CA1228038A - Process for producing tyrosine - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

Disclosed is a process for producing tyrosine by transforming a host microorganism belonging to the genus Corynebacterium or Brevibacterium with a recombinant DNA of a DNA fragment containing a gene involved in the biosynthesis of tyrosine and a vector DNA, culturing the transformant in a nutrient medium, accumulating tyrosine in the culture medium and recovering tyrosine therefrom.

Description

1228~8 PROCESS FOR PRODUCING TYROSINE
-Back~round of the Invention For the production of tyrosine by direct fermentation, the methods using mutant strains requiring phenylalanine for its growth or being resistant to tyro-sine of the bacteria belonging to the genus Coryne-bacterium, Brevibacterium, and the like are known Cgr.
Chem. Soc., Japan 50 (1) R79 - R87 (1976 ~.
The present inventors have constructed plasmid vectors autonomously replicable in a microorganism belonging to the genus Corynebacterium or Brevibacterium and having selectable markers and adequate cloning si~es and have developed a highly efficient transformation system (Japanese Published Unexamined Patent Application 15 Nos. 183799/82 published Nov. 12, 1982, 186492/82 and 186489/82 both published Nov. 16, 1982. Further, the present inventors have found that the plasmid vectors are useful for expressing a foreign gene in a host microorganism and increasing the productivity of amino acids by ligating a DNA fragment containing a foreign gene involved in the biosynthesis of amino acids such as glutamic acid, lysine, tryptophan, histidine and phenyl-alanine to the plasmid vectors according to the pro-cedures in recombinant DNA technology (U.S. Patent No.
25 4,237,224 and Methods in Enzymology 68, Recombinant DNA, edited by Ray Wu, ~cademic Press 1979) and transforming Corynebacterium glutamicum L-22 or its derivatives using the transformation methods (Japanese Published Unexamin-ed Patent Application No. 126789/83, and Canadian Patent 30 Applications S.N. 447,439, Feb. 15, 1984, S.N. 447,442, Feb. 15, 1984, S.N. 455,139, May 25, 1984).
Furthermore, the present inventors have found that a microorganism prepared by the same method has acquired an increased productivity of tyrosine.

- 2 - 1228038 Summary of the Invention This invention relates to a process for producing tyrosine by recombinant DNA technology. More specifically, the present invention is a process for producing tyrosine by transforming a host microorganism belonging to the genus CorYnebacterium or Brevibacterium with a recombinant DNA of a DNA fragment containing a gene involved in the biosynthesis of tyrosine and a vector DNA, culturing the transformant in a nutrient medium, accumulating tyrosine in the culture medium and recovering tyrosine therefrom.

Brief Description of the Drawinqs Fig. 1 illustrates the cleavage pattern with restriction endonucleases for plasmid pEaroF-l.
Fig. 2 illustrates the cleavage pattern with restriction endonucleases for plasmid pKmlaroFl.
In Figs. 1 and 2, the horizontal arrows show orientation of transcription of genes.

Description of the Invention The present invention provides a process for producing tyrosine by culturing in a medium a transformant which is obtained by transforming a microorganism belonging to the genus CorYnebacterium or Brevibacterium with a recombinant DNA of a DNA fragment containing a gene involved in the biosynthesis of tyrosine and a vector DNA.
As the DNA fragment containing the gene used in the present invention, the DNA fragment containing a gene involved in the biosynthesis of tyrosine derived from prokaryotes, viruses, bacteriophages or plasmids is used.
Metabolic pathway and regulation systems of aromatic amino acids in microorganisms have been studied in detail on Escherichia coli, Bacillus subtilis, and glutamic acid-producing microorganisms such as strains of the genus of CorYnebacterium 3S and Brevibacterium [Agr. Chem. Soc. Japan, 50 (1), R79 - R87 (1976) and Ann. Rev. Biochemistry 47, 533 (1978)]. The gene involved in the biosynthesis cf tyrosine of the present invention is a DNA carrying a genetic information of at least one of enzymes involved directly or indirectly in the biosynthesis of these aromatic amino acids. The genes encoding for enzymes subjected to regulation on the bio-synthetic pathway, i.e. 3-deoxy-D-arabino-heptulosonate 7-phosphate (referred to as DAHP hereinafter) synthetase (referred to as DAHPase hereinafter), chorismate mutase (referred to as CMase hereinafter), prephenate dehydro-genase (referred to as PDGase hereinafter) and pre-tyrosine aminotransferase are preferably used. Further, the genes of strains which are relieved from feed back inhibition and repression _ prlori or a posteriori are applicable.
As the desired gene of the present invention, any gene is used so long as it is expressed in the microorganisms belonging to the genus Corynebacterium or Brevibacterium and strengthens at least one enzyme activity on the biosynthesis of tyrosine. As the source of the gene, microorganisms which are used for the pro-duction of amino acids such as aromatic amino acids by Eermentation and microorganisms wherein the mechanism of biosynthesis and regulation of aromatic amino acids is clarified are preferably used. The gene derived from a bacterium belonging to the genus Escherichia, Coryne-bacterium, Brevibacterium, Microbacterium, Bacillus, -Staphylococcus, Streptococcus or Serratia and involved in the biosynthesis of tyrosine or the metabolism re-lating to the biosynthesis is preferably used. In the example of the present invention, Escherichia coli JA194 ~ roc. Natl. Acad. Sci., 74, 487-491 (1977 ~ was used.
The vector used in the present invention should autonomously replicate in cells of the host microorganism. Plasmids isolated by the present in-ventors from microorganisms belonging to the genus Corynebacterium or derivatives thereof are available.
pCGl (Japanese Published Unexamined Patent Application *
~;
.~

No. 134500/82 published Aug. 19, 1982), pCG2 (Japanese Published Unexamined Patent Application No. 35197/83), pCG4 (Japanese Published Unexamined Patent Application No. 183799/82 published Nov. 12, 1982), pCE51, pCE52 (Japanese Published Unexamined Patent Application No.
126789/83 published June 24, 1983), pCE53 (Canadian Patent Application S.N. 447,442, Feb. 15, 1984), pCE54, pCGll and pCB101 are preferably used.
Microorganisms carrying the plasmids have been deposited with the Fermentation Research Institute, Agency of Industrial Science and Technology, Ibaraki, Japan and the American Type Culture Collection, Rock-ville, Maryland, U.S.A. under the following accession numbers.
Plasmid FER~l P- ATCC
pCGl 5865 31808 pCG2 5954 31832 pCG4 5939 31830 pCE54 - 39019 pCGll - 39022 pCB101 - 39020 Of the foregoing plasmids, pCG51 is most preferred.
pCE51 is prepared as follows.
pCGl is isolated from the cultured cells of Corynebacterium glutamicum 225-57 (FERM P-5865, ATCC
31808) by the method described in the specification of Japanese Published Unexamined Patent Application No.
134500/82 published Aug. 19, 1982. pGA22 is isolated from the cultured cells of Escherichia coli harboring the plasmid by a conventional method C n, G. et al.,:
J. Bacteriol., 140, 400 (1979 ~. Plasmid pCGl is linearized with restriction endonuclease BglII and a fragment of pGA22 digested with BamHI and containing kanamycin resistant (KmR) gene is ligated to the linear-~ ~, ized pCGl using the same cohesive ends of both-pladmids.
Isolation of pCE51 from the ligated DNA mixture is achieved by selecting the transformants belonging to the genus Corynebacterium or Brevibacterium and containing KmR derived from pGA22, and analyzing the plasmid in the transformant.
Transformation with the ligated DNA mixture is carried out using protoplasts of the genus Coryne-bacterium or Brevibacterium according to the method de-10 scribed in Canadian Patent Application S.N. 401,124, April 16, 1982 and Japanese Published Unexamined Patent Application Nos. 186492/82 and 186489/82 both publish~d Nov. 16, 1982. Kanamycin is used for selection of the transformants. Transformants are recovered as a colony regenerated on a hypertonic agar medium containing 100 -800 ~g/mQ kanamycin which does not allow the reversion to normal cells of protoplast which are not treated with the ligation mixture. Alternatively, transformants are regenerated unselectively on a regeneration medium, and the resultant cells are scraped and resuspended, follow-ed by the isolation of those cells grown on an agar medium containing a drug at a concentration wherein the recipient normal cells cannot grow, that is, generally 2 - 25 ~g/mQ kanamycin.
Plasmid DNAs in the transformants can be iso-lated from cultured cells of the transformants and puri-fied according to the methods described in Canadian Patent Application S.N. 395,976, February 10, 1982 and Japanese Published Unexamined Patent Application Nos.
30 134500/82 published Aug. 19, 1982 and 186489/82 publish-ed Nov. 16, 1982. The structures of the DNAs can be determined by digesting them with various restriction endonucleases and analyzing the DNA fragments by agarose gel electrophoresis. Although several types of plasmids different in physical struct~re are obtained, the lZ2803~3 - 5a -plasmid isolated from one of the transformants is named pCE51.
pCE51 has a molecular weight of about 6 Xb and cleavage sites for HincII, HindIII, SmaI, XhoI and EcoRI
and gives Km phenotype.
Plasmids from the strains are recovered accord-ing to the methods described in Canadian Patent Appli-cations S.N. 395,976, filed February 10, 1982, S.N.
401,241, filed April 19, 1982 and S.N. 410,113, filed 10 August 25, 1982 and Japanese Published Unexamined Patent Application Nos. 134500/82 published Aug. 19, 1982, 183799/82 published Nov. 12, 1982 and 35197/83 published March 1, 1983.
Preparation of a recombinant DNA of a vector DNA with a DNA fragment containing a gene is carried out by conventional in vitro recombinant DNA technology, e.g. cleavage and ligation of a donor DNA containing a desired gene to a vector DNA (refer to Japanese Publish-ed Unexamined Patent Application No. 126789/83 published 20 June 24, 1983, USP 4,237,224).
The ligase reaction gives recombinants con-taining genes other than the desired gene. The desired recombinant DNA can be obtained by directly transforming a microorganism of the genus Corynebacterium or Brevi-bacterium with the DNA mixture, selecting the trans-formants having the phenotype derived from the desired gene and isolating the desired recombinant DNA from -'~
;~ ' - 6 - ~Z28038 the cultured cells of the transformants. Instead of cloning the desired gene directly in a microorganism of the genus Corynebacterium or BreYibacterium, the desired gene can be cloned by using another host-vector system such as Escherichia coli. Then, it is recloned ln vitro into a vector of the genus Corynebacterium or Brevibacterium to transform these microorganisms and transformants containing the desired recombinant plasmid are selected as mentioned above.
The following references are helpful for the construction of recombinant DNA:
S.N. Cohen, et al., U.S.P. No. 4,237,224;
Idenshi Sosa Jikkenho, edited by Yasuyuki Takagi~
printed by Kodansha Scientific (1980);
Methods in Enzymology 68, Recombinant DNA, edited by Ray Wu, Academic Press 1979 Japanese Published Unexamined Patent Application No. 126789/83 Microorganisms belonging to the genus Corynebacterium or Brevibacterium and which are competent for incorporating DNAs may be used as the host microorganisms in the present invention.
The following are examples of a suitable host microorganism.

Accession Number FERM P- ATCC
CorYnebacterium qlutamicum L-15 5946 31834 CorYnebacterium qlutamicum R-38 7087 CorYnebacterium qlutamicum K-43 7162 CorYnebacterium herculis 13868 CorYnebacterium herculis L-103 5947 31866 Brevibacterium divaricatum L-204 5948 31867 Brevibacterium lactofermentum 13869 Brevibacterium lactofermentum L-312 5949 31868 Brevibacterium flavum 14067 Tyrosine-producing mutant strains derived from these strains are preferably used as host microorganisms. The mutant strains are obtained as amino acid-requiring mutants, amino acid analog-resistant mutants or mutants having both the properties.

Transformation of the host microorganisms with recombinant DNAs is carried out through the following steps:
l) Preparation of protoplasts of host cells;
2) Transformation of the protoplasts with a reco~binant DNA;

3) Regeneration of the protoplasts to normal cells and sPlection of a transformant;
Practical method are described in Japanese Published Unexamined Patent Application Nos. 186492/82, 186489/82 and lOS999/83.
The thus obtained transformant is cultured in a conventional manner used in the production of tyrosine by fermentation. That is, the transformant is cultured in a conventional medium containing carbon sources, nitrogen sources, lS inorganic materials, amino acids, vitamines, etc. under aerobic conditions, with adjustment of temperature and pH. Thus, tyrosine accumulated in the medium is recovered.
As the carbon source, various carbohydrates such as glucose, fructose, sucrose, maltose, mannose, sorbitol and 2Q mannitol, sugar alcohol, glycerol, starch, starch hydrolyzate, molasses, various organic acids such as pyruvic acid, lactic acid, acetic acid, fumaric acid and gluconic acid, and lower alcohols such as ethanol may be used.
As the nitrogen source, ammonia, various inorganic or organic ammonium salts such as ammonium chloride, ammonium sulfate, ammonium carbonate and ammonium acetate, urea, and nitrogenous organic substances such as peptone, ~2-amine, meat extract, yeast textract, corn steep liquor, casein hydrolyzate, fish meal or its digested product, and chrysalis hydrolyzate are appropriate.
As the inorganic materials, potassium dihydrogenphosphate, dipotassium hydrogenphosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate and calcium carbonate may be used. Vitamines and amino acids required for the growth of microorganisms may not be added, provided that they are supplied with other components mentioned above.

- 8 ~ ~ ~28~3~

Culturing is carried out under aerobic conditions with shaking or aeration-agitation. Culturing temperature is preferably 20 to 40C. The p~ of the medium during culturing is maintained around neutral. Culturing is continued until a considerable amount of tyrosine is accumulated, generally for 2 to 5 days.
After completion of the culturing, cells are removed and tyrosine is recovered from the culture liquo~ by conventional manners such as treatment with active carbon or ion exchange resin.
In spite of the high similarity in microbiological characteristics, so called ~lutamic acid-producing microorganisms which produce glutamic acid in large amounts are classified into various species and even into different genera such as Corynebacterium and Brevibacterlum, which is probably because of their industrial importance. However, it has been pointed out that these microorganisms should belong to one species because of nearly the same composition of amino acids in the cell wall and the base composition of DNAs. Recently, it ~0 has been reported that these microorganisms have at least 70 to 80% homology in DNA-DNA hybridization, indicating that these microorganisms are closely related. See, e g., Romatsu, Y.:
Report of the Fermentation Research Institute, No. 55, 1 (1980), and Suzuki, K., Kaneko, T., and Romagata, K.: Int. J. S~st.
Bacteriol., 31, 131 11981).
In the present specifi~ation, the usefulness of the present invention is illustrated using derivatives of Corynebacterium qlutamicum as host microorganisms. However, in consideration of the facts mentioned above, it is readily assumed that the usefulness of the present invention is applicable to all the glutamic acid-producing microorganisms.
In order to stably maintain recombinant DNA molecules and express the DNA in these species, slight differences of such properties of the host microorganisms as homology in the ~A are negligible and it is sufficient for host microorqanisms to allow the autonomous replication of plasmids and expression of ~enes on them. That these microorganisms have such abilities is apparent from the fact that plasmid ?CG4 which was isolated ~rom 9 12X803~3 CorYnebacterium glutamicum 225-250 (Japanese Published Unexamined Patent Application No. 183799/82) and having an streptomycin and/or spectinomycin resistant gene could replicate in microorganisms belonging to the genus Corynebacterium or Brevibacterium and that the gene responsible for the resistance could be expressed (Japanese Published Unexamined Patent Application No. 186492/82). Further, as described in Japanese Published Unexamined Patent Application No. 126789/83, the plasmid involved in histidine production functioned in Cor~nebacterium herculis, Brevibacterium flavum and Brevibacterium lactofermentum. Therefore, the present invention is applicable to all the glutamic acid-producing microorganisms including those microorganisms belonging to the genus CorYnebacterium or Brevibacterium as well as to CorYnebacterium qlutamicum .

Certain specific embodiments of the present invention are illustrated by the following representative examples.

Example 1 (1) Preparation of the chromosomal DNA and plasmid DNA:
The chromosomal DNA of Escherichia coli (hereinafter referred to as E. coli) JA194 strain [Proc. Natl. Acad. Sci., 94, 487-491 (1977)] was prepared by the following method.
A seed culture was inoculated into 400 mQ of L-broth (pH 7.0) consisting of 10 g/Q Bacto-tryptone, 5 g/Q Bacto-yeast extract and 5 g/Q NaCl (hereinafter referred to as LB).
Culturing was carried out with shaking at 37C and continued to a latter stage of the logarithmic growth phase. Cells were harvested from the culture broth and high molecular chromosomal DNAs were isolated from the cells by the method of Saito et al., Biochim. Biophys. ~cta, 72, 619 (1963).
Separately pBR322 used as a vector was prepared from the cultured cells of E. coli JA194 strain harboring pB~322 by the following method.
The strain was grown with shaking at 37C in 400 mQ of LB containing 100 ~g/m~ ampicillin to a latter stage of the logarithmic growth phase. Cells were harvested from the culture broth and lysed by the method of Tanaka, et al., ~J. Bacteriol.
121, 35~-362 (197i)].

- 10 - 122803~3 The whole lysate was centrifuged at 4C at 23,000 rpm for one hour. The supernatant fluid was recovered and one fifth volume of 50~ (W/V) polyethyleneglycol (PEG) 6,000 aqueous solution was added. The mixture was stirred slowly and allowed to stand at 4C overnight.
Formed precipitates were collected by centrifugation at 3,000 rpm at 4C for 5 minutes and dissolved in 5 mQ of TE
buffer solution (pH 7.5) consisting of 10 mM Tris HCl and 1 mM
EDTA-Na2. Then, one mQ of 1.5 mg/mQ ethidium bromide was added, and TE buffer solution was added to the total volume of 7.5 mQ.
To the mixture was added 7.875g of CsCl, and dissolved completely. The solution was centrifuged at 105,000 x 9 at 20C
for 40 hours. A plasmid band detected under W irradiation was taken out with a injector. Ethidium bromide was extracted three times with isopropanol containing 15% (V~V) TE buffer solution.
The residue was dialysed against TE buffer solution at 4C
overnight, and used as a plasmid DNA.

(2) Cloning of a DNA fragment containing the gene coding for DAHPase, CMase and PDGase:
In this step, 5 units each of restriction enzymes EcoRI and HindIII (product of Takara Shuzo Co.) were added to 100 ~Q of a HindIII reaction solution containing 3 ~g of pBR322 plasmid DNA prepared above, and 5 units each of EcoRI and HindIII (product of Takara Shuzo Co.) was added to 100 ~Q of restriction enzyme HindIII reaction solution containing 9 l~g of the chromosomal DNA. Each of the mixtures was allowed to react at 37C for 60 minutes and heated at 65C for 10 minutes to stop the reaction. Both of the mixtures were admixed with each other.
Then, 40 ~Q of a T4 ligase buffer solution (pH 7.6) consisting of 660 mM Tris, 66 mM MgC12 and 100 mM dithiothreitol, 40 ~ of 5 mM ATP, 0.4 uQ of T4 ligase (product of Takara Shuzo Co., 1 unit/~Q) and 120 ~Q of H2O were added. The mixture was allowed to react at 12C for 16 hours.
The above ligation mixture was provided ~or the following transform~tion. As the recipient for the transformation, ~AHPase-deficient E. coli AB3248 strain [J.
Bact., 93, 237-244 (1967)] or tyrA gene (CMase)-deficient E.
coli AT2273 strain [J. Bact., 91, 1494 ~1966~ was used.

- ll - 12~8038 The seed culture of the strain was inoculated into LB
and competent cells were prepared according to the method of M. Dagert, [Gene, 6, 23-28 (1979)].
Fifty microliter of the ligation mixture was added to 0.2 mQ of a solution containing 109/m~ competent cells and the mixture was allowed to stand under ice cooling for 10 minutes.
After heating at 37C for 5 minutes, 2 m~ of LB was added, followed by standing at 37C for 90 to 120 minutes. Cells were subjected to washing with physiological saline solution and centrifugation twice. The cells were spread on M9 plate medium (pH 7.0) consisting of 1 g/Q NH4Cl, 6 g~ Na2HPO4, 3 g/Q KH2PO4, 5 g/Q NaCl, 0.1 g/Q MgSO4 7~20, 0.015 g/Q CaC12 2H2O, 3 g/~
glucose, 4 mg~ vitamine Bl and 15 g/Q agar, [J. ~act. 121, 354-362 (1975)] and containing 50 ~g/mQ each histidine, proline, arginine, isoleucine and valine. Colonies grown on M9 plate medium were plated on LB plate media containing 100 ~g/m~
ampicillin and 20 ~g/mQ tetracycline, respectively. Colonies which grew on the ampicillin-containing medium and did not grow on the tetracycline-containing medium were selected.
Plasmid DNAs were isolated from the thus selected transformants which grew on M9 plate medium containing histidine, proline, arginine, isolucine and valine and which were resistant to ampicillin and sensitive to tetracycline by the same method as described above. Plasmid pEaroFl isolated from one of the transformants was digested with various restriction endonucleases and analysed by agarose gel electrophoresis. The analysis showed that an EcoRI-HindIII cleaved DNA fragment of about 4.2 Kb was inserted into the larger EcoRI-HindIII cleaved fragment of pBR322.
E. coli. AB3248 and E. coli AT2273 were transformed using pEaroFl obtained above by the same method as described above. Both transformants grew on M9 medium containing histidine, proline, arginine, isoleucine and valine and simultaneously were resistant to ampicillin. The fact showed that the transformants had the same plasmids as pEaroFl.
The results described above showed that the genes encoding for DAHPase, CMase and PDGase exist on the DNA fragment of about 4.2 Xb cloned in pEaroFl.

- 12 ~ 1Z~8~3~

Further, pEaroFl having cleavage sites with various restriction endonucleases such as EcoRI, BamHI, HindIII and the like as shown in Fig. 1 was compared with plasmid pKB45 reported by G. Zurawski, Proc. Natl. Acad. Sci. USA 75, 4271 (1978) to show that the DNA fragment of about 4.2 Kb in pEaroFl possesses aroF, tyrA and pheA genes of E. coli.

(3) Subcloning of aroFtyrA gene:
A DNA fragment containing aroFtyrA qene was recovered from plasmid pEaroFl DNA prepared above and ligated to pCE51 which is a shuttle vector for E. coli and CorYnebacterium qlutamicum.
pCE51 is a recombinant plasmid wherein plasmid pCG1 (Japanese Published Unexamined Patent Application No. 134500/82) of CorYnebacterium qlutamicum is ligated with plasmid pGA22 of E. coli ~refer to An, G. et al: J. Bacteriol., 140, 400 (1979)]. The ligation is carried out using the same cohesive ends of BglII cleaved pCGl and BamHI fragment of pGA22 containing the kanamycin-resistant gene.
pCE51 is practically prepared by the method described in Japanese Published Unexamined Patent Application Nos. 105g99/83 and 126789/~3.
Five units of HincII (product of Takara Shuzo Co.) was added to 100 ~1 of HincII reaction solution containing 3 Yg of plasmid pEaroFl DNA and reaction was carried out at 37C for 60 minutes. Three-tenth units of HincII was added to 100 yQ of HincII reaction solution containing 3 ~9 of plasmid pCE51 DNA
prepared from pCE51-carrying E. coli JA194 strain by the same method as mentioned above and reaction was carried out at 37C
for 60 minutes to cut pCE51 at one of two HincII cleavage sites.
Both of the reaction mixtures were mixed with each other. 40 yQ
of T4 ligase buffer solution (pH 7.6) consisting of 660 mM Tris, 66 mM MgC12 and 100 mM dithiothreitol, 40 ~Q of 5 mM ATP, 0.4 yQ
of T4 ligase (product of Takara Shuzo Co., 1 unit/~Q) and 120 ~Q
of water were added. The mixture was allowed to react at 12QC
for 16 hours and reaction was stopped by heating at 55C for 10 minutes.

- 13 ~ 12 Z ~03 8 The ligation mixture was provided for the following transformation. As the recipient for the transformation, E.
coli AB3248 strain was used. Transformation was carried out by the same method as described above to obtain colonies grown on M9 plate medium containing histidine, proline, arginine, isoleucine and valine. Colonies grown on LB plate medium containing 20 ~g/mQ kanamycin were selected from the colonies obtained above.
Plasmid DNAs were isolated from the transformants which grew on M9 plate medium containing histidine, proline, arginine, isoleucine and valine and were resistant to kanamycin by the same method as described above.
Plasmid pKmlaroFl obtained from one of the transformants were digested with various restriction endonucleases and analysed by agarose gel electrophoresis. The analysis showed that a HincII cleaved DNA fragment of about 3.8 Kb bearing aroFtyrA of pEaroFl was inserted in one of two HincII cleavage sites of pCE51. Plasmid pKmlaroF1 obtained above has the restriction pattern as illustrated in Fig. 2.

(4) Preparation of tyrosine- and tyrosine analog-resistant plasmid pKmlaroFl-m-18 from pKmlaroFl-carrying strain:
pKmlaroFl-carrying AB3248 strain was grown in LB
medium containing 20 ~g/m~ kanamycin to a latter stage of the logarithmic growth phase. Cells were harvested by centrifugation and washed with 50 mM Tris-malate buffer solution IpH 6.0) twice and incubated with 400 ~g/mQ N-metyl-N'-nitro-N-nitrosoguanidine in 50 mM Tris-malate buffer solution (pH 6.0) at room temperature for 30 minutes. The treated cells were harvested by centrifugation and washed with 50 mM Tris-malate buffer solution (pH 6.0) twice. The washed cells were cultured in LB medium containing 20 ~g/mQ kanamycin at 30C for 16 hours and plasmid DNAs were isolated by the same method as mentioned above.
_. coli AB3248 was transformed using the isolated ?lasmids. Selection of transformants was carried ou~ on M9 plate medium containing 0.25 mg/mQ tyrosine and 50 ~g/mQ each histidine, ~roline, arginine, isoleucine and valine. Colonie~
grown on ~9 pl~te medium containing 0.25 mg/mQ tyrosine and lZ28038 50 ~g/mQ each histidine, proline, arginine, isoleucine and valine and on LB plate medium containing 20 ~g/mQ kanamycin were selected from the developed colonies.
The thus obtained colonies were resistant to 3-aminotyrosine (3-AT) (product of Shigma Co.) since they could grow on M9 plate medium containing 0.2 mg/mQ 3-AT and 50 ~g/mQ
each histidine, proline, arginine, isoleucine and valine.
The plasmids obtained from the thus prepared transformant can confer resistance to tyrosine or a tyrosine analog on a microorganism. E. coli AB3248 strain carrying one of such plasmids, pKmlaroF-m-18 was able to grow on M9 plate medium containing 1 mg/mQ tyrosine or 0.5 mg/mQ 3 AT, and 50 ~g/mQ each histidine, p{oline, arginine, isoleucine and valine.

(5) Transformation of CorYnebacterium qlutamicum K43 with pKmlaroFl and pKmlaroFl-m-18:
A seed culture of Corynebacterium qlutamicum K43 (FEF~I
P-7162, FERM BP-457) was inoculated in Semi-synthetic medium SSM
(pH 7.2) consisting of 20 g/Q glucose, 10 g/Q (NH4)2SO4, 3 g/Q
urea, 1 g/Q yeast extract, 1 g/Q KH2PO4, 0.4 g/Q MgC12 6H2O, 10 mg/Q FeSO4 7H2O, 0.2 mg/Q MnSO4 (4-6)H2O, 0.9 mg/Q ZnSO4 7H2O, 0.4 mg/Q CuSO4-SH2O, 0.09 mg/Q Na2B47 10H20~ 0.04 mg/Q
(NH4)6Mo7O24 4H2O, 30 ~g/Q biotin and 1 mg/Q thiamine hydrochloride and containing 50 ~g/mQ phenylalanine and culturing was carried out with shaking at 30C. NB medium (pH
7.2) consisting of 20 g/Q powdered bouillon and 5 g/Q yeast extract was used for seed culture. The optical density (OD3 at 660 nm was monitored with a Tokyo Koden colorimeter and, at OD
0.2, penicillin G was added to the broth to a final concentration of 0.5 unit/mQ. Culturing was continued to an OD
value of about 0.6.
Cells were harvested at an OD value of 0.6. The cells were suspended at about 109 cells/mQ in RCGP medium (pH 7.6) consisting of 5 g/Q glucose, 5 g/Q casamino acid, 2.5 g/Q yeast extract, 3.5 g/Q K2HPO4, 1.5 g/Q KH2PO4, 0.41 g/Q ~IgC12 6H2O, 10 mg/Q FeSO4 7H2O, 2 mgJl MnSO4 (4-5)H2O, 0.9 mg/Q ~nSO4 7H2O, 0.04 mg/Q (NH4)~Mo7O24-4H2O, 30 ~g/Q biotin, 2 mg/Q thiamine - 15 - :12Z80~

hydrochloride, 135 g/Q sodium succinate and 30 g/Q polyvinyl pyrrolidone with a molecular weight of 10,000 and containing 1 mg/mQ lysozyme~ The suspension was put in an L-tube and stirred slowly at 30C for 5 hours to obtain protoplasts.
Then, 0.5 mQ of the protoplast suspension was put in a small test tube and centrifuged at 2,500 x g for 5 minutes. The protoplasts were resuspended in 1 mQ of TSMC buffer tpH 7.5) consisting of 10 mM magnesium chloride, 30 mM calcium chloride, 50 mM Tris and 400 mM sucrose and again subjected to centrifugation and washing. The washed protoplasts were resuspended in 0.1 mQ of TSMC buffer solution. One hundred microliter of a mixture (1 : 1 by volume) of a two-~old concentrated TSMC buffer and the ligated DNA mixture described above was added to the protoplast suspension. Then, 0.8 mQ of a solution containing 20% PEG 6,000 in TSMC buffer solution was added to the mixture. Plasmid DNAs, pRmlaroFl and pRmlaroFl-m-18 were prepared from E. coli AB3248 carrying these plasmids as described above. After 3 minutesr 2 mQ of RCGP medium (pH 7.2) was added and the mixture was centrifuged at 2,500 x g for 5 minutes. The supernatant fluid was removed and the precipitated protoplasts were suspended in 1 mQ of RCGP medium. Then, 0.2 mQ
of the suspension was spread on RCGP agar medium (pH 7.2) containing 200 ~g/mQ kanamycin and 1.4% agar and incubated at 30C for 7 days. Colonies resistant to kanamycin were grown on the selection plate.

t6) Production of tyrosine by the transformant:
The thus obtained transformants carrying pKmlaroFl and pKmlaroFl-m-18 have been deposited with the Fermentation Research Institute as CorYnebacterium qlutamicum K44 (FE~M P-7163, FERM BP-458) and Corynebacterium qlutamicum K45 (FE~M P-7164, FE~M BP-460), respectively.
L-tyrosine production by pKmlaroFl and pKmlaroFl-m-18-carrying strains was carried out as follows.
The strain was cultured in NB aqueous medium at 30C
for 16 hours and 0.5 mQ of the culture broth was inoculated in 5 mQ of a production medium P4 (p~ 7.2) consisting of 100 g/Q
molasses, 20 g/Q (NH~)2SO4, 0.5 g/Q KH2PO4, 0.5 g/Q K2~PO4, - 16 ~ ~Z 2 803 8 0.25 g/Q MgSO4 7H2O and 20 g/Q CaCO3 and containing 0.25~ NZ
amine. Culturing was carried out at 30C for 96 hours.
After culturing, 6N NaOH solution was added to the broth to a concentration of 50 ~Q/mQ and the mixture was heated at 65C for 5 minutes to dissolve precipitated tyrosine completely. The culture filtrate was subjected to paper chromatography and color reaction with ninhydrin, and the amount of L-tyrosine formed was determined colorimetrically. As a control, CorYnebacterium glutamicum K43 was similarly treated.
The results are shown in Table 1.

Table 1 Amount of L-tyrosine strain (mq/m~) Corynebacterium qlutamicum K43 4.8 Corynebacterium qlutamicum K44 5.3 CorYnebacterium qlutamicum K45 7.7

Claims (12)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A process for producing tyrosine which com-prises transforming a host microorganism belonging to the genus Corynebacterium or Brevibacterium with a re-combinant DNA of a DNA fragment containing a bacterial gene involved in the biosynthesis of tyrosine and a vector DNA, culturing the transformant in a nutrient medium, accumulating tyrosine in the culture medium and recovering tyrosine therefrom.

2. The process according to claim 1, wherein the bacterial gene is derived from microorganism belonging to the genus Escherichia, Corynebacterium, Brevi-bacterium, Microbacterium, Bacillus, Staphylococcus, Streptococcus or Serratia.

3. The process according to claim 1, wherein the DNA fragment contains a gene of 3-deoxy-2-keto-D-arabino-heptulosonate 7-phosphate synthetase, chorismate mutase, prephenate dehydrogenase or pretyrosine amino-transferase.

4. The process according to claim 3, wherein the DNA fragment confers resistance to tyrosine or a tyro-sine analog on a microorganism.

5. The process according to claim 4, wherein the tyrosine analog is 3-aminotyrosine.

6. The process according to claim 1, wherein said vector is selected from plasmids, phages and derivatives thereof derived from a microorganism and autonomously replicable in the bacterium belonging to the genus Corynebacterium or Brevibacterium.

7. The process according to claim 6, wherein said plasmid and derivative thereof is selected from the group consisting of plasmids pCGl, pCG2, pCG4, pCE51, pCE52, pCE53, pCE54, pCG11 and pCB101 derived from the microorganism belonging to the genus Corynebacterium.

8. The process according to claim 1, wherein the host microorganism belongs to the genus Corynebacterium or Brevibacterium and is sensitive to lysozyme.

9. The process according to claim 1, wherein the host microorganism is a microorganism selected from the species Corynebacterium glutamicum, Corynebacterium herculis, Brevibacterium flavum and Brevibacterium lactofermentum or a derivative thereof.

10. A microorganism belonging to the genus Corynebacterium or Brevibacterium and containing a recombinant DNA of a DNA fragment containing a gene involved in the biosynthesis of tyrosine derived from a microorganism belonging to the genus Escherichia, Corynebacterium or Brevibacterium and a vector DNA.

11. Corynebacterium glutamicum K44.

12. Corynebacterium glutamicum K45.