CS265648B1 - Strain of corynebacterium glutamicum ccm 4022(vkp m b-3494) 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 high culture media carbon source conversion.

Description

The invention relates to a novel production strain of Corynebaoterium glutamioum CCM 4024, which produces L-lysine on synthetic and semi-synthetic fermentation media. The cultivation takes place in liquid nutrient media which contain sucrose or other carbohydrates as the main carbon source, as ammonium salt source, corn extract, vitamins such as biotin and mineral salts as the main source of carbon. 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 Corynebaoterium glutamicum CCM 4024, 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, J. E. Purkyně University, Tř. Obránců míru 10, Brno.

The above-mentioned production strain also has the advantage of a high carbon source conversion of between 32 and 38%. The Corynebaoterium glutamicum CCM 4024 production strain produces L-lysine on simple fermentation media without complex nitrogen sources; this makes the L-lysine preparation process cheaper and economical. The new strain was obtained by mutagenesis and selection from the original Corynebaoterium glutamicum ATCC 13 032 strain and is characterized by resistance to amino acid analogs, certain antibiotics and biologically active compounds analogs. The hybridization technique of protoplast fusion of mutant strains of Corynebacterium glutamicum was used to breed the new production strain, as shown in the following scheme:

1020-60 ^ ·

1.

ATCC 13,032

AEC _4J AEC ^r HXL ^r

100AEC AHV str _t9 AEC ^r HXL ^{r r r r} fuse /

CC 4024 (VKPM B-3494)

AEC ^{r r} HXL AHV ri ^r f ^{r r} p

The genetic characteristics of the new production strain Corynebaoterium glutamicum CCM 4024 are:

AEC ^r HXL ^r AHV ^{r r r r r} . Abbreviations used: AEC = 2-S-aminoethyl-L-oysteine, HXL = L-lysine hydroxyamate, AHV = 2-aminohydroxyvaleric acid, rif = rifamycin, str = streptomycin, r = resistance.

The morphological and culture characteristics of the production strain Corynebaoterium glutamicum CCM 4024 are characterized as follows: bacterial cells are oval in dimensions of 0.1 to 1.0 µm x 1.2 to 1.5 µm, non-sporulating, immovable 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 Corynebaoterium glutamicum CCM 4024 strain utilizes glucose, sucrose, maltose, mannose, fructose and rhamnose well; sorbitol and acetic acid. It grows weakly on arabinose, xylose

I and ethanol. Ammonium salts and urea are well utilized from nitrogen sources; gelatin is not hydrolyzed. According to the invention, L-lysine is prepared with the Corynebaoterium glutamicum CCM 4024 strain as follows.

He did

Corynebaoterium glutamioum CCM 4024 strain (VKPM B-3494) is seeded with 500 ml boiling flasks containing 50 ml inoculum medium of the following composition: sucrose. 30 g, sodium acetate crystals. 20 g, corn extract (60% by weight) 30 g, water dest. ad 1000 ml; pH of the medium 6.8 to 7.0. The flasks were further cultured on a rotary shaker (220 rpm frequency ^χ) at 28-30 ° C for 18-24 hours. A 500 ml boiling flask containing 20 ml of a fermentation medium of the following composition is inoculated with 10% vol.

100 g, biotin 50 yg / l, ammonium sulfate crystals. 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 further carried out under aerobic conditions on a rotary shaker at a temperature of 28 to 30 ° C and the pH of the medium is kept in the range of 30 to 30 ° C. 6.8 to 7.0 with 10% (v / v) ammonium hydroxide solution. After 96 hours of cultivation, production of 38 g of L-lysine / l medium is achieved in the Corynebacterium glutamicum CCM 4024 strain.

Example 1 a d 2

The Corynebacterium glutamicum CCM 4024 strain is seeded with an inoculum medium as described in Example 1. Liquid soil is used as the fermentation medium as described in Example 1 except that the biotin is replaced in the medium by corn extract (60% dry matter) in an amount of 5 g / The culture is continued as described in Example 1. After 96 hours of cultivation, production of 32 g of L-lysine / l medium is achieved in Corynebacterium glutamicum CCM 4024 strain.

Claims (1)

L-lysine-producing strain of Corynebacterium glutamicum CCM 4024 with high conversion of carbon source on synthetic and semisynthetic fermentation media.