NOVEL VARIANT OF STRESS PROTEIN AND METHOD FOR PREPARING L-AROMATIC AMINO ACID USING THE SAME

Abstract

The present invention relates to a novel stress protein variant and a method of producing an L-aromatic amino acid using the same. The stress protein variant is obtained by substituting one or more amino acids in the amino acid sequence constituting glutathione reductase or stress protein B to change the activity of the protein, and a recombinant microorganism comprising the variant is capable of efficiently producing an L-aromatic amino acid.

Claims

1. A variant selected from among: a glutathione reductase variant consisting of the amino acid sequence of SEQ ID NO: 1 in which threonine at position 74 in the amino acid sequence of SEQ ID NO: 3 is substituted with isoleucine; and a stress protein B variant consisting of the amino acid sequence of SEQ ID NO: 5 in which glutamine at position 42 in the amino acid sequence of SEQ ID NO: 7 is substituted with a stop codon.

2. A polynucleotide encoding the variant of claim 1.

3. A transformant comprising the variant of claim 1.

4. The transformant of claim 3, which is an Escherichia sp. strain.

5. The transformant of claim 3, which has ability to produce an L-aromatic amino acid.

6. A method for producing an L-aromatic amino acid, comprising steps of: culturing the transformant of claim 3 in a medium; and recovering an L-aromatic amino acid from the transformant or the medium in which the transformant has been cultured.

7. The method of claim 6, wherein the L-aromatic amino acid is at least one selected from the group consisting of L-tryptophan, L-phenylalanine, and L-tyrosine.

8. A transformant comprising the polynucleotide of claim 2.

9. The transformant of claim 8, which is an Escherichia sp. strain.

10. The transformant of claim 8, which has ability to produce an L-aromatic amino acid.

11. A method for producing an L-aromatic amino acid, comprising steps of: culturing the transformant of claim 8 in a medium; and recovering an L-aromatic amino acid from the transformant or the medium in which the transformant has been cultured.

12. The method of claim 11, wherein the L-aromatic amino acid is at least one selected from the group consisting of L-tryptophan, L-phenylalanine, and L-tyrosine.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0056] FIG. 1 shows the structure of plasmid pDSG according to one embodiment of the present invention.

[0057] FIG. 2 shows the structure of plasmid pDS9 according to one embodiment of the present invention.

MODE FOR INVENTION

[0058] Hereinafter, the present invention will be described in more detail. However, this description is merely presented by way of example to facilitate the understanding of the present invention, and the scope of the present invention is not limited by this exemplary description.

Example 1. Construction of Strains Expressing

Glutathione Reductase Variant

[0059] In order to evaluate the effect of a variant resulting from substitution of isoleucine for threonine at position 74 of the amino acid sequence of glutathione reductase (SEQ ID NO: 3) on the production of L-aromatic amino acids, a vector expressing the glutathione reductase variant and strains into which the vector has been introduced were constructed. For insertion of the gene encoding the glutathione reductase variant into the strains, plasmids pDSG and pDS9 were used, and the strains were constructed as follows.

[0060] Here, the plasmid pDSG has an origin of replication that works only in Escherichia coli, contains an ampicillin resistance gene, and has a mechanism for expressing guide RNA (gRNA). The plasmid pDS9 has an origin of replication that works only in Escherichia coli, contains a kanamycin resistance gene and A red genes (exo, bet and gam), and has a mechanism for expressing CAS9 derived from Streptococcus pyogenes.

1-1. Construction of Vector pDSG-gor (Thr74Ile) for Transformation

[0061] Using Escherichia coli MG1655 (KCTC14419BP) gDNA as a template, a fragment upstream of the amino acid mutation at position 74 of the E. coli gor gene encoding glutathione reductase was obtained by PCR using a primer pair of primer 7 and primer 9 and a primer pair of primer 8 and primer 10, and a fragment downstream of the amino acid mutation at position 74 of the E. coli gor gene was obtained by PCR using a primer pair of primer 11 and primer 13 and a primer pair of primer 12 and primer 14. At this time, each of the upstream and downstream fragments contained a sequence that modifies threonine (Thr) at amino acid residue position 74 of the gor gene into isoleucine (Ile). In the PCR, Takara PrimeSTAR Max DNA polymerase was used as polymerase, and PCR amplification was performed for 30 cycles under the following conditions: denaturation at 95 C. for 10 sec, annealing at 57 C. for 15 sec, and polymerization at 72 C. for 10 sec.

[0062] Using the plasmid pDSG as a template, four pDSG gene fragments were obtained by PCR using a primer pair of primer 3 and primer 5, a primer pair of primer 4 and primer 6, a primer pair of primer 15 and primer 1, and a primer pair of primer 16 and primer 2, respectively. At this time, each of the gene fragments contained a gRNA sequence targeting Thr at position 74 of the gor gene. The gRNA was selected as a 20 mer upstream of NGG of the sequence to be mutated. In the PCR, Takara PrimeSTAR Max DNA polymerase was used as polymerase, and PCR amplification was performed for 30 cycles under the following conditions: denaturation at 95 C. for 10 sec, annealing at 57 C. for 15 sec, and polymerization at 72 C. for 15 sec.

[0063] The obtained fragments upstream and downstream of the amino acid at position 74 of the gor gene, and the obtained four pDSG gene fragments were cloned using a self-assembly cloning method (BioTechniques 51:55-56 (July 2011)), thereby obtaining a recombinant plasmid which was named pDSG-gor (Thr74Ile).

1-2. Construction of L-Tryptophan- or L-Phenylalanine-Producing Strain into Which Glutathione Reductase Variant gor (Thr74Ile) Has Been Introduced

[0064] To construct an L-tryptophan-producing strain and an L-phenylalanine-producing strain, Escherichia coli KCCM13013P and KCCM10016 were used as parent strains.

[0065] The KCCM13013P strain or the KCCM10016 strain was primarily transformed with the plasmid pDS9 and cultured in LB-Km (containing 25 g/L of LB liquid medium and 50 mg/L of kanamycin) solid medium, and then kanamycin-resistant colonies were selected. The selected colonies were secondarily transformed with the pDSG-gor (Thr74Ile) plasmid and cultured in LB-Amp & Km (containing 25 g/L of LB liquid medium, 100 mg/L of ampicillin and 50 mg/L of kanamycin) solid medium, and then ampicillin- and kanamycin-resistant colonies were selected. Next, a gene fragment was obtained by PCR using a primer pair of primer 17 and primer 18. Here, Takara PrimeSTAR Max DNA polymerase was used as polymerase, and PCR amplification was performed for 30 cycles under the following conditions: denaturation at 95 C. for 10 sec, annealing at 57 C. for 10 sec, and polymerization at 72 C. for 15 sec. The sequence of the gene fragment obtained using the primer pair of primer 17 and primer 18 was analyzed by Macrogen.

[0066] The selected secondary transformants were passaged 7 times in LB liquid medium, and colonies were selected on LB solid medium. Each colony was selectively cultured on each of LB, LB-Amp and LB-Km solid media. Colonies that did not grow on the LB-Amp and LB-Km solid media while growing on the LB solid medium were selected. The strains constructed in this way were named KCCM13013P_gor (Thr74Ile) and KCCM10016_gor (Thr74Ile), respectively.

[0067] The primer sequences used in Example 1 are shown in Table 1 below.

TABLE-US-00001 TABLE1 Primername SEQIDNO. Primersequence(5.fwdarw.3) Primer1 9 CAATTTTATTATAGTAATTGACTATTATAC Primer2 10 TTGATACCACCAATTTTATTATAGTAATTGACTATTATAC Primer3 11 GTGGTATCAAAACCATAATCGTTTTAGAGCTAGAAATAGC Primer4 12 AACCATAATCGTTTTAGAGCTAGAAATAGC Primer5 13 GAGCCTGTCGGCCTACCTGCT Primer6 14 CGGCCGGCATGAGCCTGTCG Primer7 15 ATGCCGGCCGAATAACGTGCTGCCGTGGCT Primer8 16 AATAACGTGCTGCCGTGGCT Primer9 17 ATAATCCGGGCCGTACATAT Primer10 18 TATCAAAACCATAATCCGGG Primer11 19 GGTTTTGATATCACTATCAATAAATTCAACTGGGA Primer12 20 TCACTATCAATAAATTCAACTGGGA Primer13 21 GCGTTTTCGCGCCGAGGCCGTTAAT Primer14 22 ACAAACAGATGCGTTTTCGC Primer15 23 ATCTGTTTGTGAGCTCCTGAAAATCTCGATAAC Primer16 24 GAGCTCCTGAAAATCTCGATAAC Primer17 25 TCTGGAAGCGACCGGTATTC Primer18 26 CTGCGACCATCTTCCAGCTC

Experimental Example 1. Evaluation of L-Aromatic Amino

Acid Productivity of Mutant Strain into Which Glutathione Reductase Variant Has Been Introduced

[0068] The L-tryptophan or L-phenylalanine productivities of the parent strains (KCCM13013P and KCCM10016) and the mutant strains (KCCM13013P_gor (Thr74Ile) and KCCM10016_gor (Thr74Ile)) into which the glutathione reductase variant has been introduced were compared.

[0069] 1 vol % of each strain (parent strain or mutant strain) was inoculated into a 100-mL flask containing 10 mL of a medium for tryptophan production or a medium for phenylalanine production (the composition of each medium is shown in Table 2 below) and was cultured with shaking at 37 C. and 200 rpm for 72 hours. After completion of the culturing, the concentration of L-tryptophan or L-phenylalanine in the medium was measured using HPLC (Agilent), and the results are shown in Tables 3 and 4 below, respectively.

TABLE-US-00002 TABLE 2 Medium for Medium for tryptophan production phenylalanine 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% pH 7.0 with NaOH (33%) pH 7.0 with NaOH (33%)

TABLE-US-00003 TABLE 3 L-tryptophan L-tryptophan concentration concentration Strain (g/L) increase rate (%) KCCM13013P 4.14 KCCM13013P_gor (Thr74Ile) 5.26 27.0

TABLE-US-00004 TABLE 4 L-phenylalanine L-phenylalanine concentration concentration Strain (g/L) increase rate (%) KCCM10016 3.41 KCCM10016_gor (Thr74Ile) 4.86 42.5

[0070] As shown in Tables 3 and 4 above, it was confirmed that the mutant strains KCCM13013P_gor (Thr74Ile) and KCCM10016_gor (Thr74Ile) into which the glutathione reductase variant has been introduced showed increases in L-tryptophan production and L-phenylalanine production of about 27% and 43%, respectively, compared to the parent strains KCCM13013P and KCCM10016.

Example 2. Construction of Strains Expressing Stress Protein B Variant

[0071] In order to evaluate the effect of a variant resulting from substitution of a stop codon for glutamine at position 42 of the amino acid sequence of stress protein B (SEQ ID NO: 7) on the production of L-aromatic amino acids, a vector expressing the stress protein B variant and strains into which the vector has been introduced were constructed. For insertion of the gene encoding the stress protein B variant into the strains, the plasmids pDSG and pDS9 described in Example 1 were used, and the strains were constructed as follows.

2-1. Construction of Vector pDSG-uspB (Gln42Stop) for Transformation

[0072] Using Escherichia coli MG1655 (KCTC14419BP) gDNA as a template, a fragment upstream of the amino acid mutation at position 42 of the E. coli uspB gene encoding stress protein B was obtained by PCR using a primer pair of primer 7 and primer 9 and a primer pair of primer 8 and primer 10, and a fragment downstream of the amino acid mutation at position 42 of the E. coli uspB gene was obtained by PCR using a primer pair of primer 11 and primer 13 and a primer pair of primer 12 and primer 14. At this time, each of the upstream and downstream fragments contained a sequence that modifies glutamine (Gln) at amino acid residue position 42 of the uspB gene into a stop codon (Stop). In the PCR, Takara PrimeSTAR Max DNA polymerase was used as polymerase, and PCR amplification was performed for 30 cycles under the following conditions: denaturation at 95 C. for 10 sec, annealing at 57 C. for 15 sec, and polymerization at 72 C. for 10 sec.

[0073] Using the plasmid pDSG as a template, four pDSG gene fragments were obtained by PCR using a primer pair of primer 3 and primer 5, a primer pair of primer 4 and primer 6, a primer pair of primer 15 and primer 1, and a primer pair of primer 16 and primer 2, respectively. At this time, each of the gene fragments contained a gRNA sequence targeting Gln at position 42 of the uspB gene. The gRNA was selected as a 20 mer upstream of NGG of the sequence to be mutated. In the PCR, Takara PrimeSTAR Max DNA polymerase was used as polymerase, and PCR amplification was performed for 30 cycles under the following conditions: denaturation at 95 C. for 10 sec, annealing at 57 C. for 15 sec, and polymerization at 72 C. for 15 sec.

[0074] The obtained fragments upstream and downstream of the amino acid at position 42 of the uspB gene, and the obtained four pDSG gene fragments were cloned using a self-assembly cloning method (BioTechniques 51:55-56 (July 2011)), thereby obtaining a recombinant plasmid which was named pDSG-uspB (Gln42Stop).

2-2. Construction of L-Tryptophan- or L-Phenylalanine-Producing Strain into Which Stress Protein B Variant uspB (Gln42Stop) Has Been Introduced

[0075] To construct an L-tryptophan-producing strain and an L-phenylalanine-producing strain, Escherichia coli KCCM13013P and KCCM10016 were used as parent strains.

[0076] The KCCM13013P strain or the KCCM10016 strain was primarily transformed with the plasmid pDS9 and cultured in LB-Km (containing 25 g/L of LB liquid medium and 50 mg/L of kanamycin) solid medium, and then kanamycin-resistant colonies were selected. The selected colonies were secondarily transformed with the pDSG-uspB (Gln42Stop) plasmid and cultured in LB-Amp & Km (containing 25 g/L of LB liquid medium, 100 mg/L of ampicillin and 50 mg/L of kanamycin) solid medium, and then ampicillin- and kanamycin-resistant colonies were selected. Next, a gene fragment was obtained by PCR using a primer pair of primer 17 and primer 18. Here, Takara PrimeSTAR Max DNA polymerase was used as polymerase, and PCR amplification was performed for 30 cycles under the following conditions: denaturation at 95 C. for 10 sec, annealing at 57 C. for 10 sec, and polymerization at 72 C. for 15 sec. The sequence of the gene fragment obtained using the primer pair of primer 17 and primer 18 was analyzed by Macrogen.

[0077] The selected secondary transformants were passaged 7 times in LB liquid medium, and colonies were selected on LB solid medium. Each colony was selectively cultured on each of LB, LB-Amp and LB-Km solid media. Colonies that did not grow on the LB-Amp and LB-Km solid media while growing on the LB solid medium were selected. The strains constructed in this way were named KCCM13013P_uspB (Gln42Stop) and KCCM10016_uspB (Gln42Stop), respectively.

[0078] The primer sequences used in Example 2 are shown in Table 5 below.

TABLE-US-00005 TABLE5 Primername SEQIDNO. Primersequence(5.fwdarw.3) Primer1 9 CAATTTTATTATAGTAATTGACTATTATAC Primer2 27 GATAGAGCAACAATTTTATTATAGTAATTGACTATTATAC Primer3 28 TTGCTCTATCAATATGTTGAGTTTTAGAGCTAGAAATAGC Primer4 29 AATATGTTGAGTTTTAGAGCTAGAAATAGC Primer5 13 GAGCCTGTCGGCCTACCTGCT Primer6 14 CGGCCGGCATGAGCCTGTCG Primer7 30 ATGCCGGCCGATAGTGGTTAACCTTCTGGA Primer8 31 ATAGTGGTTAACCTTCTGGA Primer9 32 GGATCGCAGTTACGCAGTAC Primer10 33 ATAGAGCAATGGATCGCAGT Primer11 34 ATTGCTCTATTAATATGTTGATGGAGGGGG Primer12 35 TAATATGTTGATGGAGGGGG Primer13 36 ATTTTGCGTTTGTAATCAGTCTCG Primer14 37 GCATCAGGCAATTTTGCGTT Primer15 38 TGCCTGATGCGAGCTCCTGAAAATCTCGATAAC Primer16 39 GAGCTCCTGAAAATCTCGATAAC Primer17 40 AGCGTTTCTTGTGCCGTTAC Primer18 41 GATTGTGACATAGTGCGCTT

Experimental Example 2. Evaluation of L-Aromatic Amino Acid Productivity of Mutant Strain into Which Stress Protein B Variant Has Been Introduced

[0079] The L-tryptophan or L-phenylalanine productivities of the parent strains (KCCM13013P and KCCM10016) and the mutant strains (KCCM13013P_uspB (Gln42Stop) and KCCM10016_uspB (Gln42Stop)) into which the stress protein B variant has been introduced were compared. The concentrations of L-tryptophan and L-phenylalanine were measured using the same method as in Experimental Example 1, and the results are shown in Tables 6 and 7 below, respectively.

TABLE-US-00006 TABLE 6 L-tryptophan L-tryptophan concentration concentration Strain (g/L) increase rate (%) KCCM13013P 4.15 KCCM13013P_uspB (Gln42Stop) 5.01 20.7

TABLE-US-00007 TABLE 7 L-phenylalanine L-phenylalanine concentration concentration Strain (g/L) increase rate (%) KCCM10016 3.22 KCCM10016_uspB (Gln42Stop) 3.91 21.4

[0080] As shown in Tables 6 and 7 above, it was confirmed that the mutant strains KCCM13013P_uspB (Gln42Stop) and KCCM10016_uspB (Gln42Stop) into which the stress protein B variant has been introduced showed increases in L-tryptophan production and L-phenylalanine production of about 21% and 21%, respectively, compared to the parent strains KCCM13013P and KCCM10016.

[0081] So far, the present invention has been described with reference to the embodiments. Those skilled in the art will appreciate that the present invention can be implemented in modified forms without departing from the essential features of the present invention. Therefore, the disclosed embodiments should be considered in descriptive sense only and not for purposes of limitation. Therefore, the scope of the present invention is defined not by the detailed description of the present invention but by the appended claims, and all differences within a range equivalent to the scope of the appended claims should be construed as being included in the present invention.