JPH11169182A - Mutant type secretion device gene that efficiently secrets protein in the cell bodies of corynebacterium - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide the subject novel gene that comprises the DNA encoding a protein having a specific amino acid sequence in which the amino acid residue at a specific position is phenylalanine and can gives a secretory device that can efficiently secret a prescribed protein in the cell bodies of a Coryne form bacterium. SOLUTION: This DNA encodes the protein having an amino acid sequence corresponding to formula I in which the amino acid residue at the 44 position is replaced with phenylalanine. The secretor that is constituted as including the above protein is a novel DNA that can more efficiently secret the prescribed protein in Coryne form bacteria than the secretor that is constituted as including the protein having the amino acid sequence of formula II. This DNA is obtained by isolating the chromosomal DNA of a Brevibacteerium flavum MJ-233 (FERM BP-1497) through the hybridization technique using the oligo DNA probe and subjecting the isolated DNA to the site-specific mutation so that the amino acid at the 44-position becomes the residue of phenylalanine.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a gene DNA encoding a protein constituting a mutant secretory apparatus that efficiently secretes proteins in coryneform bacteria.
In a coryneform bacterium having the mutant secretory apparatus gene of the present invention, it is possible to efficiently secrete and produce a predetermined protein into a medium at an industrial level.

[0002]

2. Description of the Related Art The secretion of proteins by coryneform bacteria has not been studied so far. Coryne
DN by B. glutamicum
secretion [US Pat. No. 4,965,197] and Corynebacterium glutamic.
Only research on the secretion of PS1 and PS2, which are cell wall proteins of um [WO 93/03158], has been reported. In addition, regarding the main factors constituting the secretory apparatus (translocation machinery) derived from coryneform bacteria, SecY [JP-A-6-169780], SecE [JP-A-6-277703] and SecA [JP-A-7-207] No. 107981] has only been reported to have isolated each gene, and no systematic study has been carried out.

[0003]

Attempts to increase the secretion amount of secretory proteins using the main factors constituting these wild-type secretory apparatus have not been sufficient, and it has been considered necessary to increase the amount of secreted proteins at the industrial level. At least 2-3 times, and desirably 4 times or more the secretion amount when using a device of the type is not achieved.

[0004] It is an object of the present invention to provide a DNA derived from a coryneform bacterium which encodes a mutant secretory apparatus necessary for efficiently secreting a predetermined protein into a medium at an industrial level.

[0005]

Means for Solving the Problems The present inventors have conducted intensive studies in order to solve the above-mentioned problems, and as a result, it has become necessary to efficiently secrete a predetermined protein into a culture medium in coryneform bacteria. The present inventors have found gene DNA encoding a mutant secretion apparatus, and have completed the present invention.

Thus, according to the present invention, there is provided a protein having an amino acid sequence corresponding to the amino acid sequence of SEQ ID NO: 2 wherein the amino acid residue corresponding to the amino acid residue at position 44 in the amino acid sequence of SEQ ID NO: 2 is a phenylalanine residue. A secretory apparatus, which is a DNA encoding and comprises the protein, comprises:
DNA that secretes more efficiently than a secretion apparatus including a protein having the amino acid sequence of SEQ ID NO: 13 (hereinafter, also referred to as DNA of the present invention), a recombinant plasmid into which the DNA has been introduced, and the plasmid Coryneform bacteria are provided.

[0007] The DNA of the present invention preferably comprises SEQ ID NO: 2.
Encodes the amino acid sequence of Specifically, SEQ ID NO: 1
DNA having the base sequence of The coryneform bacterium is preferably Brevibacterium flavum.
bacterium flavum) MJ-233.

[0008] It is preferable that the recombinant plasmid further contains a DNA containing a gene responsible for the replication growth function in coryneform bacteria.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in more detail. The DNA of the present invention encodes a protein constituting a secretory apparatus necessary for efficiently secreting and producing a predetermined protein produced in a coryneform bacterium outside the cell (for example, in a culture medium). On the chromosome of a type bacterium,
Alternatively, it is used by causing it to exist outside the chromosome. That is, the DNA of the present invention refers to a gene of a protein that exists on a chromosome or extrachromosome, recognizes the N-terminal sequence of a secretory protein precursor, and constitutes a secretory apparatus that secretes and produces the secretory protein in a medium. Things.

[0010] The mutant secretory apparatus comprising the protein encoded by the DNA of the present invention comprises a gene encoding the secE gene DNA which is one of the gene DNA groups encoding the protein group constituting the protein secretory apparatus. Instead, it is considered to include the protein encoded by the DNA of the present invention.

The DNA of the present invention can be obtained from coryneform bacteria
A DNA fragment containing cE gene DNA (hereinafter referred to as “A
Fragment, which may be abbreviated as "fragment"), and then modify it.

[0012] The fragment A can be cloned from a chromosome derived from a coryneform bacterium. As a coryneform bacterium serving as a source, for example, Brevibacterium flavum MJ-233 (FER)
MBP-1498), Brevibacterium ammoniagenes, ATCC
6871, ATCC 13745, ATCC 1374
6. Brevibacterium debaricatam
um devaricatum) ATCC 14020, Brevibacterium lactofementum
rmentum) ATCC 13869, Corynebacterium
Glutamicum (Corynebacterium glutamicum) ATCC
31831 or the like can be used.

An example of the basic operation for preparing the A fragment from these source microorganisms is as follows.
The A fragment is obtained from the above-mentioned coryneform bacterium, for example, Brevibacterium flavum MJ-23.
3 (FERM BP-1497), a conventional method for isolating a protein gene that is present on the chromosome and encoded on the chromosome, that is, based on the amino acid sequence of a complementary or homologous region using a mutant strain of Escherichia coli. Can be separated and obtained by a hybridization method using an oligo DNA probe designed as described above.

By isolating the fragment containing the target gene and determining the nucleotide sequence of the isolated fragment, all the gene fragments can be confirmed. The above-mentioned A fragment is not limited to a DNA isolated from a natural coryneform bacterial chromosomal DNA, but may be a commonly used DNA after the base sequence is determined.
Synthesizers such as Applied Biosystems (App
lied Biosystems) 394 DNA / RNA synthesizer.

The A fragment obtained as described above is modified so that the amino acid residue corresponding to the amino acid residue at position 44 in the amino acid sequence shown in SEQ ID NO: 2 becomes a phenylalanine residue. This modification can be produced by introducing a mutation such as nucleotide substitution, deletion or insertion into the nucleotide sequence by a known method such as site-directed mutation.

The DNA of the present invention can be synthesized using a commonly used DNA synthesizer after its base sequence is determined. The protein encoded by the DNA of the present invention has a sequence corresponding to the amino acid sequence of SEQ ID NO: 2,
Is characterized in that the amino acid residue corresponding to the amino acid residue at position 1 has an amino acid sequence of a phenylalanine residue.
A secretion device that secretes the protein more efficiently than a secretion device comprising a protein having the amino acid sequence of, preferably, a mutation necessary for efficiently secreting a given protein into a medium at an industrial level. It constitutes a type secretory device. Therefore, the protein encoded by the DNA of the present invention may be any of SEQ ID NO: 2 if it does not substantially impair the above-mentioned functions and can secrete and produce a predetermined protein in a medium at an industrial level. Some amino acid residues in the amino acid sequence shown may be substituted or deleted with other amino acid residues, or new amino acid residues may be inserted. Some may be transposed, and any of the DNAs encoding these derivatives may be:
It is included in the DNA of the present invention as encoding a sequence corresponding to the amino acid sequence of SEQ ID NO: 2.

Particularly, those having homology of 80% or more, more preferably 90% or more with the amino acid sequence shown in SEQ ID NO: 2 are preferable. In addition, a part of the protein structure can be changed by a natural or artificial mutation without substantially changing the above function. That is, the present invention D
The protein encoded by NA also includes a protein having a structure corresponding to the homologous variant of the protein having the amino acid sequence shown in SEQ ID NO: 2. Also included are equivalent sequences that can be obtained by introducing a mutation by a normal mutagenesis method and performing selection by a normal screening method using the secretion amount as an index.

Functional expression of DNA of the present invention and DNA of the present invention
The secretion efficiency of the secretory apparatus comprising the protein encoded by the secretory protein should be confirmed and evaluated by measuring the activity of the secretory protein secreted into the medium, for example, as described in Example 2 below. Can be.

Examples of the secretory protein to be used include amylase. The amylase gene is inserted into a plasmid capable of replicating in the chromosome of the coryneform bacterium or in a coryneform bacterium, and the mutant secreted protein is expressed using the amylase activity as an index. The secretory capacity of the device can be determined. In addition, not only amylase, but also proteases, cellulases, xylanases, pullulanases, and the like that are normally secreted outside the cells can be used. The predetermined protein to be secreted is not particularly limited as long as the secretory apparatus is involved in the secretion, and is not limited to an enzyme protein whose measurement is easy.

A specific example of the DNA of the present invention is the base sequence shown in SEQ ID NO: 1. The recombinant plasmid into which the DNA of the present invention has been introduced can be obtained by introducing the DNA of the present invention into a commonly used plasmid in a conventional manner. For example, in the same manner as described for a DNA fragment containing the secE gene DNA in JP-A-6-270073, the DNA of the present invention is responsible for the replication-proliferation function of the plasmid in an appropriate plasmid, for example, a coryneform bacterium. By introducing the gene into a plasmid vector containing at least a gene, a recombinant plasmid capable of highly expressing a protein constituting a mutant secretory apparatus in coryneform bacteria can be obtained.

The coryneform bacterium having the above-mentioned recombinant plasmid can be obtained by transforming the coryneform bacterium with the above-mentioned recombinant plasmid according to a conventional method. For example, it can be obtained in the same manner as described in Japanese Unexamined Patent Publication (Kokai) No. 6-270073 for a coryneform bacterium having a recombinant plasmid.

[0022]

The present invention has been described above, and will be described more specifically with reference to the following examples. Preparation Example 1 Preparation of plasmid for evaluation (A) Construction of shuttle vector Plasmid p described in US Pat. No. 5,185,262
From CRY31, a region necessary for stabilizing the plasmid in coryneform bacteria was amplified by PCR, and pBlue
Eco of scriptIISK + (manufactured by TOYOBO)
It was cloned into RI and XhoI sites.

The sequence of the region required for stabilizing the plasmid in coryneform bacterium, which is present in the plasmid pCRY31, has been determined. Based on this sequence, the following pair of primers was used to apply Applied Bio Systems (Applie
d Biosystems) using 394 DNA / RNA synthesizer.

(A-1) 5'-TTT CTC GAG CGC ATT ACC TCC
TTG CTA CTG-3 '(SEQ ID NO: 3) (b-1) 5'-TTT GAA TTC GAT ATC AAG CTT GCA CAT CAA-
3 ′ (SEQ ID NO: 4) (In the sequence, A represents adenine, G represents guanine, C represents cytosine, and T represents thymine.)

For PCR, a DNA thermal cycler manufactured by Perkin Elmer Cetus Co., Ltd. was used, and as a reaction reagent, recombinant tack DNA / polymerase / takara.
Tack (Recombinant Taq DNA Polymerase TaKaRa Taq)
(Takara Shuzo) under the following conditions. Reaction solution: (10 ×) PCR buffer 10 μl 1.25 mM dNTP mixture 16 μl Template DNA 10 μl (DNA content 1 μM or less) 1 μl each of the above-mentioned a-1 and b-1 primers (final concentration 0.25 μM) Recombinant Tack DNA polymerase 0.5 μl (2.5 Unit) 61.5 μl or more of sterile distilled water was mixed, and 100 μl of the reaction solution was subjected to PCR. PCR cycle: Denaturation process: 94 ° C. for 60 seconds Annealing process: 52 ° C. for 60 seconds Extension process: 72 ° C. for 120 seconds One cycle was performed as described above, and 25 cycles were performed.

The above-prepared reaction solution (10 μl) was added to 0.8%
When subjected to electrophoresis on an agarose gel, about 1.
A 1 kb DNA fragment was detected. 10 μl of the reaction solution in which the amplification product was confirmed above and plasmid pBlues
Restriction enzyme Eco was added to 1 μl of the scriptIISK + solution.
RI and XhoI were added to completely cut the DNA in each solution, followed by treatment at 70 ° C. for 10 minutes to inactivate the restriction enzymes. Then, the two were mixed and buffered with T4 DNA ligase (10 ×) buffer. Solution 1µl, T4 DNA ligase 1
Each component of the unit was added, adjusted to 10 μl with sterile distilled water, reacted at 15 ° C. for 3 hours, and bound.

Using the resulting plasmid mixture, the calcium chloride method [Journal of Molecular Biology, 53, 15
9 (1970)] by Escherichia coli JM109
(Manufactured by Takara Shuzo), and a medium containing 50 mg of ampicillin [10 g of tryptone, 5 g of yeast extract, 5 g of NaCl and 16 g of agar was distilled water 1
L).

The strain grown on this medium was subjected to liquid culture by a conventional method, plasmid DNA was extracted from the culture solution, the plasmid was cut with restriction enzymes, and the inserted fragment was confirmed. As a result, an inserted DNA fragment having a length of 1.1 kb was recognized in addition to the DNA fragment having a length of 3.0 kb and the plasmid pBluescriptIISK +. This plasmid is called pBSp
ar.

Next, from the plasmid pCRY31 described in US Pat. No. 5,185,262, a region necessary for plasmid replication in coryneform bacteria was amplified by PCR, and the XhoI, Kpn of the above plasmid pBSpar was amplified.
Cloning at the I site produced a shuttle vector.

The sequence of the region required for plasmid replication in coryneform bacteria, which is present in plasmid pCRY31, has been determined. Based on this sequence, the following pair of primers was used to apply Applied Biosystems. (Applied
The DNA was synthesized using a 394 DNA / RNA synthesizer (Biosystems).

(A-2) 5'-TTT GGT ACC GAC TTA GAT AAA
GGT CTA-3 '(SEQ ID NO: 5) (b-2) 5'-TTT CTC GAG TGC TGG TAA AAC AAC TTT-3'
(In the sequence, A indicates adenine, G indicates guanine, C indicates cytosine, and T indicates thymine.)

The PCR was carried out using a DNA thermal cycler manufactured by Perkin Elmer Cetus Co., Ltd., and recombinant tuck DNA polymerase polymerase TAKARA
Tack (Recombinant Taq DNA Polymerase TaKaRa Taq)
(Takara Shuzo) under the following conditions. Reaction solution: (10 ×) PCR buffer 10 μl 1.25 mM dNTP mixture 16 μl Template DNA 10 μl (DNA content 1 μM or less) 1 μl each of the above-mentioned a-2 and b-2 primers (final concentration 0.25 μM) Recombinant Tack DNA polymerase 0.5 μl (2.5 Unit) 61.5 μl or more of sterile distilled water was mixed, and 100 μl of the reaction solution was subjected to PCR. PCR cycle: Denaturation process: 94 ° C. for 60 seconds Annealing process: 52 ° C. for 60 seconds Extension process: 72 ° C. for 120 seconds One cycle was performed as described above, and 25 cycles were performed.

10 μl of the reaction solution produced above was added to 0.8%
When subjected to electrophoresis on an agarose gel, about 1.
An 8 kb DNA fragment was detected. 10 μl of the reaction solution in which the amplification product was confirmed above and plasmid pBSpar
Restriction enzymes XhoI and KpnI were respectively added to 1 μl of the solution to completely cleave the nucleic acid in each solution.
After inactivating the restriction enzyme by treating for 0 min, the two were mixed and mixed with 1 μl of T4 DNA ligase (10 ×) buffer.
l, add each component of 1 unit of T4 DNA ligase,
The mixture was made up to 10 μl with sterile distilled water, reacted at 15 ° C. for 3 hours, and bound.

Using the resulting plasmid mixture, the calcium chloride method [Journal of Molecular Biology, 53, 15
9 (1970)] by Escherichia coli JM109
(Manufactured by Takara Shuzo), and a medium containing 50 mg of ampicillin [10 g of tryptone, 5 g of yeast extract, 5 g of NaCl and 16 g of agar was distilled water 1
1).

The strain grown on this medium was subjected to liquid culture by a conventional method, plasmid DNA was extracted from the culture, and the plasmid was cut with restriction enzymes to confirm the inserted fragment. As a result, a plasmid of 4.1 kb in length was obtained from the plasmid pBSpar.
In addition to the A fragment, an inserted DNA fragment having a length of 1.8 kb was observed. This plasmid was designated as pBSpar-rep.

The plasmid pBSpar-
Restriction enzymes KpnI and EcoRI were added to 1 μl of the rep solution and 1 μl of pHSG298 (manufactured by Takara Shuzo Co., Ltd.), respectively, to completely cut the DNA in each solution.
After inactivating restriction enzymes by treating for 10 minutes,
The two were mixed, and T4 DNA ligase (10 ×) buffer 1
1 μl of each unit of 1 unit of T4 DNA ligase was added, made up to 10 μl with sterile distilled water, reacted at 15 ° C. for 3 hours, and bound.

Using the resulting plasmid mixture, the calcium chloride method [Journal of Molecular Biology, 53, 15
9 (1970)] by Escherichia coli JM109
(Manufactured by Takara Shuzo), and a medium containing 50 mg of kanamycin [10 g of tryptone, 5 g of yeast extract, 5 g of NaCl, and 16 g of agar were distilled water 1
1).

The strain grown on this medium was subjected to liquid culture by a conventional method, plasmid DNA was extracted from the culture, and the plasmid was cut with restriction enzymes to confirm the inserted fragment. This resulted in a plasmid pHSG298 with a 2.6 kb D
In addition to the NA fragment, an inserted DNA fragment of 2.9 kb in length was observed. This plasmid was transferred to pHSG298par-re.
It was named p.

(B) Insertion of the tac promoter In order to amplify the tac promoter fragment by PCR using the plasmid pTrc99A as a template, the following pair of primers was used with 394 DNA / RNA manufactured by Applied Biosystems. Synthesized using a synthesizer.

(A-3) 5'-TTT GGT ACC GAT AGC TTA CTC
CCC ATC CCC-3 '(SEQ ID NO: 7) (b-3) 5'-TTT GGA TCC CAA CAT ATG AAC ACC TCC TTT
TTA TCC GCT CACAAT TCC ACA CAT-3 '(SEQ ID NO: 8) (In the sequence, A indicates adenine, G indicates guanine, C indicates cytosine, and T indicates thymine.)

The PCR was carried out using a DNA thermal cycler manufactured by Perkin Elmer Cetus Co., Ltd.
Tack (Recombinant Taq DNA Polymerase TaKaRa Taq)
(Takara Shuzo) under the following conditions. Reaction solution: (10 ×) PCR buffer 10 μl 1.25 mM dNTP mixture 16 μl Template DNA 10 μl (DNA content 1 μM or less) 1 μl each of the above-mentioned a-3 and b-3 primers (final concentration 0.25 μM) Recombinant Tack DNA polymerase 0.5 μl (2.5 Unit) 61.5 μl or more of sterile distilled water was mixed, and 100 μl of the reaction solution was subjected to PCR. PCR cycle: Denaturation process: 94 ° C. for 60 seconds Annealing process: 52 ° C. for 60 seconds Extension process: 72 ° C. for 120 seconds One cycle was performed as described above, and 25 cycles were performed.

When 10 μl of the reaction solution produced above was subjected to electrophoresis on a 3% agarose gel, about 100 b
p DNA fragment was detected. Restriction enzymes BamHI and KpnI were respectively added to 10 μl of the reaction solution in which the amplification product was confirmed above and 5 μl of the plasmid pHSG298par-rep solution prepared in (A) above, and the DNA in each solution was completely cut. After inactivating the restriction enzyme by treating at 10 ° C. for 10 minutes, the two were mixed,
1 μl of T4 DNA ligase (10 ×) buffer, T4 D
One unit of NA ligase was added, and the mixture was made up to 10 μl with sterilized distilled water, reacted at 15 ° C. for 3 hours, and bound.

Using the obtained plasmid mixture, the calcium chloride method [Journal of Molecular Biology, 53, 15
9 (1970)] by Escherichia coli JM109
(Manufactured by Takara Shuzo), and a medium containing 50 mg of kanamycin [10 g of tryptone, 5 g of yeast extract, 5 g of NaCl, and 16 g of agar were distilled water 1
1).

The strain grown on this medium was subjected to liquid culture by a conventional method, plasmid DNA was extracted from the culture solution, the plasmid was cut with restriction enzymes, and the inserted fragment was confirmed. As a result, an inserted DNA fragment of 0.1 kb in length was observed in addition to the 5.5 kb DNA fragment of the plasmid prepared in (A) above. This plasmid was named pHSG298tac.

(C) Insertion of amylase gene Bacillus amylorequefacien
s amylase gene has been sequenced [Journa
l of Biological Chemistry, 258, 1007-1013 (198
3)], This gene portion was amplified by PCR, and inserted into the plasmid pHSG298tac prepared in the above (B) to prepare a plasmid for evaluation.

The following pair of primers were used
394 manufactured by Applied Biosystems
Synthesized using a DNA / RNA synthesizer.

(A-4) 5'-TTT CAT ATG ATT CAA AAA CGA
AAG-3 '(SEQ ID NO: 9) (b-4) 5'-TTT GGA TCC TTA TTT CTG AAC ATA-3' (SEQ ID NO: 10) (In the sequence, A is adenine, G is guanine, C is cytosine, T Represents thymine.)

For PCR, a DNA thermal cycler manufactured by Perkin Elmer Cetus was used, and as a reaction reagent, recombinant tack DNA polymerase Takara PCR was used.
Tack (Recombinant Taq DNA Polymerase TaKaRa Taq)
(Takara Shuzo) under the following conditions. Reaction solution: (10 ×) PCR buffer 10 μl 1.25 mM dNTP mixture 16 μl Template DNA 10 μl (DNA content 1 μM or less) 1 μl each of the above-mentioned a-4 and b-4 primers (final concentration 0.25 μM) Recombinant Tack DNA polymerase 0.5 μl (2.5 Unit) 61.5 μl or more of sterile distilled water was mixed, and 100 μl of the reaction solution was subjected to PCR. PCR cycle: Denaturation process: 94 ° C. for 60 seconds Annealing process: 52 ° C. for 60 seconds Extension process: 72 ° C. for 120 seconds One cycle was performed as described above, and 25 cycles were performed.

10 μl of the reaction solution produced above was added to 0.8%
When subjected to electrophoresis on an agarose gel, about 1.
A 3 kb DNA fragment could be detected. Restriction enzymes NdeI and B were added to 10 μl of the reaction solution in which the amplification product was confirmed above and 5 μl of the plasmid solution prepared in (B) above, respectively.
amHI was added, the DNA in each solution was completely cut, and then treated at 70 ° C. for 10 minutes to inactivate the restriction enzymes. Then, both were mixed, and T4 DNA ligase (10
×) Add 1 μl of buffer solution and 1 unit of T4 DNA ligase, make up to 10 μl with sterile distilled water, and add
And allowed to react for 3 hours.

Using the obtained plasmid mixture, the calcium chloride method [Journal of Molecular Biology, 53, 15]
9 (1970)] by Escherichia coli JM109
(Manufactured by Takara Shuzo), and a medium containing 50 mg of kanamycin [10 g of tryptone, 5 g of yeast extract, 5 g of NaCl, and 16 g of agar were distilled water 1
1).

The strain grown on this medium was subjected to liquid culture by a conventional method, plasmid DNA was extracted from the culture, and the plasmid was cut with restriction enzymes to confirm the inserted fragment. As a result, an inserted DNA fragment having a length of 1.3 kb was observed in addition to the DNA fragment having a length of 5.6 kb prepared in the above (B).

This plasmid was named pSec1.

Example 1 Modification of Gene DNA Fragment of Protein Constituting Secretory Device Derived from Coryneform Bacteria (A) Isolation of Gene DNA Fragment of Protein Constituting Secretory Device Derived from Coryneform Bacteria JP-A-6-169780, JP-A-6-27707
According to the methods described in Japanese Patent Publication No. 3 and JP-A-7-107981, the genes encoding the major factors SecY, SecE and SecA constituting the secretory apparatus derived from coryneform bacteria were respectively isolated.

(B) Modification of the DNA fragment of the gene constituting the protein constituting the secretory apparatus derived from coryneform bacterium The plasmid pUC118-secE containing the gene encoding SecE prepared in (A) was used as a template for PC
By performing R, the secE gene fragment was amplified. At this time, in order to reduce the fidelity of Taq polymerase, Mn ions were present in the reaction solution, and the fragment into which the mutation was randomly introduced was amplified.

The following pair of primers were used
394 manufactured by Applied Biosystems
Synthesized using a DNA / RNA synthesizer.

(A-5) 5'-TTT GGA TCC ATG GGA GAA GTC
CGT AAG -3 '(SEQ ID NO: 11) (b-5) 5'-TTT CCT GCA GGC TAC GGA GTC AGA ATC TT-
3 ′ (SEQ ID NO: 12) (In the sequence, A indicates adenine, G indicates guanine, C indicates cytosine, and T indicates thymine.)

In the PCR, a DNA thermal cycler manufactured by Perkin Elmer Cetus Co., Ltd. was used.
Tack (Recombinant Taq DNA Polymerase TaKaRa Taq)
(Takara Shuzo) under the following conditions. Reaction solution: (10 ×) PCR buffer 10 μl 1.25 mM dNTP mixture 16 μl Template DNA 10 μl (DNA content 1 μM or less) 1 μl each of the above-mentioned a-5 and b-5 primers (final concentration 0.25 μM) 100 mM MnCl ₂ 0.5 μl Recombinant tack DNA polymerase 0.5 μl (2.5 Unit) 59.5 μl or more of sterile distilled water were mixed, and 100 μl of the reaction solution was subjected to PCR. PCR cycle: Denaturation process: 94 ° C. for 60 seconds Annealing process: 52 ° C. for 60 seconds Extension process: 72 ° C. for 120 seconds One cycle was performed as described above, and 25 cycles were performed.

When 10 μl of the reaction solution formed above was subjected to electrophoresis on a 3% agarose gel, about 150 b
Was detected. Restriction enzyme Ba was added to 10 μl of the reaction solution in which the amplification product was confirmed as described above and 5 μl of the solution of plasmid pSec1 prepared in Example 1 (C).
mHI and SseI were added to completely cut the DNA in each solution, followed by treatment at 70 ° C. for 10 minutes to inactivate the restriction enzymes. Then, both were mixed and buffered with T4 DNA ligase (10 ×). 1 µl of the solution and 1 unit of T4 DNA ligase were added, and the mixture was made up to 10 µl with sterile distilled water, reacted at 15 ° C for 3 hours, and bound.

On the other hand, wild-type s
The ecE gene was amplified and the plasmid pS
It was inserted into the BamHI and SseI sites of ec1.

(C) Identification of Mutations that Increase Secretion Ability A ligation solution containing the mutated secE gene prepared in Example 1 (B) and the wild-type secE gene is described in US Pat. No. 5,185,262. According to the method of Brevibacterium flavum MJ-233
(FERM BP-1498).

The bacterial cell solution was spread on an LB medium containing 1% of corn starch and 50 μg / l of kanamycin, and a large halo formed was picked up. A plasmid was extracted from this colony and a colony transformed with the wild-type secE gene by a conventional method, and the plasmid was cut with a restriction enzyme to confirm an inserted fragment. As a result, in addition to the 6.9 kb long DNA fragment prepared in Preparation Example 1 (C),
An inserted DNA fragment of 150 b in length was observed.

The plasmid having the mutant type secE gene inserted therein was named pSec1Emt, and the plasmid having the wild type secE gene inserted therein was named pSec1Ewt. The DNA base sequence of the mutant secE gene inserted into this plasmid was determined. The determined nucleotide sequence and the amino acid sequence deduced therefrom are shown in the following Sequence Listing, SEQ ID NO: 1.
Shown in In addition, only the amino acid sequence is shown in SEQ ID NO: 2.
As a result, it was found that the amino acid Val at position 44 of the wild-type secE protein was changed to Phe. In addition,
The amino acid sequence of the wild-type secE protein is shown in SEQ ID NO: 13.

Example 2 Evaluation of Mutant Secretion Apparatus The pSec1Emt and pSEC1Ewt plasmids were prepared according to the method described in US Pat. No. 5,185,262 to Brevibacterium flavum MJ-233 (FERM).
BP-1498).

The transformed Brevibacterium flavum MJ-233 cells were placed in a loop of 10 ml of a semi-synthetic medium A medium [composition: 2 g of urea, (NH ₄ ) ₂ SO ₄ 7].
_{g, K 2 HPO 4 0. 5g} , KH 2 PO 4 0.5g, M
gSO ₄ 0.5 g, FeSO ₄ .7H ₂ O 6 mg, M
nSO ₄ · 4~6H ₂ O 6mg, yeast extract 2.5
g, casamino acid 5 g, biotin 200 μg, thiamine hydrochloride 200 μg, and glucose 20 g were dissolved in 1 liter of distilled water].
Shaking culture (pre-culture) was performed for 16 hours. Next, A medium 100
2 ml of the preculture was inoculated into each ml, shaken at 31 ° C. for 5 hours, and collected to obtain a culture for amylase activity measurement.

The amylase activity was measured according to the protocol attached to a commercially available amylase activity measurement kit [Amylase B-Test Wako (manufactured by Wako Pure Chemical Industries)]. As a result of the amylase activity measurement, the activity in the culture supernatant of the cells into which pSec1Emt was introduced was increased about 5-fold as compared with the cells into which pSec1Ewt had been introduced. It was confirmed that the activity of the fragment was remarkably high.

[0066]

Industrial Applicability The present invention provides a DNA encoding a protein constituting a mutant secretory apparatus necessary for efficiently secreting a predetermined protein derived from a coryneform bacterium into a medium, and efficiently transferring the predetermined protein into the medium. Construction of a system for secretory production,
The production method can be established.

[0067]

[Sequence list]

SEQ ID NO: 1 Sequence length: 153 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: Genomic DNA Origin Organism name: Brevibacterium flavum (Brevibacteriu)
m flavum) Strain name: MJ233 Sequence characteristics Symbol indicating characteristics: peptide Location: 1..150 Method for determining characteristics: E sequence GTG GGA GAA GTC CGT AAG GTT ATT TGG CCT ACT GCG CGC CAG ATG GTC 48 Val Gly Glu Val Arg Lys Val Ile Trp Pro Thr Ala Arg Gln Met Val 1 5 10 15 ACG TAC ACC CTT GTG GTT TTG GGA TTT TTG ATT GTT TTG ACC GCT TTG 96 Thr Tyr Thr Leu Val Val Leu Gly Phe Leu Ile Val Leu Thr Ala Leu 20 25 30 GTG TCT GGT GTG GAT TTC CTA GCT GGT CTT GGA TTT GAG AAG ATT CTG 144 Val Ser Gly Val Asp Phe Leu Ala Gly Leu Gly Phe Glu Lys Ile Leu 35 40 45 ACT CCG TAG 153 Thr Pro 50

SEQ ID NO: 2 Sequence length: 50 Sequence type: amino acid Topology: linear Sequence type: peptide sequence Val Gly Glu Val Arg Lys Val Ile Trp Pro Thr Ala Arg Gln Met Val 1 5 10 15 Thr Tyr Thr Leu Val Val Leu Gly Phe Leu Ile Val Leu Thr Ala Leu 20 25 30 Val Ser Gly Val Asp Phe Leu Ala Gly Leu Gly Phe Glu Lys Ile Leu 35 40 45 Thr Pro 50

SEQ ID NO: 3 Sequence length: 30 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other Synthetic DNA sequence TTTCTCGAGC GCATTACCTC CTTGCTACTG 30

SEQ ID NO: 4 Sequence length: 30 Sequence type: nucleic acid Number of strands: single strand Topology: linear Sequence type: other Synthetic DNA sequence TTTGAATTCG ATATCAAGCT TGCACATCAA 30

SEQ ID NO: 5 Sequence length: 27 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other Synthetic DNA sequence TTTGGTACCG ACTTAGATAA AGGTCTA 27

SEQ ID NO: 6 Sequence length: 27 Sequence type: nucleic acid Number of strands: single stranded Topology: linear Sequence type: other Synthetic DNA sequence TTTCTCGAGT GCTGGTAAAA CAACTTT 27

SEQ ID NO: 7 Sequence length: 30 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other Synthetic DNA sequence TTTGGTACCG ATAGCTTACT CCCCATCCCC 30

SEQ ID NO: 8 Sequence length: 54 Sequence type: nucleic acid Number of strands: single strand Topology: linear Sequence type: other Synthetic DNA sequence TTTGGATCCC AACATATGAA CACCTCCTTT TTATCCGCTC ACAATTCCAC ACAT 54

SEQ ID NO: 9 Sequence length: 24 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other Synthetic DNA sequence TTTCATATGA TTCAAAAACG AAAG 24

SEQ ID NO: 10 Sequence length: 24 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other Synthetic DNA sequence TTTGGATCCT TATTTCTGAA CATA 24

SEQ ID NO: 11 Sequence length: 27 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other Synthetic DNA sequence TTTGGATCCA TGGGAGAAGT CCGTAAG 27

SEQ ID NO: 12 Sequence length: 29 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other Synthetic DNA sequence TTTCCTGCAG GCTACGGAGT CAGAATCTT 29

SEQ ID NO: 13 Sequence length: 50 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Val Gly Glu Val Arg Lys Val Ile Trp Pro Thr Ala Arg Gln Met Val 1 5 10 15 Thr Tyr Thr Leu Val Val Leu Gly Phe Leu Ile Val Leu Thr Ala Leu 20 25 30 Val Ser Gly Val Asp Phe Leu Ala Gly Leu Gly Val Glu Lys Ile Leu 35 40 45 Thr Pro 50

──────────────────────────────────────────────────の Continued on front page (51) Int.Cl. ⁶ Identification code FI (C12N 1/21 C12R 1:19) (C12N 1/21 C12R 1:13) (C12P 21/02 C12R 1:19)

Claims (7)

[Claims] 1. an amino acid sequence of SEQ ID NO: 2
A DNA encoding a protein having an amino acid sequence corresponding to the amino acid sequence of SEQ ID NO: 2 wherein the amino acid residue corresponding to the amino acid residue at position is a phenylalanine residue; A DNA that allows a predetermined protein to be secreted more efficiently in a coryneform bacterium than a secretion apparatus that includes a protein having the amino acid sequence of SEQ ID NO: 13. 2. The DNA according to claim 1, which encodes the amino acid sequence of SEQ ID NO: 2. 3. The DNA according to claim 1, wherein the coryneform bacterium is Brevibacterium flavum MJ-233. 4. The DNA according to claim 1, which has the nucleotide sequence of SEQ ID NO: 1. 5. The DNA according to any one of claims 1 to 4,
A recombinant plasmid into which is introduced. 6. The DNA according to any one of claims 1 to 4.
The recombinant plasmid according to claim 5, which comprises a DNA containing a gene that controls replication and growth in coryneform bacteria. 7. A coryneform bacterium having the recombinant plasmid according to claim 5 or 6.