GB2103617A - Production of L-lysine by fermentation and new microorganisms obtained by protoplast fusion - Google Patents

Abstract

L-lysine is produced in improved yield by culturing microorganisms having the ability to produce L-lysine in a culture medium, the microorganisms being obtained by protoplast fusion. Novel microorganisms of the genus Corynebacterium and Brevibacterium are described.

Description

SPECIFICATION Process for the production of L-lysine by fermentation and microorganisms for use therein This invention relates to novel microorganisms having an ability to produce L-lysine, and to processes for producing L-lysine by fermentation using said microorganisms.

Heretofore, as processes for producing L-lysine by fermantation, there have been known processes of using strains having a nutritional requirement for various compounds, strains having sensitivity to various chemicals, or various chemicals-resistant strains, belonging to the genus Corynebacterium, Brevibacterium, Arthrobacter, Pseudomonas, Bacillus, Nocardia, Saccharomyces, Escherichia, Klebsiella, Streptomyces, Alkaligenes, Microbacterium, Acromobacter and Serratia, and processes of using mutants having a combination of the properties of the above-described microorganisms.

As a result of various studies for obtaining strains having an increased L-lysine productivity, the present inventors have found that microorganism strains belonging to the genus Corynebacterium or Brevibacterium capable of producing L-lysine and which are endowed with either a resistance to two or more antibiotics or a resistance to at least one purine analog and/or at least one pyrimidine analog have a remarkably improved ability to produce L-lysine.

In addition, the inventors have found that a strain capable of producing L-lysine in high yield can be obtained by utilizing the technology of cell fusion using protoplast to obtain recombinant strain. This process enable us to effectively obtain double mutants capable of producing L-lysine in high yield, and is an effective process which enables us to obtain mutants possessing merits of well selected parent strains.Accordingly, in one principle aspect, the invention provides a process for the production of L-lysine, which comprises culturing a microorganism capable of producing L-lysine in a culture medium, allowing the L-lysine to accumulate in the medium, and recovering the product L-lysine, wherein there is used as the microorganism, i) a mutant strain belonging to the genus Corynebacterium or Brevibacterium and which has a resistance to two or more antibiotics; ii) a mutant strain belonging to the genus Corynebacterium or Brevibacterium and which has a resistance to at least one purine analog and/or at least one pyrimidine analog; or iii) a L-lysine producing microorganism strain obtained by protoplast fusion.

The present invention will be described in more detail below.

As the microorganisms of the present invention, there are illustrated L-lysine-producing strains obtained by protoplast fusion between parent strains having different properties, and L-lysine-producing strains obtained by the technology of protoplast fusion or by conventional mutation inducing treatment or spontaneous mutation having a resistance to antibiotics of two or above, or a resistance to purine analog or pyrimidine analog.

As the strain of the present invention having both an ability to produce L-lysine and a resistance to antibiotics of two or above, there may be used a strain capable of producing L-lysine belonging to the genus Corynebacterium or Brevibacterium which is endowed with a resistance to antibiotics of two or above, or a strain having a resistance to antibiotics of two or above and belonging to the genus Corynebacterium or Brevibacterium which is endowed with an ability to produce L-lysine.

As the strain of the present invention having both an ability to produce L-lysine and a resistance to at least one of purine analog and pyrimidine analog, there may be used a strain belonging to the genus Corynebacterium or Brevibacterium and having an ability to produce L-lysine which is endowed with a resistance to at least one of purine analog and pyrimidine analog, or a strain belonging to the genus Corynebacterium or Brevibacterium and having a resistance to at least one of purine analog and pyrimidine analog which is endowed with an ability to produce L-lysine.

As the strain belonging to the genus Corynebacterium or Brevibacterium and having an ability to produce L-lysine, strains capable of producing L-lysine having one or a combination of a requirement for nutrients (for example, homoserine, methionine,threonine, histidine, proline, alanine, leucine, isoleucine, valine, serine, glutamic acid, pantothenic acid, nicotinic acid amide, acetic acid, adenine, hypoxanthine, inositol, and their combinations), a resistance to various amino acid analogs (for example, analogs of lysine, threonine, methionine, leucine, isoleucine, valine, aspartic acid, tryptophane, histidine, and their combinations), and a resistance to other chemicals (for example, sulfa drugs, penicillin type antibiotics, various organic acids, quinone compounds, quinoline compounds, and their combinations, etc.) may be mentioned.

Accordingly, a strain to be used in the present invention may be obtained by endowing such a strain capable of producing L-lysine as mentioned above with a property of resistance to antibiotics of two or above or a resistance to at least one of purine analog and pyrimidine analog. In addition, a strain capable of producing L-lysine obtained by endowing a strain belonging to the genus Corynebacterium or Brevibacterium and having a property of resistance to at least one of purine analog and pyrimidine analog or to antibiotics of two or above with various nutrients requirement, a resistance to various amino acid analogs or a resistance to other chemicals as mentioned above may also be used in the present invention. Further, the strain to be used in the present invention may have any other property of contributing to L-lysine productivity than the properties mentioned above.

As a resistance to antibiotics of two or above, a resistance to two or more of antibiotics such as penicillins, cephalosporins, streptomycin, dihydrostreptomycin, rifampicin, chloramphenicol, tetracyclines, spiramycin, erythromycin, kanamycin, kasugamycin, mitomycin C, actinomycin D, polymixin, colistin, lincomycin, gentamicin, sagamicin, fortimicin, oleandomycin, etc. is mentioned.

As a resistance to purine analog, for example, a resistance to 6-mercaptoguanine, 8-azaguanine, 2-fluoroadenine, tubercidin, 6-methylpurine, 8-azaxanthine, 8-azaadenine, 8-mercaptoguanosine, 6 mercaptoguanosine, 2-aminopurine, 2-amino-6-mercaptopurine, decoyinin, psicofuranine, etc. is mentioned and, as a resistance to pyrimidine analog, for example, a resistance to 5-bromouracil, 6-azauracil, 5-fluorouracil, 5-bromo-2-deoxyuridine, 2-thiouracil, 6-methyl-2-thiouracil, amicetin, etc. is mentioned.

Mutants useful in carrying out the present invention are derived from parent strains belonging to the genus Corynebacterium or Brevibacterium known as a glutamic acid-producing strains, such as Corynebacterium glutamicum ATCC 13032, Corynebacterium acetoacidophilum ATCC 13870, Brevibacterium lactofermentum ATCC 13869, Brevibacterium flavum ATCC 14067, etc.

Of the microorganisms to be used in the present invention, specific examples of a strain having a resistance to antibiotics of two or above are Corynebacterium glutamicum H-3127 (hereinafter referred to as H-3127) (FERM BP-153) and Brevibacterium lactofermentum H-3125 (hereinafter referred to as H-3125) (FERM BP-1 51). Specific examples of a strain having a resistance to purine analog are Corynebacterium glutamicum H-3107 (hereinafter referred to as H-3107) (FERM BP-144), Brevibacterium lactofermentum H-31 14 (hereinafter referred to as H-31 14). (FERM BP-146), and specific examples of a strain having a resistance to pyrimidine analog are Corynebacterium glutamicum H-3106 (hereinafter referred to as H-3106) (FERM BP-143) and Brevibacterium lactofermentum H-3113 (hereinafter referred to as H-3113) (FERM BP-1 45).

Specific examples of a strain having an ability to produce L-lysine obtained by the technology of protoplast fusion are a protoplastfusion strain H-3057 (hereinafter referred to as H-3057) (FERM BP-148) between Corynebacterium glutamicum H-3122 (hereinafter referred to as H-3122) (FERM BP-150) and Brevibacterium H-3126 (hereinafter referred to as H-3126) (FERM BP-152), and a protoplast fusion strain H-3055 (hereinafter referred to as H-3055) (FERM BP-147) between H-3122 and Corynebacteriumglutamicum H-3119 (hereinafter referred to as H-3119) (FERM BP-149). Further, there is mentioned a 6-azauracil-resistant strain H-1349 (hereinafter referred to as H-3149) (FERM BP-158) derived from H-3055.

An example of obtaining a protoplast fusion strain of the present invention is described below.

Protoplasts are formed from culture cells as follows. A strain is inoculated in a nutrient medium NB containing 20 g of bouillon powder and 5 g of yeast extract in 1 f of pure water and adjusted to pH 7.2, and cultured with shaking. Absorbance (OD) at 660 nm is measured by means of a turbidimeter and in the initial stage (OD = 0.1 to 0.2) of logarithmic growth phase, penicillin G is added to the culture liquor to make a final concentration of 0.1 to 0.8 u/ml.Culturing is further continued and when OD reaches 0.3 to 0.5, cells are collected and washed with SSM medium (containing 10 g of glucose, 4 g of NH4Cl, 2 g of urea, 1 g of yeast extract, 1 g of KH2PO4, 3 g of K2HPO4, 0.4 g of MgC12-6H2O,10 mg of FeSO4-7H2O,0-2 mg of MnSO44-6H2O, 0.9 mg of ZnSO4-7HzO,0.4 mg of CuSO45H2O, 0.09 mg of Na2B407-10H2O,0.04 mg of (NH4)6Mo7024-4H2O,30 ug of biotin and 1 mg of thiamine hydrochloride in 1 i of pure water and adjusted to pH 7.2; in culturing an amino acid-requiring strain, 50 g/ml of a required amino acid is further added to SSH medium).Then, the cells are again suspended in PFM medium (containing 0.4 M sucrose and 0.01 M MgCI2-6H2O in a 2-fold diluted SSM solution and adjusted to pH 7.0 to 8.5). Lysozyme (0.2 to 2 mg/ml) is added to the cell suspension to keep at 30 to 37"C for 16 hours. Formation of protoplast is confirmed under an optical microscope. (The thus prepared protoplasts of two strains to be fused are counted under an optical microscope and the suspensions are mixed in a protoplast ratio of 1 : 1, The protoplasts are separated from the mixture by centrifugation and after washing the separated protoplasts with PFM medium, the protoplasts are again suspended in 0.1 ml of PFM medium. To the suspension is added 2.5 ml of PFM medium containing 40 % polyethylene glycol (PEG) 4000, and slowly stirred for 5 minutes.In case where a streptomycin-resistant strain and a rifampicin-resistant strain are used, 0.3 ml of the solution is smeared on RCG agar plate containing 100 g/ml of streptomycin and 0.05,ug/ml of rifampicin (containing 5 g of glucose, 5 g of casamino acid, 2.5 g of yeast extract, 3.5 g of K2HPO4, 1.5 g of KH2PO4, 0.41 g of MgCl26HCl, 10 10 mg of FeSO4-7H2O,2 mg of MnSO4-4-6H2O,0.9 mg of ZnSO47H2O, 0.4 mg of CuSO4-5H2O,0.09 mg of Na2B407-10H2O,0.04 mg of (NH4)6Mo7O244H2O, 30 g of biotin, 2 mg of thiamine hydrochloride, 135 g of sodium succinate and 14 g of agar in 1 e of pure water, and adjusted to pH 7.4).After culturing at 300C for 12 days, colonies of a strain having a resistance to both streptomycin and rifampicin are obtained.

Any of synthetic medium and natural medium may be used as the medium for the present invention, so long as it properly contains a carbon source, inorganic materials and other necessary nitrients which are assimilable by the strain utilized.

As the carbon source, various carbohydrates such as glucose, fructose, sorbitol, glycerol, sucrose, starch, starch hydrolyzate, molasses, fruit juice, etc., organic acids such as acetic acid, fumaric acid, lactic acid, etc., and alcohols such as ethanol, methanol, etc. may be used.

As the nitrogen source, ammonia, inorganic and organic ammonium salts such as ammonium chloride, ammonium sulfate, ammonium acetate, ammonium phosphate, etc., urea, amines, other nitrogencontaining compounds and peptone, meat extract, yeast extract, corn steep liquor, casein hydrolyzate, soybean meal acid hydrolyzate, various microbial cells, digest of microbial cells, etc. may be used.

As the inorganic materials, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate, etc. are used. When a microorganism to be used in the present invention requires specific nutrients for growth, an appropriate amount of the nutrients are added to the medium. In some cases, these nutrients are added as components of the natural substances exemplified as the nitrogen source Further, the productivity of L-lysine by the present microorganism can be in some cases enhanced by adding other various additives, for example, various antibiotics, a-aminobutyric acid, cysteine, leucine, leucine fermentation liquor, aspartic acid, glutamic acid, etc. to the medium.

Culturing is carried out under aerobic conditions, for example, by shaking culture, agitation submerged culture, etc. The temperature for culturing is generally 20 - 40"C. The pH of the medium is in a range of 3 to 9, and is preferably maintained at around neutral, but culturing can be carried out under conditions which are out of this range so long as the microorganism used can grow. The pH of the medium is adjusted with calcium carbonate, acid or alkali solution, ammonia, pH-buffering agent, etc. Usually after culturing for 1 to 6 days, L-lysine is formed and accumulated in the resulting culture liquor.

After the completion of culturing, precipitates such as cells are removed from the culture liquor, and L-lysine can be recovered from the culture liquor by use of the conventional methods such as ion exchange resin treatment, concentration, adsorption, salting-out in combination.

Practice of specific embodiments of the invention is illustrated by the following representative examples.

EXAMPLE 1 {Preparation of 127 and H-3 125) Corynebacterium glutamicum FERM P-3634 (NRRL-B-8183) (hereinafter referred to as P-3634) (having a requirement for homoserine and leucine and a resistance to thialysine and suifamethazine) having been in advance cultured overnight in a bouillon medium is directly smeared on a bouillon agar plate medium containing 200 ug/mI of dihydrostreptomycin. After maintaining the medium at 30"C for 2 to 10 days, a spontaneous mutant strain is selected from the colonies growing, and the mutant cells are suspended in a 0.1 N tris-maleate buffer solution (pH 6.0) in a concentration of 108 cells/ml. To the suspension is added N-methyl-N'-nitro-N '-nitrosoquanidine to make a final concentration of 0.2 mg/ml.After allowing the suspension to stand for 30 minutes at room temperature, the suspension is smeared on a bouillon agar plate medium containing 1 unit/ml of penicillin G. H-3127 is obtained as the mutants selected in the colonies growing. H-3127 is clearly discriminated from the parent strain P-3634 in that the former possesses a resistance to both dihydrostreptomycin and penicillin Gas shown in Table 1. Also, a dihydrostreptomycinresistant strain obtained from Brevibacterium lactofermentum H-3056 (FERM BP-154) (hereinafter referred to as H-3056) having an ability to produce lysine (having a resistance to thialysine and a requirement for leucine and partial requirement for homoserine) is subjected to the same spontaneous mutation as described above.

H-3125 is obtained as a mutant strain selected in the colonies growing on a bouillon agar plate medium containing 50 ijg/mI of rifampicin. H-3125 is clearly discriminated from the parent strain (H-3056) in that the former has a resistance to both dihydrostreptomycin and rifampicin as shown in Table 1.

TABLE 1 P-3634 H-3127 H-3056 H-3 125 Penicillin G 0.5 u/ml - ++ 1.0 u/ml - + - Dihydrostre- 100 Fg/ml - ++ - ++ ptomycin 200sFlg/ml - ++ - ++ Rifampicin 251lg/ml - - ++ 50 Fg/ml - - - ++ Nothing added ++ ++ ++ ++ (++: sufficient growth; +: growth to some extent; -: no growth) EXAMPLE 2 {Preparation of H-3 107, H-3 106, H-3289, H-3290, H-3 114, H-3 113 and H-329 1) Cells of P-3634 are suspended in a 0.1 N trismaleate buffer solution (pH 6.0) in a concentration of 108 cells/ml. To the suspension is added N-methyl-N'-nitro-N'-nitrosoguanidine to make a final concentration of 0.2 mg/ml.After allowing the suspension to stand for 30 minutes at room temperature, the suspension is smeared on a flat agar plate of minimal medium of the following composition containing 0.4 mg/ml of 6-mercaptoguanosine or on a flat agar plate of minimal medium of the similar composition containing 0.4 mg/ml of 6-azauracil, and then selecting mutants in growing colonies. The two strains (H-3107 and H-3106) are clearly discriminated from the parent strain (P-3634) in that the two possess a resistance to 6-mercaptoguanosine and to 6-azauracil, respectively, as shown in Table 2.

The strains H-3289 and H-3290 are selected as mutants having a resistance to 5-bromouracil and to 6-methylpurine, respectively, from P-3634, and the strains H-3114, H-3113 and H-3291 are selected as mutants having a resistance to 6-mercaptoguanosine, 6-azauracil and to 5-bromouracil from Brevibacterium lactofermentum H-3056 (hereinafter referred to as H-3056) (FERM BP-154) (having a resistance to thialysine and a requirement for leucine and partial requirement for homoserine) in the same mutation treatment as described above. The strains H-3289 and H-3290 are clearly discriminated from the parent strain (P-3634) and also the strains H-3114, H-3113 and H-3291 are clearly discriminated from the parent strain (H-3056), as shown Table 2.

TABLE 2 (++: sufficient growth; +: growth to some extent; -: no growth) P- H- H- H- H- H- H- H- H 3634 3107 3106 3289 3290 3056 3114 3113 3291 Nothing added ++ ++ ++ ++ ++ ++ ++ ++ ++ 6-Mercaptoguanosine 100 g/ml - + - - ++ - 500 Fg/ml - + - - ++ 6-Azauracil 100 g/ml - - ++ - - ++ 5001lg/ml - - + - - + 6-Methylpurine 100 g/ml - ++ 400 g/ml - + 5-Bromouracil 1000 g/ml ++ ++ - - ++ 2000 Fg/ml - + - - + Compositions of the flat agar plate of minimal medium is as follows:: 10 9/4 of glucose, 4 g/l of ammonium chloride, 1 gik of KH2PO4, 3 g/ of K2HPO4, 0.4 g/l of MgSO4.7H2O, 0.01 g/ of FeSO47H2O, 0.01 g/l of MnSO44H2O, 2 g/l of urea, 50 g/l of biotin and 20 g/l of agar. pH: 7.2.

EXAMPLE 3 (Preparation of H-3057) H-3122 having an ability to produce L-lysine (having a requirement for leucine and partial requirement for homoserine and a resistance to thialysine, sulfamethazine and rifampicin) derived from P-3634 and H-3126 (having a resistance to thialysine and streptomycin) derived from Brevibacterium lactofermentum FERM P-1837 (ATCC 21888) having an ability to produce L-leucine are prepared, and protoplastfusion is conducted in the same manner as described hereinbefore to obtain a number of protoplast fusion strains having a resistance to both rifampicin and streptomycin. With respect to the parent strains H-3122 and H-31 26, the appearance frequency of the spontaneous mutant strains having a resistance to both rifampicin and streptomycin are 1.3 x 10-8 and 6.5 x 10-9, respectively, whereas that by the technology of protoplast fusion is 1.1 x 10-7. Accordingly, protoplast fusion endows the parent strains with the double resistance at least 10 times as much as spontaneous mutation. Thus, it is confirmed that strains having a resistance to both rifampicin and streptomycin appear with high frequency by the technology of the protoplast fusion.

Various properties of H-3057, selected as a typical example of the protoplast fusion strain, are shown in Table 3 in comparison with those of parent strains. As clear from the table, H-3057 has the same properties as H-3126 with respect to sensitivity to sulfamethazine and resistance to streptomycin, and has the same properties as H-3122 with respect to a requirement for leucine, sensitivity to 2-thienylalanine, resistance to rifampicin, ability to reduce NO3, and productivity of L-lysine. Such strain H-3057 cannot be obtained as a result of spontaneous mutation by single treatment, and hence H-3057 is a typical protoplast fusion strain.

TABLE 3 Sulfa metha- Reduction Produc S-Lys zine 2TA Leu STM RIF ofN03 tivity H-3122 R R S necessary S R ++ L-Lys not H-3126 R S R necessary R S + L-Leu H-3057 R S S necessary R R ++ L-Lys R :resistant S : sensitive S-Lys : S-(2-aminomethyl)-cysteine; (thialysine) 2TA : 2-thienylalanine Leu : leucine STM : streptomycin RIF : rifampicin EXAMPLE 4 (Preparation of H-3055) H-3122 and H-31 19 (having partial requirement for homoserine, no requirement for leucine and a resistance to thialysine, 2-thienylalanine, a-amino-ss-hydroxybutyric acid and streptomycin) derived from Corynebacterium glutamicum ATCC 21543 are subjected to the protoplast fusion treatment as described to obtain a strain having a resistance to both streptomycin and rifampicin.L-lysine productivity and various properties of this strain are examined. Appearance frequency of strains acquiring a resistance to both streptomycin and rifampicin as a result of spontaneous mutation from H-3122 and H-3119 are 1.0 x 10-8 and 2.0 x 10-8, respectively. On the other hand, by the above-described protoplast fusion, appearance frequency of strains acquiring the double resistance is 4 x 10-7, which is about 20 to 40 times as high as that by spontaneous mutation. Thus, it is confirmed that strains having a resistance to both rifampicin and streptomycin appear with high frequency. As a strain having a remarkably improved L-lysine productivity, there is illustrated, for example, H-3055. Various properties of this strain H-3055 are shown in Table 4 in comparison with those of parent strains.

H-3055 has the same non-requirement for leucine, and the same sensitivity to sulfamethazine and streptomycin as H-3119, and has the same sensitivity to 2-thienylalanine and the same resistance to rifampicin as H-3122. With respect to growth and productivity of L-lysine, H-3055 is more improved than the parent strains.

TABLE 4 Sulfa- Growth Produc metha- without with tivity S-Lys Leu zine 2TA STM RIF Leu Leu ofLys* nece H-3122 R ssary R S S R - ++ 36 % not nece H-3119 R ssary S R R S + + 25 H-3055 R " S S R R ++ +++ 38 R : resistant S: sensitive * yield based on sucrose (under optimal conditions) (0.5 g/e of L-leucine is added for H-3122) EXAMPLE 5 {Preparation of H-3 149) Cells of H-3055 are suspended in a 0.1 N trismaleate buffer solution (pH 6.0) in a concentration of 108 cells/ml. To the suspension is added N-methyl-N'-nitro-N'-nitrosoguanidine to make a final concentration of 0.2 mg/ml. After allowing the suspension to stand for 30 minutes at room temperature, the suspension is smeared on a bouillon agar plate medium containing 1 mg/ml of 6-azauracil.After maintaining the medium at 30 C for 2 to 10 days, H-3149 is obtained as a mutants strain selected in the colonies growing. Table 5 shows how sensitive H-3055 and H-3149 are to 6-azauracil.

TABLE 5 H-3055 H-3149 Nothing added ++ ++ 6-Azauracil 0.5 mg/ml - ++ 1.0 mg/ml - + 3.0 mg/ml (++: sufficient growth; +: growth to some extent;-: no growth) EXAMPLE 6 H-3057 is used as a seed strain.

This strain is inoculated in a 300 ml-Erlenmeyer flask containing 20 ml of a seed medium (pH 7.2) comprising 40 g/e of glucose, 3 g/e of urea, 1.5 g/l of KH2PO4, 0.5 g/l of K2HPO4, 0.5 g/l of MgSO4.7H2O, 50 g/l of biotin, 20 9ie of peptone and 5 g/e of yeast extract, and cultured at 30"C for 24 hours.Then, 2 ml of the seed culture is put into a 300 ml-Erlenmeyer flask containing 20 ml of a fermentation medium (pH 7.2) comprising 100 g/e of blackstrap molasses (as glucose), 20 g/l of soybean meal acid hydrolyzate (as soybean meal), 5 g/e of ammonium sulfate, 1 g/l of L-leucine, 3 g/l of urea, 0.5 g/l of MgSO47H2O, 0.7 g/ of KH2PO4 and 30 g/l of CaCO3, and cultured with shaking (220 r.p.m.) at 30"C for 3 days. As a result, 39.5 g/l of L-lysine (as monohydrochloride, which will be hereinafter applied) is formed and accumulated in the culture liquor.

Amounts of L-lysine in case where parent strains H-31 22 and H-31 26 cultured at the same time under the same conditions as a control are 36 gll and 0.5 g/(, respectively.

After the completion of culturing, 1 e of the culture liquor of H-3107 is centrifuged. The resulting supernatant is passed through a column of Diaion SK-1 B (H+ form, trade mark of strongly acidic ion-exchange resin made by Mitsubishi Chemical Industries, Ltd.) to adsorb thereon L-lysine. After washing the column with water, L-lysine is eluted with a dilute aqueous ammonia to collect and concentrate L-lysine-containing fractions. After pH of the concentrate is adjusted to 2 with hydrochloric acid, the concentrate is cooled, while adding ethanol thereto to thereby crystallize L-lysine. Thus, 31 g of crystals of L-lysine is obtained.

EXAMPLE 7 H-3055 is used as a seed strain The strain is inoculated in the same seed medium as used in Example 6, and cultured at 30 C for 24 hours.

Then, 2 ml of the seed culture is put into a 300 ml-Erlenmeyer flask containing 20 ml of a fermentation medium (pH 7.2) comprising 100 gTh of blackstrap molasses (as glucose), 40 g/l of ammonium sulfate, 0.5 gll of KH2PO4, 0.5 g/l of MgSO4 and 30 g/l of CaCO3, and cultured with shaking (220 r.p.m.) at 32 C for 3 days. As a result, 41 g/l of L-lysine is accumulated in the culture liquor. Amounts of L-lysine, in case where strains H-31 22 and H-31 19 are cultured at the same time under the same conditions as a control, are 2.5 g/l and 24 g/e, respectively. L-lysine productivity of H-3122 is seriously low because the culture medium does not contain L-leucine which the strain requires.

EXAMPLE 8 H-3127 and H-3125 are used as seed strains. Each ofthe strains is inoculated in the same seed medium as used in Example 6, and cultured at 30 C for 24 hours. Then, 2 ml of each seed culture is inoculated in the same fermentation medium as used in Example 5, and cultured with shaking (220 r.p.m.) at 30 C for 3 days.

As a result, L-lysine is accumulated in the culture liquor in amounts given in Table 6. Amounts of L-lysine, in case where strains P-3634 and H-3056 are cultured at the same time under the same conditions as a control, are also shown in Table 6.

TABLE 6 Amount of L-lysine (g/l) P-3634 36 H-3127 39 H-3056 31 H-3125 36 EXAMPLE 9 H-3055, H-3127, H-3125 and H-3057 are used as seed strains. Each of the strains is inoculated in the same seed medium as used in Example 6, and cultured at 30 C for 24 hours. Then, 2 ml of each seed culture is put into a 300 ml-Erlenmeyer flask containing 20 ml of a fermentation medium (pH 7.2) comprising 100 go of glucose, 20 g/l of ammonium chloride, 20 g/l of corn steep liquor, 200 g/l of biotin, 1 g/l of KH2PO4 0.5 g/l of MgSO4.7H2O, 0.2 mgK of thiamine hydrochloride, 10 mg/l of FeSO47H2O, 10 mgK of MnSO44H2O, 3 g/l of urea and 20 g/l of CaCO3, and cultured with shaking at 30 C for 3 days.Amounts of L-lysine thus accumulated are shown in Table 7 together with amounts of L-lysine accumulated in culturing parent strains P-3634, H-3122, H-3126 and H-31 19 at the same time under the same conditions as a control. Additionally, with L-leucine-requiring strains, 0.5 g/l of L-leucine is added to the medium.

TABLE 7 Amount of L-lysine (g/ H-3125 36.5 H-3127 40.0 H-3055 41.0 H-3057 39.0 P-3634 36.5 H-3122 37.0 H-3119 29.0 H-3126 0.5 EXAMPLE 10 H-3055 is used as a seed strain. The strain is inoculated in a 300 ml-Erlenmeyerflask containing 30 ml of the same seed medium as used in Example 6, and cultured with shaking (220 r.p.m.) at 300C for 24 hours.

Then, 100 ml of the seed culture is put into a 2 l-jarfermenter containing 0.8 C of a fermentation medium compising 30 g/l of glucose, 3 g/l of ammonlum acetate, 1 g/l of KH2PO4, 0.5 g/l of MgSO4 7H2O, 10 mg/l of FeSO4,7H2O, 10 mgK of MnSO4, 200 g/l of biotin, 200 g/ of thiamine hydrochloride and 2 g/l of soybean meal acid hydrolyzate (as soybean meal), and cultured with aeration stirring at an aeration rate of 1 l/min., a stirring speed of 800 r.p.m. and 35 C for 55 hours while adjusting pH of the fermentation liquor to 6.8 with a mixture of acetic acid and ammonium acetate (mixing ratio of acetic acid : ammonium acetate = 6 : 1; concentration of acetic acid in the mixture solution: 60 %). As a result, 39 g/l of L-lysine is accumulated in the culture liquor.Amounts of L-lysine, in case where parent strains H-31 22 and H-3119 are cultured at the same time under the same conditions as a control, are 35 g/l and 27 91e, respectively.

EXAMPLE 11 H-3107 is used as a seed strain.

The seed strain is inoculated in a 300 ml-Erlenmeyer flask containing 20 ml of a seed medium (pH 7.2) comprising 40 g/l of glucose, 3 g/l of urea, 1.5 g/l of KH2Po4, 0.5 g/l of K2HPO4, 0.5 g/l of MgSO4.7H2O,50 g/l of biotin, 20 g/l of peptone and 5 g/l of yeast extract, and cultured at 30 C for 24 hours. Then, 2 ml of the resulting seed culture is put into a 300 ml-Erlenmeyer flask containing 20 ml of a fermentation medium (pH 7.2) comprising 90 g/l of blackstrap molasses (as glucose), 20 g/l of soybean meal acid hydrolyzate (as soybean meal), 5 G/l of ammonium sulfate, 3 g/l of urea, 0.5 g/l of MgSO4.7H2O, 0.7 g/l of K2HPO4 and 30 g/l of CaCO3, and cultured with shaking at 300C for 3 days. As a result, 38 g/l of L-lysine is formed and accumulated in the culture liquor.Amount of L-lysine in culturing parent strain P-3634 under the same conditions as a control is 34 91e.

After the completion of culturing, 1 e of the culture liquor of H-3107 is centrifuged. The resulting supernatant is adjusted to pH 1.5 with sulfuric acid, and passed through a column of Diaion SK-1 B (H+ form, trade mark of strongly acidic ion-exchange resin made by Mitsubishi Chemical Industries, Ltd.) to adsorb thereon L-lysine. After washing the column with water, L-lysine is eluted with a dilute aqueous ammonia to collect and concentrate L-lysine-containing fractions. After pH of the concentrate is adjusted to 2 with hydrochloric acid, the concentrate is cooled while adding ethanol thereto to thereby crystallize L-lysine.

Thus, 30.4 g of crystals of L-lysine is obtained.

EXAMPLE 12 Strains shown in Table 8 are respectively inoculated in the same seed media as in Example 11 and cultured at 30 C for 24 hours. Then, 2 ml of each of the resulting seed culture liquors is put into a 300 ml-Erlenmeyer flask containing 20 ml of a fermentation medium (pH 7.2) comprising 90 g/l of glucose, 20 g/l of soybean meal acid hydrolyzate (as soybean meal), 5 g/l of ammonium sulfate, 3 g/l of urea, 0.5 g/l of MgSO4'7H2O, 1 g/l of KH2PO4, 50 g/l of biotin, 200 g/ml of thiamino hydrochloride, 10 mg/l of FeSO4 7H2O, 10 mg/l of MnSO4 4H2O and 20 g/l of CaCO3, and cultured with shaking at 30"C for 3 days. Amounts of L-lysine accumulated in the culture liquors are tabulated in Table 8.

TABLE 8 Strain L-lysine Corynebacterium glutamicum P-3634 33.0 g/l H-3106 36.0 g/l H-3289 35.0 g/l H-3290 34.5 g/l Brevibacterium lactofermentum H-3056 31.0 g/l H-3114 35.0 g/l H-3113 34.0 gq H-3291 34.0 g/J' EXAMPLE 13 H-3106 is used as a seed strain.

The seed strain is inoculated in a 2 t-Erlenmeyer flask containing 300 ml of the same seed medium as in Example 11 and cultured with shaking at 30"C for 24 hours. Then, 1 @ of the resulting seed culture is put into a 30 t-jarfermentercontaining 106 of a fermentation medium comprising 100 gk of blackstrap molasses (as glucose), 0.3 g/l of MgSO47H2O, 0.7 g/l of KH2PO4, 3 9/4 of urea, 18 g/l of soybean meal acid hydrolyzate (as soybean meal) and 1.4 % (v/v) of a leucine fermentation liquor prepared in advance as hereinafter described, and cultured with aeration stirring at an aeration rate of 10 @min., a stirring speed of 400 r.p.m., and 30"C for 48 hours while adjusting pH of the culture liquor to 6.8 with 22 % aqueous ammonia. As a result, 43 g/l of L-lysine is formed and accumulated in the culture liquor. Amount of L-lysine in case where parent strain P-3634 is cultured under the same conditions as a control, is 37 91e.

The leucine fermentation liquor used in said fermentation medium is prepared as follows.

Corynebacterium glutamicum ATCC 21885 having an ability to produce leucine is inoculated in a 5 6'-jar fermenter containing 3l of seed medium (pH 7.2) comprising 50 g/l of glucose, 10 g/l of peptone, 10 g/l of yeast extract, 5 g/l of corn steep liquor, 2.5 91e of NaCl,3 g/l of urea and 50 g/l of biotin, and cultured with aeration stirring at an aeration rate of 3 E/min., a stirring speed of 600 r.p.m., and for 17 hours.Then, 1 l of the resulting seed culture is put into a 30 t-jarfermenter containing 10 l of a fermentation medium (pH 6.8) comprising 5 g/l of ammonium acetate, 2 g/l of KH2PO4, 0.5 g/l of MgSO4.7H2O, 0.1 g/l of FeSO4-7H2O, 0.01 gk of MnSO4-4H2O, 50 g/l of biotin and 100 g/l of thiamine hydrochloride, and cultured with aeration stirring at an aeration rate of 10 l/min., a stirring speed of 400 r.p.m., and 30 C for 60 hours. In course of culturing, pH of the culture liquor is adjusted to 6.8 by using a mixed solution containing 7% ammonium acetate and 38% acetic acid as a continuous feed. Thus, a leucine fermentation liquor containing 15.3 gff of leucine is obtained, which is part of composition of said fermentation medium for L-lysine.

EXAMPLE 14 H-3149 is used as a seed strain.

The strain is inoculated in the same seed medium as used in Example 6, and cultured at 30 C for 24 hours.

Then, 2 ml of the seed culture is inoculated in a 300 ml-Erlenmeyer flask containing 20 ml of a fermentation medium (pH 7.2) comprising 100 g/l of blackstrap molasses (as glucose), 40 g/l of ammonium sulfate, 0.5 g/f of KH2PO4, 0.5 g/( of MgSo and 30 gll of CaCo, and cultured at 32"C for 3 days under shaking (220 r.p.m.).

As a result, 43 g/( of L-lysine is accumulated in the culture liquor. The amount of L-lysine, in case where parent strain H-3055 is cultured at the same time under the same condition as a control, is 41 g/(.

The specific novel microorganism strains described herein have the general characteristics of the parent strain(s) apart from the specific modifications mentioned.

Claims (36)

1. A process for producing L-lysine, which comprises culturing a microorganism having an ability to produce L-lysine in a nutrient medium, accumulating L-lysine in the resulting culture liquor, and recovering the L-lysine therefrom, wherein the microorganism is obtained by protoplast fusion.

2. A process according to claim 1, wherein said microorganism belongs to the genus Corynebacterium or Brevibacterium.

3. A process according to claim 1, wherein said microorganism is obtained by protoplast fusion between the same or different strains belonging to the species of Corynebacterium glutamicum or Brevibacterium lactofermentum.

4. A process according to claim 1,2 or 3, wherein said microorganism has a resistance two or more antibiotics.

5. A process according to claim 1,2 or 3, wherein said microorganism has a resistance to two or more antibiotics and a resistance to at least one pyrimidine analog.

6. A process according to claim 4 or 5, wherein the microorganism has resistance to two or more antibiotics selected from aminoglycoside type antibiotics, ss-lactam type antiobiotics, macrolide type antibiotics and rifamycin type antibiotics.

7. A process according to claim 4 or 5, wherein the microorganism has resistance to two or more antibiotics selected from penicillin G, cephalosporin C, streptomycin, dihydrostreptomycin, rifampicin, chloramphenicol, tetracycline, spiramycin, erythromycin, kanamycin, kasugamycin, mitomycin C, actinomycin D, polymixin, colistin, lincomycin, gentamicin, sagamicin, fortimicin and oleandomycin.

8. A process according to claim 5, wherein said microorganism has resistance to one or more of 5-bromouracil, 6-azauracil, S4luorouracil, 5-bromo-2-deoxyuridine, 2-thiouracil, 6-methyl-2-thiouracil and amicetin.

9. A process according to claim 1, wherein said microorganism is a protoplast fusion strain H-3057 (FERM BP-148) obtained by protoplastfusion between Corynebacteriumglutamicum H-3122 (FERM BP-150) and Brevibacterium lactofermentum H-3126 (FERM BP-152), a protoplastfusion strain H-3055 (FERM BP-147) obtained by protoplast fusion between Corynebacterium glutamicum H-3122 (FERM BP-150) and Corynebacterium glutamicum H-31 19 (FERM BP-149), or H-3149 (FERM BP-158) derived from the protoplast fusion strain H-3055.

10. A process according to any one of the preceding claims, wherein said culturing is conducted at 20 to 40"C for 1 to 6 days.

11. A process for producing L-lysine, which comprises culturing a microorganism having an ability to produce L-lysine in a nutrient medium, accumulating L-lysine in the resulting culture liquor, and recovering the L-lysine therefrom, wherein the microorganism is of the genus Corynebacterium or Brevibacterium and has a resistance to two or more antibiotics.

12. A process according to claim 11, wherein the microorganism has resistance to two or more antibiotics selected from aminoglycoside type antibiotics, p-lactam type antibiotics, macrolide type antibiotics and rifamycin type antibiotics.

13. A process according to claim 11, wherein the microorganism has resistance to two or more antibiotics selected from penicillin G, cephalosporin C, streptomycin, dihydrostreptomycin, rifampicin, chloramphenicol, tetracycline, spiramycin, erythromycin, kanamycin, kasugamycin, mitomycin C, actinomycin D, polymixin, colistin, lincomycin, gentamicin, sagamicin, fortimicin and oleandomycin.

14. A process for producing L-lysine by fermentation, which comprises culturing a microorganism having an ability to produce L-lysine in a nutrient medium, accumulating L-lysine in the resulting culture liquor, and recovering the L-lysine therefrom, wherein the microorganism is of the genus Corynebacterium or Brevibacterium and has a resistance to at least one purine analog and/or at least one pyrmidine analog.

15. A process according to claim 14, wherein the microorganism has resistance to one or more of the following: mercaptoguanine, 8-azaguanine, 2-fluoroadenine, tubercidin, 6-methylpurine, 8-azaxanthine, 8-azaadenine, 8-mercaptoguanosine, 6-mercaptoguanosine, 2-aminopurine, 2-amino-6-mercaptopurine, decoyinin and psicofuranine.

16. A process according to claim 14, wherein the microorganism has resistance to one or more of the following: 5-bromouracil, 6-azauracil, 5-fluorouracil, 5-bromo-2-deoxyuridine, 2-thiouracil, 6-methyl-2thiouracil and amicetin.

17. A process according to any one of claims 11-16, wherein said microorganism is of the species Corynebacterium glutamicum or Brevibacterium lactofermentum.

18. A process according to claim 17, wherein said microorgaism is Corynebacterium glutamicum H-3127 (FERM BP-153) or Brevibacterium lactofermentum H-3125 (FERM BP-1 51).

19. A process according to claim 17, wherein said microorganism is Corynebacterium glutamicum H-3107 (FERM BP-144), Corynebacterium glutamicum H-3106 (FERM BP-143), Corynebacterium glutamicum H-3290 (FERM BP-156), Corynebacterium glutamicum H-3289 (FERM BP-157), Brevibacterium lactofermen tum H-3114(FERM BP-146), Brevibacterium lactofermentum H-3113 (FERM BP-145)or Brevibacferium lactofermentum H-3291 (FERM BP-155).

20. A process according to any one of claims 11-19, wherein said culturing is conducted at 20 to 400C for 1 to 6 days.

21. A microorganism having an ability to produce L-lysine, which is obtained by the technology of protoplast fusion.

22. A microorganism according to claim 21, which belongs to the genus Corynebacterium or Brevibacterium.

23. A microorganism according to claim 21, which is obtained by protoplast fusion between the same or different strains belonging to the species of Corynebacterium glutamicum or Brevibacterium lactofermentum.

24. A microorganism according to claim 21, which has a resistance to two or more antibiotics.

25. A microorganism according to claim 21, which has a resistance to two or more antibiotics and a resistance to a pyrimidine analog.

26. A microorganism according to claim 24 or 25, which is resistant to two or more antibiotics selected from aminoglycoside type antibiotics, p-lactam type antibiotics, macrolide type antibiotics and rifamycin type antibiotics.

27. A microorganism according to claim 24 or 25, which is resistant to two or more of penicillin G, cephalosporin C, streptomycin, dihydrostreptomycin, rifam picin, chloroamphenicol, tetracycline, spiramycin, erythromycin, kanamycin, kasugamycin, mitomycin C, actinomycin D, polymixin, colistin, lincomycin, gentamicin, sagamicin, fortimicin and oleandomycin.

28. A microorganism according to claim 25, which is resistant to one or more of 5-bromouracil, 6-azauracil, S4luorouracil, 5-bromo-2-deoxyuridine, 2-thiouracil, 6-methyl-2-thiouracil and amicetin.

29. A microorganism according to claim 21, which is a protoplast fusion strain H-3057 (FERM BP-148) obtained by protoplast fusion between Corynebacterium glutamicum H-3122 (FERM BP-150) and Brevibacterium lactofermentum H-3126 (FERM BP-152); a protoplastfusion strain H-3055 (FERM BP-147) obtained by protoplast fusion between Corynebacterium glutamicum H-3122 (FERM BP-150) and Corynebacterium glutamicum H-31 19 (FERM BP-149); or H-3149 (FERM BP-158) derived from the protoplast fusion strain H-3055.

30. A biologically pure culture of the microorganism Corynebacterium glutamicum H-3107 (FERM BP-1 44),

31. A biologically pure culture of the microorganism Corynebacterium glutamicum H-3106 (FERM BP-143).

32. A biologically pure culture of the microorganism Corynebacterium glutamicum H-3290 (FERM BP-1 56).

33. A biologically pure culture of the microorganism Corynebacterium glutamicum H-3289 (FERM BP-157).

34. A biologically pure culture of the microorganism Brevibacterium lactofermentum H-31 14 (FERM BP-1 46),

35. A biologically pure culture of the microorganism Brevilbacterium lactofermentum H-31 13 (FERM BP-145).

36. A biologically pure culture of the microorganism Brevilbacterium lactofermentum H-3291 (FERM BP-155).