JPH06102024B2 - Novel promoter and gene expression method using the promoter - Google Patents

Description

The present invention relates to a DN containing a promoter and an operator region of a tryptophan operon of a coryneform glutamic acid-producing bacterium.
Regarding the A fragment, a DNA fragment obtained by ligating a DNA fragment containing a useful gene downstream of a DNA fragment containing a tryptophan operon of the coryneform glutamic acid-producing bacterium promoter and an operator region is introduced into Escherichia coli or a coryneform glutamic acid producing bacterium. Then, the present invention relates to a method for expressing a useful gene, which comprises culturing the obtained Escherichia coli or coryneform glutamate-producing bacterium.

That is, the present invention is represented by the following formula (1): AGCTGCGGAA ACTACACAAG AACCCAAAAA TGATTAATAA TTGAGA
Contains the promoter and operator region of the tryptophan operon of the coryneform glutamate-producing bacterium expressed by CAAG CTT.
DNA fragment, (2) Formula: AGCTGCGGAA ACTACACAAG AACCCAAAAA TGATTAATAA TTGAGA
Contains the promoter and operator region of the tryptophan operon of the coryneform glutamate-producing bacterium expressed by CAAG CTT.
A method for expressing a useful gene, which comprises introducing a DNA fragment obtained by ligating a DNA fragment containing a useful gene downstream of the DNA fragment into Escherichia coli, and culturing the obtained Escherichia coli, (3) Formula: AGCTGCGGAA ACTACACAAG AACCCAAAAA TGATTAATAA TTGAGA
Contains the promoter and operator region of the tryptophan operon of the coryneform glutamate-producing bacterium expressed by CAAG CTT.
Expression of a useful gene characterized by introducing a DNA fragment obtained by ligating a DNA fragment containing a useful gene downstream of the DNA fragment into a coryneform glutamic acid-producing bacterium, and culturing the obtained coryneform glutamic acid-producing bacterium Method, and (4) The medium for culturing the coryneform glutamic acid-producing bacterium is L
-It is a method of expressing the useful gene that does not contain tryptophan.

Coryneform bacteria referred to in the present invention
Is the Bargey's Manual of Determina.
tive Bacteriology) 8th edition p. 599 (1974), a group of microorganisms, which are aerobic, gram-positive, non-acidic, and spore-forming bacilli. Among such coryneform bacteria, coryneform glutamic acid-producing bacteria as described below are most preferable in the present invention.

Examples of wild strains of coryneform glutamic acid-producing bacteria include the following.

Brevibacterium divaricatum ATCC 14020 Brevibacterium saccharolyticum ATCC 14066 Brevibacterium inmaliofilm ATCC 14068 Brevibacterium lactofermentum ATCC 13869 Brevibacterium roseum ATCC 13825 Brevibacterium flavum ATCC 13826 Brevi Corynebacterium acetoacetate film ATCC 13870 Corynebacterium acetoglutamicum ATCC 15806 Corynebacterium carnae ATCC 15991 Corynebacterium glutamicum ATCC 13032, 13060 Corynebacterium lilium ATCC 15990 Corynebacterium -Meracecola ATCC 17965 Microbacterium ammoniaphilum ATCC 15354 The coryneform glutamic acid-producing bacterium of the present invention has the above-mentioned glutamic acid productivity. In addition to the wild strains that produce glutamic acid, mutant strains that have glutamic acid productivity or lose glutamic acid productivity are also included.

The tryptophan operon here means a promoter, an attenuator, a region encoding a leader peptide (trpL), an anthranilate synthase gene (trpE, trpG), a phosphoribosyl anthranilate transferase gene (trpD), N- ( 5'-phosphoribosyl) anthranilate isomerase-indole-3-glycerol phosphate synthase gene (trpC),
Each of the structural genes of the tryptophan synthase gene (trpB, trpA) is arranged adjacent to each other and functions as one transcription unit.

The method for isolating each structural gene is to first extract a chromosomal gene from a tryptophan operon of a coryneform bacterium or a strain having each structural gene (for example, H. Saito an
d K. Miura Biochem 72, method 619 (1963) can be used. ), And cut it with an appropriate restriction enzyme. Then, it is ligated to a vector that can replicate in microbial cells and has promoter activity, and the resulting recombinant DNA is used to mutate the structural gene of the tryptophan biosynthesis system in coryneform bacteria or other microorganisms. , The enzyme loses its activity, and thus transforms a mutant strain that exhibits tryptophan auxotrophy, recovers and increases the enzyme activity, and collects a strain in which tryptophan auxotrophy disappears. Composite plasmids with structural genes can be isolated.

Even with such a method, there are cases where the entire operon can be isolated fortunately. However, if the entire operon cannot be isolated (cloned), a part or all of each structural gene separated by the above method can be isolated. Can be isolated by colony hybridization from the gene bank of the chromosomal gene of coryneform bacterium prepared using a plasmid or a phage vector by labeling with a isotope or the like and using them as probes.

In order to cut the chromosomal gene, a wide variety of restriction enzymes can be used by controlling the cleavage reaction time and the like to control the degree of cleavage.

In the present invention, the vector used when the tryptophan operon or a part thereof is used for the production of tryptophan may be any vector as long as it can grow in coryneform bacterial cells. Specific examples are as follows.

(1) pAM 330 See JP-A-58-67699 (2) pAM 1519 See JP-A-58-77895 (3) pAJ 655 See JP-A-58-192900 (4) pAJ 611 Same as above (5) pAJ 1844 Same as above (6) pCG 1 JP 57-134500 (7) pCG 2 JP 58-35197 (8) pCG 4 JP 57-183799 (9) pCG 11 Same (10) pCG 1 JP 59-143591 (Mautin / Ajico) (11) pBL 100 JP-A-60-120992 (〃
) The vector DNA is cleaved by using a restriction enzyme that cuts the DNA at one site, or by partially cutting it with a restriction enzyme that cuts at multiple sites.

Vector DNA is cleaved by the restriction enzyme used when cleaving the chromosomal gene, or by connecting an oligonucleotide having a complementary base sequence to each end of the chromosomal DNA cleavage fragment and the cleaved vector DNA, Then, it is subjected to a ligation reaction between the plasmid vector and the chromosomal DNA fragment.

In order to introduce the recombinant DNA of the chromosomal DNA and the vector thus obtained into a recipient bacterium belonging to a coryneform bacterium, it has been reported that Escherichia coli K-12 has been reported (Mandel, M. and Higa, A., J. Mol., Biol., 53 , 159 (19
70) A method of treating recipient cells with calcium chloride to increase DNA permeability, or as reported for Bacillus subtilis (Duncan, CH, Wilson, GA and Youn.
g, FE, Gene, 1 , 153 (1977)) It is possible by a method of introducing into a growth stage (so-called competent cell) in which cells can take up DNA. Alternatively, Bacillus subtilis, as it has been known about the radiation fungi and yeast (Chang, S.and Choen, SN, Molec.Gen., Genet., 168 .1
11 (1979); Bibb, MJ, Ward, JMand Hopwood, OA, Nat
ure, 274 , 398 (1978); Hinnen, A., Hicks, JBand Frink,
GR, Proc.Natl.Acad.Sci.USA, 75 1929 (1978)), DNA
It is also possible to introduce the recombinant DNA into the recipient bacteria by converting the recipient bacteria into protoplasts or spheroplasts that easily incorporate the plasmid DNA.

In the protoplast method, it is possible to obtain a sufficiently high frequency even with the method used in the above Bacillus subtilis, and to the protoplasts of the genus Corynebacterium or Brevibacterium described in JP-A-57-183799, polyethylene glycol or A method of incorporating DNA in the presence of polyvinyl alcohol and a divalent metal ion can of course be used. Similar results can be obtained by a method of promoting the uptake of DNA by adding carboxymethyl cellulose, dextran, Ficoll, Bulonic F68 (Celva) instead of polyethylene glycol or polyvinyl alcohol.

To carry out molecular breeding of tryptophan-producing bacteria using the flown driptophan operon or each structural gene, use a strain transformed with the tryptophan-requiring mutant strain used during gene cloning as a host. However, strains with higher tryptophan productivity may be obtained using the following hosts.

A mutant strain that requires phenylalanine and tyrosine of Brevibacterium and has resistance to 5-methyltryptophan (I. Shiio, H. Sato, M. Nakagawa., Agric. Biol. Chem., 3
6 , 2315 (1972)), requires phenylalanine of the genus Brevibacterium, m-fluorophenylalanine, 5-
Mutant strain resistant to fluorotryptophan (I. Shii
o, S.Sugimoto, M.Nakagawa., Agric.Biol.Chemi., 39 , 627
(1975)), which requires tyrosine of the genus Brevibacterium, 5-fluorotryptophan, a mutant strain resistant to azaserine, phenylalanine and tyrosine of the genus Corynebacterium, and 5-methyltryptophan, 4.
Mutant strains resistant to -methyltryptophan, 6-fluorotryptophan, tryptophan hydroxamate, p-fluorophenylalanine, tyrosine hydroxamate, phenylalanine hydroxicomate (H. Ha
gino, K. Nakagawa., Agric. Biol. Chem., 39 , 345 (1975))
Etc.

The method of culturing the coryneform bacterium having the tryptophan-producing ability thus obtained to generate and accumulate tryptophan is not particularly different from the method conventionally used for the production of tryptophan by the coryneform bacterium. . That is, the medium is a normal medium containing a carbon source, a nitrogen source, inorganic ions, and if necessary, organic micronutrients such as amino acids and vitamins. As the carbon source, glucose, sucrose, lactose and the like and starch hydrolysates containing these, whey, molasses and the like are used.
As the nitrogen source, ammonia gas, aqueous ammonia, ammonium salt and the like can be used.

Culturing is carried out under aerobic conditions while appropriately controlling the pH and temperature of the medium until the production and accumulation of tryptophan is substantially stopped.

In addition to the molecular breeding of tryptophan-producing bacteria, another big advantage obtained by the present invention is that the promoter of the tryptophan operon of Brevibacterium is
E. coli tryptophan operon promoter or as strong as or more, and has a strong terminator as expected from the structure at the end,
It also has an operator whose expression is regulated by tryptophan in coryneform bacteria. Heterologous genes in E. coli, such as interferon,
E. coli tryptophan promoters, operators, and terminators are frequently used for expression of growth hormones, interleukins, nerve growth factors, or other physiologically active polypeptides or enzymes, or overproduction of heterologous proteins. That is, the tryptophan operon obtained by the present invention of course promotes the molecular breeding of L-tryptophan-producing bacteria in coryneform-type bacteria.
It has a promoter, an operator, and a terminator capable of strong expression of a gene in E. coli system or other coryneform type bacteria and its regulation. It is possible to express and overproduce.

Further, among the DNA sequences of the present invention, the promoter region, the operator region, the attenuator region and the base sequence of the ribosome binding region, which are parts involved in gene expression, and the base sequence of the terminator region are each used alone or in any case. When used in a combined form (removed), or the DNA sequence obtained by substituting the encoded amino acid sequence for the base sequence of the structural gene of each enzyme so that it does not differ, Derivatives obtained when the base of any part of the DNA sequence of the present invention is replaced with another base, a new base is inserted, or deleted, or when a part of the base sequence is transposed, It is believed that any protein having an amino acid sequence encoded by it gives good results for gene expression and molecular breeding of L-tryptophan-producing bacteria. Are, it is within the scope of the present invention as a technique that depends the main portion of the present invention.

Reference Example 1. Cloning of anthranilate synthase gene, phosphoribosyl anthranilate transferase gene, and tryptophan synthase β subunit gene 1-1 Preparation of chromosomal DNA containing tryptophan operon of Brevibacterium lactofermentum Brevibacterium lactofer Mental AJ11225 (F
ERM BP-1219) was inoculated into 1 CMG medium (containing peptone 1 g / dl, yeast extract 1 g / dl, glucose 0.5 g / dl, and NaCl 0.5 g / dl and adjusted to pH 7.2), Shaking culture was carried out at 30 ° C. for about 3 hours to collect cells in the logarithmic growth phase.

After lysing the cells with lysozyme / SDS, chromosomal DNA was extracted and purified by a usual phenol treatment method to finally obtain 3.5 mg of DNA.

1-2 Preparation of vector DNA Using pAJ1844 (molecular weight 5.4 sogadalton) as a vector, its DNA was prepared as follows.

First, inoculate 100 ml of CMG medium with Brevibacterium lactofermentum AJ12037 harboring pAJ1844 as a plasmid, and after culturing at 30 ° C. until the late logarithmic growth phase,
Lysiszyme was lysed by SDS treatment, and the supernatant was obtained by ultracentrifugation at 30,000 × g for 30 minutes. After phenol treatment, 2 volumes of ethanol was added to precipitate and collect DNA. Add a small amount of TEN buffer (20 mM Trif hydrochloride, 20 mM MaCl, 1 mM EDTA).
(PH8.0)), and then cesium chloride-ethidium bromide density gradient equilibrium centrifugation to separate the plasmid fraction,
Finally, about 200 μg of pAJ1844 plasmid DNA was obtained.

1-3 Insertion of Chromosomal DNA Fragment into Vector 10 μg of chromosomal DNA obtained in 1-1 and 5 μg of plasmid DNA obtained in 1-2 were each kept at 37 ° C. for 1 hour with restriction endonuclease Pst I, and cleaved. After heating for 10 minutes to 65 ° C., a mixture of both the reaction solution, ATP and dithiothreitol presence, 10 ° C. by DNA ligase from T ₄ phage
The DNA strands were ligated by holding for 24 hours. Then, the reaction solution was heated at 65 ° C. for 5 minutes, and 2 volumes of ethanol was added to the reaction solution to collect a precipitate of ligated DNA.

Cloning of 1-4 anthranilate synthase gene, phosphoribosyl anthranilate transferase gene, and tryptophan synthase β subunit gene Anthranilate synthase-deficient strain AS60 of Brevibacterium lactofermentum No., phosphoribosyl anthranilate transferase-deficient strain No. 38, Tryptophan synthase β subunit deficient strain No.30 (AJ1
1225 was used as a parent strain and isolated by mutation treatment with N-methyl-N-nitro-N-nitrosoguanidine)
Was used as a DNA recipient.

The protoplast transformation method was used as the transformation method. First, the strain was cultivated in 5 ml of CMG liquid medium until the beginning of the logarithmic growth phase, and penicillin G was added to 0.6
After adding the unit / ml, cultivate with shaking for 1.5 hours, collect the cells by centrifugation, and collect the cells with 0.5M sucrose, 20mM.
The cells were washed with 0.5 ml of SMMP medium (pH 6.5) consisting of maleic acid, 20 mM magnesium chloride and 3.5% Penassay broth (Difco). Then, the cells were suspended in SMMP medium containing 10 mg / ml lysozyme and protoplasted at 30 ° C. for 20 hours. 6000 ×
After centrifuging for 10 minutes, wash the protoplasts with SMMP.
Resuspended in 0.5 ml SMMP. The protoplasts thus obtained and 10 μg of the DNA prepared in 1-3 were mixed with 5 mM EDTA.
Mix in the presence of polyethylene glycol to a final concentration
After adding 30%, the mixture was left at room temperature for 2 minutes to incorporate DNA into protoplasts. The protoplasts were washed with 1 ml of SMMP medium, resuspended in 1 ml of SMMP medium, and cultured at 30 ° C. for 2 hours for expression of phenotype. This culture solution was applied on a protoplast regeneration medium having a pH of 7.0. Protoplast regeneration medium is 12 g of tris (hydroxymethyl) aminomethane, 0.5 g of KCl, glucose per distilled water
_{10g, Mgcl 2 · 6H 2 O8.1g} , CaCl 2 · 2H 2 O2.2g, peptone 4
g, powdered yeast extract 4 g, casamino acid (Difco) 1 g, K ₂ HP
It contains O ₄ 0.2 g, sodium succinate 135 g, agar 8 g, and chloramphenicol 3 μg / ml.

After culturing at 30 ° C for 2 weeks, about 25,000 chloramphenicol-resistant colonies appeared for each recipient bacterium, so this was used as a minimum medium (2% glucose, 1% ammonium sulfate, 0.3% urea, 0.1% phosphate diphosphate). Potassium hydrogen, 0.04%
Magnesium sulfate heptahydrate, 2 ppm iron ion, 2 ppm manganese ion, 200 μg / l thiamine hydrochloride, 50 μg / l biotin, casamino acid (Difco) 3 g / l, chloramphenicol 1
0 μg / ml, pH 7.0, agar 1.8%), chloramphenicol resistant and tryptophan auxotrophic strains disappeared from 2 strains using AS60 and 1 strain using No. 38 , 1 strain was obtained from the classification using No. 30.

When plasmids were extracted from the above 4 strains, all of the plasmids were clearly larger than the vector plasmid pAJ1844, and recombinant plasmids obtained from the section using AS60 were
The recombinant plasmid obtained from the section using ptrpE36, ptrpE4 and No.38 was named ptrpB301, and the recombinant plasmid obtained from the section using ptrpD3851 and No.30.

1-5 Retransformation 1-4 Recombinant plasmids obtained in 1-4 ptrpE36, ptrpE4, ptrpD
To confirm the presence of the anthranilate synthase gene, phosphoribosyl anthranilate transferase gene, and tryptophan synthase β subunit gene on 3851 and ptrpB301, ptrpE36 and ptrpE4 were identified as
S60, ptrpD3851 was transformed into No. 38, and ptrpB301 was transformed into No. 30 again.

Ten of the resulting chloramphenicol resistant colonies were picked up and examined for tryptophan auxotrophy.
As a result, all have lost the requirement, and ptrpE3
6, ptrpE4, the anthranilate synthase gene, p
It was revealed that trpD3851 contains the phosphoribosylanthranilate transferase gene and ptrpB301 contains the tryptophan synthase β subunit gene. However, in the transformant strain of ptrpE4, the degree of auxotrophy disappeared and the accumulation of anthranilic acid on the minimal medium was
It was suggested that ptrpE4 may lack a part of the gene for anthranilate synthase, which is worse than the transformant of ptrpE36.

1-6 Construction of restriction enzyme map of inserted DNA fragment of recombinant plasmid Recombinant plasmid ptrp was prepared by the method used in Example 1-2.
E36, ptrpE4, ptrpD3851, and ptrpB301 were prepared and cleaved with various restriction enzymes according to a conventional method to prepare a restriction enzyme map of the inserted DNA fragment (Fig. 1).

Reference Example 2. Cloning of the entire tryptophan operon of Brevibacterium lactofermentum. 5-fluorotryptophan-resistant No. 1041 isolated from Brevibacterium lactofermentum AJ11225 by natural mutation (feedback of anthranilate synthase by tryptophan). Strain from which the inhibition was released) by the method described in Example 1
Was prepared and completely digested with the restriction enzymes BamHI or SalI or XhoI, and the E. coli vector pUC18 (Messing, J., et a
l., Gene, 33 , 103-119 (1985)) and ligated to each restriction enzyme cleavage site to obtain E. coli JM109 (Messing, J., et al., Gene, 33 ,
103-119 (1985)) and transformed with X-Gal (5-bromo
−4chloro-3-indolyl-β-galactoside), 1PTG (is
opropyl-β-D-thio-galactopyranoside) and ampicillin were added to the L agar medium. At 37 ° C
A total of about 1500 white colonies that appeared after 24 hours of culture were picked up on a nitrocellulose filter. Colony hybridization (Grunstein, M., W
alls, J.: Methods in Enzymology, 68 , 379, Academic Pres
S. Inc., New York (1979)), one from the division using the restriction enzyme BamHI, and one from the division using the restriction enzyme SalI.
Two positive clones were obtained. The recombinant plasmid obtained from the BamHI section was named ptrpE97, and the plasmid obtained from the SalI section was named ptrpE42, and the inserted DNA was inserted in the method shown in Example 1.
A restriction enzyme digestion map of the fragment was prepared (Fig. 1).

As a result, ptrpE97 has a PstI fragment having the same restriction map as the inserted PstI fragment of ptrpE36, ptrpD3851, ptrpB301, and ptrpE42 has a PstI fragment of ptrpE36 and ptrpD3851.
It was revealed that it had the same restriction enzyme map as part of the PstI fragment of Escherichia coli. Also, ptrpE97 and ptrpE42 have the same BamH
It had the I-SalI fragment.

Reference Example 3. Subcloning of N- (5-phosphoribosyl) anthranilate isomerase-indole-3-glycerol phosphate synthase gene (trpC) and subcloning of tryptophan synthase subunit gene (trpA) Insertion of the recombinant plasmid shown in FIG. From the comparison of restriction enzyme maps of DNA fragments, it was thought that the trpC gene may exist between the trpD gene and the trpB gene, and the trpA gene may exist downstream of the trpB gene. Therefore, the following experiment was conducted to confirm the existence of each gene.

3-1 Subcloning of trpC gene From recombinant plasmid ptrpE97, S of about 2 kb shown in FIG.
The stI-EcoRI fragment was fractionated and pUC19 digested with SstI and EcoRI
(Messing, J., et al., Gene, 33 , 103-119, 1985) and arranged so that transcription from the lac promoter was possible. Alternatively, pUC18 (Messing, J., et., Et al.) Was prepared by fractionating the SstI-HindIII fragment of about 2.6 kb.
al., Gene, 33 , 103-119, 1985)) and arranged so that transcription from the lac promoter is possible, and E. coli C
GSCNo.5889 ( trp C60, pyr F287, his Gl, lac Z53, rps L8,
λ ⁻ ) was transformed. As a result, the SstI-EcoRI fragment,
Alternatively, a recombinant plasmid containing the SstI-HindIII fragment eliminated the E. coli requirement.

3-2 Confirmation of the presence of the trpA gene About 2.4 kb as shown in FIG. 1 from the recombinant plasmid ptrpE97.
The NruI-BamHI fragment of E. coli was fractionated and pUC digested with SmaI and BamHI
E. coli CGSC No.5644 ( trp A33, rha- 7, ligated to 18 and arranged so that transcription from the lac promoter was possible.
λ ⁻ ) was transformed. As a result, loss of tryptophan auxotrophy was observed in the transformant carrying the recombinant plasmid having the NruI-BamHI fragment.

Reference Example 4. Determination of nucleotide sequence of tryptophan operon Plasmids were prepared from the transformants having ptrpE36, ptrpD3851, ptrpB301 obtained in Example 1 and ptrpE97 obtained in Example 2. Insertion of each plasmid
For DNA fragments pUC18 or pUC19 or M13mp10 (Messin
g, J.and Vieira, J., Gene 19 , 269 (1982))
Oxy chain termination method (Sanger.F. et al., Proc.Nat
The whole tryptophan operon nucleotide sequence was determined by the strategy diagram for nucleotide sequence determination shown in FIG. 2 by I. Acad. Sci. USA 74 , 5463 (1977)). As a result, the DNA base sequence shown in Formula 1 was obtained. This base sequence was used for the expression of the tryptophan operon of Brevibacterium lactofermentum. The RNA polymerase binding site (trp promoter), ribosome binding site, anthranilate Synthase gene (trpE, trpG), phosphoribosyl anthranilate transferase gene (trpD), N- (5
-Phosphoribosyl) anthranilate isomerase-indole-3-glycerol phosphate synthase gene (tr
pC), tryptophan synthase gene (trpB, trpA)
Was found to contain a DNA sequence corresponding to the above, and a termination sequence (terminator).

In addition, between the promoter and the trpE structural gene, an operator region involved in repression, which is an expression control mechanism at the transcription level, and a leader peptide (trpL), which is involved in attenuation of the expression control mechanism at the translation level, are encoded. It was presumed that there were regions and regions where attenuator-like structures were present (Fig. 3). The structure of the terminator is shown in FIG.

Example 1. Isolation of promoter and confirmation of activity In order to isolate the putative promoter of the tryptophan operon as a result of nucleotide sequence determination and confirm its function, as shown in FIG. 4, ptrpE97 or ptrpE36 was identified.
The EcoRI-HindIII fragment (about 550 bp.) Was used as a promoter probe vector pkk175-6 of E. coli (ampicillin resistance (A
p), tetracycline (Tc) sensitive) (Brosius, J., Ge
It was subcloned into ne 27, 151 (1984)) . The resulting recombinant plasmid ptrpP01 expressed Tc resistance in E. coli.

Furthermore, a trimethoprim-resistant vector derived from pAM330,
When the recombinant plasmid ptrpP01 cleaved with PstI was ligated to the PstI cleavage site of pAJ226 and transformed with Brevibacterium lactofermentum AJ11225, expression of Tc resistance was observed. As shown in Table 1, the Tc resistance of the transformant having this recombinant plasmid ptrpP02 was
In the presence of p., the sensitivity to Tc was increased. Therefore, it is considered that a promoter and an operator are present in this region.

Next, to further limit the promoter region, EcoRI-H
The indIII fragment was cleaved with AluI or HaeIII, and each fragment was pK
It was subcloned on K175-6 (Fig. 5).

The HaeIII-HindIII fragment subcloned into pkk175-6 was named ptrpP03, and the AluI-HindIII fragment subcloned into pkk175-6 was named ptrpP04.

As shown in Table 1, AluI: HindIII fragment (51 bp.) And
It became clear that a promoter exists on HaeIII-HindIII (135 bp). Similar results (Table 1) to E. coli
Promoter probe vector pKK232-8 (Ap resistance,
Chloramphenicol sensitivity) and AluI
-It was confirmed that the promoter was present on the HindIII fragment (51 bp.).

The HaeIII-HindIII fragment subcloned into pkk232-8 was named ptrpP05, and the AluI-HindIII fragment subcloned into pkk232-8 was named ptrpP06.

Reference Example 5. By amplification of phosphoribosyl anthranilate transferase gene (trpD), N- (5-phosphoribosyl) anthranilate isomerase-indole-3-glycerol phosphate synthase gene (trpC), tryptophan synthase gene (trpB, trpA) Breeding of tryptophan-producing bacteria.

Of the cloned tryptophan operons of Brevibacterium lactofermentum, trpD, trpC, trp
L-tryptophan production was examined using a recombinant plasmid pAJ234 having 4 genes of B and trpA.

Using pAJ234, m-fluorophenylalanine and 5-
The fluorotryptophan resistant strain Brevibacterium lactofermentum M247 was transformed by the method described in 1-4, and a transformant strain was selected using chloramphenicol resistance as an index. Thus obtained AJ12195 (FERM-P
8014) was cultured and the tryptophan-producing ability was examined. The results shown in Table 2 were obtained.

Cultivation was performed using tryptophan production medium (glucose 130 g, (N
H _{4) 2} SO _4, fumaric acid 12g, acetic _{3ml, KH 2 PO 4 1g,} MnSO 4 · 7
_{H 2 O10mg, MgSO 4 · 7H} 2 O1g, d- biotin 50 [mu] g, thiamine hydrochloride 2000 [mu] g, methionine 400mg, tyrosine 650
50 ml of soybean protein acid hydrolyzate "taste liquid" and 50 g of CaCO ₃ in water 1, pH 6.5. ) 20 ml was put in a 500 ml Sakaguchi flask, the test strain was inoculated, and the mixture was shaken at 30 ° C. for 72 hours. After culturing, the L-tryptophan in the centrifugal supernatant was collected from Leuconosto mesenteroides (Leuconosto).
c mesenteroides) ATCC 8042 as a quantitative strain.

In order to obtain M247, it is possible to remove the composite plasmid in the host cell from the deposited Aj 12195 without damaging the host cell. That is, the plasmid may be naturally lost from the host or may be removed by a "removal" operation. (Bact. Rev., 36 , p361-405 (1972)). Examples of other removal operations are as follows. AJ 12195 was inoculated into a CMG liquid medium, and after overnight culture (high temperature treatment) at 37 ° C, the culture solution was appropriately diluted, and spread on CMG agar medium with or without chloramphenicol, and at 30 ° C. Incubate for 1-3 days. The strain thus isolated as a chloramphenicol sensitive strain is M247.

[Brief description of drawings]

Fig. 1 Restriction enzyme map of inserted DNA fragment of recombinant plasmid Fig. 2 Strategy diagram for determining the nucleotide sequence of tryptophan operon of Brevibacterium lactofermentum pUC18, pUC19 or DNA fragments digested with various restriction enzymes
Clone into M13mp10, and in the direction indicated by the arrow, dideoxy
The base sequence was determined by the method. Fig. 3 Restriction region of tryptophan operon of Brevibacterium lactofermentum-base sequence of 5'upstream region of trpE structural gene of Brevibacterium lactofermentum and amino acid sequence of putative leader peptide (trpL), Fig. 4 Structure of tryptophan operon of Brevibacterium lactofermentum and strategy for isolation and identification of promoter region. Fig. 5 Nucleotide sequence of promoter and operator region of tryptophan operon of Brevibacterium lactofermentum. 35 and -10 show regions corresponding to E. coli promoter, -35 and -10 region of consensus sequence. Fig. 6 Structure of terminator of tryptophan operon of Brevibacterium lactofermentum.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Office reference number FI technical display location C12R 1:13) (C12N 15/77 C12R 1:13) (C12N 1/21 C12R 1:19) (C12N 1/21 C12R 1:13) (C12N 1/21 C12R 1:15) (56) References JP-A-61-40797 (JP, A) JP-A-59-156292 (JP, A)

Claims (4)

[Claims] 1. A formula: AGCTGCGGAA ACTACACAAG AACCCAAAAA TGATTAATAA TTGAGA
Contains the promoter and operator region of the tryptophan operon of the coryneform glutamate-producing bacterium expressed by CAAG CTT.
DNA fragment. Claim 2: Formula: AGCTGCGGAA ACTACACAAG AACCCAAAAA TGATTAATAA TTGAGA
Contains the promoter and operator region of the tryptophan operon of the coryneform glutamate-producing bacterium expressed by CAAG CTT.
A method for expressing a useful gene, which comprises ligating a DNA fragment containing a useful gene downstream of the DNA fragment, introducing the obtained DNA fragment into Escherichia coli, and culturing the obtained Escherichia coli. 3. Formula: AGCTGCGGAA ACTACACAAG AACCCAAAAA TGATTAATAA TTGAGA
Contains the promoter and operator region of the tryptophan operon of the coryneform glutamate-producing bacterium expressed by CAAG CTT.
Expression of a useful gene characterized by introducing a DNA fragment obtained by ligating a DNA fragment containing a useful gene downstream of the DNA fragment into a coryneform glutamic acid-producing bacterium, and culturing the obtained coryneform glutamic acid-producing bacterium Method. 4. The method according to claim 3, wherein the medium for culturing the coryneform glutamic acid-producing bacterium does not contain L-tryptophan.