The dna fragment rhtc encoding the fusion protein rhtc, giving increased resistance to l-threonine bacteria escherichia coli, and method for producing l-amino acids

 

(57) Abstract:

The invention relates to biotechnology and genetic engineering. The DNA fragment rhtC encodes a protein synthesis RhtC, which gives increased resistance to L-threonine bacteria Escherichia coli. Based multicopying plasmids pRhtC containing this fragment, engineered strains of Escherichia coli capable of increased production of L-amino acids compared with strains that do not contain plasmid pRhtC. Amino acids are produced by cultivation of the respective strains in a suitable nutrient medium, followed by separation and purification of the target amino acids. The invention allows to increase the yield of L-amino acids. 2 S. and 4 C.p. f-crystals, 7 ill., 6 table.

The invention relates to biotechnology, in particular relates to a method of obtaining amino acids, namely L-homoserine, L-threonine, L-valine or L-leucine using bacteria belonging to the genus Escherichia.

As the closest analogue may be considered the DNA fragment encoding rhtA gene is associated with bacterial resistance to L-homoserine and L-threonine in a minimal environment, as well as obtained on the basis of mutations of this gene rhtA23 (previously designated as thrR) strains of Escherichia coli producing - 616, 1985), or L-homoserine and L-glutamic acid (Astaurov and other Applied biochemistry and Microbiology, I. 27, pp. 556 - 561, 1991).

RhtA gene wild type provides resistance to L-homoserine and L-threonine, if he cloned in multicopying plasmid, and the increase of its expression increases the production of amino acids by bacteria belonging to the genus Escherihia, and capable of producing L-threonine, L-lysine, L-valine, or L-arginine. It was found that mutation rhtA23 is located on the 18 minute maps of the chromosomes of E. coli and rhtA gene identified as orf1, an open reading frame located between genes pexB and ompX. Genetic structure expressing the protein encoded by orf1, was designated as the rhtA gene (rht - resistance to homoserine and threonine - resistant homoserine and threonine). RhtA gene consists of 5'-non-coding region, including the SD sequence, the very open-reading frames, orf1, and the terminator. Mutation rhtA23 changes the nucleotide immediately preceding the initiation codon ATG, and increases the expression of the rhtA gene (ABSTRACTS of the 17th International Congress of Biochemistry and Molecular Biology in conjugation with 1997 Annual Meeting of the American Society for Biochemistry and Molecular Biology, San Francicco, California, August 24-29, 1997, N 457).

In the cloning process rhtA gene is discovered that there are at Merano and L-threonine. One of them is the rhtA gene. As it turned out, another gene, rhtB, tells the cells of E. coli resistance only to L-homoserine (Application for the grant of a patent in Russia. N 98118425).

The present invention is to increase the level of accumulation of amino acids by cells of bacteria of the genus Escherichia, producing L-homoserine, L-threonine, L-valine or L-leucine.

The problem is solved by obtaining a DNA fragment rhtC encoding the fusion protein RhtC, giving increased resistance to L-threonine bacteria E. coli, and design based on it strains to develop a method of obtaining amino acids with a high yield of the target amino acids.

The subject of the present invention are:

1. Bacteria belonging to the genus Escherichia, in which the resistance to L-threonine increased due to the increase in bacteria activity of the protein, characterized by one of the two properties (A) or (B):

And protein, which consists of the amino acid sequence of N 2 (Fig.2); or

G - protein which consists of an amino acid sequence including deletion, substitution, insertions or additions of one or several amino acids of the sequence as N 2 and which has the activity Obispo resistance to L-homoserine improved by increasing the bacterial cells the protein activity, characterized by one of two properties (C) or (D):

(C) a protein which consists of the amino acid sequence of N 4 (Fig. 4); or

(E) protein, which consists of the amino acid sequence, including deletions, substitutions, insertions or additions of one or several amino acids of the sequence as N 4 and which has the activity, providing the bacteria containing this protein, resistance to L-homoserine;

(3). Bacteria under item (1) or (2), where the activity of the protein, determined by the properties (A) or (B) is increased as a result of transformation of bacteria with DNA that encodes a protein having properties (A) or (B);

(4). Bacteria in p. (2), where the activity of a protein having the properties (C) or (D), is increased by transformation of bacteria with DNA that encodes a protein having the properties (C) or (D);

(5). The method of obtaining amino acids, comprising the step of culturing bacteria, corresponding to any one of items 1 to 4 and having the ability to produce amino acids in the culture medium, providing products and accumulation of the corresponding amino acids in this environment, and the allocation of accumulated amino acids from the environment.

(6). The method according to p. 5, where the amino acid is one of the AB at p. (6), where the branched chain amino acids are L-valine or L-leucine.

DNA encoding a protein as defined above under item (A) or (B) can be regarded as rhtC gene and the protein encoded by the genome of rhtC, can be considered as a "protein RhtC". DNA encoding a protein as defined above under item (C) or (D) can be regarded as a rhtB gene and the protein encoded by rhtB gene can be regarded as a "RhtB protein". The activity of the protein RhtC, which is involved in making the bacteria resistance to L-threonine (i.e., the activity which makes the bacteria with protein RhtC, resistant to L-threonine) can be regarded as "Rt activity" (from the words of Resistance to threonine - resistant to threonine), and the activity of the RhtB protein, which is involved in making the bacteria resistance to L-homoserine (i.e., the activity that makes bacteria with RhtB protein, are resistant to L-homoserine), can be considered as "Rh activity" (from the words of Resistance to homoserine - resistant homoserine).

Structural genes encoding protein RhtC and RhtB protein, designated respectively as "structural rhtC gene" or "structural rhtB gene". The term "increased Rt activity or Rh activity" means giving the cells a resistance to L-threonine or L-homoserine, or improve this condition the t of these proteins, or impaired negative regulation of expression or activity of these proteins, etc., the Term "DNA encoding a protein" refers to Dunaeva DNA, one of the threads which encodes a protein. Resistance to L-threonine is an attribute bacteria to grow on minimal medium containing L-threonine concentration at which the wild-type strain carrying natural allele of the gene rhtC, can't grow, usually > 30 mg/ml Resistance to L-homoserine means property of bacteria to grow on minimal medium containing L-homoserine in concentration at which the wild-type strain carrying natural allele rhtB gene, cannot grow, usually > 5 mg/ml. the Ability to produce the amino acid means a property of bacteria to synthesize and accumulate the amino acid in the culture medium in an amount larger than a wild-type strains.

In accordance with the present invention, the resistance to high concentrations of L-threonine or L-threonine and L-homoserine can be attached bacteria belonging to the genus Escherichia, and are able to produce amino acids, in particular L-homoserine, L-threonine, L-valine or L-leucine.

The present invention is a DNA fragment (rhtC gene) that encodes a protein with Rt-activity and am having the particular, this fragment can be represented by a nucleotide sequence comprising nucleotides from 187 in 804 in the nucleotide sequences of N 1 (see claims).

The second DNA fragment used in the present invention, (rhtB gene) that encodes a protein with Rh-activity and having the amino acid sequence shown in Fig. 4 ("the Amino acid sequence of the RhtB protein"). In particular, this fragment can be represented by a nucleotide sequence comprising nucleotides from 557 in 1171 in the nucleotide sequence of the N 3 (Fig. 3).

RhtB gene having the nucleotide sequence indicated in Sequence No. 3, corresponds to part of the sequence that is complementary to the sequence of M available in GenBank, and includes well-known alleged open-reading frames f138 (nucleotides with 61959 on 61543 in the sequence M-87049), the function of which is unknown, and which is located on the chromosome of E. coli in the area 86,8 minute card, and nucleotide 201 proximal f138. It should be noted that the open reading frame f138 only with 160 5'-flanking nucleotides do not provide resistance to L-homoserine. It also turned out that this is Telenesti precedes the binding site with ribosomes (SD sequence, nucleotides with 62171 on 62166 in M). This long open reading frame (nucleotides 62160-61546) and represents a structural rhtB gene.

RhtB gene produced either by infection Lisovenko on Mucts strain of E. coli lysate, phasmida minimi d50005, as described by Groisman et al. (Groisman et al. J. Bacteriol., 158, 357-364, 1986) with subsequent isolation of plasmid DNA from colonies grown on minimal medium containing 40 μg/ml kanamycin and 10 mg/ml L-homoserine or from the E. coli chromosome by hybridization of colonies, or by using the polymerase chain reaction (PCR) (White et al. , Trends Genet., 5, 185, 1989), using oligonucleotide(s) having a sequence corresponding to the plot in the area of 86 minutes of the E. coli chromosome.

Another approach involves the synthesis of the oligonucleotide based on a sequence of N 3. Using the oligonucleotides having a sequence corresponding to a region of DNA that is located proximal to nucleotide 557 N, and the segment of DNA that is located distal to the nucleotide N 1171 in the sequence N 3 as primers for PCR, it is possible to amplify the entire coding region.

The synthesis of oligonucleotides is carried out by usual method, for example using phospholidine method (see Tetrahedron Letters, 2Applied Biosystems). PCR carried out using commercially available equipment for PCR (e.g., DNA thermal cycler, model PG 2000, manufactured by Takara Shuzo Co., Ltd) using Taq DNA polymerase in accordance with the procedure described by the supplier of the enzyme.

RhtC gene corresponds adjusted, as described below, the sequence a (nucleotide N 60860 - 61480 in the sequence M available in GenBank), which is located on the site of the chromosome, adjacent to rhtB gene. It is produced simultaneously with the rhtB gene, as shown in example 1, by infection Lisovenko on Mucts strain of E. coli lysate, phasmida minimi d50005 as described above, with subsequent isolation of plasmid DNA from colonies grown on minimal medium containing 40 μg/ml kanamycin and 50 mg/ml threonine. Another approach involves the synthesis of oligonucleotides based on a sequence of N 1 by the method described above and use them for hybridization or PCR. Using the oligonucleotides having a sequence corresponding to a region of DNA that is located proximal to the nucleotide N 187, and the segment of DNA that is located distal to the nucleotide 804 N in the sequence N 1 as primers for PCR, can amplificiation RhtB, including deletions, substitutions, insertions, or additives of one or several amino acids at one or more positions that do not violate this Rh-RhtB protein activity. Similarly, DNA encoding a protein RhtC of the present invention may encode a protein RhtC, including deletions, substitutions, insertions, or additives of one or several amino acids at one or more positions that do not violate this Rt-protein activity RhtC.

DNA encoding essentially the same protein that RhtB, or the same protein that RhtC, described above, can be obtained, for example, by modifying the nucleotide sequence, in particular by using site-directed mutagenesis, so that one or more amino acid residue is deleterows, substituted, inserted or added. DNA modified as described above can be obtained by the known methods using mutational effects. The mutation treatment includes methods of processing DNA coding RhtB protein or protein RhtC, in vitro, for example, with hydroxylamine, or processing methods microorganism, particularly a bacterium belonging to the genus Escherichia and bearing DNA encoding RhtB protein or protein RhtC, UV radiation or mutagenic agents such as N-methyl-N'-nitro-PC, encoding options RhtB protein or RhtC selected by expression of plasmid DNA carrying genes rhtC or rhtB and subjected to in vitro mutagenic effects, as described above, in the respective cells with subsequent determination of their resistance to L-threonine or L-homoserine and selection of DNA that provides this stability. The invention also pertains to variants of the protein RhtC, which are found in different species, strains and variants of bacteria of the genus Escherichia and due to natural diversity. DNA encoding these variants, and hybridize in stringent conditions with a DNA having the nucleotide sequence from nucleotide 187 on 804 nucleotide in the sequence N 1.

Similarly, the invention also pertains to variants RhtB protein, which are found in different species, strains and variants of bacteria of the genus Escherichia and due to natural diversity. DNA encoding these variants, and hybridize in stringent conditions with a DNA having the nucleotide sequence from nucleotide 557 on 1171 nucleotide in the sequence N 3. The term "stringent conditions" means conditions under which so-called specific hybridization occurs, and nonspecific does not occur. It is difficult to clearly exp and which DNA having high homology, for example at least 70% homology with respect to each other, - hybridize, and DNA having homology lower than indicated.

In the present invention consider bacteria belonging to the genus Escherichia, represented here by E. coli, in which the Rt activity increased. In addition, the present invention includes bacteria E. coli, which also increased Rh-activity.

The increase in Rt activity occurs, for example, by amplification of the copy number of the structural rhtC gene in the cells in the transformation of their recombinant DNA, which includes the fragment containing the structural rhtC gene that encodes a protein RhtC, legirovannye with a promoter sequence that functions efficiently in bacteria of the genus Escherichia. Rt-activity can also be enhanced by replacing the promoter sequence rhtC gene on chromosome promotor sequence, which more effectively function in bacteria of the genus Escherichia.

Increasing Rh activity occurs, for example, by amplification of the copy number of the structural rhtB gene in the cells in the transformation of their recombinant DNA, which includes the fragment containing the structural rhtB gene that encodes a protein RhtB, lagunet can also be enhanced by replacing the promoter sequence rhtB gene on chromosome promoter sequence, which is more effectively function in bacteria of the genus Escherichia.

Amplification of the number of copies of the structural gene rhtC, or structural rhtB gene in the cells can be carried out also by introducing multicopying vector, which carries the structural gene rhtC or structural rhtB gene in cells of bacteria belonging to the genus Escherichia. In particular, the number of copies can be increased by introducing a plasmid, phage or transposon (Berg, D. E. and Berg, C. M., Bio/Technol., 1, 417, 1983) containing the structural gene rhtC or structural rhtB gene in cells of bacteria belonging to the genus Escherichia. Multicabinet vector can be represented plasmid vectors such as pBR322, pMW118, pUC19 or the like, or fagbemi vectors, such as 1059, BF 101, m13MP9 or similar. Transposons can be represented by the phage Mu transposons Tn10, Tn5 or similar. The introduction of DNA into bacteria belonging to the genus Escherichia can be implemented, for example, using the method of Morrison (Methods in Enzymology, 68, 326, 1979) or the method in which recipient cells of bacteria is exposed to calcium chloride to increase permeability in relation to DNA (Mandel and Higa, J. Mol. Biol., 53, 159, 1970) or other similar methods.

Found the link between chia to increase the production of L-amino acids inventors is used for strains, with increased productivity of amino acids. When you do this you have two options:

1. A sign of increased Rt activity, or simultaneously increased Rt activity and Rh activity, is introduced into strains that are already capable of producing the desired amino acids.

2. The ability to produce amino acids attached to the strains that have increased Rt activity or simultaneously increased Rt activity and Rh-activity.

Designed based on the amplification of a DNA fragment rhtC the E. coli strain MG442/pRhtC - producer of homoserine, the E. coli strain MG442/pVIC40, pRhtC producing threonine and strains of E. coli NZ10/pRhtBC and E. coli NZ10/pRhtB, pRhtC - producers of homoserine, valine and leucine are capable of increased production of these amino acids compared with strains that do not contain amplified DNA fragment rhtC.

New strains deposited in Russian national collection of industrial microorganisms. The E. coli strain MG442/pRhtC deposited under number VKPM B-7700; E. coli strain MG442/pVIC40, pRhtC deposited under number VKPM B-7680; E. coli strain NZ10/pRhtB, pRhtC deposited under number VKPM B-7681 and E. coli strain NZ10/pRhtBC deposited under number VKPM B-7682.

The E. coli strain MG442/pRhtC (VKPM B-7700) has the following cultural-morphological and biochemical traits.

Cultural characteristics.

Mastopathy agar. After 24 hours growth at 37oC forms a round whitish translucent colonies with a diameter of 1.5-3.0 mm; surface smooth colonies, edges smooth or slightly wavy, the center of the colonies raised, the structure is homogeneous, pasty consistency, easy to emulsify.

Agar Luria. After 24 h growth at 37oC forms a whitish translucent colonies with a diameter of 1.5-2.5 mm; surface colonies smooth, smooth edge, the structure is homogeneous, pasty consistency, easy to emulsify.

Agar Wednesday Adams.

After 40-48 h of growth at 37oC forms colonies with a diameter of 0.5-1.5 mm; grayish-white translucent, slightly convex with a shiny surface. In the presence of L-isoleucine (0.1-0.5 g/l) growth is stimulated and similar colonies formed after 18-20 h

Growth in mesopatamia broth. After 24 h of growth - strong uniform turbidity, characteristic odor.

Physiological and biochemical characteristics.

Growth injection in mesopatamia agar. Good growth throughout the injection. The microorganism is optional gone anaerobic.

Gelatino not thins.

Growth in milk good sleep broth at temperatures 20-42oC. the Optimal temperature for growth is temperature 33-37oC.

Relation to the pH of the medium. Grows in environments with a pH from 6.0 to 8.0. The optimum pH value is 7.2.

The sources of carbon. Grows well on glucose, fructose, lactose, mannose, galactose, xylose, glycerol, mannitol with the formation of acid and gas.

The sources of nitrogen. Absorb nitrogen in the form of ammonium, nitrate and some organic nitrogen compounds.

Resistant to ampicillin.

The content of the plasmids. Cells contain mnogoseriynuyu hybrid plasmid pRhtC bearing rhtC gene that tells cells resistance to L-threonine (50 mg/ml) and determinants of resistance to ampicillin.

The E. coli strain MG442/pVIC40, pRhtC (VKPM B-7680) has the same cultural-morphological and biochemical evidence that the strain VKPM B-7700 (MG442/pRhtC), except that he along with plasmid pRhtC contains mnogoseriynuyu hybrid plasmid pVIC40, carrying the genes of the threonine operon, and the determinants of resistance to streptomycin. Strain resistant to ampicillin and streptomycin.

The E. coli strain NZ10/pRhtB, pRhtC (VKPM B-7681) has the same cultural-morphological and biochemical evidence that the strain VKPM B-7700 (MG442 other and it contains mnogoseriynuyu plasmid pRhtB bearing rhtB gene that tells cells resistant to L-homoserine (10 mg/ml), and the determinants of resistance to kanamycin. Strain resistant to kanamycin and ampicillin.

The E. coli strain NZ10/pRhtBC (VKPM B-7682) has the same cultural-morphological and biochemical evidence that the strain VKPM B-7681 (NZ10/pRhtB, pRhtC), except that it contains mnogoseriynuyu plasmid pRhtBC, carrying genes simultaneously rhtB and rhtC, as well as determinants of resistance to ampicillin. Strain resistant to ampicillin.

A method of producing amino acids by culturing strains-producers is as follows.

The amino acid is produced by culturing bacteria, in which the Rt-activity, or at the same time Rt-activity and Rh-activity is increased, for example, by amplification of the copy number of the gene rhtC or rhtB, as described above, and which have the ability to produce amino acids by culturing them in a culture medium, where the accumulation of amino acids, with subsequent isolation of this amino acid from the medium (culture fluid). The amino acid represented mainly L-homoserine, L-threonine, L-alanine, L-valine or L-leucine. In accordance with present the C culture fluid carried out by the known methods. For cultivation use synthetic or natural environment. This environment includes a source of carbon, nitrogen, mineral salts and additives in quantities that are optimal for growth and biosynthesis. As a carbon source use various carbohydrates such as glucose, sucrose, various organic acids. Depending on assimilative abilities it is possible to use alcohols, including ethanol or glycerol. As a source of nitrogen using ammonia, various ammonium salts such as ammonium sulfate, or other nitrogen-containing compounds such as amines, a natural nitrogen sources such as peptone, soybean hydrolysate, or a hydrolysate of microbial cells. As the mineral components are phosphate one-deputizing potassium, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, calcium carbonate. Cultivation is mainly carried out in aerobic conditions, such as cultivation on a mixer, or with aeration and agitation culture. The cultivation temperature from 30 to 40oC, mainly 30-38oC. the pH of the culture - 5-9, mainly of 6.5 to 7.2. the pH of the culture was adjusted to desired values by using ammonium, calcium carbonate is tipirovaniya selection of amino acids is carried out by removal of solid particles, such as cells from the medium by centrifugation or filtration through membrane filters with subsequent isolation and purification of target amino acids using ion-exchanger fractionation using concentration and crystallization.

List of figures.

Fig. 1. Cloning and identification of a gene rhtB and rhtC gene.

Fig. 2. The amino acid sequence of the protein RhtC (sequence No. 2).

Fig. 3. The nucleotide sequence containing the rhtB gene (sequence No. 3).

Fig. 4. Amino acid sequence RhtB protein (sequence No. 4).

Fig. 5. The structure of the plasmid pRhtB carrying rhtB gene.

Fig. 6. The structure of the plasmid pRhtC carrying rhtC gene.

Fig. 7. The structure of the plasmid pRhtBC carrying genes rhtB and rhtC.

The present invention more specifically explain the following examples.

Example 1. Obtaining DNA fragments rhtB and rhtC

Step 1. Cloning of genes associated with resistance to L-threonine and L-homoserine, phasmida minimi.

Genes associated with resistance to L-threonine and L-homoserine, clone in vivo in phasmida minimi (Groisman et al. J. Bacteriol., 168, 357-364, 1986). As a donor using the strain MG442, lysogenize the Amma PMBC-513 Mu cts62 (Hfr K10 metB) and plated on minimal medium with methionine (50 µg/ml), L-homoserine (10 mg/ml) and kanamycin (40 μg/ml) and cultured at 30oC. From growing up in 48 hours, colonies produce plasmid DNA, which transform the E. coli strain VKPM B-513 according to standard methods. Transformants are selected on plates with L-agar and kanamycin (40 μg/ml). From the transformants, which are resistant to L-homoserine, isolated plasmid DNA, which is analyzed by restrictive analysis. From the donor strain were otkalibrovani insert (DNA fragments), belonging to two different regions of the chromosome. Thus, on the chromosome of E. coli found at least two genes that when amplification tell the cells of E. coli resistance to L-homoserine. One type inserts contains rhtA gene, which has been reported (ABSTRACTS of the 17th International Congress of Biochemistry and Molecular Biology in conjugation with 1997 Annual Meeting of the American Society for Biochemistry and Molecular Biology, San Francicco, California, August 24-29, 1997). The second type of inserts contains a fragment of the SacII-SacII, indicating resistance to L-homoserine and L-threonine, or minimum MluI fragment-M1uI length of 0.8 kb, indicating stability only to L-homoserine (Fig. 1).

Stage 2: Identification of genes rhtB and rhtC.

Received MluI-M1uI fragment is sequenced in two circuits the method of Singer (Sanger et al. Proc. Natl. Acad. Sci. USA, 74, 5463, 1977) and the nucleotide 201, located in front of her. The insert containing the open reading frame f138 only with 160 5'-flanking nucleotides do not provide resistance to L-homoserine. The specified sequence above f138 does not contain a stop codon in frame f138. In addition, one of the ATG codons in the sequence is preceded by a binding site with ribosomes (SD sequence, nucleotides with 62171 on 62166 in M). This long open reading frame (nucleotides 62160-61546) is a structural rhtB gene. Coded them RhtB protein (Fig. 4) is a highly hydrophobic protein and contains a potential transmembrane segments.

A plasmid containing the rhtB gene tells the cell resistance only to high concentrations of homoserine (Fig. 1). At the same time, the DNA fragment SacII-SacII, indicating to the cells at the same time the resistance to high concentrations of homoserine and threonine, contains the second unidentified open reading frame, a. Subclavian o at a minimum ClaI fragment-Eco47III shows that the plasmid carrying the gene that tells the cell resistance only to high (50 mg/ml) concentrations of L-threonine (Fig. 1). Subcloned fragment was sequenced, and found that it contains additional is in the specified sequence eliminates the shift of the reading frame and extends the open reading frame in the direction of 5'-flanking region to 60860 nucleotides, inclusive. This new gene (nucleotide N 60860-61480 in M) designated as rhtC. Both genes, rhtB and rhtC, encode proteins homologous to the conveyor associated with the transport of lysine cells Corynbacterium glutamicum.

Example 2. The effect of amplification rhtB gene, or rhtC gene products L-homoserine.

<1> Constructing L-homoserine-producing E. coli strain NZ10/pAL4, pRhtB (VKPM B-7658) and obtain L-homoserine with it.

The DNA fragment rhtB clone in mnogoopytny plasmid vector pUK21 (Vieira, Messing, Gene, 100, 189-194, 1991). The result is the plasmid pRhtB (Fig. 5).

The E. coli strain NZ10, which is Leu+revertants known strain C600 (thrB, leuB) (Appleyard, Genetics., 39, 440-452, 1954), transform the plasmid pAL4, which is a vector pBR322 carrying the thrA gene, encoding aspartokinase-gomoserinlaktonazy I. the result is an E. coli strain NZ10/pAL4, capable of production of L-homoserine. The resulting strain transformed by the plasmid pRhtB or vector pUK21 and the result of the strains of E. coli NZ10/pAL4, pRhtB (VKPM B-7658) and E. coli NZ10/pAL4, pUK21 (VKPM B-7661). The E. coli strain NZ10/pAL4, pRhtB tells the cells resistance to high concentrations of homoserine (10 mg/ml), to which the E. coli strain NZ10/pAL4, pUK21 remains sensitive. Each of the obtained strains cultivar is 95), containing 50 mg/l kanamycin and 100 mg/ml ampicillin. 0.3 ml of the obtained culture liquid make in 3 ml of a fermentation medium having the composition specified below, and containing 50 mg/ml kanamycin and 100 mg/ml ampicillin contained in test tubes 20 x 200 mm, and cultured at 37oC 46 hours on a rotary shaker.

The composition of the fermentation medium, g/l:

Glucose - 80

(NH4)2S04- 22

TO2HPO4- 2

NaCl - 0,8

MgSO47H2O - 0,8

FeSO47H2O - 0.02

MnSO45H2O - 0.02

Thiamine HCl - is 0.0002

Yeast extract - 1,0

CaCO3- 30 (add after autoclaving)

After culturing to determine the number accumulated in the environment L-homoserine and the optical density of the culture liquid at 560 nm by the known methods. As shown in the table. 1, the strain NZ10/pAL4, pRhtB accumulates L-homoserine more than the strain NZ10/pAL4, pUK21, in which the number of copies of the rhtB gene is not increased.

<2> Constructing L-homoserine-producing E. coli strain MG442, pRhtC (VKPM B-7700) and obtain L-homoserine with it.

The DNA fragment rhtC clone in mnogoopytny plasmid vector pUC21 (Vieira, Messing, Gene, 100, 189-194, 1991). The result PLA is Midol pRhtC or vector pUC21 and the result of the strains of E. coli MG442/pRhtC (VKPM B-7700) and E. coli MG442/pUC21. The E. coli strain MG442/pRhtC is resistant to high concentrations of threonine (50 mg/ml), to which the E. coli strain MG442/pUC21 remains sensitive.

Each of the thus obtained strains were cultured at 37oC 18 hours in LB broth with 100 mg/l ampicillin. Then 0.3 ml of the obtained culture liquid make in tubes 20 x 200 mm with 3 ml of a fermentation medium described above containing 100 mg/l ampicillin, and cultured at 37oC 72 hours on a rotary shaker. After cultivation, the amount accumulated in the environment L-homoserine, and the optical density of the culture liquid at 560 nm was measured with known methods. The results are presented in table. 2.

As shown in the table. 2, the strain MG442 after introducing plasmids pRhtC from producer threonine turns into a producer of homoserine and accumulates this amino acid is greater than the strain MG442/pUC21, in which the number of copies of the rhtC gene is not increased.

Example 3. The effect of amplification rhtB gene or gene rhtC on the production of L-threonine.

<1> Designing strain-producer of L-threonine E. coli MG442/pVIC40, pRhtB (VKPM B-7660) and obtain L-threonine.

To obtain a new strain-producer of L-threonine E. coli MG442/pVIC40, pRhtB as the U.S. 5175107, 1992). Transformants are selected on plates with LB agar containing 100 mg/l streptomycin, and receive the strain MG442/pVIC40. The strain MG442/pVIC40 transform plasmids pRhtB or pUK21 and get the strains MG442/pVIC40, pRhtB (VKPM B-7660) and MG442/pVIC40, pUK21 (VKPM B-7663). The E. coli strain MG442/pVIC40, pRhtB is resistant to high concentrations of homoserine (10 mg/ml), to which the E. coli strain MG442/pUC21 remains sensitive.

Each of these strains were cultured at 37oC 18 hours in LB broth as in example 2 with 50 mg/l kanamycin and 100 mg/l streptomycin. Then 0.3 ml of the obtained culture liquid make in tubes 20 x 200 mm with 3 ml of a fermentation medium described in example 2, containing 50 mg/l kanamycin and 100 mg/l streptomycin, and cultured at 37oC 46 hours on a rotary shaker. After cultivation, the amount accumulated in the environment of L-threonine and the optical density of the cultures at 560 nm was measured by known methods. The results are presented in table. 3.

As shown in table. 3, the strain MG442/pVIC40, pRhtB accumulate L-threonine in larger quantities than the strain MG442/pVIC40, pUK21, in which the number of copies of the rhtB gene is not increased.

<2> the Design of strain-producer of L-threonine E. coli MG442/pVIC40, pRhtC (VKPM B-7680) and obtain L-threonine.

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The strain MG442/pVIC40, pRhtC is resistant to high concentrations of threonine (50 mg/ml), to which the E. coli strain MG442/pVIC40, pUC21 remains sensitive.

Each of these strains were cultured at 37oC 18 hours in LB broth as in example 2 with 100 mg/l ampicillin and 100 mg/l streptomycin. Then 0.3 ml of the obtained culture liquid make in tubes 20 x 200 mm with 3 ml of a fermentation medium described in example 2, containing 100 mg/l ampicillin and 100 mg/l streptomycin, and cultured at 37oC 46 hours on a rotary shaker. After cultivation, the amount accumulated in the environment of L-threonine and the optical density of the cultures at 560 nm was measured by known methods. The results are presented in table. 4.

As shown in the table. 4, the strain MG442/pVIC40, pRhtC accumulate L-threonine in larger quantities than the strain MG442/pVIC40, pUC21, in which the number of copies of the rhtC gene is not increased.

Example 4. The influence of the joint amplification of gene rhtB and rhtC gene products of amino acids.

The DNA fragment SacII-SacII containing both genes rhtB and rhtC, clone in mnogoopytny plasmid vector pUC21 (Vieira, Messing, Gene, 100, 189-194, 1991). The result is the plasmid pRhtBC (Fig. 1, figs. 7).

The E. coli strain NZ10 transform vector pUC21 or PL is In-7684), E. coli NZ10/pRhtB, pRhtC (VKPM B-7681) and E. coli NZ10/pRhtBC (VKPM B-7682). The E. coli strain NZ10/pRhtB has a high resistance to high concentrations of homoserine (10 mg/ml), E. coli strain NZ10/pRhtC has a high resistance to L-threonine (50 mg/ml), and the strains of E. coli NZ10/pRhtB, pRhtC and E. coli NZ10/pRhtBC have both a high resistance to high concentrations of homoserine (10 mg/ml) and threonine (50 mg/ml), to which the E. coli strain NZ10/pUC21 remains sensitive.

Each of the obtained strains were cultured as described above, making cultivation and fermentation environment appropriate antibiotics. After cultivation for 46 hours number accumulated in the environment of the amino acid and the optical density of the cultures at 560 nm was measured by known methods. The results are presented in table. 5.

As shown in the table. 5, the simultaneous amplification of genes rhtB and rhtC in the cells of the strain NZ10 increases the accumulation in the culture liquid L-homoserine, L-valine and L-leucine. This result shows that in cells the gene products rhtB and rhtC can interact.

Example 5. The effect of amplification of the gene rhtB and rhtC gene for resistance of E. coli to certain amino acids and analogs of amino acids.

As shown above, pliney liquids of different strains-producers. It also turned out that the nature of the accumulated amino acids depends on the genotype of the strain. Homology gene products rhtB and rhtC with lysine Transporter LysE engaged in export of L-lysine from the cells of Corynebacterium glutamicum (Vr1jic et al. , Mol. Environ., 22, 815-826, 1996), indicates a similar function of proteins RhtB and RhtC. It is known that the increase in the activity of genes that control the transport of cells to various growth inhibitors, increases their resistance to the corresponding compounds. In this regard, determine the impact of plasmids pRhtB and pRhtC on the stability of E. coli strain N 99, which is StrRa mutant of a known strain of E. coli W3350 (VKPM B-1557), some amino acids and analogs of amino acids. To this end, the strain N 99 transform plasmids pRhtB, pRhtC and vectors pUC21 and pUK21. Night of culture the strains obtained N 99/pRhtB, No. 99/pRhtC, No. 99/pUK21 and N 99/pUC21 grown in minimal medium M9 on the rocking chair (about 109cells/ml) was diluted 1:100 and pokasivaut for 5 hours in the same environment. Then the obtained culture in the logarithmic growth phase is diluted to about 104viable cells applied on the dried plates with agar (2% agar) medium M9 containing different concentrations of amino acids or amino acid analogues. Growth or absence of the compounds (table. 6).

As can be seen from the table, the rhtB gene amplification significantly increases the resistance of bacteria not only to homoserine, but similar to threonine, -amino--oxovalerate acid (AOW), and to a lesser extent, increases resistance to L-threonine and analogue L-lysine, (S)-2-amino-ethyl-L-cysteine (AFC). In addition, there is some increase in resistance of bacteria to the analogue of leucine, 4-Aza-DL-Latino. The rhtC gene amplification in addition to L-threonine significantly increases the resistance of bacteria to AOW, L-histidine and L-valine. These results indicate that each of the alleged conveyors, RhtB and RhtC have specificity towards several substrates (amino acids) or may detect nonspecific effect of amplification.

1. The DNA fragment rhtC encoding the fusion protein RhtC, giving increased resistance to L-threonine bacteria Escherichia coli containing the regulatory elements of the rhtC gene and the structural part of the rhtC gene and having the nucleotide sequence N1, shown in the graphics part.

2. The method of obtaining L-amino acids by culturing resistant to L-threonine producer strains of bacteria of the genus Escherichia in a suitable nutrient medium with th the E. coli bacteria with high resistance to L-threonine, which is due to high concentration in the cells of these bacteria protein RhtC encoded by the DNA fragment rhtC under item 1.

3. The method according to p. 2, wherein receiving the amino acid L-threonine.

4. The method according to p. 2, wherein receiving the amino acid L-homoserine.

5. The method according to p. 2, wherein receiving the amino acid L-valine.

6. The method according to p. 2, wherein receiving the amino acid L-leucine.

 

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