E. COLI VARIANT STRAIN OR CORYNEBACTERIUM GLUTAMICUM VARIANT STRAIN PRODUCING L-AMINO ACIDS, AND METHOD FOR PRODUCING L-AMINO ACIDS USING SAME

Abstract

The present disclosure relates to an L-amino acid-producing E. coli mutant strain or Corynebacterium glutamicum mutant strain having enhanced L-amino acid productivity, and a method of producing L-amino acid using the same. The L-amino acid-producing E. coli mutant strain and Corynebacterium glutamicum mutant strain according to the present disclosure exhibit an enhanced ability to produce L-amino acid, such as L-tryptophan, L-phenylalanine, L-tyrosine, L-glutamine, L-lysine, L-arginine, L-valine, L-leucine, L-isoleucine, L-threonine, L-histidine, L-serine, or L-citrulline, compared to parent strains thereof, and are capable of producing a high concentration of L-amino acid in high yield.

Claims

1. A mutant strain having enhanced L-amino acid productivity due to overexpression of at least one gene selected from the group consisting of yicL, ydiN, ydhK, aaeB, yeeA, rhtC and emrD, the mutant strain being Escherichia coli or Corynebacterium glutamicum.

2. The mutant strain of claim 1, wherein the yicL consists of the nucleotide sequence of SEQ ID NO: 1, the ydiN consists of the nucleotide sequence of SEQ ID NO: 2, the ydhK consists of the nucleotide sequence of SEQ ID NO: 3 or 37, the aaeB consists of the nucleotide sequence of SEQ ID NO: 4, the yeeA consists of the nucleotide sequence of SEQ ID NO: 5, the rhtC consists of the nucleotide sequence of SEQ ID NO: 6, and the emrD consists of the nucleotide sequence of SEQ ID NO: 7 or 39.

3. The mutant strain of claim 1, wherein the L-amino acid is selected from the group consisting of L-tryptophan, L-phenylalanine, L-tyrosine, L-glutamine, L-lysine, L-arginine, L-valine, L-leucine, L-isoleucine, L-threonine, L-histidine, L-serine and L-citrulline.

4. A method for producing L-amino acid, the method comprising steps of: (a) culturing the mutant strain of claim 1 in a medium; and (b) recovering L-amino acid from the mutant strain or the medium.

5. The method of claim 4, wherein the L-amino acid is selected from the group consisting of L-tryptophan, L-phenylalanine, L-tyrosine, L-glutamine, L-lysine, L-arginine, L-valine, L-leucine, L-isoleucine, L-threonine, L-histidine, L-serine and L-citrulline.

Description

EXPERIMENTAL EXAMPLE 1-1

Construction of yddG Gene-Deleted Mutant Strains

[0036] In order to examine the effect of the yddG gene on L-amino acid production, yddG gene-deleted mutant strains were constructed.

[0037] The yddG gene-deleted mutant strains were constructed using an L-tryptophan-producing E. coli W0G strain (accession number: KFCC11660P) and an L-phenylalanine-producing E. coli MWTR42 strain (accession number: KFCC10780) as parent strains with reference to the experimental method described in the literature (One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products, Datsenko K A, Wanner B L., Proc Natl Acad Sci USA, 2000 Jun. 6; 97(12):6640-5).

[0038] First, to construct DNA fragments for disruption of the yddG gene, polymerase chain reaction (PCR) was performed using the chromosome of each parent strain and a pKD13 plasmid as a template and the primers shown in Table 1 below. At this time, the PCR was performed for a total of 30 cycles, each consisting of (i) denaturation at 95° C. for 30 sec, (ii) annealing at 58° C. for 30 sec, and (iii) extension at 72° C. for 2 min. The PCR products were electrophoresed on 0.8% agarose gel, and bands of desired size were obtained. Using the fragments corresponding to the bands, overlap PCR was performed under the above-described PCR conditions, thereby constructing single DNA fragments for disruption of the yddG gene. The DNA fragments were transformed into the respective parent strains which were then streaked on kanamycin-containing solid media and cultured at 37° C. for 24 hours. The resultant colonies were subjected to PCR under the above-described PCR conditions.

TABLE-US-00001 TABLE 1 Nucleotide sequence Primer (5′ .fwdarw. 3′) yddG H1F CAATGCCGCTACTGTTGTTCCAGCC (SEQ ID  NO: 15) yddG H1R CAGTGGTGCGTTTTTCTACCGCTAT (SEQ ID  NO: 16) yddG H2F AATAACTGCCGGGTCTACGGCC (SEQ ID  NO: 17) yddG H2R AACGTATTTTCTAAACGAATTTTAAACGGCGTC (SEQ ID  NO: 18) yddG CF CGGAACAGTATGTGCAGGTGTTACGG (SEQ ID  NO: 19) yddG CR AACAAACCAGTTACAACCACCGCAAC (SEQ ID  NO: 20)

[0039] As a result, it was confirmed that the yddG gene was deleted in each colony.

[0040] In addition, the mutant strain obtained by deleting the yddG gene from the E. coli W0G strain was named W1G strain, and the mutant strain obtained by deleting the yddG gene from the E. coli MWTR42 strain was named MWTR5 strain.

EXPERIMENTAL EXAMPLE 1-2

Examination of L-Tryptophan and L-Phenylalanine Productivities of yddG Gene-Deleted Mutant Strains

[0041] The L-tryptophan productivities of the W0G and W1G strains and the L-phenylalanine productivities of the MWTR42 and MWTR5 strains were examined.

[0042] Each of the strains was 1% inoculated into a flask containing 10 ml of each medium having the composition shown in Table 2 below, and was cultured with shaking at 200 rpm at 37° C. for 70 hours. Each culture was measured for absorbance at OD.sub.610nm, and the amount of L-tryptophan or L-phenylalanine produced was compared between the strains. The results are shown in Table 3 below.

TABLE-US-00002 TABLE 2 Medium for tryptophan Medium for phenylalanine production production Component Content Component Content Glucose 80.0 g/L Glucose 80.0 g/L (NH.sub.4).sub.2SO.sub.4 20.0 g/L (NH.sub.4).sub.2SO.sub.4 20.0 g/L K.sub.2HPO.sub.4 0.8 g/L K.sub.2HPO.sub.4 1.0 g/L K.sub.2SO.sub.4 0.4 g/L KH.sub.2PO.sub.4 1.0 g/L MgCl.sub.2 0.8 g/L K.sub.2SO.sub.4 0.4 g/L Fumaric acid 1.0 g/L MgCl.sub.2 1.0 g/L Yeast extract 1.0 g/L Fumaric acid 0.5 g/L (NH.sub.4) .sub.6Mo.sub.7O.sub.24 0.12 ppm Yeast extract 1.0 g/L H.sub.3BO.sub.3 0.01 ppm Glutamic acid 0.5 g/L CuSO.sub.4 0.01 ppm CaCl.sub.2 5.00 ppm MnCl.sub.2 2.00 ppm MnCl.sub.2 2.00 ppm ZnSO.sub.4 0.01 ppm ZnSO.sub.4 1.00 ppm CoCl.sub.2 0.10 ppm CoCl.sub.2 0.10 ppm FeCl.sub.2 10.00 ppm FeCl.sub.2 10.00 ppm Thiamine_HCl 20.00 ppm Thiamine_HCl 20.00 ppm L-tyrosine 200.00 ppm L-tyrosine 200.00 ppm L-phenylalanine 300.00 ppm CaCO.sub.3 3% CaCO.sub.3 3% — —

TABLE-US-00003 TABLE 3 Strain L-tryptophan (g/L) Strain L-phenylalanine (g/L) W0G 4.21 ± 0.113 MWTR42 21.3 ± 0.115 W1G 2.53 ± 0.132 MWTR5  11.7 ± 0.141

[0043] As shown in Table 3 above, it was confirmed that the yddG-gene mutant strains (W1G strain and MWTR5 strain) still produced L-tryptophan and L-phenylalanine, respectively. That is, it could be seen that, even if yddG known as an aromatic amino acid exporter gene was deleted, the L-amino acid productivity of each of the mutant strains was retained, indicating that genes involved in amino acid export are present in addition to yddG.

EXPERIMENTAL EXAMPLE 2-1

Construction of Mutant Strains Containing the Amplified Genes yicL, ydiN, ydhK, aaeB, yeeA, rhtC and emrD, Respectively

[0044] E. coli mutant strains into which yicL, ydiN, ydhK, aaeB, yeeA, rhtC and emrD genes have been inserted, respectively, were constructed.

[0045] As a parent strain, an L-tryptophan-producing E. coli W0G strain (accession number: KFCC11660P) was used.

[0046] First, each of yicL, ydiN, ydhK, aaeB, yeeA, rhtC and emrD genes was amplified by PCR using the W0G strain as a template and the primers shown in Table 4 below, and then the PCR products were treated with the restriction enzymes SacI and XbaI, thereby preparing the genes.

[0047] As a result of sequencing, it was confirmed that the yicL gene has the nucleotide sequence of SEQ ID NO: 1, the ydiN gene has the nucleotide sequence of SEQ ID NO: 2, the ydhK gene has the nucleotide sequence of SEQ ID NO: 3 or 37, the aaeB gene has the nucleotide sequence of SEQ ID NO: 4, the yeeA gene has the nucleotide sequence of SEQ ID NO: 5, the rhtC gene has the nucleotide sequence of SEQ ID NO: 6, and the emrD gene has the nucleotide sequence of SEQ ID NO: 7 or 39.

[0048] The vector pTrc99A was digested by treatment with the restriction enzymes SacI and XbaI, and each of the prepared genes was inserted into the digested vector, thereby constructing the expression vectors pTrc99A::yicL, pTrc99A::ydiN, pTrc99A::ydhK, pTrc99A::aaeB, pTrc99A::yeeA, pTrc99A::rhtC, and pTrc99A::emrD. Each of the expression vectors was transformed into the W0G strain, thereby constructing the mutant strains W0G/pTrc99A::yicL, W0G/pTrc99A::ydiN, W0G/pTrc99A::ydhK, W0G/pTrc99A::aaeB, W0G/pTrc99A::yeeA, W0G/pTrc99A::rhtC, and W0G/pTrc99A::emrD, which contain each of the amplified genes.

TABLE-US-00004 TABLE 4 Nucleotide sequence Primer (5′ .fwdarw. 3′) yicL_F GCGAGCTCATGGGTTCCACCAGAAAGGG (SEQ ID NO: 21) yicL_R GCTCTAGATCACTTATGCCGCGCCGGA (SEQ ID NO: 22) ydiN_F GCGAGCTCATGTCTCAAAATAAGGCTTTCAGCA (SEQ ID NO: 23) ydiN_R GCTCTAGAGGCCATCAACCCAATCAATT (SEQ ID NO: 24) ydhK_F GCGAGCTCATGAACGCATCGTCATGGTCCTTGC (SEQ ID NO: 25) ydhK_R GCTCTAGATCACTTATGCCGCGCCGGA (SEQ ID NO: 26) aaeB_F GCGAGCTCATGGGTATTTTCTCCATTGCT (SEQ ID NO: 27) aaeB_R GCTCTAGATTTTGACTTAACTATCGGTca (SEQ ID NO: 28) yeeA_F GCGAGCTCGTGCGTGCCGATAAGTC (SEQ ID NO: 29) yeeA_R GCTCTAGATTATTTGCGCAAGGCCCG (SEQ ID NO: 30) rhtC_F GCGAGCTCATGTTGATGTTATTTCTCACCGT (SEQ ID NO: 31) rhtC_R gctctagaCTGGCATCACCGCGAAATAA (SEQ ID NO: 32) emrD_F GCGAGCTCATGAAAAGGCAAAGAAACG (SEQ ID NO: 33) emrD_R GCTCTAGACGGTGACGTGCGCTTAAAC (SEQ ID NO: 34)

EXPERIMENTAL EXAMPLE 2-2

Examination of L-Tryptophan Productivities of Mutant Strains Containing the Amplified Genes yicL, ydiN, ydhK, aaeB, yeeA, rhtC and emrD, Respectively

[0049] The L-tryptophan productivities of W0G/pTrc99A obtained by inserting only the vector pTrc99A into W0G, and the mutant strains W0G/pTrc99A::yicL, W0G/pTrc99A::ydiN, W0G/pTrc99A::ydhK, W0G/pTrc99A::aaeB, W0G/pTrc99A::yeeA, W0G/pTrc99A::rhtC, and W0G/pTrc99A::emrD, were examined.

[0050] Each of the strains was 1% inoculated into a flask containing 10 ml of each medium having the composition shown in Table 2 above, and was cultured with shaking at 200 rpm at 34° C. for 72 hours. Each culture was measured for absorbance at OD.sub.610nm, and the amount of L-tryptophan produced was compared between the strains. The results are shown in Table 5 below.

TABLE-US-00005 TABLE 5 Strain L-tryptophan (g/L) W0G/pTrc99A::(control) 4.21 ± 0.113 W0G/pTrc99A::yicL 4.83 ± 0.102 W0G/pTrc99A::ydiN 4.86 ± 0.169 W0G/pTrc99A::ydhK 4.88 ± 0.133 W0G/pTrc99A::aaeB 4.91 ± 0.101 W0G/pTrc99A::yeeA 4.95 ± 0.123 W0G/pTrc99A::rhtC 5.02 ± 0.131 W0G/pTrc99A::emrD 5.26 ± 0.156

[0051] As shown in Table 5 above, it was confirmed that the L-tryptophan productivities of the E. coli mutant strains containing the amplified genes yicL, ydiN, ydhK, aaeB, yeeA, rhtC and emrD, respectively, increased compared to that of the control.

EXPERIMENTAL EXAMPLE 3-1

Construction of Mutant Strains Containing Amplified Genes yicL, ydiN, ydhK, aaeB, yeeA, rhtC and emrD, Respectively

[0052] E. coli mutant strains into which yicL, ydiN, ydhK, aaeB, yeeA, rhtC and emrD genes were inserted, respectively, were constructed.

[0053] Expression vectors were constructed in the same manner as in Experimental Example 2-1 above, except that an L-phenylalanine-producing E. coli MWTR42 strain (accession number: KFCC10780) was used instead of the W0G strain. Each of the expression vectors was transformed into the MWTR42 strain, thereby constructing the mutant strains MWTR42/pTrc99A::yicL, MWTR42/pTrc99A::ydiN, MWTR42/pTrc99A::ydhK, MWTR42/pTrc99A::aaeB, MWTR42/pTrc99A::yeeA, MWTR42/pTrc99A::rhtC, and MWTR42/pTrc99A::emrD, which contain each of the amplified genes.

EXPERIMENTAL EXAMPLE 3-2

Examination of L-Phenylalanine Productivities of Mutant Strains Containing the Amplified Genes yicL, ydiN, ydhK, aaeB, yeeA, rhtC and emrD, Respectively

[0054] The L-phenylalanine productivities of MWTR42/pTrc99A obtained by inserting only the vector pTrc99A into MWTR42, and the mutant strains MWTR42/pTrc99A::yicL, MWTR42/pTrc99A::ydiN, MWTR42/pTrc99A::ydhK, MWTR42/pTrc99A::aaeB, MWTR42/pTrc99A::yeeA, MWTR42/pTrc99A::rhtC, and MWTR42/pTrc99A::emrD, were examined.

[0055] The amount of L-phenylalanine produced was compared between the strains in the same manner as in Experimental Example 2-2, and the results are shown in Table 6 below.

TABLE-US-00006 TABLE 6 Strain L-phenylalanine (g/L) MWTR42/pTrc99A (control) 21.3 ± 0.115 MWTR42/pTrc99A::yicL 27.1 ± 0.081 MWTR42/pTrc99A::ydiN 27.2 ± 0.165 MWTR42/pTrc99A::ydhK 27.3 ± 0.105 MWTR42/pTrc99A::aaeB 27.0 ± 0.111 MWTR42/pTrc99A::yeeA 27.3 ± 0.103 MWTR42/pTrc99A::rhtC 26.7 ± 0.122 MWTR42/pTrc99A::emrD 27.1 ± 0.085

[0056] As shown in Table 6 below, it was confirmed that the L-phenylalanine productivities of the E. coli mutant strains containing the amplified genes yicL, ydiN, ydhK, aaeB, yeeA, rhtC and emrD, respectively, increased compared to that of the control.

EXPERIMENTAL EXAMPLE 4-1

Construction of Mutant Strains Containing the Amplified Genes yicL, ydiN, ydhK, aaeB, yeeA, rhtC and emrD, Respectively

[0057] Corynebacterium glutamicum mutant strains into which yicL, ydiN, ydhK, aaeB, yeeA, rhtC and emrD genes have been inserted, respectively, were constructed.

[0058] As a parent strain, L-tryptophan-producing Corynebacterium glutamicum DW28G (accession number: KTCT13769BP) was used.

[0059] First, each of yicL, ydiN, ydhK, aaeB, yeeA, rhtC and emrD genes was amplified by PCR using each of the expression vectors (pTrc99A::yicL, pTrc99A::ydiN, pTrc99A::ydhK, pTrc99A::aaeB, pTrc99A::yeeA, pTrc99A::rhtC, and pTrc99A::emrD) constructed in Experimental Examples 2-1 as a template and the primers shown in Table 7 below, and then the PCR products were 5′-phosphorylated, thereby preparing the genes. The vector pEKO was digested by treatment with the restriction enzyme Eco53KI, and each of the prepared genes was inserted into the digested vector, thereby constructing the expression vectors pEKO::yicL, pEKO::ydiN, pEKO::ydhK, pEKO::aaeB, pEKO::yeeA, pEKO::rhtC, and pEKO::emrD. Each of the expression vectors was transformed into the DW28G strain, thereby constructing the mutant strains DW28G/pEKO::yicL, DW28G/pEKO::ydiN, DW28G/pEKO::ydhK, DW28G/pEKO::aaeB, DW28G/pEKO::yeeA, DW28G/pEKO::rhtC, and DW28G/pEKO::emrD, which contain each of the amplified genes.

TABLE-US-00007 TABLE 7 Nucleotide sequence Primer (5′ .fwdarw. 3′) Primer_F gcgccgacatcataacggttctgg (SEQ ID NO: 35) Primer_R cgcaacgttcaaatccgctcccg (SEQ ID NO: 36)

EXPERIMENTAL EXAMPLE 4-2

Examination of L-Tryptophan Productivities of Mutant Strains Containing the Amplified Genes yicL, ydiN, ydhK, aaeB, yeeA, rhtC and emrD, Respectively

[0060] The L-tryptophan productivities of DW28G/pEKO obtained by inserting only the vector pEKO into DW28G, and the mutant strains DW28G/pEKO::yicL, DW28G/pEKO::ydiN, DW28G/pEKO::ydhK, DW28G/pEKO::aaeB, DW28G/pEKO::yeeA, DW28G/pEKO::rhtC, and DW28G/pEKO::emrD, were examined.

[0061] The amount of L-tryptophan produced was compared between the strains in the same manner as in Experimental Example 2-2, except that the composition shown in Table 8 was used as a medium. The results are shown in Table 9 below.

TABLE-US-00008 TABLE 8 Component Content Cane molasses (glucose) 100.00 g/L KH.sub.2PO.sub.4 5.00 g/L K.sub.2HPO.sub.4 5.00 g/L K.sub.2SO.sub.4 0.40 g/L MgSO.sub.4 0.25 g/L (NH.sub.4) .sub.2SO.sub.4 20 g/L Corn steep liquor 10 g/L CaCO.sub.3 3%

TABLE-US-00009 TABLE 9 Strain L-tryptophan (g/L) DW28G/pEKO (control) 2.64 ± 0.107 DW28G/pEKO::yicL 3.57 ± 0.115 DW28G/pEKO::ydiN 3.05 ± 0.089 DW28G/pEKO::ydhK 3.11 ± 0.095 DW28G/pEKO::aaeB 2.95 ± 0.102 DW28G/pEKO::yeeA 3.21 ± 0.113 DW28G/pEKO::rhtC 3.31 ± 0.091 DW28G/pEKO::emrD 3.45 ± 0.115

[0062] As shown in Table 9 above, it was confirmed that the L-tryptophan productivities of the Corynebacterium glutamicum mutant strains containing the amplified genes yicL, ydiN, ydhK, aaeB, yeeA, rhtC and emrD, respectively, increased compared to that of the control.

[0063] In conclusion, in the present disclosure, the E. coli mutant strain or Corynebacterium glutamicum mutant strain having enhanced L-tryptophan or L-phenylalanine productivity compared to the parent strain was obtained by inducing overexpression of the amino acid exporter gene yicL, ydhK, aaeB, yeeA, rhtC or emrD, and it was confirmed that a high concentration of L-tryptophan or L-phenylalanine could be produced in high yield by using this mutant strain.

[0064] So far, the present disclosure has been described with reference to the embodiments thereof. Those of ordinary skill in the art to which the present disclosure pertains will appreciate that the present disclosure may be embodied in modified forms without departing from the essential characteristics of the present disclosure. Therefore, the disclosed embodiments should be considered from an illustrative point of view, not from a restrictive point of view. The scope of the present disclosure is defined by the claims rather than the foregoing description, and all differences within the scope equivalent thereto should be construed as being included in the present disclosure.