SU1362021A1 - Strain of eschericia coli bacteria as producer of l-treonin - Google Patents

Abstract

The invention relates to microm; - biological flux and relates to a NEW bacterial strain 5 producing an amino acid L-threonine, which can be used in various sectors of the national economy. A new producer strain Ij-threonine Escherichia coli VKPM B-3420 (VNIIgenetika-TDG-6) has a hybrid pusmid containing the gene of the threonine operon, blocking the biochemical degradation of L-threonine. Using new prod; a1, enta allows you to increase the level of kolesch L-threonine in the fermentation medium to 85 g / l, and also c1shzit specific consumption of carbohydrate raw materials by 15-22% compared to the best of the best a1№1 strain a; -production of L -treonine. 2 tab. up A new bacterial strain PJscherichia coli VKPM is capable of pumping up to 85 g / l of threonine in 54 h of fermentation. The specific consumption of sugars is 2 g / l. The new strain was obtained on the basis of the E. coli strain of HSE 472 T-23 and, preserving its genetic features, differs from it in its inability to threonine enzymatic degradation, the first stage of which is catalyzed by threonine dehydrogenase. Strain E, coli VKPM B-3420 was obtained by transferring 472 T-23 VNIIgenetiki 472 T-23 to the original E. coli strain, inactivated by transposon Tn5, the gene encoding threonine dehydrogenase, using the s method:

Description

common transduction by bacteriophage F1, duplicated on cells of the mutant strain of E. coli iso, which is defective in triad vid e ni another o n e e.5

The transposon TPn5 reports cells resistance to manamycin.

Thus, the new strain, in contrast to all other threo-1-shna producers, created on the basis of E, coli,. 10 is not capable of converting threonine synthesized by them or added to the medium into degradation products (aminacetone, glycine, acetyl CoA), which contributes to an increase in the level of HaKoraie-fS or threonine in the culture fluid and reduces the consumption of carbon raw materials,

Strain E, coli. BKPM-3420 (VSHigen.ethics TDG-b) has the following cultures — morphological and physiological — 20 biochemical traits,

Morphological and cultural signs, Gram-negative, weakly moving rods with zakpyryglenny and ends of 1.5-2 microns in length, 25

 After 24 hours of growth at 37 ° C, the mi-peptone agar forms whitish, translucent cololes with a diameter of mm, the surface of the colony is smooth, the edges are even or uneven, the center of the colony is raised, the structure is uniform, paste-like consistency, is easily emulsified.

On Adams agar medium after 40–48 h of growth, when or it forms coliiiJi 0.5–1.5 µm in diametr – rb, grayish-white, translucent, slightly convex, with glitter 1, its surface.

On mi-peptone broth, specific Q growth rate at 37 ° C 1.2–1.3 After 24 h of growth, there is a strong equimensional turbidity, a small precipitate, and a characteristic odor.

On a medium medium A, cs-45a, the growth rate at 37 ° C is 0.1–0.2 hours; After 2 days of growth with aeration, a strong uniform turbidity, 3 an ax X ar ary,

By injecting with m-peptone agar, a good increase is given for a BceNjy injection,

 11izia11ogo-bio, chemical signs, Gelatin does not dilute. Good growth with coagulation of milk. Indole forms. It grows well on sucrose, glucose, lactose, mannose, galactose, coshosis, fructose, glycerin. MaiiiDiTe with the formation of acid and gas.

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Resistant to penicillin and iamycin.

Cells contain a multi-copy plasmid pYN7 (mol, May, 5.8 megadalton), which provides resistance to penicillin and carries the genes for the threonine operon.

Resistant to L-threonine and L-hemoserine ,.

With growth in the presence of L-threonine, aminoacetone does not form.

The production of L-threonine using a new producer strain is carried out as follows.

Cells of producer strain E, coli are grown on Adams agar medium in the presence of penicillin, then a suspension of cells washed off from the skewed agar is seeded with a liquid nutrient medium containing sources of carbon, nitrogen, necessary mineral salts, nutritional supplement in the form of hydrolysis protein containing substrates and penicillin. The inoculum is grown in a fermenter equipped with a pK-statisation system at pH 6.8-7.2, a temperature of 36-38 0 and continuous aeration of agitation for 20-24 h. The prepared inoculum in 10-15% is used for sowing fermentation medium containing a carbon source, for example, sugar or molasses, a nitrogen source and optional mineral salts. Fermentation is carried out in fermenters equipped with a pH-setting system at pH 6.8-7.2, temperature 36-38 0 and continuous aeration stirring, as pH-stabilizer cient applied ammonia water or sbalansgprovashguyu of carbon and nitrogen am miachno carbohydrate-feeding. The duration of fermentation is 40-60 hours. By the end of the cultivation, up to 85 g / l of threonine accumulates in the culture fluid. The specific consumption of the carbon source for the synthesis of G g threonine is 2 g. There is no accumulation of aminoacetone in the culture fluid,

Example 1, Culture E, coli VNIIgenetika 472 T-23 and strain E, coli VKGSh B-3420 in parallel are grown on Adams agar medium containing sucrose (0.2%) and penicillin (500 units / ml) and sow them with liquid seed medium the next composes 7 9 s / o

Sucrose Ammonium sulfate Ammon phosphate acid disubstituted 7-water sulphate Potassium chloride Iron (C) sulphate T-water Manganese (II) sulphate 5-water Yeast extract Benzylpenicillinate sodium Water:

The seeding material is grown in fermenters with a capacity of 0.5 liters with aeration of 0.5 liters of air per minute and stirring at a speed of 900 rpm at 37 ° C. The pH value is maintained within 6.8-7.2 by automatically feeding the signal from the pH sensor of the sugar-ammonia feed, which is prepared by mixing 6.5 volumes of a sterile 40% sucrose solution with one volume of 14.7 n ammonia water. After 20 h of cultivation, 50 ml of the culture is introduced into the fermentation medium of the following composition, Z: Sucrose3.0

Ammonium sulphate 0.5

Ammonium phosphate disubstituted 0.12

Potassium chloride0.06

Sodium chloride0.06

7-water magnesium sulfate 0.04

Iron (II) sulfur,., - acidic 7-water0.002

Manganese (II) 5-water sulfate, 0.002 Water, the remaining fermentation is carried out in fermenters with a capacity of 0.5 l with aeration of 0.5 l of air per minute and mixing at 900 rpm at

ABOUT

temperature 37 C. pH value is supported by the editor L, Velsk

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Compiled by V.Varlamov Tehred M. Mrgrental

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VNSHSHI of the State Committee for Inventions and Discoveries at the State Committee on Science and Technology of the USSR 4/5, Moscow, Zh-35, Raushsk nab. 113035

Production and publishing plant Patent, Uzhgorod, st. Gagarin, 101

620216

lives in the range of 6.8-7.2. by automatic feeding on a signal from the pH sensor of sugar ammonia feed, the preparation of which is indicated above. Fermentation is carried out for 48 h. Indicators of the process of threonine biosynthesis after 48 h of fermentation are given in tab, 1,

10 Example 2. The cultivation of strains on a seed and fermentation media is carried out under conditions similar to Example I. The difference is that in the fermentation medium is added.

15 yeast extract in the amount of 0.1% Fermentation lasts 54 hours. Indicators of the process of threonine biosynthesis under these conditions are given in Table. 2,

20Form of the invention

The strain of the bacterium Escherichia coli VKPM B-3420 - producing b-threonine with

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Table}

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Table 2

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Claims (1)

20 Formula of the invention Escherichia coll bacteria strain VKPM B-3420 - producer of 1-threonine _with Table! Pcs amm The accumulation of threonine, g / l The consumption of sucrose in the calculation . those on | g synthesis of roven threonine, g / g 472 T-23 '57, 0 2,38 TDG-6 75-9 2.01 f Table 2 Strain The accumulation of threonine, g / l Sucrose consumption per 1 g of synthesized threonine, g / g 472-T-23 66.9 2,55 TDG-6 85 ₀ 0 1.99