JPH0351396B2 - - Google Patents

Description

[Detailed description of the invention]

(Industrial Application Field) The present invention relates to a novel yeast strain Saccharomyces cerevisiae M9-36 that secretes proteins to a high degree. (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 when producing a heterologous protein by genetic recombination technology using the yeast Saccharomyces cerevisiae as a host, it is possible to efficiently secrete the produced substance. As a result of research aimed at obtaining a host,
A mutant obtained by inserting an integrated plasmid into the chromosome of the well-known yeast Saccharomyces cerevisiae YNN27, which itself has a small ability to secrete proteins, and then treating it with ethyl methanesulfonate has been identified. The present invention was achieved by confirming that the heterologous protein was highly secreted in the medium. That is, the gist of the present invention is a mutant strain of Satucharomyces cerevisiae YNN27, which in a medium containing low and high concentrations of glucose,
Satucharomyces cerevisiae M9, which secretes proteins to a high degree and was deposited as FIKEN Bacteria No. 9406
- Present in 36 strains. To explain the present invention in detail, the yeast Saccharomyces cerevisiae M9-36 strain of the present invention has been deposited as FEIRM P-9406 (FERM P-9406), and the well-known yeast Saccharomyces cerevisiae YNN27 [Journal of Molecular Biology,
158, pp. 157-179 (1982)] as the parent strain, and is a mutant strain prepared by the method described below. (1) [Insertion of linear DNA of plasmid pSAK011 into Satucharomyces cerevisiae YNN27 chromosome] First, Satucharomyces cerevisiae YNN27
The linear DNA of plasmid pSAK011 prepared by the method described later was introduced into
Insert into YNN27 chromosome. The plasmid pSAK011 used here is
As shown in Figure 1, the SUC2 gene, known as the invertase gene of Satucharomyces cerevisiae [Nucleic Acids Research;
Volume 11, No. 6, Pages 1943-1953] Promoter;
Secretion signal of invertase, corresponding to the N-terminal 12 amino acid residues of mature invertase
It is obtained by inserting the DNA sequence, the mouse α-amylase gene, and the terminator region of the SUC2 gene into the well-known plasmid Ylp5, and is prepared, for example, by the following steps. [Preparation of plasmid pSAK011] (a) Preparation of plasmid pSAK009 Well-known plasmid Ylp5 [Proceedings of
the National Academy of Sciences of
the USA, Vol. 76, pp. 1035-1039 (1979)]
was cut with BamH, the ends were blunted with Mung bean nuclease to destroy the BamH cleavage site, and the plasmid was self-ligated.
Prepare pSAK009 (see Figure 1). (b) Preparation of plasmid pSAK010 A well-known plasmid containing the promoter and terminator regions of the SUC2 gene
pSMF38T［Agricultural and Biological
Chemistry, Vol. 51, pp. 515-521 (1987) and Japanese Unexamined Patent Publication No. 62-96086] by BamH and
SUC2 promoter fragment (approximately 900 bp) obtained by cutting with EcoR, BamH and
The terminator fragment (approximately 550 bp) obtained by cutting with Hind was isolated separately, and these two fragments were added to the plasmid pSAK009.
Approx. obtained by cutting with EcoR and Hind
Plasmid by ligation with 5.5kb fragment
Obtain pSAK010 (see Figure 1). Plasmid pSAK010 contains the promoter of the SUC2 gene, the secretion signal peptide of invertase, and the N-terminal 12
DNA sequence corresponding to the amino acid residue of and
Contains the terminator region of the SUC gene,
It has BamH as a cloning site. (c) Preparation of plasmid pSAK011 The well-known plasmid pSMF38TMA [Agricultural and
Biological Chemistry, Vol. 51, pp. 515-521)
(1987)] with BamH, and the resulting approximately 1.5 kb mouse α-amylase gene fragment was extracted with BamH of the plasmid pSAK010.
A plasmid in which the promoter and amylase gene are linked in the same direction is selected and designated as plasmid pSAK011 (see Figure 1). [Introduction of pSAK011 into YNN27] Plasmid obtained as above
pSAK011 was cut with Mlu to make a linear DNA, and this was introduced into YNN27, resulting in a bacterial strain in which one copy of the linear DNA of pSAK011 was integrated into the chromosome No. of YNN27 (hereinafter referred to as YNN27/
pSAK011/M) is obtained. The linear DNA of pSAK011 is introduced into YNN27 by a method known per se. For example, according to the method described in JP-A No. 59-28478, YNN27 is mixed with an appropriate medium,
For example, after culturing using YPD medium, collect the bacteria, suspend in a buffer solution, add metal ions such as lithium, rubidium, or cesium, and then use the linear DNA obtained by cutting the pSAK011 with Mlu.
and adding a polyethylene glycol aqueous solution,
Next, it is heat-treated at about 40°C for a short time and washed 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 way are suspended in sterile water and plated on a plate from which uracil has been removed from the CSM medium and cultured. After culturing some colonies grown on the plate in YPD medium,
DNA was extracted and the obtained DNA was analyzed by Southern hybridization method, pSAK011
One copy of the linear DNA of
Select YNN27/pSAK011/M. (2) [Treatment with ethyl methanesulfonate] YNN27/YNN27 prepared as above
pSAK011/M was then treated with ethyl methanesulfonate (hereinafter referred to as EMS), which is known as a yeast cell mutagenic agent.
Methods in yeast genetics, a laboratory
manual, Cold Spring Harbor Laboratory,
The S. cerevisiae strain M9-36 is prepared by the method described in Cold Spring Harbor, NY (1978). That is, YNN27/pSAK011/ obtained above
After culturing M in YPD medium and washing with buffer, the cell concentration was adjusted to a predetermined concentration, and a predetermined amount of EMS was added to this.
YPD by diluting the EMS-treated bacteria.
M9-36 of the present invention by culturing on a medium
Stocks can be obtained. The M9-36 strain of the present invention obtained as described above has a low concentration (0.2%), as shown in the Examples below.
and secretes amylase with high efficiency in a medium containing a high concentration (5%) of glucose. In addition, YNN27/pSAK011/ before processing by EMS
M also secretes amylase, but the amount of amylase secreted by the M9-36 strain far exceeds that of YNN27/pSAK011/M. By introducing a plasmid containing a gene for another heterologous protein into the M9-36 strain of the present invention, the heterologous protein can be secreted. For example, if the plasmid pSAK025 containing the human β-endorphin gene, prepared by the method described below, is introduced into the M9-36 strain of the present invention, low concentration (0.2%) and high concentration (5%) glucose β-endorphin is secreted with high efficiency in a medium containing . Plasmid pSAK025 was obtained from the above-mentioned plasmid pSMF38T (Japanese Unexamined Patent Publication No. 62-96086) and the well-known plasmid pUC8-βE (Japanese Unexamined Patent Publication No. 61-271988) containing the human β-endorphin gene. It is prepared as follows. That is, pSMF38T was cut with BamH and ClAP
to prevent DNA self-ligation, and then
By inserting the BamH-BamH fragment obtained by cutting pUC8-βE with BamH into the BamH cleavage site, plasmid pSAK025 in which the β-endorphin gene (β-E) is inserted into the BamH site of pSMF38T is prepared. (See Figure 2). pSAK025 prepared in this way was transferred to M9-36
Introduce into stocks. Introduction of pSAK025 into M9-36 strain was performed as described above.
It was carried out by the same method as the introduction of linear DNA of pSAK011 into YNN27, and cultured by plating on a plate from which tryptophan had been removed from CSM medium.
Colonies that grow on this plate are isolated. The M9-36 strain into which pSAK025 was introduced as described above showed high efficiency in medium containing low (0.2%) and high (5%) concentrations of glucose, as shown in the Examples below. secretes amylase and β-endorphin. In addition, in the above YNN27/pSAK011/M,
If pSAK025 is introduced in the same manner as above,
In a medium containing a high concentration (5%) of glucose,
No secretion of β-endorphin was observed, and β-endorphin was secreted only in medium containing a low concentration (0.2%) of glucose;
When pSAK025 is introduced, the amount of β-endorphin secreted reaches approximately 10 times or more. 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, unless otherwise specified. [Cleavage and recovery of DNA using restriction enzymes] Use the following three types of buffers for cutting with restriction enzymes.
Bacterial Genetics (1981) (Cold spring
Harbor, New York). The cutting conditions were as follows: using a restriction enzyme of 2 units/μg DNA;
Process at 37°C or 65°C for 30 minutes. Then TE buffer (10mM Tris-HCl PH
Extract once with phenol saturated with 8.0 and 1mM EDTA), remove the phenol with ether, add 2 volumes of ethanol, and incubate at -20°C for 30 minutes.
After allowing it to stand for a minute, centrifuge it to collect the DNA. (1) Low salt concentration buffer consisting of 10mM Tris-HCl (PH7.4), 10mM magnesium sulfate, and 1mM dithiothreitol. (2) Medium salt concentration buffer 50mM NaCl, 10mM Tris-HCl (PH
7.4), 10mM magnesium sulfate and 1mM
dithiothreitol. (3) High salt concentration buffer 100mM NaCl, 50mM Tris-HCl (PH
7.4) and 10mM magnesium sulfate. [Treatment of DNA fragment with calf small intestine alkaline phosphatase (ClAP)] In order to prevent self-ligation of plasmid DNA by ligase reaction, the fragment of plasmid DNA cut by a restriction enzyme is treated with ClAP prior to the ligase reaction. Plasmid DNA (10p) cut with restriction enzymes
mol5' end) was dissolved in 50μ of ClAP buffer [consisting of 50mM Tris-HCl (PH9.0), 1mM magnesium chloride, 0.1mM zinc chloride and 1mM spermidine], and per 1pmol end,
After adding 0.01 units of ClAP and reacting at 37℃ for 30 minutes,
10μ 0.1M Tris-HCl (PH8.0), 1M NaCl,
Add 10mM EDTA, 5μ of 10% SDS, 40μ of water and keep at 65 °C for 15 min. After cooling, extract with phenol saturated with TE buffer, remove phenol with ether, and collect plasmid DNA by ethanol precipitation. [Removal of DNA sticky ends using Mung bean nuclease] DNA with sticky ends was treated with 30mM sodium acetate (PH4.6), 50mM NaCl and 1ml.
Dissolve in 100μ of a buffer consisting of ZnCl ₂ and add to this
After adding 1μ (2 units/μ) of Mung bean nuclease and treating at 37℃ for 10 minutes, 2μ
Add 25% SDS solution, 10μ of 0.5M Tris-HCl buffer (PH9.5) and 10μ of 8M LiCl. Next, 150μ of phenol and chloroform mixture (1:1) was added to extract the aqueous layer, 10μ of 3M sodium acetate and 200μ of ethanol were added, cooled to -20°C, and centrifuged. Collect the precipitated DNA. [Legation using T4 DNA ligase] The two DNA fragments to be ligated are 1μg/10μ
Linking buffer [66mM Tris-HCl (PH7.5), 6.6mM magnesium chloride,
consisting of 10mM dithiothreitol] and treated at 65°C for 10 minutes, followed by 66μM dithiothreitol at 4°C.
Add ATP (adenosine triphosphate) and T4 DNA ligase at 0.1 unit/μg DNA for sticky ends or 1 unit/μg DNA for blunt ends.
After reacting at 18°C for 18 hours, treat at 65°C for 10 minutes. [Transformation of yeast (KU method) Transform 10 ml of the yeast Satucharomyces cerevisiae into
Culture the cells overnight at 30°C in YEPD medium, collect the bacteria, wash once with TE buffer, and suspend in the same buffer until the cell number is 2 x 10 cells/ml. After adding the same amount of 0.2M lithium acetate (PH7.5) to 500μ of this suspension and keeping it at 30℃ for 1 hour,
Dispense the DNA into 100μ test tubes at 0°C.
and mix for 30 minutes at 0°C. 70 of 100μ
Add TE buffer containing 4000% polyethylene glycol, mix well, hold at 30°C for 1 hour, then at 42°C for 5 minutes, centrifuge to collect bacteria,
Wash with sterile water. Suspend this in 500μ of sterilized water, inoculate 100μ onto a selective medium, and culture at 30°C for 3 to 4 days to obtain a transformed strain. Example 1 (1) Preparation of plasmid pSAK011 (a) Preparation of plasmid pSAK009 Plasmid Ylp5 was cut with BamH,
After blunting the ends with Mung bean nuclease to destroy the BamH cleavage site, T4
Plasmid pSAK009 was prepared by self-ligation using DNA ligase (Figure 1). (b) Preparation of plasmid pSAK010 Plasmid pSMF38T containing the promoter and terminator region of SIC2 was incubated with BamH.
and EcoR to obtain a SUC2 promoter fragment (approximately 900 bp). Separately, pSMF38T was added to BamH and Hind
Cut the terminator fragment (about 550bp) with
I got it. The two fragments obtained above were added to the pSAK009
obtained by cutting with EcoR and Hind
The 5.5 kb fragment was ligated using T4 DNA ligase to obtain plasmid pSAK010 (Figure 1). (c) Preparation of plasmid pSAK011 Plasmid pSMF38TMA containing the mouse α-amylase gene was cut with BamH, and the obtained α-amylase gene fragment of approximately 1.5 kb was inserted into the plasmid pSAK010.
Ligate to the BamH site using T4 DNA ligase, select a plasmid in which the promoter and amylase gene are linked in the same direction.
pSAK011 was obtained (Figure 1). (2) Introduction of plasmid pSAK011 into Satucharomyces cerevisiae YNN27 pSAK011 was cut with Mlu to obtain linear DNA. YNN27 was cultured in YPD medium until the middle of the logarithmic growth phase, and after washing once with 1M lithium acetate aqueous solution, the cell number was determined to 1 × 10 ⁷ cells/12μ.
1M lithium acetate aqueous solution was added so that 12 μg (1×10 ⁷ cells) were collected and transferred to an Eppendorf tube, and 2 μg of the
Add linear DNA of pSAK011 and 45μ of 50% polyethylene glycol aqueous solution and mix for 30 minutes.
It was kept at 45°C for 45 minutes, then heat treated at 42°C for 5 minutes, and then washed with sterile water. The cells thus treated were suspended in 100μ of sterile water, plated on a plate from which uracil had been removed from the CSM medium, and cultured at 30°C for 3 days.
5 colonies grown on the plate
After culturing overnight in ml of YPD medium, DNA was extracted, and the resulting DNA was analyzed by Southern hybridization to determine whether YNN27/YNN27/1 had one copy of the linear DNA of pSAK011 incorporated.
pSAK011/M was selected. (3) Processing of YNN27/pSAK011/M by EMS YNN27/pSAK011/M obtained in (2) above
After culturing overnight in YPD medium and washing with 0.1M sodium phosphate buffer (PH7.0), 5 × 10 ⁸ cells/
Add 0.1M sodium phosphate buffer to a concentration of 1.0ml, then add 30μ of EMS to 30ml.
Hold at ℃, take 100μ pieces at 5 minute intervals,
ml of 5% aqueous sodium thiosulfate solution.
Neutralized EMS. 10,000 EMS treated bacteria each collected at 5 minute intervals
It was diluted twice, and 200μ of the solution was spread on YPD medium (plate) and cultured at 24°C for 2 days. During this time, the cells suspended in the sodium thiosulfate aqueous solution were protected from light with aluminum foil and stored at 8°C. After 2 days, the number of colonies that appeared on the YPD plate was counted to determine the survival rate. Determine the EMS treatment time at which the survival rate is 40-50%, dilute the sample at that time (EMS-treated bacteria suspended in sodium thiosulfate aqueous solution) 5000 times with sterilized water, and spread 200μ of this onto a YPD plate. 24℃
Cultured with Satucharomyces cerevisiae M9
−36 stocks were obtained. (4) Secretion of amylase by M9-36 strain The M9-36 strain obtained in (3) above was cultured overnight at 24°C in YPD medium containing 5% glucose, and then cultured in YPD medium containing 5% glucose. The cells were transferred to YPD medium containing 20% and cultured at 24°C. Quantify glucose in the culture solution over time,
1 hour after glucose is consumed
The α-amylase activity contained in the culture solution after 2 and 3 hours was quantified by the method described below. For comparison, the YNN27/
pSAK011/M was cultured in exactly the same manner as above, and α-amylase activity was quantified. The results were as shown in Table 1. [Method for quantifying amylase] Add 0.5 ml of the culture solution at each time point to 0.5 ml of 20 mM phosphate buffer (PH7.0) containing 1% starch.
0.2 ml samples were collected after 0 minutes, 15 minutes, and 30 minutes.
To each sample was added 0.5 ml of DNS solution (0.5 ml of dinitrosalicylic acid suspended in 50 ml of water, and then added with 20 ml of 2N-NaOH, 30 g of Rothsiel's salt, and water to make 100 ml). Boil for 5 minutes. After cooling, add 1.3ml of water and adjust the sample to 525nm.
Measure the absorbance (A ₅₂₅ ) at The _A525 value after 15 minutes of enzyme reaction and the sample taken at 0 minutes
A Calculate the difference from the ₅₂₅ value. On the other hand, a calibration line is determined using glucose instead of the enzyme reaction solution, and the amount of glucose is determined from the measured value of the sample. One unit of secreted amylase is expressed as the amount that produces 1 μg of glucose per minute.

[Table] (5) Preparation of plasmid pSAK025 Cut plasmid pSMF38T with BamH,
DNA self-ligation was then blocked by treatment with ClAP. Plasmid pUC8-βE containing the human β-endorphin gene was cut with BamH, and the obtained BamH-BamH fragment containing β-E was
pSMF38T above using T4 DNA ligase.
It was inserted into the BamH site to obtain pSAK025 (Figure 2). (6) Introduction of plasmid pSAK025 into M9-36 strain M9-36 strain was cultured in YPD medium until the mid-logarithmic growth phase, and after washing once with 1M lithium acetate aqueous solution, the number of cells was measured. ⁷ cells/
A 1M aqueous lithium acetate solution was added to the solution to a volume of 12μ. 12 μg (1×10 ⁷ cells) were collected and transferred to an Eppendorf tube, and 2 μg of the
A 50% polyethylene glycol aqueous solution of pSAK025 and 45μ was added and mixed, held at 30°C for 45 minutes, then heat-treated at 42°C for 5 minutes, and then washed with sterile water. The cells treated in this manner were suspended in a 100μ solution, spread on a plate from which tryptophan had been removed from the CSM medium, and cultured at 30°C for 3 days, and several colonies that had grown on the plate were isolated. (7) Secretion of β-endorphin by M9-36 strain introduced with pSAK025
36 strains were cultured using CSM medium containing 5% glucose without tryptophan.
After culturing overnight at °C, β-
The amount of endorphin secreted was measured. Next, after washing the bacterial cells in this culture twice with water, they were transferred to CSM medium containing 0.2% glucose and excluding tryptophan, and cultured at 24°C for 3 hours, so that the cells contained in the culture supernatant were The amount of β-endorphin secreted was measured. Note that the measurement of the amount of β-endorphin secreted above was performed using a β-endorphin [RlA] kit.
Radioimmunoassay was performed using New England, catalog number NEK-003, according to the method described in the catalog. For comparison, the YNN27/pSAK011/M
Next, a strain into which pSAK025 had been introduced was cultured using the same method as above, and the amount of β-endorphin secreted was measured using the same method. The results were as shown in Table 2.

[Table] (Effects of the invention) Satucharomyces cerevisiae M9- of the present invention
Strain 36 is a well-known mutant strain of Satucharomyces cerevisiae YNN27 that does not have protein secretion ability.
It secretes proteins with high efficiency in medium containing low (0.2%) and high (5%) concentrations of glucose. Therefore, it is extremely useful when using this as a host to commercially produce useful heterologous proteins by genetic recombination technology.

[Brief explanation of drawings]

Figures 1 and 2 show plasmids, respectively.
FIG. 2 is a schematic diagram showing the configuration routes of pSAK011 and pSAK025. Symbols in the diagram: B is BamH, E is EcoR
, H represents Hind, and M represents Mlu.

Claims (1)

[Claims] 1. Satucharomyces cerevisiae strain M9-36, which is a mutant strain of Satucharomyces cerevisiae YNN27 and is deposited as Microtech Research Institute Deposit No. 9406, which secretes protein to a high degree in medium containing low and high concentrations of glucose.