CS265647B1 - Strain of corynebacterium glutamicum ccm 4023(vkp m b-3493)microorganism,producing l-lysine - Google Patents

Abstract

A method for preparing prototrophic is described strain Corynebacterium glutamicum na based on hybridization using protoplast techniques for constructing a new strain that is characterized by the production of L-lysine in simple culture media and high conversion carbon source.

Description

The present invention relates to a novel production strain of Corynebacterium glutamicum CCM 4023, which produces L-lysine, a variety of synthetic and semi-synthetic fermentation media. The cultivation takes place in liquid nutrient media containing as the main carbon source sucrose or other carbohydrates as the nitrogen source as ammonium salts, corn extract, vitamins such as biotin and mineral salts. Fermentation media do not contain complex nitrogen sources, such as protein substrate hydrolysates, eg peanut flour, etc.

The present invention provides a new production strain of Corynebacterium glutamicum CCM 4023, characterized by the production of L-lysine with high conversion of the carbon source on synthetic and semi-synthetic fermentation media. The strain is deposited in the Czechoslovak Collection of Microorganisms of J. E. Purkyně University, Tř. Obránců míru 10, Brno.

Further, the advantage of the above production strain is the high conversion of the carbon source, which is between 33 and 35%. The production strain Corynebacterium glutamicum CCM 4023 produces L-lysine on simple fermentation media without complex nitrogen sources; this makes the fermentation process of L-lysine preparation cheaper and economical. The new strain was obtained by mutagenesis and selection from the original strain of Corynebacterium glutamicum ATCC 13 032 and is characterized by resistance to amino acid analogs, certain antibiotics and biologically active compounds analogs.

The hybridization technique of fusion of protoplast-mutant strains of Corynebacterium glutamicum was used to breed the new production strain, as shown in the following scheme:

1.

/

AEC

-¼ 49ATCC 13,032

1020-60AEC

1002.

->

49-172.

> X-24-23 ^;

AEC AHV str fuse /

-> CCM 4023 (VKPM B-3493) AEC ^r HXL ^r AHV ^r aza ^r str ^r

The genetic characteristics of the new production strain Corynebacterium glutamicum CCM 4023 are:

AEC ^r HXL ^r AHV ^r aza ^r str ^r . Abbreviations used: AEC = 2-S-aminoethyl-L-cysteine, HXL = L-lysine hydroxyamate, AHV = 2-aminohydroxyvaleric acid, aza = azauracil, str = streptomycin, r = resistance.

The morphological and culture characteristics of the production strain of Corynebacterium glutamicum CCM 4023 are characterized as follows: bacterial cells are oval in dimensions of 0.1 to 1.0 µm xx 1.2 to 1.5 µm, non-sporulating, immovable, and gram-positive bacteria. When cultivated at 30 ° C on masopepton agar, colonies have a diameter of 3 to 4 mm, colonies are convex with a straight edge, cream colors with a pasty consistency. The strain grows optimally under aerobic conditions at pH 7.0 and 30 ° C. The Corynebacterium glutamicum CCM 4023 strain utilizes glucose, sucrose, maltose, mannose, fructose and rhamnose well; sorbitol and acetic acid. It grows poorly on arabinose, xylose and ethanol. Ammonium salts and urea are well utilized from nitrogen sources; gelatin is not hydrolyzed. According to the invention, L-lysine is prepared using the Corynebacterium glutamicum CCM 4023 strain as follows. ¹

Example 1

Corynebacterium glutamicum CCM 4023 (VKPM B-3493) is seeded with 500 ml boiling flasks containing 50 ml of inoculum medium of the following composition: sucrose. 30 g, sodium acetate crystals. 20 g, corn extract (60% dry matter) 30 g, water dist. ad 1000 ml; pH of the medium 6.8 to 7.0. The flasks were further cultured on a rotary shaker (220 rpm) at 28-30 ° C for 18-24 hours. 500 ml boiling flasks containing 20 ml of a fermentation medium of the following composition are inoculated with 10% vol. 100 g, biotin 50 pg / l, ammonium sulfate. 25 g, potassium hydrogen phosphate 1 g, magnesium sulfate crystals. 0.1 g, calcium carbonate ground 35 g, water dest. ad 1000 ml; pH of the medium 6.8 to 7.0; the cultivation is carried out under aerobic conditions on a rotary shaker at a temperature of 28 to 30 ° C and the pH of the medium is maintained in the range of 6.8 to 7.0 with a solution of 10% by volume ammonium hydroxide. After 96 hours of cultivation, production of 33 g of L-lysine / l medium is achieved in the production strain Corynebacterium glutamicum CCM 4023.

Example 2

The Corynebacterium glutamicum CCM 4023 strain is seeded with the inoculation medium as described in Example 1. Liquid soil is used as the fermentation medium as described in Example 1, except that the biotin in the medium is replaced with corn extract (60% dry matter) in an amount of 5 g. / 1 media. The cultivation is continued as described in Example 1. After 96 hours of cultivation, the production of 35 g of L-lysine / l medium is achieved.

Claims (1)

L-lysine-producing strain of Corynebacterium glutamicum CCM 4023 with high conversion of carbon source on synthetic fermentation media.