U.S. patent application number 13/666116 was filed with the patent office on 2013-05-16 for yeast host, transformant and method for producing heterologous proteins.
This patent application is currently assigned to ASAHI GLASS COMPANY, LIMITED. The applicant listed for this patent is Asahi Glass Company, Limited. Invention is credited to Yuko HAMA, Alimjan IDIRIS, Hideki TOHDA.
Application Number | 20130122547 13/666116 |
Document ID | / |
Family ID | 37708750 |
Filed Date | 2013-05-16 |
United States Patent
Application |
20130122547 |
Kind Code |
A1 |
IDIRIS; Alimjan ; et
al. |
May 16, 2013 |
YEAST HOST, TRANSFORMANT AND METHOD FOR PRODUCING HETEROLOGOUS
PROTEINS
Abstract
A method of constructing a host for expression of an exogenous
gene which comprises deleting or inactivating at least one gene
selected from the protease-associated genes of Schizosaccharomyces
pombe such as psp3 (SPAC1006.01), sxa2 (SPAC1296.03c), ppp51
(SPAC22G7.01c) or ppp52 (SPBC18A7.01). An isolated or purified host
cell in which a protease-associated gene is deleted or inactivated,
a transformant obtained by introducing an exogenous gene into the
host cell and a method of producing an exogenous or heterologous
protein using the transformant.
Inventors: |
IDIRIS; Alimjan;
(Chiyoda-ku, JP) ; TOHDA; Hideki; (Chiyoda-ku,
JP) ; HAMA; Yuko; (Chiyoda-ku, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Asahi Glass Company, Limited; |
Chiyoda-ku |
|
JP |
|
|
Assignee: |
ASAHI GLASS COMPANY,
LIMITED
Chiyoda-ku
JP
|
Family ID: |
37708750 |
Appl. No.: |
13/666116 |
Filed: |
November 1, 2012 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
12025138 |
Feb 4, 2008 |
8329448 |
|
|
13666116 |
|
|
|
|
PCT/JP2006/315173 |
Jul 31, 2006 |
|
|
|
12025138 |
|
|
|
|
Current U.S.
Class: |
435/69.4 ;
435/254.2; 435/471; 435/69.1 |
Current CPC
Class: |
C12N 15/815 20130101;
C12P 21/02 20130101; C07K 14/61 20130101 |
Class at
Publication: |
435/69.4 ;
435/471; 435/254.2; 435/69.1 |
International
Class: |
C12N 15/81 20060101
C12N015/81; C07K 14/61 20060101 C07K014/61 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 3, 2005 |
JP |
2005-225638 |
Jun 8, 2006 |
JP |
2006-160347 |
Claims
1. A method of constructing a Schizosaccharomyces pombe host cell
for expression of a recombinantly introduced foreign gene,
comprising: deleting or inactivating at least one target gene
selected from the groups consisting of genes encoding serine
proteases (serine protease gene family), genes encoding amino
peptidases (amino peptidase gene family), genes encoding carboxy
peptidases (carboxy peptidase gene family) and genes encoding
dipeptidases (dipeptidase gene family).
2. The method according to claim 1, wherein the target gene is at
least one gene selected from the group consisting of psp3
(SPAC1006.01), sxa2 (SPAC1296.03c), ppp51 (SPAC22G7.01c) and ppp52
(SPBC18A7.01).
3.-5. (canceled)
6. The method according to claim 1, wherein the genes are deleted
or inactivated by replacing their open reading frames (ORFs) with
marker genes.
7. A Schizosaccharomyces pombe host for expression of a
recombinantly introduced foreign gene, in which at least one gene
selected from the group consisting of psp3 (SPAC1006.01), sxa2
(SPAC1296.03c), ppp51 (SPAC22G7.01c) and ppp52 (SPBC18A7.01) is
deleted or inactivated.
8.-10. (canceled)
11. A transformant obtained by introducing a gene encoding a
heterologous protein into the host as defined above in claim 7.
12. The transformant according to claim 11, wherein a secretion
signal gene is introduced with the gene encoding a heterologous
protein.
13. A method of producing a heterologous protein, which comprises
culturing the transformant as defined in claim 11 to allow it to
produce the heterologous protein, and recovering the heterologous
protein.
14. A method of producing a heterologous protein, which comprises
culturing the transformant as defined in claim 12 to allow it to
produce the heterologous protein and secret the heterologous
protein in the culture, and recovering the heterologous protein
from the culture.
15. The method according to claim 13, wherein according to 13
mentioned above, wherein the heterologous protein is human growth
hormone (hGH).
16. An isolated or purified Schizosaccharomyces pombe cell produced
by deleting or inactivating from a corresponding
Schizosaccharomyces pombe parent strain at least one gene selected
from the group consisting of: a metalloprotease family gene
selected from cdb4 (SPAC23H4.09), mast (SPBC18E5.12c), pgp1
(SPCC1259.10), ppp20 (SPAC4F10.02), ppp22 (SPBC14C8.03), ppp51
(SPC22G7.01c), ppp52 (SPBC18A7.01) and ppp53 (SPAP14E8.04); a
serine protease family gene selected from isp6 (SPAC4A8.04), ppp16
(SPBC1711.12), psp3 (SPAC1006.01) and sxa2 (SPAC1296.03c); a
cysteine protease family gene selected from ppp80 (SPAC19B12.08),
pca1 (SPCC1840.04), cut1 (SPCC5E4.04) and gpi8 (SPCC11E10.02c); and
a aspartyl protease family gene selected from sxa1 (SPAC26A3.01),
yps1 (SPCC1795.09) and ppp81 (SPAC25B8.17).
17. The isolated or purified Schizosaccharomyces pombe cell of
claim 16 produced by deleting or inactivating at least one gene
selected from the group consisting of psp3 (SPAC1006.01), sxa2
(SPAC1296.03c), ppp51 (SPAC22G7.01c) and ppp52 (SPBC18A7.01) from a
corresponding Schizosaccharomyces pombe parent strain.
18. The isolated or purified Schizosaccharomyces pombe cell of
claim 16, wherein said at least one gene is deleted.
19. The isolated or purified Schizosaccharomyces pombe cell of
claim 16, wherein said at least one gene is inactivated.
20. The isolated or purified Schizosaccharomyces pombe cell of
claim 16, wherein said at least one gene comprises psp3
(SPAC1006.01).
21. The isolated or purified Schizosaccharomyces pombe cell of
claim 16, wherein said at least one gene consists of psp3
(SPAC1006.01).
22. The isolated or purified Schizosaccharomyces pombe cell of
claim 16, wherein said at least one gene comprises sxa2
(SPAC1296.03c).
23. The isolated or purified Schizosaccharomyces pombe cell of
claim 16, wherein said at least one gene consists of sxa2
(SPAC1296.03c).
24. The isolated or purified Schizosaccharomyces pombe cell of
claim 16, wherein said at least one gene comprise ppp51
(SPAC22G7.01c).
25. The isolated or purified Schizosaccharomyces pombe cell of
claim 16, wherein said at least one gene consists of ppp51
(SPAC22G7.01c).
26. The isolated or purified Schizosaccharomyces pombe cell of
claim 16, wherein said at least one gene comprises ppp52
(SPBC18A7.01).
27. The isolated or purified Schizosaccharomyces pombe cell of
claim 16, wherein said at least one gene consists of ppp52
(SPBC18A7.01).
28. The isolated or purified Schizosaccharomyces pombe cell of
claim 16, further comprising a polynucleotide sequence or vector
encoding an exogenous polypeptide.
29. A method for making an exogenous polypeptide comprising
transforming the isolated or purified Schizosaccharomyces pombe
cell of claim 16 with a polynucleotide or vector encoding said
exogenous proteins under conditions suitable for expression of said
exogenous polypeptide and recovering said exogenous polypeptide.
Description
TECHNICAL FIELD
[0001] The present invention relates to an improved eukaryotic host
microorganism in which part of the chromosomes are modified for the
purpose of improving the productivity of a heterologous protein by
a transformant of the eukaryotic host microorganism, a method of
constructing the host, a transformant of the host and a method of
producing a protein using the transformant, in particular, wherein
the eukaryotic microorganism is Schizosaccharomyces pombe
(hereinafter referred to as S. pombe) called a fission yeast.
BACKGROUND ART
[0002] Recombinant DNA technology is used for production of
heterologous proteins in various host microorganisms and animals
including Escherichia coli (hereinafter referred to as E. coli).
The target products are various biogenous proteins (herein,
inclusive of polypeptides), and many of them have already been
produced industrially for medical and other uses so far.
[0003] Among various hosts developed for production of heterologous
proteins, yeasts seem favorable for expression of animal and plant
proteins because of their eukaryotic similarity in the
transcription and translation systems to animals and plants, and
the baker's yeast (Saccharomyces cerevisiae) is a widely used
host.
[0004] Among yeasts, S. pombe is known to be close to animal cells
in nature as is evident from the fact that it grows by fission not
by budding as a result of the different evolution process it has
followed since it diverged from other yeasts at early stages.
Therefore, the use of S. pombe as the host for expression of
heterologous proteins is expected to provide a gene product closer
to its natural form in animal cells.
[0005] Though studies of gene expression in S. pombe are delayed,
the recent discovery of potent promoters functional in S. pombe has
accelerated the development of expression systems using S. pombe as
the host, and various improvements have been added to expression
vectors to develop more stable and efficient expression systems
(Patent Documents 1 to 8). As a result, expression systems using S.
pombe as the host show high production efficiency now.
[0006] Production systems for heterologous proteins using
eukaryotic microorganisms such as yeasts can be realized easily by
conventional microbiological techniques and recombinant DNA
technology with high productivity. Large cultures are already
available and are acceleratingly used for actual production. Even
after the scale is enlarged for actual production, cells retain the
high production efficiency per cell obtained in the laboratory.
[0007] Considering that cost reduction is often demanded in actual
production, it is necessary to improve the production efficiency of
heterologous proteins through improvement in cell growth
efficiency, suppression of degradation of the heterologous protein
of interest, more efficient eukaryotic modifications in the
microorganisms or more efficient utilization of the nutrition
sources. For example, increase in the conversion of the carbon
sources added to the medium for culture growth into the
heterologous protein of interest is expected to drastically improve
cell growth efficiency and therefore production efficiency of the
heterologous protein, because efficient utilization of the carbon
sources in the medium for production of the heterologous protein of
interest seems to be sacrificed for their consumption by metabolic
systems unnecessary for cell growth or production of the
heterologous protein of interest (such as the ethanol fermentation
system for production of ethanol).
[0008] Therefore, attempts have been made to improve production
efficiency of heterologous proteins by a host by deleting or
inactivating part or all of the genome of the host unnecessary or
detrimental to production of heterologous proteins (Patent
Documents 9 and 10).
[0009] The present inventors reported about the invention described
in the patent applications from which the present application
claims the earlier priority date, in an article published after the
earlier priority application (before the later priority date)
(Non-patent Document 1) [0010] Patent Document 1: Japanese Patent
No. 2776085 [0011] Patent Document 2: JP-A-07-163373 [0012] Patent
Document 3: JP-A-10-215867 [0013] Patent Document 4: JP-A-10-234375
[0014] Patent Document 5: JP-A-11-192094 [0015] Patent Document 6:
JP-A-2000-136199 [0016] Patent Document 7: JP-A-2000-262284 [0017]
Patent Document 8: WO96/023890 [0018] Patent Document 9:
WO02/101038 [0019] Patent Document 10: WO04/090117 [0020]
Non-patent Document 1: Yeast, vol. 23, pp. 83-99, 2006
DISCLOSURE OF THE INVENTION
Problems that the Invention is to Solve
[0021] The use of an improved host in which all or part of the
regions in the genome detrimental to production of heterologous
proteins have been deleted or inactivated improves the efficiency
of production of heterologous proteins as disclosed in the
above-mentioned patent documents. However, it is necessary to
further investigate where on the chromosomes (especially, which
genes) to modify in order to attain a higher production efficiency
because efficiency of production of heterologous proteins depends
on which parts of the chromosomes (especially which genes) are
deleted or inactivated in what combinations.
Means of Solving the Problems
[0022] Under the above-mentioned circumstance, the present
inventors studied from the above-mentioned aspects, and, as a
result, found that deletion or inactivation of at least one
protease-related gene drastically improves the production
efficiency of heterologous proteins. Namely, is the present
invention provides:
[0023] 1. A method of constructing an improved Schizosaccharomyces
pombe host for expression of a recombinantly introduced foreign
gene, which is characterized by deleting or inactivating at least
one gene selected from the groups consisting of genes encoding
serine proteases (serine protease gene family), genes encoding
amino peptidases (amino peptidase gene family), genes encoding
carboxy peptidases (carboxy peptidase gene family) and genes
encoding dipeptidases (dipeptidase gene family), as a target
gene.
[0024] 2. The method according to 1, wherein the target gene is at
least one gene selected from the group consisting of psp3
(SPAC1006.01), sxa2 (SPAC1296.03c), ppp51 (SPAC22G7.01c) and ppp52
(SPBC18A7.01).
[0025] 3. A method of constructing an improved Schizosaccharomyces
pombe host for expression of a recombinantly introduced foreign
gene, which is characterized by deleting or inactivating two or
more gene selected from the groups consisting of genes encoding
metalloproteases (metalloprotease gene family), genes encoding
serine proteases (serine protease gene family), genes encoding
cysteine proteases (cysteine protease gene family) and genes
encoding aspartyl proteases (aspartyl protease gene family), as
target genes.
[0026] 4. The method according to 3 mentioned above, wherein the
two or more genes are a total of at least three genes consisting of
at least one gene selected from the metalloprotease gene family and
at least two genes selected from the serine protease gene
family.
[0027] 5. The method according to 3 or 4 mentioned above, wherein
the two or more genes are a total of at least three genes
consisting of at least one gene selected from the group consisting
of cdb4 (SPAC23H4.09), ppp22 (SPBC14C8.03) and ppp53 (SPAP14E8.04)
and at least two genes selected from the group consisting of isp6
(SPAC4A8.04), ppp16 (SPBC1711.12), psp3 (SPAC1006.01) and sxa2
(SPAC1296.03c).
[0028] 6. The method according to any one of 1 to 5 mentioned
above, wherein the genes are deleted or inactivated by replacing
the ORF(s) (open reading frame(s)) of the gene(s) with marker
genes.
[0029] 7. An improved Schizosaccharomyces pombe host for expression
of a recombinantly introduced foreign gene, in which at least one
gene selected from the group consisting of psp3 (SPAC1006.01), sxa2
(SPAC1296.03c), ppp51 (SPAC22G7.01c) and ppp52 (SPBC18A7.01) is
deleted or inactivated.
[0030] 8. An improved Schizosaccharomyces pombe host for expression
of a recombinantly introduced foreign gene, in which two or more
genes selected from the group consisting of genes encoding
metalloproteases (metalloprotease gene family), genes encoding
serine proteases (serine protease gene family), genes encoding
cysteine proteases (cysteine protease gene family) and genes
encoding aspartyl proteases (aspartyl protease gene family).
[0031] 9. The host according to 8 mentioned above, wherein the two
or more genes are a total of at least three genes consisting of at
least one gene selected from the metalloprotease gene family and at
least two genes selected from the serine protease gene family.
[0032] 10. The host according to 8 or 9 mentioned above, wherein
the two or more genes are a total of at least three genes
consisting of at least one gene selected from the group consisting
of cdb4 (SPAC23H4.09), ppp22 (SPBC14C8.03) and ppp53 (SPAP14E8.04)
and at least two genes selected from the group consisting of isp6
(SPAC4A8.04), ppp16 (SPBC1711.12), psp3 (SPAC1006.01) and sxa2
(SPAC1296.03c).
[0033] 11. A transformant obtained by introducing a gene encoding a
heterologous protein into the host as defined above in any one of 7
to 10.
[0034] 12. The transformant according to 11 mentioned above,
wherein a secretion signal gene is introduced with the gene
encoding a heterologous protein.
[0035] 13. A method of producing a heterologous protein, which
comprises culturing the transformant as defined above in 11 or 12
to allow it to produce the heterologous protein, and recovering the
heterologous protein.
[0036] 14. A method of producing a heterologous protein, which
comprises culturing the transformant as defined above in 12 to
allow it to produce the heterologous protein and secret the
heterologous protein in the culture, and recovering the
heterologous protein from the culture.
[0037] 15. The method according to 13 or 14, wherein according to
13 mentioned above, wherein the heterologous protein is human
growth hormone (hGH).
Effects of the Invention
[0038] The present invention is based on the discovery that
gene-disrupted host strains constructed by deleting or inactivating
(hereinafter sometimes referred to collectively as disrupting) one
or more protease-related genes in the fission yeast S. pombe
putatively associated with degradation of heterologous proteins can
produce heterologous proteins more efficiently when transformed.
These protease-related gene disruptants can be used widely for
production of protease-sensitive heterologous proteins.
BRIEF DESCRIPTION OF THE DRAWINGS
[0039] FIG. 1 A graph showing the relative growth rates of
protease-related gene disruptants from S. pombe.
[0040] FIG. 2 The structure of a multiple expression cassette
vector for expression of r-hGH.
[0041] FIG. 3 (A) SDS-PAGE showing r-hGH secretions from a
transformant ARC001(hGH) at various times. (B) SDS-PAGE showing
r-hGH secretions from a transformant ARC001(hGH) in the presence of
protease inhibitors in culture at various times.
[0042] FIG. 4 SDS-PAGE showing r-hGH secretions at various times
from protease gene disruptants and an ARC001 transformed with a hGH
expression vector.
[0043] FIG. 5 A graph showing the relative growth rates of multiple
protease-related gene disruptants from S. pombe.
[0044] FIG. 6 SDS-PAGE showing hGH secretions from multiple
protease-related gene disruptants from S. pombe at various
times.
[0045] FIG. 7 SDS-PAGE showing hGH sections from a sextuple and
septuple protease-related gene disruptants from S. pombe at various
times.
BEST MODE FOR CARRYING OUT THE INVENTION
[0046] In the present invention, the improved host is a S. pombe
strain. Hereinafter, the host means a S. pombe strain unless
otherwise noted. In the present invention, protease-related genes
include genes which are presumed to be protease-related genes from
their DNA sequences (or from the structures or amino acid sequences
of the polypeptides or proteins encoded by the genes).
[0047] When a transformant produces a heterologous protein in
culture, part of the genome is unnecessary or detrimental to
production of the heterologous protein. The part of the genome may
or may not be a gene. It is believed that a lot of such unnecessary
or detrimental genes exist in a genome.
[0048] It is generally presumed that some protease-related genes
tend to inhibit production of a heterologous protein. Because the
heterologous protein is a product fundamentally unnecessary to the
host, the transformant tends to degrade the produced heterologous
protein by proteases. Therefore, degradation of the heterologous
protein is considered as a factor of reduction in the production
efficiency of the heterologous protein. However, not all proteases
are unnecessary or detrimental to the host, and some have
unfavorable effect when inactivated. Therefore, the present
invention resides in the discovery that selective disruption of
genes which produce unnecessary or detrimental proteases improves
production efficiency of a heterologous protein.
[0049] In the present invention, the efficiency of production of a
heterologous protein by a transformant is successfully improved by
deleting or inactivating at least one protease-related gene
selected from the group consisting of four protease-related gene
families, the serine protease gene family, the amino peptidase gene
family, the carboxypeptidase gene family and the dipeptidase gene
family, as the target(s) in the genome unnecessary or detrimental
to production of the heterologous protein. In the improved yeast
host of the present invention, at least one gene selected from the
above-mentioned four groups of protease-related genes is deleted or
inactivated, and further, at least one other gene may be deleted or
inactivated.
[0050] The target gene selected from the above-mentioned four
protease-related gene families is preferably at least one gene
encoding a protease or a putative protease protein selected from
psp3 (SPAC1006.01), sxa2 (SPAC1296.03c), ppp51 (SPAC22G7.01c) and
ppp52 (SPBC18A7.01). psp3 (SPAC1006.01) and sxa2 (SPAC1296.03c) are
classified as serine protease genes, while ppp51 (SPAC22G7.01c) and
ppp52 (SPBC18A7.01) are genes putatively encoding amino peptidase
proteins (amino peptidase genes). They are also classified as
metalloprotease genes (genes of proteases containing metal
ions).
[0051] However, the above-mentioned object is often difficult to
attain satisfactorily by deleting or inactivating only one
protease-related gene. Deletion or inactivation of a single
protease-related gene can be insufficient (i.e., is not versatile
to improve various heterologous proteins). Further, because various
proteases in a living organism often have overlapping functions,
deletion or inactivation of a single protease-related gene may
improve productivity somewhat, but not drastically. Therefore, in
the present invention, it is preferred to delete or inactivate at
least two, preferably at least three genes. Deletion or
inactivation of the two or more protease-related genes of choice
drastically improves production efficiency of a heterologous
protein.
[0052] Therefore, in the present invention, the efficiency of
production of a heterologous protein by a transformant is
successfully improved also by deleting or inactivating at least two
genes selected from the group consisting of four protease-related
gene families, the metalloprotease gene family, the serine protease
gene family, the cysteine protease gene family and the aspartyl
protease gene family, as the targets in the genome unnecessary or
detrimental to production of the heterologous protein. In the
improved yeast host of the present invention, at lease two genes
selected from the above-mentioned four groups of protease-related
genes, and, further, at least one other gene may be deleted or
inactivated.
[0053] The construction method of the present invention comprising
deleting or inactivating two or more genes selected form the
above-mentioned four groups of protease-related genes and the host
of the present invention in which such two or more genes are
deleted or inactivated will be described. Deletion or inactivation
of at least one gene can be carried out similarly.
[0054] The two or more genes to be deleted or inactivated is which
are selected from the above-mentioned four protease-related gene
families may be two or more genes selected from the same single
gene family or from different gene families. In the latter case,
they may be a total of three or more genes consisting of at least
two genes selected from one family and at least one gene selected
from another family. Further, at least two genes selected from the
above-mentioned four protease-related gene families and a gene
selected from another gene family (which may not be
protease-related genes) may be deleted or inactivated in
combination.
[0055] The target protease-related genes may be deleted or
inactivated by known methods. Further, the regions of the
protease-related genes to be deleted or inactivated may be the open
reading frames (ORFs) or the expression regulatory regions. The
method for carrying out the deletion or inactivation is preferably
the PCR-mediated homologous recombination (Yeast, vol. 14, pp.
943-951, 1998), which was used later in the Examples, but is not
restricted to it.
[0056] The deletion or inactivation of protease-related genes may
be deletion of the entire genes or inactivation of the genes by
partial deletion. The inactivation of protease-related genes means
not only partial deletion of the genes but also modification of the
genes without deletion. Further, it may be insertion of other genes
or DNA into the base sequences of the protease-related genes. In
either case, the inactivated protease-related genes encode inactive
proteins or are unable to be transcribed or translated. When two or
more genes for the same protease are present in the cell, all the
genes may be deleted, or some of the genes may be left as long as
the protease encoded by the gene shows reduced activity in the
cell.
[0057] The genes in the protease-related gene families in the
present invention are at least two genes selected from the group
consisting of genes encoding metalloproteases (the metalloprotease
gene family), genes encoding serine proteases (the serine protease
gene family), genes encoding cysteine proteases (the cysteine
protease gene family) and genes encoding aspartyl proteases (the
aspartyl protease gene family), preferably at least two genes
selected from the metalloprotease gene family and the serine
protease gene family. A combination of at least one gene in the two
gene families and at least one gene selected from the cysteine
protease gene family and the aspartyl protease gene family is also
preferred. Examples of these genes are given below (see Table 1,
which appears later).
[0058] The metalloprotease gene family: cdb4 (SPAC23H4.09), mas2
(SPBC18E5.12c), pgp1 (SPCC1259.10), ppp20 (SPAC4F10.02), ppp22
(SPBC14C8.03), ppp51 (SPC22G7.01c), ppp52 (SPBC18A7.01) and ppp53
(SPAP14E8.04).
[0059] The serine protease gene family: isp6 (SPAC4A8.04), is ppp16
(SPBC1711.12), psp3 (SPAC1006.01) and sxa2 (SPAC1296.03c).
[0060] The cysteine protease gene family: ppp80 (SPAC19B12.08),
pca1 (SPCC1840.04), cut1 (SPCC5E4.04) and gpi8 (SPCC11E10.02c).
[0061] The aspartyl protease gene family: sxa1 (SPAC26A3.01), yps1
(SPCC1795.09) and ppp81 (SPAC25B8.17).
[0062] In the present invention, the protease-related genes as the
targets for deletion or inactivation are selected from the
metalloprotease gene family and the serine protease gene family,
and are preferably a combination of two or more genes selected from
the two gene families or a combination of at least one gene
selected from the two gene families and at least one gene selected
from the other families. The former combination is particularly
preferred. More preferably, they are a total of at least three
genes consisting of at least one gene selected from the
metalloprotease gene family and at least two genes selected from
the serine protease gene family. When the targets are at least four
genes, they preferably consists of at least 50%, in number, of
genes in the serine protease gene family and at least one gene
(preferably at least two genes) in the metalloprotease gene family,
and other genes, if any, in the cysteine protease gene family.
[0063] The target genes in the metalloprotease gene family are
preferably cdb4 (SPAC23H4.09), pgp1 (SPCC1259.10), ppp20
(SPAC4F10.02), ppp22 (SPBC14C8.03), ppp52 (SPBC18A7.01) and ppp53
(SPAP14E8.04), and particularly cdb4 (SPAC23H4.09), ppp22
(SPBC14C8.03) and ppp53 (SPAP14E8.04).
[0064] The target genes in the serine protease gene family are
preferably isp6 (SPAC4A8.04), ppp16 (SPBC1711.12), psp3
(SPAC1006.01) and sxa2 (SPAC1296.03c).
[0065] The target gene in the other gene families is preferably
ppp80 (SPAC19B12.08).
[0066] More specifically, the target genes are preferably a
combination of a total of at least three genes consisting of at
least one gene selected from the group consisting of cdb4
(SPAC23H4.09), ppp22 (SPBC14C8.03) and ppp53 (SPAP14E8.04) and at
least two genes selected from the group consisting of isp6
(SPAC4A8.04), ppp16 (SPBC1711.12), psp3 (SPAC1006.01) and sxa2
(SPAC1296.03c), particularly preferably a combination of a total of
at least three genes consisting of at least one gene selected from
the group consisting of ppp53 (SPAP14E8.04) and cdb4 (SPAC23H4.09),
and isp6 (SPAC4A8.04) and psp3 (SPAC1006.01). For example, at least
three genes comprising psp3 (SPAC1006.01), isp6 (SPAC4A8.04) and
ppp53 (SPAP14E8.04) are preferred (see Table 3, which appears
later).
[0067] A particularly preferred combination consists of at least
four genes comprising ppp53 (SPAP14E8.04), isp6 (SPAC4A8.04), psp3
(SPAC1006.01) and ppp16 (SPBC1711.12), more preferably at least
five genes comprising ppp53 (SPAP14E8.04), isp6 (SPAC4A8.04), psp3
(SPAC1006.01), ppp16 (SPBC1711.12) and ppp22 (SPBC14C8.03). When
the targets are at least 6 genes, it is further preferred to
combine sxa2 (SPAC1296.03c) with these five genes (see Table 3,
which appears later).
[0068] The maximum number of genes to be disrupted is not limited,
as long as the object of the present invention is attained.
However, disruption of too many genes tends to produce unfavorable
effects such as a low growth rate. In the present invention, the
relative growth rate of the gene-disrupted host (the growth rate in
relation to the intact S. pombe strain before gene disruption) is
preferably at least 0.6, particularly at least 0.8. In the present
invention, it has little significance to disrupt genes whose
disruption hardly improves the expression efficiency of foreign
genes, though their disruption may not reduce the growth rate so
much. For these reasons, it is appropriate to estimate that the
maximum number of genes to be disrupted is 20, preferably 10.
[0069] The present invention further provides a host (i.e., a
transformant) carrying a gene (hereinafter referred to as a foreign
gene) encoding a protein extrinsic to the host (hereinafter
referred to as a heterologous protein) recombinantly introduced
therein, and a method of producing a heterologous protein which
comprises culturing the transformant to allow it to produce the
heterologous protein and recovering the heterologous protein.
[0070] Though there are no restrictions on the heterologous protein
to be produced by the improved host of the present invention, it is
preferably a protein produced by multicellular organisms such as
animals and plants, especially a protein produced by a mammal
(inclusive of human) such as human growth hormone. Such a protein
is rarely obtained with high activity from a prokaryotic host
microorganism such as E. coli, but is usually obtained from an
animal cell line such as CHO used as the host with low production
efficiency. The use of the genetically modified eukaryotic host
microorganism of the present invention is considered to solve these
problems.
[0071] For genetic transformation using yeasts as the host, various
expression systems, especially expression vectors and expression
vectors with a secretion signal gene, which allow efficient and
stable production of heterologous proteins, have been developed,
and they are widely available to genetically transform the improved
host of the present invention. For example, expression systems
disclosed in Japanese Patent No. 2776085, JP-A-07-163373,
JP-A-10-215867, JP-A-10-234375, JP-A-11-192094, JP-A-2000-136199,
JP-A-2000-262284 and WO96/023890 can be widely used in the method
of producing a heterologous protein of the present invention.
[0072] Now, the present invention will be described in further
detail by reference to specific Examples. The following Examples
exemplify deletion of protease-related genes in S. pombe through
replacement with marker genes, and hereinafter deletion of genes
will be referred to as disruption.
[0073] Hereinafter, percentages (%) are expressed in weight percent
unless otherwise noted.
Example 1
Transformation of S. Pombe Strains and Cultivation Conditions
[0074] All S. pombe strains were derived from ARC001 (h-leu1-32)
and ARC010 (h.sup.-leu1-32ura4-D18) and transformed by the lithium
acetate transformation method (Okazaki K et al. 1990, Nucleic Acids
Res 18:6485-8489.). Transformant mixtures were plated onto MMA
(minimal medium agar, Qbiogene) or MMA+Leu (supplemented with
leucine) and incubated at 32.degree. C. for 3 to 4 days. The
cultures were grown in YES medium [yeast extract with supplements,
0.5% Bactoyeast extract, (Becton, Dickinson and Company), 3%
glucose and SP supplements (Qbiogene)], YPD medium [1% Bactoyeast
extract, 2% Bacto peptone (Becton, Dickinson and Company) and 2%
glucose] and SDC-Ura and SDC-Ura-Leu media (synthetic complete
dextrose media lacking uracil or both uracil and leucine
Qbiogene).
<Preparation of Recombinant DNA>
[0075] Recombinant DNA procedures were followed as described in
Sambrook et al. (Sambrook J et al. 1989. Molecular Cloning. A
Laboratory Manual. 2.sup.nd ed. Cold Spring Harbor Laboratory
Press, Cold Spring Harbor). Restriction enzymes and DNA
modification enzymes were obtained from Takara Bio, Toyobo, Nippon
Gene and Roche Diagnostics. Gene disruption fragments were prepared
by PCR amplification using KOD Dash DNA polymerase (TOYOBO). All
enzymes were used according to the suppliers' protocols.
Escherichiacoli strain DH5 (Toyobo) was used for plasmid
preparation. DNA sequencing was carried out with the DNA sequencer
ABI Prism 3100 genetic analyzer (Applied Biosystems). Yeast genomic
DNA was prepared using a DNeasy genomic DNA kit (Qiagen).
<Construction of Protease Gene-Deficient S. Pombe
Strains>
[0076] 62 genes were listed as putative S. pombe proteases (ppp) on
the basis of the chromosome sequence data (Wood et al., 2002,
http://www.sanger.ac.uk./Projects/Spombe/) and the S. pombe Gene DB
(http://www.genedb.org/genedb.pombe/). Table 1 shows known and
putative S. pombe proteases. The ORFs (open reading frames) of the
listed genes were disrupted by PCR-mediated homologous
recombination (Bahler J et al. 1998. Yeast 14: 943-91) using a ura4
gene cassette as the selection marker. 200 to 300-bp sequences
flanking the 5'- and 3'-termini of each target ORF were amplified
from the genomic DNA of the parental S. pombe strain ARC001 by two
separate PCRs using an appropriate gene adapter pair designed so
that the 5' and 3' termini fuse separately. Then, by fusion
extension PCR, the ura4 gene cassette was sandwiched between each
pair of the resultant fusion PCR products to obtain vectors for
protease gene disruption (hereinafter referred to as gene
disruption vectors).
[0077] S. pombe strain ARC010 was transformed with the gene
disruption vectors. The transformed cells were grown on minimal
medium, and colonies of uracil-unrequiring strains formed in the
minimal medium (MMA+Leu plates) were obtained. Disruption of
protease genes in the strains was confirmed by colony PCR and DNA
sequencing.
TABLE-US-00001 TABLE 1 S. pombe putative proteases selected for
gene disruption. Protease S. cerevisiae Deficient strain No. Gene
Systematic name Description of gene products family homologue name
1 ape1 SPBC1921.05 Aminopeptidase I M1 APE2 MGF0006 2 cdb4
SPAC23H4.09 Metallopeptidase M24X no MGF0071 3 cps1 SPAC24C9.08
Vacuolar carboxypeptidase S* M20E CPS1 MGF0014 4 cpy1 SPAC19G12.10C
Carboxypeptidase Y S10 PRC1 MGF0030 5 cut1 SPCC5E4.04 Separin,
(caspase-like protease) C50 ESP1 no disruption 6 gpi8 SPCC11E10.02c
GPI anchor biosynthesis protease C13 GPI8 no disruption 7 isp6
SPAC4A8.04 Subtilase-type proteinase* S8A PRB1 MGF0056 8 krp1
SPAC22E12.09C Kexin, (dibasic processing endoprotease) S8B KEX2
lethal** 9 mas2 SPBC18E5.12C Mitochondrial processing peptidase a
M16B MAS2 MGF0029 10 mde10 SPAC17A5.04C Zinc metallopeptidase* M12B
no MGF0018 11 oct1 SPAC1F3.10C Mitochondrial intermediate
peptidase* M3A OCT1 MGF0037 12 pca1 SPCC1840.04 Cysteine protease*
C14 YOR197W MGF0058 13 pgp1 SPCC1259.10 Endopeptidase* M22 YDL104C
MGF0034 14 pgp2 SPBC16D10.03 Glycoprotease family M22 KAE1 no
disruption 15 psp3 SPAC1006.01 Subtilase-type peptidase* S8A PRB1
MGF0031 16 qcr1 SPBP23A10.15C Mitochondrial processing peptidase
.beta. M16B MAS1 MGF0025 17 qcr2 SPCC613.10 Mitochondrial signal
processing protease M16B YPR191W MGF0157 18 sxa1 SPAC26A3.01
Aspartic proteinase A1 no MGF0005 19 sxa2 SPAC1296.03C Serine
carboxypeptidase* S10 no MGF0008 20 yps1 SPCC1795.09 Aspartic
proteinase* A1 YPS1 MGF0004 21 ppp10 SPAC1687.02 CAAX prenyl
protease 2* U48 RCE1 no disruption 22 ppp11 SPAC3H1.05 CAAX prenyl
protease 1* M48 STE24 MGF0019 23 ppp16 SPBC1711.12 Dipeptidyl
peptidase* S9C no MGF0020 24 ppp17 SPCC965.12 Dipeptidase* M19 no
MGF0021 25 ppp18 SPAC13A11.05 Cytosol aminopeptidase M17 no MGF0011
26 ppp20 SPAC4F10.02 Aspartyl aminopeptidase* M18 APE1 MGF0007 27
ppp21 SPAC14C4.15C Dipeptidyl aminopeptidase S9B DAP2 MGF0012 28
ppp22 SPBC14C8.03 Methionine metallopeptidase* M24A MAP2 MGF0022 29
ppp23 SPBC3E7.10 Methionine aminopeptidase* M24A MAP1 MGF0023 30
ppp28 SPBC337.07C Carboxypeptidase* M14A ECM14 MGF0013 31 ppp33
SPBC16G5.09 Serine carboxypeptidase* S10 KEX1 MGF0009 32 ppp34
SPACUNK4.12C Zinc-protease* M16A STE23 MGF0015 33 ppp36 SPCC965.04C
Metallopeptidase* M41 YME1 MGF0017 34 ppp37 SPBC119.17 Zinc
metallopeptidase* M16C YDR430C MGF0032 35 ppp39 SPAC22F3.06C
ATP-dependent protease* S16 YBL022C MGF0033 36 ppp43 SPAC12B10.05
Metallopeptidase* M24B YER078C MGF0038 37 ppp44 SPAC3A11.10C
Microsomal dipeptidase* M19 no MGF0039 38 ppp45 SPBC1685.05 Serine
protease* S1C YNL123W MGF0057 39 ppp50 SPACUNK4.08 Dipeptidyl
aminopeptidase S9B DAP2 MGF0059 40 ppp51 SPAC22G7.01C
Aminopeptidase* M24B YLL029W MGF0062 41 ppp52 SPBC18A7.01
Aminopeptidase* M24B no MGF0144 42 ppp53 SPAP14E8.04 Zinc
metallopeptidase* M48B YKR087C MGF0063 43 ppp54 SPAC3H1.02C Zinc
metallopeptidase* M16C YOL098C MGF0068 44 ppp57 SPAC607.06C Zinc
metallopeptidase* M10B Y1L108W MGF0069 45 ppp58 SPBC1198.08
Metallopeptidase* M20A YFR044C MGF0065 46 ppp59 SPBC354.09C
Metalloprotease* M28X YJR126C MGF0066 47 ppp60 SPCC1919.12C
Metalloprotease* M28X YBR074W MGF0067 48 ppp61 SPCC1259.02C
Metallopeptidase* M28X YBR074W no disruption 49 ppp62 SPAP8A3.12C
Tripeptidylpeptidase* S8A no MGF0226 50 ppp63 SPBC23E6.05
Metallopeptidase* M24X YDR101C MGF0070 51 ppp67 SPBC2D10.07C
Mitochondrial protease subunit 2 S26A IMP2 no disruption 52 ppp68
SPBC336.13C Mitochondrial protease subunit 1 S26A IMP2 MGF0072 53
ppp69 SPBC1685.03 Signal sequence processing peptidase S26B SEC11
no disruption 54 ppp72 SPBC13E7.11 Mitochondrial signal processing
protease S54 PCP1 MGF0088 55 ppp73 SPBP4H10.10 Mitochondrial signal
processing protease S54 PCP1 MGF0089 56 ppp75 SPCC790.03 Rhomboid
family protease S54 YPL246C MGF0153 57 ppp76 SPBC543.09
Mitochondrial signal processing protease M41 YMR089C no disruption
58 ppp78 SPCC757.05c Metallopeptidase M20A no MGF0154 59 ppp79
SPAC19B12.06c Rhomboid family protease S54 YPL246C MGF0158 60 ppp80
SPAC19B12.08 Peptidase* C54 YNL223W MGF0159 61 ppp81 SPAC25B8.17
Signal peptide peptidase A22B YKL100C MGF0160 62 ppp85 SPCC1322.05c
Metalloprotease* M1 YNL045W no disruption *Putative proteases.
**Ref.: Davey et al., 1994
<Measurement of Cell Growth Rate>
[0078] The growth rates of the resulting protease gene-deficient S.
pombe strains were measured. Growth curves for the S. pombe strains
were obtained using a biophotorecorder (TN-1506, Advantec). Cells
were cultured in 5 ml YTS medium in L-tubes at 32.degree. C. with
shaking. Turbidity was monitored every 5 minutes at an absorbance
of 660 nm. The relative maximum growth rates (.mu..sub.max) of 52
protease disruptants were calculated using the .mu..sub.max value
(0.26-0.30/H) for ARC001 strain as a control. FIG. 1 shows the
relative maximum growth rates of the protease gene disruptants.
[0079] The results in FIG. 1 indicate that some protease genes
affected cell growth rates. For example, disruption of nine
protease genes (qcr2, oct1, ppp23, ppp37, ppp72m ppp73, ppp79 and
ppp81) resulted in an over 20% decrease in .mu..sub.max as compared
with the ARC001 control. A decrease exceeding 40% was obtained by
deleting three mitochondrial signal processing proteases (qcr2,
ppp72 and ppp73), indicating that all these protease genes are very
important in the cell respiration process in S. pombe and that
their disruption does not favor protein expression. Such growth
rate reductions hinder efficient protein production. On the other
hand, a .mu..sub.max increase exceeding 20% was obtained with
disruption of cdb4, ppp11, ppp17, ppp51, ppp54, ppp57, ppp60 and
ppp63, indicating that such growth rate increases do not hinder
protein production.
Example 2
Construction of r-hGH-Producing Transformant ARC001(hCH)
[0080] A multicassette vector for secretory expression of r-hGH was
prepared and used to construct a r-hGH-producing transformant
ARC001(hGH). A 594-bp hGH-ORF was artificially synthesized (Gene.
Art) according to a codon table (highly biased) favorable for
translation in S. pombe obtained from the ORF sequences of the
high-expression genes, adh, tpi and gdp1, in S. pombe. By using
restriction enzymes AflII and BamHI, from the integrative vector
pXL4 (Isoai et al., 2002 Biotechnol Bioeng 80: 22-32.), the
synthetic hGH gene fragment was integrated with the frame with a
downstream P3 secretion signal sequence (WO96/023890). A
multicassette expression vector, pTL2P3hGHhb(M5)-8XL, carrying
eight tandem copies of the hGH expression cassette
(hCMV-promoter/P3-signal/hGH-ORF/terminator) was then constructed
as previously described (Ikeda et al., 2004 J. Biosci Bioeng 98:
366-373). Transformation was done by inserting the eight tandem
copies of the hGH expression cassette from the expression vector
into the luel locus in the protease-deficient S. pombe strains
obtained as described previously. After 2 to 3 days of cultivation
in SDC-Leu-Ura, leucine-unrequiring strains were harvested and
incubated again in YPD medium (in 24-well plates) at 32.degree. C.
with shaking, and then secretion of r-HGH was confirmed.
[0081] FIG. 2 illustrates the structure of the multiple expression
cassette vector constructed for secretory expression of r-hGH. The
S. pombe high-bias codon-type hGH structural gene having AflII and
BamHI sites at the termini was placed downstream of the P3
secretion signal sequence and inserted into the multicloning site
(MCS) of the integrative expression vector pXL4. A SpeI site and a
NheI site were placed at either terminus of the secretory
expression cassette [hCMV-promoter-P3-hGHhb-terminator] in the
resulting vector pTL2P3hGHhb(M5)-1XL to obtain the 8XL expression
vector pTL2P3hGHhb(M5)-8XL. The two intergenic leu1+ gene sequences
in the construct were utilized to integrate the multiple expression
cassettes into the leu1 locus of the host strain ARC001.
<Detection of r-hGH Secreted from the Transformant
ARC001(hGH)>
[0082] Secretion and degradation of r-hGH by the transformant
ARC001(hGH) obtained as described above were confirmed. The
transformant was grown on YPD medium in glass tubes or 24-well
plates at 32.degree. C. with shaking, and 0.5-1.0 ml of the culture
was withdrawn at various times. The culture supernatants were
subjected to a series of SDS-PAGE analysis after precipitation with
TCA (100 trichloroacetic acid (final concentration)). The SDS-PAGE
analysis was performed according to standard procedures under
reductive conditions with 18% polyacrylamide gels (TEFCO). The gels
were stained with CBB to detect hGH. From four clones, one positive
clone was selected and stored at -80.degree. C. in 25%
glycerol.
[0083] FIG. 3A shows the time-course analysis of r-hGH secretion
from the transformant ARC001(hGH): lane 1 is a molecular weight
marker (unit: kilodalton); lane hGH is 1 g isolated intact human
hGH; lanes 24-144 are 0.5 ml supernatants harvested from the
transformant ARC001(hGH) at 24 to 144 hours subjected to
SDS-polyacrylamide gel electrophoresis (SDS-PAGE) after
precipitation with TCA. CBB (Coomassie brilliant blue) staining was
used for detection.
[0084] The time-course analysis of r-hGH secretion by SDS-PAGE
revealed that the apparent r-hGH secretion drastically decreased
after 48 hours of cultivation as shown in FIG. 3A, indicating the
possibility of proteolysis of the r-hGH secreted into the culture
medium.
<Screening of hGH-Degrading Extracellular Proteases Using
Protease Inhibitors>
[0085] Various proteases were added to a culture of the
transformant obtained above to screen extracellular proteases for
r-hGH degradation. The r-hGH-producing S. pombe strain
ARC001[pTL2P3hGHhp(M5)-8XL] was subcultured in 20 ml YPD medium at
32.degree. C. for 24 hours, and the 24-hour cell culture was
transferred to a 24-well plate (1.0 ml/well) for screening of
protease activity using a variety of protease inhibitors, except
that at this stage, a portion of the culture supernatant was stored
at -20.degree. C. as the positive control. After addition of a
variety of different protease inhibitors to the wells, the cells
were incubated at 32.degree. C. for 2 days with shaking. As the
protease inhibitors, the 10 proteases contained in a protease
inhibitor set (Roche Diagnostics) were added to the respective
wells in given amounts, except for the negative control well. A 0.5
ml supernatant was collected from the cell culture in YPD medium in
each well after 72 hours and 96 hours, concentrated by
precipitation with TCA (10% w/v) and analyzed by SDS-PAGE.
[0086] FIG. 3B shows the effect of each protease inhibitor in the
culture of the transformant ARC001(hGH) (time-course of r-hGH
secretion) analyzed by SDS-PAGE (CBB staining). Lane M is a
molecular weight marker (unit: kilodalton); Lane hGH is 0.5 .mu.g
native hGH; lane C is the control sample of the supernatant of the
24-hour culture in YPD medium withdrawn before addition of various
proteases and stored at -20.degree. C.; lane -PI is the control
sample of the culture supernatant without protease inhibitor
treatment; lanes 3-12 are samples of the culture supernatants
treated with protease inhibitors.
[0087] The results revealed that addition of the inhibitor
chymostatin to culture increased the 22-kDa major fragment from the
secretory r-hGH, as shown in FIG. 3B. Addition of antipain slightly
inhibited r-hGH degradation, too. Antipain inhibits papain-like
cysteine proteases (such as papain) and some serine proteases such
as trypsin and plasmin. Chymostatin inhibits mainly serine
proteases with chymostatin-like specificity (such as chymotripsin,
chymases and cathepsin G) and with some cysteine proteases
including cathepsin B, H and L. This suggests the possibility that
some unknown chymostatin-sensitive serine (and/or a few cysteine)
proteases secreted into the culture (or onto the cell surface) are
responsible for proteolysis during cultivation.
<Analysis of Secretory r-hGH from the Transformant
ARC001(hGH)>
[0088] The time-course of the level of secretory r-hGH from the
transformant ARC001(hGH) was analyzed. Protease gene disruptants
and ARC001 were transformed with the hGH expression vector, and 0.5
ml supernatants were withdrawn from the cultures of the
transformants in YPD medium after 72 and 96 hours of cultivation,
concentrated by TCA precipitation and analyzed by SDS-PAGE under
reductive conditions on 18% polyacrylamide gels followed by
staining with Coomassie brilliant blue R-250. Each strain is
indicated by the deleted protease gene: As the molecular weight
marker, Bench Mark prestained protein ladder (Invitrogen) was used.
The results are shown in FIG. 4.
[0089] As is shown in FIG. 4, differences in secretory r-hGH level
were observed among the ARC001(hGH) transformants. r-hGH degration
was reduced in 12 ARC001(hGH) transformants (S. pombe transformants
in which protease genes sxa2, psp3, isp6, cdb4, ppp22, ppp51,
ppp52, ppp60 or ppp79 gene was disrupted). Among these proteases,
sxa2, psp3, isp6 and ppp7 are serine proteases, while cdb4, ppp22,
ppp51, ppp52 and ppp60 belong to the metalloprotease gene family.
Therefore, it is suggested that in addition to the expected serine
proteases, some metalloproteases are involved in responsible for
extracellular proteolysis of secretory r-hGH.
[0090] As shown in FIG. 4, the level of r-hGH expression was higher
in the sxa2, psp3, pp 51 and ppp52 disruptants of S. pombe, which
are indicated by arrows, than in other protease disruptants, and
even higher than in a ppp16 disruptant (Examples in
WO02/101038).
Example 3
Construction of Multiple Protease Related Gene Disruptants of S.
Pombe
[0091] Among the 52 single protease related gene disruptants
obtained by single disruption of proteases in S. pombe in Examples
1 and 2 (Non-patent Document 1), thirteen were chosen as the target
protease genes for multiple disruption and listed in Table 2. The
ORFs (open reading frames) of the protease genes listed in Table 2
were disrupted by the PCR-mediated homologous recombination
(Non-patent Document 1) using a ura4 gene cassette as the selection
marker. 200 to 300-bp sequences flanking the 5'- and 3'-termini of
the target ORFs were amplified from the genomic DNA of the parental
S. pombe strain ARC001 by two separate PCRs using appropriate gene
adapter pairs designed so that the 5' and 3' termini fuse
separately.
[0092] Then, by fusion extension PCR, the ura4 gene cassette was
sandwiched between each pair of the resultant fusion PCR products
to obtain protease gene disruption vectors (hereinafter referred to
as gene disruption vectors).
[0093] S. pomber strain ARC010 was transformed with the gene
disruption vectors. The transformed cells were grown on minimal
medium, and colonies of uracil-unrequiring strains formed in the
minimal medium (MMA+Leu plates) were obtained. Disruption of
protease related genes in the strains was confirmed by colony PCR
and DNA sequencing.
[0094] The confirmed protease related gene disruptants were grown
on MMA+Leu+Ura+FOA medium, and colonies of uracil-requiring strains
were harvested. Protease gene disruption was repeated on the
harvested strains to give the multiple protease related gene
disruptants shown in Table 3.
TABLE-US-00002 TABLE 2 List of protease genes (including putative
genes) as targets for multiple disruption Gene Protease No. name
Systematic name Descriptions family 1 Cdb4 SPAC23H4.09
Metallopeptidase M24X 2 isp6 SPAC4A8.04 Subtilase-type proteinase
S8A 3 mas2 SPBC18E5.12c Mitochondrial M16B processing peptidase
.alpha. 4 pgp1 SPCC1259.10 Endopeptidase M22 5 ppp16 SPBC1711.12
Dipeptidyl peptidase S9C 6 ppp20 SPAC4F10.02 Aspartyl
aminopeptidase M18 7 ppp22 SPBC14C8.03 Methionine M24A
metallopeptidase 8 ppp51 SPAC22G7.01c Aminopeptidase M24B 9 ppp52
SPBC18A7.01 Aminopeptidase M24B 10 ppp53 SPAP14E8.04 Zinc
metallopeptidase M48B 11 ppp80 SPAC19B12.08 Peptidase C54 12 psp3
SPAC1006.01 Subtilase-type peptidase S8A 13 sxa2 SPAC1296.03c
Serine carboxypeptidase S10
TABLE-US-00003 TABLE 3 List of multiple protease related gene
disruptants of S. pombe Strain Groups Disrupted protease genes in
each strain name A A1 psp3 MGF241 A2 psp3- isp6 MGF242 A3 psp3-
isp6- ppp53 MGF265 A4-1 psp3- isp6- ppp53- cdb4 MGF279 A4-2 psp3-
isp6- ppp53- ppp16 MGF281 A4-3 psp3- isp6- ppp53- ppp51 MGF280 A5
psp3- isp6- ppp53- ppp16- ppp22 MGF311 A6 psp3- isp6- ppp53- ppp16-
ppp22- sxa2 MGF323 A7-1 psp3- isp6- ppp53- ppp16- ppp22- sxa2- pgp1
MGF339 A7-2 psp3- isp6- ppp53- ppp16- ppp22- sxa2- MGF340 ppp20
A7-3 psp3- isp6- ppp53- ppp16- ppp22- sxa2- MGF341 ppp80 A8 psp3-
isp6- ppp53- ppp16- ppp22- sxa2- MGF433 ppp80- ppp20 B B3 psp3-
isp6- cdb4 MGF264 B4-1 psp3- isp6- cdb4- sxa2 MGF276 B4-2 psp3-
isp6- cdb4- mas2 MGF277 B4-3 psp3- isp6- cdb4- ppp51 MGF278 B5
psp3- isp6- cdb4- sxa2- ppp52 MGF317
<Measurement of Cell Growth Rate>
[0095] The growth rates of the resulting protease related gene
disruptants of S. pombe were measured. Growth curves for the S.
pombe strains were obtained using a biophotorecorder (TN-1506,
Advantec). Cells were cultured in 5 ml YES medium in L-tubes at
32.degree. C. with shaking. Turbidity was monitored every 5 minutes
at an absorbance of 660 nm. The relative maximum growth rates
(.mu..sub.max) of 17 multiple protease disruptants were calculated
using the .mu..sub.max value (0.26-0.30/H) for ARC001 strain as a
control.
[0096] FIG. 5 shows the relative maximum growth rates of the
protease related gene disruptants. The relative .mu..sub.max value
for each disruptant was calculated from the .mu..sub.max
measurement for the disruptant using the .mu..sub.max value
(0.26-0.30/H) for the ARC001 strain (indicated as A0) as a control.
In the graph, the ordinate indicates relative .mu..sub.max, while
the abscissa indicates the strain names of the disruptants (listed
in Table 3).
[0097] The results indicate that some protease genes affected cell
growth rates. It turned out that for the sextuple and septuple
disruptants, the relative growth rates were lower by about 10 to
20%, and the decrease was significant when both of the protease
genes ppp22 and ppp20 were disrupted. Because the two genes had
little effect on cell growth rate individually, it is suggested
that multiple disruption of protease genes has combined effects.
However, the decreases in growth rate were small on the whole at a
level of from about 10 to 20% and are unlikely to affect the actual
production. In order to examine whether the decrease in relative
growth rate affects maximum cell density, the ultimate cell density
of the multiple disruptant A8 (MGF433), which showed the lowest
relative growth rate, in YES medium was actually determined after 4
days of incubation. It was found that on the contrary, the maximum
OD (660 nm) for the octuple disruptant (A8) was larger by over 10%
than that for the wild-type strain ARC001. The main reason is
probably because at the sacrifice of growth rate, the multiple
disruptant used nutrients efficiently, though slowly, for continued
cell division without wasting them in ethanolic fermentation.
Therefore, such enhanced cell growth is unlikely to hinder protein
production.
Example 4
Evaluation of Usefulness of Multiple Protease Disruptants by hGH
Production
[0098] Examples 1 and 2 (Non-patent Document 1) describe evaluation
of the usefulness of single protease disruptants using human growth
hormone (hereinafter referred to as hGH), i.e., construction of the
hGH-producing transformant ARC001(hGH) and detection of the
secretory hGH from the transformant ARC001(hGH), its usefulness as
a model heterologous protein for secretory production. In Example
4, usefulness of multiple protease disruptants was evaluated on the
basis of secretory production of hGH as a heterologous protein
model by experimentally examining inhibitory effect on multiple
protease disruption on degradation of the product. Multiple
protease disruptants were transformed with an integrative secretory
hGH expression vector pTL2P3hGHhb(M5)-8XL described in Example 2
(Non-patent Document 1) by the lithium acetate method to make the
multiple protease disruptants express hGH. Among six transformants,
one clone which produced hGH most stably was selected, and hGH
secretion was monitored. Further, reproducibility of the experiment
was confirmed using other clones.
[0099] Especially, the time courses of hGH productions by some
multiple disruptants from both groups were analyzed in detail by
SDS-PAGE. The results are shown in FIG. 6.
[0100] FIG. 6 shows the time course analysis of secretory hGH
productions by multiple protease gene disruptants of S. pombe by
SDS-PAGE. hGH secretions at various times were analyzed by SDS-PAGE
(followed by Coomassie brilliant blue staining). A 0.5 ml
supernatant was collected from the culture of each disruptant after
given cultivation times, concentrated by precipitation with TCA and
analyzed by SDS-PAGE. Above the respective lanes are the strain
names of the disruptants (previously shown in Table 3), and below
the lanes are the additively deleted protease related genes. Lane
A0 (nond: non-disrupted strain) is the strain ARC001 with no
disrupted protease genes.
[0101] The results of the SDS-PAGE analysis demonstrate that
multiple protease related gene disruption led to remarkable
increase in production of secretory hGH and clearly indicate that
hGH production was almost the same among disruptants at 24 h but
became appreciably different at 48 h and became clearly greater
with the level of gene disruption. It is suggested that the basal
hGH expression levels were almost similar among disruptants, and
the difference in apparent expression level from 72 h onward was
mainly attributed to the difference in hGH degradation. It turned
out that the secretory hGH production in the non-disruptant A0
peaked at 48 hr and then drastically decreased, while the secretory
hGH production in the multiple disruptants continued to increase
until 72 h or 96 h, and the increase enhanced with the level of
multiple disruption. Such a phenomenon was markedly observed in the
multiple disruptants in Group A, and the high hGH levels were
maintained until 120 h in the quintuple and sextuple disruptants A5
and A6. It is suggested that in multiple disruption, hGH
degradation is slowed down more effectively as the number of
disrupted protease related genes increases, but its effect is
dependent of the combination of disrupted protease genes.
Therefore, in multiple disruption of protease genes, it is
important to try many disruption combinations and choose the best
combination. In this respect, the present approach is proven to be
useful.
[0102] Because in the above hGH expression experiments, there was
little difference in effect between disruptants with more than five
disrupted genes in Group A, experiments with the three septuple
disruptants in Group A were carried out for an incubation time
prolonged to 216 h. The SDS-PAGE results are shown in FIG. 7.
[0103] FIG. 7 shows the results of time course analysis of the hGH
secretions by sextuple and septuple protease disruptants of S.
pombe by SDS-PAGE. A 0.5 ml supernatant was collected from the
culture of each disruptant was collected after given cultivation
times, concentrated by precipitation with TCA and analyzed by
SDS-PAGE (followed by Coomassie brilliant blue staining). Above the
respective lanes are the strain names of the disruptants
(previously shown in Table 3). Lane A0 (nond: non-disrupted strain)
is the strain ARC001 with no disrupted protease genes.
[0104] The results indicate that the septuple disruptants and the
sextuple disruptant were similarly effective and did not differ
much in secretory hGH production until 216 h. The experimental
system employed has its detection limit and could hardly prove the
difference between sextuple and higher disruptants in
effectiveness. The difficulty may be solved by an experimental
system using a more protease-sensitive heterologous protein as the
model protein.
INDUSTRIAL APPLICABILITY
[0105] In the present invention, the efficiency of production of a
heterologous protein by a transformant of the fission yeast S.
pombe is improved by deleting or inactivating one or more
protease-related genes in the host cells. Such a protease
disruptant can be widely used for production of protease-sensitive
heterologous proteins.
[0106] The entire disclosures of Japanese Patent Application No.
2005-225638 filed on Aug. 3, 2005 and Japanese Patent Application
No. 2006-160347 filed on Jun. 8, 2006 including specifications,
claims, drawings and summaries are incorporated herein by reference
in their entireties.
* * * * *
References