 495 257, EP 0 531 372, WO
96/11262, WO 96/29397, WO 98/08940. Other examples are cellulase
variants such as those described in WO 94/07998, EP 0 531 315, U.S.
Pat. No. 5,457,046, U.S. Pat. No. 5,686,593, U.S. Pat. No.
5,763,254, WO 95/24471, WO 98/12307 and PCT/DK98/00299.
[0285] Commercially available cellulases include Celluzyme.TM., and
Carezyme.TM. (Novozymes A/S), Clazinase.TM., and Puradax HA.TM.
(Genencor International Inc.), and KAC-500(B).TM. (Kao
Corporation).
[0286] Proteases:
[0287] Suitable proteases include those of animal, vegetable or
microbial origin. Microbial origin is preferred. Chemically
modified or protein engineered mutants are included. The protease
may be a serine protease or a metalloprotease, preferably an
alkaline microbial protease or a trypsin-like protease. Examples of
alkaline proteases are subtilisins, especially those derived from
Bacillus, e.g., subtilisin Novo, subtilisin Carlsberg, subtilisin
309, subtilisin 147 and subtilisin 168 (described in WO 89/06279).
Examples of trypsin-like proteases are trypsin (e.g., of porcine or
bovine origin) and the Fusarium protease described in WO 89/06270
and WO 94/25583.
[0288] Examples of useful proteases are the variants described in
WO 92/19729, WO 98/20115, WO 98/20116, and WO 98/34946, especially
the variants with substitutions in one or more of the following
positions: 27, 36, 57, 76, 87, 97, 101, 104, 120, 123, 167, 170,
194, 206, 218, 222, 224, 235, and 274.
[0289] Preferred commercially available protease enzymes include
Alcalase.TM., Savinase.TM., Primase.TM., Duralase.TM.,
Esperase.TM., and Kannase.TM. (Novozymes A/S), Maxatase.TM.,
Maxacal.TM., Maxapem.TM., Properase.TM., Purafect.TM., Purafect
OxP.TM., FN2.TM., and FN3.TM. (Genencor International Inc.).
[0290] Lipases:
[0291] Suitable lipases include those of bacterial or fungal
origin. Chemically modified or protein engineered mutants are
included. Examples of useful lipases include lipases from Humicola
(synonym Thermomyces), e.g., from H. lanuginosa (T. lanuginosus) as
described in EP 258 068 and EP 305 216 or from H. insolens as
described in WO 96/13580, a Pseudomonas lipase, e.g., from P.
alcaligenes or P. pseudoalcaligenes (EP 218 272), P. cepacia (EP
331 376), P. stutzeri (GB 1,372,034), P. fluorescens, Pseudomonas
sp. strain SD 705 (WO 95/06720 and WO 96/27002), P. wisconsinensis
(WO 96/12012), a Bacillus lipase, e.g., from B. subtilis (Dartois
et al., 1993, Biochemica et Biophysica Acta, 1131: 253-360), B.
stearothermophilus (JP 64/744992) or B. pumilus (WO 91/16422).
[0292] Other examples are lipase variants such as those described
in WO 92/05249, WO 94/01541, EP 407 225, EP 260 105, WO 95/35381,
WO 96/00292, WO 95/30744, WO 94/25578, WO 95/14783, WO 95/22615, WO
97/04079 and WO 97/07202.
[0293] Preferred commercially available lipase enzymes include
Lipolase.TM., Lipolase Ultra.TM., and Lipex.TM. (Novozymes
A/S).
[0294] Amylases:
[0295] Suitable amylases (.alpha. and/or .beta.) include those of
bacterial or fungal origin. Chemically modified or protein
engineered mutants are included. Amylases include, for example,
.alpha.-amylases obtained from Bacillus, e.g., a special strain of
Bacillus licheniformis, described in more detail in GB
1,296,839.
[0296] Examples of useful amylases are the variants described in WO
94/02597, WO 94/18314, WO 96/23873, and WO 97/43424, especially the
variants with substitutions in one or more of the following
positions: 15, 23, 105, 106, 124, 128, 133, 154, 156, 181, 188,
190, 197, 202, 208, 209, 243, 264, 304, 305, 391, 408, and 444.
[0297] Commercially available amylases are Duramyl.TM.,
Termamyl.TM., Fungamyl.TM. and BAN.TM. (Novozymes A/S),
Rapidase.TM. and Purastar.TM. (from Genencor International
Inc.).
[0298] Peroxidases/Oxidases:
[0299] Suitable peroxidases/oxidases include those of plant,
bacterial or fungal origin. Chemically modified or protein
engineered mutants are included. Examples of useful peroxidases
include peroxidases from Coprinus, e.g., from C. cinereus, and
variants thereof as those described in WO 93/24618, WO 95/10602,
and WO 98/15257.
[0300] Commercially available peroxidases include Guardzyme.TM.
(Novozymes A/S).
[0301] The detergent enzyme(s) may be included in a detergent
composition by adding separate additives containing one or more
enzymes, or by adding a combined additive comprising all of these
enzymes. A detergent additive of the invention, i.e., a separate
additive or a combined additive, can be formulated, for example, as
a granulate, liquid, slurry, etc. Preferred detergent additive
formulations are granulates, in particular non-dusting granulates,
liquids, in particular stabilized liquids, or slurries.
[0302] Non-dusting granulates may be produced, e.g., as disclosed
in U.S. Pat. Nos. 4,106,991 and 4,661,452 and may optionally be
coated by methods known in the art. Examples of waxy coating
materials are poly(ethylene oxide) products (polyethyleneglycol,
PEG) with mean molar weights of 1000 to 20000; ethoxylated
nonylphenols having from 16 to 50 ethylene oxide units; ethoxylated
fatty alcohols in which the alcohol contains from 12 to 20 carbon
atoms and in which there are 15 to 80 ethylene oxide units; fatty
alcohols; fatty acids; and mono- and di- and triglycerides of fatty
acids. Examples of film-forming coating materials suitable for
application by fluid bed techniques are given in GB 1483591. Liquid
enzyme preparations may, for instance, be stabilized by adding a
polyol such as propylene glycol, a sugar or sugar alcohol, lactic
acid or boric acid according to established methods. Protected
enzymes may be prepared according to the method disclosed in EP
238,216.
[0303] The detergent composition of the invention may be in any
convenient form, e.g., a bar, a tablet, a powder, a granule, a
paste or a liquid. A liquid detergent may be aqueous, typically
containing up to 70% water and 0-30% organic solvent, or
non-aqueous.
[0304] The detergent composition comprises one or more surfactants,
which may be non-ionic including semi-polar and/or anionic and/or
cationic and/or zwitterionic. The surfactants are typically present
at a level of from 0.1% to 60% by weight.
[0305] When included therein the detergent will usually contain
from about 1% to about 40% of an anionic surfactant such as linear
alkylbenzenesulfonate, alpha-olefinsulfonate, alkyl sulfate (fatty
alcohol sulfate), alcohol ethoxysulfate, secondary alkanesulfonate,
alpha-sulfo fatty acid methyl ester, alkyl- or alkenylsuccinic
acid, or soap.
[0306] When included therein the detergent will usually contain
from about 0.2% to about 40% of a non-ionic surfactant such as
alcohol ethoxylate, nonylphenol ethoxylate, alkylpolyglycoside,
alkyldimethylamineoxide, ethoxylated fatty acid monoethanolamide,
fatty acid monoethanolamide, polyhydroxy alkyl fatty acid amide, or
N-acyl N-alkyl derivatives of glucosamine ("glucamides").
[0307] The detergent may contain 0-65% of a detergent builder or
complexing agent such as zeolite, diphosphate, triphosphate,
phosphonate, carbonate, citrate, nitrilotriacetic acid,
ethylenediaminetetraacetic acid, diethylenetriaminepentaacetic
acid, alkyl- or alkenylsuccinic acid, soluble silicates, or layered
silicates (e.g., SKS-6 from Hoechst).
[0308] The detergent may comprise one or more polymers. Examples
are carboxymethylcellulose, poly(vinylpyrrolidone), poly (ethylene
glycol), poly(vinyl alcohol), poly(vinylpyridine-N-oxide),
poly(vinylimidazole), polycarboxylates such as polyacrylates,
maleic/acrylic acid copolymers, and lauryl methacrylate/acrylic
acid copolymers.
[0309] The detergent may contain a bleaching system which may
comprise a H.sub.2O.sub.2 source such as perborate or percarbonate
which may be combined with a peracid-forming bleach activator such
as tetraacetylethylenediamine or nonanoyloxybenzenesulfonate.
Alternatively, the bleaching system may comprise peroxyacids of,
for example, the amide, imide, or sulfone type.
[0310] The enzyme(s) of the detergent composition of the invention
may be stabilized using conventional stabilizing agents, e.g., a
polyol such as propylene glycol or glycerol, a sugar or sugar
alcohol, lactic acid, boric acid, or a boric acid derivative, e.g.,
an aromatic borate ester, or a phenyl boronic acid derivative such
as 4-formylphenyl boronic acid, and the composition may be
formulated as described in, for example, WO 92/19709 and WO
92/19708.
[0311] The detergent may also contain other conventional detergent
ingredients such as, e.g., fabric conditioners including clays,
foam boosters, suds suppressors, anti-corrosion agents,
soil-suspending agents, anti-soil redeposition agents, dyes,
bactericides, optical brighteners, hydrotropes, tarnish inhibitors,
or perfumes.
[0312] In the detergent compositions, any enzyme may be added in an
amount corresponding to 0.01-100 mg of enzyme protein per liter of
wash liquor, preferably 0.05-5 mg of enzyme protein per liter of
wash liquor, in particular 0.1-1 mg of enzyme protein per liter of
wash liquor.
[0313] In the detergent compositions, a polypeptide of the present
invention may be added in an amount corresponding to 0.001-100 mg
of protein, preferably 0.005-50 mg of protein, more preferably
0.01-25 mg of protein, even more preferably 0.05-10 mg of protein,
most preferably 0.05-5 mg of protein, and even most preferably
0.01-1 mg of protein per liter of wash liquor.
[0314] A polypeptide of the present invention may also be
incorporated in the detergent formulations disclosed in WO
97/07202, which is hereby incorporated by reference.
Signal Peptide
[0315] The present invention also relates to nucleic acid
constructs comprising a gene encoding a protein operably linked to
a nucleotide sequence comprising or consisting of nucleotides 1 to
72 of SEQ ID NO: 1, nucleotides 1 to 72 of SEQ ID NO: 3,
nucleotides 1 to 57 of SEQ ID NO: 5, nucleotides 1 to 54 of SEQ ID
NO: 7, nucleotides 1 to 72 of SEQ ID NO: 9, nucleotides 1 to 57 of
SEQ ID NO: 11, nucleotides 1 to 72 of SEQ ID NO: 13, or nucleotides
1 to 75 of SEQ ID NO: 15 encoding a signal peptide comprising or
consisting of amino acids 1 to 24 of SEQ ID NO: 2, amino acids 1 to
24 of SEQ ID NO: 4, amino acids 1 to 19 of SEQ ID NO: 6, amino
acids 1 to 18 of SEQ ID NO: 8, amino acids 1 to 24 of SEQ ID NO:
10, amino acids 1 to 19 of SEQ ID NO: 12, amino acids 1 to 24 of
SEQ ID NO: 14, or amino acids 1 to 25 of SEQ ID NO: 16,
respectively, wherein the gene is foreign to the nucleotide
sequence.
[0316] The present invention also relates to recombinant expression
vectors and recombinant host cells comprising such nucleic acid
constructs.
[0317] The present invention also relates to methods for producing
a protein comprising (a) cultivating such a recombinant host cell
under conditions suitable for production of the protein; and (b)
recovering the protein.
[0318] The protein may be native or heterologous to a host cell.
The term "protein" is not meant herein to refer to a specific
length of the encoded product and, therefore, encompasses peptides,
oligopeptides, and proteins. The term "protein" also encompasses
two or more polypeptides combined to form the encoded product. The
proteins also include hybrid polypeptides which comprise a
combination of partial or complete polypeptide sequences obtained
from at least two different proteins wherein one or more may be
heterologous or native to the host cell. Proteins further include
naturally occurring allelic and engineered variations of the above
mentioned proteins and hybrid proteins.
[0319] Preferably, the protein is a hormone or variant thereof,
enzyme, receptor or portion thereof, antibody or portion thereof,
or reporter. In a more preferred aspect, the protein is an
oxidoreductase, transferase, hydrolase, lyase, isomerase, or
ligase. In an even more preferred aspect, the protein is an
aminopeptidase, amylase, carbohydrase, carboxypeptidase, catalase,
cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase,
deoxyribonuclease, esterase, alpha-galactosidase,
beta-galactosidase, glucoamylase, alpha-glucosidase,
beta-glucosidase, invertase, laccase, another lipase, mannosidase,
mutanase, oxidase, pectinolytic enzyme, peroxidase, phytase,
polyphenoloxidase, proteolytic enzyme, ribonuclease,
transglutaminase or xylanase.
[0320] The gene may be obtained from any prokaryotic, eukaryotic,
or other source.
[0321] The present invention is further described by the following
examples which should not be construed as limiting the scope of the
invention.
EXAMPLES
Materials
[0322] Chemicals used as buffers and substrates were commercial
products of at least reagent grade.
[0323] Strains
[0324] Aspergillus fumigatus PaHa34, Magnaporthe grisea FGSC 8958
(Fungal Genetics Stock Center), and Aspergillus nidulans A1000
(Fungal Genetics Stock Center, Kansas City, Mo.) were used as
sources for the lipase genes.
Media
[0325] Potato dextrose medium was composed per liter of 24 grams of
potato dextrose.
[0326] YP medium was composed per liter of 10 g of yeast extract
and 20 g of Bacto peptone.
[0327] SOC medium was composed per liter of 20 g of tryptone, 5 g
of yeast extract, 2 ml of 5 M NaCl, and 2.5 ml of 1 M KCl.
[0328] NZY.sup.+ medium was composed per liter of 10 g of NZ amine,
5 g of yeast extract, 5 g of NaCl, 12.5 mM MgCl.sub.2, 12.5 mM
MgSO.sub.4, and 10 ml of 2 M glucose.
[0329] LB medium was composed per liter of 10 g of tryptone, 5 g of
yeast extract, and 5 g of NaCl.
[0330] MY25 medium was composed per liter of 25 g of maltodextrin,
2 g of MgSO.sub.4.7H.sub.2O, 10 g of KH.sub.2PO.sub.4, 2 g of
citric acid, 2 g of K.sub.2SO.sub.4, 2 g of urea, 10 g of yeast
extract, and 1.5 ml of AMG trace metals solution, adjusted to pH
6.
[0331] AMG trace metals solution was composed per liter of 14.3 g
of ZnSO.sub.4.7H.sub.2O, 2.5 g of CuSO.sub.4.5H.sub.2O, 0.5 g of
NiCl.sub.2.6H.sub.2O, 13.8 g of FeSO.sub.4.7H.sub.2O, 8.5 g of
MnSO.sub.4.H.sub.2O, and 3 g of citric acid.
[0332] CM was composed per liter of 6 g of yeast extract, 6 g of
casein acid hydrolysate, and 10 g of sucrose.
[0333] 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.
[0334] COVE salt solution was composed per liter of 26 g of KCl, 26
g of MgSO.sub.4.7H.sub.2O, 76 g of KH.sub.2PO.sub.4, and 50 ml of
COVE trace metals solution.
[0335] 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.
[0336] 2X YT plates were composed per liter of 16 g of tryptone, 10
g of yeast extract, 5 g of NaCl, and 15 g of Bacto agar.
Example 1
Identification of Lipase Genes in the Partial Genomic Sequence of
Aspergillus fumigatus
[0337] A tfasty search (Pearson, W. R., 1999, in Bioinformatics
Methods and Protocols, S. Misener and S. A. Krawetz, ed., pp.
185-219) of the Aspergillus fumigatus partial genome sequence (The
Institute for Genomic Research, Rockville, Md.) was carried out
using as query a lipase sequence from Thermomyces lanuginosus
(Accession No. O59952). Several genes were identified as putative
lipases based upon an E value of less than 0.001 in the tfasty
output. Two genomic regions of approximately 900 bp and 570 bp with
greater than 33% identity to the query sequence at the amino acid
level were chosen for further study. The gene models for the
putative lipase genes were predicted based on homology to the
Thermomyces lanuginosus lipase as well as conserved sequences
present at the 5' and 3' ends of fungal introns.
Example 2
Aspergillus fumigatus Genomic DNA Extraction
[0338] Aspergillus fumigatus 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 (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 mg/liter and the lysate
was incubated at 37.degree. C. for 30 minutes. Cellular debris was
removed by centrifugation, and DNA was isolated using a QIAGEN Maxi
500 column (QIAGEN Inc., Valencia, 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., Valencia, Calif.).
DNA was precipitated in isopropanol, washed in 70% ethanol, and
recovered by centrifugation. The DNA was resuspended in TE
buffer.
Example 3
Cloning of an Aspergillus fumigatus Lipase 1 Gene
[0339] Two synthetic oligonucleotide primers shown below were
designed based on the predicted start and stop codons of the open
reading frame to PCR amplify an Aspergillus fumigatus lipase 1 gene
from the genomic DNA prepared in Example 2.
TABLE-US-00001 Forward primer: (SEQ ID NO: 17)
5'-GAGACGCATGCTTCACAAGTATAG-3' Reverse primer: (SEQ ID NO: 18)
5'-GTCACCTCTAGTTAATTAATCAGATTATCTTGC-3'
Bold Letters Represent Coding Sequence.
[0340] The fragment of interest was amplified by PCR using the
Expand High Fidelity PCR System (Roche Diagnostics, Mannheim,
Germany). One .mu.M of each of the primers above were used in a PCR
reaction containing 20 ng of Aspergillus fumigatus genomic DNA,
1.times.PCR buffer (Roche Diagnostics, Mannheim, Germany) with 1.5
mM MgCl.sub.2, 1 .mu.l of a dATP, dTTP, dGTP, and dCTP mix (10 mM
each), and 0.75 .mu.l of DNA polymerase mix (3.5 U/.mu.l; Roche
Diagnostics, Mannheim, Germany) in a final volume of 50 .mu.l. To
amplify the fragment, an Eppendorf Mastercycler Thermocycler
(Hamburg, Germany) was programmed for 1 cycle at 94.degree. C. for
2 minutes; 10 cycles each at 94.degree. C. for 15 seconds,
60.degree. C. for 30 seconds, and 72.degree. C. for 1.25 minutes;
15 cycles each at 94.degree. C. for 15 seconds, 60.degree. C. for
30 seconds, and 72.degree. C. for 1.25 minutes plus a 5 second
elongation at each successive cycle; 1 cycle at 72.degree. C. for 7
minutes; and a 10.degree. C. hold.
[0341] The reaction product was visualized on a 0.8% agarose gel
using 44 mM Tris Base, 44 mM boric acid, 0.5 mM EDTA (TBE) buffer
and a 1.3 kb product band was purified using a QIAquick PCR
Purification Kit (QIAGEN Inc., Valencia, Calif.) according to the
manufacturer's instructions.
[0342] The PCR fragment and pCR2.1-TOPO vector (Invitrogen,
Carlsbad, Calif.) were ligated using conditions specified by the
manufacturer to produce pSMO224 (FIG. 1). Two p of the reaction was
used to transform E. coli TOP10 One Shot competent cells
(Invitrogen, Carlsbad, Calif.) according to the manufacturer's
instructions. A 2 .mu.l volume of the ligation mixture was added to
the E. coli cells and incubated on ice for 20 minutes.
Subsequently, the cells were heat shocked for 30 seconds at
42.degree. C., and then placed on ice for 2 minutes. A 250 .mu.l
volume of SOC medium was added to the cells and the mixture was
incubated for 1 hour at 37.degree. C. and 250 rpm. After the
incubation the colonies were spread on 2X YT plates supplemented
with 100 .mu.g of ampicillin per ml and incubated at 37.degree. C.
overnight for selection of the plasmid. Six colonies that grew on
the plates were picked with a sterile toothpick and grown overnight
at 37.degree. C., 250 rpm in a 15 ml Falcon tube containing 3 ml of
LB medium supplemented with 100 .mu.g of ampicillin per ml. An E.
coli transformant containing a plasmid designated pSMO224 was
detected by restriction digestion and plasmid DNA was prepared
using a BioRobot 9600 (QIAGEN Inc., Valencia, Calif.).
[0343] E. coli TOP10 One Shot cells containing pSMO224 were
deposited with the Agricultural Research Service Patent Culture
Collection, Northern Regional Research Center, 1815 University
Street, Peoria, Ill., 61604, as NRRL B-30774, with a deposit date
of Sep. 16, 2004.
Example 4
Characterization of the Aspergillus fumigatus Genomic Sequence
Encoding Lipase 1
[0344] DNA sequencing of the Aspergillus fumigatus lipase 1 gene
from pSMO224 was performed with an Applied Biosystems Model 377 XL
DNA Sequencer (Perkin-Elmer/Applied Biosystems, Inc., Foster City,
Calif.) using dye-terminator chemistry (Giesecke et al., 1992,
Journal of Virology Methods 38: 47-60) and primer walking strategy.
Nucleotide sequence data were scrutinized for quality and all
sequences were compared to each other with assistance of
PHRED/PHRAP software (University of Washington, Seattle,
Wash.).
[0345] Gene models for the sequence were constructed based on the
tfasty output and alignment with a homologous lipase gene from
Thermomyces lanuginosus. The nucleotide sequence (SEQ ID NO: 1) and
deduced amino acid sequence (SEQ ID NO: 2) are shown in FIG. 2. The
genomic fragment encodes a polypeptide of 396 amino acids,
interrupted by 1 intron of 68 bp. The % G+C content of the gene is
52.6% and the mature protein coding region is 52.9%. Using the
SignalP software program (Nielsen et al., 1997, Protein Engineering
10: 1-6), a signal peptide of 24 residues was predicted. The
predicted mature protein contains 373 amino acids with a molecular
mass of 41.5 kDa.
[0346] A comparative alignment of lipase sequences was made
employing the Clustal W method (Higgins, 1989, supra) using
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 lipase 1 gene
shares 23% identity to the deduced amino acid sequence of a
Thermomyces lanuginosus lipase gene (accession number O59952).
Example 5
Construction of pAlLo1 Expression Vector
[0347] Expression vector pAlLo1 was constructed by modifying pBANe6
(U.S. Pat. No. 6,461,837), which comprises a hybrid of the
promoters from the genes for Aspergillus niger neutral
alpha-amylase and Aspergillus oryzae triose phosphate isomerase
(NA2-tpi promoter), Aspergillus niger amyloglucosidase terminator
sequence (AMG terminator), and Aspergillus nidulans acetamidase
gene (amdS). Modification of pBANe6 was performed by first
eliminating three Nco I restriction sites at positions 2051, 2722,
and 3397 bp from the amd S 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-00002 AMDS3NcoMut (2050): (SEQ ID NO: 19)
5'-GTGCCCCATGATACGCCTCCGG-3' AMDS2NcoMut (2721): (SEQ ID NO: 20)
5'-GAGTCGTATTTCCAAGGCTCCTGACC-3' AMDS1NcoMut (3396): (SEQ ID NO:
21) 5'-GGAGGCCATGAAGTGGACCAACGG-3'
[0348] 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:
TABLE-US-00003 Upper Primer to mutagenize the AMG terminator
sequence: (SEQ ID NO: 22)
5'-CACCGTGAAAGCCATGCTCTTTCCTTCGTGTAGAAGACCAGACAG- 3' Lower Primer
to mutagenize the AMG terminator sequence: (SEQ ID NO: 23)
5'-CTGGTCTTCTACACGAAGGAAAGAGCATGGCTTTCACGGTGTCTG- 3'
[0349] 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
pAlLo1 (FIG. 3).
TABLE-US-00004 Upper Primer to mutagenize the NA2-tpi promoter:
(SEQ ID NO: 24) 5'-CTATATACACAACTGGATTTACCATGGGCCCGCGGCCGCAGATC-3'
Lower Primer to mutagenize the NA2-tpi promoter: (SEQ ID NO: 25)
5'-GATCTGCGGCCGCGGGCCCATGGTAAATCCAGTTGTGTATATAG-3'
Example 6
Construction of pBM120a Expression Vector
[0350] Plasmid pBM120a was constructed to obtain a plasmid
containing the double NA2 promoter (NA2-NA2-tpi) for driving gene
expression in Aspergillus species, and containing the ampicillin
resistance gene for selection in E E. coli.
[0351] Primers were designed to PCR amplify the double NA2 promoter
from pJaL721 (WO 03/008575). Restriction enzyme sites Sal I and Nco
I (underlined) were added for cloning the double promoter into the
Aspergillus expression plasmid pAlLo1.
TABLE-US-00005 (SEQ ID NO: 26) 5'-GTCGACATGGTGTTTTGATCATTTTA-3'
(SEQ ID NO: 27) 5'-CCATGGCCAGTTGTGTATATAGAGGA-3'
[0352] The fragment of interest was amplified by PCR using the
Expand High Fidelity PCR System. The PCR amplification reaction
mixture contained 1 .mu.l of 0.09 .mu.g of pJaL721 per .mu.l, 1
.mu.l of each of the primers (50 pmol/.mu.l), 5 .mu.l of
10.times.PCR buffer with 15 mM MgCl.sub.2, 1 .mu.l of a dATP, dTTP,
dGTP, and dCTP mix (10 mM each), 37.25 .mu.l of water, and 0.75
.mu.l of DNA polymerase mix (3.5 U/.mu.l). To amplify the fragment,
an Eppendorf Mastercycler thermocycler was programmed for 1 cycle
at 94.degree. C. for 2 minutes; 10 cycles each at 94.degree. C. for
15 seconds, 55.degree. C. for 30 seconds, and 72.degree. C. for
1.25 minutes; 15 cycles each at 94.degree. C. for 15 seconds,
55.degree. C. for 30 seconds, and 72.degree. C. for 1.25 minutes
plus a 5 second elongation at each successive cycle; 1 cycle at
72.degree. C. for 7 minutes; and a 10.degree. C. hold. Ten
microliters of this PCR reaction was mixed with 1 .mu.l of
10.times.DNA loading dye (25% glycerol, 10 mM Tris pH 7.0, 10 mM
EDTA, 0.025% bromophenol blue, 0.025% xylene cyanol) and run on a
1.0% (w/v) agarose gel using TBE buffer. The 1128 bp PCR product
was observed with UV light on a Nucleotech gel visualization system
(Nucleotech, San Mateo, Calif.). The PCR product was directly
ligated into pPC2.1-TOPO according to the manufacturer's
instructions. A 1 .mu.l volume of fresh PCR product, 3 .mu.l of
double-distilled water, and 1 .mu.l of the TOPO cloning vector were
mixed with a pipette and incubated at room temperature for 5
minutes.
[0353] After the incubation, 2 .mu.l of the mixture was used to
transform OneShot competent E. coli cells. A 2 .mu.l volume of the
ligation mixture was added to the E. coli cells and incubated on
ice for 5 minutes. Subsequently, the cells were heat shocked for 30
seconds at 42.degree. C., and then placed on ice for 2 minutes. A
250 .mu.l volume of SOC medium was added to the cells and the
mixture was incubated for 1 hour at 37.degree. C. and 250 rpm.
After the incubation the colonies were spread on 2X YT plates
supplemented with 100 .mu.g of ampicillin per ml and incubated at
37.degree. C. overnight for selection of the plasmid. Eight
colonies that grew on the plates were picked with a sterile
toothpick and grown overnight at 37.degree. C., 250 rpm in a 15 ml
Falcon tube containing 3 ml of LB medium supplemented with 100
.mu.g of ampicillin per ml. The plasmids were isolated using a
QIAGEN BioRobot 9600.
[0354] Four .mu.l volumes of the resulting plasmid minipreps were
digested with Eco RI. The digestion reactions were analyzed by
agarose gel chromatography and UV analysis as previously described
for the PCR reaction. Isolated plasmids containing an insert were
sequenced using 1 .mu.l of plasmid template, 1.6 ng of M13 primer
(forward or reverse) (MWG Biotech; High Point; NC), and water to 6
.mu.l. DNA sequencing was performed with an Applied Biosystems
Model 377 Sequencer XL (Applied Biosystems, Inc., Foster City,
Calif.) using dye-terminator chemistry. The resulting plasmid was
designated pBM121b (FIG. 4).
[0355] A 5 .mu.l volume of pBM121b was digested with Sal I and Nco
I. The digestion reactions were analyzed by agarose gel
electrophoresis as described above, and ligated to the vector
pAlLo1, which had been previously digested with Sal I and Nco I.
The resulting expression plasmid was designated pBM120a (FIG.
5).
Example 7
Construction of an Aspergillus oryzae Expression Vector Expressing
Aspergillus fumigatus Lipase 1 Gene
[0356] Two synthetic oligonucleotide primers shown below were
designed to PCR amplify the Aspergillus fumigatus lipase 1 gene
from the genomic DNA prepared in Example 2.
TABLE-US-00006 Forward primer: (SEQ ID NO: 28)
5'-TACACAACTGGCCATGCTTCACAAGTATAG-3' Reverse primer: (SEQ ID NO:
29) 5'-GTCACCTCTAGTTAATTAATCAGATTATCTTGC-3'
[0357] Bold letters represent coding sequence. The remaining
sequence is homologous to the insertion sites of pBM120a.
[0358] The fragment of interest was amplified by PCR using the
Expand High Fidelity PCR System. One .mu.M of each of the primers
above were used in a PCR reaction containing 20 ng of Aspergillus
fumigatus genomic DNA, 1.times.PCR buffer (Roche Diagnostics,
Mannheim, Germany) with 1.5 mM MgCl.sub.2, 1 .mu.l of a dATP, dTTP,
dGTP, and dCTP mix (10 mM each), and 0.75 .mu.l of DNA polymerase
mix (3.5 U/.mu.l; Roche Diagnostics, Mannheim, Germany) in a final
volume of 50 .mu.l. To amplify the fragment, an Eppendorf
Mastercycler thermocycler was programmed for 1 cycle at 94.degree.
C. for 2 minutes; 10 cycles each at 94.degree. C. for 15 seconds,
60.degree. C. for 30 seconds, and 72.degree. C. for 1.25 minutes;
15 cycles each at 94.degree. C. for 15 seconds, 60.degree. C. for
30 seconds, and 72.degree. C. for 1.25 minutes plus a 5 second
elongation at each successive cycle; 1 cycle at 72.degree. C. for 7
minutes; and a 10.degree. C. hold.
[0359] The reaction product was visualized on a 0.8% agarose gel
using TBE buffer and a 1.3 kb product band was purified using a
QIAquick PCR Purification Kit according to the manufacturer's
instructions.
[0360] The 1.3 kb PCR fragment containing the Aspergillus fumigatus
lipase 1 gene was cloned into pBM120a using an InFusion Cloning Kit
(BD Biosciences, Palo Alto, Calif.) where the vector was digested
with Nco I and Pac I. The digested vector was purified by gel
electrophoresis using a 0.7% agarose gel with TBE buffer, and the
PCR fragment was extracted using a QIAquick Gel Extraction Kit
(QIAGEN Inc., Valencia, Calif.) and purified using a QIAquick PCR
Purification Kit. The gene fragment and the digested vector were
ligated together in a reaction resulting in the expression plasmid
pSMO218 (FIG. 6). 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.),
100 ng of pBM120a digested with Nco I and Pac I, and 50 ng of the
Aspergillus fumigatus lipase 1 gene purified PCR product. The
reaction was incubated at room temperature for 30 minutes. Two
.mu.l of the reaction was used to transform E. coli SoloPack.RTM.
Gold supercompetent cells (Stratagene, La Jolla, Calif.) according
to the manufacturer's instructions. One .mu.l of
.beta.-mercaptoethanol was added to the competent cells, and
incubated on ice for 10 minutes. A 2 .mu.l volume of the ligation
mixture was then added to the E. coli cells and incubated on ice
for 30 minutes. Subsequently, the cells were heat shocked for 60
seconds at 54.degree. C., and then placed on ice for 2 minutes. A
150 .mu.l volume of NZY.sup.+ medium at 42.degree. C. was added to
the cells and the mixture was incubated for 1 hour at 37.degree. C.
and 250 rpm. After the incubation the colonies were spread on 2X YT
plates supplemented with 100 .mu.g of ampicillin per ml and
incubated at 37.degree. C. overnight for selection of the plasmid.
Six colonies that grew on the plates were picked with a sterile
toothpick and grown overnight at 37.degree. C., 250 rpm in a 15 ml
Falcon tube containing 3 ml of LB medium supplemented with 100
.mu.g of ampicillin per ml. An E. coli transformant containing the
pSMO218 plasmid was detected by restriction digestion and plasmid
DNA was prepared using a QIAGEN BioRobot 9600.
Example 8
Expression of the Aspergillus fumigatus Lipase 1 Gene in
Aspergillus oryzae BECh2
[0361] Aspergillus oryzae BECh2 (.DELTA.alp, .DELTA.amy, CPA-, KA-,
.DELTA.np1) protoplasts were prepared according to the method of
Christensen et al., 1988, Bio/Technology 6: 1419-1422. Five .mu.g
of pSMO218 was used to transform Aspergillus oryzae BECh2.
[0362] The transformation of Aspergillus oryzae BECh2 with pSMO218
yielded 25 transformants. The transformants were isolated to
individual Cove plates. Confluent Cove plates of 25 transformants
were washed with 4 ml of 0.01% Tween 20. Two hundred .mu.l of spore
suspension was inoculated separately into 25 ml of MY25 medium in
125 ml plastic shake flasks and incubated at 34.degree. C., 250
rpm. Three and five days after incubation, culture supernatants
were removed for lipase assay and SDS-PAGE analysis.
[0363] Lipase activity was determined as follows: 200 .mu.l of
substrate (20 ml of 100 mM MOPS pH 7.5, 4.95 ml DMSO, and 50 .mu.l
of p-nitrophenyl butyrate) was added to 20 .mu.l of diluted enzyme
sample. The samples were diluted accordingly in 100 mM MOPS pH 7.5,
4 mM CaCl.sub.2, and 0.01% Triton X-100. The absorbance at 405 nm
was obtained after 15 minutes of incubation at room temperature
(25.degree. C.) in a 96-well microtiter plate using a SpectraMAX
250 microplate reader (Molecular Devices Corp., Sunnyvale, Calif.).
LIPOLASE.TM. (Thermomyces lanuginosus lipase; Novozymes A/S,
Bagsvaerd, Denmark)) can be used for generating a standard curve to
determine lipase units (LUs).
[0364] The lipase assay results indicated that at 3 days several of
the transformants produced lipase activity above that of the
untransformed control. SDS-PAGE analysis (BioRad Criterion 10-20%
SDS-PAGE) of 10 .mu.l of the supernatants showed a band at
approximately 41 kDa.
Example 9
Determination of Substrate Specificity of Recombinant Aspergillus
fumigatus Lipase 1
[0365] The substrate specificity of Aspergillus fumigatus lipase 1
was determined using a panel screen composed of 4-nitrophenol (PNP)
lipase substrates.
[0366] A panel screen composed of a set of 12 assays utilizing
various 4-nitrophenol (PNP) lipase substrates was prepared as
described in the Table 1.
TABLE-US-00007 TABLE 1 Panel screen of PNP substrates and buffer
conditions 1 mM PNP- 50 mM 50 mM 50 mM PNP tagged Shorthand MOPS
CHES MOPS 10 mM Triton Conversion substrate designation pH 7.0 pH
9.5 pH 7.5 CaCl.sub.2 X-100 Factors 1 Palmitate 16:0 x x 1.2% 4.466
2 Palmitate 16:0 x x 1.2% 1.1 3 Palmitate 16:0 x x 1.2% 2.037 4
Palmitate 16:0 x x 0.2% 1.0 5 Palmitate 16:0 x x 0.2% 1.495 6
Decanoate 10:0 x x 1.2% 4.466 7 Decanoate 10:0 x x 1.2% 1.1 8
Decanoate 10:0 x x 1.2% 2.037 9 Decanoate 10:0 x x 0.2% 1.0 10
Valerate 5:0 x x 0.40% 1.630 11 Valerate 5:0 x x 0% 1.370 12
Butyrate 4:0 x x 0.40% 1.630
[0367] These assays were run in 384-well plates using 8 different
dilutions of each sample (7 .mu.l) to be evaluated and 80 .mu.l of
the substrates. The assays were incubated for up to 24 hours at
ambient temperature. Assays were read at 405 nm at time points of
approximately 1, 2, 3, 5, and 24 hours. The results were calculated
as OD/hour for each individual assay. In order to make an accurate
evaluation of the amount of PNP released, it was necessary to
mathematically normalize raw OD values by using a conversion
factor. The conversion factors were values, determined
experimentally, that were necessary to compensate for the fact that
PNP has lower OD readings at low pH and in the presences of
detergent (Triton X100) than at pH 9.5. The factor normalizes the
data to the OD reading that would have been obtained were it
possible to quench the reactions to yield maximal OD for each
condition while also stopping the reaction at that time point;
i.e., PNP-fatty acid substrates are not stable at high pH, the tag
comes off without lipase present at high pH, and the tagged
substrate is particularly unstable above pH 9 and for shorter chain
length fatty acid substrates.
[0368] In Table 1 the top two rows (1 and 2) were the assays used
for the pH ratio (9.5/7.0). Rows 3 and 10 were used for the "long
chain (Slu)/short chain (Lu); comparisons.
[0369] The results for the panel screen are shown in Table 2.
TABLE-US-00008 TABLE 2 Ratio of PNP-substrates according to Table
1, Example 9, where: P = Palmitate, D = Decanoate, V = Valerate and
B = Butyrate Long chain/Short chain at pH 7.5 P/V D/V P/B D/B
ALF004 1.888 0.887 2.556 1.201 LIPOLASE .TM. 1.213 2.398 2.220
4.387 pH Ratios; (-) = 0.2% Triton data 9.5P/7.0P 9.5D/7.0D
9.5P-/7.5P- ALF004 0.045 No Data Not Tested LIPOLASE .TM. 1.527
1.881 1.452 No or Low Triton (-) compared to maximum Triton (+)
V7.5 -/+ D9.5 +/- P7.5 +/- P9.5 +/- ALF004 Not Tested Not Tested
Not Tested Not Tested LIPOLASE .TM. 8.651 6.378 9.206 9.502 No Data
= not enough activity to give sufficient signal in one of the
assays in this test ALF004 (Aspergillus oryzae BeCH2 expressing
Aspergillus fumigatus lipase 1) test data consists of the average
of up to 2 independent screening assay events; LIPOLASE .TM. test
data consists of the average of up to 17 independent screening
assay events.
[0370] In comparing the panel screen results of the Thermomyces
lanuginosus lipase (LIPOLASE.TM.) and the ALF004 the following
observations were made:
[0371] 1. The ratio of activities on PNP-palmitate at pH 9.5 versus
pH 7 is 40-fold lower for ALF004 versus LIPOLASE.TM. suggesting
that the Aspergillus fumigatus lipase 1 has much lower activity at
pH 9.5 versus Lipolase.TM. or it has a much higher activity at pH
7.0 than LIPOLASE.TM. or a combination of these two.
[0372] 2. The ratios of P/V, D/V, and D/B are also quite different
for ALF004 versus LIPOLASE.TM. suggesting there is some acyl change
length specificity differences between the two lipases.
Example 10
Cloning of an Aspergillus fumigatus Lipase 2 Gene
[0373] Two synthetic oligonucleotide primers shown below were
designed based on the predicted start and stop codons of the open
reading frame to PCR amplify an Aspergillus fumigatus lipase 2 gene
from the genomic DNA prepared in Example 2.
TABLE-US-00009 Forward primer: (SEQ ID NO: 30)
5'-GAGACACATGTTTCACCCAG-3' Reverse primer: (SEQ ID NO: 31)
5'-GTCACCTCTAGTTAATTAATCAGTTAGTTGAGC-3'
Bold letters represent coding sequence.
[0374] The fragment was amplified by PCR using the Expand High
Fidelity PCR System as described in Example 3. The reaction product
was visualized on a 0.7% agarose gel using TBE buffer and a 1.2 kb
product band was purified using a QIAquick PCR Purification Kit
according to the manufacturer's instructions. The PCR product was
then cloned into pCR2.1-TOPO according to manufacturer's
instructions to produce pSMO223 (FIG. 7). Two .mu.l of the reaction
was used to transform E. coli TOP10 One Shot competent cells
according to the manufacturer's instructions. A 2 .mu.l volume of
the ligation mixture was added to the E. coli cells and incubated
on ice for 20 minutes. Subsequently, the cells were heat shocked
for 30 seconds at 42.degree. C., and then placed on ice for 2
minutes. A 250 .mu.l volume of SOC medium was added to the cells
and the mixture was incubated for 1 hour at 37.degree. C. and 250
rpm. After the incubation the colonies were spread on 2X YT plates
supplemented with 100 .mu.g of ampicillin per ml and incubated at
37.degree. C. overnight for selection of the plasmid. Six colonies
that grew on the plates were picked with a sterile toothpick and
grown overnight at 37.degree. C., 250 rpm in a 15 ml Falcon tube
containing 3 ml of LB medium supplemented with 100 .mu.g of
ampicillin per ml. An E. coli transformant containing pSMO223 was
detected by restriction digestion and plasmid DNA was prepared
using a QIAGEN BioRobot 9600.
[0375] E. coli TOP10 One Shot cells containing pSMO223 were
deposited with the Agricultural Research Service Patent Culture
Collection, Northern Regional Research Center, 1815 University
Street, Peoria, Ill., 61604, as NRRL B-30773, with a deposit date
of Sep. 16, 2004.
Example 11
Characterization of the Aspergillus fumigatus Genomic Sequence
Encoding Lipase 2
[0376] DNA sequencing of the Aspergillus fumigatus lipase 2 gene
from pSMO223 was performed with an Applied Biosystems Model 377 XL
DNA Sequencer using dye-terminator chemistry (Giesecke et al.,
1992, supra) and primer walking strategy. Nucleotide sequence data
were scrutinized for quality and sequences were compared to each
other with assistance of PHRED/PHRAP software (University of
Washington, Seattle, Wash.).
[0377] Gene models for the sequence were constructed based on the
tfasty output and alignment with a homologous lipase gene from
Thermomyces lanuginosus. The nucleotide sequence (SEQ ID NO: 3) and
deduced amino acid sequence (SEQ ID NO: 4) are shown in FIG. 8. The
genomic fragment encodes a polypeptide of 283 amino acids,
interrupted by 2 introns of 45 and 50 bp. The % G+C content of the
gene is 47.1% and the mature protein coding region is 47.2%. Using
the SignalP software program (Nielsen et al., 1997, supra), a
signal peptide of 24 residues was predicted. The predicted mature
protein contains 259 amino acids with a molecular mass of 28.9
kDa.
[0378] A comparative alignment of lipase sequences was made
employing the Clustal W method (Higgins, 1989, supra) 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 lipase 2 gene
shares 35% identity to the deduced amino acid sequence of a
Thermomyces lanuginosus lipase gene (accession number O59952).
Example 12
Identification of Lipase Genes in the Partial Genomic Sequence of
Magnaporthe grisea
[0379] A tfasty search (Pearson, W. R., 1999, supra) of the
Magnaporthe grisea partial genome sequence (Broad Institue MIT,
Boston, Mass.) was carried out using as query a lipase sequence
from Thermomyces lanuginosus (Accession No. O59952). Several genes
were identified as putative lipases based upon an E value of less
than 0.001 in the tfasty output. Three genomic regions of
approximately 930 bp, 750 bp, and 720 bp with greater than 30%
identity to the query sequence at the amino acid level were chosen
for further study. Gene models for the putative lipase genes were
predicted based on homology to the Thermomyces lanuginosus lipase
as well as conserved sequences present at the 5' and 3' ends of
fungal introns.
Example 13
Magnaporthe grisea Genomic DNA Extraction
[0380] Four hundred .mu.l of Magnaporthe grisea (FGSC 8958, Fungal
Genetics Stock Center) spores were grown in 50 ml of CM medium in a
baffled shake flask at 25.degree. C. and 250 rpm for 4 days.
Genomic DNA was then extracted from the mycelia using a DNeasy
Plant Mini Kit (QIAGEN, Valencia, Calif.) according to
manufacturer's instructions.
Example 14
Cloning of Magnaporthe grisea Lipase 1 Gene
[0381] Two synthetic oligonucleotide primers shown below were
designed based on the genomic sequence outside of the predicted
start and stop codons of the open reading frame to PCR amplify a
Magnaporthe grisea lipase 1 gene from the genomic DNA prepared in
Example 13.
TABLE-US-00010 Forward primer: (SEQ ID NO: 32)
5'-CCTTGCCCACGCCTTTGGTTC-3' Reverse primer: (SEQ ID NO: 33)
5'-CTCATAGCAGCAGGCGAAGCC-3'
Both primers represent sequence outside of coding sequence.
[0382] Fifty picomoles of each of the primers above were used in a
PCR reaction containing 300 ng of Magnaporthe grisea genomic DNA,
1.times. Herculase reaction buffer (Stratagene, La Jolla, Calif.),
1 .mu.l of a dATP, dTTP, dGTP, and dCTP mix (10 mM each), and 2.5
units of Herculase Hotstart DNA polymerase (Stratagene, La Jolla,
Calif.) in a final volume of 50 .mu.l. The amplification was
conducted in an Eppendorf Mastercycler thermocycler programmed for
one cycle at 98.degree. C. for 2 minutes; 10 cycles each at
98.degree. C. for 15 seconds, 62.degree. C. for 30 seconds, and
72.degree. C. for 1 minute and 20 seconds; 15 cycles each at
98.degree. C. for 15 seconds, 62.degree. C. for 30 seconds, and
72.degree. C. for 1 minutes and 20 seconds plus a 5 second
elongation at each successive cycle; and 1 cycle at 72.degree. C.
for 7 minutes. The heat block then went to a 10.degree. C. soak
cycle.
[0383] The reaction products were run on a 1.0% agarose gel using
40 mM Tris base-20 mM sodium acetate-1 mM disodium EDTA (TAE)
buffer where a 1.2 kb product band was detected. PCR products were
purified using a QIAquick PCR Purification Kit according to the
manufacturer's instructions.
[0384] Two synthetic oligonucleotide primers shown below were
designed to PCR amplify a Magnaporthe grisea gene encoding a lipase
1 gene from the PCR product described above.
TABLE-US-00011 Forward primer: (SEQ ID NO: 34)
5'-ACACAACTGGCCATGAAGGTCTCGTTCGTGTCATCG-3' Reverse primer: (SEQ ID
NO: 35) 5'-AGTCACCTCTAGTTATCAGTAGCAAGCGCTAATGG-3'
Bold letters represent coding sequence. The remaining sequence is
homologous to insertion sites of pBM120a.
[0385] Fifty picomoles of each of the primers above were used in a
PCR reaction containing 2 .mu.l of the 1.2 kb PCR product described
above, 1.times. Herculase reaction buffer, 1 ptl of a dATP, dTTP,
dGTP, and dCTP mix (10 mM each), 2.5 units of Herculase Hotstart
DNA polymerase in a final volume of 50 .mu.l. The amplification was
conducted in an Eppendorf Mastercycler thermocycler programmed for
1 cycle at 98.degree. C. for 2 minutes; 10 cycles each at
98.degree. C. for 15 seconds, 62.degree. C. for 30 seconds, and
72.degree. C. for 1 minute and 20 seconds; 15 cycles each at
98.degree. C. for 15 seconds, 62.degree. C. for 30 seconds, and
72.degree. C. for 1 minutes and 20 seconds plus a 5 second
elongation at each successive cycle; and 1 cycle at 72.degree. C.
for 7 minutes. The heat block then went to a 10.degree. C. soak
cycle.
[0386] The reaction products were run on a 1.0% agarose gel using
TAE buffer where a 1.1 kb product band was detected. The 1.1 kb PCR
product was purified using a QIAquick PCR Purification Kit
according to the manufacturer's instructions.
[0387] The 1.1 kb fragment was cloned into the pCR2.1-TOPO vector.
The gene fragment was purified using a QIAquick PCR Purification
Kit according to the manufacturer's instructions. The fragment and
pCR2.1-TOPO vector were ligated by using conditions specified by
the manufacturer resulting in plasmid pHyGe026 (FIG. 9). Two .mu.l
of the reaction was used to transform E. coli One Shot competent
cell. An E. coli transformant containing the plasmid pHyGe026 was
detected by restriction digestion and plasmid DNA was prepared
using a BioRobot 9600.
[0388] E. coli TOP10 One Shot cells containing pHyGe026 were
deposited with the Agricultural Research Service Patent Culture
Collection, Northern Regional Research Center, 1815 University
Street, Peoria, Ill., 61604, as NRRL B-30772, with a deposit date
of Sep. 13, 2004.
Example 15
Characterization of the Magnaporthe grisea Genomic Sequence
Encoding a Lipase 1 Gene
[0389] DNA sequencing of the Magnaporthe grisea lipase 1 gene from
pHyGe026 was performed with an Applied Biosystems Model 377 XL DNA
Sequencer using dye-terminator chemistry (Giesecke et al., 1992,
supra) and primer walking strategy. Nucleotide sequence data were
scrutinized for quality and sequences were compared to each other
with assistance of PHRED/PHRAP software (University of Washington,
Seattle, Wash.).
[0390] Gene models for the lipase gene were predicted based on
homology to a Thermomyces lanuginosa lipase (Accession No. O59952)
as well as conserved sequences present at the 5' and 3' ends of
fungal introns. The nucleotide sequence (SEQ ID NO: 5) and deduced
amino acid sequence (SEQ ID NO: 6) are shown in FIGS. 10A and 10B.
The genomic fragment encodes a polypeptide of 318 amino acids,
interrupted by 2 introns of 54 and 77 bp. The % G+C content of the
gene is 60.9% and the mature polypeptide coding sequence
(nucleotides 58 to 1088 of SEQ ID NO: 6) is 60.5%. Using the
SignalP software program (Nielsen et al., 1997, supra), a signal
peptide of 19 residues was predicted. The predicted mature protein
contains 299 amino acids with a molecular mass of 34 kDa.
[0391] A comparative alignment of lipase sequences was determined
using the Clustal W method (Higgins, 1989, supra) 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 Magnaporthe lipase 1 gene shares 31.3%
identity to the deduced amino acid sequence of a Thermomyces
lanuginosus lipase gene (Accession No. O59952).
Example 16
Construction of an Aspergillus oryzae Expression Vector for the
Magnaporthe grisea Lipase 1 Gene
[0392] The PCR fragment containing the Magnaporthe grisea lipase 1
gene was cloned into pBM120a using an InFusion Cloning Kit. The
vector was digested with Nco I and Pac I. The digested vector was
purified by gel electrophoresis and extracted using a QIAquick Gel
Extraction Kit. The gene fragment and digested vector were ligated
together in a reaction resulting in the expression plasmid pHyGe010
(FIG. 11) in which transcription of the lipase gene was under the
control of the tandem NA2-tpi promoter. The ligation reaction (20
.mu.l) was composed of 1.times. InFusion Buffer, 1.times.BSA, 1
.mu.l of Infusion enzyme (diluted 1:10), 50 ng of pBM120a digested
with Nco I and Pac I, and 30 ng of the Magnaporthe grisea lipase
purified PCR product. The reaction was incubated at room
temperature for 30 minutes. Two .mu.l of the reaction was used to
transform E. coli SoloPack.RTM. Gold supercompetent cells according
to manufacturer's instructions. An E. coli transformant containing
pHyGe010 was detected by estriction digestion and plasmid DNA was
prepared using a QIAGEN BioRobot 9600.
Example 17
Expression of the Magnaporthe grisea Lipase 1 Gene in Aspergillus
oryzae BECh2
[0393] Aspergillus oryzae BECh2 protoplasts were prepared according
to the method of Christensen et al., 1988, supra. Sixty .mu.g of
pHyGe010 was used to transform Aspergillus oryzae BECh2.
[0394] The transformation of Aspergillus oryzae BECh2 with pHyGe010
yielded 14 transformants. Each transformant was transferred to
individual Cove plates. Confluent Cove plates of the 14
transformants were washed with 4 ml of 0.01% Tween 20. Two hundred
.mu.l of spore suspension was inoculated separately into 25 ml of
MY25 medium in 125 ml plastic shake flasks and incubated at
34.degree. C., 250 rpm. Three, four, and five days after
incubation, culture supernatants were removed for lipase assay and
SDS-PAGE analysis.
[0395] Lipase activity was determined as described in Example
8.
[0396] The lipase assay results indicated that at 3, 4, and 5 days,
several transformants produced lipase activity above that of the
untransformed control.
[0397] SDS-PAGE (BioRad Criterion 8-16% SDS-PAGE) analysis of 25
.mu.l of the supernatants showed a major band at approximately 37
kDa.
Example 18
Determination of Substrate Specificity of Recombinant Magnaporthe
grisea Lipase 1
[0398] The substrate specificity of Magnaporthe grisea lipase 1 was
determined using a panel screen composed of 4-nitrophenol (PNP)
lipase substrates as described in Example 9.
[0399] The results for the panel screen are shown in Table 3.
TABLE-US-00012 TABLE 3 Ratio of PNP-substrates according to Table
1, Example 9, where: P = Palmitate, D = Decanoate, V = Valerate and
B = Butyrate Long chain/Short chain at pH 7.5 P/V D/V P/B D/B
Hyge027 1.142 0.747 1.657 1.103 LIPOLASE .TM. 1.213 2.398 2.220
4.387 pH Ratios; (-) = 0.2% Triton data 9.5P/7.0P 9.5D/7.0D
9.5P-/7.5P- Hyge027 No Data No Data No Data LIPOLASE .TM. 1.527
1.881 1.452 No or Low Triton (-) compared to maximum Triton (+)
V7.5 -/+ D9.5 +/- P7.5 +/- P9.5 +/- Hyge027 1.101 No Data 5.123 No
Data LIPOLASE .TM. 8.651 6.378 9.206 9.502 No Data = not enough
activity to give sufficient signal in one of the assays in this
test Hyge027 (Aspergillus oryzae BECh2 expressing Magnaporthe
grisea lipase 1) test data consists of the average of up to 4
independent screening assay events; LIPOLASE .TM. (Thermomyces
lanuginosus lipase) test data consists of the average of up to 17
independent screening assay events.
[0400] In comparing the panel screen results of the Thermomyces
lanuginosus lipase (LIPOLASE.TM.) and Hyge027, the following
observations were made:
[0401] (1) There were significant differences in the ratios of
activities on valerate or palmitate at pH 7.5 (V7.5-/+ and P7.5+/-)
in the presence or absence of triton for Hyge027 versus
LIPOLASE.TM..
[0402] (2) The ratios of activities on decanoate versus valerate
(D/V), palmitate versus butyrate (P/B), and decanoate versus
butyrate (D/B) were significantly different for Hyge027 versus
LIPOLASE.TM. suggesting there is some acyl change length
specificity differences between the two lipases.
Example 19
Cloning of Magnaporthe grisea Lipase 2 Gene
[0403] Two synthetic oligonucleotide primers shown below were
designed based on the predicted start and stop codons of the open
reading frame to PCR amplify a Magnaporthe grisea lipase 2 gene
from the genomic DNA prepared in Example 13.
TABLE-US-00013 Forward primer: (SEQ ID NO: 36)
5'-CCATGGCCATGATGAGGTTCCCCAGCGTGCTCA-3' Reverse primer: (SEQ ID NO:
37) 5'-TTTAATTAAGCCACGGTCTTGTTGGCTTC-3'
Bold letters represent coding sequence. The remaining sequence is
homologous to the enzyme restriction insertion sites of
pBM120a.
[0404] The gene was amplified by PCR using the Herculase.TM.
Hotstart DNA polymerase (Stratagene, La Jolla, Calif.). Fifty ng of
each of the primers above were used in a PCR reaction containing
300 ng of Magnaporthe grisea genomic DNA. The PCR amplification
reaction mixture also contained 1.times. Herculase PCR buffer, 1
.mu.l of a dATP, dTTP, dGTP, and dCTP mix (10 mM each), and 2.5
units Herculase.TM. DNA polymerase mix in a final volume of 50
.mu.l. To amplify the fragment, a Peltien thermal cycler (MJ
Research Inc., Watertown, Mass.) was programmed for 1 cycle at
94.degree. C. for 2 minutes; 30 cycles each at 94.degree. C. for 1
minute, 50.degree. C. for 1 minute, 72.degree. C. 1 minute; 1 cycle
at 72.degree. C. for 10 minutes; and a 4.degree. C. hold.
[0405] The PCR products were size fractionated on a 1% agarose gel
using TAE buffer and a 1 kb product band was purified using a
QIAquick Gel Purification Kit according to the manufacturer's
instructions. The PCR product was then cloned into pCR2.1-TOPO
according to manufacturer's instructions resulting in plasmid
pCrAm138 (FIG. 12). Two .mu.l of the ligation reaction was used to
transform E. coli TOP10 One Shot competent cells. An E. coli
transformant containing the plasmid pCrAm138 was detected by
restriction digestion and plasmid DNA was prepared using a QIAGEN
BioRobot 9600.
[0406] E. coli TOP10 One Shot cells containing pCrAm138 were
deposited with the Agricultural Research Service Patent Culture
Collection, Northern Regional Research Center, 1815 University
Street, Peoria, Ill., 61604, as NRRL B-30781, with a deposit date
of Oct. 12, 2004.
Example 20
Characterization of the Magnaporthe grisea Genomic Sequence
Encoding Lipase 2
[0407] DNA sequencing of the Magnoporthe grisea lipase 2 gene
contained in pCrAm138 was performed with a Perkin-Elmer Applied
Biosystems Model 377 XL Automated DNA Sequencer using
dye-terminator chemistry (Giesecke et al., 1992, supra) and primer
walking strategy. Nucleotide sequence data were scrutinized for
quality and all sequences were compared to each other with
assistance of PHRED/PHRAP software (University of Washington,
Seattle, Wash.).
[0408] Gene models for the lipase gene were predicted based on
homology to a Thermomyces lanuginosus lipase (Accession No. O59952)
as well as conserved sequences present at the 5' and 3' ends of
fungal introns. The nucleotide sequence (SEQ ID NO: 7) and deduced
amino acid sequence (SEQ ID NO: 8) are shown in FIG. 13. The
genomic fragment encodes a polypeptide of 348 amino acids with a
molecular weight of 37.7 kDa. The % G+C content of the gene is 64%
and the mature polypeptide coding sequence (nucleotides 55 to 1044
of SEQ ID NO: 7) is 64%. Using the SignalP software program
(Nielsen et al., 1997, supra), a signal peptide of 18 residues was
predicted. The predicted mature protein contains 330 amino acids
with a molecular mass of 35.9 kDa.
[0409] A comparative alignment of lipase sequences was determined
using the Clustal W method (Higgins, 1989, supra) 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 Magnaporthe lipase 2 gene shares 37.8%
identity to the deduced amino acid sequence of a Thermomyces
lanuginosus lipase gene (Accession No. O59952).
Example 21
Cloning of Magnaporthe grisea Lipase 3 Gene
[0410] Two synthetic oligonucleotide primers shown below were
designed based on the predicted start and stop codons of the open
reading frame to PCR amplify a Magnaporthe grisea lipase 3 gene
from the genomic DNA described in Example 13.
TABLE-US-00014 Forward primer: (SEQ ID NO: 38)
5'-ACACAACTGGCCATGTTGTGGCGTCGGGCGGGTGGCCTCT-3' Reverse primer: (SEQ
ID NO: 39) 5'-AGTCACCTCTAGTTAATTAATTAGAGCTCATCCTGGCCAGGAGCCA
C-3'
Bold letters represent coding sequence. The remaining sequence is
homologous to the insertion sites of pBM120a.
[0411] The fragment of interest was amplified by PCR using the
Expand High Fidelity PCR System. Fifty picomoles of each of the
primers above were used in a PCR reaction containing 300 ng of
Magnaporthe grisea genomic DNA. The PCR amplification reaction
mixture also contained 1.times.PCR buffer with 1.5 mM MgCl.sub.2, 1
.mu.l of a dATP, dTTP, dGTP, and dCTP mix (10 mM each), and 0.75
.mu.l of DNA polymerase mix (3.5 U/.mu.l) in a final volume of 50
.mu.l. An Eppendorf Mastercycler thermocycler was used to amplify
the fragment programmed for 1 cycle at 94.degree. C. for 2 minutes;
10 cycles each at 94.degree. C. for 15 seconds, 62.degree. C. for
30 seconds, and 72.degree. C. for 2 minutes; 15 cycles each at
94.degree. C. for 15 seconds, 62.degree. C. for 30 seconds, and
72.degree. C. for 2 minutes plus a 5 second elongation at each
successive cycle; 1 cycle at 72.degree. C. for 7 minutes; and a
10.degree. C. hold.
[0412] The 1.2 kb reaction product was visualized on a 1.0% agarose
gel using TBE buffer. Four microliters of the product was then
cloned into pCR2.1-TOPO according to manufacturer's instructions to
produce pBM135a. Two .mu.l of the reaction was used to transform E.
coli TOP10 One Shot competent cells. An E. coli transformant
containing pBM135a was detected by restriction digestion and
plasmid DNA was prepared using a QIAGEN BioRobot 9600.
[0413] DNA sequencing of the Magnaporthe grisea lipase 3 gene from
pBM135a was performed with a Perkin-Elmer Applied Biosystems Model
377 XL Automated DNA Sequencer using dye-terminator chemistry
(Giesecke et al., 1992, supra) and primer walking strategy.
Nucleotide sequence data were scrutinized for quality and all
sequences were analyzed with assistance of ContigExpress software
(Informax, Inc., Bethesda, Md.). Sequencing results indicated the
absence of the ATG start codon in pBM135a. A second forward primer,
with sequence identical to oligo number 998205 described above was
ordered. The new oligo numbered 998524 was used in a PCR reaction
using pBM135a as DNA template to re-amplify the gene of
interest.
[0414] Using the Expand High Fidelity PCR System, 50 picomoles of
primers 998524 and 998232 were used in a PCR reaction containing 1
.mu.l of a 1:10 dilution pBM135a mini DNA. The PCR amplification
reaction mixture also contained 1.times.PCR buffer with 1.5 mM
MgCl.sub.2, 1 .mu.l of a dATP, dTTP, dGTP, and dCTP mix (10 mM
each), and 0.75 .mu.l of DNA polymerase mix (3.5 U/.mu.l) in a
final volume of 50 .mu.l. An Eppendorf Mastercycler thermocycler
was used to amplify the fragment programmed for 1 cycle at
94.degree. C. for 2 minutes; 10 cycles each at 94.degree. C. for 15
seconds, 62.degree. C. for 30 seconds, and 72.degree. C. for 1
minute, 15 seconds; 15 cycles each at 94.degree. C. for 15 seconds,
62.degree. C. for 30 seconds, and 72.degree. C. for 1 minute 15
seconds, plus a 5 second elongation at each successive cycle; 1
cycle at 72.degree. C. for 7 minutes; and a 10.degree. C. hold.
[0415] The 1.2 kb reaction product was visualized on a 1.0% agarose
gel using TBE buffer. The 1.2 kb fragment was excised from the gel
and purified using a Qiagen Gel Extraction Kit. Two microliters of
the product was then cloned into pCR2.1-TOPO according to
manufacturer's instructions to produce pBM135g (FIG. 14). Two .mu.l
of the reaction was used to transform E. coli TOP10 One Shot
competent cells. An E. coli transformant containing pBM135g was
detected by restriction digestion and plasmid DNA was prepared
using a QIAGEN BioRobot 9600.
[0416] E. coli TOP10 One Shot cells containing pBM135g were
deposited with the Agricultural Research Service Patent Culture
Collection, Northern Regional Research Center, 1815 University
Street, Peoria, Ill., 61604, as NRRL B-30779, with a deposit date
of Oct. 12, 2004.
Example 22
Characterization of the Magnaporthe grisea Genomic Sequence
Encoding Lipase 3
[0417] DNA sequencing of the Magnaporthe grisea lipase 3 gene from
plasmid, pBM135g, was performed with a Perkin-Elmer Applied
Biosystems Model 377 XL Automated DNA Sequencer using
dye-terminator chemistry (Giesecke et al., 1992, supra) and primer
walking strategy. Nucleotide sequence data were scrutinized for
quality and all sequences were analyzed with assistance of
ContigExpress software.
[0418] Gene models for the lipase gene were predicted based on
homology to a Thermomyces lanuginosus lipase (Accession No. O59952)
as well as conserved sequences present at the 5' and 3' ends of
fungal introns. The genomic coding sequence (SEQ ID NO: 9) and
deduced amino acid sequence (SEQ ID NO: 10) for pBM135g and is
shown in FIG. 15. The genomic fragment encodes a polypeptide of 393
amino acids. The % G+C content of the gene is 58.71% and the mature
polypeptide coding sequence (nucleotides 73 to 1179 of SEQ ID NO:
9) is 58.4%. Using the SignalP software program (Nielsen et al,
1997, supra), a signal peptide of 24 residues was predicted. The
predicted mature protein contains 369 amino acids with a molecular
mass of 40.8 kDa.
[0419] A comparative alignment of lipase sequences was determined
using the Clustal W method (Higgins, 1989, supra) 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 Magnaporthe grisea lipase 3 gene shares
20.5% identity to the deduced amino acid sequence of the
Thermomyces lanuginosus lipase gene (Accession No. O59952).
Example 23
Identification of Lipase Genes in the Genomic Sequence of
Aspergillus nidulans
[0420] A tfasty search (Pearson, W. R., 1999, supra) of the
Aspergillus nidulans partial genome sequence (The Institute for
Genomic Research, Rockville, Md.) was carried out using as query a
lipase protein sequence from Thermomyces lanuginosus (Accession No.
O59952). Several genes were identified as putative homologs based
upon an E value of less than 0.001 in the tfasty output. Three
genomic regions of approximately 780 bp, 600 bp, and 960 bp with
45.8, 34, and 33% identity to the query sequence at the amino acid
level were identified. Gene models for the putative lipase genes
were predicted based on homology to the Thermomyces lanuginosus
lipase as well as conserved sequences present at the 5' and 3' ends
of fungal introns.
Example 24
Aspergillus nidulans Genomic DNA Extraction
[0421] Four hundred .mu.l of Aspergillus nidulans A1000 (Fungal
Genetics Stock Center, Kansas City, Mo.) spores were grown in 50 ml
of YP medium in a baffled shake flask at 37.degree. C. and 250 rpm
for 24 hours. Genomic DNA was then extracted from the mycelia using
a DNeasy Plant Mini Kit according to manufacturer's
instructions.
Example 25
Cloning of the Aspergillus nidulans Lipase 1 Gene
[0422] Two synthetic oligonucleotide primers shown below were
designed based on the predicted start and stop codons of the open
reading frame to PCR amplify an Aspergillus nidulans gene encoding
a lipase from the genomic DNA prepared in Example 24.
TABLE-US-00015 Forward primer: (SEQ ID NO: 40)
5'-ACACAACTGGCCATGATCCGTTTGGGGTATTCTGCC-3' Reverse primer: (SEQ ID
NO: 41) 5'-AGTCACCTCTAGTTAATTAATTACTGGCAGGCAGTGATAT-3'
Bold letters represent coding sequence. The remaining sequence is
homologous to the insertion sites of pBM120a (see Example 6).
[0423] The fragment of interest was amplified by PCR using the
Expand High Fidelity PCR System. One .mu.M of each of the primers
above were used in a PCR reaction containing 20 ng of Aspergillus
nidulans genomic DNA, 1.times.PCR buffer with 1.5 mM MgCl.sub.2, 1
.mu.l of a dATP, dTTP, dGTP, and dCTP mix (10 mM each), and 0.75
.mu.l of DNA polymerase mix (3.5 U/.mu.l) in a final volume of 50
.mu.l. To amplify the fragment, an Eppendorf Mastercycler
thermocycler was programmed for 1 cycle at 94.degree. C. for 2
minutes; 10 cycles each at 94.degree. C. for 15 seconds,
59.5.degree. C. for 30 seconds, and 72.degree. C. for 1.25 minutes;
15 cycles each at 94.degree. C. for 15 seconds, 59.5.degree. C. for
30 seconds, and 72.degree. C. for 1.25 minutes plus a 5 second
elongation at each successive cycle; 1 cycle at 72.degree. C. for 7
minutes; and a 10.degree. C. hold.
[0424] The reaction product was visualized on a 0.7% agarose gel
using TBE buffer and the 1.1 kb product band was purified using a
QIAquick PCR Purification Kit according to the manufacturer's
instructions. The PCR fragment and pCR2.1-TOPO were ligated using
conditions specified by the manufacturer resulting in plasmid
pJLin171 (FIG. 16).
[0425] Two .mu.l of the reaction was used to transform E. coli
TOP10 One Shot competent cells according to the manufacturer's
instructions. A 2 .mu.l volume of the ligation mixture was added to
the E. coli cells and incubated on ice for 20 minutes.
Subsequently, the cells were heat shocked for 30 seconds at
42.degree. C., and then placed on ice for 2 minutes. A 250 .mu.l
volume of SOC medium was added to the cells and the mixture was
incubated for 1 hour at 37.degree. C. and 250 rpm. After the
incubation the colonies were spread on 2X YT plates supplemented
with 100 .mu.g of ampicillin per ml and incubated at 37.degree. C.
overnight for selection of the plasmid. Twelve colonies that grew
on the plates were picked with a sterile toothpick and grown
overnight at 37.degree. C., 250 rpm in a 15 ml Falcon tube
containing 3 ml of LB medium supplemented with 100 .mu.g of
ampicillin per ml. An E. coli transformant containing the pJLin171
was detected by restriction digestion and plasmid DNA was prepared
using a BioRobot 9600.
[0426] E. coli TOP 10 One Shot cells containing pJLin171 were
deposited with the Agricultural Research Service Patent Culture
Collection, Northern Regional Research Center, 1815 University
Street, Peoria, Ill., 61604, as NRRL B-30755, with a deposit date
of Jul. 21, 2004.
Example 26
Characterization of the Aspergillus nidulans Genomic Sequence
Encoding Lipase 1
[0427] DNA sequencing of the Aspergillus nidulans lipase 1 gene
from pJLin171 was performed with an Applied Biosystems Model 377 XL
DNA Sequencer using dye-terminator chemistry (Giesecke et al.,
1992, supra) and primer walking strategy. Nucleotide sequence data
were scrutinized for quality and all sequences were compared to
each other with assistance of PHRED/PHRAP software (University of
Washington, Seattle, Wash.).
[0428] Gene models for the lipase gene were predicted based on
homology to the Thermomyces lanuginosus lipase as well as conserved
sequences present at the 5' and 3' ends of fungal introns. The
nucleotide sequence (SEQ ID NO: 11) and deduced amino acid sequence
(SEQ ID NO: 12) are shown in FIG. 17. The genomic fragment encodes
a polypeptide of 294 amino acids, interrupted by 3 introns of 47
bp, 59 bp, and 50 bp. The % G+C content of the gene is 54.5% and of
the mature protein coding region (nucleotides 58 to 1038 of SEQ ID
NO: 11) is 54.5%. Using the SignalP software program (Nielsen et
al., 1997, supra), a signal peptide of 19 residues was predicted.
The predicted mature protein contains 275 amino acids with a
molecular mass of 29.4 kDa.
[0429] A comparative alignment of lipase sequences was made
employing the Clustal W method (Higgins, 1989, supra) 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 nidulans lipase 1 gene
shares 48% identity to the deduced amino acid sequence of a
Thermomyces lanuginosus lipase gene (accession number O59952).
Example 27
Construction of an Aspergillus oryzae Expression Vector Expressing
Aspergillus nidulans Lipase 1 Gene
[0430] The 1.1 kb PCR fragment (Example 25) containing the
Aspergillus nidulans lipase 1 gene was cloned into pBM120a using an
InFusion Cloning Kit where the vector was digested with Nco I and
Pac I. The digested vector was purified by gel electrophoresis
using a 0.7% agarose gel with TBE buffer, and the PCR fragment was
extracted using a QIAquick Gel Extraction Kit and purified using a
QIAquick PCR Purification Kit. The gene fragment and the digested
vector were ligated together in a reaction resulting in the
expression plasmid pJLin167 (FIG. 18). The ligation reaction (50
.mu.l) was composed of 1.times. InFusion Buffer, 1.times.BSA, 1
.mu.l of Infusion enzyme (diluted 1:10), 100 ng of pBM120a digested
with Nco I and Pac I, and 50 ng of the Aspergillus nidulans lipase
1 gene purified PCR product. The reaction was incubated at room
temperature for 30 minutes. Two .mu.l of the reaction was used to
transform E E. coli SoloPack.RTM. Gold supercompetent cells
according to the manufacturer's instructions. One .mu.l of
.beta.-mercaptoethanol was added to competent cells, and incubated
on ice for 10 minutes. A 2 .mu.l volume of the ligation mixture was
then added to the E. coli cells and incubated on ice for 30
minutes. Subsequently, the cells were heat shocked for 60 seconds
at 54.degree. C., and then placed on ice for 2 minutes. A 150 .mu.l
volume of NZY.sup.+ medium at 42.degree. C. was added to the cells
and the mixture was incubated for 1 hour at 37.degree. C. and 250
rpm. After the incubation the colonies were spread on 2X YT plates
supplemented with 100 .mu.g of ampicillin per ml and incubated at
37.degree. C. overnight for selection of the plasmid. Twelve
colonies that grew on the plates were picked with a sterile
toothpick and grown overnight at 37.degree. C., 250 rpm in a 15 ml
Falcon tube containing 3 ml of LB medium supplemented with 100
.mu.g of ampicillin per ml. An E E. coli transformant containing
the pJLin167 plasmid was detected by restriction digestion and
plasmid DNA was prepared using a QIAGEN BioRobot 9600.
Example 28
Expression of the Aspergillus nidulans Lipase 1 Gene in Aspergillus
oryzae BECh2
[0431] Aspergillus oryzae BECh2 (Aalp, Damy, CPA-, KA-, .DELTA.np1)
protoplasts were prepared according to the method of Christensen et
al., 1988, Bio/Technology 6: 1419-1422. Five .mu.g of pJLin167 was
used to transform Aspergillus oryzae BECh2.
[0432] The transformation of Aspergillus oryzae BECh2 with pJLin167
yielded 34 transformants. The transformants were isolated to
individual Cove plates. Confluent Cove plates of 28 transformants
were washed with 4 ml of 0.01% Tween 20. Two hundred .mu.l of spore
suspension was inoculated separately into 25 ml of MY25 medium in
125 ml plastic shake flasks and incubated at 34.degree. C., 250
rpm. Three and five days after incubation, culture supernatants
were removed for lipase assay and SDS-PAGE analysis.
[0433] Lipase activity was determined as described in Example 8.
The lipase assay results indicated that at both 3 and 5 days, 27 of
the 28 transformants produced lipase activity well above that of
the untransformed control.
[0434] SDS-PAGE (BioRad Criterion 10-20% SDS-PAGE) analysis of 10
.mu.l of the supernatants showed a major band at approximately 30
kDa.
Example 29
Purification and Characterization of Recombinant Aspergillus
nidulans Lipase 1
[0435] One of the Aspergillus oryzae transformants producing the
highest yield of Aspergillus nidulans lipase 1 was grown in 500 ml
of MY25 medium for 4 days at 30.degree. C., 250 rpm for
purification. Supernatant was sterile filtered under pressure using
SEITZ-EKS filters (PALL Corporation, Waldstetten, Germany). The
sterile filtered supernatant was then adjusted to pH 9 and sodium
chloride was added to a final concentration of 2 M.
[0436] Decylamine agarose was custom made by UpFront Chromatography
A/S, Lerso Parkalle Denmark. The decylamine agarose matrix was
packed into a 50 ml column and then washed and equilibrated with 50
mM borate pH 9 buffer containing 2 M sodium chloride. Filtered
fermentation supernatant was then applied to the column using an
Akta explorer system. Unbound material was washed with 50 mM borate
pH 9 buffer containing 1 M sodium chloride. The bound proteins were
eluted with 50 mM borate pH 9 buffer containing 30% 2-propanol as
an eluent.
[0437] Fractions of 10 ml were collected and analyzed for lipase
activity according to the assay described by Svendsen et al., in
Methods in Enzymology, Lipases Part a Biotechnology Vol. 284 pages
317-340 Edited by Byron Rubin and Edward A. Dennis, Academic Press,
1997, New York. Fractions containing lipase activity were then
pooled and diluted to adjust the ionic strength below 4 mSi.
[0438] A 50 ml FFQ Sepharose (Pharmacia Amersham, Uppsala, Sweden)
column was washed and equilibrated with 50 mM borate pH 9 buffer.
The decylamine agarose pool containing activity was then applied
onto the column and washed with the same buffer to remove unbound
material. Bound proteins were then eluted using linear salt
gradient using 50 mM borate pH 9 buffer containing 0.5 M sodium
chloride.
[0439] Fractions of 10 ml were collected and analyzed for activity
as described above and fractions were also analyzed by SDS-PAGE for
purity. Best fractions contain highest lipase activity and best
purity judged by SDS-PAGE was pooled. SDS-PAGE showed a pure
protein band with molecular weight between 33 to 35 kDa protein
which is usually seen due to glycosylation.
[0440] Substrate specificity of the Aspergillus nidulans lipase 1
was evaluated at pH 7 according to WO 2005/040410. The results
showed that the Aspergillus nidulans lipase 1 efficiently degrades
phospholipids such as lecithin (and alkylated
phosphatidylethanolamins) and well as triliolein.
Example 30
Determination of Thermostability of Recombinant Aspergillus
nidulans Lipase 1
[0441] The thermostability of purified recombinant Aspergillus
nidulans lipase 1 (Example 29) was determined by Differential
Scanning Calorimetry (DSC). The thermal denaturation temperature,
Td, was taken as the top of the denaturation peak (major
endothermic peak) in thermograms (Cp vs. T) obtained after heating
of enzyme solutions at a constant programmed heating rate. Cp
refers to heat capacity (at constant pressure). T refers to
temperature.
[0442] A VP-DSC Differential Scanning Calorimeter (MicroCal Inc.,
Northampton, Mass.) was used for the thermostability determination
according to the manufacturer's instructions. Sample enzyme and
reference solutions were carefully degassed immediately prior to
loading of samples into the calorimeter (reference: buffer without
enzyme). Sample enzyme (approximately 1 mg/ml) and reference
solutions (approximately 0.5 ml) were thermally pre-equilibrated
for 20 minutes at 5.degree. C. The DSC scan was performed from
5.degree. C. to 95.degree. C. at a scan rate of approximately 90
K/hr. Denaturation temperatures were determined at an accuracy of
approx. +/-1.degree. C.
[0443] The results as shown in Table 4 indicated that the
Aspergillus nidulans lipase 1 had thermal denaturation temperatures
of 63.degree. C. in 50 mM acetate pH 5.0 buffer and 55.degree. C.
in 50 mM glycine pH 10.0 buffer.
TABLE-US-00016 TABLE 4 Thermostability Determination Buffer pH Td
(.degree. C.) 50 mM Acetate 5.0 63 50 mM Glycine 10.0 55
Example 31
Cloning of the Aspergillus nidulans Lipase 2 Gene
[0444] Two synthetic oligonucleotide primers shown below were
designed based on the predicted start and stop codons of the open
reading frame to PCR amplify an Aspergillus nidulans lipase 2 gene
from the genomic DNA prepared in Example 24.
TABLE-US-00017 Forward primer: (SEQ ID NO: 42)
5'-ACACAACTGGCCATGTATTTCCTTCTCTCCGTCATC-3' Reverse primer: (SEQ ID
NO: 43) 5'-AGTCACCTCTAGTTAATTAATCAGCCTAGTGGGCAAGCAT-3'
Bold letters represent coding sequence. The remaining sequence is
homologous to the insertion sites of pBM120a.
[0445] The fragment was amplified by PCR using the Expand High
Fidelity PCR System as described in Example 25. The reaction
product was visualized on a 0.7% agarose gel using TBE buffer and a
1.2 kb product band was purified using a QIAquick PCR Purification
Kit according to the manufacturer's instructions. The PCR product
was then cloned into pCR2.1-TOPO according to manufacturer's
instructions to produce pJLin170 (FIG. 19). Two p of the reaction
was used to transform E. coli TOP10 One Shot competent cells as
described in Example 25. An E. coli transformant containing the
pJLin170 plasmid was detected by restriction digestion and plasmid
DNA was prepared using a QIAGEN BioRobot 9600.
[0446] E. coli TOP 10 One Shot cells containing pJLin170 were
deposited with the Agricultural Research Service Patent Culture
Collection, Northern Regional Research Center, 1815 University
Street, Peoria, Ill., 61604, as NRRL B-30754, with a deposit date
of Jul. 21, 2004.
Example 32
Characterization of the Aspergillus nidulans Genomic Sequence
Encoding Lipase 2
[0447] DNA sequencing of the Aspergillus nidulans lipase 2 gene
from pJLin170 was performed with an Applied Biosystems Model 377 XL
DNA Sequencer using dye-terminator chemistry (Giesecke et al.,
1992, supra) and primer walking strategy. Nucleotide sequence data
were scrutinized for quality and sequences were compared to each
other with assistance of PHRED/PHRAP software (University of
Washington, Seattle, Wash.).
[0448] Gene models for the lipase gene were predicted based on
homology to the Thermomyces lanuginosus lipase as well as conserved
sequences present at the 5' and 3' ends of fungal introns. The
nucleotide sequence (SEQ ID NO: 13) and deduced amino acid sequence
(SEQ ID NO: 14) are shown in FIG. 20. The genomic fragment encodes
a polypeptide of 308 amino acids, interrupted by 3 introns of 49,
73 and 70 bp. The % G+C content of the gene is 51.7% and the mature
protein coding region (nucleotides 73 to 1116 of SEQ ID NO: 13) is
51.9%. Using the SignalP software program (Nielsen et al., 1997,
supra), a signal peptide of 24 residues was predicted. The
predicted mature protein contains 284 amino acids with a molecular
mass of 30.5 kDa.
[0449] A comparative alignment of lipase sequences was made
employing the Clustal W method (Higgins, 1989, supra) 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 nidulans lipase 2 gene
shares 37% identity to the deduced amino acid sequence of a
Thermomyces lanuginosus lipase gene (accession number O59952).
Example 33
Cloning of the Aspergillus nidulans Lipase 3 Gene
[0450] Two synthetic oligonucleotide primers shown below were
designed based on the predicted start and stop codons of the open
reading frame to PCR amplify an Aspergillus nidulans lipase 3 gene
from the genomic DNA prepared in Example 24.
TABLE-US-00018 Forward primer: (SEQ ID NO: 44)
5'-ACACAACTGGCCATGACGGTGTCTCTTGACAGTTTATTCC-3' Reverse primer: (SEQ
ID NO: 45) 5'-AGTCACCTCTAGTTAATTAATCACCCCCCTGACAAATCTCCCTTGG-
3'
Bold letters represent coding sequence. The remaining sequence is
homologous to the insertion sites of pBM120a.
[0451] The fragment of interest was amplified by PCR using the
Expand High Fidelity PCR System. Fifty picomoles of each of the
primers above were used in a PCR reaction containing 25 ng of
Aspergillus nidulans genomic DNA. The PCR amplification reaction
mixture also contained 1.times.PCR buffer with 1.5 mM MgCl.sub.2, 1
.mu.l of a dATP, dTTP, dGTP, and dCTP mix (10 mM each), and 0.75
.mu.l of DNA polymerase mix (3.5 U/.mu.l) in a final volume of 50
.mu.l. An Eppendorf Mastercycler thermocycler was used to amplify
the fragment programmed for 1 cycle at 94.degree. C. for 2 minutes;
10 cycles each at 94.degree. C. for 15 seconds, 62.degree. C. for
30 seconds, and 72.degree. C. for 3 minutes; 15 cycles each at
94.degree. C. for 15 seconds, 62.degree. C. for 30 seconds, and
72.degree. C. for 3 minutes plus a 5 second elongation at each
successive cycle; 1 cycle at 72.degree. C. for 7 minutes; and a
10.degree. C. hold.
[0452] The reaction product was visualized on a 1.0% agarose gel
using TBE buffer and the 1.3 kb product band was purified using a
QIAquick PCR Purification Kit according to the manufacturer's
instructions.
Example 34
Construction of an Aspergillus oryzae Expression Vector Expressing
Aspergillus nidulans Lipase 3 Gene
[0453] The PCR fragment containing the Aspergillus nidulans lipase
3 gene described in Example 33 was cloned into the pBM120a
expression vector using an InFusion Cloning Kit. The vector was
digested with restriction endonucleases Nco I and Pac I (using
conditions specified by the manufacturer). The digested vector was
purified by gel electrophoresis and extracted using a QIAquick Gel
Extraction Kit, and the PCR fragment was purified using a QIAquick
PCR Purification kit. The gene fragment and the digested vector
were ligated together in a reaction resulting in the expression
plasmid pJSF8c (FIG. 21). The ligation reaction (20 .mu.l) was
composed of 1.times. InFusion Buffer, 1.times.BSA, 1 .mu.l of
Infusion enzyme (diluted 1:10), 100 ng of pBM120a digested with Nco
I and Pac I, and 50 ng of the Aspergillus nidulans lipase 3 gene
purified PCR product. The reaction was incubated at room
temperature for 30 minutes. Two .mu.l of the reaction was used to
transform E. coli SoloPack.RTM. Gold supercompetent cells. An E.
coli transformant containing the pJSF8c plasmid was detected by
restriction digestion and plasmid DNA was prepared using a QIAGEN
BioRobot 9600.
Example 35
Construction of an Aspergillus nidulans Lipase 3 Gene Cloning
Vector
[0454] The two synthetic oligonucleotide primers described in
Example 33 were used to PCR amplify the genomic coding region of
the Aspergillus nidulans lipase 3 gene from plasmid pJSF8c.
[0455] The fragment of interest was amplified by PCR using the
Expand High Fidelity PCR System. Fifty picomoles of each of the
primers above were used in a PCR reaction containing 1:10 dilution
of plasmid, pJSF8c, mini DNA. The PCR amplification reaction
mixture also contained 1.times.PCR buffer with 1.5 mM MgCl.sub.2, 1
.mu.l of a dATP, dTTP, dGTP, and dCTP mix (10 mM each), and 0.5
.mu.l DNA polymerase mix (3.5 U/.mu.l) in a final volume of 50
.mu.l. An Eppendorf Mastercycler thermocycler was used to amplify
the fragment programmed for 1 cycle at 94.degree. C. for 2 minutes;
10 cycles each at 94.degree. C. for 15 seconds, 62.degree. C. for
30 seconds, and 72.degree. C. for 1 minute, 15 seconds; 15 cycles
each at 94.degree. C. for 15 seconds, 62.degree. C. for 30 seconds,
and 72.degree. C. for 1 minute, 15 seconds plus a 5 second
elongation at each successive cycle; 1 cycle at 72.degree. C. for 7
minutes; and a 10.degree. C. hold.
[0456] The 1.3 kb PCR product was then cloned into pCR2.1-TOPO
according to manufacturer's instructions to produce pBM141 (FIG.
22). Two .mu.l of the reaction was used to transform E. coli TOP10
One Shot competent cells. An E. coli transformant containing pBM141
was detected by restriction digestion and plasmid DNA was prepared
using a QIAGEN BioRobot 9600.
[0457] E. coli TOP 10 One Shot cells containing pBM141 were
deposited with the Agricultural Research Service Patent Culture
Collection, Northern Regional Research Center, 1815 University
Street, Peoria, Ill., 61604, as NRRL B-30780, with a deposit date
of Oct. 12, 2004.
Example 36
Characterization of the Aspergillus nidulans Genomic Sequence
Encoding Lipase 3
[0458] DNA sequencing of the Aspergillus nidulans lipase 3 gene
from pJSF8c and pBM141 was performed with a Perkin-Elmer Applied
Biosystems Model 377 XL Automated DNA Sequencer
(Perkin-Elmer/Applied Biosystems, Inc., Foster City, Calif.) using
dye-terminator chemistry (Giesecke et al., 1992, supra) and primer
walking strategy. Nucleotide sequence data were scrutinized for
quality and all sequences were analyzed with assistance of
ContigExpress software (Informax, Inc., Bethesda, Md.).
[0459] Gene models for the putative lipase genes were predicted
based on homology to a Thermomyces lanuginosus lipase as well as
conserved sequences present at the 5' and 3' ends of fungal
introns. The genomic coding sequence (SEQ ID NO: 15) and deduced
amino acid sequence (SEQ ID NO: 16) are identical for pJSF8c and
pBM141 and are shown in FIG. 23. The genomic fragment encodes a
polypeptide of 404 amino acids, interrupted by 1 intron of 68 bp.
The % G+C content of the gene is 52.14% and the mature protein
coding region (nucleotides 76 to 1280 of SEQ ID NO: 15) is 52.3%.
Using the SignalP software program (Nielsen et al., 1997, supra), a
signal peptide of 25 residues was predicted. The predicted mature
protein contains 380 amino acids with a molecular mass of 42.4
kDa.
[0460] A comparative alignment of lipase sequences was determined
using the Clustal W method (Higgins, 1989, supra) 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 nidulans lipase 3 gene
shares 21.6% identity to the deduced amino acid sequence of a
Thermomyces lanuginosus lipase gene (accession number O59952).
Deposit of Biological Material
[0461] The following biological materials have been deposited under
the terms of the Budapest Treaty with the Agricultural Research
Service Patent Culture Collection, Northern Regional Research
Center, 1815 University Street, Peoria, Ill., 61604, and given the
following accession number:
TABLE-US-00019 Deposit Accession Number Date of Deposit E. coli
(pSMO223) NRRL B-30773 Sep. 16, 2004 E. coli (pSMO224) NRRL B-30774
Sep. 16, 2004 E. coli pHyGe026 NRRL B-30772 Sep. 13, 2004 E. coli
pBM135g NRRL B-30779 Oct. 12, 2004 E. coli pCrAm138 NRRL B-30781
Oct. 12, 2004 E. coli pJLin170 NRRL B-30754 Jul. 21, 2004 E. coli
pJLin171 NRRL B-30755 Jul. 21, 2004 E. coli pBM141 NRRL B-30780
Oct. 12, 2004
[0462] The strains have been deposited under conditions that assure
that access to the cultures will be available during the pendency
of this patent application to one determined by the Commissioner of
Patents and Trademarks to be entitled thereto under 37 C.F.R.
.sctn.1.14 and 35 U.S.C. .sctn.122. The deposits represent
substantially pure cultures of the deposited strains. The deposits
are available as required by foreign patent laws in countries
wherein counterparts of the subject application, or its progeny are
filed. However, it should be understood that the availability of a
deposit does not constitute a license to practice the subject
invention in derogation of patent rights granted by governmental
action.
[0463] 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.
[0464] Various references are cited herein, the disclosures of
which are incorporated by reference in their entireties.
Sequence CWU 1
1
4511259DNAAspergillus fumigatus 1atgcttcaca agtatagcct gttctgtctc
actattttct cctgcctctt cgttgtgagc 60gtggatgggg caatactcgg tagggatgat
gaaggccgac agcagatacc tgatgaactc 120tttgaatcac tcgaagagct
ctcacgtatc gttgatgttt cgtactgtgt tgggaccact 180gaaattcgga
agccattcaa gtgtctcagt cattgtagtg aattccaggg cttcgaactg
240gtcaccgtat gttgaaagcc tctcacttat atgttgtctt tctgtccaag
cttgatggct 300tagatcgtct ccagacatgg aataccggtc ctttcctttc
cgattcctgt ggctatgtaa 360ccctctccca cgaaccatct ccgaagcgga
tcatcgttgc ctttcgcggc acttactcga 420tcgccaacac gatcatcgat
ctatccgcct atcctcaggc ctatgtaccg tatcatcccg 480aggatggaaa
agtgtccgac catttacaat gtctgaactg cacagttcat gcggggtttt
540tggcctcgtg gagcaatgct cgcgccatag tactcgagca cgtagctgtg
gcaagggccc 600ggtacccgga ttacagcttg gttttgaccg gccactcgct
tggcggcgcc gtcgccgctc 660ttgccggggt cgaaatgcaa ctgcgcgggt
gggagccaca agtgaccact ttcggagagc 720caagaatcgg aaacaaggct
tttgtggaat ttcttgatcg gatctttgat ctagatggct 780tgggagctga
tgcccaggac accagatttc gaagagtcac acatatcaac gatccggtcc
840ccctactccc attgtcagaa tggggttatg agatgcatgc gggagagatc
tttatcgcta 900aggaggagct ctcgcctcta ccccatgata tcagactgtg
ccagggcgac aacgatgcgc 960gatgcattgc aggaacggat ggagctgtaa
cgcgcatgct gaacgagttg gatgataccg 1020ttttgcccaa gcagcctcta
gcgaaacgag tccagtcacc ccatcaggcc gttttggcag 1080acgtggatcc
tcatagtagt gcagatgtcg acgagcaagt acagacgcca ttcagcctgc
1140cttggcatct tattccctcc agataccgac tgtgggagct gttcttcgct
catcgtgatt 1200atttctggcg gcttgggctt tgtgtaccag gcggtgatcc
gactggcaag ataatctga 12592396PRTAspergillus fumigatus 2Met Leu His
Lys Tyr Ser Leu Phe Cys Leu Thr Ile Phe Ser Cys Leu 1 5 10 15 Phe
Val Val Ser Val Asp Gly Ala Ile Leu Gly Arg Asp Asp Glu Gly 20 25
30 Arg Gln Gln Ile Pro Asp Glu Leu Phe Glu Ser Leu Glu Glu Leu Ser
35 40 45 Arg Ile Val Asp Val Ser Tyr Cys Val Gly Thr Thr Glu Ile
Arg Lys 50 55 60 Pro Phe Lys Cys Leu Ser His Cys Ser Glu Phe Gln
Gly Phe Glu Leu 65 70 75 80 Val Thr Thr Trp Asn Thr Gly Pro Phe Leu
Ser Asp Ser Cys Gly Tyr 85 90 95 Val Thr Leu Ser His Glu Pro Ser
Pro Lys Arg Ile Ile Val Ala Phe 100 105 110 Arg Gly Thr Tyr Ser Ile
Ala Asn Thr Ile Ile Asp Leu Ser Ala Tyr 115 120 125 Pro Gln Ala Tyr
Val Pro Tyr His Pro Glu Asp Gly Lys Val Ser Asp 130 135 140 His Leu
Gln Cys Leu Asn Cys Thr Val His Ala Gly Phe Leu Ala Ser 145 150 155
160 Trp Ser Asn Ala Arg Ala Ile Val Leu Glu His Val Ala Val Ala Arg
165 170 175 Ala Arg Tyr Pro Asp Tyr Ser Leu Val Leu Thr Gly His Ser
Leu Gly 180 185 190 Gly Ala Val Ala Ala Leu Ala Gly Val Glu Met Gln
Leu Arg Gly Trp 195 200 205 Glu Pro Gln Val Thr Thr Phe Gly Glu Pro
Arg Ile Gly Asn Lys Ala 210 215 220 Phe Val Glu Phe Leu Asp Arg Ile
Phe Asp Leu Asp Gly Leu Gly Ala 225 230 235 240 Asp Ala Gln Asp Thr
Arg Phe Arg Arg Val Thr His Ile Asn Asp Pro 245 250 255 Val Pro Leu
Leu Pro Leu Ser Glu Trp Gly Tyr Glu Met His Ala Gly 260 265 270 Glu
Ile Phe Ile Ala Lys Glu Glu Leu Ser Pro Leu Pro His Asp Ile 275 280
285 Arg Leu Cys Gln Gly Asp Asn Asp Ala Arg Cys Ile Ala Gly Thr Asp
290 295 300 Gly Ala Val Thr Arg Met Leu Asn Glu Leu Asp Asp Thr Val
Leu Pro 305 310 315 320 Lys Gln Pro Leu Ala Lys Arg Val Gln Ser Pro
His Gln Ala Val Leu 325 330 335 Ala Asp Val Asp Pro His Ser Ser Ala
Asp Val Asp Glu Gln Val Gln 340 345 350 Thr Pro Phe Ser Leu Pro Trp
His Leu Ile Pro Ser Arg Tyr Arg Leu 355 360 365 Trp Glu Leu Phe Phe
Ala His Arg Asp Tyr Phe Trp Arg Leu Gly Leu 370 375 380 Cys Val Pro
Gly Gly Asp Pro Thr Gly Lys Ile Ile 385 390 395 3947DNAAspergillus
fumigatus 3atgtttcacc cagatatttc ggctgggctt ctagccaatc taacactatt
ttctgaatat 60gccgctgcct cgacgtgtgc agcaaacttc aactcacata cattatcgaa
ggtagtgtgt 120gatccaggtg tatgcccaac cctggagcag acagacacaa
atgtcatggt tgggttcatg 180gggtatgatt ccatcaacag ggcatgaact
gaaaagctaa cgccaagtat acatcatccg 240ggtaacgtca ctggcttcgt
ggcaattgac aacacaaatc aattgatcgt tctgtcattc 300cgcggtagcc
ggaccctagg caactatatc actgattcca aataccagca ggtgcctgct
360atttgcccag gttgccaagt gcataaaggc tattactggg cctggggaaa
cttttcagca 420tttataatgc aacctataaa ccagcttgct gctatatatc
caagctatca gattgtcttc 480actggccaca gttttggagg tgcactagct
acgcttgggg cagcacttga gggaggaaat 540cctagcagac ctattgatct
ggtaagtacc tcagtcaccg caaatgtttc cctagagaat 600gcattaatca
gatacagtac acttttggat gtccccaact gggcaatcat gattttgctg
660agtttgtcac tgctgtaacg gcaggctctg ggtacagagt cacacattcg
gatgatccag 720ttccaagggt cttttctact cagccttgga tcaacaagac
ttggcagtat agcacaactt 780ctcctgagtt ttggattacc acaggaaatg
gcgtgccagt cacagccagt gatatacaag 840tcatcgaggg cattgacaac
aagagtggga accttggcac cactggttct gatacttcag 900ctcatatttg
gtatattggc aacatgagcg ggtgctcaac taactga 9474283PRTAspergillus
fumigatus 4Met Phe His Pro Asp Ile Ser Ala Gly Leu Leu Ala Asn Leu
Thr Leu 1 5 10 15 Phe Ser Glu Tyr Ala Ala Ala Ser Thr Cys Ala Ala
Asn Phe Asn Ser 20 25 30 His Thr Leu Ser Lys Val Val Cys Asp Pro
Gly Val Cys Pro Thr Leu 35 40 45 Glu Gln Thr Asp Thr Asn Val Met
Val Gly Phe Met Gly Ile His His 50 55 60 Pro Gly Asn Val Thr Gly
Phe Val Ala Ile Asp Asn Thr Asn Gln Leu 65 70 75 80 Ile Val Leu Ser
Phe Arg Gly Ser Arg Thr Leu Gly Asn Tyr Ile Thr 85 90 95 Asp Ser
Lys Tyr Gln Gln Val Pro Ala Ile Cys Pro Gly Cys Gln Val 100 105 110
His Lys Gly Tyr Tyr Trp Ala Trp Gly Asn Phe Ser Ala Phe Ile Met 115
120 125 Gln Pro Ile Asn Gln Leu Ala Ala Ile Tyr Pro Ser Tyr Gln Ile
Val 130 135 140 Phe Thr Gly His Ser Phe Gly Gly Ala Leu Ala Thr Leu
Gly Ala Ala 145 150 155 160 Leu Glu Gly Gly Asn Pro Ser Arg Pro Ile
Asp Leu Ile Gln Tyr Thr 165 170 175 Phe Gly Cys Pro Gln Leu Gly Asn
His Asp Phe Ala Glu Phe Val Thr 180 185 190 Ala Val Thr Ala Gly Ser
Gly Tyr Arg Val Thr His Ser Asp Asp Pro 195 200 205 Val Pro Arg Val
Phe Ser Thr Gln Pro Trp Ile Asn Lys Thr Trp Gln 210 215 220 Tyr Ser
Thr Thr Ser Pro Glu Phe Trp Ile Thr Thr Gly Asn Gly Val 225 230 235
240 Pro Val Thr Ala Ser Asp Ile Gln Val Ile Glu Gly Ile Asp Asn Lys
245 250 255 Ser Gly Asn Leu Gly Thr Thr Gly Ser Asp Thr Ser Ala His
Ile Trp 260 265 270 Tyr Ile Gly Asn Met Ser Gly Cys Ser Thr Asn 275
280 5 1088DNAMagnaporthe grisea 5atgaaggtct cgttcgtgtc atcgctcctc
gcgctcccgc tccttgcggc cgcggccccc 60aagcccgagg ccgctgagct gtcgagccgc
gacgtgaccg tgacgcagca ggagctcaac 120ggcttcatgt acatgcggca
gctcgcctcg gccgcctact gcaactccaa ggacagcctg 180gtcggccaaa
aggtcacatg cagcaacaac gcctgcccgg acattggcgc cgctaacgtg
240gtcaactttg cccatctcga gtatgttttt ttccctcttt tttcttgtga
aactcttgtt 300gtataaaaaa aaaggtcaaa tcatctaaca gatacccccc
acgatggggc caacagcacc 360gacatcggca tcaaggccga cggcgctgtc
ttcatcgacc acaccagggg cggcatcgtc 420atgtccttca tgggctccaa
gtcgtggcag tccttcatga ccgagtaagc aaaacacccc 480acttactcta
gatttttttt tatcttgtca tccacatgac tcacatttca cctccaaaca
540gcctcgactt caccggcagc gactcctccg agatctgcag cggctgcacg
gtccactacg 600gcatcaagct cacctacgac atcatcgagg gcgcgctgat
caacgccctc aactcggccc 660gcgcccagtg gccgtcgtac caggtcgtcg
cgacgggcca ctccatcgga gccggcgtcg 720ccaccgtcgc cgccgcccgc
ctgcgcaacc gcctcaacgt cgacatccag ctctacacct 780ttggcagccc
ccgcgtcggc aacgacgcct ttgccacctt tgtcaccaac cagaaccgcg
840gccgcaacta ccgcatcacc cactacgacg acgtcgtcgc cgccttgccc
ccgtcctggg 900ccggcttcgc ccacgtcagc cccgagtact ggctgcgcag
gaaggacgcc agcgacttca 960actacccgct cagcgaggtc gtcgtctgcg
agggcatcaa ccccaagggt tgcaggaaca 1020gcatgggcac caccctcagc
ggcaaggccc acggagagta ctttggcgcc attagcgctt 1080gctactga
10886318PRTMagnaporthe grisea 6Met Lys Val Ser Phe Val Ser Ser Leu
Leu Ala Leu Pro Leu Leu Ala 1 5 10 15 Ala Ala Ala Pro Lys Pro Glu
Ala Ala Glu Leu Ser Ser Arg Asp Val 20 25 30 Thr Val Thr Gln Gln
Glu Leu Asn Gly Phe Met Tyr Met Arg Gln Leu 35 40 45 Ala Ser Ala
Ala Tyr Cys Asn Ser Lys Asp Ser Leu Val Gly Gln Lys 50 55 60 Val
Thr Cys Ser Asn Asn Ala Cys Pro Asp Ile Gly Ala Ala Asn Val 65 70
75 80 Val Asn Phe Ala His Leu Glu Ser Asn His Leu Thr Asp Thr Pro
His 85 90 95 Asp Gly Ala Asn Ser Thr Asp Ile Gly Ile Lys Ala Asp
Gly Ala Val 100 105 110 Phe Ile Asp His Thr Arg Gly Gly Ile Val Met
Ser Phe Met Gly Ser 115 120 125 Lys Ser Trp Gln Ser Phe Met Thr Glu
Leu Asp Phe Thr Gly Ser Asp 130 135 140 Ser Ser Glu Ile Cys Ser Gly
Cys Thr Val His Tyr Gly Ile Lys Leu 145 150 155 160 Thr Tyr Asp Ile
Ile Glu Gly Ala Leu Ile Asn Ala Leu Asn Ser Ala 165 170 175 Arg Ala
Gln Trp Pro Ser Tyr Gln Val Val Ala Thr Gly His Ser Ile 180 185 190
Gly Ala Gly Val Ala Thr Val Ala Ala Ala Arg Leu Arg Asn Arg Leu 195
200 205 Asn Val Asp Ile Gln Leu Tyr Thr Phe Gly Ser Pro Arg Val Gly
Asn 210 215 220 Asp Ala Phe Ala Thr Phe Val Thr Asn Gln Asn Arg Gly
Arg Asn Tyr 225 230 235 240 Arg Ile Thr His Tyr Asp Asp Val Val Ala
Ala Leu Pro Pro Ser Trp 245 250 255 Ala Gly Phe Ala His Val Ser Pro
Glu Tyr Trp Leu Arg Arg Lys Asp 260 265 270 Ala Ser Asp Phe Asn Tyr
Pro Leu Ser Glu Val Val Val Cys Glu Gly 275 280 285 Ile Asn Pro Lys
Gly Cys Arg Asn Ser Met Gly Thr Thr Leu Ser Gly 290 295 300 Lys Ala
His Gly Glu Tyr Phe Gly Ala Ile Ser Ala Cys Tyr 305 310 315
71047DNAMagnaporthe grisea 7atgaggttcc ccagcgtgct cacccttttg
gccacagccc tcacctgctc ggcatcggtt 60ctccctgccg gcctgaccta caccaagact
gtcgagggcc gcgatgtgac cgtcagcgag 120acagacctag acaacttccg
tttctatgcg cagtacagcg cggcgaccta ctgcaacgat 180gccgccgcct
caggggccgc cgtcgcctgc agcaacgacg gatgtcccgc cgtcgtggcc
240aacggagcca agatcatccg ttcgctgaac caagacacgt ccaccaacac
tgccggctac 300cttgcactcg accccaagcg gaagaacatc gtgctcgccc
tccgtggctc cacgagcctc 360cggaactgga tcaccaacct gactttcctg
tggacccgct gcgactttgt ccaggactgc 420aagctgcaca cgggctttgc
cacagcctgg tcccaggtgc aggccgatgt tctggccgcc 480atcgccgacg
ccaaggccca gaaccccgac tacaccgtcg tcgtgacggg ccactccctc
540ggcggcgccg tcgccaccgt cgcgggagtc tacctccgcc agctgggcta
ccccgtcgag 600gtttacacgt acggcagccc gcgcatcggc aatcaggagt
ttgtgcagtg ggtttccacg 660caggccggca acgtcgagta ccgcgtcacg
cacatcgacg accccgtccc ccgcctgccg 720cccatcttcc tcggctacag
gcacgtcacc cccgagtact ggctcaactc tggcacctcc 780aacacggtca
actacaccgt cgccgacatc aaggtctgcg agggcttcgc caacatcaac
840tgcaacggcg gcagcctcgg cctcgacaca aacgcccacc tctactacct
caccgacatg 900atcgcctgcg gctccaacaa gttcgtcttc cgccgcgacg
acgccaacgc catcagtgac 960gccgagctcg agcagaggct gaccatgtac
gctcaaatgg atcgcgagtt tgttgctgcg 1020cttgaagcca acaagaccgt ggcttaa
10478348PRTMagnaporthe grisea 8Met Arg Phe Pro Ser Val Leu Thr Leu
Leu Ala Thr Ala Leu Thr Cys 1 5 10 15 Ser Ala Ser Val Leu Pro Ala
Gly Leu Thr Tyr Thr Lys Thr Val Glu 20 25 30 Gly Arg Asp Val Thr
Val Ser Glu Thr Asp Leu Asp Asn Phe Arg Phe 35 40 45 Tyr Ala Gln
Tyr Ser Ala Ala Thr Tyr Cys Asn Asp Ala Ala Ala Ser 50 55 60 Gly
Ala Ala Val Ala Cys Ser Asn Asp Gly Cys Pro Ala Val Val Ala 65 70
75 80 Asn Gly Ala Lys Ile Ile Arg Ser Leu Asn Gln Asp Thr Ser Thr
Asn 85 90 95 Thr Ala Gly Tyr Leu Ala Leu Asp Pro Lys Arg Lys Asn
Ile Val Leu 100 105 110 Ala Leu Arg Gly Ser Thr Ser Leu Arg Asn Trp
Ile Thr Asn Leu Thr 115 120 125 Phe Leu Trp Thr Arg Cys Asp Phe Val
Gln Asp Cys Lys Leu His Thr 130 135 140 Gly Phe Ala Thr Ala Trp Ser
Gln Val Gln Ala Asp Val Leu Ala Ala 145 150 155 160 Ile Ala Asp Ala
Lys Ala Gln Asn Pro Asp Tyr Thr Val Val Val Thr 165 170 175 Gly His
Ser Leu Gly Gly Ala Val Ala Thr Val Ala Gly Val Tyr Leu 180 185 190
Arg Gln Leu Gly Tyr Pro Val Glu Val Tyr Thr Tyr Gly Ser Pro Arg 195
200 205 Ile Gly Asn Gln Glu Phe Val Gln Trp Val Ser Thr Gln Ala Gly
Asn 210 215 220 Val Glu Tyr Arg Val Thr His Ile Asp Asp Pro Val Pro
Arg Leu Pro 225 230 235 240 Pro Ile Phe Leu Gly Tyr Arg His Val Thr
Pro Glu Tyr Trp Leu Asn 245 250 255 Ser Gly Thr Ser Asn Thr Val Asn
Tyr Thr Val Ala Asp Ile Lys Val 260 265 270 Cys Glu Gly Phe Ala Asn
Ile Asn Cys Asn Gly Gly Ser Leu Gly Leu 275 280 285 Asp Thr Asn Ala
His Leu Tyr Tyr Leu Thr Asp Met Ile Ala Cys Gly 290 295 300 Ser Asn
Lys Phe Val Phe Arg Arg Asp Asp Ala Asn Ala Ile Ser Asp 305 310 315
320 Ala Glu Leu Glu Gln Arg Leu Thr Met Tyr Ala Gln Met Asp Arg Glu
325 330 335 Phe Val Ala Ala Leu Glu Ala Asn Lys Thr Val Ala 340 345
91182DNAMagnaporthe grisea 9atgttgtggc gtcgggcggg tggcctctgt
ctgttgctgt gttgggcctg ggcaacaccg 60gcacaagctg cagcatttag tcatgacatc
acccagctga gctacacgga tgtcgactcg 120cctctccaaa aacacctaca
atcacaacaa caacaaaaac aagaacacaa acaaaaacct 180atcaccacca
caaccatttc ttcaatactc ttcacgtctc tagagcgcct ggcccgcctc
240gtagacatag cctactgcgt gggaagtctg cccggcatct cgcggccctt
cacctgcgcc 300tcgcgctgcg ccgatttccc ccacgtttca ctcgtcaaca
cctgggacac gggcccactc 360ctgacagaca gctgcggcta cgtcgccatc
gaccacgccg atgaagccat agtggtcgcc 420tttcggggca cctacagcat
cgccaacacc gtgattgacc tcagcacggt gccgcaggag 480tacgtgccct
atccggagcc cgacgacgat ggcgacaggg agcgctgcga caactgcacc
540gtgcacctag gattcctagc cagctggaag gtcgccagga atctggtcct
gcccgccatc 600gaggaggcga ggcagaagca ccccggcttc agcatcaacc
tcgtcggcca cagcctcggc 660ggtgccgtcg ctgcgctggc ggcgctcgag
ctgaagctca tcagcggcta cgatgtcgta 720gtcacgactt ttggtgagcc
gagggttggc aacagcgggc tggcaaagtt cattgatcgc 780gtgttcggct
tagaccagga agcaaaagag gacatggcgt accgcagggt cacgcacgcc
840gaggatccgg taccgttgct gccgctcgag gagtggggat acaggtcaca
cgccggcgag 900attcacattg agaagccggc gctaccacca gcaccgactg
acataaagtt gtgcaaaggc 960gaaagggatc ccgattgcag caacgggaac
tctgacgcgg cgctgactac cttgctgctt 1020ggagaggaac attatctaaa
gaaacagcat ctgaagctgt ggcagctgtt ctttgcccat 1080cgagactact
tttggaggct tgggctttgc gttcccggtg gtgatccggc tgattggggt
1140agagacaagt atgatgtggc tcctggccag gatgagctct aa
118210393PRTMagnaporthe grisea 10Met Leu Trp Arg Arg Ala Gly Gly
Leu Cys Leu Leu Leu Cys Trp Ala 1 5 10 15 Trp Ala Thr Pro Ala Gln
Ala Ala Ala Phe Ser His Asp Ile Thr Gln 20 25 30 Leu Ser Tyr Thr
Asp Val Asp Ser Pro Leu Gln Lys His Leu Gln Ser 35 40 45 Gln Gln
Gln Gln Lys Gln Glu His Lys Gln Lys Pro Ile Thr Thr Thr 50
55 60 Thr Ile Ser Ser Ile Leu Phe Thr Ser Leu Glu Arg Leu Ala Arg
Leu 65 70 75 80 Val Asp Ile Ala Tyr Cys Val Gly Ser Leu Pro Gly Ile
Ser Arg Pro 85 90 95 Phe Thr Cys Ala Ser Arg Cys Ala Asp Phe Pro
His Val Ser Leu Val 100 105 110 Asn Thr Trp Asp Thr Gly Pro Leu Leu
Thr Asp Ser Cys Gly Tyr Val 115 120 125 Ala Ile Asp His Ala Asp Glu
Ala Ile Val Val Ala Phe Arg Gly Thr 130 135 140 Tyr Ser Ile Ala Asn
Thr Val Ile Asp Leu Ser Thr Val Pro Gln Glu 145 150 155 160 Tyr Val
Pro Tyr Pro Glu Pro Asp Asp Asp Gly Asp Arg Glu Arg Cys 165 170 175
Asp Asn Cys Thr Val His Leu Gly Phe Leu Ala Ser Trp Lys Val Ala 180
185 190 Arg Asn Leu Val Leu Pro Ala Ile Glu Glu Ala Arg Gln Lys His
Pro 195 200 205 Gly Phe Ser Ile Asn Leu Val Gly His Ser Leu Gly Gly
Ala Val Ala 210 215 220 Ala Leu Ala Ala Leu Glu Leu Lys Leu Ile Ser
Gly Tyr Asp Val Val 225 230 235 240 Val Thr Thr Phe Gly Glu Pro Arg
Val Gly Asn Ser Gly Leu Ala Lys 245 250 255 Phe Ile Asp Arg Val Phe
Gly Leu Asp Gln Glu Ala Lys Glu Asp Met 260 265 270 Ala Tyr Arg Arg
Val Thr His Ala Glu Asp Pro Val Pro Leu Leu Pro 275 280 285 Leu Glu
Glu Trp Gly Tyr Arg Ser His Ala Gly Glu Ile His Ile Glu 290 295 300
Lys Pro Ala Leu Pro Pro Ala Pro Thr Asp Ile Lys Leu Cys Lys Gly 305
310 315 320 Glu Arg Asp Pro Asp Cys Ser Asn Gly Asn Ser Asp Ala Ala
Leu Thr 325 330 335 Thr Leu Leu Leu Gly Glu Glu His Tyr Leu Lys Lys
Gln His Leu Lys 340 345 350 Leu Trp Gln Leu Phe Phe Ala His Arg Asp
Tyr Phe Trp Arg Leu Gly 355 360 365 Leu Cys Val Pro Gly Gly Asp Pro
Ala Asp Trp Gly Arg Asp Lys Tyr 370 375 380 Asp Val Ala Pro Gly Gln
Asp Glu Leu 385 390 111041DNAAspergillus nidulans 11atgatccgtt
tggggtattc tgccatcttc gtagcccttg ctgggttagc cgttgccgct 60ccagcgccgc
tgaatcgtcg tggtatgtca ccttgcatgg ggatatggta caggactaac
120gttgtgtaga cgtttcgacg gaggccctaa atcaactgac cctgttcgcg
gagtattctg 180cggcatcgta ctgcacaccc aatattgggt cagtcgggga
caagctgact tgcgcatctg 240gaaactgccc gacagttgag gcagcagaca
cgacaacgct ggctgaattc tatcagtgcg 300ttaagccctg cattcggggt
tccaaacaga tccaactagt ctgacgagtc acagggagaa 360cgaatacggg
gatgtagcag gcttccttgc cgcagataca accaacgagt tactcgtctt
420gtccttccgt gggagccgga cgattgacac gtggattgca aacctcgact
ttggcctgga 480gtcggtcgag gagatctgta gcggatgcaa agcccacggc
gggttctgga aggcatggca 540ggttgttgca gactcgttga cctcagcaat
tgagtctgct actgccacat atcccggcta 600cgccattgtc ttcacaggcc
acagctttgg aggagcattg gctactctag gcgcagcgca 660gctgcgaaaa
gcaggttatg ccatcgaact tgtaagaatc cagtgtccag ctggtggcta
720gctgtctgct gacgagcgta gtacccctat ggtagcccgc gtgttggcaa
cgaagctttg 780gcgcaataca tcacagacca gggggcaaac tatcgagtga
cgcacactaa cgatatcgtt 840cccagacttc ctcccatgtt gttgggcttc
agccacttga gccctgagta ttggattacc 900agcgacaatg aggttacccc
gacgacgaca gatatccagg tgattgaagg cgttgggtcg 960agggacggaa
atgcgggtga ggctgcccag tcagtggagg cacacagttg gtatctgata
1020gatatcactg cctgccagta a 104112294PRTAspergillus nidulans 12Met
Ile Arg Leu Gly Tyr Ser Ala Ile Phe Val Ala Leu Ala Gly Leu 1 5 10
15 Ala Val Ala Ala Pro Ala Pro Leu Asn Arg Arg Asp Val Ser Thr Glu
20 25 30 Ala Leu Asn Gln Leu Thr Leu Phe Ala Glu Tyr Ser Ala Ala
Ser Tyr 35 40 45 Cys Thr Pro Asn Ile Gly Ser Val Gly Asp Lys Leu
Thr Cys Ala Ser 50 55 60 Gly Asn Cys Pro Thr Val Glu Ala Ala Asp
Thr Thr Thr Leu Ala Glu 65 70 75 80 Phe Tyr Gln Glu Asn Glu Tyr Gly
Asp Val Ala Gly Phe Leu Ala Ala 85 90 95 Asp Thr Thr Asn Glu Leu
Leu Val Leu Ser Phe Arg Gly Ser Arg Thr 100 105 110 Ile Asp Thr Trp
Ile Ala Asn Leu Asp Phe Gly Leu Glu Ser Val Glu 115 120 125 Glu Ile
Cys Ser Gly Cys Lys Ala His Gly Gly Phe Trp Lys Ala Trp 130 135 140
Gln Val Val Ala Asp Ser Leu Thr Ser Ala Ile Glu Ser Ala Thr Ala 145
150 155 160 Thr Tyr Pro Gly Tyr Ala Ile Val Phe Thr Gly His Ser Phe
Gly Gly 165 170 175 Ala Leu Ala Thr Leu Gly Ala Ala Gln Leu Arg Lys
Ala Gly Tyr Ala 180 185 190 Ile Glu Leu Tyr Pro Tyr Gly Ser Pro Arg
Val Gly Asn Glu Ala Leu 195 200 205 Ala Gln Tyr Ile Thr Asp Gln Gly
Ala Asn Tyr Arg Val Thr His Thr 210 215 220 Asn Asp Ile Val Pro Arg
Leu Pro Pro Met Leu Leu Gly Phe Ser His 225 230 235 240 Leu Ser Pro
Glu Tyr Trp Ile Thr Ser Asp Asn Glu Val Thr Pro Thr 245 250 255 Thr
Thr Asp Ile Gln Val Ile Glu Gly Val Gly Ser Arg Asp Gly Asn 260 265
270 Ala Gly Glu Ala Ala Gln Ser Val Glu Ala His Ser Trp Tyr Leu Ile
275 280 285 Asp Ile Thr Ala Cys Gln 290 131119DNAAspergillus
nidulans 13atgtatttcc ttctctccgt catcttccac tttcctgtct tctgtgccgg
ctttccacct 60gccgtatcca gaggtacgtg attcaatgtg ctcagtaatc cggcttgaac
caaccgcaat 120agaaatatcc acaactctcc tcaccaaact caccctcatg
tctcaatact ctgctgcttc 180aggttgcagc gaaaacaata actcttctgt
agggagttct gtttattgcg gggctgaaat 240gtgtccgctt atcgacagtg
ccaatacaga actcctttat gcattctcag agtattcccc 300tttcgatatc
tcattggatt accatactca atagacgcta actctttact ttctcattac
360acaggattta ccccggcgat acggctggct acattgccgc cgaccacaca
aacgcccttc 420tgatcatctc gtttcgcaat agcgtgaccc ccacaaactt
catcaccgat tgggcattcc 480ttcaagtcag cgcgcctacc gcgtgctccg
gatgccgagc acataaaggg ttctggtcgg 540cggccgtggc cgccgacaag
gctttagatg gttccatcag ggaggcaaag gccagatacc 600cagagtacga
actgacgttg actgggcata gtttgggagg tgcacttgca acgcttcatg
660caattttcct gaggaataga ggagttgctg ttgattctgt aagttgaggc
tttgccgcaa 720tgacgacccg agcaacttga tggctgctga tgctgactgg
actgctagta taccttcggc 780gcgccatcgg ttggtgacta cgcaatggcc
gattacatca cgaacgggcc cggtagcgac 840aatgggagga actatcgcgt
tacgcacctg aatgacgtct ttccaaaaat gctctaccgt 900gcgtctagga
tgccggttgc agatcggctg gtacaagagt acagccagtc cgggccagag
960tactggatta cgtctggctt cggcgagcct gttacaactg cggatgtgca
catccttgag 1020ggcgtggata atgagcaggg caatctggga agagaacctg
gcagtctgag ggaccatatg 1080tggtatttgg gggcgacaga tgcttgccca
ctaggctga 111914308PRTAspergillus nidulans 14Met Tyr Phe Leu Leu
Ser Val Ile Phe His Phe Pro Val Phe Cys Ala 1 5 10 15 Gly Phe Pro
Pro Ala Val Ser Arg Glu Ile Ser Thr Thr Leu Leu Thr 20 25 30 Lys
Leu Thr Leu Met Ser Gln Tyr Ser Ala Ala Ser Gly Cys Ser Glu 35 40
45 Asn Asn Asn Ser Ser Val Gly Ser Ser Val Tyr Cys Gly Ala Glu Met
50 55 60 Cys Pro Leu Ile Asp Ser Ala Asn Thr Glu Leu Leu Tyr Ala
Phe Ser 65 70 75 80 Glu Ile Tyr Pro Gly Asp Thr Ala Gly Tyr Ile Ala
Ala Asp His Thr 85 90 95 Asn Ala Leu Leu Ile Ile Ser Phe Arg Asn
Ser Val Thr Pro Thr Asn 100 105 110 Phe Ile Thr Asp Trp Ala Phe Leu
Gln Val Ser Ala Pro Thr Ala Cys 115 120 125 Ser Gly Cys Arg Ala His
Lys Gly Phe Trp Ser Ala Ala Val Ala Ala 130 135 140 Asp Lys Ala Leu
Asp Gly Ser Ile Arg Glu Ala Lys Ala Arg Tyr Pro 145 150 155 160 Glu
Tyr Glu Leu Thr Leu Thr Gly His Ser Leu Gly Gly Ala Leu Ala 165 170
175 Thr Leu His Ala Ile Phe Leu Arg Asn Arg Gly Val Ala Val Asp Ser
180 185 190 Tyr Thr Phe Gly Ala Pro Ser Val Gly Asp Tyr Ala Met Ala
Asp Tyr 195 200 205 Ile Thr Asn Gly Pro Gly Ser Asp Asn Gly Arg Asn
Tyr Arg Val Thr 210 215 220 His Leu Asn Asp Val Phe Pro Lys Met Leu
Tyr Arg Ala Ser Arg Met 225 230 235 240 Pro Val Ala Asp Arg Leu Val
Gln Glu Tyr Ser Gln Ser Gly Pro Glu 245 250 255 Tyr Trp Ile Thr Ser
Gly Phe Gly Glu Pro Val Thr Thr Ala Asp Val 260 265 270 His Ile Leu
Glu Gly Val Asp Asn Glu Gln Gly Asn Leu Gly Arg Glu 275 280 285 Pro
Gly Ser Leu Arg Asp His Met Trp Tyr Leu Gly Ala Thr Asp Ala 290 295
300 Cys Pro Leu Gly 305 151283DNAAspergillus nidulans 15atgacggtgt
ctcttgacag tttattcctt acacttatta tattcctcac gaggctatgc 60agcgtctcga
ccgctcacgt ggtacccctt gaggccagca aggatcccga aaatatcacg
120ccagggaggc aaatctccca ggaactattt gactctattg aggagctggc
tcatattgtc 180gatatcgcct actgcattgg gactactggc attagaaagc
cgttccaatg cctcagtcac 240tgtgatgagc taaaagggtt tgaactaatc
aacgtgcgct tttccacaga cgatctaccc 300gagccgtttg agagatagta
gctgactaga tagaaactca gacatggcat acaggtccct 360ttctctctga
ttcctgcggc tacatcgccc tctcgcatcc cccctcaccg aagcgaatca
420tagtcgcttt ccgcggtaca tactcaatcc cgaacgcaat agttgacctt
tccatgtatc 480cccaggaata cataccgttt tccccaggca acgatactga
cggcgatgca ccgaagtgcg 540aggactgttg ggtccattta ggcttcatga
acgcatggcg tttaacccgc gcaacaatcc 600tagacaccat ctccgcagca
agagaccaat accctgatta cgctctaacc ctagtaggcc 660actctctcgg
cggcgcagtt gccgctctcg caggaacaga aatgcagctc cgcggatggg
720aacccgtcgt gacgactttc ggggaaccaa gggtagggaa taaggcgttt
gtcgactatc 780tagacaccgt gttccgcctg gaatctggca atgagcgggt
gtggaaattc cgccgggtga 840cgcatgtgaa tgaccctgta cccctaatcc
cgcttacaga atggggctac gagatgcaca 900gcggagagat ttatattgac
cgcgttgagc ttccattttc tgttgacgat gtcaggtact 960gccagggcgg
gtccgatcca aactgcattt cagacgcgga ggggaagagc acaactttct
1020ccccatatag ctcgcagggc tttgatctct ccgaatccaa catggagcag
caagtccttt 1080cgcgctcgcc gcaccagtcg aaggatcagc aacaggagaa
tgagaaggga gcttttccat 1140atctggaatc ccagagtacc tcgtgtctgc
catggggtat acttccacct aggtttcgac 1200tgtgggagct attctactct
catcgtgact actttattcg tttggggctt tgcgttccca 1260agggagattt
gtcagggggg tga 128316404PRTAspergillus nidulans 16Met Thr Val Ser
Leu Asp Ser Leu Phe Leu Thr Leu Ile Ile Phe Leu 1 5 10 15 Thr Arg
Leu Cys Ser Val Ser Thr Ala His Val Val Pro Leu Glu Ala 20 25 30
Ser Lys Asp Pro Glu Asn Ile Thr Pro Gly Arg Gln Ile Ser Gln Glu 35
40 45 Leu Phe Asp Ser Ile Glu Glu Leu Ala His Ile Val Asp Ile Ala
Tyr 50 55 60 Cys Ile Gly Thr Thr Gly Ile Arg Lys Pro Phe Gln Cys
Leu Ser His 65 70 75 80 Cys Asp Glu Leu Lys Gly Phe Glu Leu Ile Asn
Thr Trp His Thr Gly 85 90 95 Pro Phe Leu Ser Asp Ser Cys Gly Tyr
Ile Ala Leu Ser His Pro Pro 100 105 110 Ser Pro Lys Arg Ile Ile Val
Ala Phe Arg Gly Thr Tyr Ser Ile Pro 115 120 125 Asn Ala Ile Val Asp
Leu Ser Met Tyr Pro Gln Glu Tyr Ile Pro Phe 130 135 140 Ser Pro Gly
Asn Asp Thr Asp Gly Asp Ala Pro Lys Cys Glu Asp Cys 145 150 155 160
Trp Val His Leu Gly Phe Met Asn Ala Trp Arg Leu Thr Arg Ala Thr 165
170 175 Ile Leu Asp Thr Ile Ser Ala Ala Arg Asp Gln Tyr Pro Asp Tyr
Ala 180 185 190 Leu Thr Leu Val Gly His Ser Leu Gly Gly Ala Val Ala
Ala Leu Ala 195 200 205 Gly Thr Glu Met Gln Leu Arg Gly Trp Glu Pro
Val Val Thr Thr Phe 210 215 220 Gly Glu Pro Arg Val Gly Asn Lys Ala
Phe Val Asp Tyr Leu Asp Thr 225 230 235 240 Val Phe Arg Leu Glu Ser
Gly Asn Glu Arg Gly Trp Lys Phe Arg Arg 245 250 255 Val Thr His Val
Asn Asp Pro Val Pro Leu Ile Pro Leu Thr Glu Trp 260 265 270 Gly Tyr
Glu Met His Ser Gly Glu Ile Tyr Ile Asp Arg Val Glu Leu 275 280 285
Pro Phe Ser Val Asp Asp Val Arg Tyr Cys Gln Gly Gly Ser Asp Pro 290
295 300 Asn Cys Ile Ser Asp Ala Glu Gly Lys Ser Thr Thr Phe Ser Pro
Tyr 305 310 315 320 Ser Ser Gln Gly Phe Asp Leu Ser Glu Ser Asn Met
Glu Gln Gln Val 325 330 335 Leu Ser Arg Ser Pro His Gln Ser Lys Asp
Gln Gln Gln Glu Asn Glu 340 345 350 Lys Gly Ala Phe Pro Tyr Leu Glu
Ser Gln Ser Thr Ser Cys Leu Pro 355 360 365 Trp Gly Ile Leu Pro Pro
Arg Phe Arg Leu Trp Glu Leu Phe Tyr Ser 370 375 380 His Arg Asp Tyr
Phe Ile Arg Leu Gly Leu Cys Val Pro Lys Gly Asp 385 390 395 400 Leu
Ser Gly Gly 1724DNAAspergillus fumigatus 17gagacgcatg cttcacaagt
atag 241833DNAAspergillus fumigatus 18gtcacctcta gttaattaat
cagattatct tgc 331922DNAAspergillus fumigatus 19gtgccccatg
atacgcctcc gg 222026DNAAspergillus fumigatus 20gagtcgtatt
tccaaggctc ctgacc 262124DNAAspergillus fumigatus 21ggaggccatg
aagtggacca acgg 242245DNAAspergillus fumigatus 22caccgtgaaa
gccatgctct ttccttcgtg tagaagacca gacag 452345DNAAspergillus
fumigatus 23ctggtcttct acacgaagga aagagcatgg ctttcacggt gtctg
452444DNAAspergillus fumigatus 24ctatatacac aactggattt accatgggcc
cgcggccgca gatc 442544DNAAspergillus fumigatus 25gatctgcggc
cgcgggccca tggtaaatcc agttgtgtat atag 442626DNAAspergillus
fumigatus 26gtcgacatgg tgttttgatc atttta 262726DNAAspergillus
fumigatus 27ccatggccag ttgtgtatat agagga 262830DNAAspergillus
fumigatus 28tacacaactg gccatgcttc acaagtatag 302933DNAAspergillus
fumigatus 29gtcacctcta gttaattaat cagattatct tgc
333020DNAAspergillus fumigatus 30gagacacatg tttcacccag
203133DNAAspergillus fumigatus 31gtcacctcta gttaattaat cagttagttg
agc 333221DNAMagnaporthe grisea 32ccttgcccac gcctttggtt c
213321DNAMagnaporthe grisea 33ctcatagcag caggcgaagc c
213436DNAMagnaporthe grisea 34acacaactgg ccatgaaggt ctcgttcgtg
tcatcg 363535DNAMagnaporthe grisea 35agtcacctct agttatcagt
agcaagcgct aatgg 353633DNAMagnaporthe grisea 36ccatggccat
gatgaggttc cccagcgtgc tca 333729DNAMagnaporthe grisea 37tttaattaag
ccacggtctt gttggcttc 293840DNAMagnaporthe grisea 38acacaactgg
ccatgttgtg gcgtcgggcg ggtggcctct 403947DNAMagnaporthe grisea
39agtcacctct agttaattaa ttagagctca tcctggccag gagccac
474036DNAAspergillus nidulans 40acacaactgg ccatgatccg tttggggtat
tctgcc 364140DNAAspergillus nidulans 41agtcacctct agttaattaa
ttactggcag gcagtgatat 404236DNAAspergillus nidulans 42acacaactgg
ccatgtattt ccttctctcc gtcatc 364340DNAAspergillus nidulans
43agtcacctct agttaattaa tcagcctagt gggcaagcat 404440DNAAspergillus
nidulans 44acacaactgg ccatgacggt gtctcttgac agtttattcc
404546DNAAspergillus nidulans 45agtcacctct agttaattaa tcacccccct
gacaaatctc ccttgg
46
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