RU2209248C2 - Method for preparing l-methionine, strain of bacterium escherichia coli vkpm b-8125 as producer of l-methionine - Google Patents

Method for preparing l-methionine, strain of bacterium escherichia coli vkpm b-8125 as producer of l-methionine Download PDF

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RU2209248C2
RU2209248C2 RU2001117332A RU2001117332A RU2209248C2 RU 2209248 C2 RU2209248 C2 RU 2209248C2 RU 2001117332 A RU2001117332 A RU 2001117332A RU 2001117332 A RU2001117332 A RU 2001117332A RU 2209248 C2 RU2209248 C2 RU 2209248C2
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methionine
strain
escherichia coli
bacterium
resistant
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RU2001117332A
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Russian (ru)
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RU2001117332A (en
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М.Х. Зиятдинов
Э.Б. Ворошилова
М.М. Гусятинер
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Закрытое акционерное общество "Научно-исследовательский институт Аджиномото-Генетика"
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Abstract

FIELD: biotechnology, microbiology, amino acids. SUBSTANCE: L-methionine is prepared by culturing bacterium Escherichia coli that shows ability to produce at least 0.5 g/l of L-methionine in the process of culturing in minimal medium and this strain is resistant to effect of methionine analogue. The strain Escherichia coli 218 (VKPM B-8125) exhibits such properties. Accumulated amino acid is isolated from cultural fluid. Invention allows preparing L-methionine with high degree of effectiveness. EFFECT: improved preparing method. 6 cl, 1 tbl, 3 ex

Description

Technical field
The invention relates to a bacterium belonging to the genus Escherichia, a producer of L-methionine and a method for producing L-methionine by fermentation using the specified bacteria.

 Currently, DL-methionine production is second only to glutamic acid in total amino acid production. Most DL-methionine is chemically produced.

 Optically active methionine (L-methionine) is required for pharmaceutical purposes, for mixtures of amino acids as a vitamin, and for similar purposes.

 Description of the prior art.

 It is known that some mutants of Escherichia coli resistant to methionine analogues can produce L-methionine.

 It was found that mutants of Escherichia coli that are resistant to ethionine and have lost the ability to inhibit the final product are capable of producing methionine (Abelberg E.A., J. Bacteriol., 76, 326-328 (1958)). The production of methionine by Escherichia coli mutants resistant to norleucine has also been described (Rowbury R. J., Nature, 206, 962 (1965)). But only trace amounts of L-methionine were obtained.

 A microorganism is described, obtained by introducing into a recipient strain of Escherichia a hybrid plasmid containing a DNA fragment isolated from the donor strain of Escherichia resistant to ethionine (UK patent GB2075055). A variation type of the metJ gene is also described with a specific sequence encoding the methionine biosynthesis-related repressor protein from Escherichia coli, in which repression activity is reduced and which is useful in the production of L-methionine (JP157267A2). But the yields in the production process of L-methionine by the indicated mutants (about 0.3-0.5 g / l) cannot satisfy the growing demand for this amino acid.

SUMMARY OF THE INVENTION
The aim of the present invention is the provision of bacteria belonging to the genus Escherichia and having the ability to produce L-methionine, and a method for producing L-methionine using this bacterium.

 It is difficult enough to obtain a producer capable of excreting significant amounts of L-methionine from a wild-type strain by selection. The authors of the present invention decided to obtain a mutant strain producing L-methionine, using a strain producing L-homoserine as the parent strain. L-homoserine is known to be a precursor of L-methionine. Thus, the authors of the present invention have found that such a bacterium belonging to the genus Escherichia, resistant to the methionine analogue, has an increased ability to produce L-methionine. Based on this finding, the present invention has been completed.

 The present invention provides a bacterium belonging to the genus Escherichia that is resistant to the L-methionine analogue and capable of producing at least 0.5 g / L of L-methionine when grown in minimal medium. (Hereinafter referred to as "the bacterium according to the present invention").

 The specified bacterium according to the present invention is resistant to the methionine analogue.

 The specified bacterium according to the present invention is obtained by selection among bacteria belonging to the genus Escherichia, a strain resistant to the methionine analogue.

 Said bacterium according to the present invention is preferably resistant to norleucine.

 The present invention also provides a method for producing L-methionine, comprising the steps of growing a bacterium according to the present invention in a culture medium in order to produce and accumulate in the culture medium L-methionine and isolate L-methionine from the culture fluid (hereinafter referred to as "the method according to the present invention ").

 Further, the present invention will be explained in more detail.

 Detailed description of the present invention.

 The bacterium according to the present invention is a bacterium belonging to the genus Escherichia, possessing the ability to produce L-methionine and resistant to the L-methionine analogue, preferably norleucine.

 The bacteria belonging to the genus Escherichia include Escherichia coli.

The ability to produce L-methionine means the ability to accumulate a significant amount of L-methionine in a nutrient medium during the cultivation of the specified bacteria in a nutrient medium. Usually this means the ability to accumulate at least 0.5 g / l of L-methionine when growing bacteria on minimal medium. The minimum environment is one that contains nothing but the components necessary for bacterial growth. Removing any of the components stops the growth of bacteria. For example, if the bacterium is auxotrophic for threonine, a limited amount of threonine should be added to the culture medium. In another example, the limiting component is a carbon source, such as glucose. Examples of cultivation on a minimal medium are given in the Examples section (see below). The amount of L-methionine produced by the bacterium can be further increased by adding additional nutrients to the medium, such as vitamin B 12 , yeast extract or the like, or by increasing the concentration of compounds used as carbon or nitrogen sources. For example, growing the bacteria of the present invention in a minimal medium containing 4% glucose results in an accumulation of 1.1 g / L of L-methionine. And growing the bacteria according to the present invention on a minimal medium containing 6% glucose leads to the accumulation of 1.6 g / l of L-methionine (see Examples below).

 Resistance to the L-methionine analogue means the ability of the bacterium to grow on minimal medium in the presence of the L-methionine analogue, present in an amount in which the wild-type strain or the parent strain cannot grow. Examples of L-methionine analogs are α-methylmethionine, selenomethionine, ethionine, L-homoserin, norleucine, selenoethionine and the like. The amount of L-methionine analogue in the culture medium depends on the type of analogue. In the case of norleucine, it is usually 5 mg / ml under the conditions described in the Examples below.

 A bacterium belonging to the genus Escherichia, which is resistant to the L-methionine analogue, can be obtained by growing a bacterium belonging to the genus Escherichia on a minimal medium containing the L-methionine analogue in a growth inhibitory concentration and subsequent selection of growing strains. The selection of strains resistant to the L-methionine analogue can be carried out with one type of analogue or with a large number of analogues. The selection of the strain can be carried out once or more times for an analogue of the same type.

 A bacterium belonging to the genus Escherichia, before selection can be subjected to processing in order to obtain mutations. Mutagenesis can be carried out, for example, by UV irradiation or by treatment with a reagent commonly used for artificial mutagenesis, such as N-methyl-N'-nitro-N-nitrosoguanidine (NTG) and nitrous acid. Mutagenesis and selection of mutant strains can be repeated two or more times.

 In a bacterium according to the present invention, the activity of one or more L-methionine biosynthesis enzymes can be increased by mutagenesis or by genetic engineering methods. In addition, the bacterium of the present invention can be improved by introducing a DNA encoding a protein with a reduced ability to repress methionine biosynthesis (metJ gene, JP157267A2).

 The method according to the present invention relates to a method for producing L-methionine, comprising the steps of growing a bacterium according to the present invention in a culture medium in order to produce and accumulate L-methionine in a culture medium and isolate L-methionine from the culture fluid.

 The cultivation of the bacteria according to the present invention, the isolation and purification of L-methionine from the culture fluid can be carried out in a manner similar to traditional methods using fermentation, in which L-methionine is produced using E. coli. The nutrient medium used in the present invention can be either synthetic or natural, provided that the specified nutrient medium contains a carbon source, a nitrogen source, mineral additives and, if necessary, other trace elements.

 Sugars such as glucose, lactose, galactose, fructose or starch hydrolyzate can be used as a carbon source; alcohols such as glycerol or sorbitol; or organic acids, such as acetic, fumaric, citric, succinic acids.

 Inorganic ammonium salts such as ammonium sulfate, ammonium chloride or ammonium phosphate can be used as a nitrogen source; organic nitrogen compounds such as soybean hydrolyzate; gaseous ammonia or aqueous ammonia.

 Preferably, compounds such as L-threonine and sulfur source compounds are present in suitable amounts as organic nutrients. In addition, if necessary, potassium phosphate, magnesium sulfate, iron, manganese ions and other similar compounds can be added in small quantities.

Cultivation is preferably carried out under aerobic conditions, such as mixing the culture with aeration or the like. Cultivation usually ends in 16-72 hours. The temperature during the growing process is maintained in the range from 20 o C to 40 o C, the pH is maintained in the range of 5-8. Inorganic and organic, acidic and basic compounds, such as gaseous ammonia or the like, can be used to maintain a certain pH level.

 The culture consists of cells and culture fluid, preferably a culture fluid.

 Isolation of L-methionine from the culture fluid is usually carried out by a combination of ion exchange chromatography and precipitation methods, or other known methods.

BEST MODE FOR CARRYING OUT THE INVENTION
The present invention will be explained in more detail with reference to the following examples.

 Example 1. Induction of mutant strains of E. coli resistant to norleucine.

A plasmid-free E. coli C600 strain deficient in threonine and leucine was used as the initial strain. First, by transduction with phage P1 grown on the E. coli K-12 strain, Leu + variants of the E. coli C600 strain were obtained. Then, after processing the obtained variants with N-methyl-N'-nitro-N-nitrosoguanidine (NTG) (0.1 mg / ml), a mutant strain 44, resistant to 8 g / L of L-homoserine, was isolated. The specified strain 44 was deficient in L-threonine, was resistant to high concentrations of L-homoserin and was able to produce 4 g / l of L-homoserin, the precursor of L-methionine. Strain 44 was deposited in the All-Russian collection of industrial microorganisms (VKPM) under inventory number VKPM B-2175.

 Then, using mutagenesis using NTG from strain 44, mutants resistant to the methionine analog, norleucine, were obtained.

The night culture cells of strain 44 grown in L-broth were precipitated and resuspended in physiological saline (0.9% Nad) containing 50 μg / ml NTG. After treatment with NTG for 30 minutes at 37 ° C., the cells were pelleted, washed 4 times with physiological saline and placed on M9 minimal agar medium containing 0.5 mg / ml threonine and 2.5 or 5.0 mg / ml norleucine. The plates were incubated for 5 days at 37 ° C. The colonies grown on the plates were collected and purified by sieving to individual colonies on L-agar plates.

 Example 2. Production of L-methionine by new mutant strains - producers of L-methionine in vitro fermentation.

 232 purified strains resistant to different amounts of norleucine were tested for their ability to produce L-methionine (Table 1).

 Among all the obtained mutants, only 4 strains accumulated significant amounts of L-methionine. Among them, strain 218 turned out to be the best producer of L-methionine. Strain 218 was deposited in the All-Russian Collection of Industrial Microorganisms (VKPM) on May 14, 2001 under accession number VKPM B-8125.

The new strain 218 was grown in a nutrient medium for fermentation. As a nutrient medium for fermentation, a minimal medium containing 15 g / l (NH 4 ) 2 SO 4 , 1.5 g / l KH 2 PO 4 , 1.0 g / l MgSO 4 , 0.1 mg / l was used thiamine, 40 g / l glucose, 0.5 g / l threonine and 20 g / l chalk. Glucose and chalk were sterilized separately.

2 ml of the nutrient medium were placed in test tubes, inoculated with one loop of the tested microorganisms and grown at 32 ° C for 3 days on a shaker. The amount of methionine accumulated in the culture fluid was determined by thin layer chromatography (TLC). The composition of the mobile phase for TLC is as follows: isopropanol - 80 ml, ethyl acetate - 80 ml, NH 4 OH (30%) - 15 ml, H 2 O - 45 ml. Growing a new strain of 218 led to the accumulation of 1.1 g / l of L-methionine (4% glucose) in the nutrient medium.

 Example 3. Production of L-methionine by a new mutant strain 218 - producer of L-methionine in vitro fermentation.

 The new strain 218 was grown in a nutrient medium for fermentation, as described in Example 2, except that the glucose concentration was increased to 60 g / L.

 Growing a new strain of 218 led to the accumulation in the nutrient medium of 1.6 g / l of L-methionine (6% glucose).

Claims (6)

 1. A method of producing L-methionine, comprising the steps of growing the bacterium Escherichia coli, a producer of L-methionine in a nutrient medium, and isolating L-methionine from the culture fluid, characterized in that the bacterium Escherichia coli, which is capable of producing at least 0.5 g / l of L-methionine during cultivation on minimal medium and resistant to the methionine analogue.
 2. The method according to p. 1, characterized in that the specified methionine analogue is norleucine.
 3. The method according to p. 2, characterized in that said bacterium is obtained by selection of a strain of resistance to norleucine among Escherichia coli bacteria, said selection being carried out at least once for said methionine analogue.
 4. The method according to any one of claims 1 to 3, characterized in that the strain of said bacterium is obtained from the bacterium Escherichia coli resistant to L-homoserin.
5. The method according to claim 4, characterized in that the strain of the specified bacteria is derived from a strain of Escherichia coli C600 (Leu + ).
 6. The bacterial strain Escherichia coli 218 (VKPM B-8125) - producer of L-methionine.
RU2001117332A 2001-06-26 2001-06-26 Method for preparing l-methionine, strain of bacterium escherichia coli vkpm b-8125 as producer of l-methionine RU2209248C2 (en)

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Cited By (14)

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FR2862067A1 (en) * 2003-11-06 2005-05-13 Metabolic Explorer Sa New evolved microorganisms with altered metabolic pathways, useful e.g. for production of amino acids, are selected as mutants able to grow on defined media
WO2011043485A1 (en) 2009-10-05 2011-04-14 Ajinomoto Co.,Inc. A METHOD FOR PRODUCING AN L-CYSTEINE, L-CYSTINE, A DERIVATIVE OR PRECURSOR THEREOF OR A MIXTURE THEREOF USING A BACTERIUM OF Enterobacteriaceae FAMILY
WO2011065469A1 (en) 2009-11-30 2011-06-03 味の素株式会社 L-cysteine-producing bacterium, and process for production of l-cysteine
WO2012036151A1 (en) 2010-09-14 2012-03-22 味の素株式会社 Sulfur amino acid-producing bacteria and method for producing sulfur amino acids
WO2012114802A1 (en) 2011-02-22 2012-08-30 味の素株式会社 L-cysteine-producing bacterium and method for producing l-cysteine
WO2012137689A1 (en) 2011-04-01 2012-10-11 味の素株式会社 Method for producing l-cysteine
EP2796560A1 (en) 2013-04-23 2014-10-29 Ajinomoto Co., Inc. A method for producing an L-amino acid using a bacterium of the family Enterobacteriaceae having attenuated expression of the yjjK gene
WO2015060314A1 (en) 2013-10-21 2015-04-30 味の素株式会社 Method for producing l-amino acid
WO2015122544A1 (en) 2014-02-14 2015-08-20 Ajinomoto Co.,Inc. A METHOD FOR PRODUCING AN L-AMINO ACID USING A BACTERIUM OF THE FAMILY ENTEROBACTERIACEAE HAVING OVEREXPRESSED THE yajL GENE
RU2598276C2 (en) * 2010-12-29 2016-09-20 СиДжей ЧеилДжеданг Корпорейшн Method of producing l-methionine and related products
EP3098319A1 (en) 2015-05-28 2016-11-30 Ajinomoto Co., Inc. A method for producing an l-amino acid using a bacterium of the family enterobacteriaceae having an attenuated expression of a gsha gene
EP3385389A1 (en) 2017-04-03 2018-10-10 Ajinomoto Co., Inc. Method for producing l-amino acid from fructose
WO2020067487A1 (en) 2018-09-28 2020-04-02 Ajinomoto Co., Inc. Method for producing l-methionine using a bacterium
WO2020071538A1 (en) 2018-10-05 2020-04-09 Ajinomoto Co., Inc. Method for producing target substance by bacterial fermentation

Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2862067A1 (en) * 2003-11-06 2005-05-13 Metabolic Explorer Sa New evolved microorganisms with altered metabolic pathways, useful e.g. for production of amino acids, are selected as mutants able to grow on defined media
WO2011043485A1 (en) 2009-10-05 2011-04-14 Ajinomoto Co.,Inc. A METHOD FOR PRODUCING AN L-CYSTEINE, L-CYSTINE, A DERIVATIVE OR PRECURSOR THEREOF OR A MIXTURE THEREOF USING A BACTERIUM OF Enterobacteriaceae FAMILY
WO2011065469A1 (en) 2009-11-30 2011-06-03 味の素株式会社 L-cysteine-producing bacterium, and process for production of l-cysteine
WO2012036151A1 (en) 2010-09-14 2012-03-22 味の素株式会社 Sulfur amino acid-producing bacteria and method for producing sulfur amino acids
RU2598276C2 (en) * 2010-12-29 2016-09-20 СиДжей ЧеилДжеданг Корпорейшн Method of producing l-methionine and related products
WO2012114802A1 (en) 2011-02-22 2012-08-30 味の素株式会社 L-cysteine-producing bacterium and method for producing l-cysteine
WO2012137689A1 (en) 2011-04-01 2012-10-11 味の素株式会社 Method for producing l-cysteine
EP2796560A1 (en) 2013-04-23 2014-10-29 Ajinomoto Co., Inc. A method for producing an L-amino acid using a bacterium of the family Enterobacteriaceae having attenuated expression of the yjjK gene
WO2015060314A1 (en) 2013-10-21 2015-04-30 味の素株式会社 Method for producing l-amino acid
WO2015122544A1 (en) 2014-02-14 2015-08-20 Ajinomoto Co.,Inc. A METHOD FOR PRODUCING AN L-AMINO ACID USING A BACTERIUM OF THE FAMILY ENTEROBACTERIACEAE HAVING OVEREXPRESSED THE yajL GENE
EP3098319A1 (en) 2015-05-28 2016-11-30 Ajinomoto Co., Inc. A method for producing an l-amino acid using a bacterium of the family enterobacteriaceae having an attenuated expression of a gsha gene
EP3385389A1 (en) 2017-04-03 2018-10-10 Ajinomoto Co., Inc. Method for producing l-amino acid from fructose
WO2020067487A1 (en) 2018-09-28 2020-04-02 Ajinomoto Co., Inc. Method for producing l-methionine using a bacterium
WO2020071538A1 (en) 2018-10-05 2020-04-09 Ajinomoto Co., Inc. Method for producing target substance by bacterial fermentation

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