PROCESSES FOR PRODUCING L-PROLINE BY FERMENTATION

Abstract: L-glutamic acid and L-proline are produced by culturing, in a nutrient medium, an L-glutamic acid or L-proline producing mutant microorganism belonging to the genus Corynebacterium or Brevibacterium and which has or is endowed with resistance to a purine or pyrimidine analog or both. Also disclosed are novel mutants derived from mic­ roorganisms of the genus Corynebacterium and Brevibacter­ ium and having the specified resistance.

Description: PROCESSES FOR PRODUCING L-GLUTAMIC ACID AND L-PROLINE BY FERMENTATION AND NOVEL MICROORGANISM STRAINS FOR USE THEREIN The present invention relates to the production of L-glutamic acid and L-proline by fermentation and to microorganisms for use therein. L-glutamic acid and L-proline are important amino acids which are commercially useful as food additives. It is already known that L-glutamic acid and L-proline can be produced by fermentation using microorganisms of the genera Corynebacterium and Brevibacterium. However, the production yields of the known processes are comparatively low from a commercial point of view. Thus, a need exists for processes for producing L-glutamic acid and L-proline in high yield and at low cost. To this end, it has now been found that L-glutamic acid or L-proline producitivity of an L-glutamic acid or L-proline producing microorganism belonging to the genus Corynebacterium or Brevibacterium is greatly improved if the microorganism is endowed with a resistance to a purine or pyrimidine analog. Further, it has been found that L-proline is produced in good yield by culturing the mutant in the presence of the specified amount of NH4 ion. In accordance with the invention, therefore, L-glutamic acid and Lproline are produced by culturing, respectively an L-glutamic acid or Lproline producing microorganism belonging to the genus Corynebacterium or Brevibacterium in a nutrient medium until L-glutamic acid or L-proline is accumulated in the culture liquor and thereafter recovering said L-glutamic acid or L-proline, wherein a mutant strain of the microorganism has or has been endowed with a resistance to at least one purine or pyrimidine analog is used. In a preferred embodiment, L-proline is produced by culturing the mutant strain in a culture medium containing from 0.05 to 0.5 M of the NH4+ ion. The purine analog or pyrimidine analog resistant mutants used in this invention can be obtained by endowing an L-glutamic acid- or L-prolineproducing strain of a microorganism of the genus Corynebacterium or Brevibacterium with a resistance to a purine analog or a pyrimidine analog or both, or by endowing a microorganism strain already having a resistance to a purine analog or a pyrimidine analog or both with the ability to produce L-glutamic acid or L-proline. The purine analog to which the mutant has resistance may be any of the following: 6-mercaptoguanine, 8-azaguanine, 2-fluoroadenine, tubercidin, 6-methylpurine (hereinafter referred to as "6MP"), 8azaxanthine, 8-azaadenine, 8-mercaptoguanosine, 6-mercaptoguanosine (hereinafter referred to as "6MG"), 2-aminopurine, 2-amino-6mercaptopurine, decoyinin and psicofuranine. Likewise, the pyrimidine analog to which the mutant has resistance may be any of the following: 5bromouracil (hereinafter referred to as "5BU"), 6-azauracil (hereinafter referred to as "6AU"), 5-fluorouracil, 5-bromo-2-deoxyuridine, 2-thiouracil, 6-methyl-2-thiouracil and amicetin. Moreover, mutant strains having other properties such as various nutrient requirements, drug resistance, drug sensitivity and drug dependence in addition to the above-mentioned resistance may be employed. Suitably mutated strains for use in this invention can be obtained by culturing and screening suitable parent strains in a nutrient medium containing a pyrimidine or purine analog and selecting the strain or strains having the ability to produce L-glutamic acid or L-proline in greater yields than the parent strain. For example, a selected parent strain in suspension in a 0.1 N tris-maleate buffer solution (pH 6.0) in a concentration of lO8 cells/ml, is treated with a suitable analog e.g. N-methyl-N'-nitro-N-nitrosoguanidine to make a final concentration of 0.2 mg/ml. The suspension is allowed to stand at room temperature for 30 minutes and then smeared on a flat agar plate of minirnal medium of the following composition containing 0.4 mg/ml of 6MG, 0.2 mg/ml of 6MP, 0.1 mg/ml of 5BU and 0.1 mg/ml of 6AU. The desired mutants can then be selected from the growing colonies. The composition of the flat agar plate of minimal medium is as follows: 10 g/l of glucose, 4 g/l of ammonium chloride, 1 g/l of KH2PO4, 3 g/l of K2HPO4, 0.4 gll of MgSO4. 7H20, 0.01 gil of FeSO4.7H20, 0.01 g/l of MnSO4. 4H20, 2 gll of urea, 50 pg/l of biotin and 20 g/l of agar. pH: 7.2. Examples of preferred strain used in the present invention are as follows. Table 1 EMI4.1 <tb> <SEP> No. <SEP> Strain <SEP> FERM <SEP> Property <SEP> Product <SEP> Parent <tb> <SEP> P-No. <SEP> of <SEP> *1 <SEP> strain <tb> <SEP> mutant <SEP> * <tb> <SEP> 1 <SEP> Corynebacterium <tb> <SEP> glutamicum <SEP> H-3280 <SEP> 6573 <SEP> 6 <SEP> MPR <SEP> G <SEP> 1 <tb> <SEP> 2 <SEP> n <SEP> H-3281 <SEP> 6574 <SEP> 6 <SEP> MGR <SEP> G <SEP> 1 <tb> <SEP> 3 <SEP> " <SEP> H-3282 <SEP> 6575 <SEP> 5 <SEP> AUR <SEP> G <SEP> 1 <tb> <SEP> 4 <SEP> " <SEP> H-3283 <SEP> 6576 <SEP> 6 <SEP> AUR <SEP> G <SEP> 1 <tb> <SEP> 5 <SEP> " <SEP> H-3334 <SEP> 6823 <SEP> 6 <SEP> MGR <SEP> P <SEP> 1 <tb> <SEP> 6 <SEP> Corynebacterium <tb> <SEP> acetophilum <SEP> H-3336 <SEP> 6824 <SEP> 6 <SEP> MPR <SEP> P <SEP> 2 <tb> <SEP> 7 <SEP> " <SEP> H-3337 <SEP> 6825 <SEP> 5 <SEP> BUR <SEP> P <SEP> 2 <tb> <SEP> 8 <SEP> Corvnebacterium <tb> <SEP> acetoacidphilum <SEP> H-3338 <SEP> 6826 <SEP> 6 <SEP> AUR <SEP> P <SEP> 3 <tb> <SEP> 9 <SEP> Brevibacterium <tb> <SEP> lactofermentum <SEP> H-3284 <SEP> 6577 <SEP> 6 <SEP> MGR <SEP> G <SEP> 4 <tb> 10 <SEP> | <SEP> " <SEP> H-3285 <SEP> 6578 <SEP> 6 <SEP> MPR <SEP> G <SEP> 4 <tb> <SEP> 11 <SEP> " <SEP> H-3286 <SEP> 6579 <SEP> 6 <SEP> AUR <SEP> G <SEP> 4 <tb> <SEP> 12 <SEP> " <SEP> H-3287 <SEP> 6580 <SEP> 5 <SEP> BU@ <SEP> G <SEP> 4 <tb> <SEP> 13 <SEP> " <SEP> H-3356 <SEP> 6827 <SEP> 5 <SEP> BUR <SEP> P <SEP> 4 <tb> <SEP> 14 <SEP> " <SEP> H-3357 <SEP> 6828 <SEP> 6 <SEP> MPR <SEP> P <SEP> 4 <tb> *1 G | L-glutamic acid, P | L-proline *2 1 Corynebacterium glutamicum ATCC 13032 2 Corynebacterium acetophilum FERM-P 4045 3 Corynebacterium acetoacidphilum ATCC 13870 5 Brevibacterium lactofermentum ATCC 13869 The strains listed in Table 1 are clearly discriminated from the parent strain, as shown in Tables 2 and 3. Table 2 EMI5.1 <tb> <SEP> Additive <SEP> No <SEP> 6MP <SEP> 6MG <SEP> 5BU <SEP> 6AU <tb> <SEP> ( g/ml) <SEP> addition <SEP> 100 <SEP> 400 <SEP> 1000 <SEP> 2000 <SEP> 1000 <SEP> 2000 <SEP> 100 <SEP> 500 <tb> <SEP> ATCC <SEP> 13032 <SEP> ++ <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> <tb> <SEP> H <SEP> - <SEP> 3280 <SEP> ++ <SEP> ++ <SEP> + <tb> <SEP> H <SEP> - <SEP> 3281 <SEP> ++ <SEP> ++ <SEP> + <tb> <SEP> H <SEP> - <SEP> 3282 <SEP> ++ <SEP> ++ <SEP> + <tb> <SEP> H <SEP> - <SEP> 3283 <SEP> ++ <SEP> ++ <SEP> + <tb> <SEP> ATCC <SEP> 13869 <SEP> ++ <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> <tb> <SEP> H <SEP> - <SEP> 3284 <SEP> ++ <SEP> ++ <SEP> + <tb> <SEP> H <SEP> - <SEP> 3285 <SEP> ++ <SEP> ++ <SEP> + <tb> <SEP> H <SEP> - <SEP> 3286 <SEP> ++ <SEP> ++ <SEP> + <tb> ; ; <SEP> H <SEP> - <SEP> 3287 <SEP> + <SEP> | <SEP> ++ <SEP> + <tb> Table 3 EMI6.1 <tb> <SEP> Additive <SEP> No <SEP> 6MG <SEP> 6MP <SEP> 6AU <SEP> 5BU <tb> <SEP> ( g/ml) <SEP> addition <SEP> 1000 <SEP> 200 <SEP> 200 <SEP> 50 <SEP> 100 <SEP> 50 <SEP> 10 <SEP> 1000 <SEP> 200 <tb> <SEP> ATCC <SEP> 13032 <SEP> ++ <SEP> - <SEP> + <tb> <SEP> H <SEP> - <SEP> 3334 <SEP> ++ <SEP> | <SEP> ++ <SEP> ¯ <SEP> . <SEP> <tb> <SEP> FERM-P <SEP> 4045 <SEP> ++ <SEP> - <SEP> + <SEP> i <SEP> + <tb> <SEP> H <SEP> - <SEP> 3336 <SEP> ++ <SEP> ++ <SEP> ++ <tb> H <SEP> - <SEP> 3337 <SEP> ++ <SEP> ++ <SEP> ++ <tb> <SEP> ATCC <SEP> 13870 <SEP> ++ <SEP> - <SEP> + <SEP> <SEP> + <tb> <SEP> H <SEP> - <SEP> 3338 <SEP> ++ <SEP> ++ <SEP> ++ <SEP> ++ <tb> <SEP> ATCC-13869 <SEP> I <SEP> ++ <SEP> - <SEP> + <SEP> | <SEP> <tb> <SEP> 11 <SEP> - <SEP> 3356 <SEP> ++ <tb> <SEP> H <SEP> - <SEP> 3357 <SEP> ++ <SEP> | <SEP> ++ <SEP> ++ <SEP> ++ <SEP> ++ <tb> ( ++: sufficient growth; +: growth to some extent; *: a little growth; -: no growth The aforesaid mutants H-3280 - H-3287 were depos ited on June 14, 1982 with the Fermentation Research Insti tute, the Agency of Industrial Science and Technology Japan as FERM-P numbers shown in Table 1. The mutants H-3334, H-3336 - H-3338, H-3356 and H-3357 were deposited on December 9, 1982 with the same depository described above as FERM-P numbers shown in Table 1. All of the mutants are directly transferred from the Fermentation Research Institute described above to Agricultural Research Cultural Correction (NRRL) and are deposited under the terms of the Budapest Treaty. Moreover, biologically pure cultures of the mutant strains identified above are intended to be included within the scope of the present invention. As the medium used for the culturing process of the present invention, either a natural medium or synthetic medium may be employed as long as it contains an assimilable carbon source, a nitrogen source and the inorganic materials and other nutrients which may be required by the specific mutant used. As suitable carbon sources, there may be used carbohydrates such as sucrose, fructose, glucose, maltose, mannose, starch, starch hydrolyzate and molasses; sugar alcohols such as glycerine and sorbitol; organic acids such as formic acid, acetic acid, lactic acid, fumaric acid and malic acid,; and alcohols such as ethanol and methanol. These carbon sources may be employed either alone or as mixtures at various weight ratios. The total amount may be initially supplied in the medium or may be supplied by incremental addition. As suitable nitrogen sources there may be used inorganic and organic ammonium salts such as ammonium chloride, ammonia, ammonium sulfate, ammonium carbonate, ammonium acetate, ammonium nitrate and ammonium phosphate; urea; natural nitrogen containing substances such as peptone, meat extract, corn steep liquor, casein hydrolyzate and soybean meal hydrolyzate. Inorganic materials which may be required include potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate and calcium carbonate. When the strain used requires nutrients such as amino acids, nucleic acids and vitamins, it is, of course, also necessary to include appropriate amounts of these substances in the medium. In some cases, such nutrients may be supplied by another medium component and thus specific supplementation is not required. Culturing is generally carried out at 20-40 ' C, under aerobic conditions, for example, by shaking culture or aeration agitation culture until a recoverable quantity of L-glutamic acid or L-proline is produced in the culture liquor, usually within 1-8 days. After the completion of culturing, the product L-glutamic acid or L-proline may be recovered from the culture liquor by conventional methods such as by ion exchange resin treatment. The process of the invention is illustrated by the following representative examples. Example 1 A main fermentation medium (pH 7.0) comprising 50 g/l glucose, I g/l K112 PO4, 0.5 g/l MgSO4.7H20, 200 ugiml thiamine hydrochloride, 5 g/l urea, 10 mg/l FeSO4. 7H20, 10 mg/l MnSO4.4H20, l mg/l CuSO4.4H20 and 3 ugll biotin is prepared, and 20 ml of the fermentation medium is put into each of 250 ml-Erlenmeyer flasks and sterilized by heating at 120 ' C for 10 minutes. In a separate step, each strain identified in Table 4 is cultured in a seed medium which is prepared by adding the above medium to 100 ugll biotin. Then, 0.5 ml of each resulting seed culture liquor is inoculated in 20 ml of the main fermentation medium, and cultured with shaking at 32 C for 30 hours. In the course of culturing, pH of the culture liquor is adjusted to 6.5-8.0 by using a sterile urea solution as a continuous feed. Amounts of L-glutamic acid accumulated in the culture liquors are given in Table 4. Table 4 Strain L-glutamic acid (g/l) ATCC 13032 23.5 H - 3281 25.5 H - 3280 26.0 H - 3283 , 26.0 H -.3282 25.0 ATCC 13869 23.0 H - 3284 24.5 H - 3285 25.5 H - 3286 25.0 H - 3287 25.5 ExamPle 2 A main fermentation medium (pH 6.5) comprising 60 g/l blackstrap molasses (as glucose), 5 g/l urea, 2 g/l (NH4)2SO4, 1 g/l KH2PO4, 1 g/l K2HDOfl and 20 mg/l MnSO4. H20 is prepared, and 16 ml of the fermentation medium is put into each of 250 ml-Erlemmeyer flasks and sterilized by heating at 1200C for 10 hours. Separately, each strain identified in Table 5 is cultured in a seed medium comprising the same components as the fermentation medium. Then, 3 ml of each resulting seed culture liquor is inoculated in 16 ml of the growth medium, and penicillin C is added thereto to a final concentration of 5 U/ml. Culturing is carried out with shaking at 340C for 30 hours. In the course of culturing, pH of the culture liquor is adjusted to 6.5-8.0 while adding 0.5 ml of sterile 10% urea solution thereto. Amounts oj L-glutamic acid accumulated in the culture liquors are @iven in Table 5. Table 5 Strain L-glutamic acid (g/l) ATCC 13032 29.0 H - 3280 33.5 H - 3282 34.0 ATCC 13869 27.5 H - 3285 32.0 H - 3287 33.5 Example 3 Each strain identified in Table 6 is cultured in the same seed medium as used in Example 2, and 3 ml of each resulting seed culture liquor is inoculated in 16 ml of a medium prepared by adding the same fermentation medium as used in Example 2 to 0.08 g/l polyoxyethylenesorbitanmono- palmitate. Culturing is carried out at 340C for 30 hours. In the course of culturing, pH of the culture liquor is adjusted to 6.5-8.0 while adding 0.5 ml of sterile 10% urea solution thereto. Amounts of L-glutamic acid accumulated in the culture liquors are given in Table 6 Table 6 Strain L-glutamic acid (g/l) ATCC 13869 28.5 H - 3284 32.0 H - 3285 32.5 H - 3286 31.5 H - 3287 33.0 Example 4 Each strain identified in Table 7 is cultured in the same seed medium as in Example 2. Then, 3 ml of each resulting seed culture liquor is inoculated in 16 ml of the same fermentation medium as used in Example 2, and cultured at 340C for 30 hours. In the course of culturing, pH of the culture liquor is adjusted to 6.5-8.0 while adding 0.5 ml of sterile 10% urea solution thereto. Amount o L-glutamic acid accumulated in the culture liquors are given in Table 7. Table 7 Strain L-glutamic acid (g/l) ATCC 13032 2.0 H - 3283 31.0 ATCC 13869 3.0 H - 3286 30.5 Example 5 Each strain identified in Table 8 is inoculated in a 250 ml-Erlenmeyer flask containing 30 ml of a sterile seed medium (pH 7.2) comprising 10 g/l glucose, 5 g/l meat extract, 10 g/l peptone and 3 g/l yeat extract, and cultured with shaking (220 rpm) at 300C for 24 hours. Then, 30 ml of the resulting seed culture liquor is put into a 2 Q Erlenmeyer flask provided with baffle containing 300 ml of a sterile fermentation medium (pH 7.4) comprising 100 g/l glucose, 3 g/l XH2PO4, 0.5 g/l MgSO4.7H2O, 10 g/l ammonium sulfate, 10 mg/l FeSO4 7H2O, 10 mg/l nicotinic acid, 100 pg/l thiamine hydrochloride, 100 pg/l biotin, 20 g/l corn steep liquor, 20 g/l sodium L-glutamate and 30 g/l CaCO3, and cultured with shaking (220 rpm) at 320C for 4 days. Amounts of L-proline accumulated in the culture liquors are given in Table 8. Table 8 Strain L-proline (g/l) ATCC 13032 0.5 H - 3334 18.5 FERM-P 4045 24.0 H - 3336 38.0 H - 3337 37.0 ATCC 13870 1.0 H - 3338 38.5 ATCC 13869 0.5 H - 3356 25.5 H - 3357 27.0 Example 6 In this example, using each strain identified in Table 9 as a seed strain and the same seed culture medium as obtained in Example 5, the resulting culture liquor is subjected to a main fermentation in the same manner and in the seme culture medium as in Example 5 except that the concentration of ammonium sulfate to be contained therein is changed to those shown in Table 9. Fount of L-proline accumulated in the culture liquors are given in Table 9. Table 9 Ammonium sulfate (NH4 concentra- H-3338 H-3357 F@RM-P 4045 tion M) 0 15.0 11.0 2.0 0.02 21.5 17.5 8.0 0.05 36.0 25.0 13.0 0.2 38.0 27.0 21.0 0.5 37.0 26.5 33.0 1.0 32.0 20.0 35.5 2.0 19.0 12.0 29.0 Example 7 Strain H-3336 or H-3338 is inoculated in a 2 @ - Erlenmeyer flask provided with baffle containing 200 ml of a sterile seed medium comprising the following components, and cultured with shaking (22C rpm) at 300C for 24 hours. Seed Culture tedium Sucrose 60 g/l KH2PO4 2 g/l MgSO4-7H2O 0.5 g/l FeSO4.7H2O 10 mg/l MnSO4-4H20 10 mg/l Corn steep liquor 10 g/l Thiamine hydrochloride 100 g/l Biotin 100 pg/l Urea 3 g/l pH 7.4 Then, 100 ml of the seed culture liquor is put into a 2 l-jar fermenter containing 700 ml of a sterile fermentation medium comprising the following components, and cultured with aeration-stirring at 320C. In the course of culturing, pH of the culture liquor is adjusted to 7.0 by using acetic acid solution as a continuous weed. Fermentation.Medium Sucrose 20 g/1 Ammonium acetate 7 g/l MgSO4.7H2O 0.6 g/l KH2PO4 4 g/l Ammonium sulfate 5 g/l FeSO4.7H2O 10 mg/l MnSO4-4H2O ' 10 mg/l CuSO4.5H2O 1 mg/l Thiamine hydrochloride 100 g/l Biotin 100 g/l pH 7.4 When culturing is continued for 72 hours1 acetic acid is consumed at a rate of 15% to the starting culture liquor, and amounts of L-proline accumulated in the culture liquors of H-3336 and H-3338 are 45 g/l and 43 g/l, respectively. Amounts of L-proline, in case where corresponding parent strains FERM P-4045 and ATCC 13870 are cultured at the same time under the same conditions as a control, are 28.5 g/l and 0.7 g/l.

Claims: CLAIMS 1 A process for producing L-glutamic acid or L-proline by fermentation which comprises culturing an L-glutamic acid or L-proline producing microorganism belonging to the genus Corynebacterium or Brevibacterium in a nutrient medium until L-glutamic acid or L-proline is accumulated in the culture liquor and thereafter recovering said L-glutamic acid or Lproline therefrom, characterized in that the microorganism used is a mutant strain having or having been endowed with a resistance to at least one purine analog and/or at least one pyrimidine analog. 2 A process according to claim 1, wherein said microorganism is a mutant strain of Corynebacterium glutamicum or Brevibacterium lactofermentum. 3 A process according to claim l or 2, wherein the mutant microorganism has resistance to one or more of the following purine analogs: 6-mercaptoguanine, 8-azaguanine, 2-fluoroadenine, tubercidin, 6methylpurine, 8-azaxanthine, 8-azaadenine, 8-mercaptoguanosine, 6mercaptoguanosine, 2-aminopurine, 2-amino-6-mercaptopurine, decoyinin or psicofuranine and/or resistance to one or more of the following pyrimidine analogs: 5-bromouracil, 6-azauracil, 5-fluorouracil, 5-bromo-2-deoxyuridine, 2-thiouracil, 6-methyl-2-thiouracil or amicetin. 4 A process according to any one of claims 1-3, wherein said culturing is conducted at 20 to 40&commat;C for l to 8 days. 5 A process according to any one of claims 1-4, wherein the mutant microorganism strain is an L-proline producing mutant and the culture medium contains from 0.05 to 0.5 M of NH4+ ion. 6 A process according to any one of the preceding claims, wherein said mutant microorganism strain is: Corynebacterium glutamicum H-3280, Corynebacterium glutamicum H-3281, Corynebacterium glutamicum H-3282, Corynebacterium glutamicum H-3283, Corynebacterium acetophilum H3336, Corynebacterium acetophilum H-3337, Corynebacterium acetoacidphilum H-3338, Brevibacterium lactofermentum H3284, Brevibacterium lactofermentum H-3285, Brevibacterium lactofermentum H-3286, Brevibacterium lactofermentum H-3287, Brevibacterium lactofermentum H-3356, or Brevibacterium lactofermentum H-3357. 7 A biologically pure culture of a microorganism having the characteristics of and identifiable with any of the following: Corynebacterium glutamicum H-3280, Corynebacterium glutamicum H 3281, Corynebacterium glutamicum H-3282, Corynebacterium glutamicum H-3283, Corynebacterium acetophilum H-3336, Corynebacterium acetophilum H-3337, Corynebacterium acetoacidphilum H-3338, Brevibacterium lactofermentum H-3284, Brevibacterium lacto fermentum H-3285, Brevibacterium lactofermentum H-3286, Brevibacterium Aactofermentum H-3287, Brevibacterium lacto fermentum H-3356, and Brevibacterium lactofermentum H-3357.

Inventors: AZUMA, TOMOKI, HATTORI, KIYOJI, HIRAO, TOSHIHIKO, NAKANISHI, TOSHIHIDE, YONEKURA, HIDEAKI
Applicants: KYOWA HAKKO KOGYO CO., LTD.
Application year: 1983
Main classification: C12P13
IPC codes: C12P13