RU2549690C1 - L-LYSINE PRODUCING Corynebacterium glutamicum OR Brevibacterium flavum BACTERIA, RESISTANT TO FUSIDIC ACID, AND METHOD FOR MICROBIOLOGICAL SYNTHESIS OF L-LYSINE USING SAME - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology and microbiology and provides a method of producing Corynebacterium glutamicum or Brevibacterium flavum bacteria with a high level of synthesis of L-lysine, which includes seeding a culture of Corynebacterium glutamicum or Brevibacterium flavum bacteria, capable of producing L-lysine, on a medium containing fusidic acid, and collecting from the grown colonies, those colonies with a high level of synthesis of L-lysine. The invention also relates to a method for microbiological synthesis of L-lysine by culturing bacteria obtained using said method.

EFFECT: invention enables to obtain L-lysine with high efficiency.

2 cl, 1 tbl, 8 ex

Description

The invention relates to biotechnology and relates to strains of Corynebacterium glutamicum or Brevibacterium flavum producing L-lysine, as well as a method for producing L-lysine using a fermentation process using bacteria-producers of L-lysine resistant to fusidic acid.

L-lysine belongs to the group of essential amino acids and is the most popular feed additive for livestock and poultry farming.

The main method for producing L-lysine is the fermentation of sugars using bacteria, such as Corynebacterium glutamicum (US 3687810, US 3929571), Brevibacterium sp. (US 3687810, US 3929571) and Escherichia coli (RU 2347807). In the known methods for producing L-lysine, mainly various genetically modified bacterial strains with increased L-lysine productivity are obtained that are obtained either by genetic engineering or using traditional breeding methods. One approach to increasing the productivity of strains is to obtain mutant strains that are resistant to cell metabolites (antibiotics).

Known methods for producing L-lysine, which use bacteria that are resistant to rifampicin antibiotics (US 4,623,623), streptomycin (US 3,929,571, US 4,623,623), kanamycin (US 7736880, US 8361758) or simultaneously to several antibiotics (US 3687810, US 4623623) . These antibiotics belong to the groups of ansamycins and aminoglycoside antibiotics, respectively. The effect of antibiotics from other groups on the production of L-lysine was not reported.

The task of the invention is to expand the arsenal of bacteria with the ability to increase the level of synthesis of L-lysine as a result of acquiring antibiotic resistance, and the development of a method of microbiological synthesis of L-lysine using these bacteria.

The problem is solved by:

- obtaining L-lysine-producing strain of Corynebacterium glutamicum VKPM B-11609, resistant to fusidic acid;

- obtaining L-lysine-producing strain of Brevibacterium flavum VKPM B-11635, resistant to fusidic acid;

- obtaining L-lysine-producing strain of Brevibacterium flavum VKPM B-11404, resistant to fusidic acid;

- development of a method for microbiological synthesis of L-lysine by culturing under suitable conditions L-lysine-producing bacteria Corynebacterium glutamicum or Brevibacterium flavum resistant to fusidic acid.

The characteristics of the claimed strains:

Strain Brevibacterium flavum VKPM B-11635

A mutant of the strain Brevibacterium flavum 90 VKPM B-5593, resistant to fusidic acid and having increased L-lysine productivity, was deposited in the All-Russian Collection of Industrial Microorganisms (VKPM) as Brevibacterium flavum VKPM B-11635.

Cultural and morphological signs:

- oval cells with a size of 1.0-1.5 × 0.6-0.6-0.8 μm, motionless, gram-positive, do not form spores;

- after 2-4 days of growth at 30 ° C on MPA (GOST 29112-91) forms colonies with a diameter of 2-4 mm, yellowish-cream, the surface is smooth, the shape is convex, the edge is even, the structure is homogeneous pasty;

- when sowing with a stroke after 2-4 days, growth is moderate, the edge is smooth, the surface is dull.

Physiological and biochemical signs:

- aerob;

- grows well on glucose, sucrose, maltose, fructose, mannose, ethanol, acetic acid, does not grow on xylose, arabinose, lactose, raffinose;

- assimilates nitrogen in the form of ammonium and urea salts;

- grows at a temperature of 24-37 ° C, the optimum temperature is 30-32 ° C;

- grows on media with a pH of 6 to 8.5, the optimum pH is 6.8-7.2;

- when growing in a minimal environment, it needs biotin, thiamine and L-leucine;

- resistant to the lysine analog S- (2-aminoethyl) -L-cysteine (AEC);

- resistant to fusidic acid.

Strain Brevibacterium flavum VKPM B-11404

A mutant of the strain Brevibacterium flavum NF410, resistant to fusidic acid and having increased L-lysine productivity, was deposited in VKPM as Brevibacterium flavum VKPM B-11404.

Strain NF410, in turn, was obtained using genetic methods from a strain of Brevibacterium flavum 90 (VKPM B-5593). Strain NF410, unlike VKPM strain B-5593, is not able to use acetate as the sole carbon source and has increased activity of aspartate kinase and diaminopimelate dehydrogenase.

Cultural and morphological signs:

- oval cells with a size of 1.0-1.5 × 0.6-0.6-0.8 μm, motionless, gram-positive, do not form spores;

- after 2-4 days of growth at 30 ° C on an MPA medium, it forms colonies with a diameter of 2-4 mm, yellowish-cream, the surface is smooth, the shape is convex, the edge is even, the structure is homogeneous pasty;

- when sowing with a stroke after 2-4 days, growth is moderate, the edge is smooth, the surface is dull.

Physiological and biochemical signs:

- aerob;

- grows well on glucose, sucrose, maltose, fructose, mannose, does not grow on xylose, arabinose, lactose, raffinose and acetate;

- assimilates nitrogen in the form of ammonium and urea salts;

- grows at a temperature of 24-37 ° C, the optimum temperature is 30-32 ° C;

- grows on media with a pH of 6 to 8.5, the optimum pH is 6.8-7.2;

- when growing in a minimal environment, it needs biotin, thiamine and L-leucine;

- resistant to the lysine analog S- (2-aminoethyl) -L-cysteine (AEC);

- resistant to fusidic acid;

- has increased, compared with the strain of Brevibacterium flavum VKPM B-5593, the activity of aspartate kinase and diaminopimelate dehydrogenase.

Strain Corynebacterium glutamicum VKPM B-11609

A mutant of the strain Corynebacterium glutamicum VKPM B-11608, resistant to fusidic acid and having increased L-lysine productivity, was deposited in VKPM under the number VKPM B-11609.

The strain Corynebacterium glutamicum VKPM B-11608, in turn, was obtained from the strain Corynebacterium glutamicum ATCC 13032 as resistant to S- (2-aminoethyl) -cysteine.

Cultural and morphological signs:

- oval cells with a size of 1.0-1.5 × 0.6-0.6-0.8 μm, motionless, gram-positive, do not form spores;

- after 1-3 days of growth at 30 ° C on MPA it forms colonies with a diameter of 2-4 mm, cream, the surface is smooth, the shape is convex, the edge is even, the structure is homogeneous pasty;

- when sowing with a stroke after 1-2 days, growth is moderate, the edge is smooth, the surface is dull.

Physiological and biochemical signs:

- aerob;

- grows well on glucose, sucrose, maltose, fructose, mannose and acetate, does not grow on xylose, arabinose, lactose, raffinose;

- assimilates nitrogen in the form of ammonium and urea salts;

- grows at a temperature of 24-40 ° C, the optimum temperature is 32-34 ° C;

- grows on media with a pH of 6 to 8.5, the optimum pH is 6.8-7.2;

- when growing in a minimal environment, it needs biotin and thiamine;

- resistant to the lysine analog S- (2-aminoethyl) -L-cysteine (AEC);

- resistant to fusidic acid.

Fusidic acid resistant strains are obtained by plating on an agarized nutrient medium containing fusidic acid, intact L-lysine-producing cells of Corynebacterium glutamicum or Brevibacterium flavum, or L-lysine-producing cells of Corynebacterium glutamicerum pre-known Brev or Brev (in particular, nitrosoguanidine), followed by incubation at 30 ° C for 72 hours. Among the mutants resistant to fusidic acid, strains with increased L-lysine productivity are at least 20%.

The method in General

The method of microbiological synthesis of L-lysine is carried out by culturing an L-lysine-producing strain of bacteria Corynebacterium glutamicum or Brevibacterium flavum resistant to fusidic acid. As a nutrient medium for cultivation, a mineral medium is used, including sodium or potassium phosphates, magnesium salts, and, if necessary, manganese and iron salts, and containing any sugars as a carbon source, such as glucose, glucose syrups or sucrose, ammonium as a nitrogen source salts or urea, as well as amino acids and vitamins necessary for growth. As components that accelerate the growth of bacteria, corn extract, gluten or soy hydrolysates are additionally added. The cultivation of bacteria is carried out at 24-42 ° C, mainly at 30-32 ° C, and the pH of the medium in the range of 6.0-8.5, mainly in the range of 6.8-7.2.

The cultivation of bacteria is carried out in test tubes, flasks or special reactors under aerobic conditions.

The inventive method of microbiological synthesis allows you to select the synthesized L-lysine from the culture fluid in one of the known ways, including adding sulfuric or hydrochloric acid to the culture fluid, using ion-exchange chromatography, crystallization, etc.

Example 1. Obtaining L-lysine-producing strain of Brevibacterium flavum VKPM B-11635 resistant to fusidic acid

The selection of fusidic acid-resistant mutants of the L-lysine-producing strain of bacteria Brevibacterium flavum VKPM B-5593 is carried out by plating the culture on LB agar medium of the following composition (wt.%): Tryptone - 1.0, yeast extract - 0.5, sodium chloride - 0.5, water - the rest, with the addition of biotin up to 0.00001%, glucose up to 0.5% and fusidic acid up to 0.0005%. Cultures of the strain Brevibacterium flavum VKPM B-5593 were preliminarily grown on a shaker (250 rpm) at 30 ° C for 24 h in LB liquid medium with the addition of biotin up to 0.00001% and glucose up to 0.5%.

After 3-5 days of incubation of the dishes at 30 ° C, colonies of clones resistant to fusidic acid grow on the surface of the agar medium. The level of L-lysine biosynthesis in selected clones is tested in a cup test (modified auxanographic method) (General bacteriology methods in 3 volumes, 1984), which is based on the ability of strains producing L-lysine to ensure auxotrophic growth in L on an agarized minimal medium -lysine of strains, for example, E. coli strain VKPM B-558. To this end, the biomass of strain VKPM B-558, previously grown at 37 ° C in LB medium with stirring (240 rpm) for 18 hours, is separated by centrifugation, washed twice with 0.9% NaCl solution, suspended in 0.9% NaCl solution and then 0.1 ml of the resulting cell suspension are plated on a plate with a minimum medium of the following composition (wt.%): monosubstituted potassium phosphate - 0.1, disubstituted potassium phosphate - 0.6, seven-water magnesium sulfate - 0.05, chloride ammonium - 0.5, glucose - 2.0, biotin - 0.00001, thiamine - 0.00002, L-leucine - 0.025, agar - 1.5, water - the rest, pH 7.2-7.4. Cells of the mutant Brevibacterium flavum VKPM B5593 clones grown on medium containing fusidic acid (about 25 pcs per cup) are transferred to the same cup with a toothpick. As a control, the cellular material of the original Brevibacterium flavum VKPM B-5593 strain is transferred to the plate with a toothpick. Plates are incubated at 30 ° C for 2 days.

As a result of the test, a fusidic acid resistant (5 μg / ml) mutant of the Brevibacterium flavum VKPM B-5593 strain was selected, around the colonies of which the maximum growth zone of E. coli VKPM B-558 strain was observed in a cup test. The resulting mutant was deposited in VKPM as Brevibacterium flavum VKPM B-11635.

Example 2. Obtaining L-lysine-producing strain of Brevibacterium flavum VKPM B-11404, resistant to fusidic acid

Obtaining mutants is carried out as described in example 1, but using the original strain of Brevibacterium flavum NF410.

One of the mutants of the strain Brevibacterium flavum NF410, resistant to fusidic acid (5 μg / ml) and forming the maximum growth zone of the strain E. coli VKPM B-558 when co-cultured on minimal medium in a cup test, was deposited in VKPM as a strain of Brevibacterium flavum VKPM B -11404.

Example 3. Obtaining L-lysine-producing strain of Corynebacterium glutamicum VKPM B-11609 resistant to fusidic acid

The mutant is obtained as described in Example 1, but using VKPM B-11608 VKPM B-11608 as the initial strain of Corynebacterium glutamicum, which in turn was obtained from C. glutamicum ATCC13032 strain as resistant to the lysine analogue S- (2-aminoethyl) -cysteine.

One of the mutants of the strain Corynebacterium glutamicum VKPM B-11608, resistant to fusidic acid (5 μg / ml) and forming the maximum growth zone of the E. coli strain VKPM B-558 when co-cultured on minimal medium in a cup test, was deposited in VKPM as a Corynebacterium strain glutamicum VKPM B-11609.

Example 4. Assessment of the level of synthesis of L-lysine obtained strains

Evaluation is carried out in vitro tests. To do this, 0.3 ml of the culture of one of the claimed strains, previously grown for 22 hours with stirring (240 rpm) at 30 ° C, is introduced into test tubes with 3 ml of medium of the following composition (wt.%): Glucose - 10, 0 ammonium chloride - 2.5, potassium phosphate monosubstituted - 0.1, magnesium sulphate - 0.1, chalk - 2.5, biotin - 0.00001, thiamine - 0.00002, with the addition of wheat gluten acid hydrolyzate * 50 , 0 ml / l, water - the rest. * Wheat gluten hydrolyzate is obtained by hydrolysis of wheat gluten with sulfuric acid according to known methods of hydrolysis of various raw materials in order to obtain a source of growth factors in the microbiological production of amino acids (Biotechnology and Bioengineering, Riga, 1978). Before entering the medium, the hydrolyzate is neutralized with concentrated ammonia. Tubes with cultures are incubated for 24 or 48 hours with stirring (250 rpm) at 30 ° C and then the level of accumulation of L-lysine by thin layer chromatography is determined in the culture fluid. The results are presented in the table.

Table. The level of L-lysine synthesis by the obtained strains of Brevibacterium flavum VKPM B-11635, Brevibacterium flavum VKPM B-11404 and Corynebacterium glutamicum VKPM B-11609 in vitro tests. Culture The concentration of L-lysine in the culture fluid after 24 hours (g / l) The concentration of L-lysine in the culture fluid after 48 h (g / l) The increase in the level of synthesis of L-lysine compared with the parent strain (%) Brevibacterium flavum KPM B-11635 (mutant) 22.2 39.5 10 Brevibacterium flavum.
VKPMV-5593 (parent strain) 17.6 36.0 Corynebacterium lutamicum VKPM B-11609 (mutant) Not determined 18.0 9 Corynebacterium glutamicum VKPM B-11608 (parent strain) Not determined 16.5 Brevibacterium flavum VKPM B-11404 (mutant) Not determined 41.5 10 Brevibacterium flavum NF410 (parent strain) Not determined 37.5

From the results presented in the table, it follows that fusidic acid-resistant mutants of L-lysine-producing strains of Brevibacterium flavum and Corynebacterium glutamicum have an increased level of L-lysine synthesis compared to the parent strains by at least 9%.

Example 5. Synthesis of L-lysine by culturing a strain of Brevibacterium flavum VKPM B-11635 in a fermenter

The synthesis of L-lysine is carried out by culturing a strain of Brevibacterium flavum VKPM B-11635 in a 3 L fermenter. To do this, 0.450 l of a 70% sterile glucose solution and 0.15 l of culture previously grown at 31 ° C for 16 hours are added to a fermenter containing 0.600 l of fermentation medium. The composition of the fermentation medium (wt.%): Potassium phosphate monosubstituted - 0.90, magnesium sulfate of seven-water - 0.32, ammonium sulfate - 5.5, biotin - 0.000045, thiamine - 0.000045, ferrous sulfate - 0.0045, manganese pentahydrate - 0.0045, antifoam propinol B -400 - 0.10, with the addition of wheat gluten acid hydrolyzate - 167 ml / l, the rest is water, the pH of the medium is adjusted to 7.0 by the addition of 16% ammonia. Cultivation in the fermenter is carried out at 32 ° C, with stirring (900 rpm), aeration of 1.2 l / min and a pH of 7.0, which is supported by the addition of 16% ammonia.

The cultivation is completed with the complete exhaustion of glucose in the culture fluid. The level of accumulation of L-lysine in the culture fluid is determined using thin layer chromatography.

After 70 hours of cultivation, the concentration of L-lysine in the culture fluid was 115 g / L.

Example 6. Synthesis of L-lysine by culturing a strain of Brevibacterium flavum VKPM B-11404 in a fermenter

The synthesis of L-lysine is carried out by culturing a strain of Brevibacterium flavum VKPM B-11404 in a 3 L fermenter. For this, 0.15 L of culture previously grown at 31 ° C for 16 hours in a seed fermenter is added to a fermenter containing 0.550 L of fermentation medium. The composition of the fermentation medium (wt.%): Potassium phosphate monosubstituted - 0.90, magnesium sulphate - 0.32, ammonium sulfate - 5.50, glucose - 2.50, biotin - 0.000045, thiamine - 0.000045, ferrous sulfate seven-water - 0.0045, manganese five-water sulfate - 0.0045, antifoam propinol B-400 - 0.10, with the addition of wheat gluten acid hydrolyzate - 167 ml / l, water - the rest, the pH of the medium was adjusted to 7.0 by adding 16% ammonia. Cultivation in the fermenter is carried out at 32 ° C, with stirring (900 rpm), aeration of 1.2 l / min and a pH of 7.0, which is supported by the addition of 16% ammonia.

During fermentation, the residual concentration of glucose in the culture fluid is maintained in the range of 5 to 60 g / l by the addition of a feed containing glucose and wheat gluten hydrolyzate. The total supply of glucose (taking into account the initial concentration) is 310 g / l, and the hydrolyzate of wheat gluten is 45 ml / l.

The cultivation is completed with the complete exhaustion of glucose in the culture fluid. The level of accumulation of L-lysine in the culture fluid is determined using thin layer chromatography.

After 70 hours of cultivation, the concentration of L-lysine in the culture fluid was 150 g / L.

Example 7. Obtaining L-lysine monohydrochloride from the culture fluid of a strain of Brevibacterium flavum VKPM B-11635 or a strain of Brevibacterium flavum VKPM B-11404

To 1 liter of L-lysine-containing culture fluid obtained as described in examples 5 or 6, concentrated sulfuric acid in an amount of? necessary to bring the pH to 1.5. Then the culture fluid is passed through a column with sulfonic cation exchanger KU-2-8 (GOST 20298-74) in ammonium form. After absorption of L-lysine, the column is immediately washed with demineralized water at room temperature. L-lysine adsorbed on sulfocationite is eluted with a solution containing 2.0 mol / L ammonium hydroxide. The fractions containing L-lysine are concentrated by evaporation in vacuo, acidified with concentrated hydrochloric acid to pH 4.5 and cooled to 10 ° C. The precipitated crystals are separated from the mother liquor by centrifugation, washed with a small amount of demineralized water and dried. The resulting product contains at least 98% L-lysine monohydrochloride.

In the case of using 1 liter of culture fluid, the preparation of which is described in Example 5, 103 g of L-lysine monohydrochloride are obtained, and in the case of a culture fluid obtained as described in Example 6, 135 g of L-lysine monohydrochloride.

Example 8. Obtaining L-lysine sulfate from the culture fluid of the claimed strain of Brevibacterium flavum VKPM B-11635 or strain Brevibacterium flavum VKPM B-11404

To 1 L of L-lysine-containing culture fluid obtained as described in Examples 5 or 6, 3.5 ml of concentrated sulfuric acid was added to pH 5.0. Then, the resulting liquid is sent to spray drying. Drying is carried out at an inlet air temperature of 120 ° C for 3 hours.

The result is a product containing 65% L-lysine sulfate. In the case of using 1 l of the culture fluid obtained as described in example 5, 157.5 g of product is obtained, and in the case of using 1 l of the culture fluid obtained as described in example 6, 205 g of product.

Thus, the introduction of mutations of resistance to fusidic acid in L-lysine-producing strains of Corynebacterium glutamicum or Brevibacterium flavum leads to an increase in the level of production of L-lysine by at least 9%. The inventive method for the synthesis of L-lysine using L-lysine-producing strains of Corynebacterium glutamicum or Brevibacterium flavum resistant to fusidic acid, allows you to get two of the most common commodity forms of L-lysine - lysine hydrochloride and lysine sulfate.

Information sources

1. Methods of general bacteriology. Ed. F. Gerhardt, in 3 volumes, 1984.

2. Biotechnology and bioengineering, Riga, 1978.

3. US 3687810.

4. US 3929571.

5. RU 2347807.

6. US 4,623,623.

7. US 7736880.

8. US 8361758.

Claims (2)

1. A method of producing bacteria Corynebacterium glutamicum or Brevibacterium flavum with an increased level of L-lysine synthesis, including plating cultures of the bacteria Corynebacterium glutamicum or Brevibacterium flavum, capable of producing L-lysine, on a medium containing fusidic acid, and selection among those grown in colonies increased levels of L-lysine synthesis. 2. The method of microbiological synthesis of L-lysine by culturing the bacteria obtained by the method according to claim 1, under suitable conditions on a mineral medium containing sources of carbon, nitrogen, as well as amino acids and vitamins necessary for bacterial growth.