U.S. patent application number 10/000845 was filed with the patent office on 2004-10-07 for methods for modifying the production of a polypeptide.
This patent application is currently assigned to Novozymes Biotech, Inc.. Invention is credited to Brody, Howard, Hansen, Kim, Lamsa, Michael H., Yaver, Debbie S..
Application Number | 20040197854 10/000845 |
Document ID | / |
Family ID | 24865645 |
Filed Date | 2004-10-07 |
United States Patent
Application |
20040197854 |
Kind Code |
A1 |
Brody, Howard ; et
al. |
October 7, 2004 |
Methods for modifying the production of a polypeptide
Abstract
The present invention relates to methods for modifying the
production of a polypeptide, comprising: (a) introducing a nucleic
acid construct into a cell, wherein the cell comprises a DNA
sequence encoding a polypeptide, under conditions in which the
nucleic acid construct integrates into the genome of the cell at a
locus not within the DNA sequence encoding the polypeptide to
produce a mutant cell, wherein the integration of the nucleic acid
construct modifies the production of the polypeptide by the mutant
cell relative to the cell when the mutant cell and the cell are
cultured under the same conditions; and (b) identifying the mutant
cell with the modified production of the polypeptide.
Inventors: |
Brody, Howard; (Davis,
CA) ; Yaver, Debbie S.; (Davis, CA) ; Lamsa,
Michael H.; (Davis, CA) ; Hansen, Kim;
(Bjaeverskov, DK) |
Correspondence
Address: |
NOVOZYMES BIOTECH, INC.
1445 DREW AVE
DAVIS
CA
95616
US
|
Assignee: |
Novozymes Biotech, Inc.
Davis
CA
Novozymes A/S
Bagsvaerd
|
Family ID: |
24865645 |
Appl. No.: |
10/000845 |
Filed: |
October 24, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10000845 |
Oct 24, 2001 |
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08928692 |
Sep 12, 1997 |
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5958727 |
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08928692 |
Sep 12, 1997 |
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08713312 |
Sep 13, 1996 |
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Current U.S.
Class: |
435/69.1 ;
435/320.1; 435/325 |
Current CPC
Class: |
C12N 9/20 20130101; C12N
9/2434 20130101; C12Y 302/01004 20130101; C12P 21/02 20130101; C12N
15/80 20130101; C12N 15/67 20130101 |
Class at
Publication: |
435/069.1 ;
435/320.1; 435/325 |
International
Class: |
C12P 021/02; C12N
005/06 |
Claims
What is claimed is:
1. A method of producing a polypeptide, comprising (a) cultivating
a mutant cell under conditions conducive for production of the
polypeptide, wherein (i) the mutant cell is related to a parent
cell, which comprises a first DNA sequence encoding the
polypeptide, by the introduction of a nucleic acid construct into
the genome of the parent cell at a locus which is not within the
first DNA sequence, not within a second DNA sequence encoding a
protein that negatively regulates transcription, translation or
secretion of the polypeptide, and not within a third DNA sequence
encoding a protease capable of hydrolyzing the polypeptide under
the conditions; and (ii) the mutant cell produces more of the
polypeptide than the parent cell when both cells are cultivated
under the conditions; and (b) recovering the polypeptide.
2. The method of claim 1, wherein the nucleic acid construct has
less than 40% homology with the first DNA sequence.
3. The method of claim 1, wherein the nucleic acid construct has
less than 40% homology with the locus.
4. The method of claim 1, wherein the locus is on a different
chromosome than the first DNA sequence or on the same chromosome
but at least 3,000 bps from the 5' or 3' terminus of the first DNA
sequence.
5. A method of producing a polypeptide, comprising (A) cultivating
a mutant cell under conditions conducive for production of the
polypeptide, wherein (i) the mutant cell is related to a parent
cell, which comprises a first DNA sequence encoding the
polypeptide, by the introduction of a nucleic acid construct into
the genome of the parent cell at a locus which is not within the
first DNA sequence, wherein the introduction of the nucleic acid
construct disrupts a gene encoding an oxidoreductase, a
transferase, a hydrolase, a lyase, an isomerase, a ligase, or
regulatory or control sequences thereof, other than a gene encoding
a protease which is capable of hydrolyzing the polypeptide under
the conditions; and (ii) the mutant cell produces more of the
polypeptide than the parent cell when both cells are cultivated
under the conditions; and (B) recovering the polypeptide.
6. A method of producing a polypeptide, comprising (a) cultivating
a mutant cell under conditions conducive for production of the
polypeptide, wherein (i) the mutant cell is related to a parent
cell, which comprises a first DNA sequence encoding the
polypeptide, by the introduction of a nucleic acid construct into
the genome of the parent cell at a locus which is not within the
first DNA sequence, not within a second DNA sequence encoding a
protein that negatively regulates transcription of the polypeptide,
and not within a third DNA sequence encoding a protease capable of
hydrolyzing the polypeptide under the conditions; and (ii) the
mutant cell expresses more of the polypeptide than the parent cell
when both cells are cultivated under the conditions; and (b)
recovering the polypeptide.
7. The method of claim 6, wherein the nucleic acid construct has
less than 40% homology with the first DNA sequence.
8. The method of claim 6, wherein the nucleic acid construct has
less than 40% homology with the locus.
9. The method of claim 6, wherein the locus is on a different
chromosome than the first DNA sequence or on the same chromosome
but at least 3,000 bps from the 5' or 3' terminus of the first DNA
sequence.
10. A method of producing a polypeptide, comprising (A) cultivating
a mutant cell under conditions conducive for production of the
polypeptide, wherein (i) the mutant cell is related to a parent
cell, which comprises a first DNA sequence encoding the
polypeptide, by the introduction of a nucleic acid construct into
the genome of the parent cell at a locus which is not within the
first DNA sequence, wherein the introduction of the nucleic acid
construct disrupts a gene encoding an oxidoreductase, a
transferase, a hydrolase, a lyase, an isomerase, a ligase, or
regulatory or control sequences thereof, other than a gene encoding
a protease which is capable of hydrolyzing the polypeptide under
the conditions; and (ii) the mutant cell expresses more of the
polypeptide than the parent cell when both cells are cultivated
under the conditions; and (B) recovering the polypeptide.
11. A method of producing a polypeptide, comprising (a) cultivating
a mutant cell under conditions conducive for production of the
polypeptide, wherein (i) the mutant cell is related to a parent
cell, which comprises a first DNA sequence encoding the
polypeptide, by the introduction of a nucleic acid construct into
the genome of the parent cell at a locus which is not within the
first DNA sequence, not within a second DNA sequence encoding a
protein that negatively regulates translation of the polypeptide,
and not within a third DNA sequence encoding a protease capable of
hydrolyzing the polypeptide under the conditions; and (ii) the
mutant cell synthesizes more of the polypeptide than the parent
cell when both cells are cultivated under the conditions; and (b)
recovering the polypeptide.
12. The method of claim 11, wherein the nucleic acid construct has
less than 40% homology with the first DNA sequence.
13. The method of claim 11, wherein the nucleic acid construct has
less than 40% homology with the locus.
14. The method of claim 11, wherein the locus is on a different
chromosome than the first DNA sequence or on the same chromosome
but at least 3,000 bps from the 5' or 3' terminus of the first DNA
sequence.
15. A method of producing a polypeptide, comprising (A) cultivating
a mutant cell under conditions conducive for production of the
polypeptide, wherein (i) the mutant cell is related to a parent
cell, which comprises a first DNA sequence encoding the
polypeptide, by the introduction of a nucleic acid construct into
the genome of the parent cell at a locus which is not within the
first DNA sequence, wherein the introduction of the nucleic acid
construct disrupts a gene encoding an oxidoreductase, a
transferase, a hydrolase, a lyase, an isomerase, a ligase, or
regulatory or control sequences thereof, other than a gene encoding
a protease which is capable of hydrolyzing the polypeptide under
the conditions; and (ii) the mutant cell synthesizes more of the
polypeptide than the parent cell when both cells are cultivated
under the conditions; and (B) recovering the polypeptide.
16. A method of producing a polypeptide, comprising (a) cultivating
a mutant cell under conditions conducive for production of the
polypeptide, wherein (i) the mutant cell is related to a parent
cell, which comprises a first DNA sequence encoding the
polypeptide, by the introduction of a nucleic acid construct into
the genome of the parent cell at a locus which is not within the
first DNA sequence, not within a second DNA sequence encoding a
protein that negatively regulates secretion of the polypeptide, and
not within a third DNA sequence encoding a protease capable of
hydrolyzing the polypeptide under the conditions; and (ii) the
mutant cell secretes more of the polypeptide than the parent cell
when both cells are cultivated under the conditions; (b) recovering
the polypeptide.
17. The method of claim 16, wherein the nucleic acid construct has
less than 40% homology with the first DNA sequence.
18. The method of claim 16, wherein the nucleic acid construct has
less than 40% homology with the locus.
19. The method of claim 16, wherein the locus is on a different
chromosome than the first DNA sequence or on the same chromosome
but at least 3,000 bps from the 5' or 3' terminus of the first DNA
sequence.
20. A method of producing a polypeptide, comprising (A) cultivating
a mutant cell under conditions conducive for production of the
polypeptide, wherein (i) the mutant cell is related to a parent
cell, which comprises a first DNA sequence encoding the
polypeptide, by the introduction of a nucleic acid construct into
the genome of the parent cell at a locus which is not within the
first DNA sequence, wherein the introduction of the nucleic acid
construct disrupts a gene encoding an oxidoreductase, a
transferase, a hydrolase, a lyase, an isomerase, a ligase, or
regulatory or control sequences thereof, other than a gene encoding
a protease which is capable of hydrolyzing the polypeptide under
the conditions; and (ii) the mutant cell secretes more of the
polypeptide than the parent cell when both cells are cultivated
under the conditions; and (B) recovering the polypeptide.
21. A method of producing a polypeptide, comprising (a) cultivating
a mutant cell under conditions conducive for production of the
polypeptide, wherein (i) the mutant cell is related to a parent
cell, which comprises a first DNA sequence encoding the
polypeptide, by the random integration of a nucleic acid construct
into the genome of the parent cell at a locus wherein the nucleic
acid construct is not homologous with the locus and wherein the
locus is not within the first DNA sequence nor within a second DNA
sequence encoding a protease capable of hydrolyzing the polypeptide
under the conditions; and (ii) the mutant cell produces more of the
polypeptide than the parent cell when both cells are cultivated
under the conditions; and (b) recovering the polypeptide.
22. The method of claim 1, wherein the nucleic acid construct is
introduced by restriction enzyme-mediated integration.
23. The method of claim 1, wherein the nucleic acid construct
comprises a selectable marker.
24. The method of claim 23, wherein the selectable marker is amdS,
argB, bar, hygB, niaD, pyrG, sC, or trpC.
25. The method of claim 1, wherein the parent cell is a mammalian
cell.
26. The method of claim 1, wherein the parent cell is a bacterial
cell.
27. The method of claim 1, wherein the parent cell is a fungal
cell.
28. The method of claim 27, wherein the fungal cell is a
filamentous fungal cell.
29. The method of claim 28, wherein the filamentous fungal cell is
selected from the group consisting of Acremonium, Aspergillus,
Fusarium, Humicola, Mucor, Myceliophthora, Neurospora, Penicillium,
Scytalidium, Thielavia, Tolypocladium, and Trichodenna.
30. The method of claim 27, wherein the fungal cell is a yeast
cell.
31. The method of claim 1, wherein the polypeptide is a recombinant
polypeptide.
32. The method of claim 1, wherein the polypeptide is a
heterologous polypeptide.
33. The method of claim 1, wherein the polypeptide is a hormone, a
hormone variant, an enzyme, a receptor or portions thereof, an
antibody or portions thereof, or a reporter.
34. The method of claim 33, wherein the polypeptide is an
oxidoreductase. a transferase, a hydrolase, a lyase, an isomerase,
or a ligase.
35. The method of claim 34, wherein the polypeptide is an
aminopeptidase, an amylase, a carbohydrase, a carboxypeptidase, a
catalase, a cellulase, a chitinase, a cutinase, a
deoxyribonuclease, an esterase, an alpha-galactosidase, a
beta-galactosidase, a glucoamylase, an alpha-glucosidase, a
beta-glucosidase, an invertase, a laccase, a lipase, a mannosidase,
a mutanase, an oxidase, a pectinolytic enzyme, a peroxidase, a
phytase, a polyphenoloxidase, a proteolytic enzyme, a ribonuclease,
or a xylanase.
36. The method of claim 1, wherein the mutant cell has an increased
uptake of an inorganic cofactor compared to the parent cell.
37. The method of claim 1, wherein the mutant cell has a more
desirable morphology than the parent cell.
38. The method of claim 1, wherein the mutant cell produces higher
yields of one or more secreted proteins than the parent cell.
39. The method of claim 1, wherein the mutant cell which has lost
its ability to synthesize one or more essential metabolites.
40. The method of claim 1, wherein a phenotype of the mutant cell
is observed only under certain conditions.
41. The method of claim 1, wherein the mutant cell exhibits an
altered growth rate relative to the parent cell.
42. The method of claim 1, wherein the growth of the mutant cell is
not inhibited by the overproduction of a desired polypeptide or
metabolite when grown under conditions that induce high level
production of the polypeptide or metabolite.
43. The method of claim 1, wherein the mutant cell is able to
tolerate lower oxygen conditions than the parent cell.
44. The method of claim 1, wherein the mutant cell exhibits altered
production of a transcriptional activator of a promoter than the
parent cell.
45. The method of claim 1, wherein the mutant cell has a mutation
in on e or more of the genes of the signal transduction pathway of
the parent cell.
46. The method of claim 1, wherein the mutant cell does not
recognize and erroneously splice a cryptic intron.
47. The method of claim 1, wherein the nucleic acid construct is
pDSY109, pDSY112, pMT1936, pDSY138, pDSY162, pDSY163, pDSY141,
pSMO1204, pSMOH603, p4-8.1, p7-14.1, pHB220, pSMO717, pSMO321,
pHowB571 or pSMO810.
48. The method of claim 1, wherein the locus is SEQ ID NO:9, SEQ ID
NO: 16, SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:34, SEQ ID NO:39, SEQ
ID NO:50, SEQ ID NO:56, SEQ ID NO:63, SEQ ID NO:66, SEQ ID NO:71,
SEQ ID NO:76, or a fragment thereof.
49. The method of claim 1, wherein the locus encodes a glucose
transporter, mannitol-1-phosphate dehydrogenase, chitin synthase,
heat shock protein, manganese superoxide dismutase, or a gene
required for activation of pacC,
50. The method of claim 49, wherein the locus is a palB gene.
51. A method of producing a polypeptide, comprising (a) cultivating
a mutant cell under conditions conducive for production of the
polypeptide, wherein (i) the mutant cell is related to a parent
cell, which comprises a first DNA sequence encoding the
polypeptide, by the introduction of a nucleic acid construct into
the genome of the parent cell at a locus which is not within the
first DNA sequence and a second DNA sequence encoding a protein
that positively regulates transcription, translation or secretion
of the polypeptide; and (ii) the mutant cell produces less of the
polypeptide than the parent cell when both cells are cultivated
under the conditions; and (b) recovering the polypeptide.
52. A method of producing a metabolite, comprising (A) cultivating
a mutant cell under conditions conducive for production of the
metabolite, wherein (i) the mutant cell is related to a parent
cell, which comprises one or more first DNA sequences encoding
first polypeptides in the biosynthetic pathway of the metabolite,
by the introduction of a nucleic acid construct into the genome of
the parent cell at a locus which is not within (a) the first DNA
sequences, (b) a second DNA sequence encoding a protein that
negatively regulates transcription, translation or secretion of the
first polypeptides, (c) a third DNA sequence encoding a protease
capable of hydrolyzing any of the first polypeptides under the
conditions, and (d) one or more fourth DNA sequences encoding a
second polypeptide in the second biosynthetic pathway of a second
metabolite wherein the biosynthetic pathway and the second
biosynthetic pathway involve the production of the same
intermediate and the second polypeptide catalyzes a step after the
production of the intermediate; and (ii) the mutant cell produces
more of the metabolite than the parent cell when both cells are
cultivated under the conditions; and (B) recovering the
metabolite.
53. A method of producing a first polypeptide, comprising (a)
forming a mutant cell by introducing a nucleic acid construct into
the genome of the parent cell, which comprises a first DNA sequence
encoding the polypeptide, at a locus which is not within the first
DNA sequence, a second DNA sequence encoding a protein that
negatively regulates transcription, translation or secretion of a
second polypeptide, and a third DNA sequence encoding a protease
capable of hydrolyzing the polypeptide under conditions conducive
to the production of the first polypeptide; (b) isolating the
mutant cell which produces more of the polypeptide than the parent
cell when both cells are cultivated under the conditions; (c)
identifying the locus wherein the nucleic acid construct has been
integrated; (d) producing a cell in which a corresponding locus has
been disrupted; (e) culturing the cell under the conditions; and
(f) recovering the first polypeptide.
54. A method of producing a polypeptide, comprising (a) cultivating
a mutant cell under conditions conducive for production of the
polypeptide, wherein (i) the mutant cell is related to a parent
cell, which comprises a first DNA sequence encoding the
polypeptide, by the introduction of a nucleic acid construct into
the genome of the parent cell at a locus which is not within the
first DNA sequence and a second DNA sequence encoding a protease
capable of hydrolyzing the polypeptide under the conditions,
wherein the introduction of the nucleic acid construct specifically
enhances transcription, translation or secretion of the
polypeptide; and (ii) the mutant cell produces more of the
polypeptide than the parent cell when both cells are cultivated
under the conditions; and (b) recovering the polypeptide.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of pending U.S.
application Ser. No. 08/713,312 filed on Sep. 13, 1996, which
application is fully incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] This invention relates to methods for modifying the
production of a polypeptide by a cell.
[0004] 2. Description of the Related Art
[0005] Several methods have been used to modify the production of
polypeptides by mutagenizing cells. For example, the production of
proteins has been altered by producing mutant cells by classical
mutagenesis which involves treating cells with chemical, physical,
and biological agents as mutagenic (mutation inducing) agents to
increase the frequency of mutational events.
[0006] Production of proteins also has been modified by mutagenesis
of a cell with short sections of double-stranded DNA, consisting of
more than 2000 base pairs, called transposons which usually code
for resistance to one or sometimes several antibiotics. Transposons
are able to move or jump within the genome, even between a
bacterial chromosome and a plasmid, and they are able to become
integrated in a number of different sites on the genome. An
insertion of a transposon within a structural gene interrupts the
normal nucleotide sequence of the gene so that it can no longer
deliver the information for the synthesis of the normal, functional
polypeptide (Seifert et al., 1986, Proceedings of the National
Academy of Sciences USA 83: 735-739). An insertion also may disrupt
a gene whose gene product is required for expression (Mrquez-Magaa
and Chamberlin, 1994, Journal of Bacteriology 176: 2427-2434). In
addition, Errede et al. (1980, Cell 22: 427-436) disclose the
insertion of a transposable element adjacent to the structural gene
coding for iso-2-cytochrome c causing overproduction. Furthermore,
WO 96/29414 discloses that transposable elements may be constructed
containing a transposon and a DNA sequence capable of regulating a
targeted gene where upon introduction into a cell the transposable
element integrates into the genome of the cell in a manner which
regulates the expression of the gene.
[0007] A widely used method for increasing production of a
polypeptide is amplification to produce multiple copies of the gene
encoding the polypeptide. For example, U.S. Pat. No. 5,578,461
discloses the inclusion via homologous recombination of an
amplifiable selectable marker gene in tandem with the gene where
cells containing amplified copies of the selectable marker can be
selected for by culturing the cells in the presence of the
appropriate selectable agent.
[0008] In addition, the production of polypeptides has been
increased by replacing one promoter with a different promoter or
one signal peptide coding region with another. See, e.g., U.S. Pat.
No. 5,641,670.
[0009] Methods for altering gene expression by disrupting genes
encoding various regulatory elements have also been described. For
example, Toma et al. (1986, Journal of Bacteriology 167: 740-743)
showed that a deletion from -156 to -90 in the npr promoter region
caused overexpression of the neutral protease encoded by the npr
gene. Pero and Sloma (1993, In A. L. Sonensheim, J. A. Hoch, and R.
Losick, editors, Bacillus subtilis and Other Gram-Positive
Bacteria, pp. 939-952, American Society for Microbiology,
Washington, D.C.) disclose that mutating the sporulation gene spoOA
results in deficient synthesis of proteases and that mutations in
the abrB gene restore synthesis.
[0010] The production of polypeptides also has been increased by
disrupting DNA sequences encoding a protease capable of hydrolyzing
the polypeptide under the conditions for producing the
polypeptide.
[0011] The secretion of polypeptides has also been modified by
overproduction of secretion proteins (Ruohonen et al., 1997, Yeast,
13: 337-351), and producing a super-secreting cell (U.S. Pat. No.
5,312,735).
[0012] Methods for increasing the production of metabolites have
also been described. For example, WO 96/41886 discloses that
increased production of clavam produced by an organism having at
least part of the clavam pathway and at least part of a
cephalosporin pathway by interfering with the conversion of
L-lysine to L-alpha-aminoadipic acid in the cephalosporin pathway.
WO 94/13813 discloses the disruption of gene which encodes a
protein which degrades betaine, an enzyme inducer.
[0013] It is an object of the present invention to provide new and
improved methodologies for altering production of polypeptides and
metabolites.
BRIEF SUMMARY OF THE INVENTION
[0014] The present invention is drawn to methods for modifying the
production of a polypeptide by a cell. In the methods of the
present invention, a nucleic acid construct is introduced into a
cell which contains a DNA sequence encoding a specific polypeptide.
The introduced nucleic acid construct integrates into the host
genome at a locus not within the DNA sequence of interest to
produce a mutant cell. The integration of the nucleic acid
construct into the locus modifies the production of the polypeptide
by the mutant cell relative to the parent cell. Mutant cells are
then identified in which the polypeptide's production is modified
by the mutant cell relative to the parent cell. Modification is
determined by comparing production of the polypeptide when the
mutant cell and the parent cell are cultured under the same
conditions.
[0015] An advantage of the present invention is that the mutation
can be recovered and leads to a modification of the production of a
polypeptide encoded by a DNA sequence which does not contain the
mutation.
BRIEF DESCRIPTION OF THE FIGURES
[0016] FIG. 1 is a restriction map of pJaL292.
[0017] FIG. 2 is a restriction map of pKS6.
[0018] FIG. 3 is a restriction map of pBANe13.
[0019] FIG. 4 is a restriction map of pBANe6.
[0020] FIG. 5 is a restriction map of pMHan37.
[0021] FIG. 6 is a restriction map of pBANe8.
[0022] FIG. 7 is a restriction map of pSO2.
[0023] FIG. 8 is a restriction map of pSO122 and shows the
construction of pDSY81 and pDSY82 from pSO122.
[0024] FIG. 9 is the nucleic acid sequence and the deduced amino
acid sequence of the rescued locus of mutant Aspergillus oryzae
DEBY599.3 (SEQ ID NOS:9 and 10, respectively).
[0025] FIG. 10 is the nucleic acid sequence and the deduced amino
acid sequence of the rescued locus of mutant Aspergillus oryzae
DEBY10.3 (SEQ ID NOS: 16 and 17).
[0026] FIG. 11 is a restriction map of pJaL400.
[0027] FIG. 12 is the construction of pMT1935.
[0028] FIG. 13 is a restriction map of pJaL394.
[0029] FIG. 14 is a restriction map of pMT1931.
[0030] FIG. 15 is a restriction map of pMT1936.
[0031] FIG. 16 is the nucleic acid sequence and the deduced amino
acid sequence of the rescued locus of mutant Aspergillus oryzae
DEBY932 (SEQ ID NOS:25 and 26).
[0032] FIG. 17 is the nucleic acid sequence and deduced amino acid
sequence of the rescued locus of mutant Aspergillus oryzae DEBY1058
(SEQ ID NOS:29 and 30).
[0033] FIG. 18 is a restriction map of pDSY161.
[0034] FIG. 19 is a restriction map of pDSY162.
[0035] FIG. 20 is the nucleic acid sequence of the rescued locus of
mutant Aspergillus oryzae 1204.3.3 (SEQ ID NO:34).
[0036] FIG. 21 is the nucleic acid sequence of the rescued locus of
mutant Aspergillus oryzae H603 (SEQ ID NO:39).
[0037] FIG. 22 is a restriction map of pGAG3.
[0038] FIG. 23 is a restriction map of pJaL389.
[0039] FIG. 24 is a restriction map of pJaL335.
[0040] FIG. 25 is a restriction map of pJaL399.
[0041] FIG. 26 is a restriction map of pDM176.
[0042] FIG. 27 is a restriction map of pHB218.
[0043] FIG. 28 is a restriction map of pSE39.
[0044] FIG. 29 is a restriction map of pDSY153.
[0045] FIG. 30 is a restriction map of pCaHj505.
[0046] FIG. 31 is a restriction map of pMStr107.
[0047] FIG. 32 is the nucleic acid sequence and deduced amino acid
sequence of the rescued locus of mutant Aspergillus oryzae P4-8.1
(SEQ ID NOS:50 and 51).
[0048] FIG. 33 is the nucleic acid sequence and deduced amino acid
sequence of the rescued locus of mutant Aspergillus oryzae P7-14.1
(SEQ ID NOS:56 and 57).
[0049] FIG. 34 is a restriction map of pMT1612.
[0050] FIG. 35 is the nucleic acid sequence and deduced amino acid
sequence of the rescued locus of mutant Aspergillus oryzae
DEBY7-17.2 (SEQ ID NOS:63 and 64).
[0051] FIG. 36 is the nucleic acid sequence of the rescued locus of
mutant Aspergillus oryzae DEBY3-2.1 (SEQ ID NO:66).
[0052] FIG. 37 is the nucleic acid sequence of the rescued locus of
mutant Aspergillus oryzae DEBY5-7.1 (SEQ ID NO:71).
[0053] FIG. 38 is the nucleic acid sequence of the rescued locus of
mutant Aspergillus oryzae DEBY8-10.1 (SEQ ID NO:76).
DETAILED DESCRIPTION OF THE INVENTION
[0054] In a first embodiment, the present invention relates to
methods of producing a polypeptide, comprising
[0055] (a) cultivating a mutant cell under conditions conducive for
production of the polypeptide, wherein
[0056] (i) the mutant cell is related to a parent cell, which
comprises a first DNA sequence encoding the polypeptide, by the
introduction of a nucleic acid construct into the genome of the
parent cell at a locus which is not within the first DNA sequence,
not within a second DNA sequence encoding a protein that negatively
regulates transcription, translation or secretion of the
polypeptide, and not within a third DNA sequence encoding a
protease capable of hydrolyzing the polypeptide under the
conditions; and
[0057] (ii) the mutant cell produces more of the polypeptide than
the parent cell when both cells are cultivated under the
conditions; and
[0058] (b) recovering the polypeptide.
[0059] A "protein that negatively regulates transcription" is
defined herein as a repressor that negatively affects the process
of RNA synthesis by RNA polymerase to produce a single-stranded RNA
complementary to a DNA sequence, or as a protein that degrades an
enzyme inducer which is generally a chemical agent produced by a
biosynthetic or catabolic pathway of a cell. The repressor consists
of distinct domains that are required for DNA-binding,
transcription repression, and inducer or repressor binding.
[0060] A "protein that negatively regulates translation" is defined
herein as a protein or a substance, the production of which is
catalyzed by the protein, that negatively affects the process of
protein synthesis carried out by ribosomes which de-code the
information contained in mRNA derived from transcription of a gene.
For example, the substance may be a cap-dependent translation
initiation factor, e.g., p20 (Altmann et al., 1997, EMBO Journal
16: 1114-1121); or a sex-lethal protein, e.g., the sex-lethal
protein of Drosophila which regulates the translation of msl-2
(Bashaw and Baker, 1997, Cell 89: 789-798).
[0061] A "protein that negatively regulates secretion" is defined
herein as a protein or a substance, the production of which is
catalyzed by the protein, that negatively affects the process of
transferring a protein molecule through a membrane into (i) an
intracellular compartment, e.g., a vacuole or mitochrondrion, (ii)
the periplasmic space, or (iii) the culture medium and, in
eukaryotic cells, the process of vesicular transport that
ultimately results in exocytic release of secreted proteins from
the cell. The secretory process oversees and promotes correct
protein folding, mediates any required post-translational
modifications (such as glycosylation), and sorts, processes, and
targets proteins to specific cellular sites all at a rate
consistent with the function of the cell as a whole. Such
substances include a protein with Ca.sup.2+-ATPase activity which
upon inactivation increase levels of secreted heterologous or
mutant proteins (for example, see Rudolph et al., 1989, Cell 58:
133-145); or the binding protein BiP which is an ATP-dependent
hsp70-class chaperone found in the endoplasmic reticulum of
eukaryotic cells which when decreased in mammalian cells through
the use of anti-sense RNA results in up to a three-fold increase in
secreted levels of a mutant protein (Dorner et al., 1988, Molecular
and Cellular Biology 8: 4063-4070). In a specific embodiment, the
substance is a protein with ATPase activity or the binding protein
BiP.
[0062] In a second embodiment, the present invention relates to
methods of producing a polypeptide, comprising
[0063] (A) cultivating a mutant cell under conditions conducive for
production of the polypeptide, wherein
[0064] (i) the mutant cell is related to a parent cell, which
comprises a first DNA sequence encoding the polypeptide, by the
introduction of a nucleic acid construct into the genome of the
parent cell at a locus which is not within the first DNA sequence,
wherein the introduction of the nucleic acid construct disrupts a
gene encoding an oxidoreductase, a transferase, a hydrolase, a
lyase, an isomerase, a ligase, or regulatory or control sequences
thereof, other than a gene encoding a protease which is capable of
hydrolyzing the polypeptide under the conditions; and
[0065] (ii) the mutant cell produces more of the polypeptide than
the parent cell when both cells are cultivated under the
conditions; and
[0066] (B) recovering the polypeptide.
[0067] A mutant cell that "produces" more of a polypeptide is
defined herein as a cell from which more of the polypeptide is
recovered relative to the parent cell.
[0068] In a third embodiment, the present invention relates to
methods of producing a polypeptide, comprising
[0069] (a) cultivating a mutant cell under conditions conducive for
production of the polypeptide, wherein
[0070] (i) the mutant cell is related to a parent cell, which
comprises a first DNA sequence encoding the polypeptide, by the
introduction of a nucleic acid construct into the genome of the
parent cell at a locus which is not within the first DNA sequence,
not within a second DNA sequence encoding a protein that negatively
regulates transcription of the polypeptide, and not within a third
DNA sequence encoding a protease capable of hydrolyzing the
polypeptide under the conditions; and
[0071] (ii) the mutant cell expresses more of the polypeptide than
the parent cell when both cells are cultivated under the
conditions; and
[0072] (b) recovering the polypeptide.
[0073] In a fourth embodiment, the present invention relates to
methods of producing a polypeptide, comprising
[0074] (A) cultivating a mutant cell under conditions conducive for
production of the polypeptide, wherein
[0075] (i) the mutant cell is related to a parent cell, which
comprises a first DNA sequence encoding the polypeptide, by the
introduction of a nucleic acid construct into the genome of the
parent cell at a locus which is not within the first DNA sequence,
wherein the introduction of the nucleic acid construct disrupts a
gene encoding an oxidoreductase, a transferase, a hydrolase, a
lyase, an isomerase, a ligase, or regulatory or control sequences
thereof, other than a gene encoding a protease which is capable of
hydrolyzing the polypeptide under the conditions; and
[0076] (ii) the mutant cell expresses more of the polypeptide than
the parent cell when both cells are cultivated under the
conditions; and
[0077] (B) recovering the polypeptide.
[0078] A mutant cell that "expresses" more of a polypeptide is
defined herein as a cell that contains an increase in functional
mRNA encoding the polypeptide relative to the parent cell. It will
be understood that an increase in functional mRNA may result from
an increase in the absolute rate of transcription of the gene
encoding the polypeptide and/or from alterations in
post-transcriptional processing or modification of the transcripts,
including nuclear-cytoplasmic transport and/or cytoplasmic
stabilization of the mRNA. Such mutant cells may be identified
using conventional techniques, including without limitation
Northern blot analysis, run-off transcription assays, and the
like.
[0079] In a fifth embodiment, the present invention relates to
methods of producing a polypeptide, comprising
[0080] (a) cultivating a mutant cell under conditions conducive for
production of the polypeptide, wherein
[0081] (i) the mutant cell is related to a parent cell, which
comprises a first DNA sequence encoding the polypeptide, by the
introduction of a nucleic acid construct into the genome of the
parent cell at a locus which is not within the first DNA sequence,
not within a second DNA sequence encoding a protein that negatively
regulates translation of the polypeptide, and not within a third
DNA sequence encoding a protease capable of hydrolyzing the
polypeptide under the conditions; and
[0082] (ii) the mutant cell synthesizes more of the polypeptide
than the parent cell when both cells are cultivated under the
conditions; and
[0083] (b) recovering the polypeptide.
[0084] In a sixth embodiment, the present invention relates to
methods of producing a polypeptide, comprising
[0085] (A) cultivating a mutant cell under conditions conducive for
production of the polypeptide, wherein
[0086] (i) the mutant cell is related to a parent cell, which
comprises a first DNA sequence encoding the polypeptide, by the
introduction of a nucleic acid construct into the genome of the
parent cell at a locus which is not within the first DNA sequence,
wherein the-introduction of the nucleic acid construct disrupts a
gene encoding an oxidoreductase, a transferase, a hydrolase, a
lyase, an isomerase, a ligase, or regulatory or control sequences
thereof, other than a gene encoding a protease which is capable of
hydrolyzing the polypeptide under the conditions; and
[0087] (ii) the mutant cell synthesizes more of the polypeptide
than the parent cell when both cells are cultivated under the
conditions; and
[0088] (B) recovering the polypeptide.
[0089] A mutant cell that "synthesizes" more of a polypeptide is
defined herein as a cell that accumulates larger amounts of the
polypeptide relative to a parent cell. Accumulation refers to the
total amount of the polypeptide in the culture as a whole, i.e., in
both intracellular and extracellular compartments taken together.
Such mutant cells may be identified using any suitable technique,
including without limitation pulse-labelling or steady-state
labelling using radiolabelled amino acids; immunoblot analysis of
cell and medium fractions using an antibody specific to the
polypeptide; assays of biological activity; separation by
conventional chromatographic methods; and the like.
[0090] In a seventh embodiment, the present invention relates to
methods of producing a polypeptide, comprising
[0091] (a) cultivating a mutant cell under conditions conducive for
production of the polypeptide, wherein
[0092] (i) the mutant cell is related to a parent cell, which
comprises a first DNA sequence encoding the polypeptide, by the
introduction of a nucleic acid construct into the genome of the
parent cell at a locus which is not within the first DNA sequence,
not within a second DNA sequence encoding a protein that negatively
regulates secretion of the polypeptide, and not within a third DNA
sequence encoding a protease capable of hydrolyzing the polypeptide
under the conditions; and
[0093] (ii) the mutant cell secretes more of the polypeptide than
the parent cell when both cells are cultivated under the
conditions;
[0094] (b) recovering the polypeptide.
[0095] In an eighth embodiment, the present invention relates to
methods of producing a polypeptide, comprising
[0096] (A) cultivating a mutant cell under conditions conducive for
production of the polypeptide, wherein
[0097] (i) the mutant cell is related to a parent cell, which
comprises a first DNA sequence encoding the polypeptide, by the
introduction of a nucleic acid construct into the genome of the
parent cell at a locus which is not within the first DNA sequence,
wherein the introduction of the nucleic acid construct disrupts a
gene encoding an oxidoreductase, a transferase, a hydrolase, a
lyase, an isomerase, a ligase, or regulatory or control sequences
thereof, other than a gene encoding a protease which is capable of
hydrolyzing the polypeptide under the conditions; and
[0098] (ii) the mutant cell secretes more of the polypeptide than
the parent cell when both cells are cultivated under the
conditions; and
[0099] (B) recovering the polypeptide.
[0100] A mutant cell that "secretes" more of a polypeptide is
defined herein as a cell in which the amount of the polypeptide
released into the extracellular medium is increased relative to the
parent cell. Such mutant cells may be identified using, e.g.,
pulse-chase labelling in conjunction with immunoprecipitation to
quantify the proportion of the newly synthesized polypeptide that
is externalized as well as the absolute amount released in the
mutant cell relative to the parent cell. Immunoblot analysis,
biological activity assays, and physical-chemical separation
methods may also be used to quantify the absolute amounts of the
polypeptide released in mutant vs. parent cells.
[0101] In a ninth embodiment, the present invention relates to
methods of producing a polypeptide, comprising
[0102] (a) cultivating a mutant cell under conditions conducive for
production of the polypeptide, wherein
[0103] (i) the mutant cell is related to a parent cell, which
comprises a first DNA sequence encoding the polypeptide, by the
random integration of a nucleic acid construct into the genome of
the parent cell at a locus wherein the nucleic acid construct is
not homologous with the locus and wherein the locus is not within
the first DNA sequence nor within a second DNA sequence encoding a
protease capable of hydrolyzing the polypeptide under the
conditions; and
[0104] (ii) the mutant cell produces more of the polypeptide than
the parent cell when both cells are cultivated under the
conditions; and
[0105] (b) recovering the polypeptide.
[0106] In a tenth embodiment, the present invention relates to
methods of producing a polypeptide, comprising
[0107] (a) cultivating a mutant cell under conditions conducive for
production of the polypeptide, wherein
[0108] (i) the mutant cell is related to a parent cell, which
comprises a first DNA sequence encoding the polypeptide, by the
introduction of a nucleic acid construct into the genome of the
parent cell at a locus which is not within the first DNA sequence
and a second DNA sequence encoding a protein that positively
regulates transcription, translation or secretion of the
polypeptide; and
[0109] (ii) the mutant cell produces less of the polypeptide than
the parent cell when both cells are cultivated under the
conditions; and
[0110] (b) recovering the polypeptide.
[0111] A mutant cell that "produces" less of a polypeptide is
defined herein as a cell from which less of the polypeptide is
recovered relative to the parent cell.
[0112] A "protein that positively regulates transcription" is
defined herein as an activator or an inducer that positively
affects the process of RNA synthesis by RNA polymerase to produce a
single-stranded RNA complementary to a DNA sequence. The activator
consists of distinct domains that are required for DNA-binding,
transcription activation, and inducer or repressor binding. An
inducer is generally a chemical agent produced by a biosynthetic or
catabolic pathway of a cell. In a specific embodiment, the
substance is an activator or an inducer.
[0113] A "protein that positively regulates translation" is defined
herein as a protein or a substance, the production of which is
catalyzed by the protein, that positively affects the process of
protein synthesis carried out by ribosomes which de-code the
information contained in mRNA derived from transcription of a gene.
In a specific embodiment, the substance is an initiation factor or
an elongation factor.
[0114] A "protein that positively affects secretion" is defined
herein as a protein or a substance, the production of which is
catalyzed by the protein, that positively affects the process of
transferring a protein molecule through a membrane into (i) an
intracellular compartment, e.g., a vacuole or mitochrondrion, (ii)
the periplasmic space, or (iii) the culture medium or positively
affects vesicular transport as described above. Such substances
include folding proteins, e.g., protein disulfide isomerase and
peptidyl prolyl isomerase isoforms; chaperones, e.g., heat shock
proteins, signal recognition particles, PrsA, SecD, SecF, and BiP;
translocating chain-associating membrane proteins (TRAM);
translocase complexes; and processing enzymes, e.g., glycosylating
enzymes; signal peptidases; pro region peptidases. In a specific
embodiment, the substance is a folding protein, a chaperone, a
signal recognition particle, PrsA, SecD, SecF, BiP, a translocating
chain-associating membrane, a translocase complex, or a processing
enzyme.
[0115] Other embodiments of the present invention relate to methods
for producing polypeptides as described in the tenth embodiment,
except that the mutant cells express, synthesize or secrete less of
the polypeptide than the parent cell when both cells are cultivated
under the conditions.
[0116] A mutant cell that "expresses" less of a polypeptide is
defined herein as a cell that contains a decrease in functional
mRNA encoding the polypeptide relative to the parent cell. It will
be understood that a decrease in functional mRNA may result from a
decrease in the absolute rate of transcription of the gene encoding
the polypeptide and/or from alterations in post-transcriptional
processing or modification of the transcripts, including
nuclear-cytoplasmic transport and/or cytomplasmic stabilization of
the mRNA. Such mutant cells may be identified using conventional
techniques, including without limitation Northern blot analysis,
run-off transcription assays, and the like.
[0117] A mutant cell that "synthesizes" less of a polypeptide is
defined herein as a cell that accumulates smaller amounts of the
polypeptide relative to a parent cell. Accumulation refers to the
total amount of the polypeptide in the culture as a whole, i.e., in
both intracellular and extracellular compartments taken together.
Such mutant cells may be identified using any suitable technique,
including without limitation pulse-labelling or steady-state
labelling using radiolabelled amino acids; immunoblot analysis of
cell and medium fractions using an antibody specific to the
polypeptide; assays of biological activity; separation by
conventional chromatographic methods; and the like.
[0118] A mutant cell that "secretes" less of a polypeptide is
defined herein as a cell in which the amount of the polypeptide
released into the extracellular medium is decreased relative to the
parent cell. Such mutant cells may be identified using, e.g.,
pulse-chase labelling in conjunction with immunoprecipitation to
quantify the proportion of the newly synthesized polypeptide that
is externalized as well as the absolute amount released in the
mutant cell relative to the parent cell. Immunoblot analysis,
biological activity assays, and physical-chemical separation
methods may also be used to quantify the absolute amounts of the
polypeptide released in mutant vs. parent cells.
[0119] The present invention also relates to methods of producing a
metabolite, comprising
[0120] (A) cultivating a mutant cell under conditions conducive for
production of the metabolite, wherein
[0121] (i) the mutant cell is related to a parent cell, which
comprises one or more first DNA sequences encoding first
polypeptides in the biosynthetic pathway of the metabolite, by the
introduction of a nucleic acid construct into the genome of the
parent cell at a locus which is not within (a) the first DNA
sequences, (b) a second DNA sequence encoding a substance that
negatively regulates transcription, translation or secretion of the
polypeptides, (c) a third DNA sequence encoding a protease capable
of hydrolyzing any of the first polypeptides under the conditions,
and (d) one or more fourth DNA sequences encoding a second
polypeptide in the second biosynthetic pathway of a second
metabolite wherein the biosynthetic pathway and the second
biosynthetic pathway involve the production of the same
intermediate and the second polypeptide catalyzes a step after the
production of the intermediate; and
[0122] (ii) the mutant cell produces more of the metabolite than
the parent cell when both cells are cultivated under the
conditions; and
[0123] (B) recovering the metabolite.
[0124] The present invention also relates to methods of producing a
metabolite, comprising
[0125] (A) cultivating a mutant cell under conditions conducive for
production of the metabolite, wherein
[0126] (i) the mutant cell is related to a parent cell, which
comprises one or more first DNA sequences encoding first
polypeptides in the biosynthetic pathway of the metabolite, by the
introduction of a nucleic acid construct into the genome of the
parent cell at a locus which is not within (a) the first DNA
sequences, (b) a second DNA sequence encoding a protein that
negatively regulates transcription, translation or secretion of the
polypeptides, and (c) one or more third DNA sequences encoding a
second polypeptide in the second biosynthetic pathway of a second
metabolite wherein the biosynthetic pathway and the second
biosynthetic pathway involve the production of the same
intermediate and the second polypeptide catalyzes a step prior to
the production of the intermediate; and
[0127] (ii) the mutant cell produces less of the metabolite than
the parent cell when both cells are cultivated under the
conditions; and
[0128] (B) recovering the metabolite.
[0129] The present invention also relates to methods of producing a
first polypeptide, comprising
[0130] (a) forming a mutant cell by introducing a nucleic acid
construct into the genome of the parent cell at a locus which is
not within the first DNA sequence, a second DNA sequence encoding a
protein that negatively regulates transcription, translation or
secretion of a second polypeptide, and a third DNA sequence
encoding a protease capable of hydrolyzing the polypeptide under
conditions conducive to the production of the first
polypeptide;
[0131] (b) isolating the mutant cell which produces more of the
polypeptide than the parent cell when both cells are cultivated
under the conditions;
[0132] (c) identifying the locus wherein the nucleic acid construct
has been integrated;
[0133] (d) producing a cell in which a corresponding locus has been
disrupted;
[0134] (e) culturing the cell under the conditions conducive;
and
[0135] (f) recovering the first polypeptide.
[0136] A corresponding locus is defined herein as a locus which
encodes a polypeptide with has the same function as the polypeptide
encoded by the rescued locus.
[0137] The present invention also relates to methods of producing a
polypeptide, comprising
[0138] (a) cultivating a mutant cell under conditions conducive for
production of the polypeptide, wherein
[0139] (i) the mutant cell is related to a parent cell, which
comprises a first DNA sequence encoding the polypeptide, by the
introduction of a nucleic acid construct into the genome of the
parent cell at a locus which is not within the first DNA sequence
and a second DNA sequence encoding a protease capable of
hydrolyzing the polypeptide under the conditions, wherein the
introduction of the nucleic acid construct specifically enhances
transcription, translation or secretion of the polypeptide; and
[0140] (ii) the mutant cell produces more of the polypeptide than
the parent cell when both cells are cultivated under the
conditions; and
[0141] (b) recovering the polypeptide.
[0142] "Specific" enhancement of transcription, translation, or
secretion as used herein refers to an enhancement of one or more
aspects of the biogenesis and production of the polypeptide that is
limited to the polypeptide of interest and is not accompanied by a
global effect on other polypeptides in the cell. Preferably,
specific enhancement affects only a small number of polypeptides,
including the polypeptide of interest. Most preferably, specific
enhancement affects only the polypeptide of interest.
[0143] Global enhancement of these biogenetic processes can be
distinguished from specific enhancement using conventional methods
that are well known in the art. For example, biosynthetic
pulse-labelling with .sup.3H-uridine (followed by quantitation of
total radioactivity incorporated into RNA) can be used to determine
that a mutant cell does not generally synthesize mRNA at a higher
or lower rate than the parent cell to which it is related.
Similarly, pulse-labelling with, e.g., .sup.35S-methionine
(followed by quantitation of total radioactivity incorporated into
protein) can be used to determine that a mutant cell does not
generally synthesize proteins at a higher or lower rate than the
parent cell to which it is relate. General rates of secretion can
be compared between mutant and parent cells by pulse-chase
labelling using radioactive amino acids or sugars followed by
quantitation of extracellular vs. intracellular radioactivity.
[0144] These methods can also be used to determine if
transcription, translation, or secretion of a limited number of
other polypeptides might also be affected in the mutant cell. For
example, capture of specific radiolabelled RNA transcripts by
hybridization to an immobilized oligonucleotide probe can be used
to assess transcription rates of individual genes. For translation
and secretion, resolution of radiolabelled nascent proteins by,
e.g., SDS-PAGE (with or without immunoprecipitation of individual
proteins) can be used to compare instantaneous rates of translation
and/or secretion of individual proteins.
[0145] Polypeptides
[0146] The term "polypeptide" encompasses peptides, oligopeptides,
and proteins and, therefore, is not limited to a specific length of
the encoded product. The polypeptide may e native to the cell or
may be a heterologous polypeptide. Preferably, it is a heterologous
polypeptide. The polypeptide may also be a recombinant polypeptide
which is a polypeptide native to a cell, which is encoded by a
nucleic acid sequence which comprises one or more control sequences
foreign to the gene. The polypeptide may be a wild-type polypeptide
or a variant thereof. The polypeptide may also be a hybrid
polypeptide which contains a combination of partial or complete
polypeptide sequences obtained from at least two different
polypeptides where one or more of the polypeptides may be
heterologous to the cell. Polypeptides further include naturally
occurring allelic and engineered variations of the above mentioned
polypeptides.
[0147] In a preferred embodiment, the polypeptide is an antibody or
portions thereof.
[0148] In a preferred embodiment, the polypeptide is an
antigen.
[0149] In a preferred embodiment, the polypeptide is a clotting
factor.
[0150] In a preferred embodiment, the polypeptide is an enzyme.
[0151] In a preferred embodiment, the polypeptide is a hormone or a
hormone variant.
[0152] In a preferred embodiment, the polypeptide is a receptor or
portions thereof.
[0153] In a preferred embodiment, the polypeptide is a regulatory
protein.
[0154] In a preferred embodiment, the polypeptide is a structural
protein.
[0155] In a preferred embodiment, the polypeptide is a
reporter.
[0156] In a preferred embodiment, the polypeptide is a transport
protein.
[0157] In a more preferred embodiment, the polypeptide is an
oxidoreductase,
[0158] In a more preferred embodiment, the polypeptide is a
transferase.
[0159] In a more preferred embodiment, the polypeptide is a
hydrolase.
[0160] In a more preferred embodiment, the polypeptide is a
lyase.
[0161] In a more preferred embodiment, the polypeptide is an
isomerase.
[0162] In a more preferred embodiment, the polypeptide is a
ligase.
[0163] In an even more preferred embodiment, the polypeptide is an
aminopeptidase
[0164] In an even more preferred embodiment, the polypeptide is an
amylase.
[0165] In an even more preferred embodiment, the polypeptide is a
carbohydrase.
[0166] In an even more preferred embodiment, the polypeptide is a
carboxypeptidase.
[0167] In an even more preferred embodiment, the polypeptide is a
catalase.
[0168] In an even more preferred embodiment, the polypeptide is a
cellulase.
[0169] In an even more preferred embodiment, the polypeptide is a
chitinase.
[0170] In an even more preferred embodiment, the polypeptide is a
cutinase.
[0171] In an even more preferred embodiment, the polypeptide is a
deoxyribonuclease.
[0172] In an even more preferred embodiment, the polypeptide is a
dextranase.
[0173] In an even more preferred embodiment, the polypeptide is an
esterase.
[0174] In an even more preferred embodiment, the polypeptide is an
alpha-galactosidase.
[0175] In an even more preferred embodiment, the polypeptide is a
beta-galactosidase.
[0176] In an even more preferred embodiment, the polypeptide is a
glucoamylase.
[0177] In an even more preferred embodiment, the polypeptide is an
alpha-glucosidase.
[0178] In an even more preferred embodiment, the polypeptide is a
beta-glucosidase.
[0179] In an even more preferred embodiment, the polypeptide is a
haloperoxidase.
[0180] In an even more preferred embodiment, the polypeptide is an
invertase.
[0181] In an even more preferred embodiment, the polypeptide is a
laccase.
[0182] In an even more preferred embodiment, the polypeptide is a
lipase.
[0183] In an even more preferred embodiment, the polypeptide is a
mannosidase.
[0184] In an even more preferred embodiment, the polypeptide is a
mutanase.
[0185] In an even more preferred embodiment, the polypeptide is an
oxidase.
[0186] In an even more preferred embodiment, the polypeptide is a
pectinolytic enzyme.
[0187] In an even more preferred embodiment, the polypeptide is a
peroxidase.
[0188] In an even more preferred embodiment, the polypeptide is a
phytase.
[0189] In an even more preferred embodiment, the polypeptide is a
polyphenooxidase.
[0190] In an even more preferred embodiment, the polypeptide is a
proteolytic enzyme.
[0191] In an even more preferred embodiment, the polypeptide is a
ribonuclease.
[0192] In an even more preferred embodiment, the polypeptide is a
transglutaminase.
[0193] In an even more preferred embodiment, the polypeptide is a
xylanase.
[0194] In an even more preferred embodiment, the polypeptide is
human insulin or an analog thereof.
[0195] In an even more preferred embodiment, the polypeptide is
human growth hormone.
[0196] In an even more preferred embodiment, the polypeptide is
erythropoietin.
[0197] In an even more preferred embodiment, the polypeptide is
insulinotropin.
[0198] The polypeptide also may be an enzyme involved in the
biosynthesis of a specific metabolite. The biosynthesis of a
metabolite generally involves a biosynthetic pathway containing an
array of enzyme-catalyzed chemical reaction steps in which one or
more steps may be rate-limiting. In this embodiment of the present
invention, the integration of the nucleic acid construct into the
cell's genome modifies the production of the metabolite by
modifying one or more of these enzyme-catalyzed steps.
[0199] The metabolite may be any organic compound of a cell which
has been produced by transformation of a precursor organic compound
by an enzyme-catalyzed chemical reaction of the cell. The
metabolite may be a primary metabolite or a secondary metabolite.
Furthermore, the metabolite may be a biosynthetic pathway
intermediate or a biosynthetic pathway product. Preferably, the
metabolite is an alkaloid, an amino acid, an antibiotic, a
cofactor, a drug, a fatty acid, a fungicide, a herbicide, an
insecticide, an organic acid, a prosthetic group, a rodenticide, a
sweetener, a vitamin, a deoxysugar, a surfactant, a mycotoxin, an
organic acid, a sugar alcohol, a toxic metabolite, or a toxin.
[0200] Nucleic Acid Constructs
[0201] The nucleic constructs used in the methods of the present
invention may be termed "tagged nucleic acid constructs". "A tagged
nucleic acid construct" is a nucleic acid molecule containing an
identifiable nucleic acid sequence which integrates into the cell's
genome at one or more loci thereby marking the loci. The genome is
the complete set of DNA of a cell including chromosomal and
artificial chromosomal DNA and ;extrachromosomal DNA, i.e.,
self-replicative genetic elements.
[0202] The nucleic acid constructs may be any nucleic acid
molecule, either single- or double-stranded, which is synthetic
DNA, isolated from a naturally occurring gene, or has been modified
to contain segments of nucleic acid which are combined and
juxtaposed in a manner which would not otherwise exist in nature.
The nucleic acid constructs may be circular or linear. Furthermore,
the nucleic acid constructs may be contained in a vector, may be a
restriction enzyme cleaved linearized fragment, or may be a PCR
amplified linear fragment.
[0203] The nucleic acid constructs may contain any nucleic acid
sequence of any size. In one embodiment, the nucleic acid
constructs are between about 10-20,000 bp in length, preferably
100-15,000 bp in length, more preferably 500-15,000 bp in length,
even more preferably 1000-15,000 bp in length, and most preferably
1,000-10,000 bp in length.
[0204] Preferably, the nucleic acid constructs have less than 40%
homology, preferably less than 30% homology, more preferably less
than 20% homology, even more preferably less than 10% homology, and
most preferably no homology with the locus.
[0205] Preferably, the nucleic acid constructs have less than 40%
homology, preferably less than 30% identity, more preferably less
than 20% identity, even more preferably less than 10%, and most
preferably no homology with the DNA sequence encoding the
polypeptide of interest.
[0206] The nucleic acid construct can be introduced into a cell as
two or more separate fragments. In the event two fragments are
used, the two fragments share DNA sequence homology (overlap) at
the 3' end of one fragment and the 5' end of the other. Upon
introduction into a cell, the two fragments can undergo homologous
recombination to form a single fragment. The product fragment is
then in a form suitable for recombination with the cellular
sequences. More than two fragments can be used, designed such that
they will undergo homologous recombination with each other to
ultimately form a product suitable for recombination with a
cellular sequence.
[0207] It will be further understood that two or more nucleic acid
constructs may be introduced into the cell as circular or linear
fragments using the methods of the present invention, wherein the
fragments do not contain overlapping regions as described above. It
is well known in the art that for some organisms, the introduction
of multiple constructs into a cell results in their integration at
the same locus.
[0208] The nucleic acid constructs can contain coding or non-coding
DNA sequences. Coding sequences are sequences which are capable of
being transcribed into mRNA and translated into a polypeptide when
placed under the control of the appropriate control sequences. The
boundaries of a coding sequence are generally determined by a
translation start codon ATG at the 5'-terminus and a translation
stop codon at the 3'-terminus. A coding sequence can include, but
is not limited to, DNA, cDNA, and recombinant nucleic acid
sequences.
[0209] In a preferred embodiment, the nucleic acid constructs
contain a selectable marker as the identifiable nucleic acid
sequence. 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. Integration of a
selectable marker into the genome of a host cell permits easy
selection of transformed cells. Selectable marker genes for use in
the methods of the present invention include, but are not limited
to, acetamidase (amdS), 5-aminolevulinic acid synthase (hemA),
anthranilate synthase (trpC), glufosinate resistance genes,
hygromycin phosphotransferase (hygB), nitrate reductase (niaD),
ornithine carbamoyltransferase (argB), orotidine-5'-phosphate
decarboxylase (pyrG), phosphinothricin acetyltransferase (bar), and
sulfate adenyltransferase (sC), as well as equivalents from other
species. In a more preferred embodiment, the selectable marker is
the amdS gene of Aspergillus nidulans or Aspergillus oryzae, the
bar gene of Streptomyces hygroscopicus, the hemA gene of
Aspergillus oryzae or the pyrG gene of Aspergillus nidulans or
Aspergillus oryzae. Other selectable markers for use in the methods
of the present invention are the dal genes from Bacillus subtilis
or Bacillus licheniformis, or markers which confer antibiotic
resistance such as ampicillin (amp), kanamycin (kan),
chloramphenicol (cam) or tetracycline resistance (tet). A
frequently used mammalian marker is the dihydrofolate reductase
gene (dfhr). Suitable markers for yeast host cells are ADE2, HIS3,
LEU2, LYS2, MET3, TRP1, and URA3.
[0210] In another preferred embodiment, the constructs comprise
vector sequences alone or in combination with a selectable marker,
including vector sequences containing an origin of replication,
e.g., E. coli vector sequences such as pUC19, pBR322, or
pBluescript. For example, an E. coli vector sequence containing an
origin of replication can facilitate recovery of the construct from
the host genome after integration due to the E. coli origin of
replication. The construct can be recovered from the host genome by
digestion of the genomic DNA with a restriction endonuclease
followed by ligation of the recovered construct and transformation
of E. coli.
[0211] In a preferred embodiment, the nucleic acid constructs do
not contain the coding sequence of the DNA sequence for the
polypeptide or portions thereof. In another preferred embodiment,
the nucleic acid constructs contain a sequence which is not
homologous to the DNA sequence encoding the polypeptide in order to
block the construct from integrating or disrupting the DNA sequence
of interest.
[0212] In another preferred embodiment, the nucleic acid constructs
contain one or more copies of the DNA sequence coding for the
polypeptide operably linked to control sequences. In this
embodiment, the production of the polypeptide will be modified by
both gene inactivation and the introduction of one or more copies
of the DNA sequence.
[0213] In another preferred embodiment, the nucleic acid constructs
do not contain transposable elements, i.e., transposons. A
transposon is a discrete piece of DNA which can insert itself into
many different sites in other DNA sequences within the same cell.
The proteins necessary for the transposition process are encoded
within the transposon. A copy of the transposon may be retained at
the original site after transposition. The ends of a transposon are
usually identical but in inverse orientation with respect to one
another.
[0214] In another preferred embodiment, the nucleic acid constructs
may contain one or more control sequences, e.g., a promoter alone
or in combination with a selectable marker, wherein the control
sequences upon integration are not operably linked to the DNA
sequence encoding the polypeptide of interest. The term "operably
linked" is defined herein as a configuration in which a control
sequence is appropriately placed at a position relative to the
coding sequence of the DNA sequence such that the control sequence
directs the production of a primary RNA transcript. Such control
sequences are a promoter, a signal sequence, a propeptide sequence,
a transcription terminator, a polyadenylation sequence, an enhancer
sequence, an attenuator sequence, and an intron splice site
sequence. Each control sequence may be native or foreign to the
cell or to the polypeptide-coding sequence.
[0215] The presence of a strong promoter in the nucleic acid
construct allows for additional genetic effects in addition to gene
inactivation via insertion into a structural gene (or functional
transcriptional promoter or mRNA termination regions). The promoter
may insert upstream of a structural gene so as to enhance its
transcription. Alternatively, if the promoter sequences insert in
reverse gene orientation so as to generate antisense RNA, there is
the possibility of gene inactivation in diploid or higher ploidy
cells. By the same mechanism, insertion of the promoter sequences
in reverse orientation may result in inactivation of multiple gene
family encoded gene product activities.
[0216] In another preferred embodiment, the nucleic acid constructs
contain a control sequence other than a promoter.
[0217] In another preferred embodiment, the nucleic acid constructs
do not contain control sequences.
[0218] Locus
[0219] In the methods of the present invention, the nucleic acid
constructs are introduced at a "locus not within the DNA sequence
of interest" or a "locus not within DNA sequences encoding
polypeptides in the biosynthetic pathway of a metabolite" which
means that the nucleic acid construct is not introduced into the
polypeptide-coding sequence, the control sequences thereof, and any
intron sequences within the coding sequence.
[0220] Control sequences include all components which are operably
linked to the DNA sequence and involved in the expression of the
polypeptide-coding sequence. Such control sequences are a promoter,
a signal sequence, a propeptide sequence, a transcription
terminator, a polyadenylation sequence, an enhancer sequence, an
attenuator sequence, and an intron splice site sequence. Each of
the control sequences may be native or foreign to the coding
sequence.
[0221] The promoter sequence contains transcriptional control
sequences which mediate the expression of the polypeptide. The
promoter may be any promoter sequence including mutant, truncated,
and hybrid promoters.
[0222] The signal peptide coding region codes for an amino acid
sequence linked to the amino terminus of the polypeptide which can
direct the expressed polypeptide into the cell's secretory
pathway.
[0223] The propeptide coding region codes for an amino acid
sequence positioned at the amino terminus of the 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 mature active polypeptide by catalytic or
autocatalytic cleavage of the propeptide from the
propolypeptide.
[0224] The terminator is a sequence operably linked to the 3'
terminus of the polypeptide coding sequence, and is recognized by
the cell to terminate transcription of the polypeptide coding
sequence.
[0225] The polyadenylation sequence is a sequence which is operably
linked to the 3' terminus of the DNA sequence and which, when
transcribed, is recognized by the cell as a signal to add
polyadenosine residues to the transcribed mRNA.
[0226] The enhancer sequence is a sequence which can increase
transcription from a gene when located up to several kilobases from
the gene. The enhancer sequencer is usually upstream of the
gene.
[0227] The attenuator sequence is a sequence which regulates the
expression of a gene by determining whether the mRNA molecule
containing its transcript will be completed or not.
[0228] The intron sequence is a sequence of a gene which is not
represented in the protein product of the gene. Intron sequences
are transcribed into RNA and must be excised and the RNA molecule
religated through a process called intron splicing before it can be
translated.
[0229] The locus may be noncontiguous or contiguous with the
above-noted sequences. Preferably the locus is noncontiguous. The
locus may be on the same chromosome or the same extrachromosomal
element or on a different chromosome or a different
extrachromosomal element as that of the DNA sequence of interest.
Furthermore, the locus may be native or foreign to the cell.
[0230] In a preferred embodiment, the locus is at least 1,000 bp,
more preferably at least 2,000 bp, and even more preferably at
least 3,000 bp, even more preferably at least 4,000 bp, even more
preferably at least 5,000 bp, and most preferably at least 10,000
bp from the 5' or 3' terminus of the DNA sequence of interest.
[0231] In another preferred embodiment, the locus is on a different
chromosome than the DNA sequence encoding the polypeptide of
interest.
[0232] In various methods of the present invention, the nucleic
acid constructs are introduced at a locus not within a DNA sequence
encoding a protease capable of hydrolyzing the polypeptide under
physiological conditions, which means that the nucleic acid
construct is not introduced into the protease-coding sequence, the
control sequences thereof, any intron sequences within the coding
sequence, and any DNA sequences encoding proteins that positively
regulate transcription, translation or secretion of the
protease.
[0233] In another preferred embodiment, the locus encodes a
polypeptide different from the polypeptide encoded by the DNA
sequence.
[0234] In another preferred embodiment, the locus encodes a glucose
transporter. Preferably, the locus has at least 60% homology, more
preferably at least 70% homoloy, even more preferably at least 80%
homology, even more preferably at least 90% homology, and most
preferably at least 95% homology with the nucleic acid sequence of
SEQ ID NO:9.
[0235] In another preferred embodiment, the locus encodes a
mannitol-1-phosphate dehydrogenase. Preferably, the locus has at
least 60% homology, more preferably at least 70% homoloy, even more
preferably at least 80% homology, even more preferably at least 90%
homology, and most preferably at least 95% homology with the
nucleic acid sequence of SEQ ID NO:25.
[0236] In another preferred embodiment, the locus encodes a chitin
synthase. Preferably, the locus has at least 60% homology, more
preferably at least 70% homoloy, even more preferably at least 80%
homology, even more preferably at least 90% homology, and most
preferably at least 95% homology with the nucleic acid sequence of
SEQ ID NO:56.
[0237] In another preferred embodiment, the locus encodes a heat
shock protein. Preferably, the locus has at least 60% homology,
more preferably at least 70% homoloy, even more preferably at least
80% homology, even more preferably at least 90% homology, and most
preferably at least 95% homology with the nucleic acid sequence of
SEQ ID NO:50.
[0238] In another preferred embodiment, the locus encodes a
manganese superoxide dismutase. Preferably, the locus has at least
60% homology, more preferably at least 70% homoloy, even more
preferably at least 80% homology, even more preferably at least 90%
homology, and most preferably at least 95% homology with the
nucleic acid sequence of SEQ ID NO:29.
[0239] In another preferred embodiment, the locus is a gene
required for activation of pacC, preferably a palB gene.
Preferably, the locus has at least 60% homology, more preferably at
least 70% homoloy, even more preferably at least 80% homology, even
more preferably at least 90% homology, and most preferably at least
95% homology with the nucleic acid sequence of SEQ ID NO:16.
[0240] In another preferred embodiment, the locus has at least 60%
homology, more preferably at least 70% homoloy, even more
preferably at least 80% homology, even more preferably at least 90%
homology, and most preferably at least 95% homology with the
nucleic acid sequence of SEQ ID NO:34.
[0241] In another preferred embodiment, the locus has at least 60%
homology, more preferably at least 70% homoloy, even more
preferably at least 80% homology, even more preferably at least 90%
homology, and most preferably at least 95% homology with the
nucleic acid sequence of SEQ ID NO:39.
[0242] In another preferred embodiment, the locus has at least 60%
homology, more preferably at least 70% homoloy, even more
preferably at least 80% homology, even more preferably at least 90%
homology, and most preferably at least 95% homology with the
nucleic acid sequence of SEQ ID NO:63.
[0243] In another preferred embodiment, the locus has at least 60%
homology, more preferably at least 70% homoloy, even more
preferably at least 80% homology, even more preferably at least 90%
homology, and most preferably at least 95% homology with the
nucleic acid sequence of SEQ ID NO:66.
[0244] In another preferred embodiment, the locus has at least 60%
homology, more preferably at least 70% homoloy, even more
preferably at least 80% homology, even more preferably at least 90%
homology, and most preferably at least 95% homology with the
nucleic acid sequence of SEQ ID NO:71.
[0245] In another preferred embodiment, the locus has at least 60%
homology, more preferably at least 70% homoloy, even more
preferably at least 80% homology, even more preferably at least 90%
homology, and most preferably at least 95% homology with the
nucleic acid sequence of SEQ ID NO:76.
[0246] In another preferred embodiment, the locus encodes an
aminopeptidase.
[0247] In another preferred embodiment, the locus encodes an
amylase.
[0248] In another preferred embodiment, the locus encodes a
carbohydrase.
[0249] In another preferred embodiment, the locus encodes a
carboxypeptidase.
[0250] In another preferred embodiment, the locus encodes a
catalase.
[0251] In another preferred embodiment, the locus encodes a
catalase.
[0252] In another preferred embodiment, the locus encodes a
cellulase.
[0253] In another preferred embodiment, the locus encodes a
chitinase.
[0254] In another preferred embodiment, the locus encodes a
cutinase.
[0255] In another preferred embodiment, the locus encodes a
deoxyribonuclease.
[0256] In another preferred embodiment, the locus encodes a
dextranase.
[0257] In another preferred embodiment, the locus encodes an
esterase.
[0258] In another preferred embodiment, the locus encodes an
alpha-galactosidase.
[0259] In another preferred embodiment, the locus encodes a
beta-galactosidase.
[0260] In another preferred embodiment, the locus encodes a
glucoamylase.
[0261] In another preferred embodiment, the locus encodes a n
alpha-glucosidase.
[0262] In another preferred embodiment, the locus encodes a
beta-galactosidase.
[0263] In another preferred embodiment, the locus encodes a
glucoamylase.
[0264] In another preferred embodiment, the locus encodes an
alpha-glucosidase.
[0265] In another preferred embodiment, the locus encodes a
beta-glucosidase.
[0266] In another preferred embodiment, the locus encodes a
haloperoxidase.
[0267] In another preferred embodiment, the locus encodes an
invertase.
[0268] In another preferred embodiment, the locus encodes a
laccase.
[0269] In another preferred embodiment, the locus encodes a
lipase.
[0270] In another preferred embodiment, the locus encodes a
mannosidase.
[0271] In another preferred embodiment, the locus encodes a
mutanase.
[0272] In another preferred embodiment, the locus encodes an
oxidase.
[0273] In another preferred embodiment, the locus encodes a
pectinolytic enzyme.
[0274] In another preferred embodiment, the locus encodes a
peroxidase.
[0275] In another preferred embodiment, the locus encodes a
phytase.
[0276] In another preferred embodiment, the locus encodes a
polyphenoloxidase.
[0277] In another preferred embodiment, the locus encodes a
proteolytic enzyme.
[0278] In another preferred embodiment, the locus encodes a
ribonuclease.
[0279] In another preferred embodiment, the locus encodes a
transglutaminase.
[0280] In another preferred embodiment, the locus encodes a
xylanase.
[0281] In a more preferred embodiment, the locus is the sequence
contained in pDSY109.
[0282] In a more preferred embodiment, the locus is the sequence
contained in pDSY112.
[0283] In a more preferred embodiment, the locus is the sequence
contained in pDSY138.
[0284] In a more preferred embodiment, the locus is the sequence
contained in pDSY141.
[0285] In a more preferred embodiment, the locus is the sequence
contained in pDSY162.
[0286] In a more preferred embodiment, the locus is the sequence
contained in pMT1936.
[0287] In a more preferred embodiment, the locus is the sequence
contained in pSMO1204.
[0288] In a more preferred embodiment, the locus is the sequence
contained in pSMOH603.
[0289] In a more preferred embodiment, the locus is the sequence of
SEQ ID NO:9.
[0290] In a more preferred embodiment, the locus is the sequence of
SEQ ID NO:16.
[0291] In a more preferred embodiment, the locus is the sequence of
SEQ ID NO:25.
[0292] In a more preferred embodiment, the locus is the sequence of
SEQ ID NO:29.
[0293] In a more preferred embodiment, the locus is the sequence of
SEQ ID NO:34.
[0294] In a more preferred embodiment, the locus is the sequence of
SEQ ID NO:39.
[0295] In another more preferred embodiment, the locus is the
sequence contained in p4-8. 1.
[0296] In another more preferred embodiment, the locus is the
sequence contained in p7-14.1.
[0297] In another more preferred embodiment, the locus is the
sequence contained in pHB220.
[0298] In another more preferred embodiment, the locus is the
sequence contained in pSMO717.
[0299] In another more preferred embodiment, the locus is the
sequence contained in pSMO321.
[0300] In another more preferred embodiment, the locus is the
sequence contained in pHowB571.
[0301] In another more preferred embodiment, the locus is the
sequence contained in pSMO810.
[0302] In another more preferred embodiment, the locus is the
sequence of SEQ ID NO:50.
[0303] In another more preferred embodiment, the locus is the
sequence of SEQ ID NO:56.
[0304] In another more preferred embodiment, the locus is the
sequence of SEQ ID NO:63.
[0305] In another more preferred embodiment, the locus is the
sequence of SEQ ID NO:66.
[0306] In another more preferred embodiment, the locus is the
sequence of SEQ ID NO:71.
[0307] In another more preferred embodiment, the locus is the
sequence of SEQ ID NO:76.
[0308] In another preferred embodiment, the locus does not encode a
trans factor of the DNA sequence of interest. A "trans factor" is a
factor which is encoded by a gene separate from the DNA sequence of
interest which activates or represses transcription of the DNA
sequence. In a more preferred embodiment, the locus does not encode
a repressor of the DNA sequence of interest. In a more preferred
embodiment, the locus does not encode an activator of the DNA
sequence of interest.
[0309] Cells
[0310] The methods of the present invention may be used with any
cell containing a DNA sequence encoding a polypeptide of interest
including prokaryotic cells such as bacteria, or eukaryotic cells
such as mammalian, insect, plant, and fungal cells. The DNA
sequence may be native or foreign to the cell. The cell may be a
unicellular microorganism or a non-unicellular microorganism.
Furthermore, the cell may be wild-type or a mutant cell. For
example, the mutant cell may be a cell which has undergone
classical mutagenesis or genetic manipulation.
[0311] Useful prokaryotic cells 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 coagulans, Bacillus lautus,
Bacillus lentus, Bacillus licheniformis, Bacillus megaterium,
Bacillus stearothernophilus, Bacillus subtilis, and Bacillus
thuringiensis; or a Streptomyces cell, e.g., Streptomyces lividans
or Streptomyces urinus, or gram negative bacteria such as E. coli
and Pseudomonas sp. In a preferred embodiment, the bacterial cell
is a Bacillus lentus, Bacillus licheniformis, Bacillus
stearothermophilus, or Bacillus subtilis cell.
[0312] In a preferred embodiment, the 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). Representative groups of Ascomycota include,
e.g., Neurospora, Eupenicillium (=Penicillium), Emericella
(=Aspergillus), Eurotium (=Aspergillus), and the true yeasts.
Examples of Basidiomycota include mushrooms, rusts, and smuts.
Representative groups of Chytridiomycota include, e.g., Allomyces,
Blastocladiella, Coelomomyces, and aquatic fungi. Representative
groups of Oomycota include, e.g., Saprolegniomycetous aquatic fungi
(water molds) such as Achlya. Examples of mitosporic fungi include
Alternaria, Aspergillus, Candida, and Penicillium. Representative
groups of Zygomycota include, e.g., Mucor and Rhizopus.
[0313] In a preferred embodiment, the fungal cell is a yeast cell.
"Yeast" as used herein includes ascosporogenous yeast
(Endomycetales), basidiosporogenous yeast, and yeast belonging to
the Fungi Imperfecti (Blastomycetes). The ascosporogenous yeasts
are divided into the families Spermophthoraceae and
Saccharomycetaceae. The latter is comprised of four subfamilies,
Schizosaccharomycoideae (e.g., genus Schizosaccharomyces),
Nadsonioideae, Lipomycoideae, and Saccharomycoideae (e.g., genera
Kluyveromyces, Pichia, and Saccharomyces). The basidiosporogenous
yeasts include the genera Filobasidiella, Filobasidium,
Leucosporidim, Rhodosporidium, and Sporidiobolus. Yeast belonging
to the Fungi Imperfecti are divided into two families,
Sporobolomycetaceae (e.g., genera Bullera and Sorobolomyces) and
Cryptococcaceae (e.g., genus Candida). 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 et al., 1980, Soc. App. Bacteriol. Symposium Series
No. 9, 1980. The biology of yeast and manipulation of yeast
genetics are well known in the art (see, e.g., Biochemistry and
Genetics of Yeast, (Bacil, M., Horecker, B.J., and Stopani, A. O.
M., editors), 2nd edition, 1987; The Yeasts (Rose, A. H., and
Harrison, J. S., editors), 2nd edition, 1987; and The Molecular
Biology of the Yeast Saccharomyces, Strathern et al., editors,
1981).
[0314] In a more preferred embodiment, the yeast cell is a cell of
a species of Candida, Kluyveromyces, Pichia, Saccharomyces,
Schizosaccharomyces, or Yarrowia.
[0315] In a most preferred embodiment, the yeast cell is a
Saccharomyces carlsbergensis, Saccharomyces cerevisiae,
Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces
kluyveri, Saccharomyces norbensis, or Saccharomyces oviformis cell.
In another most preferred embodiment, the yeast cell is a
Kluyveromyces lactis cell. In another most preferred embodiment,
the yeast cell is a Yarrowia lipolytica cell.
[0316] In another preferred embodiment, the fungal cell is a
filamentous fungal cell. "Filamentous fungi" include all
filamentous forms of the subdivision Eumycota and Oomycota (as
defined by Hawksworth et al., 1995, supra). The filamentous fungi
are characterized by a mycelial wall composed of chitin, cellulose,
glucan, chitosan, mannan, and other complex polysaccharides.
Vegetative growth is by hyphal elongation and carbon catabolism is
obligately aerobic. In contrast, vegetative growth by yeasts such
as Saccharomyces cerevisiae is by budding of a unicellular thallus
and carbon catabolism may be fermentative. In a more preferred
embodiment, the filamentous fungal cell is a cell of a species of,
but not limited to, Acremonium, Aspergillus, Fusarium, Humicola,
Mucor, Myceliophthora, Neurospora, Penicillium, Scytalidium,
Thielavia, Tolypocladium, and Trichoderma.
[0317] In an even more preferred embodiment, the filamentous fungal
cell is an Aspergillus cell. In another even more preferred
embodiment, the filamentous fungal cell is an Acremonium cell. In
another even more preferred embodiment, the filamentous fungal cell
is a Fusarium cell. In another even more preferred embodiment, the
filamentous fungal cell is a Humicola cell. In another even more
preferred embodiment, the filamentous fungal cell is a Mucor cell.
In another even more preferred embodiment, the filamentous fungal
cell is a Myceliophthora cell. In another even more preferred
embodiment, the filamentous fungal cell is a Neurospora cell. In
another even more preferred embodiment, the filamentous fungal cell
is a Penicillium cell. In another even more preferred embodiment,
the filamentous fungal cell is a Thielavia cell. In another even
more preferred embodiment, the filamentous fungal cell is a
Tolypocladium cell. In another even more preferred embodiment, the
filamentous fungal cell is a Trichoderma cell.
[0318] In a most preferred embodiment, the filamentous fungal cell
is an Aspergillus awamori, Aspergillus foetidus, Aspergillus
japonicus, Aspergillus nidulans, Aspergillus niger, or Aspergillus
oryzae cell. In another most preferred embodiment, the filamentous
fungal 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 sulphureum, Fusarium
toruloseum, Fusarium trichothecioides, or Fusarium venenatum cell.
In a most preferred embodiment, the filamentous fungal cell is a
Fusarium venenatum cell (Nirenberg sp. nov.). In another most
preferred embodiment, the filamentous fungal cell is a Humicola
insolens cell or a Humicola lanuginosa cell. In another most
preferred embodiment, the filamentous fungal cell is a Mucor miehei
cell. In another most preferred embodiment, the filamentous fungal
cell is a Myceliophthora thermophila cell. In another most
preferred embodiment, the filamentous fungal cell is a Neurospora
crassa cell. In another most preferred embodiment, the filamentous
fungal cell is a Penicillium purpurogenum cell. In another most
preferred embodiment, the filamentous fungal cell is a Thielavia
terrestris cell. In another most preferred embodiment, the
filamentous fungal cell is a Trichoderma harzianum, Trichoderma
koningii, Trichoderma longibrachiatum, Trichoderma reesei, or
Trichoderma viride cell.
[0319] Useful mammalian cells include Chinese hamster ovary (CHO)
cells, HeLa cells, baby hamster kidney (BHK) cells, COS cells, or
any number of immortalized cells available, e.g., from the American
Type Culture Collection.
[0320] Introduction of Nucleic Acid Constructs into Cells
[0321] The nucleic acid construct(s) may be introduced into a cell
by a variety of physical or chemical methods known in the art
including, but not limited to, transfection or transduction,
electroporation, microinjection, microprojectile bombardment,
alkali salts, or protoplast-mediated transformation.
[0322] The introduction of the nucleic acid construct into a cell
for insertional mutagenesis is referred to as "DNA-tagged
mutagenesis". "DNA-tagged mutagenesis" is defined herein as the
introduction of a nucleic acid molecule into a cell, which leads to
one or more insertions of the nucleic acid molecule into one or
more loci of the genome of the cell thereby marking the loci into
which the nucleic acid molecule is inserted. The mutant cell
produced by DNA-tagged mutagenesis is called a tagged mutant.
[0323] Suitable procedures for transformation of Aspergillus cells
are described in EP 238 023 and Yelton et al, 1984, Proceedings of
the National Academy of Sciences USA 81: 1470-1474. A suitable
method of transforming Fusarium species is described by Malardier
et al., 1989, Gene 78: 147-156 or in WO 96/00787. Yeast may be
transformed using the procedures described by Becker and Guarente,
In Guide to Yeast Genetics and Molecular Biology, Methods of
Enzymology 194: 182-187; Ito et al., 1983, Journal of Bacteriology
153: 163; and Hinnen et al., 1978, Proceedings of the National
Academy of Sciences USA 75: 1920.
[0324] The transformation of a bacterial cell may, for instance, be
accomplished by protoplast transformation (see, e.g., Chang and
Cohen, 1979, Molecular General Genetics 168: 111-115), by using
competent cells (see, e.g., Young and Spizizin, 1961, Journal of
Bacteriology 81: 823-829, or Dubnau and Davidoff-Abelson, 1971,
Journal of Molecular Biology 56: 209-221), by electroporation (see,
e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), or by
conjugation (see, e.g., Koehler and Thorne, 1987, Journal of
Bacteriology 169: 5771-5278).
[0325] Mammalian cells may be transformed by direct uptake using
the calcium phosphate precipitation method of Graham and Van der
Eb, 1978, Virology 52: 546. Other processes, e.g., electroporation,
known to the art, may be used.
[0326] When the nucleic acid construct is a vector, integration
into the cell's genome occurs randomly by homologous and/or
non-homologous recombination depending on the cell of choice.
[0327] In a preferred embodiment, the nucleic acid construct is
introduced into the parent cell by restriction enzyme-mediated
integration (REMI). REMI, which is described in Schiestl and Petes,
1991, Proceedings of the National Academy of Sciences USA 88:
7585-7589, is the introduction of plasmid DNA digested with a
restriction enzyme along with the restriction enzyme into a cell
which subsequently leads to integration of the plasmid DNA into the
genome often at a site specified by the restriction enzyme added.
The advantage of REMI DNA-tagged mutagenesis is it can generate
mutations whose molecular basis can be easily identified.
[0328] When the nucleic acid construct is a restriction enzyme
cleaved linear DNA fragment, insertion of the construct into the
cell's genome through REMI in the presence of the appropriate
restriction enzyme is random by virtue of the randomness of the
restriction sites present in the genome. The nucleic acid construct
may insert into the cell's genome as a single copy or as multiple
copies at a single locus or at a different locus or at different
loci. It is preferable that the nucleic acid construct insert as a
single copy to facilitate the identification and recovery of the
tagged locus.
[0329] Screening of Mutant Cells
[0330] The present invention also relates to mutant cells which
produce, express, synthesize or secrete more of a polypeptide or
metabolite than the parent cell when both cells are cultivated
under the conditions.
[0331] The present invention also relates to mutant cells which
produce, express, synthesize or secrete more of a polypeptide or
metabolite than the parent cell when both cells are cultivated
under the conditions.
[0332] Following the introduction of a nucleic acid construct into
a cell, the next step is to isolate the mutant cell with the
modified production of a polypeptide from a population of
presumptive mutant cells. The isolation of the mutant cell
preferably relies on measurement of the production of the
polypeptide or the metabolite by the mutant cell relative to the
parent cell when the mutant cell and the parent cell are cultured
under the same conditions.
[0333] The phrase "modified production of a polypeptide" includes
an alteration or change of a step in the production of a
polypeptide or a metabolite by the mutant cell relative to the
parent cell. Such steps include, but are not limited to,
transcription, post-transcriptional modification, translation,
post-translational modification, secretion, fermentation,
proteolysis, down-stream processing, recovery, and
purification.
[0334] The mutant cell may be a mutant cell, for example, with
improved production of a specific polypeptide or metabolite or a
mutant cell which is no longer capable or has a diminished
capability of producing a specific polypeptide or metabolite.
Furthermore, the mutant cell may be a mutant cell having an
increased uptake of an inorganic cofactor.
[0335] The mutant cell may also have a more desirable phenotype
than the parent cell which modifies the production of a polypeptide
or a metabolite. The term "phenotype" is defined herein as an
observable or outward characteristic of a cell determined by its
genotype and modulated by its environment. Such a mutant cell
having a desired phenotype includes, but is not limited to, a
morphological mutant cell, a secretion mutant cell, an auxotrophic
mutant cell, a conditional mutant, a mutant cell exhibiting an
altered growth rate under desired conditions relative to the parent
cell, a mutant cell resulting in the relief of overexpression
mediated growth inhibition, or a mutant cell able to tolerate low
oxygen conditions.
[0336] Furthermore, the mutant cell may be characterized as being a
mutant cell exhibiting altered production of a transcriptional
activator of a promoter or a cryptic intron-splicing-deficient
mutant cell.
[0337] The isolation of a mutant cell may involve screening methods
known in the art specific to the desired phenotype and/or the
polypeptide or the metabolite of interest. In general where a
desired phenotype is involved, a method specific to the desired
phenotype may be used initially to identify the mutant cell, but
then may be followed by a method specific to the polypeptide or the
metabolite.
[0338] The population of presumptive mutants obtained by
introducing a nucleic acid construct into the cells of an organism
to produce a mutant cell are first purified using standard plating
techniques such as those used in classical mutagenesis (see, for
example, Lawrence, C. W., 1991, In Christine Guthrie and Gerald R.
Fink, editors, Methods in Enzymology, Volume 194, pages 273-281,
Academic Press, Inc., San Diego), single spore isolation, or
enrichment techniques. The standard plating techniques are
preferably conducted in combination with a means of detecting the
desired phenotype and/or the polypeptide or the metabolite.
Different enrichment techniques may be used for increasing the
percentage of mutant cells in comparison to their wild-type or
parent equivalents such as (1) direct selection which utilizes
growth conditions that greatly favor the growth of the mutant; (2)
counterselection, which makes use of conditions that kill the
parent cells; (3) physical selection, which involves unique
properties of the mutant cells that enable them to be physically
separated from their parent cells; and (4) direct measurements of
the amount of desired substances. However, whether or not a means
for identifying the mutant cell with respect to the desired
phenotype and/or the polypeptide or the metabolite of interest can
be incorporated into the plating medium, the purified presumptive
mutants may require further characterization to confirm the
identity of the mutant. Examples of the methods used to further
characterize and confirm the identity of the mutant are illustrated
below.
[0339] A mutant with improved production of a specific polypeptide
or a specific metabolite may be identified by using a detection
method known in the art that is specific for the polypeptide or the
metabolite. Detection methods for polypeptides may include, but are
not limited to, use of specific antibodies, enzymatic activity by
measuring formation of an enzyme product or disappearance of an
enzyme substrate, clearing zones on agar plates containing an
enzyme substrate, and biological activity assays. Detection methods
for metabolites may include, but are not limited to, thin layer
chromatography, high performance liquid chromatography, gas
chromatography, mass spectroscopy, biological activity assays,
bioassays, and fluorescent activating cell sorting.
[0340] In a preferred embodiment, the specifically desired mutant
cell is a mutant cell with improved production of a specific
polypeptide.
[0341] In another preferred embodiment, the specifically desired
mutant is a mutant with improved production of a specific
metabolite; more preferably an alkaloid, an amino acid, an
antibiotic, a cofactor, a drug, a fatty acid, a fungicide, a
herbicide, an insecticide, an organic acid, a pigment, a plastic
precursor, a polyester precursor, a prosthetic group, a
rodenticide, a sweetner, or a vitamin; and most preferably citric
acid or lactic acid.
[0342] A prosthetic group or an organic cofactor which is a
constituent of a polypeptide and/or required for biological
activity may be overproduced by isolating a mutant according to the
methods of the present invention. Such a mutant would be
particularly important where biosynthesis of the prosthetic group
or the cofactor is a rate-limiting event in the production of a
polypeptide in a biologically active form, e.g., a hemoprotein
containing heme including, but not limited to, a cytochrome,
specifically cytochrome P450, cytochrome b, cytochrome c.sub.1, or
cytochrome c; a globin, specifically, hemoglobin or myoglobin; an
oxidoreductase, specifically a catalase, an oxidase, an oxygenase,
a haloperoxidase, or a peroxidase; or any other polypeptide
containing a heme as a prosthetic group.
[0343] In a more preferred embodiment, the specifically desired
mutant cell is a mutant cell overproducing an adenosine phosphate,
S-adenosyl-L-methionine, biocytin, biotin, coenzyme A, coenzyme Q
(ubiquinone), 5'-deoxyadenosylcobalamine, a ferredoxin, a flavin
coenzyme, heme, lipoic acid, a nucleoside diphosphate, a
nicotinamide adenine dinucleotide, a nicotinamide adenine
dinucleotide phosphate, phosphoadenosine, phosphosulfate, pyridoxal
phosphate, tetrahydrofolic acid, thiamine pyrophosphate, or a
thioredoxin.
[0344] In another preferred embodiment, the specifically desired
mutant cell is a mutant cell characterized with an increased uptake
of an inorganic cofactor. The uptake by a cell of an inorganic
cofactor which is a constituent of a polypeptide and/or required
for biological activity may be increased by isolating a mutant
according to the methods of the present invention. Such a mutant
would be particularly important where uptake of the inorganic
cofactor is a rate-limiting event in the production of a
polypeptide in a biologically active form. In a more preferred
embodiment, the specifically desired mutant cell is a mutant cell
characterized with an increased uptake of Co.sup.2+, Cu.sup.2+,
Fe.sup.2+, Fe.sup.3+, K.sup.+, Mg.sup.2+, Mn.sup.2+, Mo, Ni.sup.2+,
Se, or Zn.sup.2+.
[0345] In a preferred embodiment, the polypeptide or the metabolite
is produced by the mutant cell in an amount which is at least 20%
greater, preferably at least 50%, more preferably at least 75%,
more preferably at least 100%, more preferably at least 100%-1000%,
even more preferably at least 200%-1000%, and most preferably at
least 500%-1000% or more greater than the cell.
[0346] In another preferred embodiment, the specifically desired
mutant cell is a mutant cell which is no longer capable or has a
diminished capability of producing a specific polypeptide. A mutant
cell which is no longer capable or has a diminished capability of
producing a specific polypeptide may be identified using the same
methods described above for polypeptides, but where no or
diminished production is measured relative to the parent cell.
[0347] In a more preferred embodiment, the specifically desired
mutant cell is a mutant cell which is no longer capable or has a
diminished capability of producing a polypeptide.
[0348] In another preferred embodiment, the polypeptide is produced
by the mutant cell in an amount which is at least 20%, more
preferably at least 50%, even more preferably at least 75%, and
most preferably 100% lower than the cell.
[0349] In another preferred embodiment, the specifically desired
mutant cell is a mutant cell which is no longer capable or has a
diminished capability of producing a specific metabolite. A mutant
cell which is no longer capable or has a diminished capability of
producing a specific metabolite may be identified using the same or
similar methods described above for metabolites, but where no or
diminished production is measured relative to the parent cell.
[0350] In a more preferred embodiment, the specifically desired
mutant cell is a mutant cell which is no longer capable or has a
diminished capability of producing a deoxysugar, a surfactant, a
mycotoxin, an organic acid, a sugar alcohol, a toxic metabolite, or
a toxin; and most preferably an aflatoxin, beta-exotoxin,
cyclopiazonic acid, an enniatin, a fusarin, kanosamine, mannitol,
oxalic acid, surfactin, a tricothecene, a zearalenol, or a
zearalenone.
[0351] In another preferred embodiment, the metabolite is produced
by the mutant cell in an amount which is at least 20% lower than
the cell, more preferably 50%, even more preferably 75%, and most
preferably 100% lower than the cell.
[0352] In another preferred embodiment, the mutant cell is a
morphological mutant cell. A "morphological mutant cell" is defined
herein as a mutant cell which has a desired morphology. A
morphological mutant cell may be identified, for example, by using
standard plating techniques employing a growth medium which elicits
the desired morphology relative to the parent cell, by microscopic
examination, or by sorting vegetatively growing cells by
fluorescence activated cell sorting. Such morphological mutants
include, but are not limited to, a mutant characterized as having
superior Theological properties, e.g., a highly-branched fungal
mutant, a restricted colonial fungal mutant, or a highly-branched
restricted colonial fungal mutant which possesses rapid growth and
low viscosity growth characteristics; a mutant which possesses a
filamentous form during fermentation in contrast to a pellet form;
a mutant which is less "sticky" preventing the colonization of
fermentor surfaces; a mutant with a predictable viscosity during
the course of a fermentation; a color mutant which aids in
monitoring and maintaining the purity of a culture and high
production of a polypeptide by the culture; a wettable cell which
lacks, for example, a cell wall or structural hydrophobic protein,
e.g., hydrophobin; an osmotic stress-insensitive mutant which
improves growth of a cell; a desiccation-insensitive mutant which
improves growth of a cell; a non-spore-forming mutant which
enhances the production of a polypeptide; and a non-slime-producing
mutant with low viscosity growth.
[0353] Preferably, the morphological mutant cell is a color mutant,
a wettable mutant cell, a mutant characterized as having superior
Theological properties, an osmotic stress-insensitive mutant, a
desiccation-insensitive mutant, a non-spore-forming mutant, or a
non-slime-producing mutant, and most preferably a highly-branched
fungal mutant, a restricted colonial fungal mutant, or a
highly-branched restricted colonial fungal mutant.
[0354] In another preferred embodiment, the mutant cell is a
secretion mutant cell. A "secretion mutant cell" is defined herein
as a mutant cell which produces higher yields of one or more
secreted proteins. A secretion mutant cell may be identified by
using a detection method known in the art that is specific for the
polypeptide and comparing the yield to one or more known secreted
polypeptides at the same time. Detection methods for polypeptides
may include, but are not limited to, use of specific antibodies,
enzymatic activity by measuring formation of an enzyme product or
disappearance of an enzyme substrate, clearing zones on agar plates
containing an enzyme substrate, biological activity assays, and
fluorescent activating cell sorting.
[0355] In another preferred embodiment, the specifically desired
mutant cell is an auxotrophic mutant cell. An "auxotrophic mutant
cell" is defined herein as a mutant cell which has lost its ability
to synthesize one or more essential metabolites or to metabolize
one or more metabolites which modifies the production of a
polypeptide by the mutant cell. An auxotrophic mutant cell may be
identified using standard plating techniques by growing the
presumptive mutant both in the absence and presence of an essential
metabolite. The auxotrophic mutant will not grow in the absence of
the essential metabolite. The auxotrophic mutant can be
advantageously used to selectively screen for a mutant producing a
specific polypeptide of interest.
[0356] In a more preferred embodiment, the specifically desired
mutant cell is an auxotrophic mutant cell unable to metabolize or
synthesize one or more of an amino acid, a fatty acid, an organic
acid, a pyrimidine, a purine, or a sugar; and more preferably
5-aminolevulinic acid, biotin, glucose, lactose, or maltose.
[0357] In another preferred embodiment, the specifically desired
mutant cell is a conditional mutant cell. A "conditional mutant
cell" is defined herein as a mutant cell which contains one or more
mutations whose phenotypes are only observed under certain
conditions and modifies the production of a polypeptide or a
metabolite by the mutant cell. Conditional mutations can occur in
virtually all genes, including those that control the steps in
macromolecular synthesis, modification, and assembly into
supermolecular structures. A conditional mutant cell may be
identified using standard plating techniques by growing the
presumptive mutant both under permissive and restrictive
conditions. For example, a mutant strain which does not produce
undesirable proteolytic activity under nitrogen limited conditions
would be desirable compared to the parent strain which produces
proteolytic activity under nitrogen limited conditions. An
additional example is an alkaline pH sensitive mutant that does not
grow at alkaline pH, but may have increased or decreased production
of a desired polypeptide. A further example is a mutant which is
unable to grow under specifc growth conditions.
[0358] In a more preferred embodiment, the conditional mutant cell
is a temperature-sensitive, acid pH sensitive, alkaline pH
sensitive, antibiotic-resistant, antibiotic-sensitive,
toxin-resistant, toxin-sensitive, virus-resistant, or
paraquat-sensitive cell; and most preferably an alkaline pH
sensitive mutant cell.
[0359] In another preferred embodiment, the specifically desired
mutant cell is a mutant cell exhibiting an altered growth rate
relative to the parent cell. A "mutant cell exhibiting an altered
growth rate" is defined herein as a mutant cell which has a
doubling time that is different than that of the parent cell. Such
a mutant cell may be identified by comparing the growth of the
mutant cell and the parent cell under controlled fermentation
conditions. Such a mutant cell may have improved fermentation
characteristics like a shorter fermentation time to increase
productivity, or a longer fermentation time to provide control of
the oxygen demand of a culture.
[0360] In another preferred embodiment, the specifically desired
mutant cell is a mutant cell resulting in the relief of
overexpression mediated growth inhibition. A "mutant cell resulting
in the relief of overexpression mediated growth inhibition" is
defined herein as a mutant cell whose growth is not inhibited by
the overproduction of a desired polypeptide or metabolite when
grown under conditions that induce high level production of the
polypeptide or the metabolite. Such a mutant may be identified by
standard plating techniques on plates with an inducing carbon
source, e.g., maltose. Mutants would be able to grow well on the
inducing carbon source while the parent cells would grow poorly.
Such a mutant would be useful since it is known in some cells that
overexpression of a polypeptide is toxic to the cells.
[0361] In another preferred embodiment, the specifically desired
mutant cell is a mutant cell able to tolerate low oxygen
conditions. A "mutant cell able to tolerate low oxygen conditions"
is defined herein as a mutant cell which is able to grow and
produce a desired polypeptide or metabolite under growth conditions
where the dissolved oxygen concentration is low. Such a mutant cell
is particularly advantageous for fermentations where the
productivity of high cell densities decreases due to oxygen
transfer. A low oxygen tolerant mutant is preferably detected by
growing the mutant cell relative to the parent cell on a solid or
in a liquid medium in the presence of low levels of oxygen.
[0362] In a more preferred embodiment, the specifically desired
mutant cell is a mutant cell able to tolerate low oxygen conditions
in the range of about 0 to about 50% saturation, preferably about 0
to about 40% saturation, even more preferably about 0% to about 30%
saturation, more preferably about 0% to about 20% saturation, most
preferably about 0% to about 10% saturation, and even most
preferably about 0% to about 5% saturation.
[0363] In another preferred embodiment, the specifically desired
mutant cell is a signal transduction pathway mutant cell. A "signal
transduction pathway mutant cell" is defined herein as a mutant
cell with a mutation in one or more of the genes of the pathway
which modifies the production of a polypeptide encoded by a DNA
sequence of interest. The term "signal transduction pathway" is
defined herein as a cascade of genes encoding polypeptides that are
all required for the activation or deactivation of another single
polypeptide. The pathway senses a signal and through the cascade of
genes, the signal is transduced and leads to the activation or
deactivation of one or more polypeptides. Such a mutant is
preferably detected using a method which is specific to the desired
phenotype which modifies the production of a polypeptide of
interest.
[0364] In a more preferred embodiment, the signal transduction
pathway mutant cell is a glucose transport signal transduction
pathway mutant or a pH signal transduction pathway mutant, even
more preferably a mutant in which gene required for activation of
pacC has been disrupted, and most preferably a gluT gene mutant or
a palB gene mutant.
[0365] In another preferred embodiment, the specifically desired
mutant cell is a mutant cell exhibiting altered production of a
transcriptional activator of a promoter. A "mutant cell exhibiting
altered production of a transcriptional activator of a promoter" is
defined herein as a mutant cell with a mutation in a gene encoding
a transcriptional activator which `turns-up` or `turns-down` a
promoter of a DNA sequence encoding a polypeptide of interest.
[0366] Examples of such promoters in a bacterial cell are promoters
of the genes of the Bacillus amyloliquefaciens alpha-amylase gene
(amyQ), the Bacillus licheniformis alpha-amylase gene (amyL), the
Bacillus licheniformis penicillinase gene (penP), the Bacillus
stearothermophilus maltogenic amylase gene (amyM), the Bacillus
subtilis levansucrase gene (sacB), the Bacillus subtilis xylA and
xylB genes, the E. coli lac operon, the Streptomyces coelicolor
agarase gene (dagA), and the 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 J. Sambrook, E. F. Fritsch, and T. Maniatus, 1989, Molecular
Cloning, A Laboratory Manual, 2d edition, Cold Spring Harbor,
N.Y.
[0367] Examples of such promoters in a filamentous fungal cell are
promoters of the genes encoding Aspergillus nidulans acetamidase,
Aspergillus niger acid stable alpha-amylase, Aspergillus niger
neutral alpha-amylase, Aspergillus awamori or Aspergillus niger
glucoamylase (glaA), Aspergillus oryzae alkaline protease,
Aspergillus oryzae TAKA amylase, Aspergillus oryzae triose
phosphate isomerase, Fusarium oxysporum trypsin-like protease (as
described in U.S. Pat. No. 4,288,627, which is incorporated herein
by reference), Rhizomucor miehei aspartic proteinase, Rhizomucor
miehei lipase, and mutant, truncated, and hybrid promoters thereof.
Particularly preferred promoters in filamentous fungal cells are
the TAKA amylase, NA2-tpi (a hybrid of the promoters from the genes
encoding Aspergillus niger neutral alpha-amylase and Aspergillus
oryzae triose phosphate isomerase), and glaA promoters.
[0368] Examples of such promoters in a yeast cell are promoters of
the genes encoding Saccharomyces cerevisiae enolase (ENO-1) gene,
the Saccharomyces cerevisiae galactokinase gene (GAL1), the
Saccharomyces cerevisiae alcohol
dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase genes
(ADH2/GAP), and the Saccharonyces cerevisiae 3-phosphoglycerate
kinase gene. Other yeast promoters are described by Romanos et al.,
1992, Yeast 8:423-488.
[0369] Examples of such promoters in a mammalian cell are viral
promoters such as those of Simian Virus 40 (SV40), Rous sarcoma
virus (RSV), adenovirus, and bovine papilloma virus (BPV).
[0370] In a more preferred embodiment, the mutant cell exhibits
altered production of a transcriptional activator of the TAKA,
TAKA/NA2, Fusarium oxysporum trypsin-like protease, or a
glucoamylase promoter.
[0371] In another preferred embodiment, the specifically desired
mutant cell is a cryptic intron-splicing-deficient mutant cell. A
"cryptic intron-splicing-deficient mutant cell" is defined herein
as a mutant cell which no longer recognizes and erroneously splices
a cryptic intron as an authentic intron during mRNA synthesis. A
cryptic intron-splicing-deficie- nt mutant cell would be
particularly useful to prevent the excision or splicing of an
erroneous cryptic nuclear pre-mRNA intron from a primary transcript
so a biologically active substance is produced. In this situation,
the cryptic intron is actually part of the coding sequence and,
therefore, is not an authentic intron but incorrectly recognized as
such and erroneously spliced during mRNA synthesis and processed by
the parent cell. The introduction of a DNA sequence encoding a
heterologous polypeptide into a parent cell such as a fungal host
cell, particularly a filamentous fungal host cell, may result in
this type of erroneous or aberrant splicing of the coding sequence.
A cryptic intron-splicing-deficient mutant cell may be identified
by screening for increased production of a polypeptide encoded by a
DNA sequence that is known to have a cryptic intron which leads to
little or no production of the polypeptide.
[0372] In another preferred embodiment, the specifically desired
mutant may be a mutant which contains two or more of the mutations
described above.
[0373] Identification of Mutant Cells of the Present Invention
[0374] The present inventors have discovered that when certain loci
in a parent cell are disrupted, the resulting mutant cell has a
modified production of a polypeptide. As described above, the
nucleic acid construct itself can have an effect on the production
of a polypeptide. For example, the nucleic acid construct may
comprise one or more copies of the nucleic acid sequence encoding
the polypeptide. In addition, the nucleic acid construct may
comprise a promoter, transcriptional activators and repressors,
etc.
[0375] When the nucleic acid construct itself can have an effect on
the amount of polypeptide produced, expressed, synthesized or
secreted, in order to determine whether a mutant cell of the
present invention has been produced, one would have to rescue the
locus as described below and introduce another nucleic acid
construct which does not have an effect, e.g., a selectable marker,
at the same locus. If the mutant cell produced by introducing the
other nucleic acid construct at the same locus also has an effect
on the amount of polypeptide produced, expressed, synthesized or
secreted, then the original mutant cell is a mutant cell of the
present invention.
[0376] Rescue of a Locus with the Inserted Nucleic Acid Construct
and Use of a Targeting Construct
[0377] The present invention further relates to methods for
rescuing a locus with the inserted nucleic acid construct
comprising isolating from the identified mutant cell (i) the
nucleic acid construct and (ii) the 3' and 5' flanking regions of
the locus of the genome where the nucleic acid construct has been
integrated; and identifying the 3' and 5' flanking regions of the
locus.
[0378] The nucleic acid construct and flanking regions can be
isolated or rescued by methods well known in the art such as
cleaving with restriction enzymes and subsequent ligation and
transformation of E. coli, inverse PCR, random primed gene walking
PCR, or probing a library of the tagged mutant. The isolated
nucleic acid construct with either or both the 3' and 5' flanking
regions is defined herein as a "targeting construct".
[0379] The targeting construct includes between 100-9,000 bp,
preferably 200-9,000 bp, more preferably 500-7,000 bp, even more
preferably 1,000-7,000 bp, and most preferably 1,000-3,000 bp
upstream and/or downstream of the integration site of the nucleic
acid construct.
[0380] The targeting construct of the invention may be introduced
into a different cell to modify the production of a polypeptide
similar or identical to or completely different from the
polypeptide modified in the original cell. The other cell may be of
the same or a different species or of a different genera as the
original cell. If the original cell was a fungal cell, the other
cell is preferably a fungal cell. If the original cell was a
bacterial cell, the other cell is preferably a bacterial cell. If
the original cell was a mammalian cell, the other cell is
preferably a mammalian cell.
[0381] When the cell is a different cell, integration of the
targeting construct preferably occurs at a target locus which is
homologous to the locus sequence of the original cell from which
the targeting construct was obtained, i.e., identical or
sufficiently similar such that the targeting sequence and cellular
DNA can undergo homologous recombination to produce the desired
mutation. The sequence of the targeting construct is preferably,
therefore, homologous to a preselected site of the cellular
chromosomal DNA with which homologous recombination is to occur.
However, it will be understood by one of ordinary skill in the art
that the likelihood of a targeting construct reinserting at a
target locus will depend on the cell since homologous recombination
frequencies range from almost 100% in the yeast Saccharomyces
cerevisiae to as low as 1% in Aspergillus. The targeting construct
may integrate by non-homologous recombination at a non-target locus
which is not within the DNA sequence encoding the polypeptide of
interest, but results in the modification of the production of the
polypeptide.
[0382] Preferably, the target locus includes DNA sequences that
have greater than 40% homology, preferably greater than 60%
homology, more preferably greater than 70% homology, even more
preferably greater than 80% homology, and most preferably greater
than 90% homology with the flanking sequences of the targeting
construct.
[0383] The targeting construct may contain either or both of the 3'
and 5' regions depending on whether a single cross-over or a
replacement is desired. Furthermore, the targeting construct may be
modified to correct any aberrant events, such as rearrangements,
repeats, deletions, or insertions, which occurred during the
introduction and integration of the original nucleic acid construct
into the cell's genome at the locus from which it was originally
rescued.
[0384] The targeting construct described above may be used as is,
i.e., a restriction enzyme cleaved linear nucleotide sequence, or
may be circularized or inserted into a suitable vector. For
example, a circular plasmid or DNA fragment preferably employs a
single targeting sequence. A linear plasmid or DNA fragment
preferably employs two targeting sequences. The targeting construct
upon introduction into a cell, in which the cell comprises a DNA
sequence encoding a polypeptide of interest, integrates into the
genome of the cell at a target locus or at a nontarget locus, but
preferably at a target locus, not within the DNA sequence encoding
the polypeptide of interest. The target locus may be on the same
chromosome or the same extrachromosomal element or on a different
chromosome or a different extrachromosomal element as that of the
DNA sequence of interest. The integration modifies the production
of the polypeptide or a metabolite by the mutant cell relative to
the parent cell when the mutant cell and the parent cell are
cultured under the same conditions. In a preferred embodiment, the
targeting construct contains a selectable marker.
[0385] Optionally, the targeting construct can be introduced into a
cell as two or more separate fragments. In the event two fragments
are used, the fragments share DNA sequence homology (overlap) at
the 3' end of one fragment and the 5' end of the other, while one
carries a first targeting sequence and the other carries a second
targeting sequence. Upon introduction into a cell, the two
fragments can undergo homologous recombination to form a single
fragment with the first and second targeting sequences flanking the
region of overlap between the two original fragments. The product
fragment is then in a form suitable for homologous recombination
with the cellular target sequences. More than two fragments can be
used, designed such that they will undergo homologous recombination
with each other to ultimately form a product suitable for
homologous recombination with the cellular target sequences.
[0386] Upon introduction of the targeting construct into a cell,
the targeting construct may be further amplified by the inclusion
of an amplifiable selectable marker gene which has the property
that cells containing amplified copies of the selectable marker
gene can be selected for by culturing the cells in the presence of
the appropriate selectable agent.
[0387] In a specific embodiment, the targeting construct is SphI
linearized pDSY109, HpaI linearized pDSY112, AsnI/PvuI linearized
pMT1936, NdeI linearized pDSY138, AsnI/PvuI linearized pDSY162,
BglII linearized p4-8.1, BglII linearized p4-8.1, NarI linearized
p7-14.1, BglII linearized pSMO717, BglII linearized pSMO321, NdeI
linearized pHowB571, or NdeI linearized pSMO810.
[0388] In a most preferred embodiment, the nucleic acid construct
is pDSY109.
[0389] In a most preferred embodiment, the nucleic acid construct
is pDSY112.
[0390] In a most preferred embodiment, the nucleic acid construct
is pMT1936.
[0391] In a most preferred embodiment, the nucleic acid construct
is pDSY138.
[0392] In a most preferred embodiment, the nucleic acid construct
is pDSY162.
[0393] In a most preferred embodiment, the nucleic acid construct
is pDSY163.
[0394] In a most preferred embodiment, the nucleic acid construct
is pDSY141.
[0395] In a most preferred embodiment, the nucleic acid construct
is pSMO1204.
[0396] In a most preferred embodiment, the nucleic acid construct
is pSMOH603.
[0397] In a most preferred embodiment, the nucleic acid construct
is p4-8.1.
[0398] In a most preferred embodiment, the nucleic acid construct
is p7-14.1.
[0399] In a most preferred embodiment, the nucleic acid construct
is pHB220.
[0400] In a most preferred embodiment, the nucleic acid construct
is pSMO717.
[0401] In a most preferred embodiment, the nucleic acid construct
is pSMO321.
[0402] In a most preferred embodiment, the nucleic acid construct
is pHowB571.
[0403] In a most preferred embodiment, the nucleic acid construct
is pSMO810.
[0404] In a preferred embodiment, one or more targeting constructs
are introduced into target loci. In another preferred embodiment,
each targeting construct modifies the production of a different
polypeptide or a different metabolite or a combination thereof, or
results in different phenotypes which modify the production of
different polypeptides or different metabolites or a combination
thereof. In another preferred embodiment, two or more targeting
constructs together when introduced into target loci act additively
or synergistically to modify the production of a polypeptide or a
metabolite.
[0405] Methods of Producing a Desired Polypeptide or Metabolite
from Mutant Cells
[0406] The present invention further relates to the mutant cells
with a desired phenotype as host cells. Mutant cells selected for
increased production of a desired polypeptide or metabolite are
cultivated in a nutrient medium suitable for production of the
polypeptide or metabolite using methods known in the art. For
example, the cell may be cultivated by shake flask cultivation,
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 or metabolite 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 (see, e.g., references for
bacteria and yeast; Bennett, J. W. and LaSure, L., editors, More
Gene Manipulations in Fungi, Academic Press, CA, 1991). Suitable
media are available from commercial suppliers or may be prepared
according to published compositions (e.g., in catalogues of the
American Type Culture Collection). If the polypeptide or metabolite
is secreted into the nutrient medium, the polypeptide can be
recovered directly from the medium. If the polypeptide or
metabolite is not secreted, it is recovered from cell lysates.
[0407] The polypeptides and metabolites may be detected using
methods known in the art that are specific for the polypeptides and
metabolites such as those methods described earlier or the methods
described in the Examples.
[0408] The resulting polypeptide or metabolite may be recovered by
methods known in the art. For example, the polypeptide or
metabolite may be recovered from the nutrient medium by
conventional procedures including, but not limited to,
centrifugation, filtration, extraction, spray-drying, evaporation,
or precipitation.
[0409] The polypeptides and metabolites 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), or extraction (see, e.g., Protein Purification, J.
-C. Janson and Lars Ryden, editors, VCH Publishers, New York,
1989).
EXAMPLES
[0410] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how to make and use various constructs and perform
the various methods of the present invention and are not intended
to limit the scope of what the inventors regard as their invention.
Unless indicated otherwise, parts are parts by weight, temperature
is in degrees centigrade, and pressure is at or near atmospheric
pressure. Efforts have been made to ensure accuracy with respect to
numbers used (e.g., length of DNA sequences, molecular weights,
amounts, particular components, etc.), but some deviations should
be accounted for.
Example 1
Strains and Materials
[0411] The starting strains were pyrG-minus Aspergillus oryzae
HowB425, pyrG-minus Aspergillus oryzae HowB101, Aspergillus oryzae
JaL250, Aspergillus niger strain JRoy3 (pyrG.DELTA.), E. coli
DH5.alpha. (GIBCO-BRL, Gaithersburg, Md.), and E. coli HB101
(GIBCO-BRL, Gaithersburg, Md.).
[0412] PDA plates contained 39 g/l Potato Dextrose Agar (Difco) and
were supplemented with 10 mM uridine for pyrG auxotrophs unless
otherwise indicated.
[0413] MY25 medium at pH 6.5 was composed per liter of 25 g of
maltose, 2.0 g of MgSO.sub.4-7H.sub.2O, 10 g of KH.sub.2PO.sub.4,
2.0 g of citric acid, 10 g of yeast extract, 2.0 g of
K.sub.2SO.sub.4, 2.0 g of urea, and 0.5 ml of trace metals
solution. MY25 shake-flask medium was diluted 1:100 or 1:1000 with
glass distilled water for use in microtiter growth experiments
(MY25/100 or MY25/1000). Cultures were grown at 34.degree. C.
2.times.MY Salts pH 6.5 solution was composed per liter of 4 g of
MgSO.sub.4-7H.sub.2O, 4 g of K.sub.2SO.sub.4, 20 g of
KH.sub.2PO.sub.4, 4 g of citric acid, 1 ml of trace metals, and 2
ml of CaCl.sub.2-2H.sub.2O (100 g/l stock solution.
[0414] Minimal medium transformation plates were composed per liter
of 6 g of NaNO.sub.3, 0.52 g of KCl, 1.52 g of KH.sub.2PO.sub.4, 1
ml of trace metals solution, 1 g of glucose, 500 mg of
MgSO.sub.4-7H.sub.2O, 342.3 g of sucrose and 20 g of Noble agar per
liter (pH 6.5). Minimal medium transfer plates (pH 6.5) were
composed per liter of 6 g of NaNO.sub.3, 0.52 g of KCl, 1.52 g of
KH.sub.2PO.sub.4, 1 ml of trace elements, 1 g of glucose, 500 mg of
MgSO.sub.4-7H.sub.2O, and 20 g Noble agar.
[0415] The trace metals solution (1000.times.) was composed per
liter of 22 g of ZnSO.sub.4-7H.sub.2O, 11 g of H.sub.3BO.sub.3, 5 g
of MnCl.sub.2-4H.sub.2O, 5 g of FeSO.sub.4-7H.sub.2O, 1.6 g of
CoCl.sub.2-5H.sub.2O, 1.6 g of (NH.sub.4).sub.6Mo.sub.7O.sub.24,
and 50 g of Na.sub.4EDTA.
[0416] COVE plates were composed per liter of 343.3 g of sucrose,
20 ml of COVE salts solution, 10 ml of 1 M acetamide, 10 ml of 3 M
CsCl, and 25 g of Nobel agar. The COVE salts (50.times.) solution
was comprised 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. COVE
trace metals solution was composed of (per liter): 0.04 g of
NaB.sub.4O.sub.7-10H.sub.2O, 0.040 g of CuSO.sub.4-5H.sub.2O, 0.70
g of FeSO.sub.4-H.sub.2O, 0.80 g of Na.sub.2MoO.sub.2-2H.sub.2O,
and 10 g of ZnSO.sub.4.
[0417] YEG medium was composed per liter of 5 g yeast extract and
20 g dextrose.
[0418] CM-1 agar plates at pH 6.5 were composed per liter of 0.25 g
of NaCl, 0.5 g of MgSO.sub.4-7H.sub.2O, 1.9 g of K.sub.2HPO.sub.4,
3.6 g of KH.sub.2PO.sub.4, 0.1 ml of trace metals solution, 30 g of
Bacto agar (Difco), pH 6.5. 11 ml of 10% urea, and 67 ml of 30%
maltose.
[0419] CD medium was composed per liter of 1 g of
MgSO.sub.4-7H.sub.2O, 1 g of K.sub.2SO.sub.4, 15 g of
KH.sub.2PO.sub.4, 0.25 ml of trace metals solution, 0.7 g of yeast
extract (Difco), 20 g of beta-cyclodextrin (Sigma C-4767). 3 ml of
50% urea, and 2 ml of 15% CaCl.sub.2-2H.sub.2O.
[0420] G1-gly medium was composed per liter of 18 g of yeast
extract (Difco), 80 g of 75% glycerol, and 0.5 g of
CaCl.sub.2-2H.sub.2O.
[0421] OL-1 medium (pH 7.0) was composed per liter of 15 g of
KH.sub.2PO.sub.4, 1 g of MgSO.sub.4-7H.sub.2O, 1 g of
K.sub.2SO.sub.4, 0.25 ml of trace metals solution, 0.3 g of
CaCl.sub.2-2H.sub.2O (autoclaved separately), 2 g of Difco yeast
extract (Difco), 0.5 g of urea (autoclaved separately), and 10 g of
glucose.
[0422] OL-6 medium (pH 7.0) was composed per liter of 15 g of
KH.sub.2PO.sub.4, 1 g of MgSO.sub.4-7H.sub.2O, 1 g of
K.sub.2SO.sub.4, 0.25 ml of trace metals solution, 0.3 g of
CaCl.sub.2-2H.sub.2O (autoclaved separately), 2 g of Difco yeast
extract (Difco), 3 g of urea (autoclaved separately), and 60 g of
glucose.
[0423] YPM medium was composed of 10 g of Bactopeptone and 5 g of
yeast extract dissolved in 500 ml of water and autoclaved, to which
50 ml of a sterilized 20% maltose solution was added.
[0424] MTBCDUY was composed per liter of 0.3 g of
MgSO.sub.4-7H.sub.2O, 0.3 g of K.sub.2SO.sub.4, 5 g of
KH.sub.2PO.sub.4, 0.013 g of urea, 0.01 g of yeast extract, 0.1 g
of maltose, 4.88 g of uridine, and 0.25 ml of trace metal solution
1 adjusted to pH 6.5.
[0425] 4.times.MTBCDUY was composed per liter of 0.3 g of
MgSO.sub.4-7H.sub.2O, 0.3 g of K.sub.2SO.sub.4, 5 g of
KH.sub.2PO.sub.4, 0.052 g of urea, 0.04 g of yeast extract, 0.4 g
of maltose, 4.88 g of uridine, and 0.25 ml of trace metal solution
1.
[0426] MDU1B was composed per liter of 45 g of Maltodextrin MD01,
1.0 g of MgSO.sub.4-7H.sub.2O, 1.0 g of NaCl, 2.0 g of
K.sub.2SO.sub.4, 12.0 g of KH.sub.2PO.sub.4, 7.0 g of yeast
extract, 0.5 ml of trace metal solution, and 0.1 ml of pluronic
acid. The trace metal solution consisted of 13.9 g of
FeSO.sub.4-7H.sub.2O, 8.45 g of MnSO.sub.4-H.sub.2O, 6.8 g of
ZnCl.sub.2, 2.5 g of CuSO.sub.4-5H.sub.2O, 2.5 g of
NiCl.sub.2-6H.sub.2O, and 3 g of citric acid. The pH of the shake
flask medium was adjusted to 5.0 before being autoclaved.
[0427] 1/5MDU2BP was composed per liter of 9 g of maltose, 0.2 g of
MgSO.sub.4-7H.sub.2O, 0.4 g of K.sub.2SO.sub.4, 0.2 g of NaCl, 2.4
g of KH.sub.2PO.sub.4, 1.0 g of urea, 1.4 g of yeast extract, and
0.1 ml of trace metal solution 1.
[0428] Trace metal solution 1 was composed per liter of 13.8 g of
FeSO.sub.4-7H.sub.2O, 8.5 g of MnSO.sub.4-H.sub.2O, 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, and 3.0 g of citric acid.
[0429] YPG plates was composed per liter of 4.0 g yeast extract,
1.0 g of K.sub.2HPO.sub.4, 0.5 g of MgSO.sub.4-7H.sub.2O, 15.0 g of
dextrose, and 20.0 g of agar.
Example 2
Construction of Aspergillus oryzae HowB430
[0430] Aspergillus oryzae HowB430 was constructed to contain a
lipase gene from Humicola lanuginosa (LIPOLASE.TM. gene, Novo
Nordisk A/S, Bagsvaerd, Denmark).
[0431] pBANe8 was constructed as described below to contain the
TAKA/NA2-tpi leader hybrid promoter, the lipase gene from Humicola
lanuginosa, the AMG terminator, and the full-length Aspergillus
nidulans amdS gene as a selectable marker.
[0432] PCR was employed to insert NsiI sites flanking the
full-length amdS gene of pToC90 (Christensen et al., 1988,
Biotechnology 6: 1419-1422) using primers 1 and 2 below and to
insert an EcoRI site at the 5' end and a SwaI site at the 3' end of
the NA2-tpi leader hybrid promoter of pJaL292 (FIG. 1) using
primers 3 and 4 below. The primers were synthesized with an Applied
Biosystems Model 394 DNA/RNA Synthesizer (Applied Biosystems, Inc.,
Foster City, Calif.) according to the manufacturer's
instructions.
1 Primer 1: 5'-ATGCATCTGGAAACGCAACCCTGA-3' (SEQ ID NO:1) Primer 2:
5'-ATGCATTCTACGCCAGGACCGAGC-3' (SEQ ID NO:2)
[0433] Amplification reactions (100 .mu.l) were prepared using
approximately 0.2 .mu.g of either pToC90 or pJaL292 as the
template. Each reaction contained the following components: 0.2
.mu.g of plasmid DNA, 48.4 pmol of the forward primer, 48.4 pmol of
the reverse primer, 1 mM each of dATP, dCTP, dGTP, and dTTP,
1.times.Taq polymerase buffer, and 2.5 U of Taq polymerase
(Perkin-Elmer Corp., Branchburg, N.J.). The reactions were
incubated in an Ericomp Thermal Cycler programmed as follows: One
cycle at 95.degree. C. for 5 minutes followed by 30 cycles each at
95.degree. C. for 1 minute, 55.degree. C. for 1 minute and
72.degree. C. for 2 minutes.
[0434] The PCR products were electrophoresed on a 1% agarose gel to
confirm the presence of a 2.7 kb amdS fragment and a 0.6 kb NA2-tpi
fragment.
[0435] The PCR products were subsequently subcloned into pCRII
using a TA Cloning Kit (Invitrogen, San Diego, Calif.) according to
the manufacturer's instructions. The transformants were then
screened by extracting plasmid DNA from the transformants using a
QIAwell-8 Plasmid Kit (Qiagen, Inc., Chatsworth, Calif.) according
to the manufacturer's instructions, and restriction digesting the
plasmid DNA with either NsiI or EcoRI/SwaI followed by agarose
electrophoresis to confirm the presence of the correct size
fragments, 2.7 kb and 0.6 kb, respectively, for the NsiI amdS
fragment and SwaI/EcoRI NA2-tpi fragment. In order to confirm the
PCR products, the products were sequenced with with an Applied
Biosystems Model 373A Automated DNA Sequencer (Applied Biosystems,
Inc., Foster City, Calif.) on both strands using the primer walking
technique with dye-terminator chemistry (Giesecke et al., 1992,
Journal of Virol. Methods 38: 47-60) using the M13 reverse (-48)
and M13 forward (-20) primers (New England Biolabs, Beverly, Mass.)
and primers unique to the DNA being sequenced. The plasmids from
the correct transformants were then digested with the restriction
enzymes for which the plasmids were designed, separated on a 1%
agarose gel, and purified using a FMC SpinBind Kit (FMC, Rockland,
Me.) according to the manufacturer's instructions.
[0436] pKS6 (FIG. 2), which contains the TAKA promoter, a
polylinker, the AMG terminator, and the Aspergillus nidulans pyrG
gene, was digested with EcoRI and SwaI to remove a portion of the
TAKA promoter. This region was replaced with the NA2-tpi PCR
product to produce pBANe13 (FIG. 3).
[0437] pBANe13 was digested with NsiI to remove the Aspergillus
nidulans pyrG gene. This region was then replaced with the full
length amdS gene PCR product described above to produce pBANe6
(FIG. 4).
[0438] PCR was used to insert SwaI and PacI flanking sites on the
full-length Humicola lanuginosa lipase gene of pMHan37 (FIG. 5)
using primers 5 and 6 below. Primers 5 and 6 were synthesized as
described above.
2 Primer 5: 5'-ATTTAAATGATGAGGAGCTCCCTTGTGCTG-3' (SEQ ID NO:5)
Primer 6: 5'-TTAATTAACTAGAGTCGACCCAGCCGCGC-3- ' (SEQ ID NO:6)
[0439] The amplification reaction (100 .mu.l) contained the
following components: 0.2 .mu.g of pMHan37, 48.4 pmol of primer 5,
48.4 pmol of primer 6, 1 mM each of dATP, dCTP, dGTP, and dTTP,
1.times.Taq polymerase buffer, and 2.5 U of Taq polymerase. The
reaction was incubated in an Ericomp Thermal Cycler programmed as
follows: One cycle at 95.degree. C. for 5 minutes followed by 30
cycles each at 95.degree. C. for 1 minute, 55.degree. C. for 1
minute, and 72.degree. C. for 2 minutes. Two .mu.l of the reaction
was electrophoresed on an agarose gel to confirm the amplification
of the lipase gene product of approximately 900 bp.
[0440] The PCR amplified lipase gene product was then subcloned
into pCRII using a TA Cloning Kit. The transformants were screened
by extracting plasmid DNA from the transformants using a QIAwell-8
Plasmid Kit, restriction digesting the plasmid DNA with SwaI/PacI,
and sequencing the DNA according to the method described above to
confirm the PCR product.
[0441] The lipase gene was excised from the pCRII plasmid by
digesting with SwaI and PacI and subsequently subcloned into
SwaI/PacI digested pBANe6 to produce pBANe8 (FIG. 6).
[0442] pBANe8 was digested with PmeI and the linear PmeI fragment
containing the NA2-tpi promoter, the lipase gene from Humicola
lanuginosa, and the AMG terminator was isolated by preparative
agarose electrophoresis using 40 mM Tris-acetate-1 mM disodium EDTA
(TAE) buffer.
[0443] Aspergillus oryzae HowB430 was generated by transformation
of Aspergillus oryzae HowB425 with the linear PmeI fragment
according to the following procedure.
[0444] Aspergillus oryzae HowB425 was grown in 100 ml of 1% yeast
extract-2% peptone-1% glucose at 32.degree. C. for 16-18 hours with
agitation at 150 rpm. The mycelia were recovered by filtration
through a 0.45 .mu.m filter until approximately 10 ml remained on
the filter, washed with 25 ml of 1.0-1.2 M MgSO.sub.4-10 mM sodium
phosphate pH 6.5, filtered as before, washed again as before until
10 ml remained, and then resuspended in 10 ml of 5 mg/ml NOVOZYM
234.TM. (Novo Nordisk A/S, Bagsvaerd, Denmark) in 1.2 M
MgSO.sub.4-10 mM sodium phosphate pH 6.5 (0.45 .mu.m filtered) in a
125 ml Ehrlenmeyer flask. The suspension was incubated with gentle
agitation at 50 rpm for approximately one hour at 37.degree. C. to
generate protoplasts. A volume of 10 ml of the protoplast/mycelia
preparation was added to a 30 ml Corex centrifuge tube, overlaid
with 5 ml of 0.6 M sorbitol-10 mM Tris-HCl pH 7.5, and centrifuged
at 3600.times.g for 15 minutes in a swinging bucket rotor to
recover the protoplasts. The protoplasts were recovered from the
buffer interface with a Pasteur pipet. The protoplasts were then
washed with five volumes of STC, centrifuged, and then rewashed and
centrifuged as before. The protoplasts were resuspended in STC to a
final concentration of 2.times.10.sup.7 protoplasts per ml.
[0445] Transformation of Aspergillus oryzae HowB425 for amdS
selection was conducted with protoplasts at a concentration of
2.times.10.sup.7 protoplasts per ml. Ten .mu.g of DNA were added to
100 .mu.l of protoplasts. A volume of 250 .mu.l of PEG solution
(60% PEG 4000-10 mM CaCl.sub.2-10 mM Tris-HCl pH 8.0) was then
added and the mixture was placed at 37.degree. C. for 30 minutes.
Three ml of 1 M sorbitol-10 mM CaCl.sub.2-10 mM Tris pH 7.5 (STC)
was added and the mixture was plated on Cove plates supplemented
with 10 mM uridine selecting for amdS. The plates were incubated
7-10 days at 34.degree. C. Transformants were transferred to plates
of the same medium and incubated 3-5 days at 37.degree. C. The
transformants were purified by streaking spores and picking
isolated colonies using the same plates of the same medium without
sucrose under the same conditions.
Example 3
Construction of Aspergillus oryzae HowB427
[0446] Aspergillus oryzae HowB425 was co-transformed with pMHan37
and pSO2 (FIG. 7) to construct Aspergillus oryzae HowB427 to
contain the lipase gene from Humicola lanuginosa behind the TAKA
promoter.
[0447] pSO2 (FIG. 7) was constructed from a genomic library of
Aspergillus oryzae 1560. The genomic library of Aspergillus oryzae
1560 was constructed by first partially digesting Aspergillus
oryzae 1560 genomic DNA with Sau3A (New England Biolabs, Beverly,
Mass.). Four units of Sau3A were used to digest 10 .mu.g of
Aspergillus oryzae 1560 genomic DNA using conditions recommended by
the manufacturer. The reaction was carried out at 65.degree. C.,
and samples were taken at 5 minute intervals (from 0 to 50
minutes). The reactions were placed on ice and stopped by the
addition of EDTA to 10 mM. These digests were then run on a 1%
agarose gel with ethidium bromide, and the region of the gel
containing DNA from 3 kb to 9 kb was excised. The DNA was then
purified from the gel slice using Beta-Agarase I using a protocol
provided by the manufacturer (New England Biolabs, Beverly, Mass.).
The size-selected DNA was then ligated into EMBL 4 arms according
to the manufacturer's instructions (Clontech, Palo Alto, Calif.) at
16.degree. C. overnight using conditions recommended by the
manufacturer. The ligation reaction was packaged and titered using
a Gigapack II Packaging Kit (Stratagene, La Jolla, Calif.)
according to the manufacturer's protocol. A total of 16,000
recombinant plaques were obtained, and the library was amplified
using a protocol provided by the manufacturer.
[0448] Appropriate dilutions of the genomic library were made to
obtain 7000 plaques per 150 mm petri plate as described in the
protocols provided with the EMBL 4 arms. The plaques were lifted to
Hybond-N plus circular filters (Amersham, Cleveland, Ohio) using
standard protocols (Sambrook et al., 1989, supra). The filters were
fixed using UV crosslinking, and prehybridized at 42.degree. C.
(5.times.SSPE, 35% formamide). The genomic library was probed at
low stringency (35% formamide, 5.times.SSPE at 42.degree. C.) with
a 500 bp fragment consisting of the Aspergillus niger pyrG gene
which was labeled with .sup.32P using a random prime DNA labeling
kit (Boehringer Mannheim, Indianapolis, Ind.). A 3.8 kb HindIII
fragment was isolated from one phage and subcloned into a pUC118
cloning vector to produce pSO2.
[0449] The co-transformation of Aspergillus oryzae HowB425 was
conducted using the procedure described in Example 2 except
selection was on Minimal medium transformation plates.
Transformants were transferred to Minimal medium transfer plates
and incubated 3-5 days at 37.degree. C. The transformants were then
purified by streaking spores and picking isolated colonies using
the same transfer plates under the same conditions.
Example 4
Construction of Plasmids pSO122, pDSY81, and pDSY82
[0450] pSO122 was constructed as described below to contain a 1.5
kb fragment of the Aspergillus oryzae pyrG gene.
[0451] PCR was used to generate pSO122 by introducing a BamHI
restriction site at the 5' end of the pyrG gene of pSO2 using
primers 7 and 8 shown below. Primers 7 and 8 were synthesized with
an Applied Biosystems Model 394 DNA/RNA Synthesizer according to
the manufacturer's instructions.
3 Primer 7: 5'-GCGGGATCCCTAGAGTAGGGGGTGGTGG-3' (SEQ ID NO:7) Primer
8: 5'-GCGGGATCCCCCCTAAGGATAGGCCCTA-3' (SEQ ID NO:8)
[0452] The amplification reaction (50 .mu.l) contained the
following components: 2 ng of pSO2, 48.4 pmoles of the forward
primer, 48.4 pmoles of the reverse primer, 1 mM each of dATP, dCTP,
dGTP, and dTTP, 1.times.Taq polymerase buffer, and 2.5 U of Taq
polymerase (Perkin-Elmer Corp., Branchburg, N.J.). The reaction was
incubated in an Ericomp Thermal Cycler programmed as follows: One
cycle at 95.degree. C. for 5 minutes followed by 30 cycles each at
95.degree. C. for 1 minute, 55.degree. C. for 1 minute and
72.degree. C. for 2 minutes. The PCR product was isolated by
electrophoresis on a 1% agarose gel.
[0453] The isolated PCR product was digested with BamHI and cloned
into the BamHI site of pBluescript SK- (Stratagene, La Jolla,
Calif.) to yield pSO122 (FIG. 8). The only homology between the
genome of Aspergillus oryzae HowB430 and pSO122 was in the 5' end
of the pyrG insert since the rest of the pyrG fragment was deleted
from Aspergillus oryzae HowB430 as described in Example 2.
[0454] In order to reduce the frequency of targeting to this
homologous region in the genome and since pSO122 contains two BamHI
sites, two derivatives of pSO122, pDSY81 and pDSY82 (FIG. 8), were
constructed in which one of the BamHI sites was destroyed. The
plasmids pDSY81 and pDSY82 were constructed by partially digesting
pSO122 with BamHI, filling-in the 5' overhangs with the Klenow
fragment, closing down the plasmid by ligation and subsequent
transformation into E. coli DH5.alpha. (Sambrook et al., 1989,
supra). The transformants were then screened by extracting plasmid
DNA from the transformants using a QIAwell-8 Plasmid Kit and
restriction digesting the plasmid DNA with BamHI to determine if
one of the BamHI sites had been destroyed. Plasmids with one of the
BamHI sites destroyed were digested with NsiI/BamHI to determine
which BamHI site had been destroyed.
Example 5
Aspergillus oryzae HowB430 Transformation with pSO122, pDSY81, or
pDSY82
[0455] Protoplasts of Aspergillus oryzae HowB430 were prepared as
described in Example A 5-15 .mu.l aliquot of DNA (circular pSO122,
pDSY81 linearized with 4 to 12 U of EcoRI, or pDSY82 linearized
with 15 U of BamHI) was added to 0.1 ml of the protoplasts at a
concentration of 2.times.10.sup.7 protoplasts per ml in a 14 ml
Falcon polypropylene tube followed by 250 .mu.l of 60% PEG 4000-10
mM CaCl.sub.2-10 mM Tris-HCl pH 7, gently mixed, and incubated at
37.degree. C. for 30 minutes. The transformations were made either
with 5 .mu.g of circular pSO122, 6 .mu.g of linearized pDSY81, or 6
.mu.g of linearized pDSY82. Three ml of SPTC (1.2 M sorbitol-10 mM
CaCl.sub.2-10 mM Tris pH 8) were then added and the suspension was
gently mixed. The suspension was mixed with 12 ml of molten overlay
agar (1.times.COVE salts, 1% NZ amine, 0.8 M sucrose, 0.6% Noble
agar) or 3 ml of STC medium and the suspension was poured onto a
Minimal medium plate. The plates were incubated at 37.degree. C.
for 3-5 days.
[0456] The transformation frequencies of the circular pSO122
transformations ranged from about 100 to 200 transformants/.mu.g. A
library of .about.120,000 DNA-tagged transformants of Aspergillus
oryzae HowB430 was obtained.
[0457] The transformation frequencies of the EcoRI REMI pDSY81
transformations ranged from about 60 to 100 per .mu.g. An EcoRI
REMI library of .about.28,000 DNA-tagged transformants of
Aspergillus oryzae HowB430 was generated.
[0458] The transformation frequencies of the BamHI REMI pDSY82
transformations ranged from about 80 to 110 transformants/tg. A
BamHI REMI library of .about.27,000 DNA-tagged transformants of
Aspergillus oryzae HowB430 was obtained.
[0459] HindIII and SalI REMI libraries of Aspergillus oryzae
HowB430 were also prepared using pDSY81 as described above.
[0460] The transformation frequencies of the HindIII REMI pDSY81
transformations ranged from about 80 to 120 per .mu.g. A HindIII
REMI library of 35,000 DNA-tagged transformants of Aspergillus
oryzae HowB430 was generated.
[0461] The transformation frequencies of the SalI REMI pDSY81
transformations ranged from about 80 to 120 per .mu.g. A SalI REMI
library of 25,000 DNA-tagged transformants of Aspergillus oryzae
HowB430 was generated.
[0462] The Aspergillus oryzae HowB430 tagged mutant library pools
were designated "h" for pSO122; "e" for pDSY81 digested with EcoRI
with subsequent transformation in the presence of EcoRI; "b" for
pDSY82 digested with BamHI with subsequent transformation in the
presence of BamHI; "hIII" for pDSY81 digested with HindIII with
subsequent transformation in the presence of HindIII; and "s" for
pDSY81 digested with SalI with subsequent transformation in the
presence of SalI. There were 123 "h" pools, 28 "e" pools, 23 "b"
pools, 55 "hIII" pools, and 25 "s" pools.
[0463] The libraries described above were pooled into groups of
.about.1000 transformants and stored in 10% glycerol at -80.degree.
C.
Example 6
Characterization of Integration Events in "REMI" Aspergillus oryzae
HowB430 Transformants
[0464] Genomic DNA was isolated from 26 of the EcoRI REMI
transformants ("e" pool) described in Example 5 according to the
following procedure. Each transformant was grown in 5 ml of YEG
medium for 24 hours at 37.degree. C. in a small Petri plate.
Mycelia were then collected from each culture by filtration through
Whatman filter paper No. 1 (Whatman, Springfield Mill, England) and
transferred to a 1.7 ml centrifuge tube. The mycelia preparations
were frozen in dry ice and dried in a SpeedVac (Savant Instruments,
Inc., Farmingdale, N.Y.) overnight at room temperature. The frozen
mycelia preparations were ground to a fine powder with a speared
spatula and then the ground mycelia were resuspended in 0.5 ml of
lysis buffer (100 mM EDTA, 10 mM Tris pH 8.0, 1% Triton X-100, 50
mM guanidine-HCl, 200 mM NaCl). RNase was added to each preparation
to a final concentration of 20 .mu.g/ml, and the preparations were
incubated at 37.degree. C. for 30 minutes. Protease K was then
added to each preparation to a final concentration of 0.1 mg/ml,
and the preparations were incubated at 50.degree. C. for 1 hour.
The preparations were centrifuged at 13,000.times.g for 15 minutes,
and the supernatants were applied to QIAprep-8-well strips (Qiagen,
Chatsworth, Calif.). The wells were washed once with 0.5 ml of PB
and 0.75 ml of PE supplied by the manufacturer (Qiagen, Chatsworth,
Calif.). After removing excess PE from each well, the DNAs were
eluted from the wells in 200 .mu.l of TE buffer (10 mM Tris-1 mM
EDTA pH 7.0).
[0465] The genomic DNA was digested with either EcoRI to determine
whether integration occurred into genomic EcoRI sites or SnaBI to
determine whether or not the integration events were random
throughout the genome by Southern hybridization according to the
procedure described by Sambrook et al., 1989, supra. Southern blots
of the digests were probed with a 1.6 kb NheI pyrG fragment
obtained from pSO122 (FIG. 8) labeled with dioxygenin using a
Genius Kit according to the manufacturer's instructions. The blot
was prehybridized for 2 hours and hybridized overnight at
42.degree. C. in DIG Easy Hyb. The blot was washed and processed as
recommended by the manufacturer.
[0466] The Southern blot demonstrated that in 13 of 26
transformants, EcoRI linearized pDSY81 integrated into an EcoRI
site in the genome, and the distribution of the integration events
appeared to be random. In 20 of the 26 transformants, only a single
copy of the plasmid was integrated while in 6 of the transformants
at least 2 copies were integrated at the same locus. In order to
determine if the bias (of 50%) towards integration at EcoRI sites
was due to REMI, genomic DNA was isolated as described above from
16 Aspergillus oryzae HowB425 transformants, in which the EcoRI
enzyme was heat inactivated before transformation with EcoRi
linearized pDSY81 according to the procedure described in Example
5, and submitted to Southern blot analysis as described above.
Southern analysis of these transformants demonstrated that in none
of the transformants did the plasmid integrate at an EcoRI site in
the genome.
Example 7
Lipase Expression Screening
[0467] The Aspergillus oryzae HowB430 tagged mutant library "h",
"e", and "b" pools described in Example 5 were assayed for lipase
expression.
[0468] For 96-well plate screens, MY25 medium was diluted 1000-fold
using a diluent made of equal volumes of sterile water and
2.times.MY Salts pH 6.5 solution. For 24-well plate methods, MY25
medium was diluted 100-fold using a diluent made of equal volumes
of sterile water and 2.times.MY Salts pH 6.5 solution.
[0469] Primary 96-well plate screens involved the dilution of
spores from distinct pools into MY25/1000 so that one spore on
average was inoculated per well when 50 .mu.l of medium was
dispensed into the wells. After inoculation, the 96-well plates
were grown for 7 days at 34.degree. C. under static conditions.
Cultures were then assayed for lipase activity as described below.
Mutants of interest were inoculated directly into 24-well plates
containing MY25/100 and were grown for 7 days at 34.degree. C.
Cultures were then assayed for lipase activity as described below.
Mutants of interest were then plated on COVE plates to produce
spores, spread on PDA plates to produce single colonies, and then 4
single colonies from each isolate were tested in the 24-well plate
method described above.
[0470] The lipase assay substrate was prepared by diluting 1:50 a
p-nitrophenylbutyrate stock substrate (21 .mu.l of
p-nitrophenylbutyrate/ml DMSO) into MC buffer (4 mM CaCl.sub.2-100
mM MOPS pH 7.5) immediately before use. Standard lipase
(LIPOLASE.TM., Novo Nordisk A/S, Bagsvaerd, Denmark) was prepared
to contain 40 LU/ml of MC buffer containing 0.02% alpha olefin
sulfonate (AOS) detergent. The standard was stored at 4.degree. C.
until use. Standard lipase was diluted {fraction (1/40)} in MC
buffer just before use. Broth samples were diluted in MC buffer
containing 0.02% AOS detergent and 20 .mu.l aliquots were dispensed
to wells in 96-well plates followed by 200 .mu.l of diluted
substrate. Using a plate reader, the absorbance at 405 nm was
recorded as the difference of two readings taken at approximately 1
minute intervals. Lipase units/ml (LU/ml) were calculated relative
to the lipase standard.
[0471] The results of the 96-well screen followed by the 24-well
screen identified for further evaluation 53 transformants from the
pSO122 transformations and 44 transformants from the pDSY81 or
pDSY82 REMI transformations. These identified transformants
produced higher levels of lipase than the control strains
Aspergillus oryzae HowB427 and Aspergillus oryzae HowB430.
Example 8
Shake Flask and Fermentation Evaluation
[0472] The highest lipase-producing DNA-tagged mutants described in
Example 7 were then plated onto COVE plates to produce spores for
shake flask and fermentation evaluations.
[0473] Shake flask evaluations were performed by inoculating
300-500 .mu.l of a spore suspension (0.02% Tween-80 plus spores
from the COVE plates) into 25 ml of MY25 medium at pH 6.5 in a 125
ml shake flask. The shake flasks were incubated at 34.degree. C.
for 3 days at 200 rpm. Samples were taken at day 2 and day 3 and
lipase activity was measured as described in Example 7.
[0474] The same DNA-tagged mutants were grown in a 2 liter lab
fermentor containing medium composed of Nutriose, yeast extract,
(NH.sub.4).sub.2HPO.sub.4, MgSO.sub.4-7H.sub.2O, citric acid,
K.sub.2SO.sub.4, CaCl.sub.2-H.sub.2O, and trace metals solution at
34.degree. C., pH 7, 1000-1200 rpm for 8 days. Lipase activity was
measured as described in Example 7.
[0475] The results obtained are shown in Table 1 below where the
lipase yield of either Aspergillus oryzae HowB427 or Aspergillus
oryzae HowB430 as a control is normalized to 1.0.
4TABLE I Lipase Expression by DNA Tagged Mutants 24-well # Screened
Plate Shake Flask Ferm. Strain in 96-well Results Results Results
Description Construction Pool Plates (LU/ml) (LU/ml) (LU/ml)
HowB427 HowB425 + pMHan37 NA NA 1.2 0.6 1.0 HowB430 HowB425 +
pBANe8 NA NA 1.0 1.0 NA DEBY10.3 pDSY81 + BamHI b1 808 1.7 2.2 3.9
DEBY203.3 pDSY81 + EcoRI e1 707 2.4 2.1 1.8 DEBY599.3 pDSY81 +
BamHI b18 443 1.5 2.4 4.1 DEBY932 pDSY81 + EcoRI e21 1092 1.9 1.9
3.6 DEBY1058 pDSY81 + BamHI b22 80 2.3 2.4 3.8 DEBY1204.3.3 pDSY81
+ EcoRI e26 1260 1.9 2.0 3.0 HINL603 pDSY81 + HindIII hi3-7 NA 2.8
2.0 3.3 HowL91.1 pSO122 h32 1134 1.9 2.3 3.3 HowL214.2 pSO122 h9
861 1.9 2.3 3.0 HowL301.4 pSO122 h58 731 2.3 2.8 3.6 HowL371.3
pSO122 h92 592 1.8 2.5 3.5 HowL442.1 pSO122 h7 1095 2.3 2.6 4.5
HowL465.2 pSO122 h8 1003 2.3 2.2 3.2 HowL500.1 pSO122 h99 885 2.3
2.7 3.4 HowL554.1 pSO122 h120 892 1.9 2.3 3.6 HowL795.4 pSO122 h29
1029 3.0 3.8 4.9
[0476] As shown in Table I, the mutants produced approximately 2-
to 4-fold more lipase than the control strain Aspergillus oryzae
HowB427 and approximately 3- to 6-fold more lipase than the control
strain Aspergillus oryzae HowB430 when grown in shake flasks. The
mutants also produced approximately 2- to 5-fold more lipase than
the control strain Aspergillus oryzae HowB427 when grown in
fermentors.
Example 9
Rescue of Plasmid DNA and Flanking DNA from High lipase Expressing
Mutants
[0477] The plasmid DNA (pSO122, pDSY81, or pDSY82) and genomic
flanking loci were isolated from mutants Aspergillus oryzae
DEBY10.3, DEBY599.3, DEBY932, DEBY1058, DEBY1204.3.3, and
HIN603.
[0478] Genomic DNA was isolated from mutants Aspergillus oryzae
DEBY10.3, DEBY599.3, DEBY932, DEBY1058, DEBY1204.3.3, and HIN603
according to the following procedure. Spore stocks of each mutant
were inoculated into 150 ml of YEG medium and were grown overnight
at 37.degree. C. and 250 rpm. The mycelia were harvested from each
culture by filtration through Miracloth (Calbiochem, La Jolla,
Calif.) and rinsed twice with TE. The mycelia preparations were
then frozen quickly in liquid nitrogen and ground to a fine powder
with a mortar and pestle. The powdered mycelia preparations were
each transferred to a 50 ml tube and 20 ml of lysis buffer was
added. RNAse was added to each preparation to a final concentration
of 20 .mu.g/ml, and the preparations was incubated at 37.degree. C.
for 30 minutes. Protease K was then added to each preparation to a
final concentration of 0.1 mg/ml, and the preparations were
incubated at 50.degree. C. for 1 hour. The preparations were then
centrifuged at 15,000.times.g for 20 minutes to pellet the
insoluble material. Each supernatant was applied to a Qiagen MAXI
column (Qiagen, Chatsworth, Calif.) which was equilibrated with QBT
provided by the manufacturer. The columns were then washed with 30
ml of QC provided by the manufacturer. DNA was eluted from each
column with 15 ml of QF provided by the manufacturer and then
recovered by precipitation with a 0.7 volume of isopropanol and
centrifugation at 15,000.times.g for 20 minutes. The pellets were
finally washed with 5 .mu.l of 70% ethanol, air-dried, and
dissolved in 200 .mu.l of TE.
[0479] Two .mu.g aliquots from each of the Aspergillus oryzae
DEBY10.3, DEBY599.3, DEBY932, DEBY1058, DEBY1204.3.3, and HIN603
genomic DNA preparations were digested separately with BglII HpaI,
NarI, NdeI, SphI, and StuI. The restriction endonucleases did not
cut pDSY82 which allowed the isolation of the integrated plasmid
and the flanking genomic DNA. The digested genomic DNAs were then
ligated in a 20 .mu.l reaction with T4 DNA ligase.
[0480] The ligated DNA preparations were each transformed into E.
coli HB101 or E. coli DH5.alpha.. The transformants were then
screened by extracting plasmid DNA from the transformants,
restriction digesting the inserts to confirm they are derived from
pDSY82, and sequencing the inserts according to the method
described above using primers specific to pDSY82.
[0481] Transformant E. coli HB101-pDSY112 contained the HpaI
rescued locus from mutant Aspergillus oryzae DEBY599.3.
Transformant E. coli HB101-pDSY109 contained the SphI rescued locus
from mutant Aspergillus oryzae DEBY10.3. Transformant E. coli
HB101-pDSY138 contained the NdeI rescued locus from mutant DEBY932.
Transformant E. coli HB101-pDSY141 contained the BglII rescued
locus from mutant DEBY1058. Transformant E. coli
DH5.alpha.-pSMO1204 contained the BglII rescued locus from mutant
Aspergillus oryzae DEBY1204.3.3. Transformant E. coli
DH5.alpha.-pSMOH603 contained the BglII rescued locus from mutant
Aspergillus oryzae HIN603.
Example 10
Characterization of Aspergillus oryzae DEBY599.3 Rescued Locus
pDSY112
[0482] The Aspergillus oryzae DEBY599.3 rescued locus pDSY112
containing 1625 bp was sequenced according to the method described
in Example 2. The nucleic acid sequence (SEQ ID NO:9) and the
deduced amino acid sequence (SEQ ID NO:10) are shown in FIG. 9. The
nucleic acid sequence suggested that integration occurred within
the promoter of a glucose transporter about 150 bp upstream of the
ATG start codon. The open reading frame was punctuated by an
intron. The predicted protein (SEQ ID NO:10) shared 31.6% and 24.8%
identity with the glucose transporters from yeast (SEQ ID NO:11)
and human (SEQ ID NO:12), respectively, and 20.1% identity with an
inositol transporter from yeast (SEQ ID NO:13). Glucose
transporters have very distinct predicted secondary structures with
12 membrane spanning domains. Kyte-Doolittle plots of the
Aspergillus oryzae DEBY599.3 rescued locus predicted 12 membrane
spanning domains similar to the yeast and human glucose
transporters.
[0483] In order to confirm that the rescued flanking DNA was the
gene disrupted in Aspergillus oryzae DEBY599.3, a Southern blot of
Aspergillus oryzae HowB101 and Aspergillus oryzae DEBY599.3 genomic
DNA preparations digested with BglII was prepared and analyzed
according to the procedure described in Example 6. The blot was
probed with the Aspergillus oryzae DEBY599.3 rescued flanking DNA
at 42.degree. C. in DIG Easy Hyb. The blot was then washed and
processed using protocols provided with a Genius Kit.
[0484] A BglII band of 2.7 kb from Aspergillus oryzae HowB101
hybridized with the probe, while an .about.8 kb BglII band from
Aspergillus oryzae DEBY599.3 hybridized to the probe. The size
difference corresponded to the length of the plasmid integrated
during REMI confirming the DNA rescued from Aspergillus oryzae
DEBY599.3 was flanking the insertion.
Example 11
Aspergillus oryzae Transformation with HpaI Linearized pDSY112 and
Lipase Expression Screening
[0485] Aspergillus oryzae HowB430 was transformed with HpaI
digested pDSY112 and the transformants were recovered using the
methods described in Example 5. Totally, 216 transformants were
grown in 24 well microtiter plates in {fraction (1/100)} strength
MY25 medium. Samples were taken at 4 and 6 days and assayed for
lipase activity as described in Example 7. An equal number of low,
average and high producing lipase transformants were spore purified
and retested in 24 well microtiter cultures as described above.
These purified transformants were also tested in shake flasks in
full-strength MY25 medium as described in Example 8. The top five
producing transformants were then grown in a 2 liter fermentor as
described in Example 8. Lipase activity was measured as described
in Example 7.
[0486] The results obtained are shown in Table 2 below where the
lipase yield of Aspergillus oryzae HowB430 is normalized to
1.0.
5 TABLE 2 Strain Fermentation Results (Relative LU/ml) HowB430 1.0
DEBY599.3 1.7 112T90.2.2 2.3 112T100.4.2 2.1 112T344.2.1 1.7
112T142.2 2.0 112T59.2 2.4
[0487] All five retransformants produced approximately the same
level of lipase activity as the original tagged strain Aspergillus
oryzae DEBY599.3 when grown under fermentation conditions. In order
to determine if the pDSY112 had integrated at the same homologous
locus in the genome, a Southern blot of Aspergillus oryzae HowB430,
Aspergillus oryzae DEBY599.3 and the pDSY112 transformants genomic
DNA preparations digested with BglII was prepared and analyzed
according to the procedure described in Example 6. The blot was
probed with the Aspergillus oryzae DEBY599.3 rescued flanking DNA
at 42.degree. C. in DIG Easy Hyb. The blot was washed and processed
using protocols provided with a Genius Kit.
[0488] A BglII band of 2.7 kb from Aspergillus oryzae HowB430
hybridized with the probe, while an .about.8 kb BgllI band from
Aspergillus oryzae DEBY599.3 hybridized to the probe. A wild-type
BglII band of 2.7 kb and a second band corresponding to the
transforming DNA hybridized to the probe in all of the
transformants. Therefore, none of the retransformants had exact
gene replacements.
Example 12
Characterization of Aspergillus oryzae DEBY10.3 Rescued Locus
pDSY109
[0489] The 3.4 and 2.2 kb regions on either side of the integration
event of the Aspergillus oryzae DEBY10.3 rescued locus pDSY109 were
sequenced according to the procedure described in Example 2. The
nucleic acid sequence suggested that the integration event occurred
within the open reading frame of a palB gene. palB genes encode a
cysteine protease involved in the signal transduction pathway that
signals ambient pH.
[0490] The genomic library of Aspergillus oryzae HowB430 was
constructed by first partially digesting Aspergillus oryzae HowB430
genomic DNA with Tsp509I. Four units of Tsp509 were used to digest
3.5 .mu.g of Aspergillus oryzae HowB430 genomic DNA using
conditions recommended by the manufacturer. The reaction was
carried out at 65.degree. C., and samples were taken at 5 minute
intervals (from 0 to 50 minutes). The reactions were placed on ice
and stopped by the addition of EDTA to 10 mM. These digests were
then run on a 1% agarose gel with ethidium bromide, and the region
of the gel containing DNA from 3 kb to 9 kb was excised. The DNA
was then purified from the gel slice using Beta-Agarase I using a
protocol provided by the manufacturer (New England Biolabs,
Beverly, Mass.). The size-selected DNA was then ligated into Lambda
ZipLox EcoRI arms according to the manufacturer's instructions at
16.degree. C. overnight using conditions recommended by the
manufacturer. The ligation reaction was packaged and titered using
a Gigapack GoldIII Packaging Kit according to the manufacturer's
protocol. 8.times.10.sup.6 recombinant plaques were obtained, and
the library was amplified using a protocol provided by the
manufacturer.
[0491] The genomic library was screened to obtain a genomic clone
of palB. Appropriate dilutions of the genomic library were made to
obtain 7000 plaques per 150 mm petri plate as described in the
protocols provided with the Lambda ZipLox arms. The plaques were
lifted to Hybond-N plus circular filters using standard protocols
(Sambrook et al., 1989, supra). The filters were fixed using UV
crosslinking, and prehybridized at 42.degree. C. in DIG Easy Hyb.
The filters were hybridized with a DIG-labeled 0.25 kb palB probe.
The probe was labeled with dioxygenin using a Genius Kit and PCR
amplified with the following primers synthesized with an Applied
Biosystems Model 394 DNA/RNA Synthesizer according to the
manufacturer's instructions:
6 5'-CTGCCGTCGAAGGTGTCCAAG-3' (SEQ ID NO:14)
5'-ATTGTGGCCCCTATGTGGATT-3' (SEQ ID NO:15)
[0492] The parameters for PCR are as described in Example 2. The
filters were washed and processed post-hybridization using
protocols provided with the Genius Kit. Several positive plaques
were identified and purified to homogeneity using standard
protocols (Sambrook et al., 1989, supra).
[0493] The nucleotide sequence was determined for the palB gene
according to the method described in Example 2. The nucleic acid
sequence (SEQ ID NO:16) and the deduced amino acid sequence (SEQ ID
NO:17) are shown in FIG. 10. The open reading frame was interrupted
by 3 introns. The Aspergillus oryzae PalB protein (SEQ ID NO:17)
shared 66.4% identity with the Aspergillus nidulans PalB protein
(SEQ ID NO:18). The site of insertion also contained a highly
conserved domain of 37 amino acids (SEQ ID NO:19) similar to that
derived from the Neurospora crassa NADH dehydrogenase (SEQ ID
NO:20) which was probably a piece of mitochondrial DNA that
inserted during transformation or rescue in E. coli.
[0494] A Southern blot of Aspergillus oryzae DEBY10.3 and
Aspergillus oryzae HowB101 genomic DNA digested with BglII was
prepared according to the emthod described in Example 6. The blot
was probed with the Aspergillus oryzae DEBY10.3 rescued flanking
DNA to confirm that the rescued flanking DNA was the gene disrupted
in Aspergillus oryzae DEBY10.3.
[0495] A BglII band of .about.7.5 kb from Aspergillus oryzae
HowB101 hybridized to the probe while a band of 12 kb from
Aspergillus oryzae DEBY10.3 hybridized to the probe. The size
difference was the expected size for one plasmid copy being
integrated confirming the locus rescued was disrupted in
Aspergillus oryzae DEBY10.3.
[0496] Because the integration event in Aspergillus oryzae DEBY10.3
would be predicted to lead to a nonfunctional PalB protein,
Aspergillus oryzae DEBY10.3 was tested for growth at pH 8.0 and pH
6.5. Aspergillus nidulans palB minus strains are unable to grow at
pH 8.0 but are able to grow at pH 6.5. Aspergillus oryzae HowB430
and Aspergillus oryzae DEBY10.3 were grown in Minimal medium with
10 mM uridine at either pH 8.0 or pH 6.5. As predicted, Aspergillus
oryzae DEBY10.3 was unable to grow at pH 8.0.
Example 13
Construction of pMT1936
[0497] pMT1936 was constructed to contain a disruption cassette of
palB using the following primers synthesized with an Applied
Biosystems Model 394 DNA/RNA Synthesizer according to the
manufacturer's instructions.
7 100752: 5'-GGTTGCATGCTCTAGACTTCGTCACCTTATTA (SEQ ID NO:21)
GCCC-3' 100753: 5'-TTCGCGCGCATCAGTCTCGAGATCGTGTGTCG (SEQ ID NO:22)
CGAGTACG-3' 100754: 5'-GATCTCGAGACTAGTGCGCGCGAACAGACATC (SEQ ID
NO:23) ACAGGAACC-3' 100755: 5'-CAACATATGCGGCCGCGAATTCACTTCATTCC
(SEQ ID NO:24) CACTGCGTGG-3'
[0498] The Aspergillus oryzae palB 5' flanking sequence and the
sequence encoding the N-terminal part of the palB product were PCR
amplified from genomic DNA of Aspergillus oryzae A1560 obtained
according to the method described in Example 2. Approximately 0.05
.mu.g of DNA template and 5 pmole of each of the two primers 100755
and 100754 were used. Amplification was performed with the
polymerase Pwo as described by the manufacturer (Boehringer
Mannheim, Indianapolis, Ind.). Amplification proceeded through 40
cycles. Part of the reaction product was phenol extracted, ethanol
precipitated, digested with restriction enzymes EcoRI and XhoI and
a fragment of approximately 1.05 kb was isolated by agarose gel
electrophoresis.
[0499] The Aspergillus oryzae palB 3' flanking sequence and the
sequence encoding the C-terminal part of the palB gene product were
obtained as described above except that primers 100753 and 100752
were used for amplification and the PCR product was digested with
restriction enzymes XhoI and XbaI before gel electrophoresis to
recover a fragment of approximately 1.50 kb.
[0500] The two digested and purified PCR fragments described above
were ligated in a three part ligation with the purified 2.7 kb
EcoRI-XbaI fragment from the vector pJaI400 (FIG. 11) to produce
pMT1935 (FIG. 12). The palB 5' and 3' flanks of pMT1935 are
separated by BssHII, SpeI, and XhoI sites introduced via PCR
primers 100754 and 100753.
[0501] To insert an Aspergillus oryzae pyrG gene between the palB
5' flank and the 3' flank of pMT1935, the 3.5 kb HindIII fragment
of pJaL394 (FIG. 13) containing the repeat flanked pyrG gene was
cloned into HindIII cut, dephosphorylated and purified pBluescript
II SK (-). Plasmids with inserts in either orientation were
obtained. One plasmid, pMT1931 (FIG. 14), was selected in which the
SpeI site of the pBluescript polylinker was downstream of the pyrG
gene and the XhoI site was upstream of the pyrG gene. The pyrG gene
was isolated as a 3.5 kb SpeI-XhoI fragment and inserted in SpeI
and XhoI digested and purified pMT1935 to produce the disruption
plasmid pMT1936 (FIG. 15).
[0502] The pyrG selectable palB disruption cassette can be isolated
from pMT1936 as a 6.2 kb NotI fragment (NotI cutting in
polylinkers) or as a 5.5 kb AseI-PvuI fragment (AseI and PvuI
cutting within the actual palB 5' and 3' flanking sequences).
Example 14
Aspergillus oryzae Transformation with AlseI/PvuI palB Disruption
Cassette from pMT1936 and Lipase Screening
[0503] Aspergillus oryzae HowB430 was transformed using the same
transformation procedure described in Example 5 with a 5.5 kb
AseI/PvuI fragment obtained from pMT1936. The linear fragment for
transformation was isolated by digestion of pMT1936 with AseI and
PvuI and separation of the fragment on a 1% agarose gel using a
QIAquick Gel Extraction Kit according to the manufacturer's
instructions. The transformants were then tested for growth on
Minimal medium plates at pH 6.5 or pH 8.0.
[0504] The results showed that 13 of the 128 transformants tested
possessed the palB minus phenotype as indicated by the inability to
grow at pH 8.0. The 13 palB minus strains and 13 of the
transformants that were able to grow at pH 8.0 were spore purified
and then evaluated in 24-well plate and shake flask cultures for
lipase production using the methods described in Examples 7 and 8,
respectively. The results are shown in Table 3 below.
[0505] Southern blots of the genomic DNA from an Aspergillus oryzae
palB minus mutant, an Aspergillus oryzae palB plus strain, and
Aspergillus oryzae HowB430 were performed to determine if the
AsnI/PvuI transforming DNA fragment had integrated as a clean
replacement into the palB locus. The genomic DNAs were prepared
according to the procedure described in Example 9, digested with
PvuI, and electrophoresed on a 0.8% agarose gel. The DNAs were
transferred to a Hybond N.sup.+ membrane using 0.4 N NaOH and
capillary action. The blot was UV crosslinked prior to
prehybridization at 65.degree. C. in Rapid Hyb. The blot was then
probed with a 0.9 kb AsnI/SpeI fragment from pMT1936. The 0.9 kb
fragment was isolated from an agarose gel slice using QiaQuick spin
column after electrophoreses on a 1% agarose gel. The fragment was
labeled using Vistra ECF Random Prime Labeling Kit. The blots were
prehybridized and hybridized at 65.degree. C. in Rapid Hyb
(Amersham, Cleveland, Ohio), and then washed twice for 5 minutes in
2.times.SSC, 0.1% SDS at 65.degree. C. and twice for 10 minutes in
0.2.times.SSC, 0.1% SDS at 65.degree. C. Following the washes, the
blot was processed for detection using the Vistra ECF Signal
Amplification Kit (Amersham, Cleveland, Ohio) and the STORM860
Imaging System (Molecular Dynamics, Sunnyvale, Calif.).
[0506] The Southern blot results demonstrated that the probe
hybridized to a band of 6 kb from Aspergillus oryzae HowB430. A
clean disruption would be expected to hybridize to about an 8 kb
PvuI band. The Southern blot results further showed that some of
the palB minus strains had clean disruptions while others did not.
The Southern blot results are summarized in Table 3.
[0507] Three of the palB minus strains were also run under
fermentation conditions according to the procedure described in
Example 8. The results obtained are shown in Table 3 below where
the lipase yield of Aspergillus oryzae HowB430 is normalized to
1.0. The three palB minus strains performed better or close to the
same as the original tagged mutant Aspergillus oryzae DEBY10.3.
8TABLE 3 Fermentation PalB 24 well Shake flasks results Southern
Strain phenotype LU/ml LU/ml LU/ml pattern HowB430 plus 1.0 1.0 1.0
wild type palB3-1 plus 1.2 1.1 1.7 wild-type and other palB4-1 plus
1.0 0.8 1.4 wild-type and other palB5-1 minus 1.4 1.4 2.0 disrupted
palB8-1 plus 0.9 1.0 NA wild-type and other palB18-1 plus 0.9 NA NA
wild-type and other palB27-1 plus 1.0 NA NA wild-type and other
palB29-1 minus 0.8 NA NA other palB30-1 plus 0.9 NA 1.0 wild-type
and other palB31-1 minus 1.3 NA NA other palB37-1 plus 0.8 NA NA
wild-type and other palB39-1 plus 0.9 NA NA wild-type and other
palB41-1 plus 1.0 0.8 NA wild-type and other palB42-1 plus 1.2 1.0
NA wild-type and other palB43-1 minus 1.3 1.2 NA other palB69-1
plus 1.2 1.4 NA wild-type and other palB71-1 minus 1.2 1.3 1.8
other palB72-1 minus 1.5 1.6 2.0 other palB75-1 minus 1.3 1.4 1.3
other palB76-1 minus 1.6 1.3 2.0 clean disruption palB79-1 plus 1.2
1.0 NA wild-type and other
Example 15
Characterization of Aspergillus oryzae DEBY932 Rescued Locus
pDSY138
[0508] The Aspergillus oryzae DEBY932 rescued locus pDSY138
containing 1625 bp was sequenced according to the method described
in Example 2. The nucleic acid sequence (SEQ ID NO:25) and deduced
amino acid sequence (SEQ ID NO:26) are shown in FIG. 16. The
nucleic acid sequence showed that the EcoRI site of the REMI
integration was 810 bp upstream of the ATG start codon for an open
reading frame and the deduced amino acid sequence (SEQ ID NO:26)
had significant identity to mannitol-1-phosphate dehydrogenases
from E. coli and Bacillus subtilis. The open reading frame coded
for a predicted protein of 319 amino acids, and shared 13.3% and
34.7% identity with the E. coli (SEQ ID NO:27) and the Bacillus
subtilis (SEQ ID NO: 28) mannitol-1-phosphate dehydrogenases,
respectively.
[0509] A Southern blot of Aspergillus oryzae DEBY932 and
Aspergillus oryzae HowB430 genomic DNA preparations digested with
NdeI was prepared and analyzed according to the method described in
Example 14. The blot was probed with the Aspergillus oryzae DEBY932
rescued flanking DNA to confirm that the rescued flanking DNA is
the gene disrupted in DEBY932.
[0510] An NdeI band of approximately 5 kb from Aspergillus oryzae
HowB430 hybridized to the rescued locus while a band of
approximately 10 kb from Aspergillus oryzae DEBY932 hybridized to
the probe confirming that the rescued locus was the disrupted locus
in Aspergillus oryzae DEBY932.
Example 16
Aspergillus oryzae Transformation with NdeI Linearized pDSY138 and
lipase Expression Screening
[0511] Aspergillus oryzae HowB430 was transformed with NdeI
digested pDSY138 and the transformants were recovered using the
methods described in Example 5. Totally, 180 recovered
transformants were grown in 24 well microtiter plates in {fraction
(1/100)} strength MY25, and samples were taken at 4 and 6 days for
lipase assays as described in Example 7. The top 11 highest lipase
producing and 1 average lipase producing transformants were spore
purified and retested in 24 well microtiter cultures. These
purified transformants were also evaluated in shake flasks in
full-strength MY25 as described in Example 8. The top two producers
were also grown in a 2 liter fermentor as described in Example 8.
Lipase activity was measured as described in Example 7.
[0512] The results obtained are shown in Table 4 below where the
lipase yield of Aspergillus oryzae HowB430 was normalized to 1.0.
The top two lipase producers produced essentially the same amount
of lipase activity as the original tagged mutant Aspergillus oryzae
DEBY932.
9TABLE 4 Fermentation Results Strain (Relative LU/ml) Southern
Results HowB430 1.0 Wild-type DEBY932.3.3 2.1 Disrupted 138T83.1.1
2.2 Disrupted 138T102.1.1 1.9 Disrupted
[0513] A Southern blot of Aspergillus oryzae DEBY932, Aspergillus
oryzae HowB430 and pDSY138 genomic DNA preparations digested with
NdeI was prepared and analyzed as described in Example 14 to
determine if pDSY138 had integrated at the homologous locus
producing gene replacements in the transformants using the
Aspergillus oryzae DEBY932 rescued flanking DNA as a probe.
[0514] The Southern blot showed that an NdeI band of approximately
5 kb from Aspergillus oryzae HowB430 hybridized to the rescued
locus while a band of approximately 10 kb from Aspergillus oryzae
DEBY932 hybridized to the probe. In Table 4, the column labeled
Southern results indicated whether the transformants had a
wild-type NdeI fragment of the size observed in the parent strain
Aspergillus oryzae HowB430 or whether the transformants had a band
corresponding to the disrupted size observed in Aspergillus oryzae
DEBY932.
Example 17
Characterization of Aspergillus oryzae DEBY1058 Rescued Locus
pDSY141
[0515] The Aspergillus oryzae DEBY1058 rescued locus pDSY141
containing approximately 1 kb was sequenced according to the method
described in Example 2. The nucleic acid sequence demonstrated that
the rescued locus contained flanking DNA from only one side of the
BamHI REMI integration event, and the pDSY141 sequence had
rearranged.
[0516] A Southern blot of Aspergillus oryzae DEBY1058 genomic DNA
digested with BamHI as probed with the Aspergillus oryzae DEBY1058
rescued flanking DNA was prepared and analyzed as described in
Example 14 to confirm that the rescued flanking DNA is the gene
disrupted in Aspergillus oryzae DEBY1058.
[0517] The Southern analysis showed that the pDSY82 DNA had
integrated as a REMI event at a BamHI site, but more than one copy
of pDSY82 had integrated which suggested why the rescued plasmid
had rearranged and only contained one side of the flanking DNA.
[0518] In order to obtain the other flanking piece, a genomic clone
(pDSY163) was isolated from the Aspergillus oryzae HowB430 genomic
library, prepared as described in Example 6, using a
.sup.32P-labeled 0.5 kb fragment of the rescued genomic DNA from
Aspergillus oryzae DEBY1058. The probe was labeled using a Prime-It
Kit according to the manufacturer's instructions (Stratagene, La
Jolla, Calif.). Five plates of approximately 7000 plaques each were
plated, and the plaques were lifted to Hybond-N.sup.+ as described
in Example 12. The filters were prehybridized at 42.degree. C. in
50% formamide, 5.times.SSPE, 0.3% SDS and 200 .mu.g/ml of sheared
and denatured salmon sperm DNA for 1 hour. The denatured probe was
added, and the filters were hybridized overnight at 42.degree. C.
The filters were washed in 1.times.SSC, 0.1% SDS for 5 minutes at
65.degree. C. twice, in 0.1.times.SSC, 0.1% SDS at 65.degree. C.
for 15 minutes twice, and in 2.times.SSC at room temperature for 10
minutes. The filters were exposed to X-ray film, and 12 positive
plaques were picked and purified using standard protocols (Sambrook
et al., 1989, supra). Plasmid DNA was isolated from the purified
genomic clones using the excision protocol provided with the Lambda
ZipLox EcoRI Arms Kit.
[0519] The nucleotide sequence of 3.6 kb of the genomic clone was
determined as described in Example 2. The nucleic acid sequence
(SEQ ID NO:29) and deduced amino acid sequence (SEQ ID NO:30) are
shown in FIG. 17. The nucleic acid sequence showed that the BamHI
site of integration in the mutant is 250 bp downstream of the stop
codon for an open reading frame that encodes a protein (SEQ ID
NO:30) which shared significant identity with manganese superoxide
dismutase from Saccharomyces cerevisiae (SEQ ID NO:31).
[0520] Since the site of integration in Aspergillus oryzae DEBY1058
was 250 bp downstream of the stop codon for the manganese
superoxide dismutase gene, the effect of this integration on
expression of the manganese superoxide dismutase was determined.
Saccharomyces cerevisiae strains lacking a functional manganese
superoxide dismutase are sensitive to paraquat when grown in the
presence of oxygen. Aspergillus oryzae DEBY1058 and Aspergillus
oryzae HowB430 were grown in 24 well microtiter plates in 1 ml of
YEG medium supplemented with 10 mM uridine and either 0, 2, 4, 6,
8, 10 or 20 mM paraquat at 34.degree. C. with shaking. Aspergillus
oryzae HowB430 grew at concentrations of paraquat up to 8 mM while
growth of Aspergillus oryzae DEBY1058 was inhibited by 2 mM
paraquat. The data indicated that the integration event 250 bp
downstream of the stop codon for manganese superoxide dismutase in
Aspergillus oryzae DEBY1058 reduced expression of manganese
superoxide dismutase.
Example 18
Construction of pDSY162
[0521] pDSY162 was constructed to contain a disruption cassette for
manganese superoxide dismutase by PCR amplification of a 3179 bp
XbaI/KpnI fragment of genomic DNA containing the manganese
superoxide dismutase gene using the following primers synthesized
with an Applied Biosystems Model 394 DNA/RNA Synthesizer according
to the manufacturer's instructions.
10 970738: 5'-GCTCTAGATCGTCGGAGCTCATGTCGGCGATT (SEQ ID NO:32)
TTAC-3' 970739: 5'-GCGGTACCACGCCTAGAGCAAAGTATAAATAA (SEQ ID NO:33)
GGAA-3'
[0522] The amplification reaction (100 .mu.l) contained the
following components: 0.2 .mu.g of the pDSY163, 48.4 pmol of primer
970738, 48.4 pmol of primer 979739, 1 mM each of dATP, dCTP, dGTP,
and dTTP, 1.times.Taq polymerase buffer, and 2.5 U of Taq
polymerase. The reaction was incubated in an Ericomp Thermal Cycler
programmed as follows: One cycle at 95.degree. C. for 5 minutes
followed by 30 cycles each at 95.degree. C. for 1 minute,
55.degree. C. for 1 minute, and 72.degree. C. for 2 minutes. Two
.mu.l of the reaction were electrophoresed on an agarose gel to
confirm the amplification of the PCR product of approximately 3179
bp.
[0523] The PCR product was subcloned into pCR.RTM.TOPO using a TOPO
TA Cloning Kit (Invitrogen, San Diego, Calif.). The transformants
were then screened by extracting plasmid DNA from the transformants
using a QIAwell-8 Plasmid Kit according to the manufacturer's
instructions, restriction digesting the plasmid DNA using XbaI/KpnI
to confirm the presence of the correct size fragment, and
sequencing the DNA according to the method described in Example 2
to confirm the PCR product.
[0524] The plasmids containing the manganese superoxide dismutase
insert were digested with XbaI and KpnI and separated on a 1%
agarose gel. A 3.1 kb manganese superoxide dismutase fragment was
purified using a QIAquick Gel Extraction Kit according to the
manufacturer's instructions. The purified fragment was ligated with
pBluescript SK-digested with XbaI and KpnI to produce pDSY161 (FIG.
18). The ligation reaction was used to transform E. coli
DH5.alpha..
[0525] The transformants were then screened by extracting plasmid
DNA from the transformants using a QIAwell-8 Plasmid Kit according
to the manufacturer's instructions and digesting the plasmids with
HindIII to determine which clones were correct.
[0526] pDSY161 was digested with HindUII to remove a 600 bp
fragment, and the digestion was electrophoresed on a 1% agarose
gel. A 5.4 kb vector fragment was isolated using a QIAquick Gel
Extraction Kit according to the manufacturer's instructions, and
ligated to the 3.5 kb HindIII fragment from pJaL394 (FIG. 13)
containing apyrG gene repeat to produce pDSY162 (FIG. 19). The
ligation reaction was used to transform E. coli DH5.alpha..
[0527] The transformants were then screened by extracting plasmid
DNA from the transformants using a QIAwell-8 Plasmid Kit according
to the manufacturer's instructions and digesting them with HindIII
to determine which plasmids contained the expected 3.5 kb HindIII
fragment in pDSY162.
Example 19
Aspergillus oryzae Transformation with AsnI/PvuI Manganese
Superoxide Dismutase Disruption Cassette and lipase Screening
[0528] Aspergillus oryzae HowB430 was transformed with a 5.8 kb
AseI/PvuI fragment containing the manganese superoxide dismutase
disruption cassette using the same transformation procedure
described in Example 5. The linear fragment for transformation was
isolated by digestion of pDSY162 with AseI and PvuI and separation
of the fragment on a 1% agarose gel using a QIAquick Gel Extraction
Kit according to the manufacturer's instructions. The transformants
were then tested for growth on Minimal medium plates at pH 6.5.
[0529] Six transformants were obtained and were tested for
sensitivity to paraquat as described in Example 17. Four of the 6
transformants were paraquat sensitive indicative of the manganese
superoxide dismutase disruption minus phenotype although the four
paraquat sensitive strains were not equally sensitive to paraquat.
As shown in Table 5 below, a dash means not sensitive to paraquat,
++ means sensitive means sensitive to intermediate levels of
paraquat and +++ means inhibited by even 2 mM paraquat. All of the
transformants were spore purified and tested in 24 well and shake
flask cultures for lipase production according to the procedures
described in Examples 7 and 8. The results tabulated in Table 5
below show that following transformation of Aspergillus oryzae
HowB430 with the manganese superoxide dismutase disruption
cassette, transformants sensitive to paraquat on average produced
higher LIPOLASE.TM. levels than Aspergillus oryzae HowB430.
[0530] Southern blots of the genomic DNA from an Aspergillus oryzae
manganese superoxide dismutase minus mutant, an Aspergillus oryzae
manganese superoxide dismutase plus strain, and Aspergillus oryzae
HowB430 were performed as described in Example 14 to determine if
the AsnI/PvuI transforming DNA fragment had integrated as a clean
replacement into the manganese superoxide dismutase locus.
[0531] The results of the Southern blot (Table 5) showed that
strains sensitive to even 2 mM paraquat were disrupted at the
manganese superoxide dismutase locus while those sensitive to
intermediate levels of paraquat have both a wild-type locus and the
disrupted cassette locus. The Souther blot and LIPOLASE.TM. yield
results together suggests that expression of both full length and
truncated manganese superoxide dismutase in the same cell leads to
an intermediate sensitivity to paraquat and an increase in
LIPOLASE.TM. production. This can be explained by the fact that
manganese superoxide dismutase is a homodimer so expression of the
wild-type and truncated forms coded for by the wild-type and
disrupted cassette, respectively, leads to heterodimers which are
either non-functional or partially functional.
11TABLE 5 Shake flasks 24 well results results Paraquat (Relative
(Relative Strain sensitivity LU/ml) LU/ml) Southern Results HowB430
- 1.0 1.0 wild-type 430162T1 - 1.0 1.1 wild-type & other
430162T2 ++ 1.3 2.5 wild-type & other 430162T3 ++ 1.4 3.0
wild-type & other 430162T4 +++ 1.2 2.1 disrupted 430162T5 +++
1.0 1.5 disrupted 430162T6 - 0.9 1.2 wild-type & other
Example 20
Characterization of Aspergillus oryzae DEBY1204.3.3 Rescued Locus
pSMO1204
[0532] The Aspergillus oryzae DEBY1204.3.3 rescued locus pSMO1204
containing 2.0 kb was sequenced according to the procedure
described in Example 2. The nucleic acid sequence (SEQ ID NO:34) as
shown in FIG. 20 had no sequence homology to any published
sequences.
[0533] Southern analysis and sequencing of a genomic clone was used
to confirm that no deletions had taken -place when the tagged
mutant was generated. A Southern blot of Aspergillus oryzae HowB430
genomic DNA digested with various restriction endonucleases (BamHI,
BglII, SalI and SphI) was prepared and analyzed as described in
Example 14. Probes from both ends of the rescued plasmid were
generated by PCR using the primers described below. The primers
were synthesized with an Applied Biosystems Model 394 DNA/RNA
Synthesizer according to the manufacturer's instructions.
12 970052 5'-CTATGATTGGCCGATAGG-3' (SEQ ID NO:35) 970053
5'-CCAGGCTCGCACGCTTTC-3' (SEQ ID NO:36) 970054
5'-CTTGCAACTAACGGGGTT-3' (SEQ ID NO:37) 970055
5'-TGAGAAAGACCAAGAATG-3' (SEQ ID NO:38)
[0534] Probe 1 was generated from one end of the rescued locus by
PCR using primer 970052 and primer 970053. Probe 2 was generated
from the other end of the rescued locus by PCR using primer 970054
and primer 970055. The amplification/labeling reaction (50 .mu.l)
contained the following conponents: 10 ng rescued plasmid pSMO1204,
50 pmole each of primer 970052 and 970053 for probe 1 or 50 pmole
each of primer 970054 and primer 970055 for probe 2, 1.times.DIG
labeling mix (Boehringer Mannheim, Indianapolis, Ind.), 1.times.Taq
polymerase buffer, and 2.5 U of Taq polymerase. The reaction was
incubated in an Ericomp Thermal Cycler programmed as follows: One
cycle at 95.degree. C. for 5 minutes followed by 30 cycles each at
95.degree. C. for 1 minute, 58.degree. C. for 1 minute, and
72.degree. C. for 1.5 minutes
[0535] Southern blots of Aspergillus oryzae HowB430 genomic DNA
digested with various restriction enzymes were prepared and
analyzed according to the procedure described in Example 14. The
blots were hybridized independently to probe 1 and probe 2.
Identical banding patterns of the digests with both probes would
suggest that a deletion had not occurred. Conditions of the
Southern analysis were as follows: Blots were prehybridized for 1
hour and hybridized overnight at 42.degree. C. in Easy Hyb. Blots
were washed in 2.times.SSC, 0.1% SDS twice at room temperature for
15 minutes each, then washed twice at 65.degree. C. in
0.1.times.SSC, 0.1% SDS for 15 minutes each. Detection continued
with buffers and reagents from Boehringer Mannheim's DIG Wash Block
Buffer System. CDP star (Boehringer Mannheim, Indianapolis, Ind.)
was used to detect the chemiluminescent reaction. Film was exposed
for approximately 1 hour.
[0536] The Southern blot results showed identical banding patterns
with the different digests suggesting a direct insertion of the
tagged plasmid. A genomic clone was obtained by probing an
Aspergillus oryzae HowB430 Ziplox library obtained as described in
Example 12 with the probe from the tagged mutant Aspergillus oryzae
DEBY1204. The Ziplox library of Aspergillus oryzae HowB430 was
screened with probe 1. A genomic clone was isolated and sequenced
(Example 2) confirming that no deletions had occurred during the
tagging event and the tagged plasmid had inserted at an EcoRI
site.
Example 21
Characterization of Aspergillus oryzae Mutant HIN603 Rescued Locus
pSMO603
[0537] The Aspergillus oryzae HIN603 rescued locus pSMO603
containing 1.0 kb was sequenced according to the procedure
described in Example 2. The nucleic acid sequence (SEQ ID NO:39) as
shown in FIG. 21 showed no homology to any published sequences.
[0538] A Southern blot of Aspergillus oryzae HowB430 genomic DNA
digested with the restriction enymes SphI, SalI, and BamHI was
hybridized to probes made from both ends of the rescued plasmid.
Probe 3 was generated by PCR using primer 970858 and primer 970859
shown below synthesized with an Applied Biosystems Model 394
DNA/RNA Synthesizer according to the manufacturer's instructions.
Probe 4 was generated by PCR using primer 970860 and primer 970861.
The template for the 50 .mu.l PCR labeling reaction was 10 ng of
the rescued plasmid pSMOH603. PCR cycles and conditions were as
described in Example 20. Southern conditions were as described in
Example 20.
13 970858: 5'-TGTAGTCTGACTAGCATG-3' (SEQ ID NO:40) 970859:
5'-GGATCTTCACCTAGATCC-3' (SEQ ID NO:41) 970860:
5'-CATAGTGTCGACCAAGC-3' (SEQ ID NO:42) 970861:
5'-CAATCGAGCTTGCCTATG-3' (SEQ ID NO:43)
[0539] Different banding patterns on the Southern suggested that a
deletion had taken place where the tagging occurred. When 500 ng of
Aspergillus oryzae HowB430 genomic DNA was used as a template in a
PCR reaction with primers 090858 and 090860, a 3 kb product was
amplified suggesting a 2.5 kb deletion had occurred. Southern
analysis of genomic DNA prepared from the tagged Aspergillus oryzae
HIN603 strain, digested with HindIII, and probed with the NheI
fragment from Aspergillus oryzae pyrG suggested that the tagged
mutant had not been generated by a REMI event.
Example 22
Construction of Aspergillus oryzae HowB432
[0540] Aspergillus oryzae HowB432 was generated by transformation
of Aspergillus oryzae JaL250 with a linear fragment containing the
NA2-tpi promoter, a cellulase gene from Humicola lanuginosa
(CAREZYME.TM. gene, Novo Nordisk A/S, Bagsvaerd, Denmark), and the
AMG terminator obtained from plasmid pGAG3 (FIG. 22).
[0541] Aspergillus oryzae JaL250 was constructed from Aspergillus
oryzae JaL142 (Christensen et al., 1988, Bio/Technology 6:
1419-1422) by deleting the neutral protease I gene (npI). The npI
deletion plasmid was constructed by exchanging a 1.1 kb BalI
fragment coding for the central part of the npI gene in plasmid
pJaL389 (FIG. 23), which contained a 5.5 kb SacI genomic fragment
encoding the npI gene, with a 3.5 kb HindIII fragment from pJaL335
(FIG. 24) containing the pyrG gene flanked by repeats, thereby
creating plasmid pJaL399 (FIG. 25). Aspergillus oryzae JaL142 was
transformed with the 7.9 kb SacI fragment. Transformants were
selected by relief of the uridine requirement on Minimal medium
plates. The transformants were analyzed by Southern analysis as
described in Example 14 and by IEF protease profile analysis
according to standard methods.
[0542] Two out of 35 transformants possessed an altered Southern
profile compared to the parent strain and displayed no neutral
protease I activity by IEF. Furthermore, Southern analysis showed
that one of the two transformants had a clean deletion of the npI
gene and was designated Aspergillus oryzae JaL228.
[0543] Totally, 2.3.times.10.sup.7 conidiospores of Aspergillus
oryzae JaL228 were spread on Minimal medium plates supplemented
with 0.1% 5-fluoro-orotic acid (FOA) and 10 mM uridine. Eight FOA
resistant colonies were obtained. A Southern blot of BamHI digested
genomic DNA from the eight colonies probed with a 401 bp pyrG
repeated region demonstrated that the pyrG gene had been excised by
recombination at the repeated regions. Aspergillus oryzae JaL228
showed two bands of the expected size of 2.7 and 3.1 kb originating
from the two copies of the repeated region. If the pyrG gene had
been lost by recombination between the repeated regions, the 3.1 kb
band would have disappeared and only the 2.7 kb would have
remained. All 8 FOA resistant colonies showed this pattern of
bands. Sequencing of a PCR fragment covering the junctions between
the npI gene and the copy of the 401 bp repeat remaining in the 8
colonies confirmed that the pyrG gene was excised by recombination
between the repeats. One of the colonies was designated Aspergillus
oryzae JaL250.
[0544] pGAG3 was constructed by isolating from pDM176 (FIG. 26) a
SwaI/PacI fragment containing the Humicola lanuginosa cellulase
gene and ligating the fragment into SwaI/PacI digested pBANe6. The
SwaI/PacI fragment from pDM176 and SwaI/PacI digested pBANe6 were
separated on a 1% agarose gel, and isolated using a QIAquick Gel
Extraction Kit (Qiagen Inc., Chatsworth, Calif.) according to the
manufacturer's instructions prior to ligation. The ligation was
used to transform E. coli DH5.alpha. cells, and the transformants
were then screened by extracting plasmid DNA from the transformants
using a QIAwell-8 Plasmid Kit according to the manufacturer's
instructions, restriction digesting the plasmid DNA to confirm the
presence of the correct size fragment, and sequencing the DNA
according to the method described in Example 2.
[0545] pGAG3 was then digested with PmeI and the linear expression
cassette was isolated by preparative agarose electrophoresis using
TAE buffer. The linear cassette was then used to transform
Aspergillus oryzae JaL250.
[0546] Transformation of Aspergillus oryzae JaL250 for amdS
selection was conducted with protoplasts at a concentration of
2.times.10.sup.7 protoplasts per ml prepared as described in
Example 2. Ten .mu.g of the linear fragment described above were
added to 100 .mu.l of protoplasts. A volume of 250 .mu.l of PEG
(60% PEG 4000-10 mM CaCl.sub.2-10 mM Tris-HCl pH 8.0) was then
added, and the mixture was placed at 37.degree. C. for 30 minutes.
Three ml of STC medium was added and the mixture was plated on Cove
plates supplemented with 10 mM uridine for amdS selection. The
plates were incubated 7-10 days at 34.degree. C. Transformants were
then transferred to plates of the same medium and incubated 3-5
days at 37.degree. C. The transformants were purified by streaking
spores and picking isolated colonies using the plates of the same
medium without sucrose.
Example 23
Aspergillus oryzae Transformation with HpaI Linearized pDSY112 and
Cellulase Expression Screening
[0547] Aspergillus oryzae HowB432 was transformed with HpaI
digested pDSY112 and the transformants were recovered using the
methods described in Example 5. Totally, 104 recovered
transformants were grown in 24 well microtiter plates in 1/4
strength MY25. Samples were taken at 5 and 7 days and assayed for
cellulase activity as described below.
[0548] Cellulase activity was measured according to the following
protocol which is derived from Novo Nordisk method AF 302.1/1-GB
which is available from Novo Nordisk A/S, Bagsvaerd, Denmark upon
request. A substrate solution containing 2%
azo-carboxymethylcellulose was prepared by dissolving the material
in 100 mM MOPS pH 7.0 buffer at 80.degree. C. for 10 minutes.
CAREZYME.TM. (Novo Nordisk A/S, Bagsvaerd, Denmark) was used as a
standard. Stock solutions of 2.5 to 25 ECU per ml were prepared to
construct a standard curve by diluting accordingly CAREZYME.TM. in
100 mM MOPS pH 7.0 buffer. Five .mu.l aliquots of the standards and
samples (diluted for shakeflasks and fermentations) were pipetted
into individual wells of a 96 well plate. A volume of 65 .mu.l of
the 2% azo-carboxymethylcellulose solution was pipetted into each
of the wells and mixed. The reactions were incubated at 45.degree.
C. for 30 minutes and then stopped by the addition of 215 .mu.l of
stop reagent followed by mixing. The stop reagent was prepared by
first suspending 0.2 g of ZnCl.sub.2 in 20 ml of 250 mM MOPS pH 7.0
and adding the suspension to 80 ml of acidified ethanol containing
1.1 ml of concentrated HCl per liter of ethanol. The plate
containing the stopped reaction was then centrifuged at 3000 rpm
for 10 minutes. A 100 .mu.l aliquot of each supernatant was
pipetted into a 96 well plate and the absorbance measured at 600
nm. Using linear regression, the slope, intercept, and correlation
coefficient were determined for the standards and samples.
[0549] The top ten cellulase producing transformants from the 24
well cultures were spore purified, and regrown in 24 well cultures
as above and assayed for cellulase activity. The purified strains
were also grown in MY25 in 125 ml shake flasks in MY25 pH 6.5 at
34.degree. C. and samples were taken at 3 and 5 days for cellulase
assays. Aspergillus oryzae HowB432 pDSY112 84-1-1 and Aspergillus
oryzae HowB432 pDSY112 94-1-1 were also grown in fermentors (2
liters) as described in Example 8. Cellulase activities were
measured as described above.
[0550] The results from the 24 well and shake flasks cultures are
presented in Table 6 where the cellulase yield of Aspergillus
oryzae HowB432 was normalized to 1.0.
14TABLE 6 24 well Shake flasks Fermentation results Strain (ECU/ml)
(ECU/ml) (ECU/ml) HowB432 1.0 1.0 1.0 C112T50.1.1 2.0 0.9 NA
C112T84.1.1 2.1 2.0 1.3 C112T86.1.1 2.3 2.4 NA C112T94.1.1 2.5 1.3
1.4 C112T95.1.1 2.1 1.3 NA C112T100.1.1 2.3 1.6 NA C112T101.1.1 1.9
1.6 NA C112T102.1.1 2.0 2.1 NA C112T103.1.1 1.5 2.0 NA C112T104.1.1
2.2 2.3 NA
[0551] A Southern blot of Aspergillus oryzae HowB432, Aspergillus
oryzae DEBY599.3 and the pDSY112 transformants genomic DNAs
digested with BglII was prepared and analyzed as described in
Example 14 performed to determine whether pDSY112 had integrated at
the homologous locus in the genome using the Aspergillus oryzae
DEBY599.3 rescued flanking DNA as a probe.
[0552] A BglII band of 2.7 kb from Aspergillus oryzae HowB432
hybridized with the probe, while an .about.8 kb BglII band from
Aspergillus oryzae DEBY599.3 hybridized to the probe. In all of the
transformants a wild-type BglII band of 2.7 kb and a second band
corresponding to the transforming DNA hybridized to the probe.
Therefore, none of the retransformants had exact gene
replacements.
Example 24
Aspergillus oryzae Transformation with NdeI Linearized pDSY138 and
Cellulase Expression Screening
[0553] Aspergillus oryzae HowB432 was transformed with NdeI
digested pDSY138 using the method described in Example 5. Totally,
240 transformants were recovered which were grown in 24 well
microtiter plates in 1/4 strength MY25 as described in Example 8
except samples were taken at days 3 and 5 and assayed for cellulase
activity as described in Example 23. The top 20 cellulase producing
transformants were spore purified and retested in 24 well
microtiter cultures. The top 8 cellulase producing once purified
transformants were spore purified a second time and tested in shake
flasks in full-strength MY25 as described in Example 8. The top 2
producers were also grown in a 2 liter fermentor as described in
Example 8. Cellulase activity was measured as described in Example
23.
[0554] The results obtained are shown in Table 7 below where the
cellulase yield of Aspergillus oryzae HowB432 is normalized to
1.0.
15 TABLE 7 Strain ECU/ml Southern Results HowB432 1.0 wild-type
C138T21.1.1 1.15 disrupted allele C138T205.1.1 1.5 wild-type and
disrupted alleles
[0555] A Southern blot of Aspergillus oryzae DEBY932, Aspergillus
oryzae HowB432, and pDSY138 genomic DNA preparations digested with
NdeI was prepared and analyzed as described in Example 14 to
determine if the pDSY138 DNA had integrated at the homologous locus
producing gene replacements in the transformants using the
Aspergillus oryzae DEBY932 rescued flanking DNA as a probe.
[0556] The results of the Southern blot demonstrated that an NdeI
band of approximately 5 kb from Aspergillus oryzae HowB432
hybridized to the rescued locus while a band of approximately 10 kb
from Aspergillus oryzae DEBY932 hybridized to the probe. In Table
7, the column labeled Southern results indicated whether the
transformants had a wild-type NdeI fragment of the size observed in
the parent strain Aspergillus oryzae HowB432 or whether the
transformants had a band corresponding to the disrupted size
observed in Aspergillus oryzae DEBY932.
Example 25
Aspergillus oryzae Transformation with AseI/PvuI palB Disruption
Cassette from pMT1936 and Cellulase Screening
[0557] Aspergillus oryzae HowB432 was transformed using the same
transformation procedure described in Example 5 with a 5.5 kb
AseI/PvuI fragment containing the palB disruption cassette. The
linear fragment for transformation was isolated by digestion of
pMT1936 with AseI and PvuI and separation of the fragment on a 1%
agarose gel using a QIAquick Gel Extraction Kit according to the
manufacturer's instructions. The transformants obtained were then
evaluated for growth on Minimal medium plates at pH 6.5 or pH
8.0.
[0558] The results showed that 10 of the 312 transformants tested
were unable to grow at pH 8.0 indicative of the palB minus
phenotype. The 10 palB minus transformants and 10 of the
transformants that were able to grow at pH 8.0 were spore purified
and tested in 24 well and shake flask cultures for cellulase
production according to the procedures described in Example 23. The
results tabulated in Table 8 below demonstrated that palB minus
strains were better cellulase producers than the palB plus
strains.
[0559] Southern blots of the genomic DNA from an Aspergillus oryzae
palB minus mutant, an Aspergillus oryzae palB plus strain, and
Aspergillus oryzae HowB432 were performed as described in Example
14 to determine if the AseI/PvuI transforming DNA fragment had
integrated as a clean replacement into the palB locus.
[0560] The results of the Southern blot (Table 8) demonstrated that
some of the palB minus strains had clean disruptions while others
did not.
16TABLE 8 Shake flasks Strain PalB phenotype (ECU/ml) Southern
pattern HowB432 plus 1.0 wild-type CpalB5-1 plus 1.0 wild-type and
other CpalB6-1 plus 1.2 wild-type and other CpalB7-1 plus 1.1
wild-type and other CpalB24-1 plus 1.7 wild-type and other
CpalB28-1 minus 1.6 other CpalB34-1 minus 1.6 disruption CpalB45-1
minus 1.4 disruption CpalB47-1 plus 1.1 wild-type and other
CpalB72-1 plus 1.3 wild-type and other CpalB76- 1 minus 1.6
disruption CpalB89-1 minus 1.6 disruption CpalB153-1 minus 1.1
disruption CpalB161-1 plus 1.2 wild-type and other CpalB163-1 minus
1.9 other CpalB185-1 minus 1.8 other CpalB190-1 minus 1.4
disruption
Example 26
Aspergillus oryzae Transformation with AsnI/PvuI Manganese
Superoxide Dismutase Disruption Cassette and Cellulase
Screening
[0561] Aspergillus oryzae HowB432 was transformed with a 5.8 kb
AsnI/PvuI fragment containing the manganese superoxide dismutase
disruption cassette according to the same procedure described in
Example 5.
[0562] Twenty transformants were obtained and tested for
sensitivity to paraquat as described in Example 17. Seven of the 20
transformants were paraquat sensitive indicative of the manganese
superoxide dismutase minus phenotype although they are sensitive to
different levels of paraquat as indicated in Table 9 below. Those
indicated as +++ for paraquat sensitivity are sensitive to as low
as 2 mM paraquat, while those labeled - and ++ are not sensitive to
paraquat and sensitive to intermediate levels of paraquat,
respectively. All of the transformants were spore purified and
tested in 24 well cultures for cellulase production as described in
Example 23. The strains were also tested in 125 ml shake flasks
cultures as described in Example 23. The results are shown in Table
9 below. The strains that are paraquat sensitive produce on average
more CAREZYME.TM. than those strains that are not paraquat
sensitive.
[0563] Southern blots of the transformants and Aspergillus oryzae
HowB432 were prepared and analyzed as described in Example 14 to
determine if the AsnI/PvuI manganese superoxide dismutase
disruption cassette had integrated to give a clean replacement into
the manganese superoxide dismutase disruption cassette locus.
[0564] The results of the Southern blot shown in Table 9 below
indicate that the strains sensitive to 2 mM paraquat have only the
disrupted locus of manganese superoxide dismutase, while those
sensitive to intermediate levels of paraquat have both the
wild-type locus and the disrupted cassette. The intermediate
sensitivity to paraquat may be explained by the fact that manganese
superoxide dismutase is a homodimer, and those cells expressing the
wild-type and truncated manganese superoxide dismutase coded for by
the disruption cassette would be producing heterodimers that are
probably not functional.
17TABLE 9 Shake flasks Strain Paraquat sensitivity (Relative LU/ml)
Southern Results HowB432 - 1.00 wild-type 432162T3 +++ 1.26
disrupted 432162T7 ++ 1.24 wild-type & other 432162T8 - 1.21
wild-type & other 432162T9 - 0.61 wild-type & other
432162T10 ++ 1.14 wild-type & other 432162T11 ++ 1.04 wild-type
& other 432162T12 ++ 1.12 wild-type & other 432162T15 -
0.44 wild-type & other 432162T16 ++ 1.10 wild-type & other
432162T17 +++ 1.53 -- 432162T18 - 1.12 -- 432162T19 +++ 1.44 --
Example 27
Construction of Glucose Transporter Gene Overexpression Plasmids
pHB218 and pDSY153 and Stop Control Plasmids pDSY152 and
pDSY155
[0565] Plasmids to overexpress the glucose transporter rescued
locus from Aspergillus oryzae DEBY599.3 were constructed to
determine if overexpression of the glucose transporter would lead
to an increase in the yields of Humicola lanuginosa lipase and
cellulase. The glucose transporter open reading frame was PCR
amplified to place SwaI and PacI sites at the 5' and 3' end of the
ORF, respectively. The following primers synthesized with an
Applied Biosystems Model 394 DNA/RNA Synthesizer according to the
manufacturer's instructions were used in combination with 0.2 .mu.g
of pDSY112 in the amplification:
18 961176: 5'-ATTTAAATGGTCCTCGGTGGATCAAGC-3' (SEQ ID NO:44) 961177:
5'-TTAATTAATTAGTCCTGTCTGCGCTGGT-3' (SEQ ID NO:45)
[0566] The conditions and parameters used for the amplification are
described in Example 2. Ten .mu.l of the PCR reaction was
electrophoresed on an agarose gel, and a 1.5 kb product was
obtained as expected. The PCR product was cloned using a
pPCR-Script.TM. Kit (Stratagene, La Jolla, Calif.) according to the
manufacturer's protocols. The ligation reaction was used to
transform E. coli DH5.alpha. cells, and plasmid DNA was isolated
from several of the tramsformants using the QIAWell-8 Plasmid Kit.
The plasmids were digested with NotI and EcoRi to determine which
clones had the 1.5 kb insert. Six of the 11 clones analyzed had the
correct size insert as determined by electrophoreses on an agarose
gel. One of the clones, pDSY119, was digested with PacI and SwaI,
and the digest was run on an agarose gel. The 1.5 kb SwaI/PacI band
was excised from the gel, and DNA was purified from the gel slice
using the QIAQuick Gel Extraction Kit. The 1.5 kb fragment was
ligated with SwaI/PacI cut pBANe13 (FIG. 3) using standard
conditions (Sambrook et al., 1989, supra). The ligation was used to
transform E. coli DH5.alpha. cells, and plasmid DNA was isolated
from several of the transformants. The plasmids were digested with
SwaI/PacI to determine which clones had the expected 1.5 kb insert.
The final plasmid was designated pHB218 (FIG. 27).
[0567] As a control for the overexpression experiments, a
derivative of pHB218 in which a stop codon was inserted at amino
acid 9 in the glucose transporter open reading frame was made using
site-directed mutagenesis. A MORPH.TM. Site-Specific Plasmid DNA
Mutagenesis kit from 5 Prime.fwdarw.3 Prime was used for the
mutagenesis, and protocols provided with the kit were followed. The
reaction contained pHB218 as template, and the mutagenic primer
used was:
970545: 5'-CGGTGGATCAAGCGGTTAATTAATCACTCCGTACCTGAT-3' (SEQ ID
NO:46)
[0568] Several E. coli colonies were obtained after following the
protocols, and plasmid DNA was isolated from the colonies using the
QIA-Well8 plasmid kit. The plasmids were digested with PacI since
the mutagenic primer introduced a PacI site which served as a
marker for the mutagenesis. Two of the plasmids with the extra PacI
site indicative of a successful mutagenesis were sequenced as
described in Example 2 to confirm the presence of the stop codon at
amino acid 9 in the ORF. The pHB218 derivative with the stop codon
at amino acid 9 was designated pDSY152.
[0569] Versions of pHB218 and pDSY152 in which the selectable
marker was the bar gene were constructed for transformation of
strains which are pyrG plus. The SwaI/PacI inserts from pHB218 and
pDSY152 were isolated by restriction digestion, electrophoresed on
an agarose gel, and purified using QIAQuick Gel Extraction Kit. The
inserts were ligated into pSE39 (FIG. 28) and digested with
SwaI/PacI. The ligation reaction was used to transform E. coli
DH5.alpha., and plasmid DNA was isolated from the colonies as
described above. The plasmids were digested with SwaI/PacI to
determine which clones contained the expected 1.5 kb insert. The
plasmids were sequenced as described in Example 2 to confirm the
presence or absence of the stop codon at amino acid 9 in pDSY155
and pDSY153 (FIG. 29), respectively. The only difference between
pDSY155 and pDSY153 was the stop codon at amino acid 9 of the
glucose transporter ORF in pDSY155.
Example 28
Transformation of Aspergillus oryzae HowB430 and Aspergillus oryzae
HowB432 with pHB218 and pDSY152 and lipase and cellulase Screening,
Respectively.
[0570] Aspergillus oryzae HowB430 was transformed with pHB218 or
pDSY152, and the transformants were recovered using the methods
described in Example 5. One hundred and twenty transformants each
with pHB218 and pDSY152 were recovered, grown in 24-well microtiter
plates in {fraction (1/100)} strength MY25 and assayed for lipase
activity after 3 and 5 days as described in Example 8. The assay
results showed that there was a slight shift towards higher lipase
production in the pHB218 transformants versus the pDSY152
transformants supporting the idea that overexpression of the
glucose transporter has a positive effect on lipase expression.
[0571] Aspergillus oryzae HowB432 was transformed with pHB218 and
pDSY152, and the transformants were recovered using the methods
described in Example 5. One hundred transformants each with pHB218
and pDSY152 were recovered, grown in 24-well microtiter plates in
1/4 strength MY25 and assayed for cellulase activity after 3 and 5
days as described in Example 23. The assay results showed that
there was a shift towards higher cellulase production in the pHB218
transformants versus the pDSY152 transformants indicating that
overexpression of the glucose transporter had a positive effect on
cellulase expression.
Example 29
Transformation of Aspergillus oryzae DEBY10.3 with pDSY153 and
pDSY155 and lipase Screening.
[0572] Aspergillus oryzae DEBY10.3 was transformed with pDSY153 and
pDSY155, and the transformants were recovered using the methods
described in Example 5. Two hundred sixteen and 144 transformants
with pDSY153 and pDSY155, respectively, were recovered, grown in
24-well microtiter plates in {fraction (1/100)} strength MY25, and
assayed for lipase activity on days 4 and 6 as described in Example
8. There was shift towards higher lipase production in the pDSY153
transformants when compared to the pDSY155 transformants indicating
that overexpression of the glucose transporter led to an increase
in lipase production and also suggesting that the palB minus effect
and the glucose transporter overexpression were additive.
Example 30
Identification of Tagged Event in Aspergillus oryzae HowL795
[0573] Genomic DNA was prepared from Aspergillus oryzae HowL795
according to Example 9. One .mu.g of DNA was digested with either
SnaB1 or NsiI. Both enzymes cleave within the pyrG gene contained
on the tagging construct. The DNA was then diluted to 4 ng/.mu.l
and recircularized with T4 Ligase at 22.degree. C. for 18 hours.
Inverse PCR was then performed using approximately 500 ng of
recircularized DNA using the primers shown below which were
synthesized with an Applied Biosystems Model 394 DNA/RNA
Synthesizer according to the manufacturer's instructions. Both were
located downstream of the NsiI and SnaBI sites.
19 Primer x: 5'-GCACTCGAATGACTACT-3' (SEQ ID NO:47) Primer y:
5'-CGCATCATACTTGCGACA-3' (SEQ ID NO:48)
[0574] The inverse PCR amplification reaction contained the
following components: 500 ng of recircularized DNA, 150 pmoles of
primer x, 150 pmoles of primer y, 1 mM each of dATP, dCTP, dGTP,
and dTTP, 1.times.Taq polymerase buffer, and 2.5 U of Taq
polymerase. The reaction was incubated in an Ericomp Thermal Cycler
programmed as follows: One cycle at 95.degree. C. for 5 minutes
followed by 30 cycles each at 95.degree. C. for 1 minute,
55.degree. C. for 1 minute and 72.degree. C. for 2 minutes. The PCR
product was isolated by electrophoresis on a 1% agarose gel.
[0575] PCR of the religated SnaBI DNA amplified a 4 kb fragment
whereas PCR of the religated NsiI DNA amplified a 2 kb fragment.
PCR confirmed that the smaller NsiI fragment was contained within
the larger SnaBI fragment.
[0576] DNA sequence analysis was performed according to the
procedure described in Example 2 using primer A. The analysis
identified that the insertion of pSO122 had occurred in the 3'
non-translated region of the amdS gene contained within plasmid
pBANe8.
Example 31
Construction of Aspergillus oryzae MStr107
[0577] Aspergillus oryzae MStr107 was constructed to contain extra
copies of one of the native alpha-amylase (TAKA) genes
(FUNGAMYL.TM. gene, Novo Nordisk A/S, Bagsvaerd, Denmark), by
transforming Aspergillus oryzae HowB101 with a DNA fragment from
pMStr15. pMStr15 was constructed from pCaHj505 and pTAKA17 as
described below. Standard methods were employed (Sambrook et al.,
1989, supra) except where noted.
[0578] pCaHj505 (FIG. 30) was constructed to contain the
Aspergillus oryzae NA-14 alpha-amylase (TAKA) promoter, the
Aspergillus niger glucoamylase (AMG) terminator, and the
Aspergillus nidulans amdS gene from the following fragments:
[0579] a) The vector pToC65 (WO 91/17243) digested with EcoRI and
XbaI.
[0580] b) A 2.7 kb XbaI fragment from Aspergillus nidulans carrying
the amdS gene (Corrick et al., 1987, Gene 53: 63-71). The amdS gene
was used as a selective marker in fungal transformations. The amdS
gene was modified so that the BamHI site normally present in the
gene was destroyed. This was done by introducing a silent point
mutation using the primer: AGAAATCGGGTATCCTTTCAG (SEQ ID
NO:49).
[0581] c) A 0.6 kb EcoRI-BamHI fragment carrying the Aspergillus
oryzae NA-14 alpha-amylase promoter.
[0582] d) A 675 bp xbaI fragment carrying the Aspergillus niger
glucoamylase transcription terminator. The fragment was isolated
from the plasmid pICAMG/Term (EP 238 023).
[0583] The BamHI site of fragment c was connected to the XbaI site
in front of the transcription terminator on fragment d via the
pIC19R linker (BamHI to XbaI) (Boehringer Mannheim, Indianapolis,
Ind.).
[0584] pMStr15 (FIG. 31) was constructed to contain the Aspergillus
oryzae NA-14 alpha-amylase promoter, gene and terminator and the
Aspergillus nidulans amdS gene. The alpha-amylase gene with
promoter and terminator was excised from pTAKA17 (European patent
0238 023) as a 2.9 kb EcoRI-HindIII fragment and cloned adjacent to
the amdS gene in the vector pCaHj505 by replacing the EcoRI-XbaI
promoter/terminator fragment in pCaHj505. To facilitate cloning,
the recessed 3' termini generated by HindIII and XbaI digestion
were filled in.
[0585] A single linear DNA fragment containing both the
alpha-amylase gene and the amdS gene was obtained by digesting
pMStr15 with NotI, resolving the vector and insert sequences using
agarose gel electrophoresis, excising the appropriate DNA band from
the gel, and purifying the DNA from the agarose using GenElute.TM.
Agarose Spin Columns according to the manufacturer's directions
(Supelco, Bellefonte, Pa.). This 5.6 kb NotI fragment was used to
transform Aspergillus oryzae HowB101 to construct Aspergillus
oryzae MStr107, using the transformation protocol and selective
medium described in Example 2. Transformants were propagated from
single colonies twice in succession on COVE medium with 0.1% Triton
X100 before performing additional screens.
[0586] Aspergillus oryzae MStr107 was selected from among the
transformants based on its ability to produce more alpha-amylase
thanAspergillus oryzae HowB101. The ability of the transformants to
produce alpha-amylase was determined by culturing them in 10 ml of
YPM medium for 4 days at 30.degree. C. with shaking and resolving 5
.mu.l of the culture medium by SDS-PAGE according to standard
methods. The strain producing the most alpha-amylase under these
conditions was selected as Aspergillus oryzae MStr107, and was
compared in a 3 liter fermentation culture to Aspergillus oryzae
HowB101. The medium was composed of maltose syrup, yeast extract,
KH.sub.2PO.sub.4, K.sub.2SO.sub.4, (NH.sub.4).sub.2SO.sub.4, citric
acid, MgSO.sub.4, trace metals and uridine. Under these conditions,
Aspergillus oryzae MStr107 produced 360% of Aspergillus oryzae
HowB101.
Example 32
Aspergillus oryzae Mstr107 Transformation with Linearized
pDSY82
[0587] Protoplasts of Aspergillus oryzae Mstr107 were prepared as
described in Example 2. A 5-15 .mu.l aliquot of pDSY82 (6 .mu.g)
linearized with 1.25 U of XbaI was added to 0.1 ml of the
protoplasts at a concentration of 2.times.10.sup.7 protoplasts per
ml in a 14 ml Falcon polypropylene tube followed by 250 .mu.l of
60% PEG 4000-10 mM CaCl.sub.2-10 mnM Tris-HCl pH 7, gently mixed,
and incubated at 37.degree. C. for 30 minutes. Three ml of SPTC
were then added and the suspension was gently mixed. The suspension
was mixed with 12 ml of molten overlay agar (1.times.COVE salts, 1%
NZ amine, 0.8 M sucrose, 0.6% Noble agar) or 3 ml of STC medium and
the suspension was poured onto a Minimal medium plate. The plates
were incubated at 37.degree. C. for 3-5 days.
[0588] The transformation frequency of Aspergillus oryzae MStr107
with pDSY82 and XbaI was approximately 200 transformants/.mu.g of
DNA. A library of approximately 30,000 transformants was obtained.
Spores from 70 pools with approximately 400 transformants in each
pool were collected and stored in a 20% glycerol, 0.1% Tween 80 at
-80.degree. C. The pools and transformants from these libraries
were designated with the letter "x".
Example 33
Characterization of Integration Events in "REMI" Aspergillus oryzae
MStr107 Transformants
[0589] Transformants of Aspergillus oryzae MStr107 with pDSY82 and
XbaI (library "x") were analyzed as described in Example 6. Genomic
DNA was isolated from 40 transformants, 20 from one pool (x15) and
20 from 20 various pools. DNA samples were cut with HindIII,
resolved, blotted and probed with radiolabeled pDSY82. Thirty-three
of 40 displayed apparently novel band patterns, suggesting that
plasmid integrations were distributed to different sites in the
genome. For 19 of the 40 transformants the band patterns suggested
that only one copy of pDSY82 integrated in the genome, while more
than one copy was observed in the remaining 21 transformants. DNA
from the 20 transformants taken from various pools was also cut
with XbaI, resolved, blotted and probed with radiolabeled pDSY82 as
described in Example 6. A single, plasmid-sized band was observed
indicating REMI had occurred at an XbaI site in 9 of the
transformants.
Example 34
FUNGAMYL.TM. Expression Screening
[0590] The Aspergillus oryzae MStr107 tagged mutant library "x"
pools described in Example 32 were assayed for FUNGAMYL.TM.
expression.
[0591] For 96-well plate screens, MTBCDYU medium was used. For
24-well plate methods, 4.times.MTBCDYU medium was used.
[0592] Primary 96-well plate screens involved the dilution of
spores from distinct pools into MTBCDYU so that one spore on
average was inoculated per well when 100 .mu.l of medium was
dispensed into the wells. After inoculation, the 96-well plates
were grown for 3-4 days at 34.degree. C. under static conditions.
Cultures were then assayed for FUNGAMYL.TM. activity as described
below. Mutants of interest were isolated twice on YPG or Cove
plates, and single colonies transferred to Cove agar slants. Spores
from Cove slants were inoculated into 24-well plates containing
4.times.MTBCDYU with approximately 103 spores per well and grown
under static conditions for 4 days at 34.degree. C. Cultures were
then assayed for FUNGAMYL.TM. activity as described below.
[0593] The FUNGAMYL.TM. assay substrate
(4-nitrophenyl-alpha-D-maltoheptas- id-4,6-O-ethyliden, EPS) was
prepared as a 1/2 strength solution relative to the instructions
given by the manufacturer (Boehringer Mannheim, Indianapolis,
Ind.). The substrate was prepared in HEPES pH 7.0 buffer. A
FUNGAMYL.TM. standard (FUNGAMYL.TM., Novo Nordisk A/S, Bagsvaerd,
Denmark) was prepared to contain 2 FAU/ml in HEPES pH 7.0 buffer.
The standard was stored at -20.degree. C. until use. FUNGAMYL.TM.
stock was diluted appropriately to obtain a standard series ranging
from 0.02 to 0.2 FAU/ml just before use. Broth samples were diluted
in HEPES buffer and 25 .mu.l aliquots were dispensed to wells in
96-well plates followed by 180 .mu.l of diluted substrate. Using a
plate reader, the absorbance at 405 nm was recorded as the
difference of two readings taken at approximately 1 minute
intervals. FUNGAMYL.TM. units/ml (FAU/ml) were calculated relative
to the FUNGAMYL.TM. standard solutions.
[0594] The results of the 96-well screen followed by the 24-well
screen identified for further evaluation 51 transformants from the
pDSY82 and XBaI transformations. These identified transformants
produced higher levels of FUNGAMYL.TM. than the control strain
Aspergillus oryzae MStr107.
Example 35
Shake Flask and Fermentation Evaluation
[0595] The highest FUNGAMYL.TM.-producing DNA-tagged mutants
described in Example 34 were evaluated in shake flasks and
fermentors.
[0596] Shake flask evaluations were performed by inoculating half
the spore content of a COVE slant suspended in a suitable volume of
sterile water containing 0.02% TWEEN-80 into 100 ml of G1-gly
medium at pH 7.0 in a 500 ml shake flask. The G1-gly shake flasks
were incubated at 34.degree. C. for 1 day at 270 rpm. Next, 5 ml of
the G1-gly cultures were inoculated into 100 ml of 1/5MDU2BP at pH
6.5 in 500 ml shake flasks. Samples were taken at day 3, and
FUNGAMYL.TM. activity was measured as described in Example 34.
[0597] The DNA-tagged mutant X70-25 and 257D11 were grown in a 3
liter lab fermentor containing a medium composed of Nutriose, yeast
extract, MgSO.sub.4, KH.sub.2PO.sub.4, citric acid,
K.sub.2SO.sub.4, (NH.sub.4).sub.2SO.sub.4 and trace metals
solution. The fermentation was performed at a temperature of
34.degree. C., a pH of 7, and the agitation was maintained between
1000-1200 rpm for 5 days. FUNGAMYL.TM. activity was measured by
partial degradation of EPS to G.sub.2 and G.sub.3 derivatives
(G=glucose). The G.sub.2 and G.sub.3 derivatives were then degraded
to glucose and yellowish colored p-nitrophenolate anion by the
addition of a surplus of alpha-glucosidase. The analytical output
was determined as the change in absorbance at 405 nm per unit time
(3 minutes) at 37.degree. C. and pH 7.1 after a preincubation for
2.5 minutes. FUNGAMYL.TM. was used as standard.
[0598] The results obtained are shown in Table 10 below where the
FUNGAMYL.TM. yield of Aspergillus oryzae MStr107 as a control is
normalized to 1.0.
20TABLE 10 FUNGAMYL .TM. Expression by DNA Tagged Mutants 24-well
Shake Flask in 96-well Plate Fermentation Strain # Plates Results
Results Description Pool Screened (FAU/ml) (FAU/ml) (FAU/ml)
HowB101 NA NA 0.4 0.4 0.3 MStr107 NA NA 1.0 1.0 1.0 X70-25 x70 580
1.4 1.3 1.2 257D11 x6 480 1.5 1.4 1.0 X70-42 x70 580 1.3 1.3 ND
263A3 x6 480 1.4 1.6 ND X69-246 x69 350 1.6 1.4 ND X59-122 x59 580
1.3 1.3 ND X49-233 x49 460 1.4 1.4 ND
[0599] As shown in Table 10, the mutants produced approximately
30-60% more FUNGAMYL.TM. than the control strain Aspergillus oryzae
MStr107 when grown in 24-well plates and when grown in shake
flasks. The mutant, Aspergillus oryzae X70-25 produced
approximately 20% more FUNGAMYL.TM. than the control strain
Aspergillus oryzae MStr107 when grown in fermentors.
Example 36
Screening for Morphological Mutants
[0600] The 5 "e" pools and 5 "b" pools described in Example 5 were
screened for altered morphology by plating on CM-1 agar and
incubating at 34.degree. C. for 4 days.
[0601] Twenty-four colonies having altered plate morphology and
covering the morphological variation within the pool were
transferred to fresh CM-1 plates and incubated 5 days at 34.degree.
C. for single colony isolation. Each morphology (in most cases 1)
on a plate, was transferred from a single colony to the center of a
CM-1 plate and a PDA plate, and incubated 6-8 days at 34.degree. C.
before the morphology was evaluated, i.e., the diameter and the
appearance. A total of 218 morphological mutants was transferred to
COVE plates and incubated at 34.degree. C. for 1-2 week to generate
spores.
Example 37
Evaluation of Morphological Mutants
[0602] The morphological mutants isolated in Example 36 were
evaluated in 24-well plates for lipase production according to the
procedure described in Example 7. The highest yielding mutants were
compared with respect to plate morphology on CM-1 agar, and 23
mutants covering the observed morphological variation were further
tested in shake flasks containing CD medium to evaluate lipase
production.
[0603] Approximately 0.25 ml of spore suspension from a CM-1 plate
was inoculated into 25 ml of G1-gly medium in a 125 ml PP flask and
incubated at 34.degree. C. for 24 hours. Then 0.5 ml of the 24 hour
seed flask was transferred to 50 ml of CD medium supplemented with
1.0 .mu.l of FUNGAMYL.TM. 800L (Novo Nordisk A/S, Bagvaerd,
Denmark) in a 125 ml PP flask, and incubated at 34.degree. C., 200
rpm. The culture was sampled after 2 and 3 days and assayed for
lipase activity as described in Example 7.
[0604] The isolated mutants were also tested in the following
manner in oxygen limited media. Aspergillus oryzae HowL536.3 was
run as a control since the strain possessed the wild type
morphology and did not require uridine for growth. Approximately
250 .mu.l of spore suspension was inoculated into a 125 ml shake
flask containing 25 ml OL-1 medium and incubated at 34.degree. C.,
200 rpm until residual glucose was<<1 g/l measured using
DIASTIX.TM. (Bayer, Elkhart, Ind.). Then 75 ml OL-6 medium was
added to each flask and further incubated at 34.degree. C., 200 rpm
for approximately 25 hours until residual glucose in the
Aspergillus oryzae HowL536.3 flask was approximately 5 g/l. At that
time, all the flasks were assayed for residual glucose, and the
flasks with significantly lower glucose (0-2 g/l) were considered
positive. The majority of the flasks averaged around 5-10 g/l.
[0605] Twenty mutants converting the glucose faster than average
were considered likely to be easier to aerate and were further
tested in shake flasks containing CD medium as described above to
evaluate lipase expression.
[0606] Based on these tests, 14 mutants were identified and further
evaluated in lab fermentors according to the procedure described in
Example 8. The morphological mutants listed below in Table 11 were
identified.
21TABLE 11 Morphological mutants Strain Construction Pool
Description P2-7.1 pDSY82 + BamHI b2 colonial, easy to aerate, 50%
yield increase in fermentors P3-2.1 pDSY82 + BamHI b3 flat, yield
40% yield increase in fermentors P4-8.1 pDSY82 + BamHI b5 easy to
aerate, 30% yield increase in fermentors P5-7.1 pDSY82 + BamHI b6
easy to aerate, 60% yield increase in fermentors P7-14.1 pDSY81 +
EcoRI e2 colonial, easy to aerate, 50% yield increase in fermentors
P8-10.1 pDSY81 + EcoRI e3 easy to aerate, yield increased 50% in
fermentors
Example 38
Rescue of Plasmid DNA and Flanking DNA from Morphological
Mutants
[0607] The plasmid DNA and genomic flanking loci were isolated from
mutants Aspergillus oryzae P4-8.1 and P7-14.1 using the procedure
described in Example 9 except for the restriction endonuclease(s)
used. Transformant E. coli HB101 p4-8.1 contained a BglII rescued
locus from mutant Aspergillus oryzae P4-8.1. Transformant E. coli
HB101 p7-14.1 contained a NarI rescued locus from mutant
Aspergillus oryzae P7-14.1.
[0608] The plasmid DNA and genomic flanking loci were isolated from
mutants Aspergillus oryzae DEBY7-17.2, DEBY3-2.1, DEBY5-7.1, and
DEBY8-10.1. The rescued plasmids were generated as previously
described in Example 9 with the exception that rescues pSMO717,
pSMO321, pHowB571, and pSMO810 were isolated from transformed E.
coli DH5.alpha. cells.
[0609] Transformant E. coli DH5.alpha. pSMO717 contained the BglII
rescued locus from mutant Aspergillus oryzae DEBY7-17.2.
Transformant E. coli DH5.alpha. pSMO321 contained the BglII rescued
locus from mutant Aspergilus oryzae DEBY3-2.1. Transformant E. coli
DH5.alpha. pHowB571 contained the NdeI rescued locus from mutant
Aspergillus oryzae DEBY5-7.1. Transformant E. coli DH5.alpha.
pSMO810 contained the NdeI rescued locus from mutant Aspergillus
oryzae DEBY8-10.1.
Example 39
Characterization of Morphological Mutant Aspergillus oryzae P4-8.1
Rescued Locus p4-8.1
[0610] The Aspergillus oryzae P4-8.1 rescued locus p4-8.1
containing 915 and 665 bp regions on either side of the integration
event was sequenced according to the procedure described in Example
2. The nucleic acid sequence (SEQ ID NO:50) and the deduced amino
acid sequence (SEQ ID NO:51) are shown in FIG. 32. The nucleic acid
sequence suggested that the integration event occurred within an
open reading frame for a homologue of the Saccharomyces cerevisiae
YHM4 Heat Shock protein gene. The deduced amino acid sequence (SEQ
ID NO:51) showed 40.2% identity to the Saccharomyces cerevisiae
YHM4 Heat Shock protein (SEQ ID NO:52) and 41.8% identity to a
Schizzosaccharomyces pompe Heat Shock Protein 70 (SEQ ID
NO:53).
Example 40
Aspergillus oryzae Transformation with BglII Linearized p4-8.1 and
Morphology Screening
[0611] To verify the link between the observed plate morphology for
Aspergillus oryzae P4-8.1 and the rescued genomic locus,
Aspergillus oryzae HowB430 was transformed with the BglII
linearized rescued locus of Aspergillus oryzae P4-8.1, p4-8.1,
using the procedure described in Example 5.
[0612] Sixty-six transformants were obtained, transferred to CM-1
agar, and incubated at 34.degree. C. for 3-4 days to evaluate the
morphology. Sixteen transformants with the correct plate morphology
were transferred to fresh CM-1 plates as center colonies, and 12
transformants maintaining the plate morphology after 4 days at
34.degree. C. were analyzed by Southern blot analysis with a PCR
amplified 300 bp fragment of the rescued locus as a probe. The
fragment was PCR amplified using the primers below synthesized with
an Applied Biosystems Model 394 DNA/RNA Synthesizer according to
the manufacturer's instructions.
22 HSP-1: 5'-TACGGTTGACAGTGGAGC-3' (SEQ ID NO:54) HSP-3r:
5'-CACTGACTTCTCCGATGC-3' (SEQ ID NO:55)
[0613] The amplification reaction (50 .mu.l) contained the
following components: 0.2 ng of p4-8.1, 50 pmol of primer HSP-1, 50
pmol of primer HSP-3r, 0.25 mM each of dATP, dCTP, dGTP, and dTTP,
1.times.Taq polymerase buffer, and 2.5 U of Taq polymerase. The
reaction was incubated in an Ericomp Thermal Cycler programmed as
follows: One cycle at 95.degree. C. for 3 minutes; 30 cycles each
at 95.degree. C. for 1 minute, 58.degree. C. for 1 minute, and
72.degree. C. for 1.5 minutes; and 1 cycle at 72.degree. C. for 5
minutes. The PCR product was isolated by electrophoresis on a 1%
agarose gel.
[0614] Samples of the genomic DNA were obtained from each of the 12
transformants obtained according to the method described in Example
9. The genomic DNAs were digested with BglII and submitted to
Southern analysis according to the procedure described in Example
14.
[0615] All 12 transformants were affected at the rescued locus,
suggesting a connection between this locus and the observed plate
morphology.
Example 41
Characterization of Morphological Mutant Aspergillus oryzae P7-14.1
Rescued Locus p7-14.1
[0616] The Aspergillus oryzae P7-14. 1 rescued locus p7-14. 1
containing 1040 and 520 bp regions on either side of the
integration event was sequenced according to the procedure
described in Example 2. The nucleic acid sequence (SEQ ID NO:56)
and the deduced amino acid sequence (SEQ ID NO:57) are shown in
FIG. 33. The nucleic acid sequence suggested that the integration
event occurred within an open reading frame for a homologue of the
Aspergillus nidulans chitin synthase B (chsB) gene and the
Aspergillus fumigatus chitin synthase G (chsG) gene. Identities of
94% and 80% were found when the deduced amino acid sequences of the
two sides of the rescued locus (SEQ ID NO:57), the chsB gene (SEQ
ID NO:58), and the chsG gene (SEQ ID NO:59) were compared.
[0617] Disruption of the chsB gene in Aspergillus nidulans is known
to change the morphology significantly (Yanai et al., 1994, Biosci.
Biotech. Biochem. 58: 1828-1835), and in Aspergillus fumigatus
disruption of the chsG gene is known to cause colonial morphology
(Mellado et al., 1996, Molecular Microbiology 20: 667-679), which
is the observed phenotype of Aspergillus oryzae P7-14.1.
Example 42
Aspergillus oryzae Transformation with a Linear chs Fragment and
Morphology Screening
[0618] A 1.9 kb DNA fragment was generated by PCR using as the
template Aspergillus oryzae HowB430 genomic DNA prepared as
described in Example 6. Primer A, 5'-CACCAAGTCAGAGCGTC-3' (SEQ ID
NO:60), was derived from the rescued chs Aspergillus oryzae
homolog. Primer 5, 5'-GGICCITTYGAYGAYCCICA- -3' (SEQ ID NO:61), was
degenerate based on the consensus sequence of the Aspergillus
fumigatus chsG and Aspergillus nidulans chsB genes. The
amplification reaction (50 .mu.l) contained the following
components: 10 ng of pHB220, 48.4 pmol of each primer, 1 mM each of
dATP, dCTP, dGTP, and dTTP, and the Advantage-GC.TM. Tth Polymerase
Mix (Clontech, Palo Alto, Calif.). The reaction was incubated in an
Ericomp Thermal Cycler programmed as follows: One cycle at
95.degree. C. for 3 minutes; 30 cycles each at 95.degree. C. for 1
minute, 58.degree. C. for 1 minute, and 72.degree. C. for 3
minutes; and 1 cycle at 72.degree. C. for 5 minutes. The PCR
product was isolated by electrophoresis on a 1% agarose gel.
[0619] The DNA fragment was cloned into the PCR-Blunt Cloning
Vector (Invitrogen, San Diego, Calif.). A HindIII site in the
multicloning site was destroyed by filling in with the Klenow
fragment of DNA Polymerase I. A 2 kb HindIII-EcoRI fragment
containing the Basta gene conferring resistance to Bialaphos was
obtained from pMT1612 (FIG. 34) and inserted into the chs HindIII
site located approximately 0.7 kb within the chs fragment. The
resultant plasmid was labelled pHB220.
[0620] Using pHB220 as template, a 4 kb PCR fragment was generated
using primer A and 5'-GGGCCGTTTGACAATCCGCAT-3' (SEQ ID NO:62). The
amplification reaction was performed as described above except the
reaction was incubated in an Ericomp Thermal Cycler programmed as
follows: One cycle at 95.degree. C. for 3 minutes; 30 cycles each
at 95.degree. C. for 1 minute, 58.degree. C. for 1 minute, and
72.degree. C. for 6 minutes; and 1 cycle at 72.degree. C. for 5
minutes. The PCR product was isolated by electrophoresis on a 1%
agarose gel.
[0621] The PCR fragment was then used directly to transform
protoplasts prepared from Aspergillus oryzae HowL795 according to
the procedure described in Example 5. Of 100 transformants, three
transformants appeared to have a "colonial" plate morphology on
Minimal medium plates and PDA plates.
[0622] Southern analysis was performed on the three transformants
and Aspergillus oryzae HowB430 as a control using the 2 kb
DIG-labelled chs fragment as probe. Genomic DNA was prepared from
the three strains and control strain as described in Example 9.
Samples of the genomic DNA from each of the 3 transformants
digested with HindIII was submitted to Southern analysis according
to the procedure described in Example 14.
[0623] The Southern analysis showed that each of the three
transformants had undergone a gene replacement substituting the
chs/basta construct with the wild-type chs gene. The results
confirmed that the colonial morphology observed in the chs tagged
strain Aspergillus oryzae P7-14. 1 wag associated with a mutation
of the chs gene.
[0624] An apparent effect of the chs gene on colony morphology was
observed in shake flask cultures containing MY25 medium performed
as described in Example 8. The pellet mass of the colonies in the
broth appeared less dense in the chs mutants of Aspergillus oryzae
HowL795 compared to Aspergillus oryzae HowL795.
[0625] Fermentations were also performed as described in Example 8
on two derivatives of Aspergillus oryzae HowL795 containing gene
disruptions of the chs gene. Lipase yields in both strains were
approximately 21% greater than Aspergillus oryzae HowL795. The
kinetics of enzyme production appeared to be increased in the chs
mutants in the later stages of fermentation suggesting that these
strains exhibited a more optimal tank morphology.
Example 43
Characterization of Morphological Mutant Aspergillus oryzae
DEBY7-17.2 Rescued Locus pSMO717
[0626] The Aspergillus oryzae DEBY7-17.2 rescued locus pSMO717
containing 400 bp was sequenced according to the method described
in Example 2. The nucleic acid sequence (SEQ ID NO:63) and the
deduced amino acid sequence (SEQ ID NO:64) are shown in FIG. 35.
The deduced amino acid sequence (SEQ ID NO:64) showed 44% identity
to the deduced amino acid sequence of an ORF of Aspergillus
nidulans (AC000133) (SEQ ID NO:65).
Example 44
Characterization of Morphological Mutant Aspergillus oryzae
DEBY3-2.1 Rescued Locus pSMO321
[0627] The Aspergillus oryzae DEBY3-2.1 rescued locus pSMO321
containing 1.0 kb was sequenced according to the method described
in Example 2. The nucleic acid sequence (SEQ ID NO:66) shown in
FIG. 36 showed no homology to any published sequences.
[0628] Probes from either end of the rescued plasmid pSMO321 were
generated by PCR. Probe 5 was generated with primers 970850 and
970851 shown below. Probe 6 was generated with primers 970852 and
primer 970853 shown below. The primers were synthesized with an
Applied Biosystems Model 394 DNA/RNA Synthesizer according to the
manufacturer's instructions. The template for the 50 .mu.l PCR
labeling reaction was 10 ng of the rescued plasmid pSMO321. PCR
cycles and conditions were as described in Example 20.
23 970850: 5'-GTTCTATTGAGATACGCG-3' (SEQ ID NO:67) 970851:
5'-ACAAGCCGACCGGTTTTG-3' (SEQ ID NO:68) 970852:
5'-CGATAAGGACTCCAAGAG-3' (SEQ ID NO:69) 970853:
5'-GTCGCGCATAATATGAAG-3' (SEQ ID NO:70)
[0629] Southern blots of Aspergillus oryzae HowB430 genomic DNA
digested with SphI, SalI and BamH1 were prepared and analyzed
according to the method described in Example 14. The blots were
hybridized independently to probes 5 and 6 made from the ends of
the rescued plasmid.
[0630] Analysis of the Southern blots suggested no deletions had
occurred. When PCR was performed using 500 ng of genomic DNA from
Aspergillus oryzae HowB430 with primers 090850 and 090852, a 500 bp
product was amplified as predicted verifying that no deletions had
taken place.
[0631] Genomic DNA was prepared from the tagged mutant strain
Aspergillus oryzae DEBY3-2.1 as described in Example 9, digested
with the restriction enzyme used for REMI (BamHI), blotted and
probed with the NheI fragment from Aspergillus oryzae pyrG.
Southern analysis of this blot suggested the tagged plasmid had
inserted into a BamHI site in the genome.
Example 45
Characterization of Morphological Mutant Aspergillus oryzae DEBY
5-7.1 Rescued Locus pHowB571
[0632] The Aspergillus oryzae DEBY5-7.1 rescued locus pHowB571
containing 600 bp was sequenced according to the method described
in Example 2. The nucleic acid sequence (SEQ ID NO:71) shown in
FIG. 37 showed no homology to any published sequences.
[0633] To test if a deletion occurred during tagging, a Southern
blot was preapared and analyzed according to the method described
in Example 14 using genomic DNA from Aspergillus oryzae HowB430
digested with SphI, SalI and BamHI. Probes from either end of the
rescued tagged plasmid pSMO571 were generated by PCR. Probe 7 was
generated with primer 970936 and primer 970937 shown below. Probe 8
was generated with primer 970938 and primer 970939 shown below. The
primers were synthesized with an Applied Biosystems Model 394
DNA/RNA Synthesizer according to the manufacturer's instructions.
The template for the PCR labeling reaction was 10 ng of pSMO571.
PCR cycles and conditions were as described in Example 20.
24 970936: 5'-CTTCCTCATAAACCACCC-3' (SEQ ID NO:72) 970937:
5'-AACTGACAGGACAAGACC-3' (SEQ ID NO:73) 970938:
5'-GACTTGCATCACTTCCTC-3' (SEQ ID NO:74) 970939:
5'-TGAAGCTGAGAGTAGGTG-3' (SEQ ID NO:75)
[0634] The results showed identical banding patterns from both
probes suggesting no deletions had occurred. PCR was used to verify
that no deletions had occurred using the method described in
Example 20. A total of 500 ng of genomic DNA from Aspergillus
oryzae HowB430 was used as template with primer 970936 and primer
970939. A 550 bp product was amplified as predicted. Southern data
from genomic DNA obtained from the tagged mutant Aspergillus oryzae
DEBY5-7.1 digested with BamHI, hybridized to the NheI fragment of
pyrG suggested that the tagged plasmid had inserted into a BamHI
site in the genome. Southern blot conditions are described as
above.
[0635] Southern and PCR analysis demonstrated the tagged plasmid
had inserted directly into a BamHI site. Cloning with TOPO pCR11
vector and subsequent sequencing according to Example 2 of the PCR
product generated using Aspergillus oryzae HowB430 genomic DNA with
primers from the rescued ends of Aspergillus oryzae DEBY5-7.1
confirmed this result.
Example 46
Characterization of Morphological Mutant Aspergillus oryzae
DEBY8-10.1 pSMO810
[0636] The Aspergillus oryzae DEBY8-10.1 rescued locus pSMO810
containing 750 bp was sequenced according to the method described
in Example 2. The nucleic acid sequence (SEQ ID NO:76) shown in
FIG. 38 showed no homology to any published sequences.
[0637] Probes from either end of the rescued plasmid pSMO810 were
generated by PCR. Probe 9 was generated with primer 970854 and
primer 970855 shown below. Probe 10 was generated with primer
970856 and primer 970857 shown below. The primers were synthesized
with an Applied Biosystems Model 394 DNA/RNA Synthesizer according
to the manufacturer's instructions. The template for the PCR
labeling reaction was 10 ng of pSMO810. The PCR reaction and
conditions were as described in Example 20.
25 970854: 5'-GTTTCGGTATTGTCACTG-3' (SEQ ID NO:77) 970855:
5'-ACAGGTGAACAACTGAGG-3' (SEQ ID NO:78) 970856:
5'-CGACCAAACTAGACAAGC-3' (SEQ ID NO:79) 970857:
5'-CTTTCCTCTTGGACACAC-3' (SEQ ID NO:80)
[0638] Southern analysis was performed as described in Example 14.
Southern analysis of genomic DNA from, Aspergillus oryzae HowB430
digested with SphI, SalI, and BamHI hybridized independently with
probes 9 and 10 suggested no deletions had occurred during the
insertion of the tagged plasmid. PCR using 500 ng of Aspergillus
oryzae HowB430 genomic DNA as template with primers 090854 and
090856 amplified a 500 bp product as expected with no deletions.
Southern analysis of genomic DNA prepared from mutant Aspergillus
oryzae DEBY8-10.1, digested with EcoRI, and probed to the NheI
fragment of pyrG suggested that the plasmid integrated into an
EcoRI site in the genome. Cloning with TOPO pCR11 vector and
subsequent sequencing according to Example 2 of the PCR product
generated using Aspergillus oryzae HowB430 genomic DNA with primers
from the rescued ends of Aspergillus oryzae DEBY8-10.1 confirmed
this result.
Example 47
Screening on a Poor Carbon Source for High Producers
[0639] Eighteen pools of Aspergillus oryzae HowB430 transformants,
11 generated with HindIII digested pDSY81 and in the presence of
HindIII, and 7 generated with SalI digested pDSY81 and in the
presence of SalI (see Example 5) were screened on poor carbon
sources to identify mutants which were high producers of lipase.
Glycerol was used as poor carbon source, since the expression of
lipase is very low on glycerol, but a number of other carbon
sources, e.g., xylose, sucrose, and polyols such as mannitol and
sorbitol could be used in a similar way.
[0640] The primary 96-well plate screen was performed as described
in Example 7, but with GLY25 medium composed of 100 ml of 10% yeast
extract, 100 ml of 25% glycerol, 100 ml of 2% urea per liter
diluted 50-fold. Lipase assays were performed as described in
Example 7.
[0641] Mutants of interest were then inoculated directly into
24-well plates containing the same medium as above and grown 6 days
at 34.degree. C. and 100 rpm. Cultures were then assayed for lipase
activity as described in Example 7, and mutants of interest were
plated on COVE plates to produce spores, spread on PDA plates to
produce single colonies, and then 4 single isolates of each mutant
were grown on CM-glycerol agar (as CM-1 agar, but maltose was
replaced by glycerol as carbon source) to produce spores for
inoculation of 24-well plates as above.
[0642] After the 24-well plates, 10 transformants were identified
for further evaluation in shake flasks. The shake flasks contained
50 ml of medium at pH 6.5 composed of 1 g of MgSO.sub.4-7H.sub.2O,
1 g/l K.sub.2SO.sub.4, 15 g of KH2PO.sub.4, 0.25 ml of trace metals
solution, 0.7 g of yeast extract, 3 ml of 50% urea, 2 ml of 15%
CaCl.sub.2-2H.sub.2O, and 2% carbon source (either maltose,
glucose, sucrose, or glycerol). The shake flasks containing 50 ml
of medium in a 125 PP flask were inoculated with 0.5 ml G1-gly
overnight culture, incubated at 34.degree. C. and 200 rpm, and
sampled after 2 and 3 days. Lipase activity was measured as
above.
[0643] The results are shown in Table 12 where lipase production by
Aspergillus oryzae HowB430 grown on glycerol as carbon source is
normalized to 1.0.
26TABLE 12 Strain Pool Glycerol Maltose Glucose Sucrose HowB430 NA
1.0 152 44 1.1 HINL880.1 Hin-5 1.5 182 49 10 HINL895.3 Hin-20 8.4
140 38 11 HINL918.4 Hin-24 5.7 163 45 8.8 SALL587.4 Sal-4 1.6 178
89 22 SALL591.2 Sal-4 1.3 232 126 33 SALL631.2 Sal-5 16.6 72 35 25
SALL631.3 Sal-5 18.3 67 42 29 SALL664.2 Sal-15 6.1 70 79 9.2
SALL683.3 Sal-16 1.6 186 77 20 SALL692.2 Sal-16 2.7 148 63 24
[0644] The relative expression of lipase responded differently to
different carbon sources, suggesting that the regulation of the
lipase expression was altered in these transformants.
Example 48
Screening for .alpha.-Cyclopiazonic Acid Mutants
[0645] The pools e1-e26 from the EcoRI library described in Example
5 was used in screening for .alpha.-cyclopiazonic acid negative
strains.
[0646] The spore number in the vials containing the different pools
was determined by counting an appropriate dilution in a
haemocytometer and a dilution series was constructed in such a way
that approximately 30-50 spore derived colonies were present on
each 9 cm screening plate. The screening medium was composed per
liter of 30 g of mannitol, 10 g of glucose, 10 g of succinic acid,
3 g of Casamino acids, 1 g of KH.sub.2PO.sub.4, 0.3 g of
MgSO.sub.4-7H.sub.2O, 0.2 g of FeSO.sub.4-7H.sub.2O, 100 .mu.l of
Triton X100, and 20 g of Difco Bacto Agar. The pH was adjusted to
5.6 with 14% NH.sub.4OH before autoclaving. The ferrous ion forms a
red complex with .alpha.-cyclopiazonic acid. This complex is seen
on the reverse side of the colonies.
[0647] Approximately 2000-2500 colonies were screened from each
pool. Colonies with no red coloration on the reverse side after 7
days incubation at 34.degree. C. were reisolated on the screening
medium and incubated for 7 days at 34.degree. C. Ten colonies
originating from 6 different pools exhibited non-red reverse
coloration and were subsequently inoculated onto Cove-N slants made
as follows: 20 ml of Cove salt solution, 4.2 g of NaNO.sub.3, and
60 g of glucose were dissolved in deionised water and the volume
made up to 1000 ml. The medium was solidified by 2% Difco Bacto
Agar.
[0648] Five ml of a spore suspension, made from a Cove slant by
adding 10 ml of aqueous 0.01% Tween 80, was inoculated into 500 ml
baffled shake flasks containing 100 ml of MDU1B shake flask medium.
At the time of inoculation, 1.3 ml of 50% sterile filtered urea was
added to each shake flask. The shake flasks were incubated at 250
rpm for 5 days at 34.degree. C.
[0649] Ten .mu.l of supernatant from the 5 day old shake flask
cultures were applied to the opposite edges of a 20 cm.times.20 cm
TLC plate (Merck Silica Gel 60). The plate was then run for 15
minutes in a chloroform:acetone:propan-2-ol (85:15:20) solvent
system (CAP) allowed to dry, turned around and the opposite side
was run in a toluene:ethyl acetate:formic acid (5:4:1) solvent
system (TEF) for 15 minutes. The plate was allowed to dry
thoroughly for 1 hour in a fume hood before spraying with Ehrlich
reagent (2 g of 4-dimethylaminobenzaldehyde in 85 ml of 96% ethanol
plus 15 ml of 37% hydrochloric acid). .alpha.-Cyclopiazonic acid
was seen as bluish-violet mushroom shaped spots with a typical low
Rf value in the CAP solvent system (a neutral system) whereas the
acidic TEF solvent system yielded a typical high Rf value prolonged
smear. Solutions of 30, 15, and 7.5 ppm of .alpha.-cyclopiazonic
acid (Sigma Chemical Co., St. Louis, Mo.) in a 1:1:1 solution of
ethanol, methanol, and chloroform were used as standards.
[0650] The TLC analysis of ten putative .alpha.-cyclopiazonic
acid-free transformants showed no sign of .alpha.-cyclopiazonic
acid. The remaining contents of the shake flasks were filtered
through Miracloth and 10 ml of 0.1 M hydrochloric acid were added
to 60 ml of each filtrate. The acidified filtrates were then
vigorously shaken for 3-5 minutes with 50 ml chloroform. The bottom
phases (approximately 25 ml) were each transferred to a 300 ml
beaker after phase separation (3 hrs) and the chloroform allowed to
evaporate. The residues were each redissolved in 300 .mu.l of
chloroform and 10 .mu.l of each concentrate was analyzed by TLC as
described above.
[0651] None of the 10 strain extracts contained any detectable
.alpha.-cyclopiazonic acid.
Deposit of Biological Materials
[0652] The following strains have been deposited according to the
Budapest Treaty in the Agricultural Research Service Patent Culture
Collection (NRRL), Northern Regional Research Laboratory, 1815
University Street, Peoria, Ill. 61604, USA.
27 Strain Accession Number Deposit Date E. coli HB101 pDSY109 NRRL
B-21623 Sep. 5, 1996 E. coli DH5.alpha. pMT1936 NRRL B-21832 Sep.
8, 1997 E. coli HB101 pDSY112 NRRL B-21622 Sep. 5, 1996 E. coli
HB101 pDSY138 NRRL B-21833 Sep. 8, 1997 E. coli DH5.alpha. pDSY162
NRRL B-21831 Sep. 8, 1997 E. coli DH5.alpha. pDSY163 NRRL B-21830
Sep. 8, 1997 E. coli DH5.alpha. pSMO1204 NRRL B-21820 Sep. 8, 1997
E. coli DH5.alpha. pSMOH603 NRRL B-21821 Sep. 8, 1997 E. coli HB101
p4-8.1 NRRL B-21823 Sep. 8, 1997 E. coli HB101 p7-14.1 NRRL B-21824
Sep. 8, 1997 E. coli DH5.alpha. pHB220 NRRL B-21825 Sep. 8, 1997 E.
coli DH5.alpha. pSMO717 NRRL B-21826 Sep. 8, 1997 E. coli
DH5.alpha. pSMO321 NRRL B-21827 Sep. 8, 1997 E. coli DH5.alpha.
pHowB571 NRRL B-21828 Sep. 8, 1997 E. coli DH5.alpha. pSMO810 NRRL
B-21829 Sep. 8, 1997
[0653] The strains have been deposited under conditions that assure
that access to the culture will be available during the pendency of
this patent application to one determined by the Commissioner of
Patents and Trademarks to be entitled thereto under 37 C.F.R.
.sctn.1.14 and 35 U. S. C. .sctn.122. The deposits represent a
substantially pure culture of each deposited strain. 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.
[0654] The invention described and claimed herein is not to be
limited in scope by the specific embodiments herein disclosed,
since these embodiments are intended as illustrations of several
aspects of the invention. Any equivalent embodiments 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.
[0655] All publications mentioned herein are incorporated herein by
reference for the purpose of describing and disclosing the
particular information for which the publication was cited. The
publications discussed above are provided solely for their
disclosure prior to the filing date of the present application.
Nothing herein is to be construed as an admission that the
inventors are not entitled to antedate such disclosure by virtue of
prior invention.
[0656] It is to be understood that this invention is not limited to
the particular methods and compositions described as such may, of
course, vary. It is also to be understood that the terminology used
herein is for the purpose of describing particular embodiments
only, and is not intended to be limiting since the scope of the
present invention will be limited only by the appended claims.
[0657] Unless defined otherwise all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any materials or methods similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, the preferred methods and materials are described.
Sequence CWU 0
0
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