KR102251947B1 - Strain with Improved Aromatic Amino Acid Production Capacity by mdtK Gene Inactivation - Google Patents

Abstract

A mutant strain with improved aromatic amino acid production capacity is disclosed by weakening or inactivating the activity of the multidrug-releasing protein expressed by the mdtK gene.

Description

Strain with Improved Aromatic Amino Acid Production Capacity by mdtK Gene Inactivation}

The present invention relates to a strain having improved aromatic amino acid production capacity by mdtK gene inactivation.

Aromatic amino acids, especially L-tryptophan and L-phenylalanine, are key products of feed amino acids and are a high value-added industry with an annual market of $300 billion worldwide.

Aromatic amino acids are produced using recombinant strains, and research is being actively conducted to increase their production. Chorismate is a precursor necessary for the biosynthetic pathway of aromatic amino acids. To produce it, phosphoenolpyruvate (PEP), erythrose-4-phosphate (E4P), phosphoribosyl pyrophosphate (PRPP), serine, glutamine ), etc. are required. Therefore, in the past, studies have been conducted to enhance the biosynthetic pathway of E4P, PEP, or PRPP in order to improve L-tryptophan production capacity.

However, it has not been reported that the production of aromatic amino acids is increased by suppressing the release of serine.

Republic of Korea Registration Gazette No. 10-1830002 (2018.02.09)

According to one embodiment, by inhibiting the expression of the mdtK gene provides a strain having improved production capacity of aromatic amino acids.

One aspect provides a mutant strain having improved aromatic amino acid production ability by weakening or inactivating the activity of a multidrug efflux protein expressed by a multidrug resistance protein K (mdtK) gene.

The mdtK gene may be composed of the nucleotide sequence of SEQ ID NO: 1.

The mdtK protein is known to be involved in multidrug resistance and is a transporter involved in the excretion of serine. The association of the mdtK gene with an aromatic production pathway such as the tryptophan pathway, or its effect thereon, is unknown. Nevertheless, as a result of suppressing the release of serine by lowering the expression of the mdtK protein, the present inventors confirmed that the ability of the strain to produce aromatic amino acids was improved as serine that could not be released was used for the production of aromatic amino acids.

The term "reduced activity" as used herein means that the expression level of a gene, which is an object, is decreased compared to the original expression level. This attenuation of activity is when the activity of the enzyme itself is decreased compared to the activity of the enzyme of the original microorganism through nucleotide substitution, insertion, deletion, or a combination of nucleotide encoding the gene, and inhibition of expression or translation of the gene encoding it. In the case where the overall degree of enzyme activity in the cell is lower than that of the natural strain or the strain before modification, a combination thereof is also included.

As used herein, the term "inactivation" refers to a case in which the expression of a gene encoding a protein such as an enzyme is not expressed at all compared to a natural strain or a strain before modification, and when there is no activity even if it is expressed.

The term "increased expression" as used herein means that the expression level of a gene, which is an object, is increased compared to the original expression level. When a gene for increasing expression does not exist in the pre-mutation strain, one or more genes can be introduced into the chromosome of the strain to increase expression, and when a gene for increasing expression is present in the pre-mutation strain In the above, one or more genes may be additionally introduced into the strain or genetically engineered to increase the expression level of an existing gene.

In the present invention, the method of modifying the expression control sequence is performed by inducing a mutation in the expression control sequence by deletion, insertion, non-conservative or conservative substitution or a combination thereof in the nucleic acid sequence of the expression control sequence, or by using a weaker promoter. It can be carried out by a method such as replacement. The expression control sequence includes a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence controlling the termination of transcription and translation.

In addition, the method of modifying the gene sequence on the chromosome is performed by inducing a mutation in the sequence by deletion, insertion, non-conservative or conservative substitution, or a combination of the gene sequence so that the activity of the enzyme is further weakened, or a weaker activity is achieved. It can be carried out by replacing with a gene sequence improved to have or a gene sequence improved to have no activity.

According to one embodiment, the aromatic amino acid may be L-tyrosine, L-tryptophan, and L-phenylalanine.

According to one embodiment, the mutant strain may have all or part of the mdtK gene inserted, substituted, or deleted.

According to one embodiment, the mutant strain may be a strain of Escherichia genus.

According to one embodiment, the strain of the genus Escherichia may be Escherichia coli, for example, KFCC11660P and KCCM10016 deposited strains.

According to another aspect, there is provided a method for producing an aromatic amino acid comprising culturing the mutant strain in a medium, and recovering the aromatic amino acid from the cultured strain and the culture medium.

The strain used in the present invention may be cultured through a culture method known in the art. As a medium, natural or synthetic medium may be used. As the carbon source of the medium, for example, glucose, sucrose, dextrin, glycerol, starch, etc. may be used, and as the nitrogen source, peptone, meat extract, yeast extract, dried yeast, soybean cake, urea, thiourea, ammonium salt, nitrate And other organic or inorganic nitrogen-containing compounds may be used, but are not limited to these components.

Inorganic salts contained in the medium may include phosphates such as magnesium, manganese, potassium, calcium, iron, etc., nitrates, carbonates, chlorides, etc., but are not limited thereto. In addition to the components of the carbon source, nitrogen source and inorganic salt, amino acids, vitamins, nucleic acids and related compounds may be added to the medium.

The temperature of the culture may be 27 to 40°C, preferably 30 to 37°C, but is not limited thereto. The cultivation period may be continued until the desired production amount of the useful substance is obtained, and preferably may be 10 to 100 hours, but is not limited thereto.

In the step of recovering the aromatic amino acid, the desired amino acid can be recovered from the culture medium using a suitable method known in the art according to the method of culturing the microorganism of the present invention, for example, a batch, continuous, or fed-batch culture method. , The recovery step may include a purification process.

According to one embodiment, the aromatic amino acid may be L-tryptophan and L-phenylalanine.

According to one embodiment, the production of aromatic amino acids may be increased by weakening or inactivating the activity of the multidrug-releasing protein expressed by the mdtK gene of the strain.

Hereinafter, one or more specific examples will be described in more detail through examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

Example 1: Construction of strains in which the mdtK gene was deleted

One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products, Datsenko KA, Wanner BL., Proc Natl Acad Sci USA. 2000 Jun 6;97 12):6640-5) was used to construct a mutant strain in which the mdtK gene was inactivated.

KFCC11660P strain and KCCM10016 strain are Escherichia coli strains, and pKD46 (GenBank accession number AY048746), a red recombinase plasmid, is introduced for homologous recombination of the fourth fragment, and pKD46 is removed prior to introduction of pCP20.

The mdtK gene was deleted by homologous recombination of the mdtK gene and the DNA fragment containing the antibiotic gene, and the mdtK gene was inactivated by undergoing a process of removing the antibiotic resistance gene from the recombined DNA fragment. The specific process is as follows.

(1) Production of the first short

PCR reaction (total volume 50 μl, 95° C. 5 minutes after 1 cycle) using mdtK_PF and mdtK_PR primer pairs and pKD13 plasmid (Genbank accession number AY048744) having some sequences of the mdtK gene in Table 1 and some sequences of the pKD13 plasmid. C. 30 seconds, 58° C. 30 seconds, 72° C. 2 minutes, after a total of 30 cycles, 72° C. for 5 minutes and 12° C. for 10 minutes) to obtain an amplified first fragment of about 1.4 kb. The first fragment contains a kanamycin resistance gene derived from the pKD13 plasmid.

(2) Production of the second short

To obtain the front fragment of the mdtK gene, use the genomic DNA of E. coli MG1655 as a template, and PCR using the primers mdtK_HF1 and mdtK_HR1 in Table 1 (total volume 50 μl, 95° C. 5 minutes 1 cycle, 95° C. 30 Second, 58°C for 30 seconds, 72°C for 30 seconds, after a total of 30 cycles, 72°C for 5 minutes and 12°C for 10 minutes) to obtain a second fragment amplified by about 0.3 kb.

(3) Production of the 3rd short film

In addition, in order to obtain the rear fragment of the mdtK gene, PCR reaction using the primers mdtK_HF2 and mdtK_HR2 in Table 1 using the genomic DNA of E. coli MG1655 as a template (total volume 50 µl, 95°C for 5 minutes 1 cycle, 95°C for 30 seconds , 58°C for 30 seconds, 72°C for 30 seconds, 72°C for a total of 30 cycles, and 12°C for 10 minutes) to obtain an amplified third fragment of about 0.3 kb.

(4) Production of the 4th short film

Each of the first fragment, the second fragment, and the third fragment amplified in the above experiment may be linked into one fragment due to the complementary sequence of the primer during amplification. These fragments were subjected to a total volume of 50 µl excluding primers, 95°C for 5 minutes, 1 cycle, 95°C for 30 seconds, 58°C for 30 seconds, 72°C for 2 minutes and 30 seconds, and a total of 30 cycles at 72°C for 5 minutes and 12 PCR was performed under conditions of 10 minutes at °C to obtain one amplified fourth fragment having a size of about 2 kb. The fourth fragment contains a part of the mdtK gene and a kanamycin antibiotic resistance gene. Specifically, it consists of a part of the mdtK gene in the 5'direction, a kanamycin antibiotic resistance gene, and a part of the mdtK gene in the 3'direction. have.

(5) Injection of the fourth fragment and confirmation of mdtK deletion

The fourth fragment was injected into KFCC11660P, an Escherichia coli strain containing the red recombinase plasmid pKD46 (GenBank accession number AY048746) by electroporation. The fourth fragment is replaced with mdtK and homologous recombination by Lambda Red recombination, whereby mdtK is deleted.

Subsequently, a PCR reaction was performed on a cell line exhibiting kanamycin resistance to confirm the deletion of the mdtK gene. The reaction was carried out using mdtK_CF and mdtK_CR primers in Table 1 in a total volume of 20 µl, 95°C for 5 minutes and 1 cycle, 95°C for 30 seconds, 55°C for 30 seconds, 72°C for 3 minutes, and a total of 30 cycles at 72°C. PCR was performed under conditions of 5 minutes and 10 minutes at 12°C. Compared with the original mdtK gene, about 2.2 kb (before deletion), when the fragment is inserted into the chromosome, about 2.3 kb (including the antibiotic gene) is generated.

(6) Antibiotic resistance gene removal and selection

In order to remove the antibiotic resistance marker gene from the strain in which the mdtK gene was deleted, the pCP20 plasmid was introduced to induce FLP recombination. After that, it was confirmed that the antibiotic resistance marker gene was removed by culturing the mdtK deletion strain in LB plate medium with or without antibiotics.

Example 2: Culture of mdtK deletion strain and evaluation of aromatic amino acid production

E. coli KFCC11660PΔmdtK and KFCC11660P prepared by the method of Example 1 were cultured in the tryptophan production medium shown in Table 2 below.

Incubation was performed by inoculating 1% of the KFCC11660PΔmdtK and KFCC11660P strains, respectively, based on the volume in a flask containing 10 mL of the tryptophan production medium of the composition shown in Table 2 below, and incubating with shaking at 37°C at 200 rpm for 70 hours, from which The concentration of the obtained L-amino acid was compared.

As a result of the above experiment, it was confirmed that the production amount of L-tryptophan increased when the mdtK gene was inactivated as shown in Table 3 below.

<110> Daesang Corporation <120> Strain with Improved Aromatic Amino Acid Production Capacity by mdtK Gene Inactivation <130> PN190270 <160> 9 <170> KoPatentIn 3.0 <210> 1 <211> 1374 <212> DNA <213> Artificial Sequence <220> <223> mdtK "YDHE-MONOMER" 1743457..1744830 Escherichia coli K-12 substr. MG1655 <400> 1 gtgcagaagt atatcagtga agcgcgtctg ttattagcat tagcaatccc ggtgattctc 60 gcgcaaatcg cccaaactgc gatgggtttt gtcgataccg tgatggcggg cggctatagt 120 gccaccgaca tggcggcggt cgctatcggt acttctatct ggcttccggc gatcctcttt 180 ggtcacggac tgctgctggc attaacgccg gttatcgcgc aattaaatgg ttccggtcga 240 cgtgagcgca ttgcgcatca ggtgcgacaa ggtttctggc tggcaggttt tgtttccgtt 300 ctcattatgc tggtgctgtg gaatgcaggt tacattatcc gctccatgga aaacatcgat 360 ccggctctgg cggacaaagc cgtgggttat ctgcgtgcgt tgttgtgggg cgcgccggga 420 tatctgttct tccaggttgc ccgtaaccag tgtgaaggtc tggcaaaaac caagccgggt 480 atggtaatgg gctttatcgg cctgctggtg aacatcccgg tgaactatat ctttatttat 540 ggtcatttcg gtatgcctga gctcggtggc gttggttgtg gcgtggctac tgcggcggtg 600 tattgggtca tgttccttgc catggtttct tacattaaac gcgcccgctc catgcgcgat 660 attcgtaacg aaaaaggcac cgcaaaaccc gatcctgcgg ttatgaaacg actgattcaa 720 ctcggtttgc cgattgcgct ggcactgttc tttgaagtga cactgtttgc cgtcgtggct 780 ctgttagtgt ctccgctcgg tattgttgat gtcgcaggac accagattgc cctgaacttt 840 agttcactaa tgttcgtgct tccaatgtcg ctggcggcag cggtaactat ccgcgtaggt 900 tatcgtctgg gtcagggctc aacgctggat gcgcaaaccg ctgcgcggac cgggcttatg 960 gtgggtgtct gtatggcaac cctgacggcc attttcacgg tttcactgcg ggagcaaatc 1020 gccctgttgt acaacgacaa tcccgaggtt gtaacgctgg ctgcgcattt gatgttgctg 1080 gcggcggtat atcagatttc tgactcaatc caggtgattg gcagtgggat tttgcgtggt 1140 tataaagata cgcgttccat tttctatatt acctttacgg cttactgggt gctgggcttg 1200 ccaagcggct atattctggc actgaccgat ctggtcgttg aacctatggg gccagcaggc 1260 ttctggatag gctttattat tggcctgacg tcggcagcca ttatgatgat gttgcgtatg 1320 cggttcctgc aacgtctgcc gtcagccatc attctgcaac gagcatcccg ctaa 1374 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> mdtK_HF1 <400> 2 ccctgtacaa tccccgtaaa 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> mdtK_HR1 <400> 3 ggcgatttgc gcgagaatca 20 <210> 4 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> mdtK_PF <400> 4 tgattctcgc gcaaatcgcc gtgtaggctg gagctgcttc 40 <210> 5 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> mdtK_PR <400> 5 gacggcagac gttgcaggaa ctgtcaaaca tgagaattaa 40 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> mdtK_HF2 <400> 6 ttcctgcaac gtctgccgtc 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> mdtK_HR2 <400> 7 ctgaaagtga aacgcctgcg 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> mdtK_CF <400> 8 ggttgccgtt aatttccgtc 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> mdtK_CR <400> 9 gggagtgttg cattactgga 20

Claims (8)

E. coli mutant strain with improved L-tryptophan production ability by weakening or inactivating the activity of the multidrug-releasing protein expressed by the mdtK gene. The method of claim 1,
The mdtK gene is composed of the nucleotide sequence of SEQ ID NO: 1,
Mutant strain. delete The method of claim 1,
The mutant strain is that all or part of the mdtK gene is inserted, substituted, or deleted,
Mutant strain. delete delete Culturing the mutant strain of claim 1 in a medium; And
Comprising the step of recovering L-tryptophan from the cultured mutant strain and the culture medium,
A method for producing aromatic amino acids.

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