GB2152509A - Process for the production of L-lysine by fermentation and microorganisms for use therein - Google Patents

Abstract

L-lysine is produced in improved yield by culturing microorganisms of the genus Corynebacterium and Brevibacterium having the ability to produce L-lysine in a culture medium, the microorganisms being characterised by either a resistance to two or more antibiotics or a resistance to one or more purine analogs and/or to one or more pyrimidine analogs. Novel microorganisms of these genera are described.

Description

1 GB 2 152 509 A 1

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 5 producing L-lysine by fermentation using said microorganisms.

Heretofore, as processes for producing L-lysine by fermentation, 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 Brevibacter ium capable of producing L-lysine and which have or are endowed with either a resistance to two or more 15 antibiotics or a resistance to at least one purine analog andfor at least one pyrimidine analog have a remarkably improved ability to produce L- lysine.

Accordingly, the invention provides a process for the production of Llysine, which comprises culturing a microorganism capable of producing Llysine 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, either i) a mutant 20 strain belonging to the genus Corynebacterium or Brevibacterium and which has a resistance to two or more antibiotics; or 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.

The present invention will be described in more detail below.

The microorganisms used in the present invention, can be obtained by various techniques. In one 25 technique the L-lysine-producing strains can be obtained by protoplast fusion between parent strains having different properties and an L-lysine-producing strain. Alternatively they may be obtained by conventional mutation inducing treatment or spontaneous mutation to obtain strains having a resistance to two or more antibiotics, or a resistance to purine analogs and/or pyrimidine analogs.

For example, 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 two or more antibiotics, or conversely a strain having a resistance to two or more antibiotics and belonging to the genus Corynebacterium or Brevibacterium and which is endowed with an ability to produce L-lysine.

Alternatively, there may be used a strain belonging to the genus Corynebacterium or Brevibacterium and having an ability to produce L-lysine and which has been endowed with a resistance to at least one of purine 35 analog and pyrimidine analog which has been 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, 40 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 combina tions), 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 45 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 50 other chemicals as metnioned above may also be used in the present invention. Further, the strain to be used in the preent 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, chforamphenicol, tetracyclines, spiramycin, 55 erythromycin, kanamycin, kasugamycin, mitomycin C, actinomycin D, pofymixin, colistin, lincomycin, gentamicin, sagamicin, fortimicin, oleandomycin, etc. is mentioned.

As a resistance to purine analog, for example, a resistance to 6mercaptoguanine, 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, 5bromo-2-deoxyuridine, 2-thiouracil, 6-methy]-2thiouracil, amicetin, etc. is mentioned.

Mutants useful in carrying outthe present invention are derived from parent strains belonging to the genus Corynebacterium or Brevibacterium known as a glutamic acid- producing strains, such as Corynebac terium glutamicum ATCC 13032, Corynebacterium acetoacidophilum ATCC 13870, Brevibacterium lactofer- 65 2 GB 2 152 509 A mentum 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-1 53) 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-3114 (hereinafter referred to as H-3114) (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-145).

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 9 of bouillon powder and 5 g of yeast extract in 1 1 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 liquorto make a final 15 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 NH4C1, 2 g of urea, 1 g of yeast extract, 19 of KH2P04,3 g of K2HP04,0A g of MgC12.6H20, 10 mg of FeS04. 7H20,0.2 mg of MnS04.4-61-120, 0.9 mg of ZnS04.7H20,0A mg Of CUS04.5H20, 0.09 mg of Na2B407.1 0H20,0.04 mg of (NH4)6M07024.4H20,30 1Lg of biotin and 1 mg of thiamine hydrochloride in 1 ( of pure water and adjusted to pH 7.2; in culturing an 20 amino acid-requiring strain, 50 [ig.imi 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 M9C12.6H20 in a 2-fold diluted SSM solution and adjusted to pH 7.0 to 8.5). Lysozyme (0.2 to 2 mglml) 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 mi of PFM medium. To the suspension is added 2.5 mI 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 mi of the 30 solution is smeared on RCG agar plate containing 100 ggl-mi of streptomycin and 0.05 RgiM of rifampicin (containing 5 g of glucose, 5 9 of casamino acid, 2.5 g of yeast extract, 3.5 g of K2HP04,1.5 g of KI-12P04,0.41 9 Of M9C12.61-ICI, 10 mg of FeS04.7H20,2 mg of MnS044-61-120,0.9 mg of ZnS04.71-120, 0.4 mg of CUS04-51-120, 0.09 Mg of Na213407-1 0H20,0.04 mg of (NH4)6M07024.4H20,30 Rg of biotin, 2 mg of thiamine hydrochloride, 135 g of sodium succinate and 14 9 of agar in 1 1 of pure water, and adjusted to pH 7.4). After culturing at 30'C 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 nitrogen containing compounds and peptone, meat extract, yeast extract, corn steep liquor, casein h,,,drolyzate, 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 50 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, 55 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.

2 3 GB 2 152 509 A 3 Practice of specific embodiments of the invention is illustrated by the follwoing representative examples.

Example 1

Preparation of H-3127 and H-3125) 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 sulfamethazine) having been in advance cultured overnight in a bouillon medium is directly smeared on a bouillon agar plate medium containing 200 pg/mi of dihydrostreptomycin. After maintaining the medium at WC 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-methyi-N'-nitro-N'-nitrosoquanidine to make a final concentration of 0. 2 mglmi. 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 G as shown in Table 1. Also, a dihydrostreptomycin- is resistant strain obtained from Brevibacterium lactofermentum H-3056 (FERM BP-1 54) (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 Kg/mi of rifampicin. H-3125 is clearly discriminated from the parent strain (H-3056) in that the 20 former has a resistance to both dihydrostreptomycin and rifampicin as shown in Table 1.

TABLE 1

P3634 H-3127 H-3056 H-3125 25 Penicillin G 0.5 u/mi - ++ 1.0 U/M1 - + Dihydrostre- 100 Pg/M1 - + 4- + + 30 ptomycin 200 jig/m] - ++ ++ Rifampicin 25 50 Nothing added l,kg/M1 [tglml + - -t- + + + +: sufficient growth; +: growth to some extent;: no growth) Example 2 (Preparation of H-3107, H-3106, H-3289, W3290, H-3114, H-3113 and H-3291) Cells of P-3634 are suspended in a 0.1 N trismaleate buffer solution (pH 6.0) in a concentration of 1 U3 cells/mi. To the suspension is added N-niethy]-N'-nitro-N'- nitrosoguanidine to make a final concentration of 45 0.2 mg/mi. 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/mi of 6-mercaptoguanosine or on a flat agar plate of minimal medium of the similar composition containing 0.4 mgImI 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-mercaptoquanosine 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-mereaptoquanosine, 6-azauracil and to 5- bromouracil from Brevibacterium lactofermentum H-3056 (hereinafter referred to as H-3056) (FERM BP-1 54) (having a resistance to thialysine 55 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.

4 GB 2 152 509 A 4 TABLE 2 (±: sufficient growth; -: growth to some extent, Nothing added 6-Mercaptoguanosine [Lg/M1 500 tigi;MI 6-Azauracil 100 Rg. M1 500 Kg!rn I 6-Methyipurine Kg/m 1 400 gg, m 1 -: no growth) PH- - H- H- H- H- H- H- H- 5 3634 3107 3106 3289 3290 3056 3114 3113 3291 -- -f- + + 4- + 4- -C 5-Bromouracil 1000 Rg/m 1 2000 Kg/M + 4- + + --- + + + -- + -I- + Composition of the flat agar plate of minimal medium is as follows: 10 g, 7 of glucose, 4 g' of ammonium chloride, 1 gII of KI-12P04,3 g/1 of K2HP04,0A gl of M9S04.7H20,0.01 91' of FeS04.71-120, 0.01 91 of WS04.4H20,2 g/1 of urea, 50 Kgli of biotin and 20 g/I of 30 agar. pH: 7.2.

Example 3

H-3127 and H-3125 are used as seed strains. Each of the strains is inoculated into 20 mI of a seed medium )pH 7.2) comprising 40 g! 1 of glucose, 3 g.d of urea, 1.5 gil of KI- 12P04,0.5 g.1 of K2HP04,0.5 g/1 Of M9S04.71-120, 50 Kg/1 of biotin, 20 g, 1 of peptone and 5 g. 1 of yeast extract, and cultured at WC for 24 hours. Then, 2 mi of 35 each seed culture is inoculated into 20 mi of a fermentation medium (pH 7. 2) comprising 100 gil of blackstrap molasses (as glucose). 20 g,Il of soybean meal acid hydrolyzate (as soybean meal), 5 gil of ammonium sulfate, 1 g11 of L-leucine, 3 gil of urea, 0.5 gi 1 of MgSO.71-120, 0.7 gI of KH2P04 and 30 g, 1 of CaCO3, and cultured with shaking (220 r.p.m.) at WC for 3 days. As a result, L- lysine is accumulated in the culture liquor in amounts given in Table 3. Amounts of L-lysine, incase where strain P- 3634 and H-3056 are cultured at the 40 same time under the same conditions as a control, are also shown in Table 3.

TABLE 3

Amount of L-lysine (gU) P-3634 H-3127 H-3056 H-3125 36 39 31 36 Example 4

H-3127 and H-3125 are used as seed strains. Each of the strains is inoculated in the same seed medium as used in Example 3 and cultured at 3WC for 24 hours. Then, 2 m[ of each seed culture is put into a 300 miErlenmeyer flask containing 20 mi of a fermentation medium (pH 7.2) comprising 100 g/1 of glucose, 20 60 g,,( of ammonium chloride, 20 g/'/ of corn steep liquor, 200 lig,,1 of biotin, 1 g/i. of KH2P04, 0.5 9/1 Of M9S04-71-120,0.2 mg..( of thiamine hydrochloride, 10 mgl of FeS04.7H20, 10 Mgl of WS04.4H20,3 g/I of urea and 20 gl Of CaC03, and cultured with shaking at 30'ú for 3 days. Amounts of L-lysine thus accumulated are shown in Table 4 together with amounts of L-lysine accumulated in culturing parent strain P-3634, at the same time under the same conditions as a control. Additionally, with L-leucine-requiring 65 strains, 0.5 g/( of L-feucine is added to the medium.

GB 2 152 509 A 5 TABLE 4

Amount of L-lysine (gil) H-3125 36.5 5 H-3127 40.0 P-3634 36.5 10 Example 5

H-3107 is used as a seed strain.

The seed strain is inoculated in a 300 mi-Erlenmeyer flask containing 20 mi of a seed medium (pH 7.2) comprising 40 g/t of glucose, 3 g/ of urea, 1.5 g/( of KH2P04A.5 9/1( of K2HP04A.5 g/ Of M9S04.7H20, 50 15 tgl, of biotin, 20 g/t of peptone and 5 g/t of yeast extract, and cultured at WC for 24 hours. Then, 2 mi of the resulting seed culture is put into a 300 mi-Erienmeyer flask containing 20 mi of a fermentation medium (pH 7.2) comprising 90 gM of blackstrap molasses (as glucose), 20 gM, of soybean meal acid hydrolyzate (as soybean meal), 5 gM of ammonium sulfate, 3 gl'of urea, 0.5 g/( Of M9S04. 7H20,03 g/ pf K2HP04 and 30 glC of CaCC3, and cultured with shaking at WC for 3 days. As a result, 38 gM'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 9M.

After the completion of culturing, 1 1, 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 6

Strains shown in Table 5 are respectively inoculated in the same seed media as in Example 5 and cultured at WC for 24 hours. Then, 2 mi of each of the resulting seed culture liquors is put into a 300 mlErlenmeyer flask containing 20 mi of a fermentation medium (pH 7.2) comprising 90 gU of glucose, 20 gl of soybean meal acid hydrolyzate (as soybean meal), 5 g/1 of ammonium sulfate, 3 9/1 of urea, 0.5 g/ Of M9S04.7H20, 1 35 g/( of KH2P04, 50 [191f of biotin, 200 jig/ml of thiamine hydrochloride, 10 mgU of FeS04.71H120, 10 M91t Of WS04.4H20 and 20 gil of CaCC3, and cultured with shaking at 300C for 3 days. Amounts of L-lysine accumulated in the culture liquors are tabulated in Table 5.

TABLE 5 40

Strain L-Lysine Corynebacterium glutamicum P-3634 33.0 gli H-3106 36.0 g/t H-3289 35.0 gU H-3290 34.5 g/1 Brevibacterium lactofermentum H-3056 31.0911 H-3114 35.0 gU H-3113 34.0 9M H-3291 34.0 g/t 6 GB 2 152 509 A 6 Example 7

H-3106 is used as a seed strain.

The seed strain is inoculated in a 2 -Erlenmeyer flask containing 300 mi of the same seed medium as in Example 5 and cultured with shaking at WC for 24 hours. Then, 1 ( of the resulting seed culture is put into a 30 ( jar fermenter containing 10 of a fermentation medium comprising 100 gl of blackstrap molasses (as 5 glucose), 0.3 gU Of M9S04.7H20, 0.7 g/( of KI-12P04,3 g/,( of urea, 18 g11 of soybean meal acid hydrolyzate (as soybean meal) and 1.4 % (vlv) of a leucine fermentation liquor prepared in advance as hereinafter described, and cultured with aeration stirring at an aeration rate of 10, Imin., a stirring speed of 400 r.p.m., and WC for 48 hours while adjusting pH of the culture liquor to 6.8 with 22 % aqueous ammonia. As a result, 43 g/7 of L-lysine is formed and accumulated in the culture liquor. Amount of L-lysine in case where parent 10 strain P-3634 is cultured under the same conditions as a control, is 37 g/.

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 1 -jar fermenter containing 3 ( of seed medium (pH 7.2) comprising 50 go7 of glucose, 10 g11 of peptone, 10 gH of yeast extract, 5 g.4 of corn steep liquor, 2.5 g/( of NaCI, 3 gl of urea and 50 ligU of biotin, cultured with aeration stirring at an aeration rate of 3 1 i'min., a stirring speed of 600 r.p.m., and WC for 17 hours. Then, 1 of the resulting seed culture is put into a 30 1 -jarfermenter containing 10 of a fermentation medium (pH 6.8) comprising 5 g!( of ammonium acetate, 2 gU of KI-12P04,0.5 gU Of M9S04-71-120, 0.1 9/ of FeS04.71-120, 0.01 g; of MnS04.41-120, 50 gg.4 of biotin and 100 Kg/f ofthiamine hydrochloride, and cultured with aeration stirring at an aeration rate of 10 imin., a stirring speed of400 r.p.m., and WC for 60 hours. In 20 course ofcuituring, pH ofthe 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 g/ of leucine is obtained, which is part of composition of said fermentation medium for L-lysine.

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

Claims (16)

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 30 the L-lysine therefrom, wherein the microoganism is of the genus Corynebacterium or Brevibacterium and has either a resistance to two or more antibiotics or a resistance to at least one purine analog and/or at least one pyrimidine analog.

2. A process according to claim 1, wherein the microorganism has resistance to two or more antibiotics selected from aminoglycoside type antibiotics, p-lactam type antibiotics, macrolide type antibiotics and 35 rifamycin type antibiotics.

3. A process according to claim 1, wherein the microorganism has resistance to two or more antibiotics selected from penicillin G, cephalosporin C, streptomycin, dihydrostreptomycin, rifampicin, chlorampheni col, tetracycline, spiramycin, erythromycin, kanamycin, kasugamycin, mitomycin C, antinomycin D, polymixin, colistin, lincomycin, gentamicin, sagamicin, fortimicin and oleandomycin.

4. A process according to claim 1, 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-mercaptoquanosine, 2-aminopurine, 2- amino 6-mercaptopurine, decoyinin and psicofuranine.

5. A process according to claim 1, 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-2 thiouracil and amicetin.

6. A process according to anyone of claims 1-5, wherein said microorganism is of the species Corynebacterium glutamicum or Brevibacterium lactofermentum.

7. A process according to claim 6, wherein said microorganism is Corynebacterium glutamicum H-3127 50 (FERM BP-1 53) or Brevibacterium lactofermentum H-3125 (FERM BP-1 51).

8. A process according to claim 6. wherein said microorganism is Corynebacterium glutamicum H-3107 (FERM BP-144), Corynebacterium glutamicum H-3106 (FERM BP-143), Corynebacterium glutamicum H-3290 FERMBP-156),CorynebacteriumglutamicumH-3289(FERMBP-157), Brevibacteriumlactof ermentum H-3114(FERMBP-146)BrevibacteriumlactofermentumH-3113(FERMBP145)orBrevibacte riumlactofer- 55 mentum. H-3291 (FERM BP-155).

9. A process according to anyone of claims 1-8, wherein said culturing is conducted at 20 to WC for 1 to 6 days.

10. A biologically pure culture of the microorganism Corynebacterium glutamicum H-3107 (FERM BP-144).

11. A biologically pure culture of the microorganism Corynebacteriumglutamicum H-3106 (FERM BP-143).

12. A biologically pure culture of the microorganism Corynebacterium glutamicum H-3289 (FERM BP-1 56).

7 GB 2 152 509 A 7

13. A biologically pure culture of the microorganism Corynebacterium glutamicum H-3289 (FERM BP-1 57).

14. A biologically pure culture of the microorganism Brevibacterium lactofermentum H-3114 (FERM BP-1 46).

15. A biologically pure culture of the miroorganism Brevibacterium lactofermentum H3113 (FERM 5 BP-1145).

16. A biologically pure culture of the microoganism Brevibacterium lactofermentum H-3291 (FERM BP-155).

Printed in the UK for HMSO, D8818935, 6,85, 7102.

Published by The Patent Office, 25 Southampton Buildings, London, WC2A lAY, from which copies may be obtained.