RU2119536C1 - Strain escherichia coli - a producer of l-histidine - Google Patents

Abstract

FIELD: microbiology, biotechnology. SUBSTANCE: invention proposes a new strain Escherichia coli VKPM B-7270 as a producer of L-histidine. Strain is obtained by selection of new mutation in gene hisG of histidine operon and the following insertion of extraoperon mutations in it that enhance the expression of histidine operon. The indicated strain is resistant to sulfaguanidine, D,L-1,2,4-triazole-3-alanine and streptomycin and ensures to obtain L-histidine at yield 16-17 g/l. Conversion degree of glucose to histidine is 24-26%. EFFECT: new strain indicated above, increased yield of L-histidine. 1 tbl, 1 ex

Description

 The invention relates to the microbiological industry and is a new strain of the bacterium Escherichia coli producing histidine amino acid. L-histidine is an essential amino acid used as a component of nutritional mixtures for medical purposes and growth factor for a number of producers of primary metabolites. In the chemical and pharmaceutical industries, histidine is used to produce histamine and urocanic acid.

 Histidine producing strains belonging to various types of microorganisms (Corynebacterium glutamicum, Corynebacterium herculis, Corynebacterium acetoacidophilum, Brevibacterium flavum, Brevibacterium lactofermentum, Escherichia coli, Arthrobacterium bacillus fibrobius, Arthrobacterium bacterium bacterium US patent 3716453; US patent 5294547; Japan patent 61 - 41554; US patent 4388405; Sigiura M., Kisumi M., 1984, Appl. Environ. Microbiol. v. 48, p. 43 - 49; Mizukami T., Hamu A ., Ikeda M., Oka T., Katsumata R., 1994, Bioscience, Biotech. Biochem., V. 58, p. 635-638). The most effective histidine producers are plasmid strains of Serratia marcescens (Japanese Patent 61-271981). These producers contain a mutant histidine operon or its fragment in the composition of a low copy plasmid and are capable of accumulating up to 45 g / l histidine in 120 hours. However, the disadvantages of the plasmid strains producing histidine are the need to add antibiotics to the fermentation medium to maintain the plasmid with the cloned gene, the slow growth of plasmid producers and, as a consequence, the increase in the duration of fermentation. To date, effective histidine producers have not been obtained among representatives of E. coli. Known plasmid-free strain of E. coli 24 (RF patent 2003677), producing 10 to 12 g / l of histidine for 72 hours of fermentation in laboratory conditions. This strain is selected by us as a prototype. The disadvantage of the strain of the prototype is its low productivity and low conversion of glucose to histidine, which is 18 to 20%.

 The objective of the invention is the creation of a plasmid-free strain of E. coli with higher productivity and increased conversion of glucose to L-histidine. The objective is achieved by creating a new strain of E. coli 80, which accumulates 16-17 g / l of histidine in the laboratory for 68 hours of fermentation under laboratory conditions; the conversion of glucose to histidine is 25%.

 The strain E. coli 80 was obtained by targeted selection using N-methyl-N'-nitro-N-nitrosoguanidine as a mutagenic factor. The strain E. coli CC14, which we described earlier (Chakhmakhchyan S.S., Klyachko E.V., Shakulov R.S., Domkin V.D., 1992, Molecular Genetics, Microbiology and Virology, s. . 15 - 18). This strain contains a wild-type histidine operon and does not produce histidine. The new E. coli 80 strain differs from the initial one in three features: a mutation in the hisG gene, which determines the resistance of the first enzyme of the histidine ATP-phosphoribosyltransferase biosynthesis pathway to a mixture of histidine and sulfaguanidine, enhances the transcription of the histidine operon, and also introduces ribosome resistance.

 To obtain E. coli 80, the original strain of Escherichia coli CC14 is treated with N-methyl-N'-nitro-N-nitrosoguanidine according to the standard procedure (Miller J., Experiments in molecular genetics. - M .: Mir, 1976). Among the strains resistant to 500 μg / ml histidine and 50 μg / ml sulfaguanidine, strains capable of histidine accumulation in the culture fluid are selected. In one of them, named E. coli 70, the wild allele of the purF gene is transferred using phage P1 from E. coli CP 78 (Fiil N., Friesen J. D., 1968, J. Bacteriol., V. 95, p. 729-731). Using E. coli 70 strain (purF +) as the parent, clones resistant to the histidine analog D, L-1,2,4-triazole-3-alanine (TPA) and streptomycin are obtained. Among these clones, a histidine producing strain named E. coli 80 was selected.

 The new histidine producer E. coli 80 is deposited in the All-Union Collection of Industrial Microorganisms under registration number VKPM B-7270.

 The new strain of E. coli VKPM B-7270 has the following cultural, morphological and biochemical characteristics.

 Cultural and morphological characters. Gram-negative, slightly movable rod with rounded ends 1.5 - 2.0 microns in length. The dispute does not form.

On a full-fledged agar medium (Luria agar, Hottinger agar) after 48 h, and on a synthetic medium after 72 h incubation at 30 ^o C forms round whitish translucent colonies with a diameter of 2 mm. The surface of the colonies is smooth, the edges are even, the center of the colonies is raised, the structure is homogeneous, the consistency is pasty, easily emulsified.

 On a synthetic medium, producer cells require vitamin B1 as well as L-Proline.

The strain is maintained on plates with a dense synthetic salt medium of the following composition, g / l:
Glucose - 10.0
Tris- (hydroxymethyl) -aminomethane - 6.05
Ammonium Sulfate - 2.0
Magnesium Sulfate - 0.2
Sodium Citrate - 1.0
Potassium chloride - 2.0
Potassium phosphoric acid monosubstituted - 0.028
Thiamine - 0.002
Proline - 0.1
Streptomycin - 1.0
Agar - 20
Distilled water to 1 liter pH - 7.4 - 7.6
Physiological and biochemical characteristics. The strain is an auxotroph along the proline.

 Optional anaerobic. When sowing by injection in full agar, good growth throughout the injection. It does not dilute gelatin. Absorbs glucose, maltose, mannitol, galactose, sorbitol, glycerin. Does not consume sucrose, lactose, raffinose.

 Resistant to streptomycin.

 It contains a mutation in the hisG gene, which results in the desensitization of ATP-phosphoribosyltransferase against histidine.

 Absorbs nitrogen in the form of ammonium, nitrates, and also nitrogen of some organic compounds.

It grows at a temperature of 43 ^o C and below. The optimum temperature for growth is 37 ^o C, and for the production of histidine - 29 - 30 ^o C.

 It grows on liquid media with a pH of 6.0 to 8.0. The optimum pH value is 7.0.

 The strain accumulates 16-17 g / l histidine in 68 hours of fermentation.

Strain storage. The seed is mixed with an equal volume of 80% glycerol and stored at -70 ^o C.

Example 1. Cultures of strains of E. coli 80 and E. coli 24 are grown on a minimal agar medium at 29 ^° C for 65 hours. To obtain seed, cultures are seeded in 50 ml tubes containing 2 ml of seed medium of the following composition, g / l:
Glucose - 56
Ammonium Sulfuric Acid - 28
Potassium phosphoric acid monosubstituted - 2.25
Magnesium Sulfuric Acid, 7-water - 1.125
Iron (II) sulfuric acid, 7-water - 0.01
Manganese (II) sulfuric acid, 4-water - 0.01
Thiamine - 0.001
Yeast Autolysate - 2.8
Proline - 0.3
Streptomycin - 1.0
Distilled water to 1 liter pH - 6.0
The cultivation is carried out for 20 hours on a thermostatically controlled circular rocking chair (200 - 240 rpm.) At 20 ^o C.

The grown seed in an amount of 5 vol.% Contribute to the fermentation medium. The fermentation medium has the following composition, g / l:
Glucose - 92
Ammonium Sulfuric Acid - 28
Potassium phosphoric acid monosubstituted - 2.25
Magnesium Sulfuric Acid, 7-water - 1.125
Iron (II) sulfuric acid, 7-water - 0.01
Manganese (II) sulfuric acid, 4-water - 0.01
Thiamine - 0.001
Yeast Autolysate - 2.8
Proline - 1.0
Streptomycin - 1.0
Chalk - 65
Distilled water to 1 liter pH - 6.0
Cultivation is carried out in 50 ml microbiological tubes containing 2 ml of fermentation medium. After 68 h of cultivation on a circular shaker (350 rpm./min) at 29 ^o C in the culture medium determine the content of histidine by paper chromatography, followed by staining with ninhydrin.

 The fermentation results are shown in the table.

Claims (1)

 The bacterial strain Escherichia coli VKPM B-7270 is a producer of L-histidine.

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