NOVEL ATP SYNTHASE VARIANT AND METHOD OF PRODUCING L-AROMATIC AMINO ACID USING THE SAME

Abstract

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

Claims

1. An ATP synthase variant composed of the amino acid sequence of SEQ ID NO: 1 in which glycine at position 235 in the amino acid sequence of SEQ ID NO: 3 is substituted with serine.

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, the method 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, the method 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

[0051] FIG. 1 shows the structure of plasmid pDSG according to an embodiment of the present invention.

[0052] FIG. 2 shows the structure of plasmid pDS9 according to an embodiment of the present invention.

MODE FOR INVENTION

[0053] 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 ATP Synthase Variant

[0054] In order to evaluate the effect of a variant resulting from substitution of serine for glycine at position 235 of the amino acid sequence of ATP synthase (SEQ ID NO: 3) on the production of L-aromatic amino acids, a vector expressing the ATP synthase variant and strains into which the vector has been introduced were constructed. For insertion of the gene encoding the ATP synthase variant into the strains, plasmids pDSG and pDS9 were used, and the strains were constructed as follows.

[0055] 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-atpD(Gly235Ser) for Transformation

[0056] Using Escherichia coli MG1655 (KCTC14419BP) gDNA as a template, a upstream fragment of the amino acid mutation at position 235 of the E. coli atpD gene encoding ATP synthase 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 downstream fragment of the amino acid mutation at position 235 of the E. coli atpD 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 glycine (Gly) at amino acid residue position 235 of the atpD gene into serine (Ser). In the PCR, Takara PrimeSTAR

[0057] 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 extension at 72 C. for 10 sec.

[0058] 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 Gly at position 235 of the atpD 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 extension at 72 C. for 15 sec.

[0059] The obtained fragments upstream and downstream of the amino acid at position 235 of the atpD 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-atpD (Gly235Ser).

1-2. Construction of L-Tryptophan- or L-Phenylalanine-Producing Strain into which ATP Synthase Variant atpD (Gly235Ser) has been Introduced

[0060] To construct an L-tryptophan-producing strain and an L-phenylalanine-producing strain, Escherichia coli KFCC11660P and KCCM10016 were used as parent strains.

[0061] The KFCC11660P 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 colonies having kanamycin resistance were selected. The selected colonies were secondarily transformed with the pDSG-atpD (Gly235Ser) 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 extension 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.

[0062] 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 KFCC11660P_atpD (Gly235Ser) and KCCM10016 atpD (Gly235Ser), respectively.

[0063] The primer sequences used in Example 1 are shown in Table 1 below.

TABLE-US-00001 TABLE1 Primer SEQ name IDNO Primersequence(5.fwdarw.3) Primer1 5 CAATTTTATTATAGTAATTGACTATTATAC Primer2 6 CGTGACGTTCCAATTTTATTATAGTAATTG ACTATTATAC Primer3 7 GAACGTCACGACCTTCGTCAGTTTTAGAGC TAGAAATAGC Primer4 8 ACCTTCGTCAGTTTTAGAGCTAGAAATAGC Primer5 9 GAGCCTGTCGGCCTACCTGCT Primer6 10 CGGCCGGCATGAGCCTGTCG Primer7 11 ATGCCGGCCGCGGCCGGGTAGATACCCAGAG Primer8 12 CGGCCGGGTAGATACCCAGAG Primer9 13 TCTGCTGTTCGTTGACAACA Primer10 14 GTCGTGACGTTCTGCTGTTC Primer11 15 ACGTCACGACTTTCGTCACGGAATTTCTCA Primer12 16 TTTCGTCACGGAATTTCTCA Primer13 17 TCCGTTCGCTAAGGGCGGTA Primer14 18 ACCTGATGTGTCCGTTCGCT Primer15 19 CACATCAGGTGAGCTCCTGAAAATCTCGAT AAC Primer16 20 GAGCTCCTGAAAATCTCGATAAC Primer17 21 TCCGTTCGCTAAGGGCGGTA Primer18 22 ACCTGATGTGTCCGTTCGCT

Experimental Example 1. Evaluation of L-Aromatic Amino Acid Productivity of Mutant Strain into which ATP Synthase Variant has been Introduced

[0064] The L-tryptophan or L-phenylalanine productivities of the parent strains (KFCC11660P and KCCM10016) and the mutant strains (KFCC11660P_atpD(Gly235Ser) and KCCM10016_atpD(Gly235Ser)) into which the ATP synthase variant has been introduced were compared.

[0065] 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 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, 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 (%) KFCC11660P 3.8 KFCC11660P_atpD(Gly235Ser) 5.1 34.2

TABLE-US-00004 TABLE 4 L-phenylalanine L-phenylalanine concentration concentration Strain (g/L) increase rate (%) KCCM10016 3.30 KCCM10016_atpD(Gly235Ser) 4.32 30.9

[0066] As shown in Tables 3 and 4 above, it was confirmed that the mutant strains KFCC11660P_atpD(Gly235Ser) and KCCM10016_atpD(Gly235Ser) into which the ATP synthase variant has been introduced showed increases in L-tryptophan and L-phenylalanine production of about 34% and 31%, respectively, compared to the parent strains KFCC11660P and KCCM10016.

[0067] 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 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.