MICROORGANISMS HAVING ENHANCED L-AMINO ACIDS PRODUCTIVITY AND PROCESS FOR PRODUCING L-AMINO ACIDS USING THE SAME

Abstract

Disclosed are a recombinant microorganism having enhanced L-amino acid productivity, wherein the recombinant microorganism is transformed to have removed or decreased activity of at least one of adenosine deaminase and AMP nucleosidase, and a method of producing an L-amino acid using the recombinant microorganism. The use of the recombinant microorganism may enable the production of the L-amino acid in a highly efficient manner.

Claims

1. A recombinant microorganism having enhanced producibility of an L-amino acid, wherein activity of at least one of adenosine deaminase comprising an amino acid sequence of SEQ ID NO: 14 and AMP nucleosidase comprising an amino acid sequence of SEQ ID NO: 16 removed or decreased.

2. The recombinant microorganism of claim 1, wherein the L-amino acid is L-threonine or L-tryptophan.

3. The recombinant microorganism of claim 1, the recombinant microorganism belongs to the genus Escherichia.

4. The recombinant microorganism of claim 3, wherein the recombinant microorganism is Escherichia coli.

5. A method of producing L-amino acid, the method comprising: culturing the recombinant microorganism of claim 1; and collecting an L-amino acid from the culture.

6. The method of claim 5, wherein the L-amino acid is L-threonine or L-tryptophan.

7. A method of producing L-amino acid, the method comprising: culturing the recombinant microorganism of claim 2; and collecting an L-amino acid from the culture.

8. A method of producing L-amino acid, the method comprising: culturing the recombinant microorganism of claim 3; and collecting an L-amino acid from the culture.

9. A method of producing L-amino acid, the method comprising: culturing the recombinant microorganism of claim 4; and collecting an L-amino acid from the culture.

Description

DESCRIPTION OF THE DRAWINGS

[0030] FIG. 1 is a graph showing ATP levels in an L-threonine-producing strain upon gene deletion performed according to the present invention.

[0031] FIG. 2 is a graph showing ATP levels in an L-tryptophan-producing strain upon gene deletion performed according to the present invention.

MODE OF THE INVENTION

[0032] Hereinafter, the present invention will be described in further detail with reference to the following examples. These examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

Example 1

Preparation of L-Threonine-Producing Strain and L-Tryptophane-Producing Strain, Each has Weakened Activity of a Protein Encoded by Add Gene or Amn Gene

[0033] In L-threonine-producing strains, i.e., KCICM10910P (Korean Patent No: 2009-0076389) and KCCM-10132 (Korean Patent NO: 2000-0013853), and L-tryptophane-producing strain, i.e., KCCM10812P (Korean Patent Publication No: 10-0792095), genes that each encode the Add and the Amn were deleted by homologous recombination. The add and amn genes to be deleted each include a base sequence of SEQ ID NO: 13 and a base sequence of SEQ ID NO: 15.

[0034] In detail, one step inactivation, which is a technique of constructing a mutant using lambda Red recombinase developed by Datsenko K A et al. (Proc Natl Acad Sci USA., (2000) 97:6640-6645), was used. To confirm the insertion of amplification product into the gene, a chloramphenicol-resistant gene of pUCprmfmloxC was used as a marker (Korean Patent No: 2009-007554). Then, polymerase chain reaction (hereinafter, referred to as PCR) was performed by using pUCprmfmloxC as a template, a primer set of SEQ ID NOS: 1 and 2 having a part of the base sequences of these two genes and a part of the base sequence of the chloramphenicol-resistant gene of pUCprmfmloxC, and a primer set of SEQ ID NOS: 7 and 8 under the following conditions: 30 cycles of denaturation at 94 C. for 30 seconds, annealing at 55 C. for 30 seconds, and elongation at 72 C. for 1 minute, resulting in the amplification of a gene fragment of approximately 1,200 bp.

[0035] The DNA fragment obtained by the PCR amplification was electrophoresed on a 0.8% agarose gel, eluted, and used as a template for secondary PCR. The secondary PCR was performed by using the eluted primary PCR product as a template, a primer set of SEQ ID NOS: 3 and 4 having 20 bp of a complementary sequence to the 5 and 3 regions of the primary DNA fragment and further having the 5 and 3 regions of the genes, a primer set of SEQ ID NOS: 9 and 10 under the following conditions: 30 cycles of denaturation at 94 for 30 seconds, annealing at 55 for 30 seconds, and elongation at 72 for 1 minute, resulting in the amplification of 4 types of a gene fragment of approximately 1,300 bp. The DNA fragments obtained therefrom were electrophoresed on a 0.8% agarose gel, eluted, and used in recombination.

[0036] E. coli, which was transformed with a pKD46 plasmid according to the method developed by Datsenko K A et al (Proc Natl Acad Sci USA., (2000) 97:6640-6645), was prepared as a competent strain, and transformation was performed by introducing the gene fragment of 1,300 bp that was obtained by PCR. The obtained strains were selected on a LB medium supplemented with chloramphenicol. Accordingly, a deletion of the genes was confirmed by a PCR product of approximately 1,440 bp and 2,104 bp obtained by PCR using a primer set of SEQ ID NOS: 5 and 6 and a primer set of SEQ ID NOS: 11 and 12.

[0037] After removal of the pKD46 plasmid, the primary recombinant E. coli strain having chloramphenicol resistance was introduced with a pJW168 plasmid so as to remove the chloramphenicol marker gene from the strain (Gene, (2000) 247,255-264). In the microbial cells that were finally obtained, a deletion of the genes was confirmed by a PCR product of approximately 340 bp and 1,004 bp obtained by PCR using a primer set of SEQ ID NOS: 5 and 6 and a primer set of SEQ ID NOS: 11 and 12.

[0038] A deletion of the amm gene was performed in the same manner as described above by using a strain where the add gene was deleted, a primer set of SEQ ID NOS: 5 and 6, and a primer set of SEQ ID NOS: 11 and 12, and accordingly, a double deletion of these two genes were confirmed.

[0039] According to the method described above, 6 types of L-threonine-producing strains, i.e., a KCCM10910Padd strain, a KCCM10910Pamn strain, a KCCM10910Paddamn strain, a KCCM-10132add strain, a KCCM-10132amn strain, and a KCCM-10132addamn strain, were prepared. In addition, 3 types of L-tryptophan-producing strains, i.e., a KCCM10812Padd strain, a KCCM10812Pamn strain, and a KCCM10812Paddamn strain, were prepared.

Example 2

Measurement of ATP Levels in L-Threonine-Producing Strain and L-Tryptophan-Producing Strain

[0040] In order to quantify actual ATP levels found in the strains of Example 1, the Efficient Method for Quantitative determination of Cellular ATP Synthetic Activity developed by KIYOTAKA Y et al (J Biom Scre, (2006) V11: No. 3: PP310-17) in accordance with the use of luciferase was used. In a glucose-containing LB liquid medium, the strains of Example 1 each having different genetic transformation were cultured overnight. After removal of the supernatant by centrifugation, the microbial cells were washed with a solution of 100 mM Tris-Cl (pH 7.5), and then, treated with a permeable (PB) buffer solution (40%[v/v] Glucose, 0.8%[v/v] Triton X-100) for 30 minutes, thereby transporting the intracellular ATP to the outside. After separation of the supernatant by centrifugation again, the resultant was mixed with luciferin, which is used as a substrate of luciferase. After 10 minutes of a reaction, the degree of color development of the luciferase was measured by using a luminometer, so as to quantify ATP levels. The results are shown in FIGS. 1 and 2. All the resulting values were mean values obtained by experiments that were repeated 3 times.

[0041] As shown in FIGS. 1 and 2, in comparison between the mother strains without the gene deletion (i.e., the L-threonine-producing strain and the L-tryptophan-producing strain) and the strains of Example 1, it was confirmed that the ATP levels in the strains of Example 1 were found to be increased. In addition, it was confirmed that the ATP levels were further increased in the strains having the deletion of the add and amn genes in combinations rather than the strains having the deletion of a single gene.

Example 3

Confirmation of Effects of L-Threonine-Producing Strain Having Weakened Activity of Proteins Encoded by E. coli Add and Amn Genes in Glucose-Containing Medium

[0042] In the L-threonie-producing strain (KCCM10910P) of Example 1, the add and amn genes were deleted separated or in combination, so as to proceed a potency test with respect to the strains having increased intracellular ATP levels by using glucose as a carbon source.

[0043] The strains each having different genetic transformation were cultured in the LB solid medium overnight in an incubation at 33 C. Afterwards, 1 platinum loop of each of the microbial cells was inoculated in 25 ml of titer medium containing glucose as shown in the composition of Table 1 below, and then, was cultured in an incubator at 33 C. and at 200 rpm for 50 hours. The results are shown in Table 2 below. All the resulting values were mean values obtained from 3 flasks.

TABLE-US-00001 TABLE 1 Composition Concentration (per liter) Glucose 70 g KH.sub.2PO.sub.4 2 g (NH.sub.4).sub.2SO.sub.4 25 g MgSO.sub.4H.sub.2O 1 g FeSO.sub.4H.sub.2O 5 mg MnSO.sub.4H.sub.2O 5 mg Yeast extract 2 g Calcium carbonate 30 g pH 6.8

TABLE-US-00002 TABLE 2 Glucose consumption L-threonine Strain OD (g/L)* (g/L)** KCCM10910P 25.8 30.3 31.5 KCCM10910Padd 23.7 33.9 32.7 KCCM10910Pamn 22.9 35.7 33.4 KCCM10910Paddamn 22.7 36.0 33.6 *30-hr measured value **50-hr measured value

[0044] As shown in Table 2 above, it was confirmed that the strains having the gene deletion according to the present invention resulted in the increased glucose consumption by about 18.8% as compared with the glucose consumption of the mother strain. It was also confirmed that the amounts of threonine produced in the strains were increased by about 6.6% as compared with the amount of threonine produced in the mother strain. These results denote that, in consideration of the ATP levels as shown in FIG. 1, the activity of the transformed strains was increased by the increased ATP levels thereof, and accordingly, the glucose consumption rates or the producibility of the amino acid of the transformed strains were improved.

[0045] In this regard, the E. coli KCCM10910Paddamn strain having enhanced glucose consumption rates and threonine producibility was named CA03-8254P (Accession No: KCCM11494P, deposited at the Korean Culture Center of Microorganisms (KCCM) on Dec. 9, 2013).

Example 4

Confirmation of Effects of L-Threonine-Producing Strain Having Weakened Activity of Proteins Encoded by E. coli Add and Amn Genes in Glucose-Containing Medium

[0046] In the L-threonie-producing strain (KCCM-10132) of Example 1, the add and amn genes were deleted separated or in combination, so as to proceed a potency test with respect to the strains having increased intracellular ATP levels by using glucose as a carbon source.

[0047] The strains each having different genetic transformation were cultured in the LB solid medium overnight in an incubation at 33 C. Afterwards, 1 platinum loop of each of the microbial cells was inoculated in 25 ml of titer medium containing glucose as shown in the composition of Table 1 below, and then, was cultured in an incubator at 33 C. and at 200 rpm for 50 hours. The results are shown in Table 3 below. All the resulting values were mean values obtained from 3 flasks.

TABLE-US-00003 TABLE 3 Glucose consumption L-threonine Strain OD (g/L)* (g/L)** KCCM-10132 25.8 32.0 20.2 KCCM-10132add 22.7 34.0 21.0 KCCM-10132amn 22.7 35.5 21.5 KCCM-10132add amn 23.0 36.2 21.5 *30-hr measured value **50-hr measured value

[0048] As shown in Table 3 above, it was confirmed that the strains having the gene deletion according to the present invention resulted in the increased glucose consumption by about 13% as compared with the glucose consumption of the mother strain. It was also confirmed that the amounts of threonine produced in the strains were increased by about 6.4% as compared with the amount of threonine produced in the mother strain.

Example 5

Confirmation of Effects of L-Tryptophan-Producing Strain Having Weakened Activity of Proteins Encoded by E. coli Add and Amn Genes in Glucose-Containing Medium

[0049] In the L-tryptophan-producing strain (KCCM10812P) of Example 1, the add and amn genes were deleted separated or in combination, so as to proceed a potency test with respect to the strains having increased intracellular ATP levels by using glucose as a carbon source.

[0050] In order to proceed the potent test, 1 platinum loop of each of the microbial cells was inoculated in 25 ml of titer medium containing glucose as shown in the composition of Table 4 below, and then, was cultured in an incubator at 37 C. and at 200 rpm for 48 hours. The results are shown in Table 5 below. All the resulting values were mean values obtained from 3 flasks.

TABLE-US-00004 TABLE 4 Composition Concentration (per liter) Glucose 60 g KH.sub.2HPO.sub.4 1 g (NH.sub.4).sub.2SO.sub.4 10 g NaCl 1 g MgSO.sub.4H.sub.2O 1 g Sodium citrate 5 g Yeast extract 2 g Calcium carbonate 40 g Phenylalanine 0.15 g Thyrosine 0.1 g pH 6.8

TABLE-US-00005 TABLE 5 Glucose consumption L-tryptophan Strain OD (g/L)* (g/L)** KCCM10812P 18.2 34.5 5.92 KCCM10812Padd 17.7 36.8 6.90 KCCM10812Pamn 17.9 38.0 7.15 KCCM10812Paddamn 18.0 38.0 7.50 *33-hr measured value **48-hr measured value

[0051] As shown in Table 5 above, it was confirmed that the strains having the gene deletion according to the present invention resulted in the increased glucose consumption by about 10% as compared with the glucose consumption of the mother strain. It was also confirmed that the amounts of tryptophan produced in the strains were increased by about 26.6% as compared with the amount of tryptophan produced in the mother strain. These results denote that, in consideration of the ATP levels as shown in FIG. 2, the activity of the transformed strains was increased by the increased ATP levels thereof, and accordingly, the glucose consumption rates or the producibility of the amino acid of the transformed strains were improved.

[0052] It should be understood that the exemplary embodiments described therein should be considered in a descriptive sense only and not for purposes of limitation. Descriptions of features or aspects within each embodiment should typically be considered as available for other similar features or aspects in other embodiments. While one or more embodiments of the present invention have been described with reference to the figures, it will be understood by those of ordinary skill in the art that various changes in form and details may be made therein without departing from the spirit and scope of the present invention as defined by the following claims.

[0053] [Accession number]

[0054] Depositary institution: Korean Culture Center of Microorganisms (international)

[0055] Accession number: KCCM11494P

[0056] Depositary date: Dec. 9, 2013