JPH02156881A - Saccharomyces cereviciae pop2 - Google Patents

Abstract

NEW MATERIAL:Saccharomyces cereviciae POP2 (FERM 10418) which is a mutant of Saccharomyces cerevisiae YNN27 and excretes protein in a high degree. USE:It is useful as a host which can excrete the protein in a high degree, when different kinds of proteins are produced through a gene recombination technique. PREPARATION:At first, PGK gene promoter, secretion signal of alpha-factor of yeast (MF alpha1) in a yeast, mouse alpha-amylase gene and the terminator area of MF alpha1 gene are inserted into a known plasmid Ylp5 to prepare plasmid pSAKO 31. The straight chain DNA of the plasmid is inserted into Saccharomyces cerevisiae YNN27 chromosome and treated with ethyl methanesulfonate to induce mutation.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to a novel yeast, Saccharomyces cerevisiae pop2, which is highly dichotomous.

(Conventional technology and problems) Yeast is a useful microorganism with cell structures and functions that are characteristic of higher organisms, and is closely related to human daily life as a raw material for foods, medicines, feeds, etc. It is expected to be developed as a host in genetic engineering.

However, when producing useful substances (heterologous proteins) using gene recombination technology using yeast as a host, it is difficult to purify the produced heterologous proteins because the yeast cell surface layer has a strong cell wall outside the cell membrane. Various attempts have been made to secrete the product outside the bacterial cells in order to simplify purification, but no satisfactory results have yet been obtained.

(Means for Solving the Problems) The present inventors have discovered that the yeast Satucharomyces cerevisiae (Sa
When producing heterologous proteins using genetic recombination technology using C. ccharomyces cerevisiae as a host, research aimed at obtaining a host that can efficiently secrete the produced substances, and as a result, it was found that the host itself does not have the ability to secrete proteins. Mutants obtained by inserting certain integrative plasmids into the chromosome of the small well-known yeast Saccharomyces cerevisiae YNN27 and subsequent treatment with ethyl methanesulfonate highly secrete heterologous proteins in specific media. The present invention was achieved by confirming this.

That is, the gist of the present invention resides in Satucharomyces cerevisiae pop2, which is a mutant strain of Satucharomyces cerevisiae YNN27 and has been deposited as Microtech Research Institute No. 10418, which secretes proteins to a high degree.

To explain the present invention in detail, the yeast Satucharomyces cerevisiae pop2 of the present invention is
No. (FERM P-10418), the yeast Saccharomyces cerevisiae YNN27 [Jo
Urnaof Molecular Biology,
Volume 158, pp. 157-179 (1982 Blasmi I
'Insertion of linear DNA of psAKO31] First, insert linear DNA of 031 into Saccharomyces cerevisiae YNN27 into plasmid pSA prepared by the method described later.
NA is introduced and inserted into the chromosome of YNN27.

As shown in Figure 2, the plasmid psAKO3+ used here contains PGK, which is known as the phosphoglycerate kinase gene of Satucharomyces cerevisiae.
Gene [Nucleic Ac1ds Re5earc
h, Vol. 10, No. 23, pp. 7791-7808 (198
3) A promoter obtained by inserting the secretion signal of α-factor (MFαl), a columnar mating factor of yeast, and the terminator region of mouse α-amylase gene and anal α1 gene into the well-known plasmid V195. and is prepared, for example, by the following steps.

[Preparation of plasmid pSA031 (a) Plasmid psAKOO9 (7) Preparation of well-known plasmid YIp5 [Proceedings of th
eNational Academy of 5cie
nces of the U, S, A,.

76, pp. 1035-1039 (1979)] B
After cutting with amHI and blunting the ends with Mung bean nuclease to destroy the BamH■ cleavage site, plasmid psAKOO9 is prepared by self-ligation (see Figure 2).

(b) Preparation of plasmid ρREI078 (7.5 kb) Japanese Patent Application Laid-Open No. 63-133987 and [Mo1ecul
ar and Cellular Biology, Volume 7,
3185-3193 (1987)] of Satucharomyces cerevisiae.
Promoter sequence, leader (secretion signal) sequence, terminator sequence and T of the pheromone gene MFα1
The known plasmid I"pRE+05 has the ori of RPI, 2 μm and pBR322, and the ampicillin resistance gene (Ap'), and the human β-endorphin gene is inserted between the leader sequence and the terminator sequence.
Plasmid pRE+078 is prepared by using 9 and replacing its promoter sequence with the promoter sequence of phosphoglycerate kinase (PG).

That is, the known plasmid pMA91 containing the promoter sequence and terminator sequence of PG [Gene, Vol. 24.

pages 1-14 (1983) cut with BglII and D
After making blunt ends with NA polymerase I, cut with EcoRI to create 1.5 kb containing the promoter sequence in PG.
EcoRI-(BglII) fragment (1) is isolated.

On the other hand, the above pRE+059 was cut with Hinfl, the ends were filled in, and then cut with 5alI to create a 337 bp fragment (I
I) is isolated. In addition, the SatI-AatII fragment (m) containing the human β-endorphin gene of pRE+059 and the ori of TRPI, 2Bm and pBR322 were used.
and EcoRI-AatII containing the A +1 r region
The fragment (rV) is obtained by cleaving with the respective restriction enzymes. By simultaneously ligating (1) to (IV) obtained above, pRE+078 (7.5 kb) is produced (see Figure 1).

(c) Preparation of plasmid ρ5AKO28 pREI078
psAKO27 from which the human β-endorphin gene has been removed is prepared by cleaving with BamHI and religating. This psAKO27 is cut with EcoRI to isolate a 2.1 kb DNA fragment containing the PG promoter, the MFα1 secretion signal sequence, and the MFα1 terminator sequence. This DNA fragment was added to the ps
5,5k obtained by cutting AKOO9 with EcoRI
028 is prepared in pSA by ligation with the DNA fragment of b (see Figure 2).

(d) Preparation of plasmid psAKO31 The well-known plasmid psAK011 [Genet
ics, Vol. 119, pp. 499-506 (1988).
It is ligated to the Bam)lE site of AK028, and one in which the promoter and amylase gene are linked in the same direction is selected and designated as 031 in plasmid 115A (see Figure 2).

[Introduction of psAKO31 into YNN27] The trasmid ρ5AKO31 obtained as above was cut with Stu I to obtain a linear DNA, and this was introduced into YNN27 to inject psAKO31 into chromosome V of YNN27. A strain containing one copy of linear DNA (hereinafter referred to as YNN)
27/psAKO31/S).

Introduction of the linear DNA of psAKO3+ into YNN27 is carried out by a method known per se. For example, according to the method described in Japanese Patent Application Laid-Open No. 59-28478, YNN27 is cultured using a suitable medium, such as YPD medium, and then the bacteria are collected, suspended in a buffer solution, and mixed with, for example, lithium, rubidium, or cesium. After adding metal ions such as
NA and polyethylene glycol aqueous solution are added, followed by heat treatment at about 40° C. for a short time and washing with sterile water. This method is particularly preferred as a method for introducing DNA into yeast because it does not require protoplastization of bacterial cells.

The cells treated in this manner are suspended in sterile water, and then plated and cultured on a plate from which uracil has been removed from the CSM medium.

After culturing several colonies grown on the plate in YPD medium, DNA was extracted, and the obtained DNA was analyzed by Southern hybridization method to create pSAKO.
'l'NN with one copy of 31 linear DNA integrated
27/psAKO31,/Select.

(2) [Treatment with ethyl methanesulfonate 1 YNN27/ psAKO31/ prepared as above
Then, using ethyl methanesulfonate (hereinafter referred to as EMS), which is known as a mutagenic agent for yeast cells, [Methods in yeast gene]
tics, laboratory manual, C
o1d Spring Harbor Labora-
tory, Colorado Spring Harbor, N.
, Y., (1978)] to prepare the S. cerevisiae pop2 strain.

That is, YNN27/ psAKO31/ obtained above
After culturing S in YPD medium and washing with a buffer solution, prepare to a predetermined cell concentration, add a predetermined amount of EMS to this, treat it, dilute the EMS-treated bacteria, and culture on YPD medium to produce. Approximately 8,000 colonies of bacteria were inoculated onto a YPD plate, the sizes of the resulting halos were compared, and mutant strains with halo diameters more than twice that of the parent strain were collected and incubated at 37°C for 2 hours.
Incubate for days. As a result, one strain that could not grow was selected and named Satucharomyces cerevisiae pop2 strain.

The pop2 strain of the present invention obtained as described above secretes amylase with high efficiency, as shown in Examples below. In addition, YNN2 before processing by EMS? / psAK
O31/S also secretes amylase, but the amount of amylase secreted by the pop2 strain is lower than that of YNN27/ psAKO31/
The amylase secretion amount far exceeds that of S.

Furthermore, by introducing a plasmid containing a gene for another heterologous protein into the pop2 strain of the present invention, the heterologous protein can be secreted.

For example, when the aforementioned plasmid pRE+078 containing the human β-endorphin gene is introduced into the pop2 strain of the present invention, β-endorphin secretion is observed with high efficiency.

The introduction of pRE+078 into the pop2 strain was carried out using the YNN2
The same method as for the introduction of psAKO31 into strain 7 is used to spread the culture on a plate from which tryptophan has been removed from the C5M medium, and isolate the colonies that have grown on this plate. The thus obtained pop2 strain into which pRE 1 078 has been introduced secretes β-endorphin with high efficiency, as shown in the Examples below.

Furthermore, when pRE1078 was introduced into YNN27/psAKO31/S by the same method as above, β
- Secretion of endorphin is observed, but when pRE1078 is introduced into the pop2 strain, the amount of β-endorphin secreted reaches approximately 5 times that amount.

Examples) The present invention will be described in more detail with reference to Examples below, but the present invention is not limited to these Examples unless the gist thereof is exceeded.

In addition, the operations in the following examples were performed according to the following methods I to (2), unless otherwise specified.

■ [DNA cutting and recovery using restriction enzymes] Use the following three types of buffers for cutting with restriction enzymes.
etics (1981) (Cold spring
Harbor, New York). The cutting conditions were as follows: using a restriction enzyme of 2 units/μg DNA;
℃ or 65℃ for 30 minutes.

Then, TE buffer (10 mM Tris-HCl pHs, o
and 1 mM EDTA), remove the phenol with ether, add 2 volumes of ethanol, leave at -20°C for 30 minutes, and then centrifuge to recover the DNA.

(1) Low salt buffer 10 mM Tris-HCl (p)I 7.4), 10
Consists of mM magnesium sulfate and 1 mM dithiothreitol.

(2) medium bulk concentration buffer 50 mM NaCl, 10 mM Tris salt vi (p
H7,4), 10mM magnesium sulfate and 1mM
dithiothreitol.

(3) High salt buffer 100 mH NaCl, 50 mM Tris-HCl (p
) I 7.4) and 10 mM magnesium sulfate.

If [Treatment of DNA fragments with calf small intestine alkaline phosphatase (CIAP) To prevent self-ligation of plasmid DNA by ligase reaction, plasmid DNA was
The fragment cut with the restriction enzyme is treated with CIAP.

Plasmid DNA cut with restriction enzyme (+Op mo1
The 5' end) was dissolved in 50 μM CIAP buffer [50 mM Tris-HCl (pH 9,0), 1 mM magnesium chloride, 0.1 mM zinc chloride, and lmM spermidine, and the 1p rigid 01 end After adding 0.01 units of CIAP and reacting at 37°C for 30 minutes, 10 μm
0.1 M) lithic hydrochloric acid (pf18°0), I M N
aCl, 10mM EDTA, 5μl lO%SD
S.

Add 40 μm water and hold at 65° C. for 15 minutes. After cooling, it is extracted with phenol saturated with TE buffer, the phenol is removed with ether, and the plasmid DNA is recovered by ethanol precipitation.

m [Removal of DNA sticky ends with Mung bean nuclease] DNA with sticky ends was dissolved in 100 μm of a buffer consisting of 30 mM sodium acetate (ptl4,6), 50 mM NaCl and 1 ml ZnCl2, Add to this the M1 of 1μ1 (2 units/μm).
After adding n g b e a n nuclease and treating at 37°C for 10 minutes, a 25% SOS solution of 2711, 1
Add 0 μm 0.5 M Tris-HCl buffer (pHL5) and 10111 8 M LiCl.

(Add 150 μm of phenol and chloroform mixture (1:I) to this for extraction treatment, collect the aqueous layer, add 10 μl of 3M sodium acetate and 200 μm of ethanol, cool to -20°C, and centrifuge. The separated and precipitated DNA is recovered.

IV [ligation by T4 tovA ligase] Ligate 2
DNA fragments were added to ligation buffer [66 mM Tris-HCl (pH 7,5), 6
．． 6 mM magnesium chloride, 10 mM dithiothreitol] and treated at 65°C for 10 minutes, then 66μ of ATP (adenosine triphosphate) was added at 4°C, and T4 DNA ligase was added to the adhesive solution. Add 0.1 unit/μg DNA for ends or 1 unit/μg DNA for blunt ends at 4°C.
After reacting for 18 hours at 65° C. for 10 minutes.

■ [Transformation of yeast (U method) Transform yeast Satucharomyces cerevisiae into 10 ml of YE
Cultured overnight at 30°C in PD medium, harvested and subjected to TE once.
! After washing with buffer, suspend in the same buffer and increase the number of cells to 2X.
Make it 10 cells/ml.

The same amount of 0.2M lithium acetate (
pH 7.5) was added and kept at 30°C for 1 hour, then
Dispense into μ test tubes, add DNA to this at 0°C, and incubate at 0°C.
Mix for 30 minutes. A total of 1 lffi solution containing 100 μm of 70% polyethylene glycol 4000 is added, mixed well, and kept at 30°C for 1 hour, then at 42°C for 5 minutes, centrifuged to collect bacteria, and washed with sterile water.

This is suspended in 500 μm sterilized water, inoculated to 100 μm onto a selective medium, and cultured at 30° C. for 3 to 4 days to obtain a transformed strain.

Example 1 (1) Preparation of plasmid psAKO31 (a) Preparation of plasmid psAKOO9 Plasmid Ylp5 (Bam)
After cutting with lI and blunting the ends with Mun3 bean nuclease to destroy the BaIIIHI cleavage site,
Plasmid psAKOO9 was prepared by self-ligation using T4 DNA ligase (Figure 2).

(b) Preparation of plasmid pRE1078 (7.5 kb) Plasmid pMA91 containing the PGK promoter sequence and terminator sequence was cut with Bg+U, blunt-ended with DNA polymerase ■, and then cut with EcoRI to remove the PGK promoter sequence. Contains 1.5 kb of Eco
Rl-(BglII) fragment (I) was isolated. On the other hand, p
RE+059 was cut with )Iinfl, the ends were filled in, and then cut with 5alI to isolate a 337 bp fragment (n) containing the 5' untranslated region and leader sequence of protein α1.

In addition, the Sat I-AatU fragment (m) containing the β-endorphin gene of pRE+059, TRPI, 2μ
Eco containing n+ and pBR322 ori, Ap'
After cutting the RI-AatII fragment (IV) with the respective restriction enzymes, they were separated by agarose gel electrophoresis,
The target band was obtained by elution.

Fragments (1) to (IV) obtained above were simultaneously combined with T4 [I
Ligate with NA ligase to create pREI078 (7.5kb)
was produced (Fig. 1).

(c) Preparation of 028 into plasmid pSA Cut pREI078 obtained in (b) above with Ban+HI and add T4DNA
psAKO in which the human β-endorphin gene was removed from pRE1078 by religation with ligase.
2? I got it. This psAKO27 was cut with EcoRI to obtain 2.1 k containing the PGK promoter, MFα1 secretion signal sequence, and MFα1 terminator sequence.
A DNA fragment of b was isolated. This DNA fragment was added to the above (
pSA obtained in a) was ligated with a 5.5 kb DNA fragment obtained by cutting 009 with EcoRI to create psAKO.
28 was prepared (Figure 2).

(d) Preparation of mouse α-amylase gene (minus its own signal sequence) into plasmid pSA Plasmid psAK011 containing the mouse α-amylase gene (minus its own signal sequence) was cleaved with Ba1I, and the resulting approximately 1.5 kb mouse α-amylase gene was cleaved with BalI. The amylase gene fragment was extracted from psAK028 obtained in (C) above.
The plasmid psAKO31 was obtained by ligating to the amfl 1 site using T4 DNA ligase and selecting one in which the promoter and amylase gene were ligated in the same direction (Fig. 2).

(2) Introduction of plasmid psAKO31 into Satucharomyces cerevisiae YNN27 psAKO31 was cut with 5 tul to obtain linear DNA.

YNN27 was cultured in YPD medium until the middle of the logarithmic growth phase, and after washing once with IM lithium acetate aqueous solution, the number of cells was measured, and IX 107! [IM aqueous lithium acetate solution was added so that the cell size was 12 μm.

The 12 μm (IX 1011i11 cells) was collected and transferred to an Eppendorf tube, and 2 μg of the above ps
AKO31 linear DNA and 45μm050% polyethylene glycol aqueous solution were added and mixed and incubated at 30℃ for 45 minutes.
The sample was held for 5 minutes, then heat-treated at 42°C for 5 minutes, and then washed with sterile water.

The cells thus treated were suspended in 100 μm sterile water, plated on a plate from which uracil had been removed from the CSM medium, and cultured at 30° C. for 3 days. After culturing several colonies grown on the plate overnight in 5 ml of YP[l medium, DNA was extracted, and the resulting DNA was analyzed by Southern hybridization, psAKO3
YNN27/in which one copy of the linear DNA of
1) SAKO31/S was selected.

(3) Treatment of YNN27/ psAKO31/S with EMS 031/S was cultured on the YNN27/pSA obtained in (2) above in YPD medium overnight, and then treated with 0.1 M sodium phosphate buffer (pH 7; O). After washing with
Add 0.1 M sodium phosphate buffer to a concentration of IQ 8m cell/1, further add 30 μm of EMS, maintain at 30°C, take 100 μm at 5 minute intervals, 41
EMS was neutralized by adding it to a 5% aqueous solution of sodium thiosulfate.

Each of the EMS-treated bacteria collected at 5-minute intervals was diluted 10,000 times, and 200 μm of the diluted solution was spread on a YPD medium (plate) and cultured at 24° C. for 2 days. The cells suspended in the aqueous sodium thiosulfate solution were protected from light with aluminum foil and stored at 8°C. The survival rate was determined by counting the number of colonies that appeared on the YPo plate after 2 days. The EMS treatment time at which the survival rate reached 40 to 50% was determined, and the sample at that time (E suspended in sodium thiosulfate aqueous solution) was
MS-treated bacteria) was diluted 5,000 times with sterile water, and 20
A 0 μm sample was spread on a YPD plate and cultured at 24° C., resulting in approximately 8,000 colonies of bacteria. Y each bacteria
The cells were inoculated onto a PD plate, and the sizes of the resulting halos were compared. As a result, nine mutant strains with halo diameters more than twice that of the parent strain were collected and cultured at 37° C. for 2 days. As a result, 2 strains of Satucharomyces cerevisiae pop that did not grow were selected and collected.

(4) Secretion of amylase by pop2 strain The pop2 strain obtained in (3) above was mixed with YPD containing 2% glucose.
The cells were cultured for two nights at 30°C using a medium, and the α-amylase activity contained in the culture solution was quantified by the method described later. For comparison, YNN27/ps before EMS treatment.
AKO31/S was cultured in exactly the same manner as above, and the α-amylase activity contained in the culture solution was quantified. The results were as shown in Table 1.

Table 1 (5) Introduction of pREI078 into pop2 strain The pop2 strain was cultured in YPD medium until the middle of the logarithmic growth phase, and after washing once with IM lithium acetate aqueous solution, the number of cells was measured. An aqueous IM lithium acetate solution was added at a ratio of cells/12 μm. Its 12 μm (1×107
Cells) were collected and transferred to an Eppendorf tube, and 2 μg of pRE1078 and 45 μm of 50% polyethylene glycol aqueous solution were added and mixed, and the mixture was incubated at 23°C for 45 minutes.
The sample was held for 5 minutes, then heat-treated at 37°C for 5 minutes, and then washed with sterile water. The thus treated cells were suspended at 100 μm and spread on a plate from which tryptophan had been removed from C5M medium, cultured at 30° C. for 3 days, and several colonies grown on the plate were isolated.

(6) β- by pop2 strain into which pRE1078 was introduced
Endorphin secretion The pop2 strain introduced with pRE+078 obtained in (5) above was cultured for two nights at 24°C using a C5M medium containing 2% glucose from which tryptophan had been removed.
The secreted amount of β-endorphin contained in the culture medium supernatant was measured.

The secreted amount of β-endorphin was measured by radioimmunoassay using β-endorphin [RIA] kit New England, catalog number NEK-003, according to the method described in the catalog.

For capture, the YNN27/psAKO31/
A strain into which pRE+078 had been introduced into S. was cultured using the same method as above, and the amount of β-endorphin secreted was measured using the same method. The results are shown in Table 2.

Table 2 (Effects of the invention) The Satucharomyces cerevisiae pop2 strain of the present invention is
This is a mutant strain of the well-known Satucharomyces cerevisiae YNN27 that does not have protein secretion ability, and secretes protein with high efficiency. Therefore, it is extremely useful when using this as a host to commercially produce useful heterologous proteins by genetic recombination technology.

[Brief explanation of the drawing]

Figures 1 and 2 show plasmid pRE l 07, respectively.
8 and psAKO31. FIG. Symbol A in the figure stands for Aatn, B stands for Bam) l I, B
g is BglII, E is EcoRI, H is l-1in
dIII, Hf is HinfI, old Mlu I,
S represents 5all, and St represents °5tuI.

Claims (1)

[Claims] (1) A mutant strain of Saccharomyces cerevisiae YNN27 that secretes proteins to a high degree.
Saccharomyces cerevisiae p deposited as No. 18
op2.