CA1228039A - Process for producing l-isoleucine - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

Disclosed is a process for producing L-isoleucine by transforming a host microorganims belonging to the genus Corynebacterium or Brevibacterium with a recombinant DNA of a DNA fragment containing a gene encoding for the enzyme involved in the biosynthesis of threonine from aspartic acid and a vector DNA, culturing the transformant in a nutrient medium, accumulating L-isoleucine in the culture medium and recovering L-isoleucine therefrom.

Description

TITLE OF THE INVENTION

PF(OCESS FOR PRODUCING L-ISOLEUCINE

Background of the Inv2ntion For the direct production of L-isoleucine by fermentation methods using glutamic acid-producing microorganisms belonging to the genus Corynebacterium or Brevibacterium, the method using L-isoleucine-producing mutant strains derived from wild-type strains are known.
As the L isoleucine-producing mutant strains, those requiring amino acids for their growth, those resistant to amino acid analogs or those having both characteristics thereof are described in Japanese Published Unexamined Patent Application Nos. 38995/72, 6237/76 and 32070/79.
The present inventors have studied the production of L-isoleucine using a microorganism belonging to the genus Corynebacterium or Brevibacterium by recombinant DNA technology different from the conventional mutational breeding t~chnology for the purpose OL improYing the L-isoleucine productivity. As the result, the present inventors have ~ound that a microorganism harboring a recombinant DNA of a gene involved in the biosynthesis of threonine which is a precursor of L-isoleucine and a vector plasmid of the microorganism belonging to the genus Corynebacterium or Brevibacterium is superior in production of L-isoleucine to a microorganism which does not harbor such recombinant~
The facts that the introduction of a recombinant DN~
containing a gene involved in the biosynthesis of threonine into an L-isoleucine-unproducing microorganism belonging to the genus Corynebacterium or revibacterium confers L-isoleucine productivity on the microorganism and that the introduction of such recombinant DNA into an L-isoleucine producing microorganism belonging to the genus Corynebacterium or Brevibacterium improve its L-isoleucine productivity have been found first by ~he present in~entors.

- 2 - ~Z~8039 In the Japanese Published Unexamined Patent Application No. 893/83, production of L-isoleucine by a microorganism belonging to the genus CorYnebacterium or Brevibacterium wherein a chromosomal gene region involved in the resistance to an S isoleucine antagonist is introduced is described. However, the gene is different from that of t~e present invention.

Summary of the Invention This invention relates to a process for producing L-isoleucine by a novel expression method of a gene. Morespecifically, the present invention is a process for producing L-isoleucine bY transforming a host microorganism belonging to the genus Corynebacterium or Brevibacterium with a recombinant DNA of a DNA ~ragment containing a gene encoding for the enzyme involved in the biosynthesis of threonine from aspartic acid and a vector DNA, cult~ring the transformant in a nutrient medium, accumulating L-isoleucine in the culture medium and recovering L-isoleucine therefrom.

Brief Description of The Drawinq Fig. 1 illustrates the process for construction of and the cleavage map for restriction endonucleases of plasmid pEthrl wherein "Bgl II~Bam HI" with a broken line indicates a recombina~ion site at the same cohesive ends formed by cleavage with both restriction endonucleases. The restriction endonucleases used in the preparation of the cleavage map are PstI, EcoRI and XhoI. Molecular weight of the plasmid is indicated as Kilobase (Kb).

Description of the Invention The present invention provides a process for producing L-isoleucine by culturing in a medium a transormant which is obtained by transforming a microorganism belonging to the genu~
Corvnebacterium or Brevibacterium with a recombinant DNA of a DNA fragment containing a gene encoding for the enzyme involved in the biosynthesis of threonine from aspartic acid and a vector DNA.

~ 3 ~ 1228039 As the host microorganism belonging to the genus CorYnebacterium or Brevibacterium, all of the microorganisms known as so-called glutamic acid-producing microorganisms are applicable. The following are examples of suitable host microorganisms.

CorYnebacterium qlutamicum ATCC 13032 CorYnebacterium acetoacidoPhilum ATCC 13870 CorYnebacterium herculis ATCC 13868 CorYnebacterium lilium ATCC 15990 Brevibacterium divaricatum ATCC 14020 Brevibacterium flavum ATCC 14067 Brevibacterium immarioPhillum ATCC 14068 Brevibacterium lactofermentum ATCC 13869 Brevibacterium thioqenitalis ATCC 19240 As the host, either wild-type strains which do not produce isoleucine or strains which already have an ability to produce isoleucine can be employed. As the latter strains, amino acid-requiring mutant strains and amino acid analog-resistant mutant strains are used.
As the enzyme involved in the biosynthesis of threonine from aspartic acid, aspartate kinase, aspartate-semialdehyde kinase, homoserine dehydrogenase, homoserine kinase and threonine synthase [Agr. Biol. Chem., 38, 993 (1974)] are mentioned.
As the gene encoding for the enzymes involved in the biosynthesis of threonine, the DNA carrying the genetic information of at least one of these enzymes is used. Any DNA
may be used so long as it is derived from prokaryotes, eukaryotes, bacteriophages, viruses or plasmids. The genes involved in the biosynthesis of threonine derived from prokaryotes, bacteria such as the microorganisms belonging to the genus Escherichia, Corvnebacterium, Brevibacterium, Microbacterium, Bacillus, StaPhylococcus, StrePtococcus or Serratia are preferable and especially the genes derived from threonine- or isoleucine-producing mutants belonging to such bacteria are preferably used. The threonine operon of Escherichia coli K12 is the most preferable example.

~ 4 ~ 12 2 ~03 9 As the vector used to incorporate the DNA, the plasmids constructed by the present inventors, pCGl, pCG2, pCG4, pCGll, pCE54 and pCB101 are preferably used. The methods of producing these vectors are described in Japanese Published Unexamined Patent Application Nos. 134500/82, 183799/82, 35197/83 and 105999/83.
The recombinant DNA of the donor DNA encoding for the enzyme involved in the biosynthesis of threonine and the vector DNA is obtained by the recombinant DNA technology which comprises cleaving ln vitro both DNAs with restriction enzymes, recombining the cleaved DNAs by DNA ligase, transforming a mutant strain belonging to the genus CorYnebacterium or Brevibacterium and defective in the gene encoding for the enzyme involved in the biosynthesis of threonine with the ligation mixture, and selecting the transformants wherein the defective phenotype is restored. The method of recombinant DNA technology is described in Japanese Published Unexamined Patent Application Nos. 186492/82 and 186489/82.
Instead of cloning the recombinant DNA directly in a microoganism belonging to the genus Corynebacterium or Brevibacterium, the recombinant DNA can also be obtained by using another well established host-vector system as exemplified with Escherichia coli system. That is, recombinant DNAs can be obtained by the method which comprises transforming an Escherichia coli mutant which lacks the gene encoding for the enzyme involved in the biosynthesis of threonine with the in vitro ligation mixture of the donor DNA encoding for the enzyme involved in the biosynthesis of threonine and the vector DNA, and selecting transformants wherein the defective phenotype is restored. Subsequently the cloned DNA isolated from the transformants and a vector DNA of the microorganism belonging to the genus Corynebacterium or Brevibacterium are cleaved with a restriction enzyme and religated with T4 ligase. With the ligation mixture, a mutant strain belonging to the genus Corynebacterium or Brevibacterium and defective in the gene encoding for the en2yme involved in the biosynthesis of threonine is transformed, and the transformants, wherein the defective phenotype is restored, are obtained. The desired recombinant DNA is isolated from such transformants.

- 5 - ~28~9 The threonine operon of Escherichia coli K12 is used as a preferable e~ample of the DNA containing the gene involved in the biosynthesis of threonine.
The present invention is explained in more detail usin~
the recombinant plasmid harboring the DNA fragment containing the threonine operon o Escherichia coli, pEthrl.
The DNA fragment containing the threonine operon oE
Escherichia coli can preliminarily be cloned by the cloning system in Escherichia coli. The method of cloning a gene in a host of Escherichia coli is described, for example, in ~ethods in Enzymology, 68, Ray Wu (Ed), Academic Press, New York (1979).
Practical cloning is carried out as follows~
The chromosomal DNA extracted ~rom Escherichia coli having wild-type threonine operon and vector plasmid pGA2~ of Escherichia coli are digested with res~riction en7yme Hlnd III, followed by T4 pha~e ligase treatment. Escherichia coli K12 variant strain GT-3, a mutant strain which is defective in three kinds of aspartate kinase and which re~uires homoserine and diaminopimelic acid is transformed by a conventional method with the ligation mixture to select transformants growing on a minimal medium containing diaminopimelic acid and kanamycin which is a selection marker of pGA22.
The plasmid which the resulting transformant harbors is isolated from the cultured cells of the transEormant by a conventional method. The structure of the plasmid is determine~
by digesting the plasmid with various restriction enzymes and analyzing the resulting DNA fragments by agarose gel electrophoresis. One of the plasmids thus obtained is pGH2 having the structure illustrated in Fig. 1. The ~NA fragment containing the threQnine operon of Escherichia coli has already been cloned and the restriction sites thereo~ ha~-e been determined [re~er to Cossart P et al, Molec. Gen. Genet., 175, 39 (1979)]. pGH2 contains the cloned DNA fragment exhibiting the same restriction sites, an~ is thus confirmed to possess the ~hreonine operon.
The defec' of aspartate kinase Oc GT-3 strain is restored by the aspartate kinase present on the threonine operon of p~H~ and G~-3 strain becomes protoproph ~or homoserine and diaminopimelic acid.

1228iO3~

pEthrl is obtained as a recombinant of pGH2 and pCGll which is a vector plasmid of the genus Coryne-bacterium and srevibacterium.
Plasmid pCGll is a plasmid constructed by the present inventors and described in Japanese Published Unexamined Patent Application No. 134500/82 published Aug. 19, 1982 and Canadian Application S.N. 395,976, filed Feb. 10, 1982. Plasmid pCGll is prepared by inserting the BamHI fragment containing a gene responsi-ble for resistance to streptomycin and/or spectinomycinof plasmid pCG4 isolated from Corynebacterium glutamicum 225-250 (ATCC 31830, FERM P-5939) into the unique BglII
cleavage site of plasmid pCGl isolated from Coryne-bacterium glutamicum 225-57 (ATCC 31808, FERM P-5865) using the same cohesive ends of both fragments.
pCGll is concentrated and isolated from the cultured cells of Corynebacterium glutamicum ATCC 39022 harboring pCGll by the method described in Japanese Published Unexamined Patent Application No. 186492/82 20 published Nov. 16, 1982. By a convantional method, pGH2 is digested with BamHI and pCGll is digested with BglII
and the digests axe mixed and treated with T4 ligase.
Corynebacterium glutamicum L~201, a mutant strain which is defective in homoserine dehydrogenase is transformed with the DNA mixture. Corynebacterium glutamicum LA201 is a mutant which is derived by a conventional mutagene-sis from lysozyme-sensitive mutant strain L-22 derived from Corynebacterium glutamicum ATCC 31833 (~apanese Published Unexamined Patent Application No. 186492/82 30 published Nov. 16, 1982 and which requires homoserine (alternatively, threonine and methionine) and leucine for its growth. The homoserine-requirement depends on the defect of homoserine dehydrogenase gene which acts upon the threonine biosynthesis pathway to metabolize aspartate-semialdehyde into homoserine.

~Z2~039 - 6a -Transformation is carried out by the trans-formation method using protoplasts of the genus Coryne-bacterium or Brevibacterium described in Japanese Published Unexamined Patent Application Nos. 186492/82 and 186489/82 both published Nov. 16, 1982. The practi-cal embodiment of the method is described in the example of the present application. Corynebacterium glutamicum LA201 is transformed by the method using protoplasts and the protoplasts are unselectively regenerated to normal cells on a regeneration ~ 7 ~ 12 Z ~03 9 medium. The regenerated cells are scraped, washed with sterile physiological saline solution and spread on a minimal medium supplemented by leucine to isolate growing colonies.
Some of the thus obtained homoserine non-requiring transformants obtain simultaneously kanamycin resistance phenotype derived from pGH2 and spectinomycin resistance phenotype derived from pCGll. The plasmid DNAs in the transformants are isolated and purified from cultured cells by the same method as in the isolation of pCGll described above.
The structure of the plasmid DNA is determined by analysis of agarose gel electrophoresis after digestion with various restriction enzymes. pEthrl is a plasmid obtained from one of the transformants. The restriction map and the process for producing the plasmid are illustrated in Fig. 1. As is apparent from Fig. 1, pEthrl is a plasmid wherein a fragment containing the threonine operon of pGH2 cleaved with 8amHI is inserted in pCGll.
From another transformant, a plasmid wherein the orientation of the BamHI fragment containing the threonine operon is opposite to that in pEthrl is obtained. When either recombinant plasmid is reintroduced into CorYnebacterium qlutamicum LA201 and transformants are selected with kanamycin or spectinomycin, the transformants restore homoserine requiring property and have the donor plasmids characterized by cleavage pattern for various restriction enzymes.
The capability to restore homoserine requiring property depends on the expression of the homoserine dehydrogenase gene on the threonine operon of Escherichia coli present in the recombinant plasmid. It is known that the threonine operon of Escherichia coli has the genes encoding for aspartate kinase, homoserine dehydrogenase, homoserine kinase and threonine synthase and the genes are transcribed as a single transcription unit l-efer to Theze, J. et al,: J. Bacteriol., 115 990 (1979)] and it is elucidated that the function of the 3S expression of all genes is located on the region between Hind III site at 5.4 Rb and BamHI site at 11.3 Rb on pGH2 illustrated in Fig. 1 ~refer to Cossart, P. et al.: Molec. Gen. Genet., 175, 39 (1979)]. Therefore, it is manifest that pEthrl has the - 8 ~ 1~ X 8 03 9 function of expression of all genes and not only homoserine dehydrogenase gene but also aspartate kinase, homoserine kinase and threonine synthase gene are expressed in CorYnebacterium qlutamicum harboring pEthrl.
An L-isoleucine-producing strain belonging to the genus CorYnebacterium or Brevibacterium and harboring pEthrl is obtained by transforming protoplasts of the genus Corynebacterium or Brevibacterium with pEthrl and selecting for spectinomycin and/or streptomycin resistance by the same method as described above.
The presence of pEthrl in the transformant is detected by isolating the plasmid from the transformant, digesting the plasmid with various restriction enzymes and analyzing DNA
fragments by agarose gel electrophoresis as described above. L-isoleucine producing strains are exemplified by pEthrl-carrying strains of CorYnebacterium qlutamicum R40 (FERM P-7160, FERM BP-455) and Brevibacterium flavum ATCC 14067 (refer tG the example below). CorYnebacterium qlutamicum K40 is an L-isoleucine producing strain derived from CorYnebacterium qlutamicum ATCC
31833 as an S-(2-aminoethyl)-cysteine resistant strain.
CorYnebacterium qlutamicum K40 has been deposited with the Fermentation Research Institute, Agency of Industrial Science and Technology, Ibaraki, Japan together with R41 strain (FERM P-7161, FE~M BP-456) prepared by introducing pEthrl into R40 strain, and Brevibacterium flavum K42 (FERM BP-355) prepared by introducing pEthrl into Brevibacterium flavum ATCC 14067.
Production of L-isoleucine by the transformant harboring pEthrl is carried out by a conventional ~ermentation method used in the production of L-isoleucine.
That is, the transformant is cultured in a conventional medium containing carbon sources, nitrogen sources, inorganic materials, amino acids, vitamins, etc. under aerobic conditions, with adjustment of temperature and pH. Isoleucine, thus accumulated in the medium, is recovered.
As the carbon source, various carbohydrates sucn as glucose, glycerol, fructose, sucrose, maltose, mannose, starch, starch hydrolyzate and molasses, polyalcohols and various organic acids such as pyruvic acid, fumaric acid, lactic acid and acetic acid may be used. Hydrocarbon and alcohols are employed in the strains which can assimilate them. Blackstrap molasses is most preferably 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, NZ-amine, meat extract, yeast extract, corn steep liquor, casein hydrolyzate, fish meal or its digested product, defatted soybean or its digested product and chrysalis hydrolyzate are available.
As the inorganic materials, potassium dihydrogenphosphate, dipotassium hydrogenphosphate, ammonium sulfate, ammonium chloride, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate and calcium carbonate may be used. Vitamins and amino acids required for the growth of microorganisms may not be added, provided that they are supplied with other components mentioned above.
Culturing is carried out under aerobic conditions with shaking or aeration-agitation. Culturing temperature is preferably 20 to 40C. The pH of the medium during culturing is maintained around neutral. Culturing is continued until a considerable amount of L-isoleucine is accumulated, generally for 1 to 5 days.
After completion of the culturing, cells are removed and L-isoleucine is recovered from the culture broth by conventional manners such as treatment with active carbon or ion exchange resin.
Higher amount of L-isoleucine is obtained using the strains of the genus CorYnebacterium and Brevibacterium harboring pEthrl compared with the strains which does not contain pEthrl.
The usefulness of the present invention lies in the fact that, in an expressible form, introduction of the recombinant DNA constructed wlth a gene involved in the biosynthesis of threonine and a vector DNA of the genus Corvnebacterium or Brevibacterium into a microorganism belonging to the genus Corvnebacterium or Brevibacterium can give or improve L-isoleucine productivity. The example of using - lo - ~228039 Escherichia coli threonine operon is provided in the present specification, but the purpose of the present invention is accomplished using a gene involved in the biosynthesis of threonine derived from other organisms. Therefore, the gene involved in the biosynthesis of threonine is not restricted to the threonine operon of Escherichia coli described in the present specification. Further, the vector plasmid merely provides its autonomously replicating ability to stably maintain the recombined gene involved in the biosynthesis of threonine.
Therefore, plasmids autonomously replicable in the genus CorYnebacterium or Brevibacterium other than pCGll described in the present specification are used in the present invention.
In spite of many common microbiological properties, microorganisms with high glutamic acid productivity (so-called glutamic acid-producing microorganisms) are classified to various species and even genera such as Corynebacterium and Brevibacterium probably because of their industrial importance.
However, it has been pointed out that these microorganisms should be classified as one species because they have homology in the amino acids in the cell walls and the CC content of DNA.
Recently, it has been reported that these microorganisms have more than 70% homology in DNA-DNA hybridization, indicating that the microorganisms are very closely related [refer to Komastsu, Y.: Report of the Fermentative Research Institute, No. 55, 1 (1980), and Suzuki, K., Kaneko, T., and Komagata, K.: Int. 3.
Sy~t. Bacteriol., 31, 131 ~1981)].
In the present specification, a case where a gene involved in the biosynthesis of threonine is introduced into CorYnebacterium qlutamicum R40 and Brevibacterium flavum ATCC
14067 and where the improvement in L-isoleucin production depends on the expression of the gene is given. Considering the above-mentioned very close relationship of glutamic acid-producing microorganisms, it is readily assumed that the present invention is applicable to all of the glutamic acid-producing microorganisms. The effect of the present invention depends on whether the recombinant DNA autonomously replicates in the glutamic acid-producing microorganism and whether the gene involved n the biosynthesis of threonine is expressed, and 50 11 - 122~039 slight difference of such DNA homology between glutamic acid-producing microorganisms are negligible. That the glutamic acid-producing microorganims have the common function to allow replication of plasmids and expression of genes is apparent from the fact that plasmid pCG4 which is isolated from CorYnebacterium qlutamicum 225-250 (Japanese Published Unexamined Patent Application No. 183799/82) and which has a spectinomycin and/or streptomycin resistance gene can be generally replicated and expressed in glutamic acid-producing microorganisms such as strains of the genera Corynebacterium and Brevibacterium tJapanese Published Unexamined Patent Application No. 186492/82). Therefore, all of the glutamic acid-producing microorganisms including the genera CorYnebacterium and Brevibacterium such as Corynebacterium qlutamicum K40 and Brevibacterium flavum ATCC 14067 fall within the scope for application of the present invention to the end that L-isoleucine-producing microorganisms are prepared by introducing a recombinant DNA containing the gene involved in the biosynthesis of threonine.
Example 1 (1) Cloning of a DNA fragment containing Escherichia coli threonine operon:
Cloning was carried out using a host-vector system of Escherichia coli. Plasmid pGA22, used as a vector, was isolated from cultured cells of a derivative of Escherichia coli K12 carrying the present plasmid according to the method of An [An, G. et al.: J. Bacteriol., 140, 400 (1979)].
The chromosomal DNA used as a donor DNA was isolated from cultured cells of Escherichia coli K12 Hfr (ATCC 23740) by the phenol-extraction method of Smith, M.G.: Methods in Enzymology, 12, part A, 545 (1967). Then, 0.4 unit of HindIII
(product of Takara Shuzo Co., 16 units/~) was added to 60 ~Q of a HindIII reaction solution (pH 7.5) consisting of 10 mM Tris-HCl, 7 mM MgC12 and 60 mM NaCl and containing 4 ~g of pGA22 plasmid DNA. The mixture was allowed to react at 37C for 30 minutes and heated at 65C for 10 minutes to stop the reaction.
pGA22 plasmid DNA was digested with HindIII under the same - 12 ~ 1 Z 2 8 03 9 conditions and subjected to agarose gel electrophoresis. It was confirmed that one of the two HindIII cleavage sites present in pGA22 was cleaved.
Separately, 4 units of HindIII was added to 140 ~Q of the HindIII reaction solution containing 8 ~g of the chromosomal DNA. The mixture was allowed to react at 37C for 60 minutes and heated at 65C for 10 minutes to stop the reaction.
Then, 40 ~Q of the T4 ligase buffer solution (pH 7.6) consisting of 660 mM Tris (hydroxymethyl) aminomethane (referred to as "Tris" hereinafter), 66 mM MgC12 and 100 mM
dithiothreitol, 40 ~Q of ATP (5 mM, 0.3 ~Q of T4 ligase (product of Takara Shuzo Co., 1 unit~Q) and 120 ~Q of H2O were added to a mixture of the above digests and reaction was carried out at 12C for 16 hours. The reaction mixture was extracted twice with 400 ~Q of phenol saturated with TES buffer solution (pH
8.0) consisting of 0.03M Tris, 0.005M EDTA and 0.05M NaCl and subjected to dialysis against TES buffer solution to remove phenol.
The ligase reaction mixture was used to transform Escherichia coli GT-3 (J. Bacteriol. 117, 133-143, 1974) which is a derivative of Escherichia coli K12 and requires homoserine and diaminopimelic acid. Competent cells of the GT-3 strain were prepared according to the method of Dagert, M. et al., Gene, 6, 23 (1979). That is, the strain was inoculated in 50 mQ
of L-medium (pH 7.2) consisting of 10 g/Q Bacto-tryptone, 5 g/Q
yeast extract, 1 g/Q glucose and 5 g/Q sodium chloride and containing 100 ~g/mQ diaminopimelic acid and cultured at 37C to an optical density ~OD) value at 660 nm of 0.5. The culture was cooled with ice water for 10 minutes and cells were recovered by centrifugation. The cells were suspended in 20 mQ of cooled O.LM calcium chloride. The suspension was allowed to stand at 0C foc 20 minutes and then centrifuged to recover the cells.
The cells were suspended in 0.S mQ of 0.lM calcium chloride and allowed to stand at 0C for 18 hours.
Then 200 ~Q of the ligase reaction mixture mentioned above was added to 400 ~Q of the cell suspension treated with calcium chloride. The mixture was allowed to stand at 0C for 10 minutes and then heated at 37C for 5 minutes. Thereafter, - 13 - ~X28039 9 mQ of the L-medium was added and the mixture was incubated with shaking at 37C for 2 hours. Cells were recovered by centrifugation and washed with a physiological saline solution twice. The cells were spread on Mg minimal agar medium (pH 7.2) consisting of 2 g/Q glucose, 1 g/Q NH4Cl, 6 g/Q Na2HPO4, 3 g/Q
KH2PO4, 0-1 g/Q MgSO4 7H20, 15 mg/Q CaC12-2H2O, 4 mg/Q thiamine hydrochloride and 15 g/Q agar and containing 12.5 ~g/mQ
kanamycin. Culturing was carried out at 37C for 3 days. Only one colony was formed and the cells from this colony could also grow on an L-agar medium containing 25 ~g/mQ ampicillin, 25 ~g/mQ chloramphenicol or 25 ~g/mQ ~anamycin, which is a selection marker of pGA22.
A plasmid DNA was isolated from cultured cells of the transformant by the same method as in the isolation of pGA22.
The plasmid DNA was digested with restriction endonucleases and analyzed by agarose gel electrophoresis. The plasmid DNA had the structure illustrated as pGH2 in Fig. 1. Since the DNA
fragment inserted in pGA22 had the same cleavage sites for restriction endonucleases as the cloned DNA fragment containing Escherichia coli operon (Cossart, P. et al.: Molec. Gen.
Genet., 175, 39, 1979), it is confirmed that pGH2 had the threonine operon.

(2) In vitro recombination of pCGll and pGH2 CorYnebacterium qlutamicum LA103/pCGll harboring pCGll ~ATCC 39022) were cultured to an OD value of 0.8 in a 400 mA of NB medium (pH 7.2) consisting of 20 g/Q powdered bouillon and 5 g/Q yeast extract. Cells were harvested from the culture broth, and washed with TES buffer solution. The cells were suspended in 10 mQ of a lysozyme solution (pH 8.0) consisting of 25% sucrose, 0.~ NaCl, 0.05M Tris and 0.8 mg/mQ lysozyme, and incubated at 37C for 4 hours. Then, 2.4 mQ of 5M NaCl, 0.6 mQ
of 0.5M EDTA (pH 8.5) and 4.4 mQ of a solution consisting of 4 sodium lauryl sulfate and 0.7M NaCl were added successively.
The mixture was stirred slowly and allowed to stand on an ice water bath for 15 hours. The whole lysate was centrifuged at 4C under 69,400 x g for 60 minutes. The supernatant fluid was recovered and 10~ (by weight) polyethyleneglycol (PEG) 6,000 (product of Nakarai Kagaku Yakuhin Co.) was added. The mixture was stirred slowly to dissolve completely and then kept on an ice water bath. After 10 hours, the mixture was subjected to centrifugation at 1,500 x g for 10 minutes to recover a pellet.
The pellet was redissolved gently in 5 mQ of TES buffer and 2.0 mQ of 1.5 mg/mQ ethidium bromide was added.
Then, cesium chloride was added to adjust the density of the mixture to 1.580. The solution was centrifuged at 18C
at 105,000 x g for 48 hours. After the density gradient centrifugation, a convalently closed circular DNA was detected under W irradiation as a high density band located in the lower part of the centrifugation tube. The band was taken out from the side of the tube with an injector to obtain a fraction containing pCGll DNA. To remove ethidium bromide, the fraction was treated five times with an equal amount of cesium chloride saturated isopropyl alcohol solution consisting of 90% by volume isopropyl alcohol and 10~ TES buffer solution. The residue was dialysed against TES buffer solution. Thus, a plasmid pCG11, was obtained.
The plasmid pCGll was digested with restriction endonucleases and analysed by agarose gel electrophoresis to determine the molecular weight and clea~age sites for restriction endonucleases. Fig. 1 illustrates the cleavage map for various restriction endonucleases of plasmid pCGll.
2 units of BglII (product of Takara Shuzo Co., 6 units/~Q) was added to 100 yQ of the BglII reaction buffer solution (pH 7.5) consisting of 10 mM Tris-HCl, 7 mM MgC12, 60 mM NaCl and 7 mM 2-mercaptoethanol and containing 2 ~g of pCGll plasmid DNA. The mixture was allowed to react at 37C for 60 minutes. Separately, 2 units of BamHI (product of Takara Shuzo Co., 6 units/yQ) was added to 100 ~Q of BamHI reaction buffer solution (pH 8.0) consisting of 10 mM Tris-MCl, 7 mM
MgC12, 100 mM NaCl, 2 mM mercaptoethanol and 0.01% bovine serum albumin and containing 2 ~g of pGH2 plasmid DNA. The mixture was allowed to react at 37C for 60 minutes. Both digests were heated at 65C for 10 minutes, and mixed. Then, 40 ~Q of T4 ligase buffer solution, 40 yQ of ATP (5 mM), 0.2 yQ of T4 ligase and 120 ~Q of H2O were added to the whole mixture of both the 1;~28039 digests. Reaction was carried out at 12C for 16 hours. The reaction mixture was extracted twice with 400 ~Q of phenol saturated with TES buffer solution and subjected to dialysis against TES buffer solution to remove phenol.

(3) Recovery of plasmid pEthrl Protoplasts of CorYnebacterium qlutamicum LA201 which requires homoserine and leucine were used for transformation. A
seed culture of Corynebacterium qlutamicum LA201 was inoculated in the NB medium and cultured with shaking at 30C. Cultured cells were collected at an OD value of 0.6 and suspended in an RCGP medium (pH 7.6) containing 1 mg/mQ lysozyme at a concentration of about 109 cells/mQ. The RCGP medium consists 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 MgC12 6H2O, 10 mg/Q
FeSO4 7H2O, 2 mg/Q MnSO4 (4-6) H2O, 0.9 mg/Q ZnSO4 7H2O, 0.04 mg/Q (NH4)6Mo7O24 4H2O, 30 ~g/Q biotin, 2 mg/Q thiamine hydrochloride, 135 g/Q sodium succinate and 30 g/Q
polyvinylpyrrolidone of a molecular weight of 10,000. The suspension was put into an L-tube and allowed to react with gentle shaking at 30C for 5 hours to make protoplasts.
Then, 0.5 mQ of the protoplast suspension was transferred into a small tube and centrifuged at 2,500 x g for 5 minutes to obtain pellets. The pellets was resuspended in 1 mQ
of a TSMC buffer solution (pH 7.5) consisting of 10 mM magnesium chloride, 30 mM calcium chloride, 50 mM Tris and 400 mM sucrose and centrifuged. The protoplasts were resuspended in 0.1 mQ of the TSMC buffer solution. Then, 100 ~Q of a mixture of a two-fold concentrated TSMC buffer solution and the above-described DNA mixture treated with ligase (1:1) was added to the suspension and 0.8 mQ of a TSMC buffer solution containing 20%
PEG 6,000 was added. After 3 minutes, 2 mQ of the RCGP medium (pH 7.2) was a~ded and the mixture was centrifuged at 2,500 x g for 5 minutes. The supernatant fluid was removed and the precipîtated protoplasts were suspended in 1 mQ of the RCGP
medium. The suspension was slowly shaken at 30C for 2 hours.
Then, 0.1 mQ of the suspension was spread on RCGP agar medium (pH 7.2), the RCGP medium supplemented b~y 1.4% agar, - 16 ~ 1 2 2 8 O~ 9 containing 400 ~g/mQ kanamycin, and culturing was carried out at 30C for 6 days to regenerate the transformants resistant to kanamycin. Cells grown over the whole surface of the agar medium were scraped, washed with physiological saline solution and subjected to centrifugation. The cells were spread on a minimal agar medium Ml (pH 7.2) consisting of 10 g/Q glucose, l g/Q NH4H2PO4, 0.2 g/Q KCl, 0.2 g/Q ~gSO4 7H2O, 10 mg/Q FeSO4 7H20, 0.2 mg/Q MnSO4 (4-6)H20, 0.9 mg/Q ZnSO4 7H2O, 0.4 mg/Q
CuSO4-5H2O, 0.09 mg/Q Na2B4O7 10H2O, 0.04 mg/Q
(NH4) 6Mo7O24 4H2O, 50 ~g/Q biotin, 2.5 mg/Q p-amino-benzoic acid, l mg/Q thiamine hydrochloride and 16 g/Q agar and containing 50 ~g/mQ leucine. Culturing was carried out at 30C
for 3 days. Among colonies developed, those able to grow on the NB agar medium containing 20 ~g/mQ kanamycin and 100 ~g/mQ
15 spectinomycin were obtained. Three strains selected at random were grown in 400 m~ of the NB medium to an OD value of about 0.8. Cells were recovered and the plasmids were isolated from the cells by ethidium bromide-cesium chloride density gradient centrifugation described in Example l (2) whereby 40 to 55 ~g of 20 plasmid DNA were recovered from each strain.
These plasmid DNAs were digested with restriction endonucleases and analyzed by agarose gel electrophoresis to determine the molecular weights and cleavage sites for PstI, EcoRI and XhoI. The plasmid obtained from one strain is named pEthrl and the structure is illustrated in Fig. l. It was confirmed that pEthrl has the structure wherein a BamHI fragment containing pGH2 threonine operon is inserted into pCGll at its BglII site. One of the remaining strains has the same plasmid as pEthrl and the other has a plasmid wherein the BamHI fragment containing pGH2 threonine operon is combined at the opposite orientation.
Corynebacterium qlutamicum LA201 strain was again transformed with these plasmid DNAs as mentioned above. As a result, strains which do not require homoserine were obtained at 35 high frequency, about 10-3 cell/regenerated cell. All of them are endowed with the phenotypes of the resistance to kanamycin and spectinomycin and have the same plasmid as the donor plasmid characterized by the cleavage pattern for various restriction endonucleases.

(4) Production of L-isoleucine by the pEthrl-carrying strain:
Corynebacterium qlutamicum ~40 and Brevibacterium flavum ATCC 14067 were transformed with pEthrl. The thus obtained transformants were cultured with shaking in NB medium at 30C for 16 hours, and 0.1 mQ of the seed culture was inoculated into 10 mQ of SSM medium (pH 7.2) comprising 10 g/Q
glucose, 4 g/Q NH4Cl, 2 g/Q urea, 1 g/Q yeast extract, 1 g/Q
KH2PO4~ 3 g/Q K2HPO4, 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 5H20, 0.09 mg/Q Na2B4O7-10H2O~ 0.04 mg/Q (NH4)6Mo7O24-4H2O, 30 ug/Q biotin and 1 mg/Q thiamine hydrochloride in an L-tube.
Culturing was carried out at 30C in a Monod-type culture bath, and penicillin G was added at an OD value of 0.15 to a concentration of 0.5 unit/mQ. Culturing was continued to an OD
value of about 0.6. Cells were harvested and suspended in 2 mQ
of RCGP medium (pH 7.6) containing 1 mg/mQ lysozyme. The suspension was put in an L-tube and stirred slowly at 30C for 14 hours to obtain protoplasts.
Then, 1 mQ of the protoplast suspension was put in a small test tube and centrifuged at 2,500 x g for 15 minutes.
The protoplasts were resuspended in 1 mQ of TSMC buffer and centrifuged at 2,500 x g. 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-fold concentrated TSMC buffer and the pEthrl DNA described above was added to the protoplast suspension. Transformation was carried out using PEG 6,000 by the same method described in Example 1 ~3) for expression of the desired gene. Then, ~ Q of the mixture was spread on RCGP agar medium containing 400 ~g/mQ
spectinomycin and incubated at 30C for 10 days. From the colonies developed, the transformants which grow on N8 agar medium containing 100 ug/mQ spectinomycin and 20 ug/mQ kanamycin were obtained. The strains resistant to spectino~ycin and kanamycin were cultured with shaking in 400 mQ of SSM medium, and penicillin G was added at an OD value of 0.15 to a concentration of 0.5 unit/mQ. Culturing was continued to an CD
value of 0.55, and cells were harvested. From the cells, plasmids were isolated by the same method as the isolation method of pCGll in Example 1 (2). These plasmids were digested with restriction endonucleases and analyzed by agarose gel electrophoresis. The analysis showed that some of the plasmids have the same physical structure as pEthrl characterized by the cleavage pattern for various restriction endonucleases. Such transformants are CorYnebacterium qlutamicum K41 (FERM P-7161) ~FERM BP-456) and Brevibacterium flavum K42 (FERM BP-355).
Corynebacterium qlutamicum K40, Brevibacterium flavum ATCC 14067 and their pEthrl-carrying strains were tested for L-isoleucine production as follows.
The strain was cultured in NB medium at 30C ror 16 hours and 0.5 mQ of the culture liquor was inoculated in a production medium adjusted to pH 7.2 consisting of 100 g/Q
glucose, 20 g/Q (NH4)2SO4, 0.5 g/Q KH2PO4, 0.5 g/Q K2HPO4, 1 g/Q
MgSO4 7H20, la mg/Q FeSO4 7H2O, 10 mg/Q MnSO4 (4-6)H2O, 100 ~g/Q biotin and 30 g/Q CaCO3. Culturing was carried out with shaking at 30C for 72 hours. The culture filtrate was subjected to paper chromatography, color reaction with ninhydrin and the amount of L-isoleucine formed was determined colorimetrically. The results are shown in Table 1.

Table 1 Amount of L-isoleucine Strain (mq/mQ) CorYnebacteriu~ qlutamicum K40 1.2 CorYnebacterlum qlutamicum K41 2.7 Brevibacterium flavum ATCC 14067 0 Brevibacterium flavum K42 0.8

Claims (4)

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

1. A process for producing L-isoleucine which comprises transforming a host microorganism belonging to the genus Corynebacterium or Brevibacterium with a re-combinant DNA of a DNA fragment containing a bacterial gene encoding for the enzyme involved in the bio-synthesis of threonine from aspartic acid and a vector DNA, culturing the transformant in a nutrient medium, accumulating L-isoleucine in the culture medium and re-covering L-isoleucine therefrom.

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

3. The process according to Claim 1, wherein the vector is a vector derived from a microorganism belonging to the genus Corynebacterium or Brevibacterium or a derivative thereof.

4. The process according to Claim 1, wherein the vector is selected from pCG1, pCG2, pCG4, pCG11, pCE54 and pCB101.