JP3341286B2 - Repetitive DNA sequence from coryneform bacteria - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a base sequence (hereinafter referred to as "repeated sequence") repeatedly present in the chromosomal DNA of a coryneform bacterium useful in the amino acid production industry, and a homologous recombination system using the repetitive sequence. The present invention relates to a method for incorporating a useful gene into the chromosomal DNA of a coryneform bacterium by the method of application, and a coryneform bacterium in which the useful gene is incorporated into the chromosomal DNA by the method, which can be used for breeding microorganisms useful for fermentative production of amino acids and the like. Things.

[0002]

2. Description of the Related Art There have been many reports on gene manipulation techniques for coryneform bacteria and production of various amino acids by recombinant coryneform bacteria. For example, (1) a report on the transformation method using protoplasts (J. Bacteriol. 159 (1984) 304-311), (2) a report on the development of various vectors (Agric. Biol. Chem. 48 (1984) )
2901-2903, Bacteriol. 159 (1984) 306-311, Gene 47 (19
86) 301-306), (3) Report on development of gene expression control method (Bio / Technology 6 (1988) 428-430), (4) Report on increase in yield of various amino acids (Agric. Biol. Chem. 51).
(1987) 597-599). However, at present, sufficient results have not been obtained in terms of stabilizing the introduced foreign gene and constantly producing substances such as amino acids.

[0003]

Accordingly, an object of the present invention is to provide a method for stabilizing a transgene and for constantly producing a substance such as an amino acid.

[0004]

Means for Solving the Problems The present inventors have conducted intensive studies to solve the above problems, and as a result, focused on the repetitive sequences present in the chromosomal DNA of coryneform bacteria, and found that the repetitive sequences and homologous recombination The present inventors have found that a large number of target genes can be stably introduced into chromosomal DNA by using the combination of the present invention and the present invention, thereby completing the present invention.

Specifically, the present invention relates to a repetitive sequence represented by SEQ ID NO: 1, 2, 3, 4 or 5 in the chromosomal DNA of a coryneform bacterium, and a gene for an amino acid biosynthesis system of a coryneform bacterium using the sequence. Is incorporated into the chromosomal DNA of a coryneform-type bacterium, and a coryneform bacterium obtained by the method. The term "repeated sequence" refers to a base sequence that is repeatedly present in chromosomal DNA and has a mutually high homology. Since this sequence exists abundantly in the chromosome, by utilizing homologous recombination with this portion (Saibo Kogaku 9 (1990) 269-279), a useful gene of interest (eg,
Amino acid biosynthesis gene) can be inserted in multiple copies.

The present inventors cultured various coryneform bacteria and extracted their chromosomal DNA. And chromosomal DNA
Was cloned and its DNA sequence was determined. As a result, five types of repetitive sequences were found, and the present invention was completed. The present invention will be described in detail below.

First, a solution containing several hundred micrograms of DNA obtained from various coryneform bacteria is adjusted to about pH 12 using an alkali. Then, using an appropriate acid
Return pH to near neutral. After further adjusting the salt concentration to about 0.3 M by adding salt, the mixture is heated at about 65 ° C. for about 1 minute and then rapidly cooled.
The fraction thus prepared is reacted with S1 nuclease to remove a single-stranded DNA portion. The nuclease used at this time can be used other than S1 nuclease as long as it is an enzyme specific to a single strand of DNA. Next, after the enzymatic reaction, the remaining DNA fragments that have not been decomposed are detected and extracted by electrophoresis or gel filtration.
The DNA fragment thus obtained is a repetitive sequence present in the chromosome.

Next, the extracted DNA fragment is inserted into a plasmid vector and cloned. Cloned DN
The structural analysis of A is performed by the method of Sanger et al. (Proc. Nat. Acad. Sci.
74 (1977) 5463-5467). The DNA sequences determined in this manner are shown in SEQ ID NO: 1, 2, 3,
4 or 5. The present invention is not limited to the DNA sequence described in SEQ ID NOs: 1, 2, 3, 4 or 5 in the Sequence Listing, but in which one or more bases in the DNA sequence are replaced by other bases, and 5 'end or 3 of the above sequence
'Those having one or more base sequences added to the terminus,
If the effects are the same, they are included in the repetitive sequence of the present invention.

[0009] The thus obtained repetitive sequence of SEQ ID NO: 1, 2, or 3 in the sequence listing is obtained by transforming the replication function in coryneform bacteria into a temperature-sensitive mutant plasmid pHSC.
4 (Escherichia coli AJ12571 carrying this plasmid
Is FERM-P11763. ) And transformed into coryneform bacteria. As a result, it was observed that the plasmid was frequently inserted into the chromosome and stably maintained. This also confirms that the repetitive sequence of the present invention can stably introduce a large number of useful genes relating to amino acid production and the like into chromosomes, and that it is a useful one that can produce useful substances such as various amino acids in large quantities.

The coryneform bacterium referred to in the present invention is an aerobic gram as described in Barz's Manual of Detaminative Bacteriology, 8th edition, 599 pages (1974). It is a positive bacillus.

Examples of coryneform bacteria that can be used as host bacteria in the present invention include, for example, Brevibacterium saccharolyticum ATCC14066 and Brevibacterium inmariofilm ATCC1406.
8. Brevibacterium lactofamentum AT
CC13869, Brevibacterium roseum AT
CC13825, Brevibacterium flavum AT
Wild strains such as CC13826, Corynebacterium acetoacid film ATCC13870, Corynebacterium glutamicum ATCC13032, 13060, Corynebacterium lilium ATCC15990, and mutants derived from the above glutamic acid producing bacteria and losing glutamic acid productivity, Also include mutants that produce other products such as amino acids such as lysine and nucleic acids such as inosine.

The useful gene which can be introduced into the chromosome by using the repetitive sequence may be a gene coding for a physiologically active substance such as interleukin 2 or 6. However, various amino acids (lysine, lysine, Key to biosynthesis of threonine, isoleucine, glutamic acid, etc.
It is preferable to use a gene relating to an enzyme, specifically a gene such as homoserine dehydrogenase, threonine deaminase, aspartate kinase, and phosphoenolpyruvate carboxylase. Of course, genes other than the above may be used.

The plasmid carrying the repetitive sequence and the useful gene is not particularly limited, but usually a plasmid derived from a coryneform bacterium may be used. Specifically, pHM151
9 (Agric. Biol. Chem. 48 (1984) 2901-2903), pAM330 (Agric.
Biol. Chem. 48 (1984) 2901-2903), and drug-resistant plasmids based on these.

A recombinant plasmid having a repetitive sequence and a useful gene of interest is transformed into the above-mentioned coryneform bacterium. As a transformation method, a commonly used method such as a protoplast method (Gene, 39 (1985) 281-286) or an electroporation method (Bio / Technology, 7 (1989) 1067-1070) may be used. Further, the obtained transformant may be cultured according to a commonly used method and conditions.
Then, useful substances are accumulated inside or outside the bacteria.

[0015]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be specifically described below based on embodiments.

(Detection of repetitive sequence) Saito-Miura method (BBA, 827) was used from Brevibacterium lactofermentum and Brevibacterium flavum.
8,619-629 (1963)) to prepare chromosomal DNA,
Equivalent to 300-600 μg of them was added to 1 ml of TE buffer (10 mM Tri
s-HCl, 1 mM EDTA, pH 8.0). It is 0.2N NaO
The pH was adjusted to 12.3 with H, left at room temperature for 15 minutes, and then returned to 6.75-7.10 with 0.2N HCl. 0.3M Na + concentration with 4M NaCl
After heating at 65 ° C for 1 minute to match M,
0-fold concentration of S1 buffer (4.5 mM ZnCl2, 40 mM Sodium Acetat
eBuffer (pH 4.6)) was added to 1/9 volume of the reaction solution , then divided into three tubes, and subjected to the subsequent S1 nuclease reaction. The reaction of S1 nuclease was performed at 37 ° C. for 1 hour in three steps of 0.1 u / μg DNA, 1 u / μg DNA, and 10 u / μg DNA. The reaction was stopped by phenol treatment, and the phenol was removed with chloroform. DNA was recovered by ethanol precipitation and each was subjected to agarose gel electrophoresis. As a result, 9 bands were found from Brevibacterium lactofermentum and 3 bands were found from Brevibacterium flavum, which were not observed before the S1 nuclease reaction, and each band was recovered from the gel.

(Cloning of Repetitive Sequence) The recovered DNA was treated with T4 DNA polymerase to make both ends blunt. On the other hand, the vector pUC18 (Yanish-Perron,
C., Vieira, J. and Messing, J., Gene 33 (1985) 103-119)
Was cut with SmaI, and both were connected with T4 DNA ligase.
And trans conformation in Escherichia coli JM109, A
Npi cylinder 100 [mu] g / ml and X-gal (5-bromo- 4-chloro-3-indolyl
Those which form white colonies were selected from L-broth plates containing β-D-galactopyranoside), and clones having five types of insert DNAs were obtained from them. Using these as a probe, Southern hybridization was performed on the chromosome DNA as a source. As a result, three types (RS410, RS479, RS656) (pUC18 containing RS410 was used)
AJ12620 (FERM P-12330), RS47
Escherichia coli with pUC18 containing AJ12621 (FERM P
-12331), Escherichia coli with pUC18 containing RS656
Deposited as 12622 (FERM P-12332). ), It was confirmed that about 10 copies of them existed in the chromosome. The sequences of these three types were determined by the dideoxy method. The results are shown in SEQ ID NOs: 1, 2, and 3 in the sequence listing.

(Homology search between the obtained sequence and the DNA database) A homology search was performed between the obtained sequence and the DNA database (GENBANK) using the program ideas. RS4
It was found that 10 had strong homology with Escherichia coli IS15delta. Furthermore, by the alignment of both, this RS410 is originally an IS sequence (insertion s) having inverted repeats at both ends on the chromosome.
equence, inserted sequence) and RS4
Using 10 as a probe, screening was performed from a library of Brevibacterium lactofermentum.
When the sequence of the obtained clone was determined by the dideoxy method, it was found that this clone contained an IS-like sequence having inverted repeats at both ends shown in SEQ ID NO: 4 in the sequence listing. In addition, in the obtained clone , in addition to the sequence shown in SEQ ID NO: 4, a sequence having about 80% homology with SEQ ID NO: 1 was found. This sequence is shown in SEQ ID NO: 5. Escherichia coli holding a plasmid into which a DNA fragment containing the sequence of SEQ ID NO: 4 and SEQ ID NO: 5 was inserted was deposited as AJ12623 (FERM P-12333).

(Incorporation of Gene into Chromosome Using Repetitive Sequence) Mutant plasmid pHSC4 whose replication function has become temperature-sensitive
Both of them were inserted into the restriction enzyme PstI site located at a position unrelated to replication by blunt end ligation by DNA polymerase treatment. Details are shown in FIG. The thus prepared pHRS410 was introduced into Brevibacterium lactofermentum and Corynebacterium glutamicum by electroporation. After culturing the obtained strain at 25 ° C and then raising the temperature to 34 ° C, plasmid curing (removing the retained plasmid) was performed, and the strain retaining pHSC410 retained the vector pHSC4. Significantly more chloramphenicol resistant strains appeared than the strains (Table 1). This is considered to be because the chloramphenicol resistance gene entered the chromosome by RS410 causing homologous recombination with the chromosome. Chromosomal DNA was extracted from the emerged chloramphenicol resistant bacteria, and insertion of the chloramphenicol resistance gene was examined by Southern hybridization. As a result, it was confirmed that the gene was inserted.

[0020]

[Table 1]

[0021]

As described above, according to the present invention, a homologous recombination is produced by using a repetitive sequence existing in the chromosomal DNA of a coryneform bacterium.
Useful genes and the like introduced from foreign sources can be stably retained in the chromosomal DNA of coryneform bacteria. The present invention also provides a method for constantly producing substances such as amino acids by using coryneform bacteria bred by the above method.

[0022]

[Sequence List] SEQ ID NO: 1 Sequence length: 410 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: Genomic DNA Origin Organism name: Brevibacterium lactofermentum (Brevibacterium l)
actofermentum) Strain name: BP-734 Sequence characteristics Symbol indicating characteristics: insertion-seq Method for determining characteristics: S sequence: GCAGCGTCTT GGCCAGGAAA CGCTTGGCCG CCGCGACGTT GCGCTTCGGG GAGAGGTAAA 60 AGTCCAGGCC TGATTGCCGATCGATCGATCTGCCTGCCGGCC GGGAGCATAT TTCTGCACCC AGCGGTAGAT CGTGGTGTGA TCAACCGGCA 240 CACCACGCTC GGTCATCATT TCCTCCAGGT CGCGGTAGCT CACAAGTACG GCCAGTACCA 300 CCGCACTGCC CACAGAATGA TGTCACGGGG GAAATGACGA CCGGAGAAGA TACCCATGCTC 360 CCTGATTTCACTGCACGC

SEQ ID NO: 2 Sequence length: 479 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: Genomic DNA Origin Organism name: Brevibacterium flavu
m) strain name: ATCC 14067 sequence: GTGTAAATTA ATTCCAGTCA GCGCGACCAA CAACGCCCCA ACACATAAGA GATTATGTGG 60 ACAGTGAGGC GGATCTAGGA AAACAAACGC TCGACAAACC AACAACACTT CATCGAAGTC 120 AACCAAACAC CGATTTCGTA AGTAAAGATG AAGTAACGTT GAACAAGCTG CCAACAAGAC 180 ACCAACAAAC AAATGTTGAT GATCTTATGG TGTGCGTATG TTGTTTGAGA ACTCAATAGT 240 GTGCCATTTA TTATTTTTGT CACTACCACT CTTAACCCCT TGAGGGTTTT GGTGGTGGGT 300 CATGCCGGGT GGTGGATCGC CAATATTTAT CCATCACTGT AAACAATAAC CAATATTTGT 360 TGGCATGATT TGTGGTCTCT TGTTCCCGTC AAGGATCAGG CCCACATGAA AGACACGATG 420 AATCCTTATG TGGGTTGTGG TGTTTTTTTA AATCATTATT TTTTGATAAT GCCAGTAAC 479

SEQ ID NO: 3 Sequence length: 656 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: Genomic DNA Origin Organism name: Phage bacterium lactofermentum (Brevibacterium l)
Actofermentum) strain name: BP-734 sequence: TAGGGTTGCG CGGATCATGC GTGAACTCAA ACTGTTTGGC TACACCAAGA AACGCAAGGT 60 CACCACCACC GTGTCAGATC AGAAGAAACC AGTTGTCCCT GACCTTGTCG GTCCGTAAAT 120 TATCCGACAG CCGAACCAGG TTACGTCGTG ACATTACCTA TCTGCCGATT GCAGATGGGT 180 CGAATATGTA CTTGGCTACG GTCATTGATT GCTATTCCCG GCGGCTGGTT GGCTTCGCGA 240 TCGCCGATCA CATGCGCACA TCACTGGTGC AGGCATGCGC TGGTGATGGC CAAAGCGCGA 300 GGGAGCATGA AGGAAGCAAT TTTCCACTCG AACCACGGCA GTGTGTACAC ATCGAATGCG 360 TTCCAGGACA CCTGCACCCA GTTCCCCATC AGGCAGTCGA TGGATCAATT GGACCAGTCG 420 ANNNTTGNGG AGTCGTTCAA CGCGGCCTGA AGAGGGAAGT CCTTCAAGAT TCCAAGACCT 480 TTGCCAACCA GATGATCTGC CGGCGGGATG TTTTCCGCTG GTGTACNCGC GTTACAACAC 540 GGTCGCCGAC ATTCCAGATG TAAATATCTG CTCCTNTGTN TTTAGACNTG TCCTGCTATC 600 CTAAGATCTG CTTCTTGATT AAATCCTCCT GTCCACTTCC CGGGGGTCGG GCCCTT 656

SEQ ID NO: 4 Sequence length: 589 Sequence type: Nucleic acid Number of strands: Double-stranded Topology: Linear Sequence type: Genomic DNA Origin Organism name: Brevibacterium lactofermentum (Brevibacterium l)
Actofermentum) strain name: BP-734 sequence: GGATCCGTTT CAGCCGCCCA TGATCTCCTT CAATGACGTT ATTGAGGTAT TTCACCTGCC 60 GGTGTTCCAC GGTCTGCGGC AGATTCCCTC TGACTTCAAC TCGGCGATTG CCTGGCAGGA 120 GGGTGCTTTA TCGGTGTTGA TCACCGCGGG GACCCGGTTA TCGTGTTCGA CCTCAGGTCT 180 TGGCCAGGAA ACGCTTCGCT GCGGCGACGT TACGCTTTGG GGACAGGTAA AAATCCAGGG 240 TATTGCCCGC CCGCTGTGAT GGCCCGATAG AGGTAGCACC ACTTTCCCGA TCCGATATAG 300 GTCTCATCCA CCCGCAGGAA GTGGCCTGCC AATCCGGGAC TTGTCGGTAC CACCGGGTCT 360 GCTTGTCCAG CTCAGGAGCA TATTTCTGGA CCAGCGGTAG ATGGTGCTGT GATCGACGGT 420 ACGCAGCGTT CGGTCATTTC TTCCAGATCG CGGTAGCTGA GCNCCGTAGC GGCAGTACCA 480 CCGACCGCCC ACAGGATGAT TTCACGGGGG AAATGACGATC TGGATCGATCGATCGATCGATC CGGATCGATC CGGATCGATC CGGATCGATC CGGATCGATC CGGATCGATC CGGATCGATC CGGATCGATC CGGATCGATC CGGATCGATC CGGATCGATC CGGATCGATC CGGATCGATC CGGATCGATC CGGATCGATC CGGATCGATC CGGATCGATC CGGATCGAC

SEQ ID NO: 5 Sequence length: 656 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: Genomic DNA Origin Organism name: Brevibacterium lactofermentum (Brevibacterium l)
actofermentum) Strain name: BP-734 Sequence characteristics Symbol representing sequence: insertion-seq Method for determining characteristics: S sequence: GTGCTGTTGC AAAGTTGGGC GGAGAGCAGC GAAGACTCAG TTCTCATCCT GATCGCGCTC CGTC TG CGTC CGTC CGTC CGTC CGTC TGCCTC CGTCTCTCCGTCTCGTCCGTCTCGTCCGTCTCTCCGTCTCG AGATGTCTAA GTTCTTAAAC GCCTTTCGGC CCGAGGATCC GCTTCAGCCG 240 ACCATGGTGC CTTCCAGGAT GTTGTTGAGG TATTTCACCT GCCGGTGTTC CACTGTTAGC 300 GAGCAGATTC CCTCTGACTT CAACTCGGCG ATTGCCTGGC TAGAGACGGT GCTTATCGGT 360 GTTGATCACN CTGGGATACC CGGCTGACGC ATTGGATCTG AGGGCCTTGG CAGGAAAGCG 420 TTCGCTGCGG CCACGTTCCG TTTTGGTGAG AGGTAAAGTC CAGGGTCTTG GCCACCGGCG 480 GTATCGCAGA TCAGAGGTAG CACCACCTGC ATGCCGACCC GATATACTCT CATCACCTCA 540 GAACTTAGCT GCAGTCAGTA CTGCGTACAC GTGTTTGCTT GTCAGCTCAG ATCGCATTCT 600 GACCAGCGGT GAGATCGTGG TGTGATCAAC GGTACGCCAG CTGAAGTCAT CATTCTCAAG 660 TCGCGGTAGC TCACCGTAGC GCGAGTGACC TGACGCACGG CCACAGAATG ATGTCACGGG 720 AAACGACGAC GGAGACGATA CCCATGGCTG TGATTATTTC ACGTCGCTCT TCCTACTGCC 780 CCAACTTTGC AACAACAC 798

[Brief description of the drawings]

FIG. 1 shows a procedure in which a repetitive sequence RS410 was introduced into a PstI site of a mutant plasmid pHSC4 having a temperature-sensitive replication origin. Note that both ends of RS410 are derived from pUC18.
Multiple Cloning Site sequence is attached.

────────────────────────────────────────────────── ─── Continued on the front page (58) Fields surveyed (Int. Cl. ⁷ , DB name) C12N 15/00 C12N 1/21 BIOSIS / WPI (DIALOG) GenBank / EMBL / DDBJ / GeneSeq JICST file (JOIS)

Claims (7)

(57) [Claims] 1. The nucleotide sequence of SEQ ID NO: 1 which is present in chromosomal DNA of a coryneform bacterium. 2. The nucleotide sequence of SEQ ID NO: 2 which is present in the chromosomal DNA of a coryneform bacterium. 3. The nucleotide sequence of SEQ ID NO: 3 which is present in chromosomal DNA of a coryneform bacterium. 4. The nucleotide sequence of SEQ ID NO: 4 which is present in the chromosomal DNA of a coryneform bacterium. 5. The nucleotide sequence of SEQ ID NO: 5 which is present in chromosomal DNA of a coryneform bacterium. 6. A method for incorporating a gene of an amino acid biosynthesis system derived from a coryneform bacterium into a chromosomal DNA of a coryneform bacterium using the nucleotide sequence according to claim 1. 7. A coryneform bacterium obtained by the method according to claim 6.