CS246669B1 - Microorganism strain Corynebacterium glutamicum CCM 3800 - Google Patents

Abstract

The purpose of the solution is the production of the essential amino acid lysine in a medium with sucrose or molasses as a carbon source. Lysine is synthesized and accumulated in the medium by a mutant of the bacterial species Corynebacterium glutamicum. By the action of a mutagen and the application of selective media, a mutant resistant to S-(2-aminoethyl)-L-cysteine, dependent on homoserine and sensitive to 2-fluoropyruvate was isolated, which, when cultivated for 4 days in a medium with sucrose or molasses as a carbon source, peanut flour hydrolysate, corn steep liquor and ammonium sulfate as nitrogen sources and with mineral salts, accumulated 57 g/1 L-lysine at high conversion under aerobic conditions at 28 °C and neutral pH in an environment. Lysine, used primarily as a feed supplement, can be produced in this way using microorganisms using conventional technological procedures

Description

The invention relates to a new strain of the microorganism Corynebacterium glutamicum, resistant to a lysine analogue, dependent on homoserine and sensitive to 2-fluoropyruvate, which accumulated high amounts of lysine in a medium with a suitable carbon and nitrogen source at high conversion of the carbon source under aerobic conditions and at neutral pH.

Mutants of coryneform bacteria, which, in addition to being dependent on amino acids, were also resistant to amino acid analogues, proved to be suitable producers of lysine for its production by fermentation.

However, when it was found (Tosaka, O.-Morioka, H.-Takinami, K.: Abstracts of Papers, General Meeting of Agric. Biol. Chem. Soc. Japan, 1978, p. 176) that mutants sensitive to 2-fluoropyruvate produce relatively high amounts of lysine, the possibility of isolating mutants of the genera Brevibacterium and Corynebacterium was investigated, which, in addition to the genetic traits characteristic of lysine production, i.e. resistance to S-(2-aminoethyl)-L-cysteine and dependence on homoserine, would also show sensitivity to 2-fluoropyruvate (US Pat. 4,275,157, 157, 1981).

Thus, for example, a mutant of Corynebacterium acetoglutamicum was isolated, resistant to S-(2-aminoethyl)-L-cysteine, dependent on homoserine and alanine, sensitive to 2-fluoropyruvate and producing 39 g/l L-lysine in a medium with glucose at a conversion of 39%. With a mutant of Brevibacterium flavum resistant to S-(2-aminoethyl)-L-cysteine and sensitive to 2-fluoropyruvate, production of up to 51 g/l L-lysine was achieved in a medium where the carbon source was again glucose (Ozaki, H.-Shiio, I.: Agr. Biol. Chem. 47: 1569, 1983).

The strain Corynebacterium glutamicum, stored in the Czechoslovak Collection of Microorganisms of the J. E. Purkyně University in Brno, class Obránců míru 10, under the designation CCM 3,866, which is the subject of the invention, exceeds the previously known mentioned strains in terms of lysine production.

The main advantage of the new strain according to the invention is its ability to produce high amounts of lysine with high efficiency of carbon source conversion. When cultivating the new strain in a production medium with sucrose or molasses as carbon sources, with acid hydrolysate of peanut flour, corn steep liquor and ammonium sulfate as nitrogen sources and with mineral salts in flasks incubated on a rotary shaker at 28 °C and pH maintained at pH 7.0 with an aqueous ammonia solution, lysine production of 50 g/1 (with molasses) and 57 g/1 (with sucrose) can be achieved after 4 days of cultivation at a conversion of 30 and 35%.

The new strain Corynebacterium glutamicum CCM 3 866 is characterized by the following morphological, cultural and physiological properties:

Gram-positive, non-motile, non-spore forming cocci to short rods, occurring singly, in pairs and in irregular clusters.

On meat peptone agar, it forms round, smooth, shiny, creamy white colonies with straight edges.

On massopeptone agar slants, it forms an intense white growth over the entire agar surface; pigmentation does not occur.

When growing in broth, turbidity and sediment are formed, but no blanch is formed.

On blood agar, they form round, smooth, gray colonies with straight edges, non-hemolytic.

Physiological properties:

Optimal temperature: 27 to 32 °C.

Optimal pH: 7.0 to 7.2.

Aerobic organism.

It does not liquefy gelatin.

Litmus milk decolorizes but does not precipitate.

It ferments glucose, fructose and sucrose.

Starch hydrolyzes only weakly.

Does not break down cellulose

It does not reduce nitrates.

Hydrogen sulfide does not form.

Indole monsters.

Catalase test: positive.

Methylene blue test: negative,

Voges-Proskauer test: negative.

For growth, it requires the vitamins biotin and the amino acids homoserine.

The initial strain Corynebacterium glutamicum CCM 3 287, resistant to S-(2-aminoethyl)-L-cysteine and dependent on homoserine, is cultured submerged for 18 hours in complete medium and the cell suspension, after washing with phosphate buffer (pH 7.2), is exposed to a 0.05M solution of ethyl methanesulfonate in phosphate buffer (pH 7.2) for 18 hours.

After application of the mutagen, the cell suspension is washed with sterile distilled water and 5% (vol.) of the washed suspension is inoculated into a flask with complete medium, which is incubated for 24 h. The washed suspension, suitably diluted, is inoculated onto plates with minimal medium supplemented with homoserine. Colonies grown on the plates are transferred with sterile punches to plates with minimal medium supplemented with homoserine and plates with minimal medium with homoserine and 2-fluoropyruvate.

Fluoropyruvate-sensitive mutants are inoculated onto massopeptone agar slants. Isolates grown on slants are tested for resistance to S-(2-aminoethyl)-L-cysteine, dependence on homoserine and sensitivity to 2-fluoropyruvate and then evaluated for production in sucrose or molasses-supplemented production medium.

The details of the invention result from the following exemplary embodiments:

Example 1

60 ml of complete medium in a 500 ml boiling flask is inoculated with the microorganism Corynebacterium glutamicum CCM 3 287. The composition of the complete medium is as follows:

glucose casein hydrolyzate yeast extract KH ₂ P°4 ^K 2 ^HPO 4 salt solution

0.5%

1.0

0.5

0.3

0.1 ml/1000 ml media (The salt solution is a solution of 0.5 g FeSO^ . 7 f^O and 0.5 g MnSO^ . 4 H ₂ O in 100 ml water.)

The inoculated flask is incubated on a rotary shaker (speed frequency - 3.7, eccentric 25 mm) for 24 h at 28 ° C. The cell suspension is then washed 2x with phosphate buffer (pH 7.2) and after washing is exposed to 18 hours of 0.05M ethyl methanesulfonate solution in phosphate buffer (pH 7.2).

After application of the mutagen, the suspension is washed twice with sterile distilled water and 5% (vol.) of the suspension is inoculated into a 500ml boiling flask with 60 ml of complete medium, which is incubated for 24 h at 28 °C on a rotary shaker. The cell suspension is washed twice with sterile distilled water and the appropriately diluted suspension is inoculated into plates with minimal medium supplemented with 0.001 mg/ml L-homoserine. The composition of the minimal medium is as follows:

glucose sodium citrate

0.1% 0.04 0.7 0.2

( _NH4 ) _2I Saturday ₄ 0.1 Mgso ₄ . 7 H ₂ O 0.01 solution biotin 2mg/1000ml solution salt 1 ral/1,000 ml agar 2.5%

(The salt solution is a solution of 0.5 g FeSO ₄ . 7 HjO and 0.5 g MnSO^ . 4 H ₂ O in 100 ml of water.)

Colonies grown on plates after 2-3 days of incubation at 28 °C were transferred with sterile punches to plates with minimal medium supplemented with either homoserine or homoserine and 10 µg/ml sodium 2-fluoropyruvate. The fluoropyruvate-sensitive mutants are plated on meat peptone agar slants and the isolates grown on the agars are tested for their degree of resistance to S-(2-aminoethyl)-L-cysteine, dependence on homoserine and sensitivity to 2-fluoropyruvate, and their ability to produce lysine in production media with sucrose or molasses as carbon sources.

Example 2

A 24-hour-old culture of Corynebacterium glutamicum strain CCM 3 866 is inoculated into 60 ml of CSL-B-S inoculum medium in 500 ml boiling flasks. The composition of the inoculum medium is as follows:

sucrose 2.5% corn syrup (65% solids) 3.0 pH 7.0

The inoculated flasks are incubated on a rotary shaker (speed frequency - 3.7, eccentric 25 mm) for 24 h at 28 °C. 10% (vol.) of the inoculum is inoculated into a 500 ml boiling flask with 20 ml of BS or BM production medium of the following composition:

Medium BS: sucrose hydrolysate of peanut flour

15.0%

20.0% (vol.)

corn leach 1.5% < ^NH4 > ^2SO4 1.5 kh ₂ after ₄ 0.2 MgSO ₄ . 7 ^H 2° 0.03 CaCO ₃ 2.0 FeSO ₄ . 7 h ₂ ° 0.002% MnSO, . 4 ^H 2° 0.002 biotin (technical) 0.00005 pH 7.0

30.0%

15.0% (vol.)

Medium BM: molasses peanut flour hydrolysate

corn leach 1.5% _{( NH4} ) _2SO4 1.5 ^KH 2 ^PO 4 0.2 MgSO ₄ . 7 ^H 2° 0.03 CaCOj 2.0 FeSO, . 7 4 ^H 2° 0.002 MnS0 ₄ . 4 ^H 2° 0.002

biotin (technical) 0.00005 pH

7.0

During the 4-day cultivation, the pH is adjusted at 24-hour intervals with aqueous ammonia solution to a value of 7.0-7.2. At the end of the cultivation, the lysine and sugar content in the culture fluid is determined. Lysine is determined manometrically using lysine decarboxylase and sugar as glucose using a glucose analyzer Glucose Analyzer 2 (Beckman, U.S.A.,.

Lysine production after 4 days of cultivation in BS medium is 57 g/1 (conversion - 35%) and in BM medium 50 g/1 (conversion - 30%).

Claims (1)

Corynebacterium glutamicum strain CCM 3,866, homoserine-dependent and 2-fluoropyruvate-sensitive, S- (2-aminoethyl) -L-cysteine resistant, producing lysine at high conversion.