Microorganisms producing L-amino acids and process for producing L-amino acids using the same

Abstract

Disclosed are a recombinant microorganism having enhanced L-amino acid producibility, wherein the recombinant microorganism is transformed to have an inactivated phage receptor thereof, 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 of the genus Escherichia producing L-amino acid in which at least one of NfrA and NfrB is inactivated and tsx is inactivated, wherein the recombinant microorganism has a producibility of the L-amino acid.

2. The recombinant microorganism of claim 1, wherein the NfrA comprises the amino acid sequence of SEQ ID NO: 40, the NfrB comprises the amino acid sequence of SEQ ID NO: 42, and the Tsx comprises the amino acid sequence of SEQ ID NO: 45.

3. The recombinant microorganism of claim 1, wherein FhuA is further inactivated.

4. The recombinant microorganism of claim 3, the FhuA comprises the amino acid sequence of SEQ ID NO: 47.

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

6. The recombinant microorganism of claim 1, wherein the recombinant microorganism is Escherichia coli.

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

8. The method of claim 7, wherein the L-amino acid is L-threonine or L-tryptophan.

9. The method of claim 7, wherein the NfrA comprises an amino acid sequence of SEQ ID NO: 40, the NfrB comprises the amino acid sequence of SEQ ID NO: 42, and the Tsx comprises the amino acid sequence of SEQ ID NO: 45.

10. The method of claim 7, wherein FhuA is further inactivated.

11. The method of claim 10, wherein the FhuA comprises the amino acid sequence of SEQ ID NO: 47.

12. The method of claim 7, wherein the recombinant microorganism is Escherichia coli.

13. The recombinant microorganism of claim 1, wherein the recombinant microorganism has an increased sugar consumption rate compared to a microorganism of the genus Escherichia producing L-amino acid in which NfrA and NfrB are not inactivated.

Description

MODE FOR THE INVENTION

(1) 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 Threonine-Producing Strain Having Inactivated Phage Receptor by Using KCCM10910P

(2) In order to prepare a threonine-producing strain having an inactivated phage receptor, a KCCM10910P strain (Korean Patent No: 10-0966324) was used as a mother strain. Then, a cassette for inactivating a gene for each phage receptor was prepared, and then, was used to allow genetic transformation.

(3) 1-1. Preparation of Threonine-Producing Strain Having Inactivated nfrA Gene

(4) In order to prepare a threonine-producing strain having an inactivated nfrA gene, a cassette for inactivating an nfrA gene was prepared. The cassette used a method of 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). To confirm the insertion of the cassette into the gene, a chloramphenicol-resistant gene of pUCprmfmloxC was used as a marker (Korean Patent LaidOpen Publication NO: 2009-007554).

(5) 1.1 kb DNA fragment including a part of a sequence of the nfrA gene (SEQ ID NO: 39) and a part of a base sequence of the chloramphenicol-resistant gene of a pUCprmfmloxC was obtained by using a primer set of SEQ ID NOS: 2 and 3. Here, a polymerase chain reaction (hereinafter, referred to as PCR) was performed by using a PCR premix kit (i.e., a product of BIONEER company, hereinafter, the same product was used) under the following conditions: 27 cycles of denaturation at 95 C. for 30 seconds, annealing at 56 C. for 30 seconds, and elongation at 72 C. for 1 minute. The PCR product was electrophoresed on a 0.8% agarose gel, and then, eluted. Afterwards, PCR was performed again by using the eluted product as a template and a primer set of SEQ ID NOS: 1 and 4 under the same conditions described above, resulting in a DNA fragment of about 1.2 kb. The DNA fragment was electrophoresed on a 0.8% agarose gel, eluted, and then, was finally used to prepare the cassette for inactivating the nrfA gene.

(6) In order to prepare a threonine-producing strain having the inactivated nfrA gene, a threonin-producing strain (KCCM10910P), 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. Then, DNA of the cassette prepared for inactivating the nfrA gene was introduced to the strain to allow transformation.

(7) The obtained strain was selected on a LB plate having chloramphenicol resistance. That is, a primer set of SEQ ID NOS: 5 and 6, which has a DNA sequence lying outside of two ends of an nfrA homologous sequence of the cassette for genomic inactivation, was used to thereby select colonies where the size of the resultant PCR product was reduced from 2.8 kb to 1.5 kb.

(8) The primary recombinant strain having chloramphenicol resistance was removed from the pKD46 plasmid, and then, introduced with a pJW168 plasmid to remove the chloramphenicol marker gene from the microbial cells (Gene, (2000) 247, 255-264). Then, PCR using a primer set of SEQ ID NOS: 5 and 6 was performed to obtain 0.4 kb DNA product, indicating that the strain finally obtained had a reduced DNA size. Accordingly, the L-threonine-producing strain having the inactivated nfrA gene (KCCM10910PnfrA) was prepared.

(9) 1-2. Preparation of Threonine-Producing Strain Having Inactivated nfrB Gene

(10) In order to prepare a threonine-producing strain having an inactivated nfrB gene (SEQ ID NO: 41), a cassette for inactivating an nfrB gene was prepared in the same manner as in the preparation of the cassette for inactivating the nfrA gene of Example 1-1. 1.1 kb DNA fragment was obtained by using a primer set of SEQ ID NOS: 8 and 9, and then, 1.2 kb DNA fragment was prepared by using a primer set of SEQ ID NOS: 7 and 10.

(11) A method of preparing a threonin-producing strain having the inactivated nfrB gene was carried out by the same method described in Example 1-1, wherein a primer set of SEQ ID NOS: 11 and 12 was used to confirm the size of the resultant PCR product. Accordingly, the L-threonine-producing strain having the inactivated nfrB gene (KCCM10910PnfrB) was finally prepared.

(12) 1-3. Preparation of Threonine-Producing Strain Having Inactivated nfrAB Gene

(13) In order to prepare a threonine-producing strain having an inactivated nfrAB gene (SEQ ID NO: 43), a cassette for inactivating an nfrAB gene was prepared in the same manner as in the preparation of the cassette for inactivating the nfrA gene of Example 1-1. 1.1 kb DNA fragment was obtained by using a primer set of SEQ ID NOS: 2 and 9, and then, 1.2 kb DNA fragment was prepared by using a primer set of SEQ ID NOS: SEQ ID NO: 1 and 10.

(14) A method of preparing a threonin-producing strain having the inactivated nfrAB gene was carried out by the same method described in Example 1-1, wherein a primer set of SEQ ID NOS: 5 and 12 was used to confirm the size of the resultant PCR product. Accordingly, the L-threonine-producing strain having the inactivated nfrAB gene (KCCM10910PnfrAB) was finally prepared.

(15) 1-4. Preparation of Threonine-Producing Strain Having Inactivated Tsx Gene

(16) In order to prepare a threonin-producing strain having an inactivated tsx gene (SEQ ID NO: 44), a cassette for inactivating a tsx gene was prepared in the same manner as in the preparation of the cassette for inactivating the nfrA gene of Example 1-1. 1.1 kb DNA fragment was obtained by using a primer set of SEQ ID NOS: 13 and 14, and then, 1.2 kb DNA fragment was prepared by using a primer set of SEQ ID NOS: 15 and 16.

(17) A method of preparing the threonine-producing strain having the inactivated tsx gene was carried out by the same method described in Example 1-1, wherein a primer set of SEQ ID NOS: 17 and 18 was used to confirm the size of the resultant PCR product. Accordingly, the L-threonine-producing strain having inactivated tsx gene (KCCM10910Ptsx) was finally prepared.

(18) 1-5. Preparation of Threonine-Producing Strain Having Inactivated fhuA Gene

(19) In order to prepare a threonine-producing strain having an inactivated fhuA gene (SEQ ID NO: 46), a cassette for inactivating an fhuA gene was prepared according to the method of one-step inactivation described above. In order to obtain a DNA fragment with a base sequence having homology with a sequence of the fhuA gene, a primer set of SEQ ID NOS: 19 and 20 and a primer set of SEQ ID NOS: 21 and 22 were used, resulting in producing PCR products. In addition, in order to obtain a DNA fragment with a base sequence having chloramphenicol resistance, a primer set of SEQ ID NOS: 23 and 24 was used, resulting in producing a PCR product. Accordingly, these three resultant PCR products were electrophoresed on a 0.8% agarose gel, and then, eluted. PCR was performed by using these three eluted PCR products as templates and a primer set of SEQ ID NOS: 19 and 22 to prepare a cassette for inactivating the fhuA gene.

(20) In order to prepare a threonine-producing strain having the inactivated fhuA gene, the cassette for inactivating the fhuA gene was prepared by the same method described in Example 1-1, wherein a primer set of SEQ ID NOS: 25 and 26 was used to confirm the size of the resultant PCR products. Accordingly, the L-threonine-producing strain having the inactivated fhuA gene (KCCM10910PfhuA) was finally prepared.

(21) 1-6. Preparation of Threonine-Producing Strain Having Inactivated lamB Gene

(22) In order to prepare a threonine-producing strain having an inactivated lamB gene (SEQ ID NO: 48), a cassette for inactivating a lamB gene was prepared by the same method described in Example 1-1. 1.1 kb DNA fragment was obtained by using a primer set of SEQ ID NOS: 27 and 28, and then, 1.2 kb DNA fragment was prepared by using a primer set of SEQ ID NOS: 29 and 30.

(23) A method of preparing the threonine-producing strain having the inactivated lamB gene was carried out by the same method described in Example 1-1, wherein a primer set of SEQ ID NOS: 31 and 32 was used to confirm the size of the resultant PCR product. Accordingly, the L-threonine-producing strain having inactivated lamB gene (KCCM10910PlamB) was finally prepared.

(24) 1-7. Preparation of Threonine-Producing Strain Having Inactivated btuB Gene

(25) In order to prepare a threonine-producing strain having an inactivated btuB gene (SEQ ID NO: 50), a cassette for inactivating a btuB gene was prepared by the same method described in Example 1-1. 1.1 kb DNA fragment was obtained by using a primer set of SEQ ID NOS: 33 and 34, and then, 1.2 kb DNA fragment was prepared by using a primer set of SEQ ID NOS: 35 and 36.

(26) A method of preparing the threonine-producing strain having the inactivated btuB gene was carried out by the same method described in Example 1-1, wherein a primer set of SEQ ID NOS: 37 and 38 was used to confirm the size of the resultant PCR product. Accordingly, the L-threonine-producing strain having the inactivated btuB gene (KCCM10910PbtuB) was finally prepared.

Example 2. Comparison in L-Threonine Productivity Among Recombinant Microorganisms

(27) The recombinant microorganisms prepared according to Example 1 were cultured in a threonine titer medium containing compositions shown in Table 1 below, in an Erlenmeyer flask. Then, it was confirmed whether the recombinant microorganisms had producibility of L-threonine.

(28) TABLE-US-00001 TABLE 1 Concentration Composition (per liter) Glucose 70 g KH.sub.2PO.sub.4 2 g (NH.sub.4).sub.2SO.sub.4 27.5 g MgSO.sub.4H.sub.2O 1 g FeSO.sub.4H.sub.2O 5 mg MnSO.sub.4H.sub.2O 5 mg DL-methionine 0.15 g Yeast extract 2 g Calcium carbonate 30 g pH 6.8

(29) 1 platinum loop of each of the 7 types of the E. coli strains of Example 1 and the KCCM10910P strain that were cultured overnight in the LB solid medium in an incubator at 33 C. was inoculated in 25 ml of a titer medium containing compositions shown in Table 1 above, and then, was cultured in an incubator at 33 C. and at 200 rpm for 48 hours.

(30) TABLE-US-00002 TABLE 2 Sugar consumption (g/L) L-threonine (g/L) Strain 30 hr 48 hr KCCM 10910P (mother strain) 22 34.5 KCCM 10910PnfrA 26 34.5 KCCM 10910PnfrB 26 34.4 KCCM 10910PnfrAB 26 34.4 KCCM 10910Ptsx 25 34.4 KCCM 10910PfhuA 24 34.5 KCCM 10910PlamB 20 34.5 KCCM 10910PbtuB 21 34.5

(31) As shown in Table 2 above, it was confirmed that the sugar consumption rates of the strains each having the inactivated nfrA, nfrB, nfrAB, tsx, and fhuA genes were higher than the sugar consumption rate of the mother strain (KCCM10910P). It was also confirmed that the production rate of the strains was not reduced during a 48 hour period. Meanwhile, it was confirmed that the sugar consumption rates of the strains each having the inactivated lamB and btuB genes were similar to the sugar consumption rate of the mother strain, or slightly slower than the sugar consumption rate of the mother strain. It was also confirmed that the concentrations of L-threonine shown in the strains of the culture after 48 hours were all similar. The strains each having the inactivated nfrA, nfrB, and nfrAB genes resulted in the same culturing results. That is, the case where one of the two genes was deleted and the case where both genes were deleted generated the same results.

Example 3. Preparation of Strains with Effective Mutation Combination and Comparison in L-Threonine Producibility Thereof

(32) 3-1. Preparation of Strains Having Simultaneously Inactivated nfrAB and fhuA Genes, Simultaneously Inactivated nfrAB and Tsx Genes, and Simultaneously Inactivated nfrAB, Tsx, and fhuA Genes

(33) In order to confirm whether the case where the combined inactivation of the nfrAB, fhuA, and tsx genes having increased sugar consumption capacity has further sugar consumption capacity in the L-threonine-producing strains, a KCCM10910PnfrAB fhuA strain, a KCCM10910PnfrABtsx strain, and a KCCM10910PnfrABtsx fhuA strain were prepared. In order to prepare these strains, strains each having the inactivated fhuA and tsx genes were prepared in accordance with the KCCM10910P nfrAB strain of Example 1-3 in the same manner as described in Example 1 (resulting in KCCM10910PnfrABfhuA and KCCM10910PnfrABtsx strains). In addition, a strain having the inactivated fhuA gene was prepared in accordance with the KCCM10910PnfrABtsx strain, thereby finally preparing a KCCM10910PnfrAB tsxfhuA strain.

(34) As shown in Table 2, the strains having the inactivated nfrA, nfrB, and nfrAB genes were determined to have the same effects as one another. In this regard, in the preparation of strains with effective mutation combinations, the strains having the inactivated tsx and fhuA genes were prepared by using the strain having the inactivated nfrAB gene. However, the effects of the strains having the inactivated tsx and fhuA genes were determined to be the same as the effects of the strain having the inactivated nfrA gene only, the inactivated nfrB gene only, or the simultaneously inactivated nfrA and nfrB genes.

(35) 3-2. Comparison in L-Threonine Producibility of Strains with Effective Mutation Combinations

(36) In order to compare the L-threonine producibility of the strains with effective mutation combinations prepared above, a medium containing compositions shown in Table 1 above was used to culture strains in the same manner as described above. The results are shown in Table 3 below.

(37) TABLE-US-00003 TABLE 3 Sugar L-threonine consumption (g/L) (g/L) Strain 30 hr 48 hr KCCM10910P (mother strain) 22 34.5 KCCM10910PnfrAB 26 34.4 KCCM10910PnfrABfhuA 28 34.5 KCCM10910PnfrABtsx 28 34.4 KCCM10910PnfrABtsxfhuA 29 34.5

(38) As a result of a potency test on the KCCM10910PnfrABfhuA strain, the KCCM10910PnfrABtsx strain, and the KCCM10910PnfrABtsxfhuA strain, each prepared in accordance with the combined inactivation of the nfrAB, fhuA, and tsx genes having increased sugar consumption capacity, it was confirmed that the strain in which the fhuA gene or the tsx gene was further inactivated in addition to the mutation by the nfrAB gene only increased the sugar consumption capacity. Accordingly, the transformed KCCM10910PnfrAB strain showing increased sugar consumption capacity was named E. coli CA03-8253P, and then, was deposited at the Korean Culture Center of Microorganisms (KCCM) on Dec. 13, 2013 (Accession No: KCCM11501P).

Example 4. Preparation of Strain Having Inactivated Phage Receptor by Using KCCM-10132 and Comparison in Threonine Producibility Thereof

(39) 4-1. Preparation of Strain Having Inactivated Phage Receptor by Using KCCM10132

(40) The 10 types of strains each having an inactivated phage receptor were prepared by using a KCCM-10132 strain (see Table 4 below) in the same manner as described in Examples 1 and 3, in accordance with the 7 types of the inactivation cassettes of Example 1. The KCCM-10132 strain was disclosed in Korean Patent No: 10-0270510 as a strain having threonine producibility derived from E. coli.

(41) 4-2. Preparation of Strain Having Inactivated Phage Receptor by Using KCCM10132 and Comparison in Threonine Producibility Thereof

(42) The 10 types of the strains each having the inactivated phage receptor that were prepared by using the KCCM-10132 strain of Example 4-1 and the mother strain (KCCM-10132) were cultured in a medium containing the compositions shown in Table 1 by the same method as described in Example 2. Then, the cultured strains were evaluated by comparing the producibility of threonine thereof.

(43) TABLE-US-00004 TABLE 4 Sugar consumption (g/L) L-threonine (g/L) Strain 30 hr 48 hr KCCM-10132 (mother strain) 32 20.2 KCCM-10132nfrA 35 20.2 KCCM-10132nfrB 35 20.1 KCCM-10132nfrAB 36 20.2 KCCM-10132tsx 35 20.2 KCCM-10132fhuA 36 20.1 KCCM-10132lamB 31 20.2 KCCM-10132btuB 30 20.1 KCCM-10132nfrABfhuA 38 20.2 KCCM-10132nfrABtsx 38 20.1 KCCM-10132nfrABtsxfhuA 39 20.2

(44) As shown in Table 4 above, it was confirmed that the sugar consumption rates of the strains each having the inactivated nfrA, nfrB, nfrAB, tsx, and fhuA genes were higher than the sugar consumption rate of the mother strain (KCCM-10132). It was also confirmed that the production rate of the strains was not reduced in a 48 hour period. Meanwhile, it was confirmed that the sugar consumption rates of the strains each having the inactivated lamB and the btuB genes were similar to the sugar consumption rate of the mother strain, or slightly slower than the sugar consumption rate of the mother strain. It was also confirmed that the concentrations of L-threonine shown in the strains of the culture after 48 hours were all similar. It was also confirmed that the strains each having the simultaneously inactivated nfrAB, fhuA, nfrAB and tsx genes and the simultaneously inactivated nfrAB, tsx, and fhuA genes had improved sugar consumption rates in comparison to the sugar consumption rate of the strain having the inactivated nfrAB gene only.

Example 5. Preparation of Strain Having Inactivated Phage Receptor by Using KCCM11166P and Comparison in Threonine Producibility Thereof

(45) 5-1. Preparation of Strain Having Inactivated Phage Receptor by Using KCCM11166P

(46) 7 types of tryptophan-producing strains each having an inactivated phage receptor were prepared by using a KCCM11166P (Korean Patent NO: 10-1261147) in the same manner as described in Example 1, in accordance with the 7 types of the inactivation cassettes of Example 1.

(47) 5-2. Preparation of Strain Having Inactivated Phage Receptor by Using KCCM11166P and Comparison in Threonine Producibility Thereof

(48) In order to evaluate the producibility of the 7 types of the tryptophan-producing strains each having the inactivated phage receptor prepared by using the KCCM11166P strain of Example 5-1, a medium containing compositions shown in Table 5 below was used. That is, the microbial cells were inoculated by a platinum loop, and then, were cultured overnight in the LB solid medium. Afterwards, 1 platinum loop of each of the microbial cells was inoculated in 25 ml of titer medium containing the compositions shown in Table 5 below, and then, was cultured in an incubator at 37 C. and at 200 rpm for 48 hours. The results obtained therefrom are shown in Table 6 below.

(49) TABLE-US-00005 TABLE 5 Composition Concentration (per liter) Glucose 60 g K.sub.2HPO.sub.4 1 g (NH.sub.4).sub.2SO.sub.4 10 g MgSO.sub.4H.sub.2O 1 g NaCl 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

(50) TABLE-US-00006 TABLE 6 Sugar consumption (g/L) OD L-tryptophan (g/L) Strain 33 hr 48 hr KCCM11166P 56.8 14.0 7.2 KCCM11166PnfrA 59.5 13.5 7.2 KCCM11166PnfrB 59.5 13.5 7.2 KCCM11166PnfrAB 59.5 13.5 7.2 KCCM11166Ptsx 60.2 14.3 7.1 KCCM11166PfhuA 59.5 13.7 7.1 KCCM11166PlamB 57.0 14.0 7.2 KCCM11166PbtuB 56.2 13.0 7.1

(51) As shown in Table 6 above, in the case of the deletion of each of the nfrA, nfrB, nfrAB, tsx, and fhuA genes, it was confirmed that the amounts of tryptophan produced in the strains each having the inactivated nfrA, nfrB, nfrAB, tsx, and fhuA genes were similar while the sugar consumptions rate of the strains each having the inactivated nfrA, nfrB, nfrAB, tsx, and fhuA genes were slightly higher than others. Meanwhile, in the case of the deletion of each of the lamB and btuB genes, it was confirmed that the amounts of tryptophan produced by the strains each having the inactivated lamB and btuB genes or the sugar consumption rates of the strains each having the inactivation of lamB and btuB genes were not changed.

Example 6. Preparation of Strains with Effective Mutation Combination and Comparison in L-Tryptophan Producibility Thereof

(52) 6-1. Preparation of L-Tryptophan-Producing Strains Having Simultaneously Inactivated nfrAB and fhuA Genes, Simultaneously Inactivated nfrAB and Tsx Genes, and Simultaneously Inactivated nfrAB, tsx, and fhuA Genes

(53) In order to confirm whether the case where the combined inactivation of the nfrAB, fhuA, and tsx genes having increased sugar consumption capacity has further sugar consumption capacity in the tryptophan-producing strains, a KCCM11166PnfrAB fhuA strain, a KCCM11166PnfrABtsx strain, and a KCCM11166PnfrABtsx fhuA strain were prepared.

(54) 6-2. Comparison in L-Tryptophan Producibility of Strains with Effective Mutation Combination

(55) In order to compare the L-tryptophan producibility of the three types of the strains prepared according to Example 6-1, a medium containing compositions shown in Table 5 above was used to culture the strains in the same manner as described in Example 5. The results are shown in Table 7 below.

(56) TABLE-US-00007 TABLE 7 Sugar consumption L-tryptophan (g/L) OD (g/L) Strain 33 hr 48 hr KCCM11166P 56.8 14.0 7.2 KCCM11166PnfrAB 59.5 13.5 7.2 KCCM11166PnfrABtsx 61.0 14.0 7.2 KCCM11166PnfrABfhuA 60.5 13.8 7.1 KCCM11166PnfrABtsxfhuA 62.0 14.0 7.2

(57) As a result of a potency test on the tryptophan-producing strains with effective mutation combinations, it was confirmed that the strains in which the fhuA gene or/and the tsx gene was further inactivated in addition to the mutation by the nfrAB gene only increased the sugar consumption capacity.

(58) 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.

(59) [Accession Number]

(60) Depositary institution: Korean Culture Center of Microorganisms (international)

(61) Accession number: KCCM11501P

(62) Depositary date: Dec. 13, 2013