U.S. patent application number 17/180430 was filed with the patent office on 2021-11-18 for yeast promoters from pichia pastoris.
The applicant listed for this patent is KECK GRADUATE INSTITUTE OF APPLIED LIFE SCIENCES. Invention is credited to James M. Cregg, Ilya I. TOLSTORUKOV.
Application Number | 20210355498 17/180430 |
Document ID | / |
Family ID | 1000005740538 |
Filed Date | 2021-11-18 |
United States Patent
Application |
20210355498 |
Kind Code |
A1 |
TOLSTORUKOV; Ilya I. ; et
al. |
November 18, 2021 |
YEAST PROMOTERS FROM PICHIA PASTORIS
Abstract
In accordance with the invention, isolated nucleic acids,
expression methods, host cells, expression vectors, and DNA
constructs for producing proteins, and proteins produced using the
expression methods are described. More particularly, nucleic acids
isolated from Pichia pastoris wherein the nucleic acids have
promoter activity are described. The invention also relates to
expression methods, host cells, expression vectors, and DNA
constructs, for using the Pichia pastoris promoters to produce
proteins, and to the proteins produced using the expression
methods.
Inventors: |
TOLSTORUKOV; Ilya I.;
(Claremont, CA) ; Cregg; James M.; (Claremont,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
KECK GRADUATE INSTITUTE OF APPLIED LIFE SCIENCES |
Claremont |
CA |
US |
|
|
Family ID: |
1000005740538 |
Appl. No.: |
17/180430 |
Filed: |
February 19, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16804305 |
Feb 28, 2020 |
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17180430 |
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14773474 |
Sep 8, 2015 |
10619164 |
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PCT/US14/22135 |
Mar 7, 2014 |
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16804305 |
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61774982 |
Mar 8, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 15/815 20130101;
C12N 15/81 20130101 |
International
Class: |
C12N 15/81 20060101
C12N015/81 |
Claims
1-45. (canceled)
46. An isolated nucleic acid comprising the sequence of a
constitutive Pichia pastoris promoter, wherein the isolated nucleic
acid is operably linked to a heterologous coding sequence, wherein
the sequence of the isolated nucleic acid comprises a sequence at
least 95% identical to a fragment of SEQ ID NO:2 wherein the
fragment comprises at least 222 nucleotides upstream from the 3'
end of SEQ ID NO:2, wherein the fragment is a continuous fragment
of SEQ ID NO:2, and wherein the fragment comprises a TATA box
sequence to direct initiation of transcription.
47. The isolated nucleic acid of claim 46 wherein the sequence of
the isolated nucleic acid is at least 98% identical to the fragment
of SEQ ID NO:2.
48. The isolated nucleic acid of claim 46 wherein the sequence of
the isolated nucleic acid is the fragment of SEQ ID NO:2.
49. The isolated nucleic acid of claim 46 wherein the heterologous
coding sequence encodes a protein selected from the group
consisting of a toxin, an antibody, a hormone, an enzyme, a growth
factor, a cytokine, a structural protein, an immunogenic protein,
and a cell signaling protein.
50. The isolated nucleic acid of claim 46 wherein the heterologous
coding sequence encodes an enzyme for use in animal feed.
51. The isolated nucleic acid of claim 50 wherein the enzyme is
selected from the group consisting of a mannanase, an amylase, a
glucanase, a protease, a phytase, a galactosidase, a cellulase, and
a xylanase.
52. The isolated nucleic acid of claim 51 wherein the protein is a
phytase.
53. The isolated nucleic acid of claim 51 wherein the protein is a
galactosidase.
54. An expression vector comprising the isolated nucleic acid of
claim 46.
55. A host cell comprising the expression vector of claim 54.
56. A host cell comprising the isolated nucleic acid of claim
46.
57. The host cell of claim 56 wherein the host cell is a Pichia
species.
58. The host cell of claim 57 wherein the Pichia species is Pichia
pastoris.
59. A DNA construct comprising the isolated nucleic acid of claim
46.
60. A method of producing a protein, the method comprising the step
of culturing in a culture medium a host cell comprising a first
expression cassette comprising the isolated nucleic acid of claim
46 wherein the heterologous coding sequence encodes a protein, and
wherein the culturing is done under conditions permitting
expression of the protein.
61. The method of claim 60 wherein the protein is selected from the
group consisting of a toxin, an antibody, a hormone, an enzyme, a
growth factor, a cytokine, a structural protein, an immunogenic
protein, and a cell signaling protein.
62. The method of claim 60 wherein the protein is an enzyme for use
in animal feed.
63. The method of claim 62 wherein the enzyme is selected from the
group consisting of a mannanase, an amylase, a glucanase, a
protease, a phytase, a galactosidase, a cellulase, and a
xylanase.
64. The method of claim 63 wherein the enzyme is a phytase.
65. The method of claim 63 wherein the enzyme is a galactosidase
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 16/804,305, filed Feb. 28, 2020, which is a continuation of
U.S. application Ser. No. 14/773,474, filed Sep. 8, 2015, which is
a U.S. national application filed under 37 C.F.R. 371(b) of
International Application Serial No. PCT/US2014/022135, filed Mar.
7, 2014, which claims the benefit of U.S. Provisional Appl. Ser.
No. 61/774,982, filed Mar. 8, 2013, each of which are incorporated
herein by reference.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted in ASCII format via EFS-Web and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Dec. 10, 2015, is named 62472-242094_SL.txt and is 8,640 bytes
in size.
FIELD OF THE DISCLOSURE
[0003] The present invention is related to isolated nucleic acids,
expression methods, host cells, expression vectors, and DNA
constructs for producing proteins, and to the proteins produced
using the expression methods. More particularly, the invention
relates to nucleic acids isolated from Pichia pastoris wherein the
nucleic acids have promoter activity. The invention also relates to
expression methods, host cells, expression vectors, and DNA
constructs, for using the Pichia pastoris promoters to produce
proteins, and to the proteins produced using the expression
methods.
BACKGROUND AND SUMMARY OF THE INVENTION
[0004] Yeast expression systems can be used to effectively produce
proteins, such as enzymes, hormones, and vaccine proteins, in part,
because some yeast grow rapidly to high cell densities, are grown
in simple and inexpensive media, and are eukaryotes so they can
modify proteins in a manner similar to native proteins in mammals.
Additionally, with a proper signal sequence, the expressed protein
can be secreted into the culture medium for convenient isolation
and purification. Some yeast expression systems are also accepted
in the food and pharmaceutical industries as being safe for the
production of pharmaceuticals and food products, unlike fungal and
bacterial expression systems which may in some cases be unsafe, for
example, for human food manufacturing.
[0005] Thus, it is beneficial for a variety of industries, such as
the food and animal feed industries, the human and animal health
industries, and the like, to develop or improve yeast expression
systems that can be used to express high levels of proteins to
increase yield, reduce the expense of isolation and purification of
proteins, and reduce the costs of human and animal health products
and food products.
[0006] A variety of types of yeast expression systems have been
developed involving either the use of inducible or constitutive
expression of proteins using nucleic acids encoding homologous or
heterologous proteins, under the control of a yeast promoter.
Promoters are regulatory elements that are linked to the 5' end of
a nucleic acid encoding a protein, and may interact with various
regulatory factors in the host cell (e.g., a yeast host cell) to
control transcription of RNA from DNA. Promoters may also control
the timing of transcription of RNA from DNA. For example, the AOX 1
promoter has been identified in the yeast Pichia pastoris, and is
commonly used in yeast expression systems because it is a tightly
regulated, strong promoter.
[0007] Due to the importance of yeast expression systems for a
variety of industries, including the human pharmaceuticals
industry, and the human food and animal feed industries, the
improvement of yeast expression systems is the focus of much
research and development. Accordingly, the present inventors have
identified promoters from Pichia pastoris that are particularly
effective for use in expression of proteins in yeast. The promoters
described herein can be used, for example, in systems for the
constitutive expression of proteins.
[0008] In one illustrative embodiment of the invention, an isolated
nucleic acid is provided wherein the sequence of the isolated
nucleic acid comprises a sequence, for example, at least 90%, 95%,
or 98% identical to a sequence selected from the group consisting
of SEQ ID NO:1 and SEQ ID NO:2 as described herein, or at least
90%, 95%, or 98% identical to a fragment thereof, wherein the
isolated nucleic acid comprises the sequence of a constitutive
Pichia pastoris promoter. In other embodiments, expression vectors,
host cells, and DNA constructs comprising these promoter sequences
are provided.
[0009] In another embodiment, a method of producing a protein using
these promoter sequences is provided. The method comprises the
steps of culturing in a culture medium a host cell comprising a
first expression cassette comprising any of the above promoter
sequences operably linked to a heterologous coding sequence
encoding a protein, wherein the culturing is done under conditions
permitting expression of the protein. In another illustrative
embodiment, an isolated protein produced according to this method
is provided.
[0010] All of the embodiments described in the following clause
list are also contemplated for use in accordance with the
invention. For all of the embodiments described in the following
clauses, any applicable combination of embodiments is considered to
be in accordance with the invention.
[0011] 1. An isolated nucleic acid wherein the sequence of the
isolated nucleic acid comprises a sequence at least 90% identical
to a sequence selected from the group consisting of SEQ ID NO:1 and
SEQ ID NO:2, or at least 90% identical to a fragment thereof,
wherein the isolated nucleic acid comprises the sequence of a
constitutive Pichia pastoris promoter.
[0012] 2. The isolated nucleic acid of clause 1 wherein the
sequence of the isolated nucleic acid is at least 95% identical to
a sequence selected from the group consisting of SEQ ID NO:1 and
SEQ ID NO:2, or at least 95% identical to a fragment thereof.
[0013] 3. The isolated nucleic acid of clause 1 wherein the
sequence of the isolated nucleic acid is at least 98% identical to
a sequence selected from the group consisting of SEQ ID NO:1 and
SEQ ID NO:2, or at least 98% identical to a fragment thereof.
[0014] 4. The isolated nucleic acid sequence of clause 1 wherein
the sequence of the isolated nucleic acid is a sequence selected
from the group consisting of SEQ ID NO:1 and SEQ ID NO:2, or a
fragment thereof.
[0015] 5. The isolated nucleic acid of any one of clauses 1 to 4
operably linked to a heterologous coding sequence.
[0016] 6. The isolated nucleic acid of clause 5 wherein the
heterologous coding sequence encodes a protein selected from the
group consisting of a toxin, an antibody, a hormone, an enzyme, a
growth factor, a cytokine, a structural protein, an immunogenic
protein, and a cell signaling protein.
[0017] 7. The isolated nucleic acid of clause 6 wherein the protein
is an enzyme for use in animal feed.
[0018] 8. The isolated nucleic acid of clause 7 wherein the protein
is selected from the group consisting of a phytase, a mannanase, a
galactosidase, an amylase, a glucanase, a protease, a cellulase,
and a xylanase.
[0019] 9. The isolated nucleic acid of clause 8 wherein the protein
is a phytase.
[0020] 10. The isolated nucleic acid of clause 8 wherein the
protein is a galactosidase.
[0021] 11. An expression vector comprising the isolated nucleic
acid of any one of clauses 1 to 10.
[0022] 12. A host cell comprising the expression vector of clause
11.
[0023] 13. A host cell comprising the isolated nucleic acid of any
one of clauses 1 to 10.
[0024] 14. The host cell of any one of clauses 12 or 13 wherein the
host cell is a Pichia species.
[0025] 15. The host cell of clause 14 wherein the Pichia species is
Pichia pastoris.
[0026] 16. A DNA construct comprising the isolated nucleic acid of
any one of clauses 1 to 10.
[0027] 17. A method of producing a protein, the method comprising
the step of [0028] culturing in a culture medium a host cell
comprising a first expression cassette comprising the isolated
nucleic acid of any one of clauses 1 to 4 operably linked to a
heterologous coding sequence encoding a protein, wherein the
culturing is done under conditions permitting expression of the
protein.
[0029] 18. The method of clause 17 wherein the protein is selected
from the group consisting of a toxin, an antibody, a hormone, an
enzyme, a growth factor, a cytokine, a structural protein, an
immunogenic protein, and a cell signaling protein.
[0030] 19. The method of clause 18 wherein the protein is an enzyme
for use in animal feed.
[0031] 20. The method of clause 19 wherein the protein is selected
from the group consisting of a phytase, a mannanase, a
galactosidase, an amylase, a glucanase, a cellulase, a protease,
and a xylanase.
[0032] 21. The method of clause 20 wherein the protein is a
phytase.
[0033] 22. The method of clause 20 wherein the protein is a
galactosidase.
[0034] 23. The method of any one of clauses 17 to 22 wherein the
protein is expressed using the first expression cassette in
combination with a second expression cassette.
[0035] 24. The method of clause 23 wherein the second expression
cassette comprises the heterologous coding sequence encoding the
protein operably linked to an isolated nucleic acid having a
sequence comprising the sequence of SEQ ID NO:3 or SEQ ID NO:4
wherein SEQ ID NO:3 and SEQ ID NO:4 have promoter activity, or any
other AOX 1 or AOX 2 promoter sequence.
[0036] 25. The method of clause 24 wherein the protein is expressed
using the first expression cassette, the second expression
cassette, and a third expression cassette.
[0037] 26. The method of clause 25 wherein the third expression
cassette comprises the heterologous coding sequence encoding the
protein operably linked to an isolated nucleic acid having a
sequence at least 90% identical to a sequence selected from the
group consisting of SEQ ID NO:1 and SEQ ID NO:2, or at least 90%
identical to a fragment thereof.
[0038] 27. The method of clause 23 wherein the second expression
cassette comprises the heterologous coding sequence encoding the
protein operably linked to an isolated nucleic acid having a
sequence at least 90% identical to a sequence selected from the
group consisting of SEQ ID NO:1 and SEQ ID NO:2, or at least 90%
identical to a fragment thereof.
[0039] 28. An isolated protein produced according to the method of
any one of clauses 17 to 27.
[0040] 29. The host cell of any one of clauses 12 or 13 wherein the
host cell is a methylotrophic yeast.
[0041] 30. The host cell of clause 29 wherein the host cell is
selected from the group consisting of Hansenula species, Pichia
species, and Candida species.
[0042] 31. A host cell comprising the DNA construct of clause 16
wherein the host cell is a methylotrophic yeast.
[0043] 32. The host cell of clause 31 selected from the group
consisting of Hansenula species, Pichia species, and Candida
species.
[0044] 33. The method of any one of clauses 17 to 27 wherein the
host cell is a methylotrophic yeast.
[0045] 34. The method of clause 33 wherein the host cell is
selected from the group consisting of Hansenula species, Pichia
species, and Candida species.
[0046] 35. The method of clause 25 wherein the third expression
cassette comprises the heterologous coding sequence encoding the
protein operably linked to an isolated nucleic acid having a
sequence of SEQ ID NO:3 or SEQ ID NO:4 wherein SEQ ID NO:3 and SEQ
ID NO:4 have promoter activity, or any other AOX 1 or AOX2 promoter
sequence.
[0047] 36. A method of producing one or more proteins, the method
comprising the step of [0048] culturing in a culture medium a host
cell comprising a first expression cassette, a second expression
cassette, and one or more additional expression cassettes, wherein
each of the one or more additional expression cassettes comprises
the isolated nucleic acid of any one of clauses 1 to 4 operably
linked to a heterologous coding sequence encoding the one or more
proteins, wherein the culturing is done under conditions permitting
expression of the one or more proteins.
[0049] 37. The method of clause 17 further comprising the step of
purifying the protein from the medium of the cultured host
cell.
[0050] 38. The method of clause 36 further comprising the step of
purifying one or more of the one or more proteins from the medium
of the cultured host cell.
[0051] 39. The isolated nucleic acid, host cell, expression vector,
isolated protein, DNA construct, or method of any one of clauses 1
to 38 wherein the isolated nucleic acid consists of any one of SEQ
ID NOS. 1 to 2, or a fragment thereof.
[0052] 40. An isolated nucleic acid consisting of any one of SEQ ID
NOS. 1 to 2, or a fragment thereof.
[0053] 41. The host cell of clause 12 or 13 wherein the host cell
is selected from the group consisting of Hansenula species, Pichia
species, Saccharomyces species, Schizosaccharomyces species,
Torulaspora species, a Candida species, a Yarrowia species, and
Kluveromyces species.
[0054] 42. The isolated nucleic acid of clause 5 wherein the
heterologous coding sequence encodes a protein or polypeptide
selected from the group consisting of a toxin, an antibody, a
hormone, an enzyme, a growth factor, a cytokine, a structural
protein, an immunogenic protein, and a cell signaling protein.
[0055] 43. A method of producing a protein or a polypeptide, the
method comprising the step of [0056] culturing in a culture medium
a host cell comprising a first expression cassette comprising the
isolated nucleic acid of any one of clauses 1 to 4 operably linked
to a heterologous coding sequence encoding a protein, wherein the
culturing is done under conditions permitting expression of the
protein.
[0057] 44. The method of clause 23 wherein the second expression
cassette comprises the heterologous coding sequence encoding the
protein operably linked to an isolated nucleic acid having a
sequence comprising the sequence of SEQ ID NO:3 or SEQ ID NO:4
wherein SEQ ID NO:3 and SEQ ID NO:4 have promoter activity, or any
other promoter sequence.
[0058] 45. The method of clause 25 wherein the third expression
cassette comprises the heterologous coding sequence encoding the
protein operably linked to an isolated nucleic acid having a
sequence of SEQ ID NO:3 or SEQ ID NO:4 wherein SEQ ID NO:3 and SEQ
ID NO:4 have promoter activity, or any other promoter sequence.
BRIEF DESCRIPTION OF THE DRAWINGS
[0059] FIG. 1 shows the nucleotide sequence of the Chr1-0469
promoter (SEQ ID NO: 1).
[0060] FIG. 2 shows the nucleotide sequence of the UPP promoter
(SEQ ID NO: 2).
[0061] FIG. 3 shows the nucleotide sequence of the AOX1 promoter
sequence (SEQ ID NO:3).
[0062] FIG. 4 shows the genomic sequence 1000 bp upstream of the
AOX1 promoter ATG start codon (SEQ ID NO:4).
[0063] FIG. 5 shows the level of GFP expression for various
transformants in replica plates.
[0064] FIG. 6 shows the relative promoter activity for pUPP, TEF2,
Chr1-0469, GAP, AOX1, and DAS promoters under various
conditions.
[0065] FIG. 7 shows the map of a reporter vector. Similar vectors
were used with both the alpha and beta-Galactosidase ORF, as well
as with, or without a secretion signal (alpha mating factor)
[0066] FIG. 8 shows the nucleotide sequence of forward primers (SEQ
ID NOS: 5-13, respectively, in order of appearance) used for
isolation of the Chr1-0469 promoter (SEQ ID NO: 1) and the UPP
promoter (SEQ ID NO: 2) from Pichia pastoris genomic DNA by PCR.
The corresponding DNA fragments--products of these PCR
reactions--are introduced into the reporter vector with the
alpha-Galactosidase ORF (FIG. 7).
[0067] FIG. 9 (panels A-C) show the level of alpha-Galactosidase
expression from isolated transformants with the indicated
promoter-reporter constructs.
[0068] FIG. 10 shows the activity of the E. coli appA2 phytase in
Pichia pastoris.
DETAILED DESCRIPTION OF THE ILLUSTRATIVE EMBODIMENTS
[0069] In one illustrative embodiment of the invention, an isolated
nucleic acid is provided wherein the sequence of the isolated
nucleic acid comprises a sequence, for example, at least 90%, 95%,
or 98% identical to a sequence selected from the group consisting
of SEQ ID NO:1 and SEQ ID NO:2 as described herein, or at least
90%, 95%, or 98% identical to a fragment thereof, wherein the
nucleic acid comprises the sequence of a constitutive Pichia
pastoris promoter. In another embodiment, the isolated nucleic acid
sequence is a sequence selected from the group consisting of SEQ ID
NO:1 and SEQ ID NO:2, or a fragment thereof. In other embodiments,
expression vectors, host cells, and DNA constructs comprising these
promoter sequences are provided.
[0070] The above-described promoters have been isolated from a
yeast strain (i.e., NRRL Y11430) currently classified as a Pichia
pastoris yeast strain. However, the classification may change at
some point to a Komagataella species (e.g., Komagataella
phaffii).
[0071] In another embodiment, a method of producing a protein using
these promoter sequences is provided. The method comprises the
steps of culturing in a culture medium a host cell comprising a
first expression cassette comprising any of the above promoter
sequences operably linked to a heterologous coding sequence
encoding a protein, wherein the culturing is done under conditions
permitting expression of the protein. In another illustrative
embodiment, an isolated protein produced according to this method
is provided.
[0072] All of the embodiments described in the following clause
list are contemplated for use in accordance with the invention. For
all of the embodiments described in the following clauses, any
applicable combination of embodiments is considered to be in
accordance with the invention. Any embodiment described in the
following clause list is also contemplated for use with any
embodiment described in the Summary of Invention section of this
application or in the Detailed Description of the Illustrative
Embodiments section of this application.
[0073] 1. An isolated nucleic acid wherein the sequence of the
isolated nucleic acid comprises a sequence at least 90% identical
to a sequence selected from the group consisting of SEQ ID NO:1 and
SEQ ID NO:2, or at least 90% identical to a fragment thereof,
wherein the isolated nucleic acid comprises the sequence of a
constitutive Pichia pastoris promoter.
[0074] 2. The isolated nucleic acid of clause 1 wherein the
sequence of the isolated nucleic acid is at least 95% identical to
a sequence selected from the group consisting of SEQ ID NO:1 and
SEQ ID NO:2, or at least 95% identical to a fragment thereof.
[0075] 3. The isolated nucleic acid of clause 1 wherein the
sequence of the isolated nucleic acid is at least 98% identical to
a sequence selected from the group consisting of SEQ ID NO:1 and
SEQ ID NO:2, or at least 98% identical to a fragment thereof.
[0076] 4. The isolated nucleic acid sequence of clause 1 wherein
the sequence of the isolated nucleic acid is a sequence selected
from the group consisting of SEQ ID NO:1 and SEQ ID NO:2, or a
fragment thereof.
[0077] 5. The isolated nucleic acid of any one of clauses 1 to 4
operably linked to a heterologous coding sequence.
[0078] 6. The isolated nucleic acid of clause 5 wherein the
heterologous coding sequence encodes a protein selected from the
group consisting of a toxin, an antibody, a hormone, an enzyme, a
growth factor, a cytokine, a structural protein, an immunogenic
protein, and a cell signaling protein.
[0079] 7. The isolated nucleic acid of clause 6 wherein the protein
is an enzyme for use in animal feed.
[0080] 8. The isolated nucleic acid of clause 7 wherein the protein
is selected from the group consisting of a phytase, a mannanase, a
galactosidase, an amylase, a glucanase, a protease, a cellulase,
and a xylanase.
[0081] 9. The isolated nucleic acid of clause 8 wherein the protein
is a phytase.
[0082] 10. The isolated nucleic acid of clause 8 wherein the
protein is a galactosidase.
[0083] 11. An expression vector comprising the isolated nucleic
acid of any one of clauses 1 to 10.
[0084] 12. A host cell comprising the expression vector of clause
11.
[0085] 13. A host cell comprising the isolated nucleic acid of any
one of clauses 1 to 10.
[0086] 14. The host cell of any one of clauses 12 or 13 wherein the
host cell is a Pichia species.
[0087] 15. The host cell of clause 14 wherein the Pichia species is
Pichia pastoris.
[0088] 16. A DNA construct comprising the isolated nucleic acid of
any one of clauses 1 to 10.
[0089] 17. A method of producing a protein, the method comprising
the step of [0090] culturing in a culture medium a host cell
comprising a first expression cassette comprising the isolated
nucleic acid of any one of clauses 1 to 4 operably linked to a
heterologous coding sequence encoding a protein, wherein the
culturing is done under conditions permitting expression of the
protein.
[0091] 18. The method of clause 17 wherein the protein is selected
from the group consisting of a toxin, an antibody, a hormone, an
enzyme, a growth factor, a cytokine, a structural protein, an
immunogenic protein, and a cell signaling protein.
[0092] 19. The method of clause 18 wherein the protein is an enzyme
for use in animal feed.
[0093] 20. The method of clause 19 wherein the protein is selected
from the group consisting of a phytase, a mannanase, a
galactosidase, an amylase, a glucanase, a cellulase, a protease,
and a xylanase.
[0094] 21. The method of clause 20 wherein the protein is a
phytase.
[0095] 22. The method of clause 20 wherein the protein is a
galactosidase.
[0096] 23. The method of any one of clauses 17 to 22 wherein the
protein is expressed using the first expression cassette in
combination with a second expression cassette.
[0097] 24. The method of clause 23 wherein the second expression
cassette comprises the heterologous coding sequence encoding the
protein operably linked to an isolated nucleic acid having a
sequence comprising the sequence of SEQ ID NO:3 or SEQ ID NO:4
wherein SEQ ID NO:3 and SEQ ID NO:4 have promoter activity, or any
other AOX 1 or AOX 2 promoter sequence.
[0098] 25. The method of clause 24 wherein the protein is expressed
using the first expression cassette, the second expression
cassette, and a third expression cassette.
[0099] 26. The method of clause 25 wherein the third expression
cassette comprises the heterologous coding sequence encoding the
protein operably linked to an isolated nucleic acid having a
sequence at least 90% identical to a sequence selected from the
group consisting of SEQ ID NO:1 and SEQ ID NO:2, or at least 90%
identical to a fragment thereof.
[0100] 27. The method of clause 23 wherein the second expression
cassette comprises the heterologous coding sequence encoding the
protein operably linked to an isolated nucleic acid having a
sequence at least 90% identical to a sequence selected from the
group consisting of SEQ ID NO:1 and SEQ ID NO:2, or at least 90%
identical to a fragment thereof.
[0101] 28. An isolated protein produced according to the method of
any one of clauses 17 to 27.
[0102] 29. The host cell of any one of clauses 12 or 13 wherein the
host cell is a methylotrophic yeast.
[0103] 30. The host cell of clause 29 wherein the host cell is
selected from the group consisting of Hansenula species, Pichia
species, and Candida species.
[0104] 31. A host cell comprising the DNA construct of clause 16
wherein the host cell is a methylotrophic yeast.
[0105] 32. The host cell of clause 31 selected from the group
consisting of Hansenula species, Pichia species, and Candida
species.
[0106] 33. The method of any one of clauses 17 to 27 wherein the
host cell is a methylotrophic yeast.
[0107] 34. The method of clause 33 wherein the host cell is
selected from the group consisting of Hansenula species, Pichia
species, and Candida species.
[0108] 35. The method of clause 25 wherein the third expression
cassette comprises the heterologous coding sequence encoding the
protein operably linked to an isolated nucleic acid having a
sequence of SEQ ID NO:3 or SEQ ID NO:4 wherein SEQ ID NO:3 and SEQ
ID NO:4 have promoter activity, or any other AOX 1 or AOX 2
promoter sequence.
[0109] 36. A method of producing one or more proteins, the method
comprising the step of [0110] culturing in a culture medium a host
cell comprising a first expression cassette, a second expression
cassette, and one or more additional expression cassettes, wherein
each of the one or more additional expression cassettes comprises
the isolated nucleic acid of any one of clauses 1 to 4 operably
linked to a heterologous coding sequence encoding the one or more
proteins, wherein the culturing is done under conditions permitting
expression of the one or more proteins.
[0111] 37. The method of clause 17 further comprising the step of
purifying the protein from the medium of the cultured host
cell.
[0112] 38. The method of clause 36 further comprising the step of
purifying one or more of the one or more proteins from the medium
of the cultured host cell.
[0113] 39. The isolated nucleic acid, host cell, expression vector,
isolated protein, DNA construct, or method of any one of clauses 1
to 38 wherein the isolated nucleic acid consists of any one of SEQ
ID NOS. 1 to 2, or a fragment thereof.
[0114] 40. An isolated nucleic acid consisting of any one of SEQ ID
NOS. 1 to 2, or a fragment thereof.
[0115] 41. The host cell of clause 12 or 13 wherein the host cell
is selected from the group consisting of Hansenula species, Pichia
species, Saccharomyces species, Schizosaccharomyces species,
Torulaspora species, a Candida species, a Yarrowia species, and
Kluveromyces species.
[0116] 42. The isolated nucleic acid of clause 5 wherein the
heterologous coding sequence encodes a protein or polypeptide
selected from the group consisting of a toxin, an antibody, a
hormone, an enzyme, a growth factor, a cytokine, a structural
protein, an immunogenic protein, and a cell signaling protein.
[0117] 43. A method of producing a protein or a polypeptide, the
method comprising the step of [0118] culturing in a culture medium
a host cell comprising a first expression cassette comprising the
isolated nucleic acid of any one of clauses 1 to 4 operably linked
to a heterologous coding sequence encoding a protein, wherein the
culturing is done under conditions permitting expression of the
protein.
[0119] 44. The method of clause 23 wherein the second expression
cassette comprises the heterologous coding sequence encoding the
protein operably linked to an isolated nucleic acid having a
sequence comprising the sequence of SEQ ID NO:3 or SEQ ID NO:4
wherein SEQ ID NO:3 and SEQ ID NO:4 have promoter activity, or any
other promoter sequence.
[0120] 45. The method of clause 25 wherein the third expression
cassette comprises the heterologous coding sequence encoding the
protein operably linked to an isolated nucleic acid having a
sequence of SEQ ID NO:3 or SEQ ID NO:4 wherein SEQ ID NO:3 and SEQ
ID NO:4 have promoter activity, or any other promoter sequence.
[0121] The phrase "consists of" or "consisting of" means that the
sequence specified by the SEQ ID NO. has no additional nucleotide
sequences other than those corresponding to the SEQ ID NO.
[0122] Any yeast expression system known to those skilled in the
art can be used in accordance with the present invention. For
example, various yeast expression systems are described in U.S.
Pat. Nos. 6,451,572, 6,841,370, 6,974,690, 7,320,876, 7,078,035,
7,138,260, and PCT Publication No. WO 2007/112739, all incorporated
herein by reference. In any of the embodiments described herein,
any of these yeast expression systems can be used. Alternatively,
any yeast species or yeast expression system suitable for
expression of a protein can be used including yeast species, such
as Saccharomyces species (e.g., Saccharomyces cerevisiae),
Kluyveromyces species (e.g., Kluyveromyces lactis), Torulaspora
species, Yarrowia species (e.g., Yarrowia lipolitica),
Schizosaccharomyces species (e.g., Schizosaccharomyces pombe). In
another embodiment, methylotrophic yeast species such as Pichia
species (e.g., Pichia pastoris or Pichia methanolica), Hansenula
species (e.g., Hansenula polymorpha), Torulopsis species,
Komagataella species, Candida species (e.g., Candida boidinii), and
Karwinskia species can be used. In one embodiment the protein can
be expressed in the methylotrophic yeast Pichia pastoris.
Methylotrophic yeast are capable of utilizing methanol as a sole
carbon source for the production of the energy resources necessary
to maintain cellular function. Methylotrophic yeast contain genes
encoding enzymes for methanol utilization such as the genes
encoding alcohol oxidase. Any of these host cells can be a host
cell strain that is heterologous to the promoter described herein
(i.e., the host cell does not normally contain in nature the
promoter described herein).
[0123] A yeast expression system can be used to produce a
sufficient amount of the protein intracellularly, or secreted from
the yeast cells so that the protein can be conveniently isolated
and purified from the culture medium. As used herein, the term
"expression" means transcription and/or translation of a nucleic
acid in a host cell. A yeast expression system may include, but is
not limited to, the yeast host cell and the expression vector
(e.g., a DNA construct) used to express the protein. The expression
vector can contain a promoter described herein and, as is known in
the art, the promoter is heterologous to the expression vector
(i.e., the combination does not occur in nature). In one
embodiment, secretion of the protein into the culture medium is
controlled by a signal peptide (e.g., the yeast .alpha.-factor
signal peptide, the yeast KILM1 signal peptide, the yeast PHO1
signal peptide, or the yeast SUC2 signal peptide) incorporated into
the expression vector and which is capable of directing the
secretion of the expressed protein out of the yeast cell. In other
embodiments, other signal peptides suitable for facilitating
secretion of the protein from yeast cells are known to those
skilled in the art. In one aspect, the signal peptide is typically
cleaved from the protein after secretion.
[0124] In various embodiments, any expression vector known to the
skilled artisan (e.g., a vector that replicates autonomously or
integrates into the host genome) and compatible with a yeast
expression system can be used. As used herein, the term "vector"
means any plasmid, or other vector, in double-stranded or
single-stranded form or in linear or circular form that can
transform a yeast cell by integration into the yeast cell genome or
by existing extrachromosomally (e.g., an autonomously replicating
plasmid). As is known in the art, a vector (e.g., expression vector
or expression cassette) is a nucleic acid construct used to
transform a host cell for expression of a protein, polypeptide, or
peptide and the vector is not found in nature in the host cell it
transforms.
[0125] In one embodiment, the expression vector has restriction
endonuclease cleavage sites for the insertion of DNA fragments
(e.g., one or more cloning sites and/or a multiple cloning site),
and genetic markers for selection of transformants. For example,
the genetic markers for selection of transformants can include a
selection marker that allows a transformed yeast to grow on a
medium devoid of a necessary nutrient that cannot be produced by a
deficient strain, a selection marker that encodes an enzyme for
which chromogenic substrates are known, or a selection marker that
provides resistance to a drug, including, but not limited to, G418,
Nourseothricin (Nat), Zeocin, Blasticidin, or Hygromycin. In
another embodiment, the expression vector has a terminator sequence
for transcription termination (e.g., the AOX 1 or HSP150
terminator). In another embodiment, the expression vector has a 3'
untranslated region downstream from the protein coding sequence
with a polyadenylation site. As used herein, "3' untranslated
region" means nucleotide sequences that are not translated into
protein and are located downstream from a coding sequence for a
protein. Typically, a 3' untranslated region includes regulatory
sequences for mRNA processing. In another embodiment, the
expression vector has an origin of replication (e.g., a bacterial
origin of replication) for use in synthesizing and amplifying the
vector, for example, in a bacterial host. Various expression
vectors are described in U.S. Pat. Nos. 6,451,572, 6,841,370,
6,974,690, 7,320,876, 7,078,035, 7,138,260, and PCT Publication No.
WO 2007/112739, all incorporated herein by reference. The
construction and use of expression vectors is described in Sambrook
et al., "Molecular Cloning: A Laboratory Manual", 3rd Edition, Cold
Spring Harbor Laboratory Press, (2001), incorporated herein by
reference. In another embodiment, the expression vector, or a
fragment thereof, can be synthesized de novo or PCR can be used to
amplify and join sections of expression vectors.
[0126] As used herein, "regulatory sequences" means nucleotide
sequences that are typically upstream or downstream from the 5' or
3' end, respectively, of a protein coding sequence. Regulatory
sequences are not translated into protein. Regulatory sequences
include, but are not limited to, sequences that affect RNA
processing or stability, such as polyadenylation signal sequences,
enhancers, repressor binding sites, and promoters.
[0127] In one embodiment, the protein coding sequence can be
operably linked in the expression vector to the promoter sequence
capable of directing the expression of the protein, for example, in
yeast. As used herein, "operably linked" means functionally linked.
As described herein, the promoter can be a "constitutive promoter".
A "constitutive promoter" means a promoter that regulates
expression of a gene of interest. The term "constitutive promoter"
is known in the art. A constitutive promoter can have some
inducible activity, but the maximal activity obtained with the
promoter is not inducible.
[0128] As used herein "promoter" means a nucleotide sequence
typically located upstream from the 5' end of a coding sequence for
a protein that controls the transcription of RNA from DNA, in part,
by interacting with various regulatory factors that control
transcription. In one embodiment, the promoter may be derived from
the same species of yeast as the yeast host cell used for protein
expression. In another embodiment, the promoter may be derived from
a different yeast species than the yeast host cell used for protein
expression. In one embodiment, a promoter may include a TATA box
sequence that acts as a recognition site to direct initiation of
transcription, including, but not limited to one or more
transcriptional enhancer elements. The enhancer elements may be
proximal or distal to the TATA box sequence and may be in a normal
5' to 3' orientation or may be in a 3' to 5' orientation. In
another embodiment, an enhancer element may be an enhancer element
native to the promoter sequence or it may be a heterologous
enhancer element inserted into the expression vector construct. An
"enhancer element" as used herein is a regulatory element that can
stimulate promoter activity.
[0129] In various illustrative embodiments described herein, the
promoter can be an isolated nucleic acid wherein the sequence of
the isolated nucleic acid comprises a sequence at least 80%, at
least 85%, at least 90%, at least 92%, at least 95%, at least 96%,
at least 97%, at least 98%, or at least 99% identical to a sequence
selected from the group consisting of SEQ ID NO:1 and SEQ ID NO:2,
or at least 80%, at least 85%, at least 90%, at least 92%, at least
95%, at least 96%, at least 97%, at least 98%, or at least 99%
identical to a fragment thereof, wherein the isolated nucleic acid
comprises the sequence of a constitutive Pichia pastoris promoter.
In another embodiment, the isolated nucleic acid sequence is a
sequence selected from the group consisting of SEQ ID NO:1 and SEQ
ID NO:2, or a fragment thereof wherein the isolated nucleic acid
comprises the sequence of a constitutive Pichia pastoris
promoter.
[0130] As used herein "an isolated nucleic acid" means a nucleic
acid that is substantially free of sequences that naturally flank
the nucleic acid in the genomic DNA of the organism from which the
nucleic acid is derived. For example, in various embodiments, an
isolated nucleic acid in accordance with the invention can contain
less than about 2 kb, less than about 1 kb, less than about 0.5 kb,
less than about 0.1 kb of nucleotide sequences, less than about
0.05 kb, or no nucleotide sequences that naturally flank the
nucleic acid molecule in genomic DNA of the organism from which the
isolated nucleic acid is derived.
[0131] As used herein, "a fragment thereof" when referring to the
isolated nucleic acid molecule means a fragment of the isolated
nucleic acid of SEQ ID NOS: 1 to 2. In various illustrative
embodiments, the fragment can be about 50 nucleotides in length,
about 100 nucleotides in length, about 200 nucleotides in length,
about 300 nucleotides in length, about 400 nucleotides in length,
about 500 nucleotides in length, about 600 nucleotides in length,
about 700 nucleotides in length, about 800 nucleotides in length,
or about 900 nucleotides in length. In other embodiments, the
fragment can extend about 50 nucleotides, about 100 nucleotides,
about 200 nucleotides, about 300 nucleotides, about 400
nucleotides, about 500 nucleotides, about 600 nucleotides, about
700 nucleotides, about 800 nucleotides, or about 900 nucleotides
upstream from the 3' end of the isolated nucleic acid of any of SEQ
ID NOS: 1 to 2. In yet other embodiments, the fragment can include
about 50 nucleotides, about 100 nucleotides, about 200 nucleotides,
about 300 nucleotides, or about 350 nucleotides upstream and/or
downstream from the TATA box sequence found in each of SEQ ID NOS:
1 to 2.
[0132] In various embodiments, the isolated nucleic acids described
herein may be purified by techniques for purification of nucleic
acids (e.g., DNA) that are well-known in the art. For example, the
nucleic acids may be separated from contaminants by physical
methods including, but not limited to, centrifugation, pressure
techniques, or by using a substance with affinity for nucleic acids
(e.g., DNA), such as, for example, silica beads. After sufficient
washing, the isolated nucleic acids may be suspended in either
water or a buffer. In other embodiments, commercial kits are
available, such as Qiagen.TM., Nuclisensm.TM., and Wizard.TM.
(Promega), and Promegam.TM.. Methods for purifying nucleic acids
are described in Sambrook et al., "Molecular Cloning: A Laboratory
Manual", 3rd Edition, Cold Spring Harbor Laboratory Press, (2001),
incorporated herein by reference. An isolated nucleic acid as
described herein may be an isolated nucleic acid that is also
purified, or the isolated nucleic acid may be impure. A "purified
nucleic acid" is substantially free of other cellular material, or
culture medium when produced by recombinant techniques, or
substantially free of chemical precursors or other chemicals when
chemically synthesized.
[0133] The isolated nucleic acids described herein are capable of
specific hybridization, under appropriate hybridization conditions
(e.g., appropriate buffer, ionic strength, temperature, formamide,
and MgCl.sub.2 concentrations), to a complementary nucleic acid.
The isolated nucleic acids described herein can be modified by
substitution, deletion, truncation, and/or can be fused with other
nucleic acid molecules wherein the resulting isolated nucleic acids
hybridize specifically to the complementary nucleic acids.
[0134] Also within the scope of the invention are nucleic acids
complementary to the isolated nucleic acids, or fragments thereof,
described herein, and those that hybridize to the isolated nucleic
acids described herein or those that hybridize to their complements
under highly stringent conditions. As used herein, the term
"complementary" refers to the ability of purine and pyrimidine
nucleotide sequences to associate through hydrogen bonding to form
double-stranded nucleic acid molecules. Guanine and cytosine,
adenine and thymine, and adenine and uracil are complementary and
can associate through hydrogen bonding resulting in the formation
of double-stranded nucleic acid molecules when two nucleic acid
molecules have "complementary" sequences. The complementary DNA
sequences are referred to as a "complement."
[0135] In accordance with the invention "highly stringent
conditions" means hybridization at 65.degree. C. in 5.times.SSPE
and 50% formamide, and washing at 65.degree. C. in 0.5.times.SSPE.
Conditions for high stringency hybridization are described in
Sambrook et al., "Molecular Cloning: A Laboratory Manual", 3rd
Edition, Cold Spring Harbor Laboratory Press, (2001), incorporated
herein by reference. In some illustrative aspects, hybridization
can occur along the full-length of the isolated nucleic acid, or
along part of its length, or to a fragment thereof.
[0136] Also included are isolated nucleic acid molecules having
about 60%, about 70%, about 75%, about 80%, about 85%, about 90%,
about 92%, about 95%, 96%, 97%, 98%, and 99% identity to the
isolated nucleic acids described herein, or to a fragment thereof.
Determination of percent identity or similarity between sequences
can be done, for example, by using the GAP program (Genetics
Computer Group, software; now available via Accelrys on
http://www.accelrys.com), and alignments can be done using, for
example, the ClustalW algorithm (VNTI software, InforMax Inc.). A
sequence database can be searched using the isolated nucleic acid
sequence of interest, or the sequence of a fragment thereof.
Algorithms for database searching are typically based on the BLAST
software (Altschul et al., 1990). In some embodiments, the percent
identity can be determined along the full-length of the isolated
nucleic acid.
[0137] Techniques for synthesizing the isolated nucleic acids
described herein are well-known in the art and include chemical
syntheses and recombinant methods. Such techniques are described in
Sambrook et al., "Molecular Cloning: A Laboratory Manual", 3rd
Edition, Cold Spring Harbor Laboratory Press, (2001), incorporated
herein by reference. Isolated nucleic acid molecules can also be
made commercially. Techniques for synthesizing the isolated nucleic
acids described herein are well-known in the art. The isolated
nucleic acids described herein can be analyzed by techniques known
in the art, such as restriction enzyme analysis or sequencing, to
determine the sequence of the isolated nucleic acids. Thus,
isolated nucleic acids described herein may be synthetic.
[0138] In illustrative aspects, yeast cells are transformed with an
expression vector comprising a heterologous nucleic acid encoding
the protein of interest operably linked to one of the promoters
described herein using procedures well-known to those skilled in
the art. The term "transformation" means the transfer of a nucleic
acid, or a nucleic acid fragment, into a host cell. In illustrative
embodiments, such transformation protocols include electroporation,
lithium acetate methods, and use of spheroplasts. In illustrative
aspects, the expressed nucleic acid coding sequence can be a
heterologous nucleic acid coding sequence. As used herein, a
heterologous coding sequence is defined as an artificial or
synthetic nucleic acid or a nucleic acid originating from a
different species than the species from which the promoter sequence
was derived. Thus, a heterologous coding sequence linked to a
promoter described herein does not occur in nature.
[0139] In various embodiments, the transformed yeast cells may be
grown by techniques including batch and continuous fermentation in
a liquid medium or on a semi-solid medium, or a solid medium.
Typically, "conditions permitting expression of the protein" as
used herein means conditions for batch or continuous fermentation
of yeast in a liquid medium, but growth on a semi-solid medium,
such as agar, is not excluded. Culture media for yeast cells are
known in the art and are typically supplemented with a carbon
source (e.g., glucose). A typical yeast culture medium is YPD broth
(Sunrise Science Products, Inc.) comprising yeast extract (10
grams), Bacto peptone (20 grams), and dextrose (20 grams). In one
illustrative aspect, the transformed yeast cells can be grown
aerobically at 30.degree. C. in a controlled pH environment (a pH
of about 6) and with the carbon source (e.g., glucose) maintained
continuously at a predetermined level known to support growth of
the yeast cells to a desired density within a specific period of
time.
[0140] In one illustrative embodiment, a method of producing a
protein is provided. The method comprises the step of culturing in
a culture medium a host cell comprising a first expression cassette
comprising an isolated nucleic acid of any one of SEQ ID NOS: 1 to
2, or a fragment thereof, operably linked to a heterologous coding
sequence encoding a protein, wherein the culturing is done under
conditions permitting expression of the protein. The method can
further comprise the step of purifying the protein from the medium
of the cultured host cell. As used herein, an "expression cassette"
means the elements of an expression vector that direct the yeast
cell to make RNA. An expression cassette comprises at least
regulatory sequences (e.g., a promoter) and a coding sequence for
the RNA and protein (i.e., an open reading frame, ORF). The
isolated nucleic acid can be at least 80%, at least 85%, at least
90%, 92%, 95%, 96%, 97%, 98%, or 99% homologous to the isolated
nucleic acid of any of SEQ ID NOS: 1 to 2, or a fragment thereof.
In various illustrative aspects, the protein coding sequence can be
from a bacterium, a yeast, a fungus, or a virus.
[0141] In this method embodiment, the protein can be expressed
using the first expression cassette in combination with a second
expression cassette. In another embodiment, the second expression
cassette can comprise 1) the heterologous coding sequence encoding
the protein operably linked to an isolated nucleic acid having a
sequence comprising the sequence of SEQ ID NO:3 or SEQ ID NO:4
wherein SEQ ID NO:3 and SEQ ID NO:4 have promoter activity, or any
other known methanol-regulated promoter, such as AOX 1, AOX 2, FLD,
or DAS promoter sequences, or 2) the isolated nucleic acid of any
one of SEQ ID NOS: 1 to 2, or a fragment thereof, operably linked
to the heterologous coding sequence encoding the protein.
[0142] In yet another embodiment, the protein can be expressed
using the first expression cassette, the second expression
cassette, and a third expression cassette. In another illustrative
aspect, the third expression cassette can comprise 1) the
heterologous coding sequence encoding the protein operably linked
to an isolated nucleic acid having a sequence comprising the
sequence of SEQ ID NO:3 or SEQ ID NO:4 wherein SEQ ID NO:3 and SEQ
ID NO:4 have promoter activity, or any other known
methanol-regulated promoters, such as AOX 1, AOX 2, FLD, or DAS
promoter sequences, or 2) the isolated nucleic acid of any one of
SEQ ID NOS: 1 to 2, or a fragment thereof, operably linked to the
heterologous coding sequence encoding the protein.
[0143] In another embodiment, any number of additional expression
cassettes can be used and the expression cassettes can comprise 1)
the heterologous coding sequence encoding the protein operably
linked to an isolated nucleic acid having a sequence comprising the
sequence of SEQ ID NO:3 or SEQ ID NO:4 wherein SEQ ID NO:3 and SEQ
ID NO:4 have promoter activity, or any other known
methanol-regulated promoters, such as AOX 1, AOX 2, FLD, or DAS
promoter sequences, or 2) the isolated nucleic acid of any one of
SEQ ID NOS: 1 to 2, or a fragment thereof, operably linked to the
heterologous coding sequence encoding the protein. In another
embodiment, the first expression cassette, the second expression
cassette, and the third expression cassette as described above are
used in the method, along with a fourth expression cassette, a
fifth expression cassette, and a sixth expression cassette wherein
all of the expression cassettes comprise the isolated nucleic acid
of any one of SEQ ID NOS: 1 to 2, or a fragment thereof, operably
linked to the heterologous coding sequence encoding the
protein.
[0144] In still another embodiment, a method of producing one or
more proteins is provided. The method comprises the step of
culturing in a culture medium a host cell comprising a first
expression cassette, a second expression cassette, and, optionally,
one or more additional expression cassettes, wherein each of the
expression cassettes comprises 1) a heterologous coding sequence
encoding the one or more proteins operably linked to an isolated
nucleic acid having a sequence comprising the sequence of SEQ ID
NO:3 or SEQ ID NO:4 wherein SEQ ID NO:3 and SEQ ID NO:4 have
promoter activity, or any other known methanol-regulated promoters,
such as AOX 1, AOX 2, FLD, or DAS promoter sequences, or 2) the
isolated nucleic acid of any one of SEQ ID NOS: 1 to 2, or a
fragment thereof, operably linked to a heterologous coding sequence
encoding the one or more proteins, wherein the culturing is done
under conditions permitting expression of the one or more proteins.
The method can further comprise the step of purifying one of the
one or more proteins from the medium of the cultured host cell.
[0145] In any of the embodiments described in the preceding two
paragraphs, the isolated nucleic acid can be at least 80%, at least
85%, at least 90%, 92%, 95%, 96%, 97%, 98%, or 99% homologous to
the isolated nucleic acid of any of SEQ ID NOS: 1 to 2, or a
fragment thereof. In any of the embodiments described in the
preceding two paragraphs, the expression cassettes can be included
in one expression vector or in multiple expression vectors. In any
of the embodiments described in the preceding two paragraphs, the
expression vectors into which the expression cassettes are
incorporated can be vectors that replicate autonomously or that
integrate into the host cell genome.
[0146] In various illustrative embodiments, the protein encoded by
the any of the heterologous coding sequences described herein
operably linked to the promoter can be a protein selected from the
group consisting of a toxin, an antibody, a hormone, an enzyme, a
growth factor, a cytokine, a structural protein, an immunogenic
protein (e.g., a vaccine antigen), and a cell signaling protein. In
one embodiment, the protein is an enzyme for use in animal feed. In
this embodiment, the protein can be selected from the group
consisting of a phytase, a mannanase, a galactosidase, an amylase,
a glucanase, a cellulase, a protease, and a xylanase. In another
embodiment, the protein can be an enzyme useful for glycosylation.
In another aspect, more than one protein can be expressed by using
multiple expression cassettes, each with a heterologous coding
sequence, operably linked to a promoter. However, these protein
examples are non-limiting and any protein or peptide or polypeptide
capable of being expressed in yeast can be expressed in accordance
with the isolated nucleic acids, expression vectors, host cells,
DNA constructs, methods, and isolated proteins described herein.
The enzymes described above can be from any species (e.g., fungal
species, such as a yeast species).
[0147] The yeast-expressed proteins for use in accordance with the
present invention can be produced in purified form by conventional
techniques. As used herein, "isolated protein" means a purified
protein. A purified protein is substantially free from other yeast
cell contaminants or contaminants from the culture medium. For
example, "substantially free" from other yeast cell contaminants or
contaminants from the culture medium means that the protein is at
least about 60% pure, at least about 70% pure, at least about 80%
pure, at least about 90% pure, at least about 95% pure, at least
about 98% pure, about 60% pure, about 70% pure, about 80% pure,
about 90% pure, about 95% pure, or about 98% pure (all based on dry
weight). Typically, the protein is secreted into the yeast culture
medium and is collected from the culture medium.
[0148] In one illustrative embodiment, for purification from the
culture medium the protein can, for example, be subjected to
ammonium sulfate precipitation followed by DEAE-Sepharose column
chromatography. In other embodiments, conventional techniques known
to those skilled in the art can be used such as ammonium sulfate or
ethanol precipitation, acid extraction, gel filtration, anion or
cation exchange chromatography, DEAE-Sepharose column
chromatography, hydroxylapatite chromatography, lectin
chromatography, affinity chromatography, solvent-solvent
extraction, ultrafiltration, and HPLC.
[0149] Alternatively, purification steps may not be required
because the protein may be present in such high concentrations in
the culture medium that the protein is essentially pure in the
culture medium (e.g., 70 to 80% pure). In one embodiment, the
protein is collected from the culture medium without further
purification steps by chilling the yeast culture (e.g., to about 4
.degree. C. to about 8.degree. C.) and removing the yeast cells
using such techniques as centrifugation, microfiltration, and
rotary vacuum filtration. The protein in the cell-free medium can
then be concentrated by such techniques as, for example,
ultrafiltration and tangential flow filtration.
[0150] In some embodiments where the protein is not secreted into
the culture medium, the yeast cells can be lysed, for example, by
sonication, heat, or chemical treatment, and the homogenate
centrifuged to remove cell debris. The supernatant can then be
subjected to ammonium sulfate precipitation, and additional
fractionation techniques as required, such as gel filtration, ion
exchange chromatography, DEAE-Sepharose column chromatography,
affinity chromatography, solvent-solvent extraction,
ultrafiltration, and HPLC to purify the protein. It should be
understood that the purification methods described above for
purification of proteins from the culture medium or from lysed
yeast cells are non-limiting and any purification techniques known
to those skilled in the art can be used to purify the
yeast-expressed protein if such techniques are required to obtain a
substantially pure protein.
[0151] Various formulations of the purified protein preparations
may be prepared in accordance with the invention. In some
embodiments, the proteins can be stabilized through the addition of
other proteins (e.g., gelatin and skim milk powder), chemical
agents (e.g., glycerol, polyethylene glycol, EDTA, potassium
sorbate, sodium benzoate, and reducing agents and aldehydes),
polysaccharides, monosaccharides, lipids (hydrogenated vegetable
oils), and the like. In one embodiment, proteins for addition to
food products or animal feed blends can be dried (e.g., spray
drying, drum drying, and lyophilization) and formulated as powders,
granules, pills, mineral blocks, liquids, and gels through known
processes. In one embodiment, gelling agents such as gelatin,
alginate, collagen, agar, pectin and carrageenan can be used.
[0152] In alternate embodiments, the protein expression can be for
intracellular expression, such as for enzymatic action in the yeast
in a biotransformation process, or for display on the yeast cell
surface. For such embodiments, the protein, expressed as described
herein, is not purified.
[0153] In various embodiments, the proteins described above are
selected from the group consisting of a toxin, an antibody, a
hormone, an enzyme, a growth factor, a cytokine, a structural
protein, an immunogenic protein (e.g., a vaccine antigen), and a
cell signaling protein. In another embodiment, the proteins
described above are selected from the group consisting of an
antibody, a hormone, an enzyme, a growth factor, a cytokine, a
structural protein, an immunogenic protein (e.g., a vaccine
antigen), and a cell signaling protein. In yet another embodiment
the coding sequence for a protein, a fragment thereof, a fusion
protein (e.g., a chimeric protein), or a peptide, can be used in
accordance with the invention. In another embodiment, a modified
protein can be expressed, such as a mutated protein or a protein
with non-natural amino acids.
[0154] In one embodiment, the toxins can be proteins such as, for
example, botulinum toxin or verotoxin-1, and after preparation
using the methods, isolated nucleic acids, expression vectors, host
cells, and DNA constructs described herein, the toxins can be
modified using a targeting agent so that they are directed
specifically to diseased cells. In another illustrative aspect, the
antibody can be a humanized antibody, an antibody that is not
humanized, a nanobody, or an antibody fragment, such as an Fab
fragment of an antibody or a single-chain antibody. In another
embodiment, the hormone can be, for example, a gonadotropin, an
adrenocorticotrophic hormone, a growth hormone, vasopressin,
oxytocin, somatostatin, gastrin, or leptin. In another illustrative
embodiment, the growth factor can be insulin, epidermal growth
factor, fibroblast growth factor, vascular endothelial growth
factor, erythropoietin, platelet-derived growth factor,
thrombopoietin, or a bone morphogenic protein. In one aspect, the
cytokine can be IL-2, IFN-.alpha., IFN-.gamma., or GM-CSF. In one
embodiment, the protein is not a silk toxin. In another
illustrative aspect, the vaccine proteins can be any suitable
vaccine proteins that are immunogenic in a patient or an animal,
including, but not limited to, HPV proteins (e.g., HPV 16 and HPV
18), and tetanus vaccine proteins, as examples. In another
illustrative embodiment, the enzymes can be, for example, enzymes
for animal feeds as discussed herein, acetylcholinesterase, or
cyclooxygenase, or any other useful enzyme that can be expressed in
yeast. In another embodiment, structural proteins can be expressed,
for example, netrins, actin-binding proteins, or myosin, and, in
another embodiment, cell signaling proteins such as ras proteins,
kinases, the ErbB2 protein (the Her-2 receptor) can be expressed
using the methods, isolated nucleic acids, expression vectors, host
cells, and DNA constructs described herein.
[0155] In one embodiment, the protein is an enzyme for use in
animal feed. In this embodiment, the protein can be selected from
the group consisting of a phytase, a mannanase, a galactosidase, an
amylase, a glucanase, a cellulase, a protease, and a xylanase, or a
combination thereof. For example, a variety of phytases may be
expressed according to the methods described herein. Exemplary of
phytase genes (i.e., a phytase coding sequence) that can be
expressed in accordance with the invention are phytase genes
derived from bacteria, filamentous fungi, plants, and yeast, such
as the appA (Gene Bank accession number M58708) and appA2 (Gene
Bank accession number 250016) genes derived from Escherichia coli
and the phyA and phyB genes derived from the fungus Aspergillus
niger, or any mutant of these genes that retains or has improved
myo-inositol hexakisphosphate phosphohydrolase activity (see, for
example, Rodriguez et al., Arch. of Biochem. and Biophys. 382:
105-112 (2000), incorporated herein by reference). Exemplary alpha-
and beta-galactosidase genes are from Aspergillus species (e.g.,
Aspergillus alliaceus, Aspergillus orzae, Aspergillus nidulans,
Aspergillus parasiticus, and Aspergillus niger). Substituted,
deleted, and truncated phytase genes, or a fragment thereof, can
also be expressed in accordance with the invention.
[0156] In one embodiment, the protein expressed using the methods
described herein can be used in animal feed comprising an animal
feed blend. In various embodiments, any animal feed blend known in
the art can be used such as rapeseed meal, cottonseed meal, soybean
meal, and cornmeal. Optional ingredients of the animal feed blend
include sugars and complex carbohydrates such as both water-soluble
and water-insoluble monosaccharides, disaccharides and
polysaccharides. Optional amino acid ingredients that can be added
to the feed blend are arginine, histidine, isoleucine, leucine,
lysine, methionine, phenylalanine, threonine, tryptophan, valine,
tyrosine ethyl HCl, alanine, aspartic acid, sodium glutamate,
glycine, proline, serine, cysteine ethyl HCl, and analogs, and
salts thereof. Vitamins that can be optionally added are thiamine
HCl, riboflavin, pyridoxine HCl, niacin, niacinamide, inositol,
choline chloride, calcium pantothenate, biotin, folic acid,
ascorbic acid, and vitamins A, B, K, D, E, and the like. Minerals,
protein ingredients, including protein obtained from meat meal or
fish meal, liquid or powdered egg, fish solubles, whey protein
concentrate, oils (e.g., soybean oil), cornstarch, calcium,
inorganic phosphate, copper sulfate, salt, and limestone can also
be added. Antioxidants can also be added.
[0157] In another embodiment, a kit comprising an expression vector
comprising the isolated nucleic acid of SEQ ID NO: 1 to SEQ ID NO:
2, or a fragment thereof, is provided. In one illustrative aspect,
the isolated nucleic acid, or the fragment, can be 80%, 85%, 90%,
92%, 95%, 96%, 97%, 98%, or 99% identical to the isolated nucleic
acid of SEQ ID NO: 1 to SEQ ID NO: 2, or to the fragment thereof.
In various illustrative embodiments, the fragment can be about 50
nucleotides in length, about 100 nucleotides in length, about 200
nucleotides in length, about 300 nucleotides in length, about 400
nucleotides in length, about 500 nucleotides in length, about 600
nucleotides in length, about 700 nucleotides in length, about 800
nucleotides in length, or about 900 nucleotides in length. In other
embodiments, the fragment can extend about 50 nucleotides, about
100 nucleotides, about 200 nucleotides, about 300 nucleotides,
about 400 nucleotides, about 500 nucleotides, about 600
nucleotides, about 700 nucleotides, about 800 nucleotides, or about
900 nucleotides upstream from the 3' end of the isolated nucleic
acid of any of SEQ ID NOS: 1 to 2. In yet other embodiments, the
fragment can include about 50 nucleotides, about 100 nucleotides,
about 200 nucleotides, about 300 nucleotides, or about 350
nucleotides upstream and/or downstream from the TATA box sequence
found in each of SEQ ID NOS: 1 to 2.
[0158] In one embodiment, the expression vector (i.e., with the
heterologous promoter) included in the kit can have any of the
other elements described herein, such as a selection marker, a
cloning site, such as a multiple cloning site, an enhancer, a
termination sequence, a signal peptide sequence, and the like. In
another aspect, the expression vector can be a vector that
replicates autonomously or integrates into the host cell genome. In
another embodiment, the expression vector can be circularized or
linearized (i.e., digested with a restriction enzyme so that a gene
of interest can easily be cloned into the expression vector). In
another embodiment, the kit can include an expression vector and a
control ORF encoding a marker or control gene for expression (e.g.,
an ORF encoding a LacZ-.alpha. fragment) for use as a control to
show that the expression vector is competent to be ligated and to
be used with a gene of interest.
[0159] In another illustrative embodiment, the kit can contain a
tube containing a circular expression plasmid. In another
embodiment, the kit can contain a tube with a linear expression
vector that is digested with a restriction enzyme so it is ready to
clone a gene of interest. In one embodiment, the tube can be
sterilized. In either of these embodiments, the kit can also
include a circular or linear expression vector and a control ORF
encoding a marker or control gene for expression (e.g., an ORF
encoding a LacZ-.alpha. fragment) for use as a control in showing
that the expression vector can be ligated with a gene of interest,
and/or to show that the host cell is competent for
transformation.
[0160] In yet another embodiment, the kit can contain multiple
different expression vectors. In another embodiment, the multiple
different expression vectors can contain the same promoter, but
different selectable markers, such as genes for resistance to the
drugs G418, Nourseothricin (Nat), Zeocin, Blasticidin, or
Hygromycin. In this embodiment, the kit may also contain aliquots
of the drugs (e.g., G418, Nourseothricin (Nat), Zeocin,
Blasticidin, or Hygromycin) in tubes, or other containers, separate
from the corresponding vectors.
[0161] In any of the kit embodiments described above, the isolated
nucleic acid can consist of any one of SEQ ID NOS. 1 to 2, or a
fragment thereof. The phrase "consists of" means that the sequence
specified by the SEQ ID NO. has no additional nucleotide sequences
other than those corresponding to the SEQ ID NO.
[0162] In another illustrative aspect, the kit can include other
components for use with the expression vector, such as components
for transformation of yeast cells, restriction enzymes for
incorporating a protein coding sequence of interest into the
expression vector, ligases, components for purification of
expression vector constructs, buffers (e.g., a ligation buffer),
instructions for use (e.g., to facilitate cloning), and any other
components suitable for use in a kit for making and using the
expression vectors described herein. In another embodiment, the
expression vector or any other component of the kit can be included
in the kit in a sealed tube (e.g., sterilized or not sterilized) or
any other suitable container or package (e.g., sterilized or not
sterilized). The kits described in the preceding paragraphs that
include the expression vector comprise the expression vector
comprising a promoter described herein operably linked to the
vector which is heterologous to the promoter (i.e., the combination
does not occur in nature).
[0163] The following examples provide illustrative methods for
carrying out the practice of the present invention. As such, these
examples are provided for illustrative purposes only and are not
intended to be limiting.
EXAMPLES
Example 1
Preparation of Promoter Sequences and Promoter-Containing
Plasmids
[0164] Microarray "chips" to measure Pichia pastoris mRNA levels
were created by Affymetrix (Affymetrix, Inc.) using Pichia genomic
DNA sequence provided by Dr. James Cregg. Microarray chips were
probed with RNA samples from yeast cultures grown on different
carbon sources, including methanol, glycerol and ethanol. Cultures
were harvested at various time points in mid-log phase and frozen
for the subsequent RNA isolation as recommended by Affymetrix.
Standard Affymetrix protocols for probing Affymetrix microarrays
were followed.
[0165] Total RNA from each cell sample was extracted by a glass
bead breakage/phenol extraction/LiCl precipitation method. RNA was
converted to cDNA and subsequently amplified and labeled to probe
Affymetrix chips. Labeled cDNAs were hybridized to Affymetrix
Pichia pastoris microarray chips and evaluated using Affymetrix
software. Data analysis identified genes induced by methanol
(relative to glycerol) or genes expressed under most conditions
(i.e., constitutively expressed) as shown in Table 1.
[0166] Table 1 lists the relative mRNA levels from the indicated
genes comparing promoter activity from Pichia pastoris cells grown
on media with methanol, glycerol or ethanol as the carbon
source.
TABLE-US-00001 TABLE 1 Relative Activity of Promoters Relative
Promoter Activity MeOH Protein Gene Methanol starvation Glycerol
Ethanol Hypothetical UPP 9942 8297 9512 10673 protein Protein TEF2
8910 5626 9309 9471 Translation Elongation Factor 1A Hypothetical
Chr1-0469 6904 192 7673 7618 protein Glyceraldehyde GAP 5685 4688
6701 6069 3-phosphate dehydrogenase AOX1 Average AOX1 9127 4974 610
361 Dihydroxy- DAS 8533 2591 1211 796 acetone synthase
[0167] FIG. 6 shows the relative promoter activity for pUPP, TEF2,
Chr1-0469, GAP, AOX1, and DAS using the carbon sources methanol
(leftmost bar in each set of bars), methanol starvation (bar second
from the left in each set of bars), glycerol (bar third from the
left in each set of bars), and ethanol (rightmost bar in each set
of bars). The hypothetical proteins are the ORFs for the UPP and
Chr1-0469 promoters.
[0168] Promoters from two constitutively expressed genes were
cloned and tested with reporter vectors. All of the reporter
plasmids were missing promoter elements to drive expression of the
reporter ORF; alternatively, a cloning scheme utilizing Type II
restriction enzymes (i.e. BsaI) was put in place to introduce DNA
fragments (promoters) upstream of the ORF. All constructs contained
the same 3'AOX1 transcriptional terminator sequence after the stop
codon of the reporter ORF.
[0169] One reporter plasmid, pTZ-GFP, is a derivative of pJAZ
(zeocin resistance as a selection marker) without the AOX1
promoter, but with a green fluorescent protein (GFP) ORF as a
reporter gene for intracellular expression. A second group of
reporter plasmids contains selectable marker genes for zeocin or
G418 resistance and the Aspergillus alpha-galactosidase or
beta-galactosidase ORFs as reporters for intracellular expression.
The third group of reporter plasmids has the S. cerevisiae
pre-pro-alpha Mating Factor (aMF) signal for secretion fused in
frame with the E. coli appA2 coding sequence for phosphatase
(phytase), as the reporter ORF. None of the reporter plasmid
sequences are expressed in Pichia pastoris without the introduction
of a sequence that has promoter activity upstream of the reporter
ORF.
[0170] Two of the most active constitutive promoters, from the UPP
and Chr1-0469 genes, consisted of the DNA 1000 bp "upstream" of
ORF's of unknown function. These promoter sequences were amplified
using PCR primers and Pichia pastoris genomic DNA. The sequence of
the PCR products was confirmed, prior to ligation into reporter
plasmids. The UPP promoter was cloned using strategically placed
BsaI sites in the primers flanking the promoter. The Chr1-0469
promoter contains recognition sites for BsaI and BsmBI; therefore,
cloning ends were created by a PCR-denaturation-annealing
procedure. Briefly, the promoter was amplified with two sets of
primers. The PCR products represent two nearly identical
double-stranded DNA molecules except that the ends are off-set by 4
bp (one is four nucleotides shorter from the 5'-end and four
nucleotides longer from the 3'-end, and the other four nucleotides
longer from the 5'-end and four nucleotides shorter from the
3'-end). The two different PCR products are mixed, denatured and
annealed to create promoter molecules with the proper 4 base single
strand extensions at each end of the molecule ("sticky" ends to
facilitate cloning). Cloning ends, generated by BsaI digestion or
by PCR-denaturation-annealing, were complementary to ends created
in the reporter plasmids. Promoters regulating the Pichia pastoris
AOX1 and GAP1 genes were introduced into the same reporter
constructs as controls.
[0171] The promoter specific plasmid constructs were linearized by
digestion with specific restriction enzymes and introduced into
wild type Pichia pastoris cells by electroporation. Transformants
containing the plasmid DNA integrated into the genome of the host
strain were selected on YPD agar with either zeocin or G418. Single
colonies from the transformation were purified (again on selective
plates), then, multiple clones were collected on YPD master plates.
These master plates were replica-plated to a series of diagnostic
plates with agar media containing one of the following compounds as
the only carbon source for growth of the Pichia pastoris cells:
glucose (i.e., dextrose), glycerol, methanol, or ethanol (all at 1%
w/v). Plates were incubated at 30.degree. C. for 20 to 44 hours
before scoring.
Example 2
Determination of GFP Expression in Transformants
[0172] The relative level of GFP expression between the various
transformants was compared. In this example, replica plates were
exposed to UV light using a transilluminator and the fluorescence
intensity of the different strains was compared by direct
visualization. Pichia cells without GFP expression, negative
colonies, did not demonstrate GFP fluorescence under UV
illumination. The intensity of the observed fluorescence correlated
with an increase in GFP expression.
[0173] FIG. 5 is an example of GFP expression for various
transformants on replica plates with different carbon sources. The
location of the different Pichia promoter strains in each panel is
the same, and the "Master YPD" panel identifies the location of the
strains by their promoter designation: AOX1, pUPP, GAP1, 0001,
0002, 0003, 0004, 0005, 0006, 0007, and Vector (mock control). The
lower part of FIG. 5, labeled as "Expression of GFP induced by Dex,
Gly, MeOH, or EtOH" shows that the carbon source used in each panel
is dextrose (D), glycerol (G), methanol (M) or ethanol (E),
respectively. The data show that the UPP promoter is as strong or
stronger than the GAP promoter under the conditions tested, and is
stronger than the AOX promoter under most conditions tested.
Example 3
Determination of Relative Activity of Alpha- and
Beta-Galactosidases of Transformants
[0174] The UPP promoter and the Chr1-0469 promoter sequences were
amplified using PCR primers and Pichia pastoris genomic DNA (the
sequences of the forward primers used for amplification of the
promoter fragments are shown on FIG. 8). The sequences of the PCR
products were confirmed, prior to ligation into the alpha-GAL
reporter plasmid (FIG. 7). Promoters regulating the Pichia pastoris
GAP, TEF2, DAS and AOX1 ORFs were introduced into the same reporter
plasmid as controls. The sequences of the inserted promoter
fragments were confirmed by sequencing the entire insertions, in
both directions, using plasmid specific primers located upstream of
the inserted fragment (forward primers) and in the 5' region of the
alpha-Galactosidase ORF (reverse primers). The promoter specific
plasmid constructs were linearized by digestion with specific
restriction enzymes and introduced into wild type Pichia pastoris
cells by electroporation, and transformants containing the plasmid
DNA integrated into the genome of the host strain were selected.
Cells from well isolated, single-colony transformants were purified
and collected on a YPD master plate and incubated at 30.degree. C.
The master plate was subsequently replica-plated to a series of
diagnostic plates with agar media containing one of the following
compounds as the only carbon source for growth of the Pichia
pastoris cells: glucose (i.e., dextrose), glycerol, methanol, or
ethanol (all at 1% w/v) and alpha-X-Gal chromogenic substrate.
These plates were then incubated at 30.degree. C. for 44 hours
(FIG. 9).
[0175] The expression level from the different
promoter-alpha-galactosidase reporter gene constructs, was
determined by the observation of the alpha-galactosidase from YPD
agar plates made with 100 mM phosphate buffer, pH 6.5, and
alpha-X-GAL as a chromogenic substrate. Reporter activity was
detected as a blue colored product in, or around the cells.
Reporter activity was demonstrated with the UPP and Chr1-0469
promoters.
[0176] The promoter designations in FIG. 9 correspond to the
Chr1-0469 promoter (SEQ ID NO: 1) and the UPP promoter (SEQ ID NO:
2). The length of each promoter fragment is indicated with the +1
position equal to the A in the ATG start codon of the corresponding
gene in the Pichia pastoris genome (therefore the promoter
sequences are from -1 to -1500). Fragments of other Pichia pastoris
promoters as well as a reporter vector without any promoter
sequences are used as controls. The transformants were incubated on
agar plates containing X-alpha-GAL, a chromogenic substrate for
alpha-Galactosidase, and one of four carbon sources as indicated.
Plates A and B in FIG. 9, panel A, were replica-plated on different
carbon sources to plates A and B, respectively, shown in FIG. 9,
panels B and C, respectively. Thus, the designations in FIG. 9,
panel A of plates A and B refer to FIG. 9, panels B and C,
respectively. The notations of NO:1 and NO:2 refer to SEQ ID NOS: 1
and 2 (i.e., to the promoter used to express a-galactosidase). The
level of .alpha.-galactosidase expression with the promoters is
shown in the tables labeled Plate A and Plate B below on the
various carbon sources. The level of .alpha.-galactosidase
expression is shown as 0, +1, +2, +3, +4, and +5, with the level of
expression increasing from 0 to +5.
[0177] Similarly, the beta-Galactosidase ORF was used for analysis
of expression of the reporter constructs with the Chr1-0469
promoter and the UPP promoter sequences in the Pichia pastoris
transformants. In these experiments the expression level from the
beta-galactosidase reporter gene was determined on plates with
beta-X-GAL as a chromogenic substrate. Reporter activity with
beta-Galactosidase ORF was also demonstrated with both the UPP and
Chr1-0469 promoters.
TABLE-US-00002 Relative Alpha-Galactosidase Expression, Plate A,
from FIG. 9. Promoter FIG. 9 Carbon Relative A-gal (seq ID/name)
(Panel/row/column) Source expression (1-5) Control A/C/1 Glucose 0
Control A/E/1 Glucose 0 Control A/E/2 Glucose 0 UPP-513 (No: 2)
A/F/5 Glucose 4 UPP-354 (No: 2) A/G/1 Glucose 4 DAS A/G/2 Glucose 0
1-0469 (No: 1) A/G/3 Glucose 4 GAP A/G/4 Glucose 3 Control A/C/1
Glycerol 0 Control A/E/1 Glycerol 0 Control A/E/2 Glycerol 0
UPP-513 (No: 2) A/F/5 Glycerol 3 UPP-354 (No: 2) A/G/1 Glycerol 4
DAS A/G/2 Glycerol 0 1-0469 (No: 1) A/G/3 Glycerol 3 GAP A/G/4
Glycerol 2 Control A/C/1 Methanol 1 Control A/E/1 Methanol 0
Control A/E/2 Methanol 1 UPP-513 (No: 2) A/F/5 Methanol 3 UPP-354
(No: 2) A/G/1 Methanol 4 DAS A/G/2 Methanol 2 1-0469 (No: 1) A/G/3
Methanol 3 GAP A/G/4 Methanol 3 Control A/C/1 Ethanol 0 Control
A/E/1 Ethanol 0 Control A/E/2 Ethanol 0 UPP-513 (No: 2) A/F/5
Ethanol 3 UPP-354 (No: 2) A/G/1 Ethanol 4 DAS A/G/2 Ethanol 0
1-0469 (No: 1) A/G/3 Ethanol 3 GAP A/G/4 Ethanol 2 Relative
Alpha-Galactosidase Expression, Plate B, from FIG. 9 Promoter FIG.
9 (Plate Carbon Relative A-gal (seq ID/name) B/row/column) Source
expression (1-5) UPP-513 (No: 2) B/F/1 Glucose 4 UPP-345 (No: 2)
B/F/2 Glucose 4 DAS B/F/3 Glucose 0 1-0469 (No: 1) B/F/4 Glucose 3
GAP B/F/5 Glucose 4 UPP 222 (No: 2) B/G/2 Glucose 3 UPP-513 (No: 2)
B/F/1 Glycerol 4 UPP-345 (No: 2) B/F/2 Glycerol 4 DAS B/F/3
Glycerol 0 1-0469 (No: 1) B/F/4 Glycerol 3 GAP B/F/5 Glycerol 4 UPP
222 (No: 2) B/G/2 Glycerol 3 UPP-513 (No: 2) B/F/1 Methanol 5
UPP-345 (No: 2) B/F/2 Methanol 5 DAS B/F/3 Methanol 3 1-0469 (No:
1) B/F/4 Methanol 3 GAP B/F/5 Methanol 3 UPP 222 (No: 2) B/G/2
Methanol 4 UPP-513 (No: 2) B/F/1 Ethanol 4 UPP-345 (No: 2) B/F/2
Ethanol 4 DAS B/F/3 Ethanol 0 1-0469 (No: 1) B/F/4 Ethanol 3 GAP
B/F/5 Ethanol 4 UPP 222 (No: 2) B/G/2 Ethanol 3
Example 4
Determination of Phytase Activity of Transformants
[0178] The level of phytase activity was determined in the various
transformants Agar medium on plates was supplemented with 100 mM
phosphate buffer, pH 6.5, and calcium phytate. The activity of
phytase acts to convert the non-transparent colloid calcium phytate
substance into transparent material, forming a transparent halo
around colonies expressing and secreting a phosphatase (phytase).
An increased expression of the phytase by the cells leads to
increased secretion of the enzyme, as demonstrated by a wider halo
observed around the cell colony. Reporter activity was demonstrated
with the UPP and Chr1-0469 promoters (FIG. 10). In FIG. 10, the
transformants harboring promoter sequences GAP, Chr1-0469 promoter
(SEQ ID NO: 1) and the UPP promoter (SEQ ID NO: 2) are cloned
upstream of the ORF consisting of the S. cerevisiae pre-pro alpha
factor fused in frame with DNA encoding the E. coli appA2 phytase.
Cells were spotted on diagnostic plates with calcium phytate and
either glucose or methanol and incubated for 27 hours at 30.degree.
C. The level of phytase expression with the promoters is noted by
0, +1, +2, +3, +4, and +5, with the level of expression increasing
from 0 to +5. The relative levels of phytase expression are shown
in the table below with No:1 and No:2 representing SEQ ID NO: 1 and
SEQ ID NO: 2, respectively.
TABLE-US-00003 TABLE X Relative Phytase Activity from FIG. 10
Promoter Carbon Relative activity 1-5 (seq: ID) Source (5 is
highest activity) GAP glucose 2 No: 1 glucose 2 No: 2, c glucose 5
No: 2, d glucose 5 No: 2, e glucose 5 GAP methanol 1 No: 1 methanol
2 No: 2, c methanol 2 No: 2, d methanol 2 No: 2, e methanol 2
Sequence CWU 1
1
1311003DNAPichia pastoris 1tgccaggatc tgaccactac aagaagcgta
attaagtatt tattatgtag cttgaggaat 60tgatttctaa aggccaaaga tgatgtagag
caatagtaga gtgattagca agtttctgaa 120catgttttgt ggtcgggtat
taggcgccgt acctctgtat tttttttttt tggcagatgg 180gatgaccact
gtgcttacat aattttcgat gggccctttt ggaaacgacc actccttccg
240atcacgtaat actaggcgag tatcactacg cgtcggtatc cgcattttag
ccgctttaac 300gacaaggtca gggtaaatac tgtgttgcat gggcgatgcc
gtaggatctg gccaacttgg 360aacagagcag cccaccgcgt aggcagcgct
tgggaccagt ttgttagcca atcagttccc 420ccgtctgagt ccctacacgc
tcaggcttgc atggatatgc atgaaaaatg tcaactcagt 480cgcacacagg
gatgctgttg tcgtggtgag aaaaagctaa aatagcaaac ccatagcaat
540cgagagactg tgattggaag caaaagaacc aggcattgac taggaaagac
tgacaagaaa 600attagacggt gccagtggta tttcattgaa ttgcactgaa
cacgcctccc atttccttcc 660ctcaatccat tcaaagggct aattaaacct
tatctcggcc tacattagtt gtcatggaaa 720cgtctcactt tctctctgtg
tcccacagta gcgagtaggc ggagagacca aaaatggtct 780ctcggcccta
atcaaaacat cctttgtcgc gttgttttca gatcgattcg caatctgaga
840gaaaaacaat aacaccaaaa taaaaagctt ggaaaaagaa gacatgattc
acatccacta 900gtaaaatata aatactcctg ccgcctcccc ctttcctttc
ttcttattcc tcaactcact 960gtttcagttt attccaacta ctttcactca
cttatcaaaa atg 10032773DNAPichia pastoris 2gggtgaaagc caaccatctt
tgtttcgggg aaccgtgctc gccccgtaaa gttaattttt 60ttttcccgcg cagctttaat
ctttcggcag agaaggcgtt ttcatcgtag cgtgggaaca 120gaataatcag
ttcatgtgct atacaggcac atggcagcag tcactatttt gctttttaac
180cttaaagtcg ttcatcaatc attaactgac caatcagatt ttttgcattt
gccacttatc 240taaaaatact tttgtatctc gcagatacgt tcagtggttt
ccaggacaac acccaaaaaa 300aggtatcaat gccactaggc agtcggtttt
atttttggtc acccacgcaa agaagcaccc 360acctctttta ggttttaagt
tgtgggaaca gtaacaccgc ctagagcttc aggaaaaacc 420agtacctgtg
accgcaattc accatgatgc agaatgttaa tttaaacgag tgccaaatca
480agatttcaac agacaaatca atcgatccat agttacccat tccagccttt
tcgtcgtcga 540gcctgcttca ttcctgcctc aggtgcataa ctttgcatga
aaagtccaga ttagggcaga 600ttttgagttt aaaataggaa atataaacaa
atataccgcg aaaaaggttt gtttatagct 660tttcgcctgg tgccgtacgg
tataaataca tactctcctc ccccccctgg ttctcttttt 720cttttgttac
ttacatttta ccgttccgtc actcgcttca ctcaacaaca aaa 7733939DNAPichia
pastoris 3gatctaacat ccaaagacga aaggttgaat gaaacctttt tgccatccga
catccacagg 60tccattctca cacataagtg ccaaacgcaa caggagggga tacactagca
gcagaccgtt 120gcaaacgcag gacctccact cctcttctcc tcaacaccca
cttttgccat cgaaaaacca 180gcccagttat tgggcttgat tggagctcgc
tcattccaat tccttctatt aggctactaa 240caccatgact ttattagcct
gtctatcctg gcccccctgg cgaggttcat gtttgtttat 300ttccgaatgc
aacaagctcc gcattacacc cgaacatcac tccagatgag ggctttctga
360gtgtggggtc aaatagtttc atgttcccca aatggcccaa aactgacagt
ttaaacgctg 420tcttggaacc taatatgaca aaagcgtgat ctcatccaag
atgaactaag tttggttcgt 480tgaaatgcta acggccagtt ggtcaaaaag
aaacttccaa aagtcggcat accgtttgtc 540ttgtttggta ttgattgacg
aatgctcaaa aataatctca ttaatgctta gcgcagtctc 600tctatcgctt
ctgaaccccg gtgcacctgt gccgaaacgc aaatggggaa acacccgctt
660tttggatgat tatgcattgt ctccacattg tatgcttcca agattctggt
gggaatactg 720ctgatagcct aacgttcatg atcaaaattt aactgttcta
acccctactt gacagcaata 780tataaacaga aggaagctgc cctgtcttaa
accttttttt ttatcatcat tattagctta 840ctttcataat tgcgactggt
tccaattgac aagcttttga ttttaacgac ttttaacgac 900aacttgagaa
gatcaaaaaa caactaatta ttcgaaacg 93941001DNAPichia pastoris
4catgttggta ttgtgaaata gacgcagatc gggaacactg aaaaataaca gttattattc
60gagatctaac atccaaagac gaaaggttga atgaaacctt tttgccatcc gacatccaca
120ggtccattct cacacataag tgccaaacgc aacaggaggg gatacactag
cagcagaccg 180ttgcaaacgc aggacctcca ctcctcttct cctcaacacc
cacttttgcc atcgaaaaac 240cagcccagtt attgggcttg attggagctc
gctcattcca attccttcta ttaggctact 300aacaccatga ctttattagc
ctgtctatcc tggcccccct ggcgaggttc atgtttgttt 360atttccgaat
gcaacaagct ccgcattaca cccgaacatc actccagatg agggctttct
420gagtgtgggg tcaaatagtt tcatgttccc caaatggccc aaaactgaca
gtttaaacgc 480tgtcttggaa cctaatatga caaaagcgtg atctcatcca
agatgaacta agtttggttc 540gttgaaatgc taacggccag ttggtcaaaa
agaaacttcc aaaagtcggc ataccgtttg 600tcttgtttgg tattgattga
cgaatgctca aaaataatct cattaatgct tagcgcagtc 660tctctatcgc
ttctgaaccc cggtgcacct gtgccgaaac gcaaatgggg aaacacccgc
720tttttggatg attatgcatt gtctccacat tgtatgcttc caagattctg
gtgggaatac 780tgctgatagc ctaacgttca tgatcaaaat ttaactgttc
taacccctac ttgacagcaa 840tatataaaca gaaggaagct gccctgtctt
aaaccttttt ttttatcatc attattagct 900tactttcata attgcgactg
gttccaattg acaagctttt gattttaacg acttttaacg 960acaacttgag
aagatcaaaa aacaactaat tattcgaaac g 1001520DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
5atccctacac gctcaggctt 20633DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primermodified_base(1)..(3)a, c, t,
g, unknown or othermodified_base(10)..(10)a, c, t, g, unknown or
other 6nnnggtctcn atcctcgctg ggtgaaagcc aac 33736DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
primermodified_base(1)..(3)a, c, t, g, unknown or
othermodified_base(10)..(10)a, c, t, g, unknown or other
7nnnggtctcn atccgcagat acgttcagtg gtttcc 36834DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
primermodified_base(1)..(3)a, c, t, g, unknown or
othermodified_base(10)..(10)a, c, t, g, unknown or other
8nnnggtctcn atccagtacc tgtgaccgca attc 34933DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
primermodified_base(1)..(3)a, c, t, g, unknown or
othermodified_base(10)..(10)a, c, t, g, unknown or other
9nnnggtctcn atcctgcctc aggtgcataa ctt 331037DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
primermodified_base(1)..(3)a, c, t, g, unknown or
othermodified_base(10)..(10)a, c, t, g, unknown or other
10nnnggtctcn atccaatgga ccaaattgtt gcaaggt 371129DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
primermodified_base(1)..(3)a, c, t, g, unknown or other
11nnnggtctcc atccctttgt tgagcaaca 291228DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
primermodified_base(1)..(3)a, c, t, g, unknown or other
12nnnggtctcg atccgcccaa acgaacag 281334DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
primermodified_base(1)..(3)a, c, t, g, unknown or other
13nnnggtctcc atccaaagac gaaaggttga atga 34
* * * * *
References