Process for producing L-leucine
BACKGROUND OF THE INVENTION 
     This invention relates to a process for producing L-leucine by bacterial fermentation. More particularly, it relates to a process for producing L-leucine by fermentation with mutant microorganisms of the genus Brevibacterium or Corynebacterium. 
     L-leucine is one of the essential amino acids. It has been used as an additive to human and animal diets and as a raw material for pharmaceutical products. 
     It has been reported heretofore that L-leucine can be obtained by using a microorganism which requires an amino acid such as isoleucine and methionine for its full growth (U.S. Pat. No. 3,668,073) and a mutant which is resistant to 2-thiazolealanine (British Pat. No. 1,335,210). However, the L-leucine producing ability of these known strains is generally considered to be insufficient for commercial production. 
     THE INVENTION 
     Processes have now been discovered for producing L-leucine in remarkably large quantities by fermentation. More specifically a process has been discovered for producing L-leucine which comprises culturing an L-leucine producing microorganism of the genus Brevibacterium or Corynebacterium under aerobic conditions in an aqueous medium containing a source of assimilable carbon and nitrogen, inorganic salts and nutriments at a pH or 5 to 9 until L-leucine accumulates in the medium, and recovering the accumulated L-leucine; said microorganism being characterized by: 
     1. requiring at least one of isoleucine, threonine or methionine as a growth nutriment, and 
     2. being resistant to feedback inhibition by leucine and analogs of leucine. 
     The presently preferred L-leucine producing microorganisms are artificially produced mutants of strains of Brevibacterium and Corynebacterium obtained by conventional mutant inducing procedures utilizing irradiation by X-rays or ultraviolet light or chemical treatment with nitrosoquanidine, diethyl sulfate or diethyl nitrite. The preferred strains for producing such mutants are Brevibacterium lactofermentum and Cornebacterium glutamicum. As aforesaid the useful microorganisms are identified by screening for requirements of isoleucine, threonine or methionine and for resistance to feedback inhibition by leucine and its analogs. 
     The microorganisms useful in the invention can be isolated by the replica method from growth colonies on the plate according to specific nutrient requirements, which are determined by standard methods known in the art. The mutant strains having resistance to feedback inhibition and/or repression of leucine can be isolated from the parent strains and identified by resistance to feedback by leucine or analogs such as β-hydroxyleucine, trifluoroleucine, 2-thiazolealanine, 2-amino-4-methyl-hexanoic acid, glycylleucine, α-aminobutyric acid, β-hydroxyvaline, thiaisoleucine. The compounds 2-thiazoleanaline and β-hydroxyleucine are particularly useful for such screening. 
     Particularly suitable mutant strains for use in the process according to the present invention are Brevibacterium lactofermentum AJ-3718 (FERM-P 2516) which is resistant to 2-thiazolealanine and β-hydroxyleucine and requires isoleucine and methionine, Brevibacterium lactofermentum AJ-3452 (FERM-P 1965) which is resistant to 2-thiazolealanine and β-hydroxyleucine and requires threonine, and Brevibacterium lactofermentum AJ-3719 (FERM-P 2517) which is resistant to 2-thiazolealamine and β-hydroxyleucine and requires isoleucine and methionine. These three mutants were all derived from the parent strain Brevibacterium lactofermentum ATCC 13869. Corynebacterium glutamicum AJ-3453 (FERM-P 1966) which is resistant to 2-thiazolealanine and β-hydroxyleucine and requires isoleucine, Corynebacterium glutamicum AJ-3455 (FERM-P 1968) which is resistant to 2-thiazolealamine and β-hydroxyleucine and requires threonine, and Corynebacterium glutamicum AJ-3720 (FERM-P 2518) which is resistant to 2-thiazolealeanine and β-hydroxyleucine and requires isoleucine (all of which were derived from Corynebacterium glutamicum ATCC 13032) are also particularly useful in this process. 
     The FERM-P number shown herein is the deposit accession number of the Fermentation Research Institute, Agency of Industrial Science of Technology, the Ministry of the Industrial Trade and Industry, Japan. 
     Conventional culture mediums may be used to produce L-leucine in accordance with the present invention. These normally include sources of assimilable carbon and nitrogen and the usual minor constituents such as inorganic salts and organic nutrients. Examples of the carbon source are carbohydrates glucose, fructose, maltose, starch hydrolysate, cellulose hydrolysate or molasses, organic acids such as acetic, propionic or succinic, alcohols such as glycerol, methanol or ethanol, any hydrocarbons such as n-paraffin. Useful nitrogen sources include ammonium sulfate, urea, ammonium nitrate, ammonium chloride or gaseous ammonia. Inorganic salts, such as phosphates, magnesium, calcium, ferrous, manganese and other minor metallic salts are generally present. Amino acids, vitamins, &#34;Aji-Eki&#34; (Brand Name of soybean protein hydrolysate), yeast extracts, peptone and casamino acid are preferably employed for good bacterial growth. Of course, isoleucine, threonine or methionine will be utilized. 
     The fermentation of the present invention is performed at a pH of from 5 to 9, at a temperature of 24° to 37°C under aerobic conditions for 2 to 7 days or until sufficient L-leucine accumulates. The pH of the culture medium can be adjusted by adding sterile calcium carbonate, urea, aqueous or gaseous ammonia, mineral acid or organic acid during the fermentation. 
     The L-leucine is recovered from the cultured broth by conventional methods. It may be identified by its RF values in paper chromatography, its ninhydrin reaction and by bioassay with Leuconostoc mesenteroides ATCC 8042. 
    
    
     The following experiment shows the difference in degree of feedback inhibition and repression by leucine analogue between the mutant strains employed in the present process and strains which do not have resistance to leucine analogues. It illustrates one of the steps employed in identifying microorganisms useful in this invention. 
     EXPERIMENT 
     1. A culture medium containing 10% glucose, 4% (NH 4 ) 2  SO 4 , 0.1% KH 2  PO 4 , 0.04% MgSO 4 .7H 2  O, 2ppm Fe +   + , 2ppm Mn +   + , 100γ/l biotin, 350γ/l thiamine hydrochloride, 2ml/dl soybean protein hydrolyzate and 5% CaCO 3  (sterilized separately), at pH 7.0 was prepared, 20ml aliquots of the medium were each placed in 500ml shaking flasks, and autoclaved at 110°C for 5 minutes. The media were supplemented with required nutrients as shown in Table 1, and were respectively inoculated with the seven strains listed in the table and cultured at 31°C for 24 hours. The strains had been cultured previously on a bouillon slant at 30°C for 24 hours. The microbial cells obtained from the broth after 24 hours&#39; cultivation were examined according to the modified method of Burns et al (J. Bacteriol, 91, 1570-1576, 1966) to determine how the activity of α-isopropylmalate synthetase was influenced by the presence or absence of L-leucine. Specifically, the cells were harvested, washed twice with 0.05M tris (hydroxymethyl) amino methane-HCl (pH 7.2) buffer, and suspended in the same buffer. The suspended cells were disrupted by treatment with a 10-KC sonic Oscillator (Toyo-Riko Type 50-S, 80W) for 30 min. The cell debris was removed by centrifugation at 13,000 g. Dialyzed crude extracts were prepared by dialyzing 10ml of the supernatant from the centrifugation in viscose tubing against 1000ml of the same buffer for 16 hours at 4°C with stirring. These dialyzed crude extracts were assayed for α-isopropylmalate synthetase activity. The mixture for determining activity contained (in 0.5ml); sodium α-ketoisovalerate, 5 μmoles; acetyl CoA, 0.5 μmoles; tris (hydroxymethyl) aminomethane-HCl (pH 8.5), 50 μmoles; potassium chloride, 35 μmoles; and dialyzed crude extract. After 8 min. at 30°C, the reaction was stopped by adding 3.0ml of 0.2% metaphosphoric acid in saturated sodium chloride. Acetyl CoA cleavage was determined by using the 5,5&#39;-dithiobis-2-nitrobenzoic acid assay of Srere and Brazil for free --SH groups [Srere, P., Brazil, H. and Gonen, L., Acta. Chem. Scand., 17, S129 (1963)]. 
     The 5,5&#39;-dithiobis-2-nitrobenzoic acid assay was standardized with CoA. A reaction mixture from which α-ketoisovalerate had been omitted served to correct for the acetyl CoA cleavage due to other activities in the dialyzed crude extracts. 
     One activity unit was defined as that amount of enzyme which catalyzes the formation of 1 μmole of CoA per hour under the standard assay conditions. The specific activity was expressed in units per mg of protein. 
     Protein concentration was determined by the method of Lowry et al., [Lowry, O. H., N. J. Rosebrough; A. L. Farr, and R. J. Randall, J. Biol. Chem., 193, 265 (1951)]. 
     The results are shown in Table 1 from which it is apparent that the α-isopropylmalate synthetase of the preferred strains of this invention are resistant to feedback inhibition by L-leucine. 
     
                       Table 1                                                     
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         Amino Acids added                                                
         to the medium                                                    
                      Specific activity                                   
Strains               Amount   Without                                    
                                      With                                
employed   Amino Acids                                                    
                      (mg/dl)  Leu.   Leu.                                
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Brevibacterium                                                            
Lactofermentum                                                            
           threonine  20       100    50                                  
FERM-P 1841                                                               
Brevibacterium                                                            
Lactofermentum                                                            
           isoleucine &amp;                                                   
                      10                                                  
FERM-P 1858                                                               
           methionine 10       100    65                                  
Brevibacterium                                                            
Lactofermentum                                                            
           isoleucine &amp;                                                   
                      10       100    95                                  
FERM-P 2516                                                               
           methionine 10                                                  
Brevibacterium                                                            
Lactofermentum                                                            
           threonine &amp;                                                    
                      20       100    98                                  
FERM-P 1965                                                               
           isoleucine 20                                                  
Brevibacterium        30       100    100                                 
Lactofermentum                                                            
           methionine                                                     
FERM-P 2517                                                               
Corynebacterium                                                           
           isoleucine --       100    40                                  
glutamicum                                                                
ATCC 13032                                                                
Corynebacterium                                                           
glutamicum threonine  --       100    98                                  
FERM-P 1966                                                               
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     2. The repressive effects of L-leucine on α-isopropylmalate synthetase in leucine producers were examined according to the modified method of Burns et al. 
     The strains of Table 1 were separately innoculated in each of the following media: 
     a. The amino acid supplemented medium of Table 1. 
     b. The same medium containing an additional 0.2% L-leucine by weight. 
     c. The same medium containing an additional 0.5% L-leucine by weight. 
     Each innoculum was cultured for 24 hours at 30°C. The activity was determined as explained above in connection with Table 1. The results are shown in Table 2. 
     
                       Table 2                                                     
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   Amount of Leu.    Specific Activity                                    
Strains employed     0       0.2 %   0.5 %                                
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Brevibacterium lacto-                                                     
fermentum FERM-P 1841                                                     
                     1.80    0.70    0.48                                 
Brevibacterium lacto-                                                     
fermentum FERM-P 1858                                                     
                     2.00    0.81    0.30                                 
Brevibacterium lacto-                                                     
fermentum FERM-P 2516                                                     
                     5.00    4.90    4.85                                 
Brevibacterium lacto-                                                     
fermentum FERM-P 1965                                                     
                     4.51    4.25    4.26                                 
Brevibacterium lacto-                                                     
fermentum FERM-P 2517                                                     
                     4.80    4.75    4.70                                 
Corynebacterium                                                           
glutamicum ATCC 13032                                                     
                     2.25    0.28    0.05                                 
Corynebacterium                                                           
glutamicum FERM-P 1966                                                    
                     3.80    3.35    3.34                                 
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     It is apparent that the α-isopropylmalate activity of the preferred mutants of this invention is not significantly repressed even in the presence of large amounts of leucine. 
     The following non-limiting example is illustrative of the invention. 
     EXAMPLE 1 
     A culture medium containing 10% glucose, 4% (NH 4 ) 2  SO 4 , 0.1% KH 2  PO 4 , 0.04% MgSO 4 .7H 2  O, 2ppm Fe +   + , 2ppm Mn +   + , 100γ/l biotin, 350γ/l thiamine hydrochloride, 2 ml/dl soybean protein hydrolysate, and 5% CaCO 3  (sterilized separately, at pH 7.0 was prepared, 20ml aliquots of the medium were placed in 500 ml shaking flasks, and autoclaved at 100°C for 5 minutes. 
     The media were supplemented with the required amino acids as listed in Table 3, and were thereafter inoculated with strains shown in the table. 
     After 72 hours&#39; cultivation at 30°C, each culture broth was found to contain L-leucine in amounts shown. 
     
                       Table 3                                                     
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              Amino Acids Required                                        
                            L-leucine                                     
            Resis-  Amino Acids                                           
                               Amount Produced                            
Strains employed                                                          
            tance              (mg/dl)                                    
                                      (g/dl)                              
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Brevibacterium                                                            
lactofermentum                                                            
            --      Threonine  20     0                                   
FERM-P 1841                                                               
Brevibacterium                                                            
lactofermentum                                                            
            --      Isoleucine &amp;                                          
                               10     0                                   
FERM-P 1858         Methionine 10                                         
Brevibacterium                                                            
lactofermentum                                                            
            2-TA,   Isoleucine &amp;                                          
                               10                                         
FERM-P 2516 β-HL                                                     
                    Methionine 10     2.21                                
Brevibacterium                                                            
lactofermentum                                                            
            2-TA,   Threonine  20     2.10                                
FERM-P 1965 β-HL                                                     
Brevibacterium                                                            
lactofermentum                                                            
            2-TA,   Isoleucine &amp;                                          
                               20                                         
FERM-P 2517 β-HL                                                     
                    Methionine 30     2.20                                
Coryne. gluta-                                                            
micum ATCC 13032                                                          
            --      Isoleucine --     0                                   
Coryne. gluta-                                                            
micum ATCC 14296                                                          
            --      Threonine  --     0                                   
Coryne. gluta-                                                            
            2-TA,                                                         
micum FERM-P                                                              
            β-HL                                                     
                    Isoleucine 15     1.80                                
1966                                                                      
Coryne. gluta-                                                            
            2-TA,   Threonine  15     1.70                                
micum FERM-P                                                              
            β-HL                                                     
1968                                                                      
Coryne. gluta-                                                            
            2-TA,   Isoleucine 15     1.95                                
micum FERM-P                                                              
            β-HL                                                     
2518                                                                      
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 Note:                                                                    
 2-TA: 2-Thiazolealanine                                                  
 β-HL: β-Hydroxyleucine