KR102778362B1 - A gene of cold shock protein from Bacillus butanolivorans and production of L-arginine by recombinant Corynebacterium glutamicum with the gene - Google Patents

Abstract

The present invention relates to the production of L-arginine using a cold shock protein gene derived from Bacillus butanolivorans and a recombinant Corynebacterium glutamicum containing the same. Corynebacterium glutamicum produced according to the present invention can be utilized for mass production of L-arginine.

Description

{A gene of cold shock protein from Bacillus butanolivorans and production of L-arginine by recombinant Corynebacterium glutamicum with the gene}

The present invention relates to a cold shock protein gene derived from Bacillus butanolivorans and L-arginine production using recombinant Corynebacterium glutamicum containing the same, and more specifically, to a cold shock protein (CSP) gene derived from Bacillus butanolivorans PAMC23377 and Corynebacterium glutamicum into which the gene has been introduced, and a method for producing L-arginine using the same.

L-arginine is found in free form in plant seeds and garlic, and is used as an amino acid strengthener and is widely used in medicines, foods, etc. For medicine, it is used as a liver function promoter, brain function promoter, male infertility treatment, and comprehensive amino acid preparation, and for food, it is a substance that has recently been in the spotlight as a fish cake additive, health drink additive, and salt substitute for hypertensive patients.

In microorganisms, L-arginine biosynthesis is accomplished through eight enzymatic reactions from L-glutamate via two different pathways, a linear step and a cyclic step. Microorganisms of the genus Corynebacterium achieve arginine biosynthesis via a cyclic step pathway. In the cyclic step, L-arginine is synthesized from L-glutamate via N-acetylglutamate, N-acetylglutamyl phosphate, N-acetylglutamate semialdehyde, N-acetylornithine, ornithine, citrulline, and argininosuccinate.

Conventionally known methods for producing L-arginine by biological fermentation are methods for producing L-arginine directly from carbon and nitrogen sources, and methods have been reported, such as methods using mutants derived from microorganisms of the genus Brevibacterium or Corynebacterium , which are glutamate-producing strains, and methods using amino acid-producing strains with improved growth through cell fusion. Recently, methods have been reported, such as methods using recombinant strains in which the gene argR, which suppresses the expression of the arginine biosynthesis operon, is inactivated, and methods for overexpressing argF of the arginine operon.

However, the existing L-arginine production method has a disadvantage in that feedback inhibition can occur due to intracellular L-arginine, making it difficult to increase the yield of L-arginine. Accordingly, there is an urgent need for the development of a strain that can further increase the production yield of L-arginine and a method for producing L-arginine using the strain.

Accordingly, the present inventors developed a method for producing L-arginine using a cold shock protein gene derived from Bacillus butanolivorans and a recombinant Corynebacterium glutamicum containing the gene, and confirmed that the L-arginine production yield thereof was significantly superior.

Accordingly, the purpose of the present invention is to provide a polynucleotide comprising a base sequence represented by sequence number 3.

Another object of the present invention is to provide a recombinant vector comprising a polynucleotide consisting of a base sequence represented by SEQ ID NO: 3.

Another object of the present invention is to provide a transformed microorganism with enhanced L-arginine productivity by introducing a polynucleotide comprising a base sequence represented by SEQ ID NO: 3.

Another object of the present invention is to provide a method for producing a microorganism with enhanced L-arginine productivity.

Another object of the present invention is to provide a method for producing L-arginine.

Another object of the present invention relates to the use of a microorganism with enhanced L-arginine productivity transformed by introducing a polynucleotide consisting of a base sequence represented by SEQ ID NO: 3 for producing L-arginine.

The present invention relates to a method for producing L-arginine using a cold shock protein gene derived from Bacillus butanolivorans and a recombinant Corynebacterium glutamicum containing the same, wherein a microorganism into which a cold shock protein gene has been introduced according to the present invention exhibits high L-arginine productivity.

The present inventors discovered a cold shock protein gene derived from Bacillus butanolivorans and transformed this gene into Corynebacterium glutamicum to evaluate L-arginine productivity.

As a result, it was confirmed that the recombinant Corynebacterium glutamicum expressing the shock protein gene not only showed superior growth compared to the wild type in a culture environment for L-arginine production, but also synthesized a high content of L-arginine.

Hereinafter, the present invention will be described in more detail.

One aspect of the present invention is a polynucleotide comprising a base sequence represented by SEQ ID NO: 3.

The term "polynucleotide" used in this specification is a DNA strand of a certain length or longer, which is a polymer of nucleotides in which nucleotide units (monomers) are covalently bonded to form a long chain.

In the present invention, the “base sequence represented by SEQ ID NO: 3” may be derived from Bacillus sp. , and specifically may refer to part or all of a cold shock protein gene derived from Bacillus butanolivorans .

In one embodiment of the present invention, the base sequence represented by SEQ ID NO: 3 may be part or all of a cold shock protein gene derived from Bacillus butanolivorans PAMC23377.

In the present invention, the cold shock protein derived from Bacillus butanolivorans PAMC23377 may include the amino acid sequence of SEQ ID NO: 4.

In the present invention, a polynucleotide comprising a base sequence represented by sequence number 3 may include a nucleotide sequence having substantial identity with the base sequence of sequence number 3.

Substantial identity may include a polynucleotide having a sequence identity of at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, for example, a polynucleotide having a sequence identity of at least 90%.

The term "cold shock protein" in this specification refers to a protein that is expressed and functions to protect an organism from damage caused by low temperature. Cold shock proteins are known to be divided into a first type (class 1) that increases more than 10 times the normal state in a low temperature environment, and a second type (class 2) that increases less than 10 times. The majority of cold shock proteins are known to have the function of a chaperone for RNA and/or DNA.

Another aspect of the present invention is a recombinant vector comprising a polynucleotide comprising a base sequence represented by SEQ ID NO: 3.

The term "vector" as used herein means a means for expressing a target gene in a host cell. For example, the vector may include a plasmid vector, a cosmid vector, and a viral vector such as a bacteriophage vector, an adenovirus vector, a retrovirus vector, and an adeno-associated virus vector.

Any vector that can be used as a recombinant vector can be used without limitation as long as it is a vector used in the art. Specifically, the vector can be produced using plasmids such as pCES208, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8/9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX series, pET series, and pUC19, phages such as λgt4λB, λ-Charon, λΔz1, and M13, or viral vectors such as SV40, and for example, pCES208GFP vector can be used, but is not limited thereto.

Another aspect of the present invention relates to a host cell transformed with a polynucleotide comprising a base sequence represented by SEQ ID NO: 3.

As used herein, the term “host cell” may be a strain cell transformed with a nucleic acid encoding a cold shock protein derived from Bacillus butanolivorans or a vector comprising the nucleic acid. Examples of the strain for transformation include, but are not limited to, Escherichia coli , Corynebacterium glutamicum , Aspergillus oryzae , Saccharomyces cerevisiae , Yarrowia lipolytica , Pichia pastoris , or Bacillus subtilis.

In a specific embodiment of the present invention, a transformant having improved L-arginine production ability can be produced by inserting an expression vector having a target protein gene inserted into a host cell, and the transformant can be obtained by introducing a recombinant vector into an appropriate host cell.

The term "transformation" as used herein means introducing a vector containing a polynucleotide encoding a target protein into a host cell so that the protein encoded by the polynucleotide can be expressed in the host cell. The transformed polynucleotide may include both a position inserted into the chromosome of the host cell or an extrachromosomal position, as long as it can be expressed in the host cell. In addition, the polynucleotide includes DNA and RNA encoding the target protein, and may be introduced in any form as long as it can be introduced into the host cell and expressed. For example, the polynucleotide can be introduced into the host cell in the form of an expression cassette, which is a genetic construct containing all elements necessary for its own expression, or in the form of a vector containing the same.

In the present invention, the delivery (introduction) of a gene encoding a cold shock protein derived from Bacillus butanolivorans or a recombinant vector containing the same into a host cell can be performed using a delivery method widely known in the art. For example, if the host cell is a prokaryotic cell, the CaCl ₂ method or the electroporation method can be used, and if the host cell is a eukaryotic cell, the microinjection method, the calcium phosphate precipitation method, the electroporation method, the liposome-mediated transfection method, the gene bombardment method, and the like can be used, but are not limited thereto.

The method for selecting transformed host cells can be easily performed by a method widely known in the art, using a phenotype expressed by a selection marker. For example, when the selection marker is a specific antibiotic resistance gene, the transformant can be easily selected by culturing the transformant in a medium containing antibiotics.

Another aspect of the present invention is a microorganism with enhanced L-arginine productivity, comprising a polynucleotide comprising a base sequence represented by SEQ ID NO: 3.

In one embodiment of the present invention, the microorganism may be transformed by introducing a polynucleotide comprising a base sequence represented by SEQ ID NO: 3.

Another aspect of the present invention is a method for producing a microorganism with enhanced L-arginine productivity, comprising the following steps:

A transformation step of transforming a microorganism with a recombinant vector containing a polynucleotide consisting of a base sequence represented by sequence number 3.

In one embodiment of the present invention, the microorganism may be transformed with a recombinant vector comprising a polynucleotide consisting of a base sequence represented by SEQ ID NO: 3.

The term "microorganism" in this specification includes both wild-type microorganisms and microorganisms that have undergone genetic modification naturally or artificially, and is a concept that includes all microorganisms whose specific mechanisms are weakened or strengthened due to causes such as the insertion of external genes or the strengthening or weakening of the activity of endogenous genes. In the present application, a microorganism may be included without limitation as long as it is a microorganism into which a polynucleotide consisting of a base sequence represented by SEQ ID NO: 3 is introduced or included.

In the present invention, the microorganism may include a polynucleotide consisting of a base sequence represented by SEQ ID NO: 3, and specifically, may include a polynucleotide consisting of a base sequence represented by SEQ ID NO: 3 and/or a vector comprising a polynucleotide consisting of a base sequence represented by SEQ ID NO: 3, and more specifically, may include a vector including a gene encoding a target protein, but is not limited thereto. In addition, the polynucleotide and the vector may be introduced into the microorganism by transformation, but are not limited thereto.

Another aspect of the present invention is a method for producing L-arginine comprising the following steps:

A transformation step of transforming a microorganism with a recombinant vector comprising a polynucleotide consisting of a base sequence represented by sequence number 3; and

The cultivation stage for cultivating microorganisms.

The term "cultivation" in this specification means growing microorganisms under appropriately controlled environmental conditions. The culturing process of the present application can be performed according to appropriate media and culturing conditions known in the art. This culturing process can be easily adjusted and used by those skilled in the art according to the selected strain. Specifically, the culturing can be batch, continuous, and/or fed-batch, but is not limited thereto.

The microorganism according to the present invention can be cultured in any medium without special restrictions as long as it is used for culturing microorganisms, but can be cultured under aerobic conditions while controlling temperature, pH, etc. in a general medium containing a carbon source, a nitrogen source, phosphorus, inorganic compounds, etc.

The carbon source may include carbohydrates such as glucose, saccharose, lactose, fructose, sucrose, maltose, etc.; sugar alcohols such as mannitol, sorbitol, etc., organic acids such as pyruvic acid, lactic acid, citric acid, etc.; amino acids such as glutamic acid, methionine, lysine, etc. In addition, natural organic nutrients such as starch hydrolysate, molasses, blackstrap molasses, rice winter, cassava, sugarcane bagasse, and corn steep liquor can be used, and specifically, carbohydrates such as glucose and sterilized pretreated molasses (i.e., molasses converted into reducing sugar) can be used, and other appropriate amounts of carbon sources can be used in various ways without limitation. These carbon sources may be used alone or in combination of two or more, but are not limited thereto.

Nitrogen sources that can be used include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, ammonium nitrate, etc.; amino acids such as glutamic acid, methionine, glutamine, etc.; peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolysate, fish or its decomposition products, defatted soybean cake or its decomposition products, etc. Organic nitrogen sources that can be used include. These nitrogen sources can be used alone or in combination of two or more, but are not limited thereto.

The human components may include potassium phosphate monobasic, potassium phosphate dibasic, or their corresponding sodium-containing salts. The inorganic compounds may include sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, and the like. In addition, amino acids, vitamins, and/or suitable precursors may be included. These components or precursors may be added to the medium in batch or continuous manner. However, the present invention is not limited thereto.

The present invention relates to the production of L-arginine using a cold shock protein gene derived from Bacillus butanolivorans and a recombinant Corynebacterium glutamicum containing the same. Corynebacterium glutamicum produced according to the present invention can be utilized for mass production of L-arginine.

Hereinafter, the present invention will be described in more detail by the following examples. However, these examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

Throughout this specification, "%", when used to indicate the concentration of a particular substance, unless otherwise noted, is (weight/weight) % for solid/solid, (weight/volume) % for solid/liquid, and (volume/volume) % for liquid/liquid.

Example 1: Bacillus butanolivorans ( Bacillus butanolivorans ) Isolation of cold shock protein gene from PAMC23377

Bacillus butanolivorans PAMC23377 was obtained from the Korea Polar Research Institute (Incheon, South Korea) and cultured using Marine broth (MB, BD Difco, Franklin Lakes, NJ, USA) medium. A single colony formed on an agar (BD, Franklin Lakes, NJ, USA) plate was inoculated into 50 mL liquid medium and cultured at 10 °C for 5 days. Genomic DNA was isolated from the isolated strain using a genomic DNA mini kit (Invitrogen, Carlsbad, CA, USA). The isolated genomic DNA was used as a template for PCR amplification using the primers shown in Table 1.

Sequence number designation Sequence (5'->3') note 1 Primer_F TTTGGATTCATGGAACAAGGTAAAGTAAAATGGTTTAACG 2 Primer_R TTTGCGGCCGCTTATGCTTTTTGAACGTTAGAAGCTTGA

The annealing temperature used for PCR was 60℃, and the elongation was performed for 30 seconds for 30 cycles. As a result of analyzing the gene sequence of the 201 bp DNA fragment obtained through PCR, a cold shock protein gene having the gene sequence and amino acid sequence in Table 2 was confirmed, and it was confirmed to have high similarity with the cold shock protein genes reported in other Bacillus species ( Bacillus sp.).

Sequence number designation Sequence (5'->3') note 3 CSP_gene ATGGAACAAGGTAAAGTAAAATGGTTTAACGCAGAAAAAGGTTTTGGATTCATCGAACGCGAAAACGGAGACGACGTATTCGTACATTTCTCAGCTATCCAAAGCGAAGGTTTCAAATCTTTAGACGAAGGCCAAGAAGTTACTTTTGAAGTAGAACAAGGTCAACGTGGACCTCAAGCTTCTAACGTTCAAAAAGCATAA 4 CSP_AA MEQGKVKWFNAEKGFGFIERENGDDVFVHFSAIQSEGFKSLDEGQEVTFEVEQGQRGPQASNVQKA

Example 2: Bacillus butanolivorans ( Bacillus butanolivorans ) Cloning of the cold shock protein gene from PAMC23377

pCES208GFP was used as a vector for cloning. DNA fragments amplified by PCR were cut with restriction enzymes BamHI (Promega, USA) and No tI (Promega) to clone. The pCES208GFP vector cut with the same restriction enzyme was also cut with the same restriction enzyme, and then the recombinant vector pCES208_CSP23377 was produced using ligase.

The recombinant vector pCES208_CSP23377 was transformed into Corynebacterium glutamicum ATCC21493 using electroporation. The transformed Corynebacterium glutamicum ATCC21493 was selected in CG medium (50 g glucose, 15 g yeast extract, 15 g _ammonium sulfate, 0.5 g KH2PO4, 0.5 g MgSO4·7H2O, 0.01 g MnSO4· _{H2O and} 0.01 g FeSO4·7H2O, 0.6 g _CaCO3 , pH 7.0 per L) containing _{30 μg /} mL kanamycin antibiotic _{, and} the selected recombinant strains were sequenced by extracting the plasmid.

Example 3: Bacillus butanolivorans ( Bacillus butanolivorans ) Corynebacterium glutamicum overexpressing cold shock protein derived from PAMC23377 ( Corynebacterium glutamicum ) L-arginine production yield confirmation

Recombinant Corynebacterium glutamicum ATCC21493 transformed with pCES208_CSP23377 containing the cold shock protein derived from Bacillus butanolivorans PAMC23377 was cultured in CG medium containing 30 μg/mL kanamycin antibiotic, and the concentration of L-arginine production was measured. The concentration of L-arginine was measured using HPLC (Agilent 1200 series, Agilent Technologies) equipped with a C18 column (Waters) and a fluorescence detector as follows. The sample for HPLC was first prepared by centrifuging the culture broth and diluting the supernatant 1:10. A mixture of buffers A and B (7:3) was used as the mobile phase. Buffer A was prepared by dissolving 71.1 g of Na ₂ HPO ₄ 12H ₂ O, 26.8 g of CH ₃ COONa 3H ₂ O, and 4 mL of acetic acid in 4 L of distilled water, and adding 260 mL of tetrahydrofolate. Buffer B was prepared by mixing methanol, acetonitrile, and water in a ratio of 450:100:450 and filtering the mixture. The column temperature was set to 60 °C and the buffer flow rate was 0.4 mL/min.

As can be seen in Table 3 of the experimental results, the L-arginine production amount of Corynebacterium glutamicum ATCC21493 strain, which is an L-arginine producing strain, was 1.7 g/L, whereas the L-arginine production amount of the recombinant Corynebacterium glutamicum transformed with the pCES208_CSP23377 vector manufactured according to one example was 2.45 g/L, confirming that it produced L-arginine with an improved yield of 0.75 g/L (44.1%) compared to the parent strain.

yield Bacteria Parent strain Corynebacterium glutamicum ATCC21493 Recombinant Corynebacterium glutamicum transformed with pCES208_CSP23377 L-Arginine (g/L) 1.7 2.45

<110> INDUSTRY FOUNDATION OF CHONNAM NATIONAL UNIVERSITY <120> A gene of cold shock protein from Bacillus butanolivorans and production of L-arginine by recombinant Corynebacterium glutamicum with the gene <130> PN210537 <160> 4 <170> KoPatentIn 3.0 <210> 1 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Primer_F <400> 1 tttggattca tggaaacaagg taaagtaaaa tggtttaacg 40 <210> 2 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Primer_R <400> 2 tttgcggccg cttatgcttt ttgaacgtta gaagcttga 39 <210> 3 <211> 201 <212> DNA <213> Artificial Sequence <220> <223> CSP_gene <400> 3 atggaacaag gtaaagtaaa atggtttaac gcagaaaaag gttttggatt catcgaacgc 60 gaaaacggag acgacgtatt cgtacatttc tcagctatcc aaagcgaagg tttcaaatct 120 ttagacgaag gccaagaagt tacttttgaa gtagaacaag gtcaacgtgg acctcaagct 180 tctaacgttc aaaaagcata a 201 <210> 4 <211> 66 <212> PRT <213> Artificial Sequence <220> <223> CSP_AA <400> 4 Met Glu Gln Gly Lys Val Lys Trp Phe Asn Ala Glu Lys Gly Phe Gly 1 5 10 15 Phe Ile Glu Arg Glu Asn Gly Asp Asp Val Phe Val His Phe Ser Ala 20 25 30 Ile Gln Ser Glu Gly Phe Lys Ser Leu Asp Glu Gly Gln Glu Val Thr 35 40 45 Phe Glu Val Glu Gln Gly Gln Arg Gly Pro Gln Ala Ser Asn Val Gln 50 55 60 Lys Ala 65

Claims (11)

A polynucleotide consisting of a base sequence represented by sequence number 3. A recombinant vector comprising a polynucleotide comprising a base sequence represented by sequence number 3. Corynebacterium glutamicum transformed with an enhanced L-arginine productivity, wherein a polynucleotide comprising a base sequence represented by SEQ ID NO: 3 is introduced. delete In the third paragraph, the polynucleotide is Corynebacterium glutamicum with enhanced L-arginine productivity derived from Bacillus butanolivorans . A method for producing Corynebacterium glutamicum with enhanced L-arginine productivity comprising the following steps:
A transformation step of transforming Corynebacterium glutamicum with a recombinant vector containing a polynucleotide consisting of a base sequence represented by SEQ ID NO: 3. delete A method for producing Corynebacterium glutamicum with enhanced L-arginine productivity, wherein the polynucleotide in claim 6 is derived from Bacillus butanolivorans . A method for producing L-arginine comprising the following steps:
A transformation step of transforming Corynebacterium glutamicum with a recombinant vector comprising a polynucleotide consisting of a base sequence represented by SEQ ID NO: 3; and
A cultivation step for culturing the above Corynebacterium glutamicum. delete A method for producing L-arginine in claim 9, wherein the polynucleotide is derived from Bacillus butanolivorans.