JPH04278093A - New recombinant plasmid, coryneform bacterium transformed with the same plasmid and production of biotin using the same - Google Patents

Abstract

PURPOSE:To provide a method for analyzing and isolating a gene participating in biosynthesis of biotin in a coryneform bacterium and efficiently producing the biotin with the coryneform bacterium utilizing the aforementioned gene. CONSTITUTION:A recombinant plasmid holding A DNA region containing a gene capable of coding diaminopelargonic aminotransferase and desthiobiotin synthetase derived from the chromosome of Brevibacterium.flavum MJ-233 strain, a DNA region containing a gene capable of coding a biotin synthetase derived from the chromosome of Brevibacterium.flavum MJ-233 strain and a DNA region containing a gene governing the replicating and proliferating function of the plasmid in the coryneform bacterium. A coryneform bacterium transformed with the aforementioned plasmid and a method for producing the biotin using the resultant coryneform bacterium are provided.

Description

[Detailed description of the invention]

0001

[Industrial Application Field] The present invention relates to genes encoding at least three enzymes involved in biotin biosynthesis derived from coryneform bacteria, and at least plasmid replication and proliferation within coryneform bacteria cells. The present invention relates to a recombinant plasmid carrying a functional gene, a coryneform bacterium transformed with the plasmid, and a method for producing biotin using the same.

Biotin is a type of vitamin that is required for the growth of humans, animals, plants, and certain microorganisms, and is used particularly as a skin metabolism regulator, as a hair growth agent to prevent hair loss in humans, or as livestock feed. It is a useful substance used as an additive.

0003

[Prior Art] Conventionally, methods for producing biotin using microorganisms include Bacillus, Escherichia, Agrobacterium, Chromobacterium, Pseudomonas ( A method using microorganisms such as Pseudomonas genus and Arthrobacter genus is known (Japanese Patent Application Laid-open No. 160998/1983). Also, methods have been proposed to impart biotin-producing ability by artificially causing mutations in these wild strains (for example, H. Yamagata et al., Agri. Bi.
al. Chem. , 47, 1611, 1983).

However, when trying to produce biotin using microorganisms, wild-type strains have a strong feedback suppression mechanism due to biotin (Y. Izumi, K.
Ogata, Adv. Appl. Microbial.
22, 155-157, 1977), biotin is produced in very small amounts. Furthermore, even with methods using mutant strains, the amount produced was not always satisfactory.

[0005] Additionally, certain strains of coryneform bacteria including Brevibacterium and Corynebacterium species, such as Brevibacterium flavum, have many advantages in industrial applications.
avum) MJ-233, Brevibacterium lactofamentum (Brevibacterium la
ctofermentum) ATCC13869, Corynebacterium glutamicum (Corynebacterium glutamicum)
erium glutamicum) ATCC3183
1. Brevibacterium ammoniagenes (Brev
ibacterium ammoniagenes)A
TCC13745 etc. have biotin requirement,
It is known that it does not produce any biotin.

[0006] As genes involved in biotin biosynthesis, genes derived from Escherichia coli have been well studied, including bioA, bioB, bioC, bioD, bio
It is known that oF and bioH genes exist. Among these, bioA is 7,8-diaminoperargonic acid aminotransferase, bioB is biotin synthetase, bioC is pimelyl-CoA synthetase, bio
It is known that D encodes desthiobiotin synthetase and bioF encodes 7-keto-8-aminopelargonic acid synthetase, and the function of bioH is not yet clear (A.J. Otsuka et. al.
, J. Biol. Chem. 263, 19577-1
9585, 1988). Also, bioA, bioB, b
ioC, bioD, bioF genes are bioABFCD
It is known that the expression of this operon is controlled by an operator located between the bioA and bioB genes. In addition, the operator is controlled by the biotin repressor encoded in the bioA gene, which binds to the operator when activated by biotin and suppresses the expression of the biotin biosynthesis operon. It is known that (J. Biol. Chem.
263, 1013-1016, 1988).

[0007]

[Problems to be Solved by the Invention] This invention analyzes and isolates genes involved in biotin biosynthesis in coryneform bacteria, and utilizes the genes to efficiently produce biotin in coryneform bacteria. It was done for a purpose.

[0008]

[Means for Solving the Problems] The present inventors have conducted extensive research in order to achieve the above object. As a result, cross-complementation tests using biotin-auxotrophic E. coli mutant strains revealed that biotin-auxotrophic coryneform bacteria have at least bioB, bioA
It has been revealed that it possesses three genes involved in biotin biosynthesis:
The present inventors have discovered that biotin can be efficiently produced by introducing this plasmid-transformed coryneform bacterium into a culture medium, and have completed the present invention.

Thus, according to the present invention, (1) a DNA region (a) containing genes encoding diaminoperargonic acid aminotransferase and desthiobiotin synthetase derived from coryneform bacteria; DNA containing a gene encoding biotin synthetase derived from type bacteria
a recombinant plasmid having at least the region (b) and the DNA region (c) containing a gene that controls the replication and propagation function of the plasmid in coryneform bacterial cells; (2) a coryneform bacteria transformed with the recombinant plasmid; (3) A method for producing biotin is provided, which comprises culturing the coryneform bacteria in a medium and collecting biotin from the culture.

[0010] Hereinafter, the structure of the plasmid newly created in the present invention, the transformation of coryneform bacteria with the plasmid, and the method for producing biotin using the coryneform bacteria obtained by the transformation will be explained in more detail. The plasmid of the present invention possesses a "DNA region containing genes encoding diaminoperargonic acid aminotransferase and desthiobiotin synthetase" (hereinafter referred to as "b").
(sometimes abbreviated as "ioA bioD area") is 7
Coating an enzyme that catalyzes the conversion reaction of -keto-8-aminopelargonic acid to 7,8-diaminoperargonic acid, that is, diaminoperargonic acid aminotransferase.
A DNA region containing both the genes encoding desthiobiotin synthetase (bioA) and the enzyme that catalyzes the conversion reaction of 7,8 diaminoperargonic acid to desthiobiotin, that is, desthiobiotin synthetase (bioD). It is.

[0011] Furthermore, the "DNA region containing the gene encoding biotin synthetase" (hereinafter sometimes referred to as the "bioB region") catalyzes the conversion reaction from desthiobiotin to biotin. This is a DNA region containing a gene (bioB) encoding an enzyme that produces biotin synthetase.

[0012] The above bioA bioD area and bioB
The microorganism that serves as the source of the area is not particularly limited as long as it is a coryneform bacterium, but generally Brevibacterium flavum MJ-233 (FERM BP-14
97) and its derived strain, Brevibacterium ammo
niagenes) ATCC6871, ATCC13
745, ATCC 13746, Brevibacterium
Brevibacterium div
aricatum) ATCC 14020, Brevibacterium lactofamentum (Brevibacte
rium lactofermentum) ATCC13
869, Corynebacterium glutamicum (Cory
nebacterium glutamicum)AT
CC31831 etc. are advantageously used.

[0013] From these source microorganisms, bioA bi
An example of the basic operation for preparing the oD region and the bioB region is as follows.

[0014] That is, the bioA bioD region and b
The ioB region is derived from the above-mentioned coryneform bacteria, such as Brevibacterium flavum strain MJ-233 (FERM BP-1
497), and can be isolated and obtained from the cleavage fragments produced by cleaving this chromosome with an appropriate restriction enzyme by the method described below.

First, Brevibacterium flavum MJ
Chromosomal DNA is extracted from the culture of -233 strain. This chromosomal DNA is partially digested using an appropriate restriction enzyme, such as Sau3AI, so that the size of the DNA fragment is about 20 to 30 kb.

[0016] The obtained DNA fragment was transformed into a cosmid vector,
For example, insert this cosmid into pWE15 and convert it into λDNA.
When preparing the bioAbioD region by transduction using an in vitro Packaging Kit, a biotin-auxotrophic E. coli mutant strain lacking bioA or bioD (Journal of Bacte.
riology, vol 94, p2065-2066
, 1967 and Journal of Bacter
iology vol 112, p830-839, 1
972), and when preparing the bioB region, a biotin-auxotrophic E. coli mutant strain lacking bioB (see
Journal of Bacteriology,v
ol 94, p2065-2066, 1967 and J
our own of Bacteriology vo
112, p. 830-839, 1972), and these E. coli mutant strains are smeared onto a biotin-free selection medium.

[0017] By extracting cosmid DNA from the obtained transformed strain and analyzing it with restriction enzymes, it is possible to confirm and obtain the inserted bioA bioD region or bioB region derived from the chromosome of Brevibacterium flavum MJ-233 strain. can. The thus obtained bioA bi
The oD region and bioB region are approximately 20 to 30 kb in size.
This is large and impractical, so it is necessary to specify it into even shorter fragments.

Next, bioA bioD obtained above
The cosmid containing the bioB region or the bioB region is cleaved using an appropriate restriction enzyme, the resulting DNA fragment is inserted into a vector plasmid that can be replicated in E. coli, and this vector plasmid is subjected to a commonly used transformation method, e.g. By transformation using calcium method and electric pulse method, bio
When preparing the A bioD region, add to the biotin-auxotrophic E. coli mutant strain lacking bioA or bioD,
In addition, when preparing bioB, it is introduced into a biotin-requiring Escherichia coli mutant strain deficient in bioB. This E. coli mutant strain is smeared onto a selection medium that does not contain biotin.

Plasmid DNA was extracted from the resulting transformed strain.
The inserted bioA bioB region and bioB region derived from the Brevibacterium flavum strain MJ-233 chromosome can be confirmed and obtained by extracting and analyzing with restriction enzymes.

[0020] The bioA bio obtained in this way
One of the D regions is the Brevibacterium flavum M
The chromosomal DNA of the J-233 strain was excised by partial digestion with the restriction enzyme Sau3AI, and further digested with the restriction enzyme Sal.
The size obtained by cutting out with I is approximately 4.0k.
The DNA fragment of b.

[0021] The number of recognition sites and the size of the cut fragments when this approximately 4.0 kb bioA bioD region DNA fragment was cut with various restriction enzymes are shown in Table 1 below.

[0022] In this specification, the "number of recognition sites" by restriction enzymes refers to the number of recognition sites obtained by completely decomposing a DNA fragment or plasmid in the presence of an excess of restriction enzymes, and then dividing the resulting decomposition products into 1 by a method known per se. % agarose gel electrophoresis and polyacrylamide gel electrophoresis, and the value determined from the number of separable fragments was adopted.

[0023] In addition, when using agarose gel electrophoresis, the "size of the cleavage fragment" and the size of the plasmid are determined by the size of the lambda phage (λphag) of Escherichia coli.
Based on the standard line drawn by the migration distance of a DNA fragment of known molecular weight obtained by cleaving the DNA of e) with the restriction enzyme HindIII on the same agarose gel, and when using polyacrylamide gel electrophoresis, Escherichia coli's phi x 174 phage (φx17
The size of each DNA fragment of the cut DNA fragment or plasmid is determined based on a standard line drawn by the migration distance on the same polyacrylamide gel of a DNA fragment of known molecular weight obtained by cutting the DNA of 4phage) with the restriction enzyme HaeIII. calculate. The size of the plasmid is determined by adding the sizes of each cut fragment. In determining the size of each DNA fragment, the results obtained by 1% agarose gel electrophoresis are used for the size of fragments of 1 kb or more, and the size of fragments from about 0.1 kb to less than 1 kb is used. Regarding the quality, the results obtained by 4% polyacrylamide gel electrophoresis were used.

[0024]

[Table 1]
Table 1 Number of restriction enzyme recognition sites Size of cleavage fragment (kb)
BamHI 1
0.8, 3.2D
raII 1
1.2, 2.8 SacI
1 1
．． 8, 2.2 XhoI
1 1.3,
2.7 In Table 1 above, the 2.7 kb XhoI fragment was also confirmed to have the function of encoding diaminoperargonic acid aminotransferase and desthiobiotin synthetase. Therefore, this cleavage fragment can also be used as the bioA bioD region of the plasmid of the present invention.

[0025] Thus, bioAbioD contained in the bioA bioD region DNA fragment described above has a size of approximately 2.7 kb obtained by cutting out Brevibacterium flavum MJ-233 chromosomal DNA with restriction enzymes SalI and XhoI. This is considered to be included in the DNA fragment of

[0026] The number of recognition sites and the size of the cut fragments when the above DNA fragment of about 2.7 kb was further cut with various restriction enzymes are shown in Table 2 below.

[0027]

[Table 2]
Table 2 Number of restriction enzyme recognition sites Size of cleavage fragment (kb)
SacI 1
0.9, 1.8D
raII 1
1.5, 1.2 BamHI
1 1
．． 9, 0.8 The size detailed above is approximately 4.0kb, approximately 2.7k
FIG. 1 shows a restriction enzyme cleavage point map of the bioA bioD region DNA fragment of b.

On the other hand, one of the bioB regions obtained as described above is the Brevibacterium flavum MJ-
The chromosomal DNA of strain 233 was cut out by partial digestion with the restriction enzyme Sau3AI, and then digested with the restriction enzyme HindI.
The size obtained by cutting with II is approximately 5.5
Kb DNA fragments can be mentioned.

Table 3 below shows the number of recognition sites and the size of the cut fragments when this bioB region DNA fragment having a size of about 5.5 kb was cut with various restriction enzymes.

[0030]

[Table 3]
Table 3 Number of restriction enzyme recognition sites Size of cleavage fragment (kb)
KpnI 1
3.0, 2.5S
acI 1
1.7, 3.8 EcoRI
1 0
．． 6, 4.9 In Table 3 above, the 3.0 kb KpnI cleavage fragment, 1.
It has been confirmed that the 7kb SacI cleavage fragment also has the function of encoding biotin synthetase, and therefore, these cleavage fragments can also be used in the biotin synthetase-encoding function of the plasmid of the present invention.
It can be used as an oB area.

[0031] Thus, bioB contained in the above-mentioned bioB region DNA fragment is obtained by converting Brevibacterium flavum MJ-233 chromosomal DNA to the restriction enzyme HindII.
A DNA fragment with a size of about 3.0 kb obtained by cutting out the same chromosomal DNA with restriction enzymes HindIII and SacI, and a DNA fragment with a size of about 1.7 kb obtained by cutting out the same chromosomal DNA with restriction enzymes HindIII and SacI. considered to be included.

[0032] The above DNA fragment of about 3.0 kb and about 1
．． The number of recognition sites and the size of the cut fragments when the 7 kb DNA fragment was further cut with various restriction enzymes are shown in Tables 4 and 5 below, respectively.

[0033]

[Table 4]
Table 4 Number of restriction enzyme recognition sites Size of cleavage fragment (kb)
SacI 1
1.7, 1.3E
coRI 1
0.6, 2.4

[0034]

[Table 5]
Table 5 Number of restriction enzyme recognition sites Size of cleavage fragment (kb)
SphI 1
0.2, 1.5 Ec
oRI 1
0.6, 1.1 SmaI
1 1.
0, 0.7 HincII
1 1.5,
0.2 The size detailed above is approximately 5.5kb, approximately 3.0k
FIG. 2 shows a restriction enzyme cut point map of the approximately 1.7 kb bioB region DNA fragment.

[0035] A DNA region (bioA bi
A plasmid vector that can incorporate a DNA region (bioB region) containing a gene encoding biotin synthetase (oD region) and a DNA region (bioB region) containing a gene encoding biotin synthetase must have at least the ability to reproduce and multiply the plasmid in coryneform bacterial cells. Those having a DNA region containing a gene that governs are preferably used, and include, for example, the following. Plasmid pCRY30 disclosed in Japanese Patent Application No. 2-4212; Plasmid p described in Japanese Patent Application Laid-Open No. 2-276575
CRY21, pCRY2KE, pCRY2KX, pCR
Y3K7, pCRY3KE, pCRY3KX; JP-A-1
-Plasmid pC described in Publication No. 191686
RY2 and pCRY3; pAM330 described in JP-A-58-67679; pHM1519 as described in JP-A-58-77895; pAJ655, pAJ611 and pAJ1 as described in JP-A-58-192900
844; pCG1 described in JP-A-57-134500
; pCG2 described in JP-A No. 58-35197; pCG4 and p as described in JP-A-57-183799;
CG11 etc.

Among these, plasmid vectors used in the host vector system of coryneform bacteria include genes that control the replication and propagation function of plasmids in coryneform bacteria and genes that control the stabilization function of plasmids in coryneform bacteria. For example, plasmid pCRY30 is preferable.
, pCRY21, pCRY2KE, pCRY2KE, p
CRY2KX, pCRY3K7, pCRY3KE, pC
RY3KX is preferably used.

[0037] As a method for preparing the above plasmid vector pCRY30, Brevibacterium stationi
s) Extract plasmid pBY503 DNA from IFO12144 (FERM BP-2515) (see JP-A-1-95785 for details of this plasmid), and use restriction enzyme XhoI to control the replication and proliferation function of the plasmid, which has a size of approximately 4.0 kb. Cut out the DNA fragment containing the gene,
The size is about 2.1 with restriction enzymes EcoRI and KpnI.
DN containing the gene that controls the stabilization function of the kb plasmid
Cut out fragment A. Both of these fragments were added to the plasmid pHS.
Plasmid vector pCRY30 can be prepared by integrating into the EcoRI, KpnI and SalI sites of G298 (manufactured by Takara Shuzo).

[0038] Next, the above-mentioned "
bioA bioD area” and “bioB area” DN
To introduce the A fragment, for example, first, a restriction enzyme site that exists only at one location in the plasmid vector is cleaved with the restriction enzyme, and the bioA bioD region DNA fragment is inserted therein.
If necessary, the DNA may be treated with S1 nuclease to make blunt ends, or the DNA may be prepared in the presence of a suitable adapter DNA.
Contact by ligase treatment.

[0039] bioA bi created in this way
The plasmid into which the oD region has been inserted is cleaved using the restriction enzyme at only one other restriction enzyme site, and the bioB region DNA fragment is treated with S1 nuclease to make it blunt. This can be done by ligating the DNA at the ends or by DNA ligase treatment in the presence of a suitable adapter DNA. Specifically, the bioA bioD region and bio
The introduction of area B can be performed as follows.

First, plasmid pCR30 was treated with restriction enzyme
cleaved with hoI, and inserted the 4.0 kb bioA therein.
The bioD region DNA fragments were ligated using DNA ligase, and the plasmid pCRYBO into which the bioA bioD region had been inserted was cleaved with the restriction enzyme EcoRI, and the 1.7 kb bioB region DNA fragment was inserted into it by S
1 to make blunt ends by treatment with nuclease, and then ligated with DNA ligase.

[0041] Plasmid pCRY30 obtained above
The plasmid into which the 4.0 kb bioA bioD region DNA fragment and the 1.7 kb bioB region DNA fragment were introduced is a plasmid containing diaminoperargonic acid aminotransferase, desthiobiotin synthetase, and biotin synthetase. This is one of the plasmids with high expression ability, and the present inventors used this plasmid pCRY30-
It was named bio4. Plasmid pCRY30-bio
Details of the method for forming No. 4 will be explained in Examples below.

[0042] A restriction enzyme cleavage point map of this plasmid pCRY30-bio4 is shown in FIG. The recombinant plasmid of the present invention constructed in this manner has a high ability to express diaminoperargonic acid aminotransferase, desthiobiotin synthetase, and biotin synthetase. A microorganism transformed with the plasmid can be suitably used for the production of biotin.

[0043] As described above, bioA bioD area D
The plasmid of the present invention, which is constructed by introducing the NA fragment and the bioB region DNA fragment into a plasmid vector that has at least "a DNA region containing a gene that controls the replication and propagation function of the plasmid in coryneform bacterial cells," Transform the bacteria. Host microorganisms that can be transformed with the plasmids of the present invention include coryneform bacteria, such as Brevibacterium flavum MJ-233 (FERM BP-1
497), Brevibacterium flavum MJ-233
-AB-41 (FERM BP-1498), Brevibacterium flavum MJ-233-ABT-11 (F
ERM BP-1500), Brevibacterium flavum MJ-233-ABD-21 (FERM BP-1
499), etc.

[0044] The above-mentioned strain FERM BP-1498 is an ethanol-assimilating microorganism that has been actively given resistance to DL-α-aminobutyric acid using the strain FERMBP-1497 as its parent strain. 59-28398, columns 3 and 4). Also, FERM BP-1500
The strain No. is a mutant strain with high L-α-aminobutyric acid transaminase activity using the FERM BP-1497 strain as the parent strain (see JP-A-62-51998). Furthermore, the strain of FERM BP-1499 is FERMBP-
It is a mutant strain with high D-α-aminobutyric acid deaminase activity using the parent strain No. 1497 (Japanese Patent Application Laid-Open No. 177993/1983).
(see publication).

[0045] In addition to these microorganisms, Brevibacterium ammoniagenes (Brevibacterium
ammoniagenes) ATCC6871, same A
TCC13745, ATCC13746, Brevibacterium devaricatum
m divaricatum) ATCC 14020, Brevibacterium lactofamentum (Brevi
bacterium lactofermentum)
ATCC13869, Corynebacterium glutamicum
cum) ATCC31831 etc. can also be used as the host microorganism.

[0046] Brevibacterium flavum MJ-
When using a strain derived from 233 as a host, the plasmid pBY502 (Japanese Patent Application Laid-Open No. 63-3678
(Refer to Publication No. 7), transformation may be difficult. In such cases, plasmid p
It is desirable to remove BY502. As a method for removing such plasmid pBY502, for example,
It is possible to delete it naturally by repeating subculturing, or it can be removed artificially [
Bact. Rev. 36 p. 361-405 (197
See 2)]. An example of a method for artificially removing the above plasmid pBY502 is as follows.

Host Brevibacterium flavum MJ-
Approximately 10 cells per ml were added to a medium containing acridine orange (concentration: 0.2 to 50 μg/ml) or ethidium bromide (concentration: 0.2 to 50 μg/ml), etc. at a concentration that completely inhibits the growth of 233. Inoculate the bacteria so that
Culture at about 35° C. for about 24 hours, with incomplete inhibition of growth. After diluting the culture solution, apply it on an agar medium and culture at about 35°C for about 2 days. A plasmid extraction operation is performed independently from each colony that appears, and a strain in which plasmid pBY502 has been removed is selected. By this operation, plasmid pBY50
Brevibacterium flavum MJ-2 with 2 removed
33-derived bacterial strains are obtained.

[0048] As a method for transforming the plasmid into the Brevibacterium flavum MJ-233-derived strain obtained in this way, as is known for Escherichia coli and Erwinia carotovora, [Cal
vin, N. M. and Hanawalt, P. C.
, Journal of Bacteriology,
170, 2796 (1988); Ito, K. , Nis
Hida, T. and Izaki. K. , Agric
Ultural and Biological Ch.
emistry, 52, 293 (1988)], D
Plasmids can be introduced by applying pulse waves to NA recipient bacteria.

Corynebacterium, such as a strain derived from Brevibacterium flavum MJ-233, which has the plasmid of the present invention obtained by transformation by the above method, can be used for diaminoperargonic acid aminotransferase, desthiobiotin synthetase, etc. Since it has a high expression ability of enzyme and biotin synthetase, biotin can be efficiently produced in the culture by culturing in a medium containing at least 7-keto-8-aminopelargonic acid. It can be accumulated.

[0050] Cultivation can be carried out in a conventional nutrient medium containing a carbon source, a nitrogen source, an inorganic salt, etc., to which an appropriate amount of at least 7-keto-8-aminopelargonic acid is added. gas, ethanol, methanol,
Blackstrap molasses and the like are used, and as the nitrogen source, for example, ammonia, ammonium sulfate, ammonium chloride, ammonium nitrate, urea, etc. are used alone or in combination. Further, as the inorganic salt, for example, potassium hydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate, etc. are used. In addition, peptone, meat extract, yeast extract, coco
Nutrients such as various vitamins such as protein precursors, casamino acids, and biotin can also be added to the medium.

7-Keto-8 that can be added to the culture medium
-Aminopelargonic acid can be used, for example, in Japanese Patent Publication No. 38-1971
It can be produced by the method described in Publication No. 6, and the amount added to the culture medium is not particularly limited, but it is usually 5 to 500 mg.
/l, preferably 10 to 100 mg/l.

[0052] Cultivation can usually be carried out under aerobic conditions such as aeration, stirring, and shaking at a temperature of about 20 to 40°C, preferably 25 to 35°C. pH during cultivation is 5-10,
The pH is preferably around 7 to 8, and the pH during cultivation can be adjusted by adding acid or alkali.

[0053] The carbon source concentration at the start of the culture is preferably 1
~5% by volume, more preferably 2-3% by volume. Further, the culture period can usually be 1 to 7 days, and the optimal period is 3 days.

[0054] After completion of the culture, when extracting and purifying biotin from the culture solution, general extraction and purification methods from natural products can be applied, making use of the various properties of biotin. That is, the bacterial cells are removed from the culture, adsorbed on activated carbon, then eluted, and purified using an ion exchange resin, or the culture filtrate is directly purified using an ion exchange resin, and then recrystallized from water or alcohol. By doing so, biotin can be obtained.

[0055] In the microorganism obtained by transforming a coryneform bacterium that does not require biotin with the plasmid of the present invention, the above method can be used without adding 7-keto-8-aminopelargonic acid to the medium. By culturing, biotin can be produced and accumulated in the culture.

[0056] In this specification, a bioA bioD region DNA fragment and a bioB region DNA fragment were isolated from Brevibacterium flavum MJ-233, and these DNA fragments were isolated from Brevibacterium flavum MJ-233.
The recombinant plasmid into which the A fragment was introduced was also introduced into a strain derived from Brevibacterium flavum MJ-233, and the improvement of biotin synthetase production ability by this microorganism was mainly described in detail. The object of the present invention can also be achieved by using other coryneform bacteria described above instead of the -233-derived strain.

Although so-called coryneform bacteria have various genus names such as the genus Corynebacterium and the genus Brevibacterium, and various bacterial names, they have the same main mycological properties. These bacterial groups are uniform in the amino acid composition of their cell walls and the base composition of their DNA, with 70 to 80% of the DNA difference between bacterial species.
It is clear that there is homology to A and that they are very closely related microorganisms (Report of the Ferme
ntation Research Institute
es No. 55, p1-5, 1980, Intern
ational Journal of System
atic Bacteriology Vol31, p.
131-138, 1981).

In addition, biotin-requiring coryneform bacteria such as Brevibacterium flavum MJ-233 (FE
RM BP-1497), Brevibacterium lactofamentum ATCC 13869, and Corynebacterium glutamicum ATCC 31831, biotin-auxotrophic Escherichia coli mutant strains lacking genes for each step involved in biotin biosynthesis (Journal of
Bacteriology, vol 112, p830
-839, 1972 and Journal of B
acteriology, vol 94, p2065-
2066, 1967) and the cross-complementarity test (Jou
rnal Bacteriology, vol 96,
p515-524, 1968), and as a result of examining the biotin biosynthesis pathway, these three strains similarly encoded the gene encoding pimeryl-CoA synthetase (bioC) and 7-keto-8-amino The gene encoding pelargonic acid synthetase (bioF) is deleted, and at least the gene encoding 7,8-diaminopelargonate aminotransferase (bioF) is deleted.
A) It was revealed that the gene encoding desthiobiotin synthetase (bioD) and the gene encoding biotin synthetase (bioB) were possessed. Based on these facts, the scope of the present invention includes not only Brevibacterium flavum MJ-233 but also recombinant plasmids into which bioA bioD region DNA fragments and bioB region fragments isolated from coryneform bacteria in general are introduced. Furthermore, it is clear that the host microorganisms that can be transformed with the plasmid of the present invention are not limited to Brevibacterium flavum MJ-233, but include all coryneform bacteria.

[0059]

EXAMPLES The present invention has been described above, and will be explained more specifically by the following examples. However, it should be understood that the examples are to be considered as an aid to concrete understanding of the present invention, and are not intended to limit the scope of the present invention.

[0060]

[Example 1] Complementation test between coryneform bacteria and biotin-auxotrophic Escherichia coli mutant strain (A) Preparation of biotin-deficient minimal medium plate containing coryneform bacteria Semi-synthetic medium A medium [Composition: 2 g of urea, ( NH4)2SO
4 7g, K2HPO4 0.5g, KH2PO4 0
．． 5g, MgSO4 0.5g, FeSO4・7H2O
6mg, MnSO4 4-6H2O 6mg, yeast extract 2.5g, casamino acid 5g, biotin 200μg,
Thiamine hydrochloride 200μg, glucose 20g, pure water 1L
]1l, Brevibacterium flavum MJ-233
(FERM BP-1497) and O. D. The cells were cultured until the number of bacteria reached approximately 2.9, and the bacterial cells were collected. The obtained bacterial cells were added to BM buffer [composition: 2 g of urea, (NH4)2SO4
7g, K2HPO4 0.5g, KH2PO4 0.
5 g, MgSO4 0.5 g] twice. This bacterial cell was suspended in 10 ml of BM buffer, and 1 ml of it was
After sterilization, C medium for biotin assay (urea 0.2%, ammonium sulfate 0.7%, KH2P
O4 0.05%, K2HPO4 0.05%, MgS
O4・7H2O 0.05%, FeSO4・7H2O
6ppm, MnSO4.4-6H2O 6ppm, thiamine/HCl 100μg/l, vitamin/assay casamino acid 0.1%, glucose 0.2%, agar 1.0
%), stirred, poured onto a plate, and solidified.

Similarly, Brevibacterium lactofamentum ATC was used instead of Brevibacterium flavum MJ-233 (FERM BP-1497).
A biotin-deficient minimal medium plate containing various coryneform bacteria was prepared using C13869 or Corynebacterium glutamicum ATCC31831. (B) Complementation test with a biotin-auxotrophic E. coli mutant strain A complementation test between a biotin-auxotrophic E. coli mutant strain lacking each step of the biotin biosynthetic pathway and various coryneform bacteria revealed that the biotin production of various coryneform bacteria was Composite routes can be estimated.

Various biotin-requiring Escherichia coli mutants were inoculated in a linear manner onto the plate of biotin-deficient minimal medium containing three types of coryneform bacteria prepared in (A) above. The biotin-auxotrophic Escherichia coli mutant strain used was Escherichia coli (Esc
herichia coli) R873 (bioA4)
, R874 (bioF12), R875 (bioB
17), R876 (bioC18), R877 (b
ioD19) [The genotype of each strain (in parentheses) is
Genotype), details of these strains, and how to obtain them can be found in the Journal of Ba
cteriology, vol 94, p2065-2
066 (1967), Journal of Bact
eriology, vol 112, p830-839
(1972)).

When these biotin-requiring Escherichia coli mutants are complemented by coryneform bacteria, the coryneform bacteria grow in the biotin-deficient minimal medium plate and form yellow colonies. Based on the presence or absence of colony formation of coryneform bacteria corresponding to various biotin-requiring Escherichia coli mutant strains, it is easy to determine which part of the gene involved in biotin biosynthesis is deleted in coryneform bacteria and which part it retains. be able to.

[0064] As a result of this complementary test, Brevibacterium
flavum MJ-233 (FERMBP-1497), Brevibacterium lactofamentum ATCC13
869, Corynebacterium glutamicum ATCC3
1831, each strain is Escherichia coli R873 (
bioA4), R875 (bioB17), R87
7 (bioD19), but the same R874 (bioD19) was complemented.
F12) did not complement R876 (bioC18). That is, each coryneform bacterium at least similarly contains 7,8-diaminoperargonic acid aminotransferase.
It is clear that it has a gene encoding desthiobiotin synthetase (bioA), a gene encoding desthiobiotin synthetase (bioD), and a gene encoding biotin synthetase (bioB). It became.

[0065]

[Example 2] Brevibacterium flavum MJ-23
Diaminopelargonic acid aminotransferase derived from 3
(A) Extraction of total DNA of Brevibacterium flavum MJ-233 Semi-synthetic medium A medium [Composition: 2 g of urea, (NH4)2SO
4 7g, K2HPO4 0.5g, KH2PO4 0
．． 5g, MgSO4 0.5g, FeSO4・7H2O
6mg, MnSO4 4-6H2O 6mg, yeast extract 2.5g, casamino acid 5g, biotin 200μg,
Thiamine hydrochloride 200μg, glucose 20g, pure water 1L
]1l, Brevibacterium flavum MJ-233
(FERM BP-1497) was cultured until the late logarithmic growth phase, and the bacterial cells were collected. The obtained bacterial cells were mixed with 10mM NaCl-20mM containing lysozyme at a concentration of 10mg/ml.
Tris buffer (pH 8.0)-1mM EDTA-2N
It was suspended in 15 ml of a solution. Next, proteinase K was added to a final concentration of 100 μg/ml and incubated at 37°C.
It was kept warm for 1 hour. Furthermore, sodium dodecyl sulfate was added to the final concentration of 0.5%, and the mixture was incubated at 50° C. for 6 hours to culture. Add an equal amount of phenol/
After adding the chloroform solution and gently shaking at room temperature for 10 minutes, the entire volume was centrifuged (5,000 x g, 20 minutes, 10-12°C), the supernatant fraction was collected, and sodium acetate was added to 0. After adding the solution to a concentration of .3M, twice the amount of ethanol was slowly added. The DNA present between the aqueous layer and the ethanol layer was collected using a glass rod, washed with 70% ethanol, and then air-dried. 10 m to the obtained DNA
M Tris buffer (pH 7.5) - 1mM EDTA・2
5 ml of Na solution was added, left standing at 4°C overnight, and used for subsequent experiments.

(B) Creation of recombinant Brevibacterium flavum MJ obtained in section (A) above
-233 total DNA (90 μl) was added with restriction enzyme Sau3AI.
Using 1 unit, the reaction was carried out at 37° C. for 20 minutes for partial decomposition. This partially degraded DNA was mixed with cosmid pWE15 (manufactured by Stradagene), which had been cut with the restriction enzyme BamHI and then dephosphorylated, and mixed with 50 mM Tris buffer (p
H7.6), 10mM dithiothreitol, 1mM A
TP, 10mM MgCl2 and T4 DNA ligase 1
Each component of the unit was added (the concentration of each component is the final concentration) and allowed to react at 4° C. for 15 hours to allow binding.

(C) Selection of cosmids containing genes encoding enzymes involved in biotin biosynthesis Using the cosmid mixture obtained in section (B) above, 7 g of the Escherichia coli R873 (bioA4) strain, K2HPO
4 0.5g, KH2PO4 0.5g, MgSO4
0.5g, FeSO4・7H2O 6mg, MnSO4
・4-6H2O 6mg, yeast extract 2.5g, casamino acid 5g, biotin 200μg, thiamine hydrochloride 200μ
Brevibacterium stathionis 1FO12144 was cultured in 1 liter of [g, 20 g of glucose, and 1 liter of pure water] until the late logarithmic growth phase, and the bacterial cells were collected. The obtained bacterial cells were divided into 10
Buffer containing lysozyme at a concentration of mg/ml [25mM
Tris(hydroxymethyl)aminomethane, 10mM EDTA, 50mM glucose]
The reaction was carried out at 7°C for 1 hour. Add 40 m of alkaline-SDS solution [0.2N NaOH, 1% (W/V) SDS] to the reaction solution.
1 was added to the mixture, mixed gently and allowed to stand at room temperature for 15 minutes. Next, this reaction solution was added with a potassium acetate solution [60 ml of 5M potassium acetate solution, 11.5 ml of acetic acid, 28 ml of pure water.
30 ml of 5 ml of mixed solution was added, thoroughly mixed, and then left to stand in ice water for 15 minutes.

The entire amount of the lysate was transferred to a centrifuge tube and transduced for 10 minutes at 4°C, and the selective medium containing 50 mg of ampicillin [K2HPO4 7g, KH2PO4 2g, (NH
4) 2SO4 1g, MgSO4・7H2O 0.1g
, 10 g of casamino acid, 2 g of glucose and 16 g of agar dissolved in 1 liter of distilled water]. For transduction, use λDNA in vitro sold by Takara Shuzo.
This was done using a Packaging Kit. The strain grown on the medium was cultured in liquid by a conventional method, cosmid DNA was extracted from the culture solution, the cosmid was cut with restriction enzymes, and examined using agarose gel electrophoresis. As a result, the length of cosmid pWE15 was 8. In addition to the .8 kb DNA fragment,
A DNA fragment with a length of approximately 30 kb was observed. This cosmid was named pWE15-bioA.

(D) Subcloning of the bioA bioD region into plasmid pBluescript II The DNA insert fragment contained in the cosmid pWE15-bioA obtained in section (C) above is as large as approximately 30 kb and is not practical. In order to miniaturize only the necessary parts of the fragment, we used the plasmid pBluescript
II (commercially available from Stratagene) contains a DNA region (bi
oA bioD region) was subcloned as follows.

Cosmid pWE15- obtained in section (C) above
bioA was cut with the restriction enzyme SalI, and plasmid pBluescript II was cut with the restriction enzyme SalI.
Mix the cut with I and add 50mM Tris buffer (p
H7.6), 10mM dithiothreitol, 1mM A
TP, 10mM MgCl2 and T4 DNA ligase 1
Each component of the unit was added (the concentration of each component is the final concentration) and allowed to react at 12° C. for 15 hours to allow binding.

Using the obtained plasmid mixture, the calcium chloride method (Journal of Molecular
Biology, 53, 159, 1970) was transformed into Escherichia coli R873 (bioA4) strain, and a selection medium containing 50 mg of ampicillin [K2HPO
4 7g, KH2PO4 2g, (NH4)2SO4
1g, MgSO4.7H2O 0.1g, Casamino acid 1
0 g, 2 g of glucose and 16 g of agar dissolved in 1 liter of distilled water].

[0072] The strain grown on this medium was cultured in liquid by a conventional method, plasmid DNA was extracted from the culture solution, the plasmid was cut with restriction enzymes, and examined using agarose gel electrophoresis.
In addition to the 3.95 kb long DNA fragment of pt II, an inserted DNA fragment of 4.0 kb length was observed.

[0073] This plasmid was used to transform the Escherichia coli R877 (bioD19) strain according to the above method, and the selection medium [K2
HPO47g, KH2PO4 2g, (NH4)2SO
4 1g, MgSO4・7H2O 0.1g, casamino acid 10g, glucose 2g and agar 16g in 1 liter of distilled water
smeared in [dissolved in]].

[0074] The strain grown on this medium was cultured in liquid by a conventional method, plasmid DNA was extracted from the culture solution, the plasmid was cut with restriction enzymes, and examined using agarose gel electrophoresis. R873(b
ioA4) strain, the plasmid pBluescript II
In addition to the 2.95 kb long DNA fragment, the approximately 4.0 kb long DNA fragment
An inserted DNA fragment of kb was observed. Length approximately 4.0kb
The number of restriction enzyme recognition sites and the size of the cut fragments when the DNA fragment was cut with various restriction enzymes were as shown in Table 1 above. A restriction enzyme breakpoint map of this DNA fragment is shown in FIG. Furthermore, the plasmids obtained above were cut with various restriction enzymes, and the sizes of the cut fragments were measured. The results are shown in Table 6 below.

[0075]

[Table 6] Table 6 Plasmid pBS-bi
oAD4 Restriction enzyme Recognition site number Cutting fragment size (kb
) HindIII
1 6.95
XhoI 2
4.25, 2.7 BamH
I 2 3
．． 65, 3.2 The plasmid characterized by the above restriction enzyme was named pBS-bioAD4.

From the above results, the bioA bioD region D, which is approximately 4.0 kb in length and contains the genes encoding diaminoperargonic acid aminotransferase and desthiobiotin synthetase, is excised with the restriction enzyme SalI.
An NA fragment could be obtained.

[0077]

[Example 3] Brevibacterium flavum MJ-23
(A) Creation of recombinant Brevibacterium obtained in Section (A) of Example 2 above.・90 μl of total DNA of flavum MJ-233 was treated with restriction enzyme Sa.
Using 1 unit of u3AI, the reaction was performed at 37° C. for 20 minutes for partial decomposition. Cosmid pWE is added to this partially degraded DNA.
15 (manufactured by Stratagene) with the restriction enzyme BamHI, the dephosphorylated product was mixed, and 50mM
Tris buffer (pH 7.6), 10mM dithiothreitol, 1mM ATP, 10mM MgCl2 and T4
Add each component of 1 unit of DNA ligase (the concentration of each component is the final concentration) and react at 4°C for 15 hours.
Combined.

(B) Selection of a cosmid containing a gene encoding an enzyme involved in biotin biosynthesis The above Escherichia coli R875 (bioB17) strain was transduced using the cosmid mixture obtained in section (A) above. ,
Selective medium containing 50 mg ampicillin [K2HPO4
7g, KH2PO4 2g, (NH4)2SO4 1g
, MgSO4・7H2O 0.1g, Casamino acid 10g
, 2 g of glucose and 16 g of agar dissolved in 1 liter of distilled water]
It was smeared on. For transduction, λDNA in vitro Packaging sold by Takara Shuzo is used.
This was done using Kit. The growing strain on the medium is cultured in liquid by a conventional method, and cosmid DNA is extracted from the culture solution.
When the cosmid was cut with a restriction enzyme and examined using agarose gel electrophoresis, it was found that in addition to a DNA fragment of cosmid pWE15 with a length of 8.8 kb, a DNA fragment with a length of about 30 kb was found.
NA fragments were observed. This cosmid was pWE15-bi
It was named o2.

(C) Plasmid pHSG of bioB region
Subcloning into 399 The DNA insert fragment contained in the cosmid pWE15-bio2 obtained in section (B) above is large at about 30 kb and is not practical, so in order to miniaturize only the necessary part of the obtained fragment Then, the DNA region containing the gene encoding biotin synthetase (bioB region) was subcloned into plasmid pHSG399 (commercially available from Takara Shuzo) as follows.
I did a ning.

Cosmid pWE15- obtained in section (C) above
bio2 cut with restriction enzyme HindIII,
Plasmid pHSG399 was cut with restriction enzyme HindIII, mixed, and mixed with 50mM Tris buffer (pH 7).
．． 6), 10mM dithiothreitol, 1mM ATP
, 10mM MgCl2 and 1un T4 DNA ligase
Each component of it was added (the concentration of each component is the final concentration) and allowed to react at 12° C. for 15 hours to allow binding.

Using the obtained plasmid mixture, the calcium chloride method (Journal of Molecular
E. coli R875 (bioB17) strain was transformed using a selective medium containing 50 mg of chloramphenicol [Biology, 53, 159, 1970].
K2HPO4 7g, KH2PO2 2g, (NH4)
1 g of 2SO4, 0.1 g of MgSO4.7H2O, 10 g of casamino acid, 2 g of glucose and 16 g of agar were dissolved in 1 liter of distilled water].

[0082] The strain grown on this medium was cultured in liquid by a conventional method, plasmid DNA was extracted from the culture solution, the plasmid was cut with restriction enzymes, and examined using agarose gel electrophoresis. In addition to the DNA fragment with a length of 2.2 kb, an inserted DNA fragment with a length of approximately 5.5 kb was observed. The number of restriction enzyme recognition sites and the size of the cleaved fragments of the approximately 5.5 kb DNA fragments obtained when cleaved with various restrictions were as shown in Table 3 above. A restriction enzyme breakpoint map of this DNA fragment is shown in FIG.

[0083] Furthermore, the plasmids obtained above were cut with various restriction enzymes, and the sizes of the cut fragments were measured. The results are shown in Table 7 below.

[0084]

[Table 7] Table 7 Plasmid pHSG399-bi
oB5.5 Restriction enzyme
Number of recognition sites Size of cleavage fragment (kb)
HindIII 2
5.5, 2.2
Sacl2
6.0, 1.7 kp
nI 2
A plasmid characterized by a restriction enzyme of 4.7, 3.0 or higher was named pHSG399-bioB5.5.

[0085] Through the above steps, biotin synthetase is coated.
A bioB region DNA fragment (HindIII fragment) with a size of approximately 5.5 kb containing the gene to be coded could be obtained.

(D) BioB region plasmid pBl
Subcloning the above into uescript II (
Plasmid pHSG399-bioB5. obtained in section C).
5 is an inserted DNA with a length of approximately 5.5 kb including bioB
The plasmid pBlues contains the fragment, but in order to further reduce the size of the obtained fragment to only the necessary part.
A DNA region (bioB region) containing a gene encoding biotin synthetase was subcloned into Ccrypt II (commercially available from Stratagene) as described below.

[0087] Plasmid pHSG3 obtained in section (C) above
99-bio5.5 cut with restriction enzymes HindIII and SacI and plasmid pBlues
cript II with restriction enzymes Hind III and S
Mix the acI cut and add 50mM Tris buffer (pH 7.6), 10mM dithiothreitol, 1mM
ATP, 10mM MgCl2 and T4 DNA ligator
1 unit of each component was added (the concentration of each component is the final concentration) and allowed to react at 12° C. for 15 hours to allow binding.

Using the obtained plasmid mixture, the Escherichia coli R875 (bioB1
7) Transform the strain and use selective medium containing 50 mg of ampicillin [7 g of K2HPO4, 2 g of KH2PO2, (N
H4) 2SO4 1g, MgSO4・7H2O0.1g
, 10 g of casamino acid, 2 g of glucose and 16 g of agar dissolved in 1 liter of distilled water].

The strain grown on this medium was cultured in liquid by a conventional method, plasmid DNA was extracted from the culture solution, the plasmid was cut with a restriction enzyme, and examined using agarose gel electrophoresis.
In addition to the 2.95 kb long DNA fragment of pt II, an inserted DNA fragment of approximately 1.7 kb length was observed. DNA with a length of approximately 1.7 kb when cut with various restriction enzymes
The number of restriction enzyme recognition sites in the fragment and the size of the cleavage fragment were as shown in Table 5 above. A restriction enzyme break point map of this DNA fragment is shown in FIG.

Furthermore, the plasmids obtained above were cut with various restriction enzymes, and the sizes of the cut fragments were measured. The results are shown in Table 8 below.

[0091]

[Table 8] Table 8 Plasmid pBS-bioB-H
S1.7 Restriction enzymes
Number of recognition sites Size of cleavage fragment (kb)
HindIII 1
4.65
SacI 1
4.65 HincII
1 4.6
5. Plasmids characterized by the above restriction enzymes were transferred to pB
It was named S-bioB-HS1.7.

[0092] As described above, biotin synthetase is coated.
A bioB region DNA fragment (Hind III-SacI fragment) with a size of about 1.7 kb containing the gene to be coded was obtained.

[0093]

[Example 4] Construction of plasmid vector pCRY30 that replicates and is stable in coryneform bacteria (A) Preparation of plasmid pBY503 Plasmid pBY503 was derived from Brevibacterium stationis IFO12144 (FERM BP-2515).
It is a plasmid with a molecular weight of about 10 megadaltons isolated from ), and was prepared as described in JP-A-1-95785. Semi-synthetic medium A medium [urea 2g, (NH4
)2SO4 7g, K2HPO4 0.5g, KH2P
O4 0.5g, MgSO4 0.5g, FeSO4・
7H2O 6mg, MnSO4・4~6H2O 6mg
, yeast extract 2.5g, casamino acids 5g, biotin 20
0 μg, thiamine hydrochloride 200 μg, glucose 20 g, and 1 liter of pure water] Brevibacterium stathionis 1FO12144 was cultured until the late logarithmic growth phase, and the bacterial cells were collected. The obtained bacterial cells were mixed with a buffer containing lysozyme at a concentration of 10 mg/ml [25 mM tris(hydroxymethyl)aminomethane, 10 mM EDTA, 50 mM
Glucose] was suspended in 20 ml and reacted at 37°C for 1 hour. Alkaline-SDS solution [0.2N NaO
40 ml of 1% (W/V) SDS] was added, mixed gently, and allowed to stand at room temperature for 15 minutes. Next, this reaction solution was added with potassium acetate solution [5M potassium acetate solution 60ml]
11.5 ml of acetic acid, 28.5 ml of pure water] 3
0 ml was added, thoroughly mixed, and then left in ice water for 15 minutes.

[0094] The entire amount of the lysate was transferred to a centrifuge tube and centrifuged at 15,000 xg for 10 minutes at 4°C to obtain a supernatant.

[0095] Add an equal amount of phenol-chloroform solution (phenol:chloroform = 1:1 mixture) to this, suspend it, transfer it to a centrifuge tube, and incubate at 15,000 ml for 5 minutes at room temperature.
The aqueous layer was collected by centrifugation at 0xg. Add twice the amount of ethanol to the aqueous layer, leave it at -20°C for 1 hour, and then heat to 4°C.
The mixture was centrifuged at 15,000 xg for 10 minutes to collect the precipitate.

After drying the precipitate under reduced pressure, TE buffer [Tris 1
0mM EDTA, 1mM EDTA, adjusted to pH 8.0 with HCl] was dissolved in 2ml. Add 15 ml of cesium chloride solution [170 g of cesium chloride dissolved in 100 ml of 5-fold concentrated TE buffer] and 1 ml of 10 mg/ml ethidium bromide solution to the solution, and adjust the density to 1.392 g/m
Adjusted to l. This solution was heated at 12°C for 42 hours at 116,
Centrifugation was performed at 000xg.

Plasmid pBY503 is found as a lower band in the centrifuge tube upon UV irradiation. By removing this band from the side of the centrifuge tube with a syringe,
A fraction containing plasmid pBY503 was obtained.

Next, this fraction was treated with an equal volume of isoamyl alcohol four times to extract and remove ethidium bromide, and then dialyzed against TE buffer. A 3M sodium acetate solution was added to the dialysate containing the plasmid pBY503 thus obtained to a final concentration of 30mM, then 2 times the volume of ethanol was added, and the mixture was allowed to stand at -20°C for 1 hour. This solution was centrifuged at 15,000 x g to precipitate the DNA, and 50 μg of plasmid pBY503 was
Obtained.

(B) Construction of plasmid vector pCRY30 0.5 μg of plasmid pHSG298 (manufactured by Takara Shuzo) was reacted with restriction enzyme SalI (5 units) at 37° C. for 1 hour to completely degrade the plasmid DNA.

[0100] Plasmid pBY prepared in section (A) above
Add restriction enzyme XhoI (1 unit) to 2 μg of 503 at 3
The reaction was carried out at 7°C for 30 minutes to partially degrade the plasmid DNA.

[0101] Both plasmid DNA digests were mixed,
After heat treatment at 65° C. for 10 minutes to inactivate the restriction enzymes, the components in the inactivation solution had a final concentration of 5% each.
0mM Tris buffer pH 7.6, 10mM MgCl2
, 10mM dithiothreitol, 1mM ATP and T
4. Strengthen each component to 1 unit of ligase,
It was kept warm at 16°C for 15 hours. Using this solution, Escherichia coli JM 109 concomitant cells (manufactured by Takara Shuzo)
was transformed.

[0102] The transformed strain is 30 μg/ml (final concentration)
kanamycin, 100 μg/ml (final concentration) IP
L containing TG (isopropyl-β-D-thiogalactobyranoside) 100 μg/ml (final concentration) of X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) Medium (10g tryptone, 5g yeast extract, 5g NaCl and 1l pure water, pH 7.2)
The cells were cultured at 37°C for 24 hours to obtain a viable strain. Among these growing strains, those growing in white colonies were selected, and each plasmid was subjected to alkaline-SDS method [T
．． Maniatis, E. F. Fritsch, J. S
ambrook, “Molecular Clonin
g" (1982) p. 90-91].

As a result, S of plasmid pHSG298
Approximately 4.0k from plasmid pBY503 at the alI site
Plasmid pHSG298-or into which the fragment of b was inserted
i was obtained.

[0104] Next, using the same method, the plasmid pBY503 DNA obtained in the above section (A) was digested with the restriction enzyme Kpn.
A DNA fragment of approximately 2.1 kb obtained by treatment with I and EcoRI was cloned into the KpnI and EcoRI sites of the above plasmid pHSG298-ori to prepare plasmid vector pCRY30.

[0105]

[Example 5] Creation of plasmid pCRY30-bio4 and introduction into coryneform bacteria 45 μg of plasmid pBS-bioAD obtained in section (D) of Example 2 was added with restriction enzyme
Using 5 units of alI, 1 µg of the plasmid pCRY30 obtained in Section (B) of Example 4 was digested by reacting at 37°C for 1 hour, and 1 µg of the restriction enzyme XhoI was added.
t for 1 hour at 37°C, the mixture was combined with the above method, and Escherichia coli R873 (bioA4) strain was transformed according to a conventional method, and a selective medium containing 50 μg/ml of kanamycin [ K2HPO4
7g, KH2PO4 2g, (NH4)2SO4 1g
, MgSO4・7H2O 0.1g, Casamino acid 10g
, 2 g of glucose and 16 g of agar dissolved in 1 liter of distilled water]
smeared on.

[0106] The strain grown on this medium was cultured in liquid by a conventional method, plasmid DNA was extracted from the culture solution, the plasmid was cut with restriction enzymes, and examined using agarose gel electrophoresis. length 8
．． In addition to the 6 kb DNA fragment, an inserted DNA fragment with a size of 4.0 kb was observed.

Plasmid DNA prepared as above
Using restriction enzyme EcoRI 1 unit, 1 μg was added to 37
Decomposed by reacting at ℃ for 1 hour and (D) of Example 3
Plasmid pBS-bioB-HS1.7 obtained in section
5 μg was digested by reacting with restriction enzymes HindIII and SacI in 5 units each at 37°C for 1 hour, mixed, treated with SI nuclease to make blunt ends, and ligated using the above method. Escherichia coli R875 (bioB17) strain was transformed according to the method, and a selection medium containing 50 μg/ml of kanamycin [K2HPO4 7g, KH2PO4 2g, (NH4
)2SO4 1g, MgSO4・7H2O 0.1g,
10 g of casamino acid, 2 g of glucose and 16 g of agar were dissolved in 1 liter of distilled water].

[0108] The strain grown on this medium was cultured in liquid by a conventional method, plasmid DNA was extracted from the culture solution, the plasmid was cut with restriction enzymes, and examined using agarose gel electrophoresis. length 8
．． In addition to the 6 kb DNA fragment, 4.0 kb and 1
．． A 7 kb inserted DNA fragment was observed.

The plasmid DNA prepared as described above was transformed into coryneform bacteria.

[0110] Transformation was carried out using the electric pulse method. Brevibacterium flavum MJ-233 (FER
M BP-1497) The plasmid pBY502 deleted strain was cultured in 100 ml of the above A medium until the early logarithmic growth phase,
Penicillin G was added to 1 unit/ml, cultured with shaking for another 2 hours, and the bacterial cells were collected by centrifugation.
rose, 7mM KH2PO4, 1mM MgCl2
: pH 7.4). Furthermore, the bacterial cells were collected by centrifugation, suspended in 5 ml of pulse solution, and 0.75 ml
cells and 50μ of the plasmid DNA solution obtained above.
1 and left to stand in water for 20 minutes. Using Gene Pulsar (manufactured by Bio-Rad), 2500 volts, 2
It was set to 5 μFD, and after applying a pulse, it was left standing on ice for 20 minutes. The entire amount was transferred to 3 ml of the above medium A and cultured at 30°C for 1 hour, then inoculated onto the above A agar medium containing 15 μg/ml (final concentration) of kanamycin and cultured at 30°C for 2 to 3 days. From the kanamycin-resistant strains that appeared, Example 4 (
Plasmids were obtained using the method described in section A). This plasmid was cut with various restriction enzymes, and the sizes of the cut fragments were measured. The results are shown in Table 9 below.

[0111]

[Table 9] Table 9 Plasmid pCRY30-b
io4 Restriction enzyme Recognition site number Cutting fragment size (kb)
BamHI 2
0.8, 13.5
EcoRI 1
14.3 KpnI
1 14
．． 3 SacI
2 2.5, 11.8
SalI 2
0.4, 13.9
XhoI 1
14.3 Convert the plasmid characterized by the above restriction enzymes into pCR
It was named Y30-Bio4. This plasmid pCRY
A restriction enzyme breakpoint map of 30-bio4 is shown in FIG. 3.

[0112] Furthermore, plasmid pCRY30-bio4
Brevibacterium flavum M transformed by
J-233-bio4 was transferred to the Institute of Microbial Technology, Agency of Industrial Science and Technology, 1-1-3 Higashi, Tsukuba City, Ibaraki Prefecture, in February 1991.
Date: February 26th: FERM No. 12042 (FERM
P-12042).

[0113]

[Example 6] Stability of plasmid pCRY30-bio4 100 ml of the above A medium was dispensed into a 500 ml Erlenmeyer flask and sterilized at 120°C for 15 minutes. Inoculate bio4,
After culturing with shaking at 30°C for 24 hours, 100ml of A medium prepared in the same manner was dispensed into a 500ml Erlenmeyer flask, sterilized at 120°C for 15 minutes, and the mixture was adjusted to a ratio of 50 cells per ml. The cells were subcultured and cultured with shaking at 30°C for 24 hours. Next, collect the bacteria by centrifugation, wash the bacteria, and then add 15μg/kanamycin.
A fixed amount of the mixture was spread on a plate culture medium prepared using A medium added and A medium without additives at a ratio of 1 ml, and after culturing at 30° C. for 1 day, growing colonies were counted.

[0114] As a result, it was confirmed that the same number of colonies grew in the medium supplemented with kanamycin and in the medium without the addition of kanamycin, and that all colonies grown in medium A grew in the medium supplemented with kanamycin, that is, the high stability of the plasmid was confirmed. .

[0115]

[Example 7] Production medium of biothion from ketoaminopelargonic acid (urea 0.2%, ammonium sulfate 0.7%, KH
2PO4 0.05%, K2HPO4 0.05%, M
gSO4・7H2O 0.05%, FeSO4・7H2
O 6ppm, MnSO4・4~6H2O 6ppm,
Thiamine/HCl 100μg/l and 7-keto-8
-aminopelargonic acid 100mg/l) 100ml
Dispense into 00ml Erlenmeyer flask and sterilize (pH 7.00ml after sterilization).
0), Brevibacterium flavum MJ-23
3-biO4 strain was inoculated, glucose was added aseptically to a final concentration of 2% (W/V), and cultured with shaking at 30°C for 3 days.

As a control, plasmid pCRY30-bi
Brevibacterium flavum MJ- which does not carry o4
233 strains were inoculated and cultured in the same manner.

After culturing, the culture solution was centrifuged to remove bacterial cells, and the biotin concentration in the supernatant was determined using Lactobacillus plantarum.
m) Quantified by microbial quantification method according to ATCC8014.

[0118] As a result, the supernatant of Brevibacterium flavum MJ-233-bio4 strain culture had a concentration of about 5. Double amount of biotin was detected.

[Brief explanation of the drawing]

[Figure 1] bioA carried by the plasmid of the present invention
Restriction enzyme breakpoint map of bioD region DNA fragment.

FIG. 2: Restriction enzyme breakpoint map of the bioB region DNA fragment carried by the plasmid of the present invention.

[Figure 3] Plasmid pCRY30-bio of the present invention
Restriction enzyme breakpoint map of 4.

Claims (6)

[Claims] Claim 1: A DNA region (a) containing a gene encoding diaminoperargonic acid aminotransferase and desthiobiotin synthetase derived from coryneform bacteria.
, a DNA region (b) containing a gene encoding biotin synthetase derived from coryneform bacteria, and D containing a gene that controls the replication and propagation function of plasmids in coryneform bacteria cells.
A recombinant plasmid containing at least the NA region (c). 2. The recombinant plasmid according to claim 1, wherein the coryneform bacterium is a biotin-auxotrophic strain. 3. The biotin-requiring coryneform bacterium is Brevibacterium flavum.
3. The recombinant plasmid according to claim 2, which is um flavum) MJ-233. 4. A coryneform bacterium transformed with the plasmid according to any one of claims 1 to 3. [Claim 5] The coryneform bacterium is Brevibacterium.
The coryneform bacterium according to claim 5, which is a strain derived from B. flavum MJ-233. 6. A method for producing biotin, which comprises culturing the coryneform bacterium according to any one of claims 4 to 5 in a medium, and collecting biotin from the culture.