NO137210B - PROCEDURES FOR THE PREPARATION OF L-TRYPTOPHANE BY CULTIVATING A CORYNEBACTERIUM GLUTAMICUM MICRO-ORGANISM - Google Patents

Description

The present invention relates to a method for producing L-tryptophan by cultivating a microorganism of the species Corynebacterium glutamicum.

L-tryptophan is an amino acid that is essential for er-

the nutrition of a variety of animals. It has found commercial use as forage and food additives and the like.

Until now, L-tryptophan has been produced by such methods as are described in US patents no. 2,999,051, no. 3,293,141,

and No. 3,385,762, Japanese Laid-Open Application No. 4632/64, French Patent No. 1,437,998 and DT-OS No. 2,037,763 (1972) in which an L-tryptophan-producing microorganism is grown in a nutrient medium containing indole or anthranilic acid. However, such a method requires an expensive precursor, i.e. indole or anthrani-

acid, and therefore the method entails disadvantageously high material costs. Since the precursors also have an inhibitory effect on the micro-organisms' growth, large amounts of them cannot be added to the culture medium, which significantly reduces the accumulated amount of final product.

Other known methods include the fermentation of histidine-requiring mutant microorganisms (US Patent No. 3,594,279); a mutant microorganism which is resistant to 5-methyltryptophan (French publication no. 2 059 715); and a mutant of Brevibacterium flavum No. 2247, i.e. Br. flavum 12-555, which requires phenylalanine and tyrosine and is resistant to 5-methyltryptophan (Nihon Nogei Kagakukai Annual Meeting for 1971, Abstract of Lectures, page 153). Said French publication No. 2,059,715 also states that L-tryptophan production by microorganisms resistant to 5-methyl-DL-tryptophan can be increased by inducing a phenylalanine and/or tyrosine requirement in the microorganism or by inducing a resistance to compounds analogous to phenylalanine or tyrosine. However, apart from the publication's practice examples which are limited to microorganisms with resistance to 5-methyl-DL-tryptophan and a requirement for phenylalanine or tyrosine for growth, there is no indication of specific mutants or of methods for obtaining mutants having such resistance to compounds analogous to phenylalanine or tyrosine. Furthermore, the yields of L-tryptophan when growing the mutants according to the above-mentioned publications are approx. 500 mg/l, when the fermentation is carried out in flasks. Even when the fermentation is carried out on a large scale under optimal conditions, the yield is only approx. 1.9 g/l (Nihon Nogei Kagakukai Annual Meeting for 1971, Abstract of Lectures, page 153). Such yields are so low that it is practically impossible to utilize the methods on an industrial scale. As a result, there is a need for an improved process for the industrial production of L-tryptophan.

According to the present invention, a method is thus provided for the production of L-tryptophan by cultivating a microorganism of the species Corynebacterium glutamicum in an aqueous nutrient medium - containing sources of carbon, nitrogen, inorganic nutrients and necessary growth factors, after which the L- tryptophan is extracted from this, characterized in that one of the strains Corynebacterium glutamicum ATCC 21842, ATCC 21843, ATCC 21844, ATCC 21845, ATCC 21846, ATCC 21847, ATCC 21848, ATCC 21849, ATCC 21850 and ATCC 21851 is used as a microorganism.

Corynebacterium glutamicum is a species of microorganism belonging to the genus Corynebacterium which is usually characterized by straight to slightly curved rods with irregularly colored segments, sometimes granules. They often show club-shaped swellings. Cleavage divisions produce angular and palisade (picket) arrangements of cells. Non-motile with exceptions among plant pathogens. Gram-positive, but sometimes young and old cells lose their color easily. Granules constantly Gram-positive. Usually completely aerobic, but microaerophilic or even anaerobic species occur. Catalase-positive. Can in some cases liquefy gelatin. In some cases produces nitriles from nitrates. In some cases ferments sugars. but rarely, if ever, produces a high degree of acidity. Many species oxidize glucose completely to C02 and H^O without

to produce noticeable gas. Corynebacterium glutamicum is a synonym for Micrococcus glutamicum stated in US patent no. 3,003,925.

The microorganisms used in the present method are mutants (analogue-resistant mutants) of L-tryptophan-producing strains of Corynebacterium glutamicum. The strains require phenylalanine and tyrosine (including mutants of a so-called "leaky" type) and are able, like their parent strains, to

grow in a minimal medium (consisting of 1% glucose, 0.1% (NH^)H2P0^, 0.02$ KC1, 0.02% MgSO^. 7H20, 30 yg/1 biotin, 10 mg/l vitamin B- ^-hydrochloride, 1 ml/l of a solution of trace metal salts

(an aqueous solution containing 88 mg Na^B^O^.lOH^O, 37 mg (NH4)6.Mo7O2l|.4H20, 72 mg MnCl2.4H20, 970' mg FeCl3.6H20,

8.8 mg ZnSOi|.7H 2 O and 20 mg CuSO^'. 5H20 per liter and 2% agar) mixed with phenylalanine and tyrosine.

In connection with the aforementioned•resistance to analogues

to tyrosine or phenylalanine, it should be noted that some of these analogues sometimes have a common structure. E.g. 4-fluorophenylalanine can be considered a tyrosine analog as well as a phenylalanine analog. I. accordance with this must be understood

that the necessary resistance may be to an analogue of both tyrosine and phenylalanine.

Any synthetic or natural medium can be used as a fermentation medium in the present method, as long as it contains a suitable carbon source, a nitrogen source, inorganic materials and trace amounts of nutrients that are necessary for the particular mutant strain.

Any carbon source and nitrogen source can be used in the medium, as long as it can be utilized by the microorganism. As a carbon source, e.g. carbohydrates, such as glucose, fructose, sucrose, maltose and mannose, sugar alcohols such as sorbitol, mannitol, glycerol, starch, starch hydrolyzate liquid and molasses are used. Furthermore, various organic acids such as pyruvic acid, lactic acid, acetic acid, fumaric acid and gluconic acid can be used, and lower alcohols such as ethanol.

As a nitrogen source, the following substances are suitable: ammonia, various inorganic and organic ammonium salts, such as ammonium chloride, ammonium sulfate, ammonium carbonate and ammonium acetate, urea and other nitrogenous materials and nitrogenous organic materials such as peptone, "NZ-Amin", meat extract, yeast extract, casting water, casein hydrolyzate, fishmeal or dissolution products thereof and chrysalis hydrolyzate.

Monopotassium dihydrogen phosphate, dipotassium monohydrogen phosphate, magnesium sulphate, sodium chloride, iron (II) sulphate, manganese sulphate and calcium carbonate can be used as inorganic materials.

With regard to the vitamins and amino acids that are necessary for the growth of the mutant strains, they must of course be present in the medium. However, it is not necessary that they are added to the medium separately, only that they are added to the medium together with other components as described above, i.e. that certain natural components can result in a satisfactory amount of the specific weight-promoting factors.

Cultivation is carried out under aerobic conditions such as shaking or aeration-stirring. A suitable cultivation temperature is usually 20° - 4d°C. It is desirable to keep the medium's pH value around neutrality during the entire cultivation in order to achieve a high yield, but these temperature and pH conditions are not decisive for the execution of the present method. Cultivation is usually carried out for 2 - 5 days, whereby a significant amount of L-tryptophan accumulates in the medium.

After completion of the cultivation, the cells are removed and L-tryptophan is recovered from the culture liquid by any well-known method such as treatment with activated carbon and treatment with ion exchange resin.

In order to obtain mutant strains suitable for use in the present method, bacteria belonging to the species Corynebacterium glutamicum are artificially mutated into phenylalanine- and tyrosine-requiring strains. These mutant strains are then mutated again by artificial means. The resulting mutant strains are then grown on minimum agar medium mixed with at least one tryptophan analog and at least one analog of phenylalanine or tyrosine at a concentration not lower than that which would normally inhibit the growth of the parent strain.

From colonies grown on this medium, the desired strains are isolated. E.g. is the development of mutant strains of the phenylalanine- and tyrosine-requiring bacterium, Corynebacterium glutamicum KY 9456, ATCC 21854, shown below.

The strain (KY 9456, ATCC 21854) undergoes growth inhibition by tryptophan analogues and phenylalanine analogues and tyrosine analogues in the concentrations indicated in Table 1 when grown in the minimum agar medium mixed with phenylalanine and tyrosine.

The values given in Table 1 show the minimal growth inhibitory growth concentrations of the above analogue. They are determined as follows. Corynebacterium glutamicum KY 9456, ATCC 21854, is grown overnight on a broth agar slant substrate. The cells thus obtained are suspended in an isotonic sodium chloride solution and a portion of the resulting suspension containing 10 6 - 10 7 cells is spread evenly over an agar plate minimum medium supplemented with one of the above analogues in a Petri dish (85 mm in diameter) and grown at 30°C for 3 days. The degree of inhibition is assessed by observing the growth on the medium.

With regard to the MIC determination on the mother strain, it is sorted

the analog-resistant strains by spreading the cells of an extreme mutant strain of Corynebacterium glutamicum KY 9456, ATCC 21854, on a minimum agar medium supplemented with a tryptophan analog and a phenylalanine analog or tyrosine analog in a concentration above growth inhibitory concentration for the mother strain. After a suitable incubation time, cells are isolated from colonies which have been able to form on the medium. These cells are then cultivated in a suitable manner for the development of microbe populations for industrial purposes.

A solid agar medium containing the analogues, a carbon source, nitrogen source, inorganic salts, tyrosine, phenylalanine, biotin, vitamin B^^ and trace amounts of other nutrients, which are necessary for the growth of the mother bacterium, is usually used as a medium for sorting out the analogue-resistant mutant. . As regards trace nutrients, natural materials can be used which normally contain the nutrients.

Mutant strains of Corynebacterium glutamicum obtained by the above screening process have been deposited in the American Type Culture Collection, Rockville, Maryland, and are freely available to the public. These strains have been assigned the accession numbers ATCC 21342 to ATCC 21851.

The mutant strains of Corynebacterium glutamicum, which are used in the present method, can produce L-tryptophan in much higher yields than other L-tryptophan-producing strains of Corynebacterium glutamicum which are used in the known methods. In particular, a comparison of the present mutant strains with the strain from Japanese laid-off application no. 14395/63 (tyrosine- and phenylalanine-requiring strain of Corynebacterium glutamicum) and the strain from US patent no. 3,594,279 (histidine-requiring strain of Corynebacterium glutamicum) shows as shown in example 1 below, that the present bacterium produces L-tryptophan in a yield that is from about 15 to about 20 times higher than that of the known strains. The French publication No. 2 059 715 states that the L-tryptophan yield by means of Corynebacterium acetoglutamicum AJ-3293 (with a need for phenylalanine and tyrosine and resistance to 5-methyltryptophan) is 0.186 mg/ml and the L-tryptophan yield by means of Micrococcus glutamicum AJ -3295 (with resistance to 5-methyltryptophan) is 0.0235 mg/ml. Nihon Nogei Kagakukai Annual Meeting for 1971j Abstract and Lectures (page 153) states that the L-tryptophan yield using Brevibacterium flavum 12-555 (which requires phenylalanine and tyrosine and has resistance to 5-methyltryptophan) is 1.9 g/l when the fermentation is carried out on a large scale under optimal conditions. In contrast, a mutant strain of Corynebacterium glutamicum used in the present method, which requires phenylalanine and tyrosine and has resistance to at least one tryptophan analog and to at least one tyrosine analog or phenylalanine analog or both, can produce L-tryptophan in yields that are unexpectedly much higher than those obtained with the known bacteria. Thus, the present method represents a very advantageous industrial process for the production of L-tryptophan.

The method according to the invention is illustrated in more detail by the following examples.

Example 1

In this example, an L-tryptophan-producing mutant strain of Corynebacterium glutamicum is used, which requires tyrosine and phenylalanine and has resistance to 5-methyltryptophan, 6-fluorotryptophan and 4-fluorophenylalanine, namely the strain Corynebacterium glutamicum Trp -1, ATCC 21842. The mutant strain is grown in an inoculum containing 2% glucose, 1% peptone, 1% yeast extract and 0.3% NaCl at 30°C in

24 hours. 1 ml of the resulting inoculum is inoculated into 10 ml of a fermentation medium of the following composition in a 250 ml Erlenmeyer flask:

The cultivation is carried out at 30°C for 4 days with shaking, whereby L-tryptophan is produced in a yield of 3.6 mg/ml.

2 liters of the resulting culture fluid is subjected to centrifugation to remove the microbe cells and CaCO^3, and the resulting supernatant is passed through a layer of a strong acid cation exchange resin, trade name "Diainon SK-104" (H+<->form), for adsorption of L-tryptophan. After washing with water, the resin is subjected to a

: elution with 0.5 N aqueous ammonia and the resulting eluate is concentrated to obtain crude crystals of L-tryptophan. The crude crystals are dissolved in a small amount of warm 50% aqueous ethanol. The resulting solution is decolorized with activated carbon and cooled, whereby 3-5 g of L-tryptophan are crystallized.

When the parent strain of this mutant strain, i.e., Corynebacterium glutamicum KY 9456, ATCC 21854, which requires tyrosine and phenylalanine, and Corynebacterium glutamicum KY 9104, ATCC 21334, which requires histidine (US Patent No. 3,594,279), are grown under under the same conditions, the yields of L-tryptophan are respectively 0.2 mg/ml and 0.2 mg/ml.

Example 2

In this example, L-tryptophan-producing mutant strains of Corynebacterium glutamicum are used which have the properties indicated in Table 2 below. Each of these strains is cultivated in the inoculum medium described in example 1 for 24 hours. One ml of each of the seed cultures thus prepared is transferred into 250 ml Erlenmeyer flasks containing 10 ml of a fermentation medium consisting of:

Cultivation is carried out at 30°C for 4 days with shaking. The results are shown in table 2.

phe : requires phenylalanine

bull : requires tyrosine

5MT : resistant to 5-methyl tryptophan

6FT : resistant to 6-fluorotryptophan

4MT : resistant to 4-methyltryptoph an

Trphx : resistant to tryptophan hydroxamate

4AP : resistant to 4-aminophenylalanine

Tyrhx : resistant to tyrosm hydroxamate

Phehx : resistant to phenylalan hydroxamate

3 AT : resistant to 3~amine ptyrosine

4FP : resistant to 4-fluorophenylalanine

3FP : resistant to 3-fluorophenylalanine

2FP<r> : resistant to 2-fluorophenylalanine

Example 3

In this example, Corynebacterium glutamicum Trp-10, ATCC 21851 is grown for 24 hours in an inoculum medium containing 7% cane sugar-molasses (b calculated as glucose), 0.3% corn molasses, 0.1% KH^PO^, 0.1% I^HPC^, 0.05 % MgSOj4.7H20 and 0.9% soybean cake compost

(obtained by decomposing soybean cake with 6N sulfuric acid and neutralizing the decomposed with aqueous ammonia, calculated as soybean cake). 300 ml of the pod medium is then inoculated into 3 liters of a fermentation medium with the following composition in a 5 liter fermentation container:

Cultivation is carried out at 30°C with an aeration rate of 3 l/min. and while stirring at 600 rpm. for 72 hours. As a result, 16.8 mg/ml L-tryptophan is produced.

Claims (1)

Process for the production of L-tryptophan by growing a microorganism of the species Corynebacterium glutamicum in an aqueous nutrient medium containing sources of carbon, nitrogen, inorganic nutrients and necessary growth factors, after which the L-tryptophan formed in the culture liquid is extracted from this, characterized in that the microorganism used is one of the strains Corynebacterium glutamicum ATCC 21842, ATCC 21843, ATCC 21844, ATCC 21845, ATCC 21846, ATCC 21847, ATCC 21848, ATCC 21849, ATCC 21850 and ATCC 21851.