Method for producing l-amino acid, strain escherichia coli tdh7Δmlc::cat/pprt614 as producer of l-threonine. RU patent 2245919.

FIELD: biotechnology, microbiology, amino acids.

SUBSTANCE: invention relates to a method for producing L-amino acids using microorganism belonging to genus Escherichia wherein gene mlc encoding the global regulator of carbohydrates metabolism is inactivated. For producing such amino acid as L-threonine the strain Escherichia coli TDH7Δmlc::cat/pPRT614 is used wherein gene mlc is inactivated. Invention provides producing L-amino acids with the high degree of effectiveness.

EFFECT: improved producing method.

5 cl, 2 dwg, 1 tbl, 2 ex

The background to the invention

The technical field

The present invention relates to the microbiological industry, in particular to a method for producing L-amino acids using bacteria belonging to the genus Escherichia in which gene ml inactivated.

Prior art

Protein l is a global regulator (repressor) of carbohydrate metabolism (Decker et al, Environ Mol 1998, 27:2:381-90; Kimata et al, Environ Mol, 1998, 29:6:1509-19; Plumbridge, Environ Mol, 1998, 27:2:369-80). It was shown that protein MS regulates the expression of several genes and operons. This ptsG (Kimata et al, Environ Mol, 1998, 29:6:1509-19; Plumbridge, Environ Mol, 1998, 29:4:1053-63; Kirn et al, J Biol Chem, 1999, 274:36:25398-402; Plumbridge, Environ Mol 1999, 33:2:260-73; Tanaka et al., Genes Cells, 1999, 4:7:391-9), encoding a subunit of IICB(Glc) glycotransferases system (PTS)associated with the membrane; operon ptsHIcrr encoding key proteins of the PTS (Kimata et al, Environ Mol, 1998, 29:6:1509-19; Plumbridge, Environ Mol, 1998, 29:4:1053-63; Kirn et al, J Biol Chem, 1999, 274:36:25398-402; Plumbridge, Environ Mol 1999, 33:2:260-73; Tanaka et al., Genes Cells, 1999, 4:7:391-9); the manXYZ operon, encoding enzyme II of the PTS mannose (Plumbridge, Environ Mol, 1998, 29:4:1053-63); gene that encoding the activator of the maltose regulon (Decker et al, Environ Mol 1998, 27:2:381-90).

Genes regulon TS also positively regulated by complex CRP-cAMP (Chapon and Colb, J. Bacteriol, 1983, 156:1135-43; Decker et al, Mol. Environ, 1998,27:2:381-90; Kimata et al, Mol. Environ., 1998, 29:6:1509-19; Plumbridge, Mol. Environ., 1998, 27:2:369-80; Plumbridge, Mol. Environ, 1998, 29:4:1053-63; Kirn et al, J. Biol. Chem. 1999, 274:36:25398-402; Plumbridge, Mol. Environ 1999, 33:2:260-73; Tanaka et al., Genes Cells, 1999,4:7:391-9).

Regulation of transcription of the gene ml is quite complex. First, the transcription is negatively regulated by protein l. Nefosfaurilirovanna protein EIICB(Glc) (product .ptsG) can remove protein l from its binding site by direct protein-protein interactions and, consequently, to induce the expression of regulon ml in response to the presence of glucose (Tanaka et al, EMBO J, 2000, 19:20, 5344-52; Lee et al, EMBO J, 2000, 19:20:5353-61; Nam et al, EMBO J, 2001, 20:3:491-8). Secondly, the mlc gene transcription from two promoters P1 and P2 (Shin et al, J. Biol. Chem. 2001, 276:28:25871-75). The P1 promoter is recognized only RNA polymerase containing Sigma factor σ 70 (Eσ 70 ), whereas the P2 promoter is recognized as Eσ 70 and Eσ 32 containing Sigma factor heat shock. Thus, mlc gene belongs to a class of genes transcribed from multiple promoters, one of which is recognized by RNA polymerase associated with alternative Sigma factor, to ensure response to various environmental conditions. Additionally, the promoter of the gene mlc is highly conserved binding site of CRP (Shin et al, J. Biol. Chem. 2001,276:28:25871-75).

The transport of carbohydrates, carried out with the help of PTS may be the limiting stage in superprodukcja some amino acids. Therefore, inactivation of mlc gene product negatively regulates the expression of genes PTS, it seems necessary to increase the production of amino acids. In a population of Escherichia coli cells grown under conditions of low glucose, were found polygenic mutations in genes mgl, mlc and malT (Munch, K., Genetics, 1999,153:1:5-12). But currently there are no reports about the use of gene inactivation mic for the production of L-amino acids.

Description of the invention

The aim of the present invention is to increase the productivity of strains producing L-amino acids and the provision of a method of producing L-amino acids using these strains.

This goal was achieved by establishing the fact that inactivation of the gene ml encoding the repressor metabolism of carbohydrates, increases the production of L-amino acids such as L-threonine. Thus was accomplished the present invention.

The present invention includes the following:

1. The bacterium belonging to the genus Escherichia, producing L-amino acids, which is modified to inactivate the gene ml.

2. The bacterium belonging to the genus Escherichia, producing L-amino acids, where the specified amino acid is L-threonine.

3. The method of obtaining L-amino acids, including the stage of growth of bacteria in accordance with 1-2 in a nutrient medium to produce and accumulate the L-amino acids in the nutrient medium and the selection of L-amino acid from the culture fluid.

4. The method according to 3, wherein the specified amino acid is L-threonine.

5. The method according to 4, in which this bacterium is modified to increase the expression of L-threonine operon.

The present invention will be described in more detail below.

1. The bacterium according to the present invention

The bacterium according to the present invention is a bacterium belonging to the genus Escherichia, producing L-amino acids, which is modified to inactivate the gene ml.

According to the present invention is a bacteria producing L-amino acids” means a bacterium which has the ability to accumulate L-amino acids in the nutrient medium, when the bacterium according to the present invention are grown in a specified medium. The capacity for production of L-amino acids can be imparted or enhanced by breeding. Used here, the term “bacteria producing L-amino acids” also means a bacterium which is able to produce and accumulate in the culture fluid of L-amino acids more than the natural or parent strain, in the case of E. coli, for example, E. coli strain K-12.

The term “bacterium belonging to the genus Escherichia”means that the bacterium belongs to the genus Escherichia according to the classification known to a specialist in the field of Microbiology. As examples of the microorganism belonging to the genus Escherichia, used in the present invention, may be mentioned the bacterium Escherichia coli (E. coli).

The range of bacteria belonging to the genus Escherichia, which can be used in the present invention is not limited in any way, however, for example, bacteria, described in Neidhardt, F.C. et al. (Escherichia coli and Salmonella typhimurium, American Society for Microbiology, Washington D.C., 1208, table 1) can be mentioned.

The term “gene ml inactivated” means that the target gene is modified so that the resulting modified gene encodes a mutant protein with reduced ability to regulate or this gene encodes a completely inactive protein. This also means that the modified DNA is unable to implement the natural expression of the protein l due to deletions of this gene or modification of the site, adjacent to the specified genome.

Inactivation of the gene ml encoding the repressor PTS, leads to an increase in revenues source of carbon, such as glucose, inside the cells of the bacteria - bacteria producing L-amino acids.

Gene mlc encodes a protein l (406 amino acid residues), a global regulator of carbohydrate metabolism. Gene mlc (gi: 16129552; non nucleotides with 1665368 on 1666588 in sequence with inventory number NC_000913.1 in GenBank) is located between genes b1593 ynfL in the chromosome of E. coli strain K-12.

Inactivation of the gene can be carried out by traditional methods, such as mutagenesis using UV-radiation or treatment nitrosoguanidine (N-methyl-N'-nitro-N-nitrosoguanidine), site-directed mutagenesis, the destruction of the gene using homologous recombination or/and insertion-deletion mutagenesis (Datsenko K.A. and Wanner B.L., Proc. Natl. Acad. Sci. USA, 2000, 97:12: 6640-45), also called “integration using Red-system”.

Bacteria producing L-threonine

As the bacteria according to the present invention can be used bacteria producing L-threonine, which is modified to inactivate the gene mlc.

The bacterium according to the present invention can be improved by increasing expression of one or more genes involved in the biosynthesis of L-threonine. Examples of such genes are the genes of the threonine operon, i.e. the thr operon, preferably comprising a mutant thrA gene, encoding aspartokinase-gomoserinlaktonazy I resistant to inhibition by threonine-type feedback; thrB gene encoding homoserine; thrC gene encoding trevisanato. Another preferred embodiment of the specified bacterium is a bacterium modified to increase the rhtA gene encoding the presumed transmembrane protein. As a preferred embodiment of the specified bacterium is a bacterium modified to increase the expression of aspC gene encoding aspartataminotransferaza (aspartatamino) (patent RF №2002104983).

The most preferred embodiment of the bacteria according to the present invention is a bacterium modified to increase the expression of aspC gene, a mutant thrA gene, gene thrB, thrC, rhtA and modified to inactivate the gene ml.

As the parent strain can be used strains - producers of L-threonine, belonging to the genus Escherichia, such as E. coli strain VKPM B-3996 (U.S. patent 5,175,107 and 5,705,371), a strain of E. coliNRRL-21593 (U.S. patent 5,939,307), a strain of E. coli FERM BP-3756 (U.S. patent 5,474,918), strains of E. coli FERM BP-3519 and FERM BP-3520 (U.S. patent 5,376,538), E. coli strain MG442 (Gustine, etc.. Genetics, 14, 947-956 (1978)), the E. coli strains VL643 and VL2055 (European patent application EP 1149911 A), and the like.

The bacterium according to the present invention can be obtained by inactivation of the gene ml in bacteria, have the ability to the production of L-amino acids. On the other hand, the bacterium according to the present invention can be obtained by giving the bacteria that already contains an inactivated gene ml, capacity for production of L-amino acids.

Methods of obtaining plasmid DNA, cutting and ligation of DNA, transformation, selection of an oligonucleotide as a nucleating and the like may be conventional methods, well known to the person skilled in the art. These methods are described, for example, in the book of Sambrook, J., Fritsch, E.F., and Maniatis, T., "Molecular Cloning A Laboratory Manual, Second Edition", Cold Spring Harbor Laboratory Press (1989).

2. The method according to the present invention

The method according to the present invention is a method of obtaining L-amino acids, including the stage of growth of bacteria according to the present invention in a nutrient medium to produce and accumulate the L-amino acids in the nutrient medium, and the selection of L-amino acid from the culture fluid. More specifically, the method according to the present invention is a method of obtaining L-threonine, comprising the stage of growth of bacteria according to the present invention in a nutrient medium to produce and accumulate L-threonine in the culture medium, and excretion of L-threonine from the culture fluid.

According to the present invention, the cultivation, isolation and purification of L-amino acid from the culture or similar fluid may be carried out in a manner similar to traditional methods of fermentation, in which the amino acid is produced using a microorganism.

The nutrient medium used for cultivation, can be both synthetic and natural, provided that the medium contains sources of carbon, nitrogen, mineral supplements and, if necessary, the appropriate amount of nutritional supplements necessary for growth of microorganisms. The carbon sources include various carbohydrates such as glucose and sucrose, and various organic acids. Depending on the nature of the assimilation of the used microorganism can be used alcohols such as ethanol and glycerol. As the nitrogen source can be used various inorganic ammonium salts such as ammonium sulfate, other nitrogen compounds such as ammonia and amines, a natural nitrogen sources such as peptone, soybean hydrolysate, fermentolizat microorganisms. As mineral additives can be used potassium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, calcium chloride and similar compounds. As vitamins can be used thiamine, yeast extract and similar substances.

The cultivation is carried out preferably under aerobic conditions, such as mixing the culture fluid on the rocking chair, stirring with aeration, at a temperature in the range from 20 to 40° C, preferably in the range from 30 to 38° C, pH maintained within the range of from 5 to 9, preferably from 6.5 to 7.2. pH can be adjusted by ammonia, calcium carbonate, various acids, bases and buffer solutions. Usually growing within 1 to 5 days, resulting in the accumulation of the target L-amino acid in the culture fluid.

After cultivation, solids such as cells can be removed from the culture fluid by centrifuging or by filtration through a membrane, and then L-amino acid can be isolated and purified by methods of ion-exchange chromatography, concentration and crystallization.

A brief description of the drawings.

Figure 1 shows the relative location of the seed mlcIL and mlcIR on the plasmid pACYC 184, used for amplification of gene cat.

Figure 2 shows the construction of the chromosomal DNA fragment containing the inactive gene ml.

The best way of carrying out the invention

In more detail the present invention will be explained below with reference to Examples.

Example 1. Construction of strains with an inactive gene ml.

1. Getting the division in the gene ml.

A deletion in the gene ml was obtained by the method, first developed by Datsenko and Wanner (Proc. Natl. Acad. Sci. USA, 2000, 97(12), 6640-6645), also called “integration using Red-system”. In accordance with the specified method were obtained for PCR priming mlcIL (SEQ ID NO: 1) and mlcIR (SEQ ID NO: 2), each of which contained areas, as homologous areas adjacent to the genome ml, and to gene, which imparts resistance to the antibiotic, located on the plasmid that was used as matrix. As the matrix used for PCR plasmid pACYC 184 (NBL Gene Sciences Ltd., UK) (inventory number H in GenBank/EMBL). Temperature conditions for PCR were as follows: initial DNA denaturation for 3 minutes at 95° C; profile for two first cycles: 1 min at 95° C, 30 seconds at 34° C, 40 seconds at 72° C; profile for 30 cycles: 30 seconds at 95° C, 30 seconds at 50° C, 40 seconds at 72° C; and a final polymerization for 5 minutes at 72° C.

The resulting PCR product length 935 BP (Figure 1, SEQ ID NO: 3) was purified on an agarose gel and used for electroporation of E. coli strain MG1655 containing plasmid pKD46 with replicon is sensitive to temperature. Plasmid pKD46 (Datsenko and Wanner, Proc. Natl. Acad. Sci. USA, 2000, 97(12), 6640-6645) contains a DNA fragment (2154 pairs of nucleotides, 31088 - 33241) phage Lamda (number J02459 in GenBank), consisting of genes λ Red-system homologous recombination (genes γ , β and Exo) under control of the promoter P Agabus induced by arabinose. Plasmid pKD46 is necessary to integrate the PCR product into the chromosome of strain MG1655.

Electrocompetent cells was prepared as follows: overnight cultures of cells of the strain E. coli MG1655 grown at 30° C in LB-medium containing ampicillin (100 mg/l)was diluted 100 times with 5 ml of medium SOB (Sambrook et al, "Molecular Cloning A Laboratory Manual, Second Edition", Cold Spring Harbor Laboratory Press (1989))containing ampicillin and L-arabinose (1 mm). The resulting culture was grown with aeration at 30° to achieve an optical density OD 600 ≈ 0.6, then concentrated 100-fold and washed 3 times with deionized water with a temperature of about 0° C. Electroporation was performed using 70 μl of cells and approximately 100 ng of PCR product. Cells after electroporation were incubated with aeration in 1 ml of SOC environment (Sambrook et al, "Molecular Cloning A Laboratory Manual, Second Edition", Cold Spring Harbor Laboratory Press (1989)) at 37° C for 2.5 hours and then sown on L-agar and grown at 37° C to select recombinant bacteria that are resistant to chloramphenicol (Cm R ). Then, to remove the plasmid pKD46, spent 2 passage 42° on L-agar with chloramphenicol and the resulting colonies were tested for sensitivity to ampicillin.

Confirmation of gene fission ml by PCR

The presence of a deletion of the gene ml in mutants bearing the gene for resistance to chloramphenicol, was checked by PCR. To confirm used locus-specific priming mlcPL (SEQ ID NO: 4) and mlcPR (SEQ ID NO: 5). Temperature conditions for PCR were as follows: initial DNA denaturation for 3 minutes at 94° C; profile for 30 cycles: 30 seconds at 94° C, 30 seconds at 52° C, 2 min at 72° C; and a final polymerization for 7 minutes at 72° C. the PCR Product obtained by amplification of DNA from the beads to the parent Mlc + strain MG1655, contained 1492 nucleotide (Figure 2, SEQ ID NO: 6). The PCR product obtained by amplification of DNA from cells of mutant strain MG1655 Δ mlc::cat, contained 1191 nucleotide (Figure 2, SEQ ID NO: 7).

3. Construction of a strain with inaktivirovannye gene mlc - producer L-20 threonine.

A deletion Δ mlc::cat was introduced into the strain E. coli TDH7/pPRT614 producing threonine (VKPM B-5318, U.S. patent 6,132,999) using standard procedures transduction by phage PI (Sambrook et al, "Molecular Cloning A Laboratory Manual, Second Edition", Cold Spring Harbor Laboratory Press (1989)). As a donor used strain MG1655 25 Δ mlc::cat. The presence of deletions Δ mlc::cat gene mic in the resulting strain TDH7Δ mlc::cat/pPRT614 was checked by PCR using nucleating rnlcPL (SEQ ID NO: 4) and rnlcPR (SEQ ID NO: 5).

Example 2. Production of L-threonine by E. coli strain with inaktivirovannye genome MS.

Each of the strains of E. coli TDH7/pPRT614 and TDH7Δ mlc::cat/pPRT614 were grown for 18-24 hours at 37° on cups with L-agar containing streptomycin (50 μg/ml). Then one loop of cells was transferred into 50 ml of L-broth of the following composition: tripton - 10 g/l, yeast extract 5 g/l, NaCl 5 g/l of Cells (50 ml, OD 540 =0.12 PU)grown on a shaker (140 rpm) at 37° C for 4 hours, used for the planting of 450 ml of 10 nutrient medium for fermentation. Fermentation until the exhaustion of the carbon source (batch fermentation) was performed in a laboratory fermenter with a volume of 1 l with aeration (1/1 vol/vol/min) with stirring at a speed of 1200 rpm at 39° C. the pH Value was maintained at 6.6 in automatic mode using an 8% aqueous solution of ammonia. The results are presented in Table 1.

The composition of the medium for fermentation (g/l):

Glucose 100.0

NH 4 Cl 1.75

KN 2 RO 4 1.0

MgSO 4 × 7H 2 O 0.8

FeSO 4 × 7H 2 O 0.01

MnSO 4 × 5H 2 O 0.01

Mameno(TN) 0.15

Betaine 1.0

Glucose and magnesium sulfate were sterilized separately. the pH was maintained at 6.6

Table 1. Strain the Amount of L-threonine, g/l Yield, % DCW, g/l production Time, h TDH7/pPRT614 12.6 13.3 16.6 22.8 TDH7Δ mlc::cat/pPRT614 16.9 18.2 16.8 21.7

As can be seen from Table 1, the inactivation of the gene ml increases the production of L-threonine with the strain TDH7/pPRT614 - producer of L-threonine.

1. The method of obtaining L-amino acids, including the stage of growth of bacteria Escherichia coli in a nutrient medium and a selection from the culture fluid obtained and accumulated in it L-amino acids, characterized in that as a producer-L-amino acid using a bacterium of Escherichia coli, modified so that the gene mlc in the specified bacteria inactivated.

2. The method according to claim 1, characterized in that the specified L-amino acid is L-threonine.

3. The method according to claim 2, characterized in that, in the bacteria further strengthened the expression of the threonine operon.

4. The method according to claim 3, characterized in that as a producer of L-threonine using a strain of Escherichia coli DH7Δmlc::cat/pPRT614.

5. The strain Escherichia coli TDH7Δmlc::cat/pPRT614 producing L-threonine.