GB1578057A - Method of producing l-valine by fermentation - Google Patents

Description

(54) IMPROVEMENTS IN OR RELATING TO A METHOD OF PRODUCING L-VALINE BY FERMENTATION (71) We, LES PRODUITS ORGANIQUES DU SANTERRE-ORSAN, a French Body Corporate, of 16, rue Ballu, 75009 PARIS, France, do hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement:- The present invention relates to a method producing L-valine by fermentation.

Numerous methods of production of L-valine have been proposed. The best results have been obtained with Brevibacterium and Corynebacterium mutants exhibiting auxotrophy in regard to isoleucine. More recently, in UK Specification No.

1433294, Ajinomoto has deseribed-and claimed a method of bioproduction of Lvaline by Brevibacterium and Corynebacterium mutants characterized mainly by their resistance to thiazolalanine. We found, however, that the amounts of L-valine produced are small and that L-valine is often produced in presence of significant amounts of other amino acids (e.g. L-threonine) resulting in an increase in extraction and purification costs.

L-valine is an amino acid that is essential for man and animals and therefore can be widely used, particularly in human and veterinary medicine. In order however, not to limit its application to pharmaceutical use, L-valine must be obtained economically and with an adequate purity.

We have discovered that considerable amounts of L-valine may be obtained in a fermentation broth if the fermentation is carried out by means of a new microorganism, as described later. In addition, we have shown the considerable advantages which can be achieved by the use of ammonium ion in the form of its acetate as a main source of nitrogen in the fermentation medium in order to obtain a high yield of L-valine. The abovementioned characteristics and advantages will appear more clearly from the following description.

The invention provides a method of producing L-valine by fermentation, wherein a Corynebacterium glutamicum strain which is characterized in that its growth is synergistically inhibited by a mixture of (i) cysteic acid and L-threonine or (ii) cysteic acid and L-valine, is cultivated in a fermentation medium containing at least one assimilable source of carbon and at least one assimilable source of nitrogen, and the organic and mineral nutrient constituents necessary for the development of the micro-organism.

Such strains can be obtained from L-glutamic acid producing strains by conventional mutation techniques by means of known mutagenic agents.

By "synergistic inhibition" we mean that the mutant strain in the presence of a mixture of cysteic acid and L-valine or cysteic acid and L-threonine exhibits practically no growth, whereas each of these compounds individually, viz. cysteic acid, L-valine or L-threonine, has a slightly unfavourable influence upon the growth of the strain. In other words if the strain is one the growth of which is inhibited to a greater extent when in the presence either of mixture (i) or (ii) then when in the presence of only one of the individual compounds of the mixture, then the strain has the characteristic feature of being synergistically inhibited by that mixture.

This synergistic phenomenon is illustrated in Example No. 1.

Another characteristic feature of the strains used is that the production of L valine is very favourably influenced if ammonium acetate is used as the main source of nitrogen.

The highly favourable influence of ammonium acetate on the production of Lvaline is illustrated in Example No. 3 wherein it is seen that high yields of L-valine are obtained when introducing from 20 to 35 g/l of ammonium acetate into the medium, a particularly high yield being obtained when using at least 25 girl.

Ammonium acetate may be used either alone or mixed with other conventional sources of nitrogen, such as other ammonium salts, urea, molasses from the sugar industry, corn-steep liquor, peptone, yeasts extract, meat extract or various hydrolyzates.

The method of the invention is accomplished by using the modified (mutant) Corynebacterium glutamicum strain deposited at the Institute Pasteur in Paris under n" 1-026 on April 14, 1976, which mutant strain has also been deposited in Japan on 21 April 1977 under nO 4035, with the FERMENTATION RESEARCH INSTITUTE - Agency of Industrial Science and Technology.

A purified preparation of the 1-026 strain has been isolated by clonage on a nutrient medium containing L-valine of the mutant I-026 strain which has itself been obtained by mutation, by means of N-methyl, N'-nitro-N-nitroso-guanidine (N T G), of a glutamic-acid producing mother strain, according to the following procedure: Glutamic-acid producing

Corynebacterium giutamicum (mother strain) 1 Mutation by mean; of NTG Corynebacterium glutamicum mutant strain I-026 Clonage on nutrient medium + L-valine Corynebacterium glutamicum purified strain I-026 The characteristics of the I-026 strain compared with those of the initial, glutamic-acid producing mothel strain are given in the Appendix. A characteristic difference between the two strains lies in their response to the acetoin production test or Voges Proskauer test.

As a source of carbon, use can be rnade of for example, carbohydrates, glucose, fructose, mannose, galactose, sucrose or molasses, either pure or mixed.

The mineral compounds added include the necessary mineral salts such as magnesium sulfate, dihydrogen potassium phosphate, monohydrogen potassium phosphate, iron sulfate and other salts employed, e.g., manganese and calcium.

It is also necessary to add vitamins, especially where the culture medium is exclusively synthetic.

The fermentation can be carried out under usual aerobic conditions, e.g. with agitation and introduction of sterile air at a temperature between 25"C and 37"C, preferably from 250C to 330C. The pH is preferably maintained between 5 and 9, more preferably between 7 and 8.5. During the cultivation, the pH tends to decrease and is therefore adjusted by introducing, for example, ammonia.

The cultivation generally last from 2 to 5 days. During that time a considerable amount of L-valine accumulates in the fermentation liquor. Other amino-acids are also found, but in negligible amounts compared with the amount of L-valine produced.

The following non-limitative examples will allow the manner in which methods embodying the invention are carried out to be better understood.

Example No. 1.

The mother strain and the purified mutant 1-026 strain are subjected to comparative growth tests in tubes, in an amount of 5 ml of medium per tube. The seeding is performed from a suspension of the strain washed once in a salt solution.

The culture is allowed to incubate for 24 hrs at 300C on a rotary agitator.

The optical densities are read after dilution to '/,ooth at 650 nm on a Beckman spectrophotometer (UV 24, l0-mm vat). Minimum media composition

Minimum medium Hypetonic minimum M.m. medium H.m.m.

Saccharose 20 g 200 g Ammonium sulfate 3 g 3 g Dihydrogen potassium phosphate 1 g 1 g Monohydrogen potassium phosphate 3 g 3 g Magnesium sulfate 0.1 g 0.1 g Calcium chloride 0.001 g 0.001 g Trace-elements solutions 1 ml 1 ml Biotin 30 g 30 g Thiamine 100 g 100 g Water 1000 ml 1000 ml pH before sterilization 8 8@, Sterilization 20 min. 1.2 kg/cm2 20 min. 1.2 kg/cm2 Composition of trace-elements solution

Zinc sulfate 8.8 g Ferric chloride 970 mg Cupric sulfate 393 mg Sodium borate 88 mg Manganous chloride 72 mg Ammonium molybdate 37 mg Water 11 Results of comparative growth test

Mutant 1-026 Mother strain strain Optical densities (per cent) (per cent) H.m.m. 100 100 H.m.m. + cysteic acid (2 g/l) 54.6 53.1 H.m.m. + L-valine (0.25 g/l.) 100 77.7 H.m.m. + L-threonine (0.25 g/l.) 104 80 H.m.m. + cysteic acid (2 g/l.) + L-valine (0.25 g/l.) 43.2 3 H.m.m. + cysteic acid (2 g,/l.) gX L-threonine (0.25 g/l.) 53.7 4.7 M.m. 100 100 M.m. + cysteic acid (2 g/l.) 59 55 M.m. + L-valine (0.25 g/l.) 122.5 98.8 M.m. + L-threonine (0.25 g/l.) 124.9 96.6 M.m. + cysteic acid (2 g/l.) +L-valine (0.25 g/l)) 65 29.7 M.m. + cysteic acid (2 g/l.) + L-threonine (0.25 g/l.) 61.7 32.2 M.m. = Minimum medium H.m.m. = Hypertonic minimum medium Example No. 2.

Laboratory production of L-valine Carbohydrate source used: Sugar molasses Use is made of the purified mutant I-026 strain cultivated on inclined nutrient gelose for 24 hrs at 300 C. After the incubation of the gelose, there is seeded a preculture medium of the following composition:

Corresponding Molasses to 30 g of sugar Ammonium sulfate 5 g Monohydrogen potassium 1 g phosphate Magnesium sulfate 0.25 g Biotin 10 jtg Thiamine 100 g Water 1000 ml Volume used 50 ml in 500-ml Erlenmeyer flask pH before sterilization 7 Sterilization 20 min at 1.2 kg/cm' pH after sterilization 6.7 After 8-hour incubation at 300C on a rotary agitator at 200 r.p.m. the following production medium is seeded (seeding rate 6 per cent).

Corresponding Molasses to 90 g of sugar Ammonium acetate 30 g Ammonium phosphate 0.5 g Monohydrogen potassium phosphate 0.5 g Magnesium sulfate 0.25 g Manganous sulfate 0.010 g Ferrous sulfate 0.010 g Biotin 30 yg Thiamine 100 118 Calcium carbonate 30 g Lactic acid 5 g Water 1000 ml Volume used 20 ml in 500-ml Erlenmeyer flask pH before sterilization 7.5 Sterilization 20 min at 1.2 kgXcm2 pH after sterilization 7.2 After 72-hr fermentation on a rotary agitator at 200 r.p.m. at 300 C, there are obtained 18.5 g of L-valine/litre.

Example No. 3.

Influence of the nitrogen source used upon the production of L-valine.

Use is again made of the purified mutant strain 1-026. The procedure is the same as in Example No. 2 until the pre-cultivation stage. For the bioproduction stage the procedure is also the same as in Example No. 2, the medium used being identical except for the source of nitrogen whose influence upon the production of L-valine is investigated.

The amounts of L-valine obtained after 72-hr fermentation, depending upon the source of nitrogen used, are summarized in the following table:

Amount of NI-14+ Used 20 g.'l 75 g'l 30 g'l 35 g I Ammonium salt used cetate Ammonium acetate 10.6 g 'l 11.7 gll 16.9 g/l 18.2 g I Ammonium sulfate 11.4 gI 10.8 g 1 12 g 'l 12.3 g,'l Ammonium chloride 11.6 g/l 11.1 g I 11.3 g '1 10.8 g 'I Example No. 4: The conditions of the experiment are similar to those described in Example No. 2. This time, however, the source of nitrogen used is a mixture of ammonium salts, vis. acetate and sulfate. The following table summarizes the amounts of Lvaline obtained depending upon the respective acetate and sulfate amounts used, the proportion of ammonium remaining constant.

Ammonium salt, g. '1 Amount of valine Ammonium and Ammonium produced, g 'I acetate sulfate 0 25 11.7 5 21.5 12.0 10 17 12.4 15 12.8 19.7 29 8.5 14.0 25 4.2 15.9 30 0 ) 16.0 Example No. 5.

The same procedure as in Example No. 2 is applied until the precultivation stage. The bioproduction is also performed as in Example No. 2. but the medium has the following composition:

Saccharose 90 g Pulverized corn-steep 10 g liquor Ammonium acetate I 30 g Dihydrogen potassium phosphate 0,5 g Monohydrogen potassium phosphate 0.5 g Magnesium sulfate 0.25 g Maganous sulfate 0.010 g Ferrous sulfate 0.010 g Biotin @ 30 g Thiamine 100 g Calcium carbonate 30 g Lactic acid 5 g Water 1000 ml After 72 hrs, 13 gfl of L-valine are obtained.

Example No. 6.

Semi-industrial production of L-valine 10) Preparation of seed The purified mutant Corynebacterium 1-026 strain is cultivated on inclined nutrient gelose for 24 hrs at 30 C. In a 6-litre Erlenmeyer baffle-flask, there is seeded, by means of two tubes of gelose, 1.5 1. of a medium having the following composition: For one litre of demineralized water

Molasses expressed as sugar 30 g Ammonium sulfate 5 g Monohydrogen potassium phosphate 1 g Magnesium sulfate 0.25 g Biotin 10 g Thiamine hydrochloride 100 g Yeast extract 3 g The culture is allowed to incubate for - 18 hrs at 300C on a rotary agitator.

2") Pre-cultivation In a 10litre vat containing 4 1. of demineralized water the following constituents are dissolved or suspended.

For one litre of demineralized water

Molasses expressed as sugar 30 g Ammonium sulfate 5 g Magnesium sulfate 0.25 g Monohydro gen potassium phosphate Biotin 30 ,g Thiamine hydrochloride 0.1 mg Silicones 0.5 g The medium is autoclave-sterilized for 30 min at 1250C. The pH after the sterilization is 5.70 and is adjusted to 7.80 with ammonia.

The 4 1. are thereafter inoculated with 320 ml of the seed described previously.

The pH is then equal to 7. Incubation Is allowed to take place for about 14 hrs with agitation at 600 r.p.m. and aeration of l vol./l vol. min. The pH is allowed to change progressively and reaches 5.40 at the end of the incubation.

3 ) Production The producing medium has the following composition: For one litre of demineralized water

Molasses expressed as sugar 90 g Ammonium acetate 30 g Magnesium sulfate 0.25 g Manganous sulfate 0.01 g Ferrous sulfate 0.01 g Biotin 30 Cig Thiamine 100 .g Monohydrogen potassium phosphate 0.5 g Dihydrogen potassium phosphate 0.5 g A small 14-l. fermentator is used for this stage.

The constituents are dissolved in 7 l. of water; and pH is then 7.20. Autoclave sterilization is performed for 30 min at 123"C; the pH after the sterilization is 6.75.

The 7-i. medium is inoculated with 700 ml of preculture. The pH rises to 7.30.

Incubation is allowed to take place for 64 hours at 30"C. Agitation -- 600 r.p.m.

Aeration - I vol/l vol.min. The pH rises spontaneously. from 7.30 to 8, i.e. the value to which it is adjusted. During the incubation, molasses are supplied so as to maintain a residual sugar proportion of 3 per cent. The supply is discontinued so that the residual sugar proportion at the end of the fermentation be less than l per cent. A silicone-based anti-froth or defoaming agent is added according to requirements.

After 64 hrs there is obtained 373 g of L-valine, i.e. 36.4 g of L-valine per litre of broth. The yield expressed as the ratio to the sugar introduced is about 19 per cent. A small amount of leucine and isoleucine is found : 3.76 g of their mixture per litre of broth, about 50 per cent of which is introduced by the sugar molasses.

APPENDIX N 1

Glutamic-acid Corynebacterium producing Coryne- glutamlcum Characteristics bacterium glutamicum 1-026 Aspect Short, straight or Short, straight or slightly incurved slightly incurved bacilli (sometimes in bacilli (sometimes in V-arrangement) V-arrangement) Spores not found not found Gram + + Oxygen requirement aerobe aerobe Growth temperature 25 - 37 C 25 - 37 C Voges Proskauer - + Nitrates reduction @+ + H2S production - Indole production Use of citrate Gelatin liquefaction Urease + + Catalase + + Arginine dihydrolase Omithine decarboxylase Lysine decarboxylase Formation of acids on: Glucose + Mannose Saccharose + + Arabinose Rhamnose Melibiose Starch Mannitol Ribose + +

Claims (11)

1. WHAT WE CLAIM IS: l. A method of producing L-valine by fermentation, wherein a Corynebacterium glutamicum mutant strain, which is characterized in that its growth is synergistically inhibited by a mixture of (i) cysteic acid and L-valine or (ii) cysteic acid and L-threonine, is cultivated in a fermentation medium containing at least one assimilable source of carbon and at least one assimilable source of nitrogen, and the organic and mineral mutrient constituents necessary for the development of the micro-organism.
2. 2. A method according to claim !, wherein the micro-organism used is the Corynebacterium glutamicum deposited in the Collection of the Institut Pasteur in Paris under n" 1-026, on April 14, 1976, and in Japan at FERMENTATION RESEARCH INSTITUTE under n" 4035 on 21 April 1977.
3. 3. The method according to claim l or claim 2, wherein a carbohydrate is used as an assimilable source of carbon.
4. 4. A method according to claim l or claim 2, wherein sugar molasses is used as an assimilable source of carbon.
5. 5. A method according to any one of the preceding claims, wherein ammonium acetate is used as a source of assimilable nitrogen.
6. 6. A method according to claim 5, wherein the amount of ammonium acetate used is between 20 and 35 g per litre of fermentation medium.
7. 7. A method according to any one of the preceding claims, wherein the fermentation is conducted at a pH between 5 and 9 and a temperature between 25 and 37"C.
8. 8. A method according to claim 7, wherein the fermentation is conducted at a pH between 7 and 8.5 and ai a temperature tetween 25 and 33"C.
9. 9. Fermented media containing L-valine and obtained by the method according to any one of claims l to 8.
10. 10. A method substantially as described.
11. l l. Fermented media containing L-valine substantially as described.