JPH02234666A - Aspergillus-sojae transformant - Google Patents

Abstract

PURPOSE:To readily produce the subject transformant containing a prescribed selective marker DNA by transforming a strain of Aspergillus.sojae deficient in a prescribed selective marker gene with a DNA vector having a DNA sequence of the above-mentioned selective marker. CONSTITUTION:A strain of Aspergillus.sojae (e.g. mutant strain of wild type Aspergilus.sojae) deficient in a prescribed selective marker gene, such as ornithine carbamoyltransferase gene, is prepared. The aforementioned strain of the Aspergillus.sojae is then transformed with a cyclic or linear DNA vector having the DNA sequence of the above-mentioned prescribed selective marker. Thereby, an Aspergillus.sojae transformant containing the prescribed selective marker DNA is obtained and usefully applied to development and production of proteins, enzymes and other products.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to Aspergillus sojae (＾spergill
ussojae).

[Conventional technology]

Aspergillus soya (hereinafter abbreviated as A) is used in the brewing industry for soy sauce and the like, as well as in the enzyme manufacturing industry, pharmaceutical manufacturing industry, food industry, and the like. Its use is based on the secretory ability of the microorganism.

A. sawyer is a microorganism used in the above-mentioned industries, in large buildings, and on a commercial scale. In order to apply genetic engineering techniques to A. sojae, it is necessary to transfer DNA to A. sojae so that the resulting transformant expresses a more useful product and secretes it in large quantities.

Many genes have been cloned in various prokaryotic vectors (eg, using E. coli) in an effort to obtain high expression levels for the encoded proteins. When genes are introduced into a host that is the same or very similar to the species to which they originally belong, these genes are expressed more efficiently and are easier to process, producing proteins that more closely resemble the native proteins. Easy to produce.

A. sojae is a microorganism that produces a variety of industrially important proteins, enzymes, and other products. It can also secrete efficiently expressed proteins. However, transformation of A. sojae has proven to be very difficult.

[Problem to be solved by the invention]

The purpose of the present invention is to develop a useful A. sawjae transformant, which is obtained by imparting a predetermined selection marker to A. sawjae using genetic engineering techniques.

[Means to solve the problem]

In the present invention, a predetermined selection marker is deleted ([Delete J is
An Aspergillus sojae strain containing a predetermined selection marker DNA obtained by transforming an Aspergillus sojae strain (including cases in which it originally has a desired predetermined selection marker) with a DNA vector containing the DNA sequence of the predetermined selection marker. It is in the Sawyer transformant.

At least a portion of the bacterial cells of the A. sawyer strain mentioned above are A.
・Sawyer protoplasts can be used, and DNA
Both circular and linear vectors can be used. Further, as the DNA of the predetermined selection marker, one derived from the genus Aspergillus is used, preferably the ornithine carbamoyltransferase gene (arg B"
) is used. This ornithine carbamoyltransferase gene is derived from A. sojae or A. nidorans.

The transformant of the present invention is characterized in that it is prepared by transforming a host A. sojae using a DNA vector containing a selection marker DNA that is not present in A. sojae cells before transformation. be. The above DNA vector is introduced into A. sojae to enhance the expression of its selectable marker protein, but this DNA vector may also contain other exogenous DNA necessary for modification. Transformants thus formed are selected from non-transformed cells based on the integrated selection marker, isolated, and obtained by propagation and culture in a conventional manner.

FIG. 1 shows the DNA vectors pSa I 43 and pSa I used for the preparation of A. sojae transformants according to the present invention.
This is a construction diagram of 123.

The selection marker used in the present invention is wild type A.
Compared to A. soja , those naturally occurring in A. soja are suitable so that their presence in the transformant does not substantially affect its properties.

For this reason, the transformant of the present invention is a transformant using a wild strain of A. sawyer, preferably a mutant strain thereof, as a host for transformation. If the wild-type A. sojae strain originally lacks (ie does not have) the desired selected genetic marker, it can be used as it is as a host for transformation. However, such cases are rare. Therefore, it is preferable to mutagenize the wild-type A. sojae strain and use a mutant strain lacking the selected genetic marker. The transformant is transformed with a vector containing a selection marker and exogenous DNA necessary for integration, and the expression of the A. sawyer selection marker protein is enhanced.

To transform wild-type A. sojae, a dominant selection marker, i.e., the ability to metabolize metabolites not normally metabolized by A. sojae, or a novel phenotype, such as resistance to the harmful effects of chemicals or antibiotics, is required. A gene that specifies can be used. Transformants of wild-type A. sojae can be selected based on the introduced dominant selection marker.

A preferred example of a selectable marker is the arg B" gene, which encodes the enzyme ornithine trans-lactamylase. This enzyme is present in wild-type A. soya.

A mutant lacking this enzyme (arg B strain)
can be prepared by conventional techniques, for example by treatment with ultraviolet irradiation, and are selected by their ability to grow on arginine-containing media but not on minimal media. According to the method of the present invention, A. Sawyer's ar.
Transformants made from the gB- strain and the vector containing the argB+ gene are therefore argB'' and can be grown by standard plating and culture techniques.
It can be easily selected and isolated from untransformed arg B-strains.

As mentioned above, although it is preferred to use a selectable marker that is naturally present in wild-type A. sojae, it is not necessary that the selectable marker used in the vector actually be derived from A. sojae. It can similarly be obtained from other similar strains that contain the desired gene under expression conditions.

In a preferred embodiment of constructing a DNA vector used to prepare the transformant of the present invention, first, the selection marker argB"r" or "mouth" in FIG.
The total DNA of the A. nitrans strain was obtained by treating with appropriate restriction enzymes, which was then transformed into A. soyer (arg B
-) vector plasmid p
BB8 or pBB29.

In a preferred embodiment of the invention, the host microorganism A.
The A. sawyer protoplast is used as the sawyer. A preferred method of preparing this protoplast is, for example,
By enzymatically dissolving the cell wall using β-1,3-glucanase and/or chidinase. The selection of enzymes suitable for enzymatically dissolving A. sojae to obtain its Brotoplus 1. is known. The enzyme mentioned above can dissolve complex polysaccharides and has a wide range of A.
It is effective for preparing fungal brotoplasts of Sawyer.

Examples of the β-1,3-glucanases include Zymolyase 20T and 100T sold by Seikagaku Corporation, and the microorganisms Hytylus circulans IA elbow 165 and Streptomyces 0143 (
Examples include two crude enzymes prepared from respective culture solutions of FERM P7276).

As the chitinase, an enzyme having chitinase activity is used, such as Chitinase N manufactured by ICN, USA.
o. 100466, Chitinase No. manufactured by Sigma, USA.
C-6137, Chitinase fungal NO. 100290 etc. are mentioned. Other suitable methods may be used to form protoplasts. Furthermore, regarding an appropriate method for incorporating the vector into cells, whole cells such as A. sawyer spores and hyphae may be used instead of protoplasts.

The transformation of the present invention is carried out using an appropriately selected DNA vector (
For example, in FIG. 1, pSa I! ．． 43 and pSa
i23). Vectors for the preparation of transformants of the present invention must not contain specific selection markers (e.g. arg B") and must also contain other useful markers to be included in the transformants. The DNA vector must contain a gene. As mentioned above, the DNA vector may be either linear or circular DNA. Preferably, the DNA vector introduces a selection marker or the like,
In order to produce appropriate amounts of the vector in E. coli, E.
・Those containing the Coli replicon (copying factor) are used.

Next, as a simple example, the present invention typically uses enzymes (e.g., β-1,3-glucanase and chitinase) to
Pro 1.
The plasts were transfected with polyethylene glycol (hereinafter referred to as PE
(abbreviated as G) 4000 or 6000 and CaC
A transformant A. sojae strain that does not require arginine can be prepared from an A. sojae arginine auxotrophic strain (A. sojae W20-4) by treatment in the presence of 1 g. These transformants have DNA arg B""
can be integrated into its chromosome and the gene can be expressed.

The transformant has pBR, which is not present in the parent strain (host strain).
As is clear from the sequence present in the transformant, the DNA
DNA other than arg B'' present in the vector is also incorporated.

Therefore, in order to carry out the transformation described in the present invention,
The recipient strain (host strain) and a part thereof (protoblast) have a selectable DNA in this strain, i.e.
Requires DNA containing a selection marker. but,
so that the transformant obtained by introducing the DNA into a recipient has an easily selectable phenotype,
The recipient strain and the transforming DNA must be selected.

The present invention will be explained in detail below.

Asbenoreginoles Sawyer (8TCC 42251)
are mutagenized with UV and the resulting isolates require citrulline or arginine for growth in defined minimal media. Among these isolates, a strain lacking ornithine carbamoyl trisferase was selected and
It is called g B-.

The method for preparing Aspergillus sojae protoplasts is the same as that described in JP-A-60-75281.

That is, a strain of A. Sawyer is inoculated into a slant medium (rice malt juice agar medium) in a test tube and cultured at room temperature until sufficient conidial attachment is achieved to obtain conidial spores.

1 l Next, the conidia are dispersed and suspended in a 0.01% sorbitan fatty acid ester solution "Sorgen TW-60 (manufactured by Dai-ichi Kogyo Seiyaku Co., Ltd.)" at a ratio of IXIO' pieces/Id.

Next, this dispersion suspension IId of conidia was mixed with a liquid nutrient medium [0.5% of yeast extract and 0.2% of casamino acids were added to Zwerbeck medium to adjust the pH to 6.0, and this was diluted with water for 10 min.
[obtained by diluting 1:3] and inoculated in a 150-fold Erlenmeyer flask at 30°C for a sufficient period of time until the length of the germinated spores becomes approximately 10 times the outer diameter of the original spores. The cells are cultured to obtain a germinated spore suspension, which is further centrifuged (4500 g, 20 minutes) to separate the germinated spores therefrom.

Next, the germinated spores thus obtained were thoroughly washed with water, and then a cell wall lysing enzyme solution IId was added thereto, and the enzyme treatment was performed by gently shaking (50 to 60 cycles per minute) at 27°C for 2 hours. Obtain protoplasts of one strain of Sawyer arg B.

The cell wall lytic enzyme used here was obtained by dissolving a crude enzyme originating from Bacillus circulans and a commercially available chitinase (manufactured by ICN, USA) at a ratio of 30 μl and 5 μl per ml of solution, respectively.

Next, the protoplasts thus obtained are separated using a G-3 standard glass filter and washed several times with a hypertonic solution (0.8M sorbitol solution) to obtain washed protoplasts.

(Method for preparing DNA vectors) The DNA vectors pSa!!43 and pSa12 shown in FIG. 1 were used in the preparation of the transformants of the present invention.
3 was prepared by DMOCHOWSKA et al.
et aLJournal of General
Microbio1ogy (1986), 13217
29-1937),
In addition, plasmids pBB29 (prepared by ligating Escherichia coli plasmid pBR327 and a yeast-derived DNA fragment) and p
The preparation of BB116 (prepared by ligating the E. coli plasmid pBB8 and the fragment with arg B+ from A. nidorans) was described by Peirce et al.
al, Gene), 25, 109-117 (1983
) can be obtained by the method described in .

l3 Also, Sal I, BamH used in the present invention
Restriction enzymes such as T and EcoR T were obtained from Takara Shuzo Co., Ltd. and used according to the manufacturer's instructions.

Next, the preparation and construction of the two types of DNA vectors useful in the method of the present invention are shown in FIG.

Figure 1 shows A by a known technique using the enzyme BamllT.
- Shows a method using the arg B'' gene extracted from Nidorans DNA.

A. nidorans fraction (DNA gene fragment [
IJ) is first ligated with the known E. coli plasmid pBB8 using T4 ligase (direct ligating enzyme) to form the known plasmid pBB116.

Blasmid pBBII6 is then treated with the restriction enzyme SalT to form a fragment "mouth" containing the arg B'' gene from A. nidorans. The fragment containing this argB gene is isolated by conventional methods, such as agarose electrophoresis and electrophoresis.
May be purified.

On the other hand, the known plasmid pBB29 (the shaded part means a yeast-derived DNA fragment) was digested with the restriction enzyme Sal
Using ligase, the fragment "mouth" was ligated to the fragment 1 obtained by cutting with plasmid p.
Sa I! ．． Get 43. In the above, the isolation of the fragments may be omitted, and the ligation step may be performed using all fragments, and a plasmid containing the desired sequence may be selected from those treated with ligase by a known method.

pSal 43 contains the arg B'' gene as a selectable marker in Aspergillus and other D.
It has an NA sequence.

The transformant thus obtained is an untransformed mutant (arg
B → can be selected, isolated and cultured.

Next, the DNA vector psa I! for transformation of A. sawyer strain arg B- is used. ．． The method for forming No. 23 will be explained.

The DNA vector pSa Q 43 obtained above was cut with the restriction enzyme EcoR T, resulting in two fragments, namely, the yeast-derived DNA fragment shown in the shaded area and the ar
g A fragment containing the B' gene is obtained.

Next, the fragment containing the “2 arg B” gene is
4 ligase to create plasmid pSa! 23
get.

The DNA vectors pSal 43 and pSal 43 thus obtained
Sa 42 23 are each about 9.2 km long and about 5
, the ambicillin resistance (Ap }l) gene from pl'lf129 and pB8116 (Ap }l) at 9 kilobase pairs.
・Contains the arg B” gene derived from Nidorans).

This can be used for protoplast transformation of the A. sawyer arg B mutant.

DNA vectors pSa j2 43 and pSa 4
Both A. 223 contain in their DNA sequences the selection marker argB' and other DNA not resident in A. sojae that is integrated into A. sojae.

This foreign DNA may contain useful genes that impart new and useful properties to the A. sawjae transformant.

In the context of the present invention, vectors containing foreign DNA include A.
It is integrated into the chromosomal DNA (genome) of the Sawyer host strain. i.e., not as a plasmid, but as a chromosome or nuclear D
Therefore, the expression of the foreign DNA becomes permanent thereafter, and the sequence is almost never deleted from the transformed cells.

The new phenotype of the transformant is therefore very stable.

〔Effect of the invention〕

The A. sojae transformant of the present invention contains other DNA other than the selection marker that does not exist in natural A. sojae, and this transformant is useful for the development and production of proteins, enzymes, and other products. It is a useful microorganism.

〔Example〕

Hereinafter, the present invention will be specifically explained with reference to Examples. However, the present invention is not limited to this example.

Example 1 - A. Sawyer's No. A spore suspension of A. Sawyer (ATCC 42251) was
The cells were subjected to ultraviolet irradiation treatment using a conventional method, and the treatment solution was appropriately diluted with sterilized water, plated on a minimal medium containing arginine, and cultured at 30° C. for 3 to 7 days.

The colonies that appear here are transplanted to a minimal medium and cultured in this medium at 30°C for 3 to 7 days. Then, the arginine auxotrophic mutant (arg
B-) Select and isolate as a strain.

In the present invention, this strain is abbreviated as A. Sawyer W20-4. And this mutant strain is Aspergillus sawjae W20
-4 has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology as Microtechnology Research Institute No. 10604.

A. Sawyer Kei 20-4 (arg B-) strain was inoculated into a slant medium (rice malt juice agar medium) in a test tube, and cultured at room temperature until sufficient conidial adhesion occurred. Obtained.

Next, add these conidia to 7 IXIO/1 conidia,
0.01% sorbitan fatty acid ester solution [Sorgen T
W-60 (manufactured by Daiichi Kogyo Seiyaku Co., Ltd.)].

Next, this dispersion suspension of conidia (1) was added to a liquid nutrient medium [0.5% yeast extract and 0.2% casamino acid were added to Zuavec medium, pH 6. diluted 10 times with water] was inoculated into 30 ml of water, and placed in a 150 ml Erlenmeyer flask at 30°C until the length of the germinated spores was the same as the original spore outer diameter. The spores were cultured with shaking for a time sufficient to increase the number of spores by about 10 times to obtain a germinated spore suspension, which was further centrifuged (45
00 g for 20 minutes), and germinated spores were isolated therefrom.

Next, the germinated spores obtained in this way were thoroughly washed with water, added with 1 sterilized cell wall-dissolving enzyme, and gently shaken (50 to 60 cycles per minute) at 27°C for 2 hours. -4 (arg B-) strain protoplasts were obtained.

The cell wall lytic enzyme used here was obtained by dissolving a crude enzyme originating from Bacillus circulans and a commercially available chitinase (manufactured by ICN, USA) at a ratio of 30 mg and 5 μm in 1 rd solution, respectively.

Next, the protoplasts thus obtained were separated using a G-3 standard glass filter, and washed with a hypertonic solution (0.8M sorbitol solution) to obtain washed protoplasts.

Next, protoblasts were diluted with 0.8M MCI, IOmM
CaCIz, 10mM }Lisu/HCI, pH 7.5
Resuspend in a final concentration of 10'/mal. This 0. 2 d of suspension to a volume of 1. 5ml
DN prepared by the above method in a plastic test tube
A vector r pSal 43' or the same r pS
a 42 23'' DNA 10 to 1
Add 00ug (total volume: in TE buffer - 10μ!) and incubate for 30 minutes on ice. 25 for this
%PEG4000, 50mM CaCI4, 10mM
} Add lm of a solution of Lith/HCI, pH 1.5, stir gently and thoroughly, then incubate for an additional 15 minutes at room temperature. Transformation is performed as described above.

DNA vector pSa I! ．． The Aspergillus sojae transformant of the present invention transformed with A.43 has been deposited as A. sojae KTR-3 at the National Institute of Microbial Technology, Agency of Industrial Science and Technology, under No. 10603 of the National Institute of Industrial Science and Technology.

Next, the entire volume of the protoplast suspension thus transformed was mixed with 0.8M KCI, 10mM CaCIz, lo
Mix and dilute with 6 d of mM Tris/HCI, pH 7.5 solution and centrifuge at 700 g for 5 minutes. Furthermore, the same volume of the above diluted solution is added to the isolated protoplasts of the transformant and centrifuged to obtain washed protoplasts.

Next, protoplasts of the washed transformants were grown at 1 m
100 μl of the above diluted solution.
, minimal medium (2.0% agar) containing 0.6M KCI
plate. Moreover, the same medium (however, 0.5%
Agar) is layered. Then, culture at 30°C for 3 to 10 days.

Transformants incorporating arg B+ and DNA vector pSal 43 and other DNA derived from pSal 23 are grown on minimal medium + MCI (potassium chloride is necessary to prevent protoplasts from bursting). .

unaltered cells, unaltered protoplasts, transformants,
All viable cells and protoplasts, including revertants and revertants, are grown in minimal culture medium containing arginine. C
Grows on I. Such a growth medium allows the viability of viable material to be determined. In these experiments, the proportion of viable substances grown in the medium containing arginine out of 10 1/ml was 30% or more. In minimal medium + arginine, only contaminated non-blast material grows. The lη staining in such non-osmotically sensitive cells was less than 1% in this experiment.

Example 2 In order to examine whether the A. sawjae W20-4 transformant obtained above was a genuine transformant rather than a revertant, the transformant was grown in a complete medium. DNA is prepared from this and tested with radiolabeled plasmids.

10 to 400 d of polypeptone/dextrin medium
The mycelia were inoculated at a rate of 5 conidia per adult, grown at 30°C for 16 hours, collected on a filter using a Falcon filter (manufactured by Falcon), and washed with distilled water to remove the medium. .

[Polypeptone Dex I Phosphorus Medium] Dextrin
2% Voripeb I・N 1.0% K}l2Po40.5% NaNO30.1% 9SQ4-71hO O. 05% casamino acids 0.1% pH 5.5-6.0 Adjusted with tap water Then, the washed mycelium was washed with 0.05% casamino acids containing 0.6M KCI.
After washing again (medium removal) with 1M phosphoric acid haphazard (pH 5.5) and draining, the bacterial cells were transferred to a Falcon tube (manufactured by Falcon), and 20 ml of phosphoric acid pamphlet containing the above KCI was added thereto. In addition, we added a cell wall lytic enzyme, a crude enzyme derived from Bacillus circulans, and a commercially available chitinase, respectively. 1 1 mg. The cells were added in amounts of 30 mg and 5 mg, and subjected to enzyme treatment by gentle shaking (50 to 60 cycles per minute) at 27'C for 2 hours to obtain digested bacterial cells.

2,000 r. of this digestive juice. p. m. The precipitate and supernatant were separated by centrifugation at 5,000 rpm for 20 minutes. p. m. The mixture was centrifuged for 30 minutes, and the resulting precipitate was mixed with the above precipitate to collect the bacterial cells. The collected cells were again washed twice with 0.6M MCI buffer.

After sterilization, the following Buff Boo A10 composition kept at 65°C is added to the washed bacterial cells and shaken at 65°C for 1 hour to destroy protoplasts and cells and elute intracellular DNA, RNA, and protein.

"Buffer A" trishydrogen xyaminomethane 50
mM NaCI
O. 15MEDTA
1. OOmMSDS
2% pH
7.5 Next, the following T prepared by dissolving proteinase K (manufactured by Boehringer Mannheim)
Add 5 ml of E buffer (10 mg/mfl). and kept at 37°C overnight to decompose the protein.

[TE Huffer] Tris/HC1 10mM EDTA
I. mm
Add 10 g of cold phenol to the pH 7.6 proteinase treatment solution and add 5,000 g
r. p. m. Centrifuge for 30 minutes and separate the aqueous phase. To this aqueous phase was added 10 ml of a mixture of phenol and chloroborum (1:1), and 5, OOOr. p. m. So 3
Centrifuge for 0 minutes and separate the aqueous phase (upper part). A mixture of chloroform and isoamyl alcohol (24:1) was added to the resulting aqueous phase, and the mixture was heated for 5,000 r. p
．． Centrifuge for 30 minutes at m, and separate the aqueous phase. 10 d of diethyl ether was added to the obtained aqueous phase, and at ice temperature,
5,000r. p. m. Centrifuge for 5 minutes and separate the aqueous phase. The obtained aqueous phase is kept at 55 to 60°C for several minutes in a hot water bath, and the diethyl ether slightly floating on the surface of the aqueous phase is evaporated and removed to obtain a protein-depleted polymeric substance solution. .

A solution LOd of 50% PEG, 2M NaC] is added to this polymeric substance solution and kept at ice temperature (in ice) for 2 hours to transfer sugars to the solution side and precipitate polymeric substances such as RNA and DNA. In order to take out the precipitate, 10,0
OOr. p. Centrifuge for 30 minutes at m, and collect the precipitate.

The precipitate is washed with cold (-20°C) 95% ethanol to thoroughly remove the PEG, and the ethanol is then removed by evaporation with suction.

Then, the obtained precipitate was added to TE buffer (10L Tris/HCI, 1mM EDTA, pH 7.6).
)5mt! Dissolve in RNasei solution (Boehringer
(manufactured by Mannheim) and kept at 37°C overnight to degrade RNA.

Add cold phenol 5- to the above retentate and add 10,000
r. p. m. Centrifuge for 20 minutes and collect the aqueous phase.

Add cold (-20″C) ethanol to the above treated material,
The temperature was maintained at 0°C for 30 minutes or more to precipitate the DNA, and the precipitate was heated to 10.00 Or. p. Centrifuge for 30 minutes to obtain purified DNA.

This purified DNA was dissolved in the TE buffer-1d and p
DNA obtained from a transformant of Sal 43 was prepared.

Southern transfood hybridization This method is developed by Southern “Janinal Molecular Biology”
other, J. Mol. Biol. +98,50
3 (1975)).

That is, a transformant of pSal 43 (A. Sawyer KT
R3) and A. Sawyer ATCC 4225 prepared in the same manner as above as a control.
and the parent strain (host strain) A. Sawyer W20-4 (arg
Regarding the DNA obtained from B-), each restriction enzyme Ba
Cut with mH T, EcoR I, or SalI.

The cut DNAs of the three strains are electrophoresed on a plate agarose gel. The conditions are 20 volts for 14 hours.

Next, in order to facilitate the formation of hybrids as described below, the electrophoresed gel was immersed in a 0.25M HCI solution for 30 minutes, followed by alkaline buffer-1.5M NaCI,
Treated with 0.5M NaOH for 30 minutes and alkalized (DN
denature A).

Then neutralization buffer y-(3M NaCl, 0.5
Soak in M Tris HCI, pH 7.0 for 30 minutes.

Next, a nitrocellulose filter is placed on top of the neutralized flat gel, and the gel is kept overnight and the migrated DNA is transferred.

The transferred nitrocellulose filter is then dried.

The dried nitrocellulose filter in a plastic bag was then coated with a radiolabeled plasmid (tip) (pBR32).
Immerse in a mixture of 5 μl of solution 2) and hybridization buffer-2, seal, and keep at 42°C overnight. The reason for using pBR322 as a radiolabeled plasmid is as follows.

That is, the Escherichia coli-derived plasmid vector in DNA vector pSal 43 used to create the transformant of the present invention is pBR327, and pBR327 is a derivative of pBR322 with a base pair deletion of 0.6 kb. be. Therefore, the base sequence of pBR327 is pBR
It matches that of 322. Therefore, pBR322
By using this as a radiolabeled plasmid, integration of the E. coli-derived plasmid vector pBR327 gene can be confirmed.

Next, a solution containing NaCl and sodium citrate (S
Wash the nitrocellulose filter with S Cm solution).

The washed nitrocellulose filter is then exposed by radioautography. In Southern hybridization, nitrocellulose filters contain
If there is DNA that is partially homologous to both, labeled plasmid/vector DNA is adsorbed thereto, a hybrid is formed, and a radioactive band is therefore formed there.

Therefore, if there are even parts of homologous base sequences, a hybrid is formed there and a band is created.

A. Sawyer ATCC 42251 and A. Sawyer W20
-4 (arg B-) showed no hybridized band.

In contrast, the transformant (A. sawya KTR-3)
exhibited separate bands, indicating different integrations at the same locus or on different sides of the genome.

Hereinafter, any strain of A. sojae, an arginine auxotrophic mutant strain (A. soya W20-4), and a transformant (A.
These are the results of Southern hybridization of E. coli-derived plasmid Hector-pBli'322 of Sawyer KTR-3).

Similarly, this time the radiolabeled plasmid pBR3
Instead of 22, the radiolabeled plasmid pSa 143 was also investigated.

That is, a transformant of pSa J2 43 (A-Sawyer KT
DNA obtained from R-3> and as a control,
A. Sawyer ATCC 422 prepared in the same manner as above
51 and the parent strain A. Sawyer 20-4, Zazan hybridization was performed in the same manner as above.

That is, the Sa volume-digested DNA of these strains was processed, and pSa
143 was cleaved at one site using BamH I and examined as a radiolabeled plasmid. A. Sawyer AT
CC 4225] indicates the hybridized hand, which was found to have a SalI fragment homologous to the arg B' gene. In the cleaning state, A. Sawyer W
20-4 did not represent a hand that formed a hybrid. In contrast, the pSa 143 transformant (A. sawya KTR-3>) exhibited separate hands, indicating different integrations at the same locus or different sides of the genome.

On the other hand, as above, pSa I! ．． The transformants obtained using pSal 23 also hybridized with radiolabeled plasmid pBR322, indicating the incorporation of the vector pBR327 gene used in preparing the pSal 23.

Therefore, A. sawyer W20-4 strain is A. nidorans arg.
Transformed with B gene. That is, this gene is also expressed in A. sojae. Vector pBR327 also a
rg B'' gene into these transformants, thus providing a method for transforming foreign DNA into A. sojae.

Example 3 The phenotypic stability of the transformed strain prepared as described above was investigated. Transformants were inoculated from minimal medium onto complete medium and cultured at 30°C for 4 days. The spores of the culture were removed and re-inoculated into complete medium and cultured as above. This was repeated 30 times in total.

The final conidia are picked from the colony, diluted appropriately and plated on minimal medium + 10mM arginine at 30°C. After 3 days, colonies are replica plated on minimal medium and minimal medium arginine. Of the 100 colonies tested from one transformant, all were prototrophic (not requiring arginine), and the transformed phenotype was maintained during this growth period, as expected for any gene integrated into the genome. It is completely stable inside. That is, the transformed arg B'' gene was completely stable, just like the original gene of Aspergillus oryzae that was originally in the genome.

[Brief explanation of the drawing]

Figure 1 shows the DNA vector pSa/. 43 and the same pSa
It is a figure showing the construction method of E23. In the figure, the shaded areas in pB829 and pSa 42 43 are yeast-derived DNA.
U) and (U) refer to a fragment containing the arg B'' gene from A. nidorans.

Claims (1)

[Scope of Claims] 1. Aspergillus containing a predetermined selection marker DNA obtained by transforming an Aspergillus sojae strain deficient in a predetermined selection marker gene with a DNA vector containing the DNA sequence of the predetermined selection marker. - Sawyer transformant. 2. The Aspergillus sojae transformant according to claim 1, wherein the Aspergillus sojae strain is a protoplast cell. 3. The Aspergillus sojae transformant according to claim 1, wherein the DNA vector is circular. 4. The Aspergillus sojae transformant according to claim 1, wherein the DNA vector is linear. 5. The Aspergillus sojae transformant according to claim 1 or 2, wherein the Aspergillus sojae strain lacking a predetermined selection marker gene is a mutant strain of wild-type Aspergillus sojae. 6. The Aspergillus sojae transformant according to claim 1, wherein the DNA of the predetermined selection marker is derived from a wild-type Aspergillus microorganism. 7. The Aspergillus sojae transformant according to claim 1 or 5, wherein the predetermined selection marker gene is the ornithine carbamoyltransferase gene (argB^+). 8. The Aspergillus sojae transformant according to any one of claims 1, 3 and 4, wherein the DNA vector contains the ornithine carbamoyltransferase gene as the selection marker DNA. 9. The Aspergillus sojae transformant according to claim 1 or 6, wherein the DNA of the predetermined selection marker contains the ornithine carbamoyltransferase gene (argB^+). 10. The ornithine carbamoyltransferase gene is present in Aspergillus sojae or Aspergillus sojae.
The Aspergillus sawjae transformant according to claim 8, which is derived from Aspergillus nidorans.