WO2016122146A1 - Novel promoter and uses thereof - Google Patents

Novel promoter and uses thereof Download PDF

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Publication number
WO2016122146A1
WO2016122146A1 PCT/KR2016/000444 KR2016000444W WO2016122146A1 WO 2016122146 A1 WO2016122146 A1 WO 2016122146A1 KR 2016000444 W KR2016000444 W KR 2016000444W WO 2016122146 A1 WO2016122146 A1 WO 2016122146A1
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peccg117
gfp
vector
promoter
host cell
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PCT/KR2016/000444
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French (fr)
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WO2016122146A8 (en
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Seung Bin Lee
Hyun Ae Bae
Ji Hye Lee
Young Lyeol Yang
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Cj Cheiljedang Corp.
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Priority to MX2017009697A priority Critical patent/MX2017009697A/en
Priority to DK16743624.5T priority patent/DK3250692T3/en
Priority to AU2016212931A priority patent/AU2016212931B2/en
Priority to ES16743624T priority patent/ES2743753T3/en
Priority to JP2017538588A priority patent/JP6469874B2/en
Priority to CA2975374A priority patent/CA2975374C/en
Priority to CN201680000214.6A priority patent/CN106459977B/en
Priority to MYPI2017001055A priority patent/MY191210A/en
Priority to SG11201705765VA priority patent/SG11201705765VA/en
Priority to EP16743624.5A priority patent/EP3250692B1/en
Priority to RU2017128034A priority patent/RU2692646C2/en
Priority to PL16743624T priority patent/PL3250692T3/en
Priority to US15/547,398 priority patent/US10273491B2/en
Priority to BR112017016301-2A priority patent/BR112017016301B1/en
Priority to UAA201707693A priority patent/UA116753C2/en
Application filed by Cj Cheiljedang Corp. filed Critical Cj Cheiljedang Corp.
Publication of WO2016122146A1 publication Critical patent/WO2016122146A1/en
Publication of WO2016122146A8 publication Critical patent/WO2016122146A8/en
Priority to PH12017501365A priority patent/PH12017501365A1/en

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    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/77—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Corynebacterium; for Brevibacterium
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/34—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Corynebacterium (G)
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
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    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine
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    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/10—Citrulline; Arginine; Ornithine
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    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/101—Plasmid DNA for bacteria

Definitions

  • One or more exemplary embodiments relate to a novel promoter and a method of producing a target product using the same.
  • target products e.g., L-amino acids, organic acids, or nucleic acid materials
  • target products e.g., L-amino acids, organic acids, or nucleic acid materials
  • a modification of an expression regulatory sequence may be performed.
  • Such a modification of the expression regulatory sequence may include, for example, a substitution a native promoter for a strong promoter, a modification of a native promoter, or a modification of a Shine-Dalgarno (SD) sequence.
  • SD Shine-Dalgarno
  • strong promoters known in the art are limited, and may be expressed only in a limited microorganism. In some cases, the strong promoters may fail to exhibit strong expression effects at various intensity levels as desired.
  • a tac promoter derived from Escherichia coli is widely known as a strong promoter in the art.
  • a new promoter has been developed by modification of a native promoter (see Gene, 102, 93-98, 1991; Microbiology, 142, 1297-1309, 1996).
  • a promoter derived from Corynebacterium ammoniagenesis had about 10% higher activity than that of the tac promoter derived from E. coli (see Biotechnol. Lett. 25, 1311-1316, 2003).
  • promoters of Pcj1 to Pcj7 with various intensity levels have been developed and had strong promoter activity that is at least 10 times as large as that of the tac promoter (see KR 10-0620092).
  • a promoter derived from Brevibacterium flavum MJ-233 (FERM BP-1497) was reported to have a stronger activity than that of the tac promoter, but had a difficulty in its expression in a microorganism other than a microorganism belonging to the genus Brevibacterium (see US 5,593,781).
  • a novel promoter synthesized according to the present inventive concept is found to be expressed in a variety of different microorganisms and to exhibit much stronger expression effects than those of the existing promoters known in the art, thereby completing the present inventive concept.
  • One or more exemplary embodiments include, a novel promoter, an expression regulatory sequence comprising the novel promoter, a vector including the novel promoter, a host cell including the vector, and a method of producing a target product using the host cell.
  • a promoter comprising a nucleotide sequence represented by SEQ ID NO: 1.
  • the promoter is named "an o2 promoter (hereinafter, referred to as "Po2”)”.
  • promoter refers to a DNA region to which an RNA polymerase is combined to allow initiation of transcription of a gene that is operatively linked to the promoter, and may be positioned at a 5'-end of an initiation site for transcription of an mRNA.
  • a polynucleotide having promoter activity as used herein may be modified to some extents according to recent studies of several techniques, such as a directed evolution technique or a site-directed mutagenesis technique.
  • a promoter having a homology of 70% or more, 80% or more, 90% or more, or 95% or more to the nucleotide sequence represented by SEQ ID NO: 1 is also included within the scope of the present inventive concept.
  • homology refers to the degree (represented in percentage) of sequence identity between polynucleotide sequences.
  • a homology of a sequence identical to a given polynucleotide sequence or a sequence having similar activity with that of a given polynucleotide sequence is represented in terms of "% homology".
  • the homology of polynucleotide sequences may be determined by using standard software, e.g., BLAST 2.0, to calculate parameters such as score, identity, and similarity.
  • the homology of polynucleotide sequences may be identified by comparing sequences according to a hybridization method performed under defined stringent conditions. The defined and appropriate conditions for the hybridization method may be determined in consideration of methods that are well known to one of person skilled in the art (see Infra in Sambrook et al., 1989).
  • an expression regulatory sequence which controls expression of a target gene, comprising the nucleotide sequence represented by SEQ ID NO: 1.
  • expression regulatory sequence refers to a DNA sequence used for expression of a coding sequence that is operatively linked to the DNA sequence in a host organism.
  • Such an expression regulatory sequence may include, a promoter required for initiating transcription, an operator sequence for regulating transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence for regulating termination of transcription and translation.
  • an expression regulatory sequence suitable for prokaryotes may include a promoter, an operator sequence, and a ribosome binding site, but is not limited thereto.
  • An expression regulatory sequence includes Po2 of the present inventive concept may constitute, if necessary, one of person skilled in the art may constitute an expression regulatory sequence as described above.
  • a vector including the expression regulatory sequence and a target gene operatively linked to the expression regulatory sequence.
  • operatively linked refers to a linking between an expression regulatory sequence which controls a target gene (e.g., a promoter) and other nucleotide sequences.
  • the expression regulatory sequence may be able to control transcription and/or translation of the other nucleotide sequences.
  • vector refers to a DNA product including base sequences of a polynucleotide to encode a target protein that is operatively linked to an appropriate expression regulatory sequence to express the target protein.
  • a plurality of nucleotide sequences may be bonded to or recombined with the vector so that a DNA sequence of a selected gene with an appropriate 3'-untranslated sequence, and a promoter may be introduced into a cell.
  • the vector may be used for transformation in an appropriate host cell, and then, may be replicated regardless of the genome of the host cell. Alternatively, the vector may be integrated into the genome of the host cell.
  • the vector may include the promoter or a variant thereof and the target gene, and furthermore, may include a replication origin, a promoter regulatory site, a ribosome binding site, a transcription termination site, a selective marker, or a combination thereof.
  • the vector used in the present inventive concept is not particularly limited so long as it can be replicated in a host cell, and any vector available in the art may be used.
  • Examples of the vector typically used in the art include a natural or recombinant plasmid vector, a cosmid vector, a viral vector, and a bacteriophage vector.
  • Examples of the bacteriophage and cosmid vectors include pWE15, M13, ⁇ MBL3, ⁇ MBL4, ⁇ IXII, ⁇ ASHII, ⁇ APII, ⁇ t10, ⁇ t11, Charon4A, and Charon21A, and examples of the plasmid vectors include pBR-based, pUC-based, pBluescriptII-based, pGEM-based, pTZ-based, pCL-based, and pET-based vectors, but the vectors are not limited thereto.
  • the target gene refers to a gene encoding a product to be expression in an excess amount.
  • the target gene may be a gene involved in the production of a product selected from the group consisting of amino acids (e.g., L-amino acid), organic acids, and a combination thereof.
  • the target gene may be a gene encoding an enzyme involved in biosynthesis of amino acids, a gene encoding an enzyme involved in biosynthesis of organic acids, or a gene encoding a protein involved in exporting a target product, but is not limited thereto.
  • a host cell including a vector, wherein the vector includes an expression regulatory sequence which controls a target gene and includes a promoter having a nucleotide sequence represented by SEQ ID NO: 1 and a target gene that is operatively linked to the expression regulatory sequence.
  • the host cell is not particularly limited so long as a microorganism used as the host cell is capable of introducing the vector including the expression regulatory sequence which controls a target gene and includes the promoter having the nucleotide sequence represented by SEQ ID NO: 1 and the target gene that is operatively linked to the expression regulatory sequence.
  • the host cell may be both a prokaryotic cell and a eukaryotic cell, but in an exemplary embodiment, may be a prokaryotic cell.
  • the host cell may include a strain of a microorganism belonging to the genus Escherichia , the genus Erwinia , the genus Serratia , the genus Providencia , the genus Corynebacterium , and the genus Brevibacterium .
  • the host cell may be a strain of a microorganism belonging to the genus Corynebacterium or the genes Escherichia .
  • the host cell may be a strain of a microorganism belonging to Escherichia coli , Corynebacterium glutamicum, Corynebacterium thermoaminogenes , Brevibacterium flavum , or Brevibacterium lactofermentum .
  • the introduction of the vector may be performed by, as described in the art, selecting a suitable technique according to the host cell.
  • the introduction of the vector may be performed by, for example, electroporation, heat-shock, calcium phosphate (CaPO 4 ) precipitation, calcium chloride (CaCl 2 ) precipitation, microinjection, polyethylene glycol (PEG), a DEAE-dextran method, a cationic liposome method, a lithium acetate-DMSO method, or a combination thereof, but is not limited thereto.
  • a method of producing a target product including: culturing a host cell in a medium, wherein the host cell comprise a vector that includes an expression regulatory sequence comprising a promoter having a nucleotide sequence represented by SEQ ID NO: 1 and a target gene that is operatively linked to the expression regulatory sequence; and recovering the target product from the host cell or the medium including the cultured host cell.
  • the target product may be selected from the group consisting of amino acids (e.g., L-amino acid), organic acids, and a combination thereof.
  • amino acids e.g., L-amino acid
  • organic acids e.g., organic acids, and a combination thereof.
  • amino acid or L-amino acid as used herein generally refers to a basic unit of a protein constituting a living body, in which an amino acid group and a carboxyl group are linked to the same carbon atom.
  • the amino acid may be selected from the group consisting of glycine, alanine, valine, leucine, isoleucine, threonine, serine, cysteine, cystine, methionine, aspartic acid, asparagine, glutamic acid, diiodotyrosine, lysine, arginine, histidine, phenylalanine, tyrosine, tryptophan, proline, oxyproline, and a combination thereof, but is not limited thereto.
  • organic acid refers to an organic compound having acidity, and for example, may include an organic compound having a carboxyl group and a sulfonic group.
  • the organic acid may include, for example, lactic acid, acetic acid, succinic acid, butyric acid, palmitic acid, oxalic acid, tartaric acid, propionic acid, hexenoic acid, capric acid, caprylic acid, valeric acid, or citric acid, but is not limited thereto.
  • the culture of the host cell may be performed according to typical methods known in the art.
  • the medium used for the culturing of the host cell may include, as a source of sugar, sugar and carbohydrates, such as glucose, saccharose, lactose, fructose, maltose, starch, and cellulose; oils and fats, such as soybean oil, sunflower oil, castor oil, and coconut oil; fatty acids, such as palmitic acid, stearic acid, and linoleic acid; alcohols, such as glycerol and ethanol; and organic acids, such as acetic acid, individually or as a mixture, but is not limited thereto.
  • the medium used for the culturing of the host cell may include, as a source of nitrogen, peptones, yeast extract, meat extract, malt extract, maize steep liquor, soybean meal, and urea, or inorganic compounds, such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, or ammonium nitrate, individually or as a mixture, but is not limited thereto.
  • the medium used for the culturing of the host cell may include, as a source of phosphorus, potassium dihydrogen phosphate or dipotassium hydrogen phosphate, or its corresponding sodium-containing salts, but is not limited thereto.
  • the medium used for the culturing of the host cell may include, salts of metals, such as magnesium sulfate or iron sulfate, which are needed for growth, but the salts of metals are not limited thereto.
  • essential growth substances such as amino acids and vitamins, or suitable precursors may be added to the culture medium. Such materials may be added to the culture medium during the culturing of the host cell in an appropriate manner, and for example, may be added in a batch or continuous manner.
  • compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid, may be added to the culture medium in a suitable manner to regulate the pH of the culture medium.
  • an anti-forming agent such as fatty acid polyglycol ester, may be used to prevent the production of foams.
  • oxygen or oxygen-containing gas e.g., air
  • a temperature of the culture medium may be in a range of about 20°C to about 45°C, for example, about 25°C to about 40°C.
  • the culturing of the host cell may be continued until a desired amount of a target product is achieved, and in this regard, the culturing of the host cell may be continues for about 10 hours to about 160 hours.
  • the recovering of the target product from the host cell or the culture medium including the cultured host cell may be performed (i.e., separated or recovered) through an appropriate reaction known in the art.
  • an appropriate reaction may be made according to a treatment using protein precipitants (i.e., a saltingout method), centrifugation, extract, sonication, ultrafiltration, dialysis, various chromatography techniques, such as molecular sieve chromatography (i.e., gel filtration), adsorption chromatography, ion-exchange chromatography, affinity chromatography, and a combination thereof, but is not limited thereto.
  • the produced target product may be collected, recovered, or separated from the host cell or the medium including the cultured host cell.
  • Example 1 Preparation of a recombinant vector comprising a novel promoter and a transformed strain using the same
  • the Po2 was used to enhance the expression of biosynthetic gene for arginine or valine.
  • PCR was performed by using the synthesized Po2 as a template and a primer set of SEQ ID NO: 2 and SEQ ID NO: 3 including Kpn I/EcoR V restriction sites.
  • the PCR was performed according to cycles of denaturation at a temperature of 94°C for 5 minutes, denaturation at a temperature of 94°C for 30 seconds, annealing at a temperature of 60°C for 30 seconds, and polymerization at a temperature of 72°C for 30 seconds, wherein the cycles were performed 30 times.
  • polymerization was performed again on the strains at a temperature of 72°C for 7 minutes, thereby consequently obtaining Po2 having a length of about 100 bp.
  • PCR was performed by using a pGFPuv vector (manufactured by Clontech, USA) as a template and a primer set of SEQ ID NO: 4 and SEQ ID NO: 5 including PstI/EcoR V restriction sites. PCR was performed according to cycles of denaturation at a temperature of 94°C for 5 minutes, denaturation at a temperature of 94°C for 30 seconds, annealing at a temperature of 55°C for 30 seconds, and polymerization at a temperature of 72°C for 1 minute, wherein the cycles were performed 30 times. Afterwards, polymerization was performed again at a temperature of 72°C for 7 minutes, thereby consequently obtaining SEQ ID NO: 6 including the ORF of the GFP gene.
  • the Po2 was treated with restriction enzymes PstI and EcoR V and the ORF of the GFP gene was treated with restriction enzymes Kpn I and EcoR V, at PstI and Kpn I sites of a shuttle vector pECCG117 (see Biotechnology letters vol 13, No. 10, p. 721-726 (1991)) that can be expressed in E. coli and Corynebacteria. Then, the treated Po2 and ORF of the GFP gene were operatively linked to each other by using a DNA conjugating enzyme, thereby manufacturing a recombinant vector in which the Po2 and the GFP gene were linked to each other.
  • the recombinant vector was named pECCG117-Po2-gfp.
  • Corynebacterium glutamicum ATCC13032 was transformed with each of a vector pECCG117 and the recombinant vector pECCG117-Po2-gfp by an electric pulse method, and then, transformed strains were obtained from a selective medium containing 25 mg/L of kanamycin.
  • the obtained strains that were transformed with the vector pECCG117 and the recombinant vector pECCG117-Po2-gfp were each named ATCC13032/pECCG117 and ATCC13032/pECCG117-Po2-gfp.
  • E. coli DH5 ⁇ was transformed with the recombinant vector pECCG117-Po2-gfp by a heat shock method, and then, transformed strains were obtained from a Luria-Bertani (LB) agar medium containing 25 mg/L of kanamycin.
  • the obtained strains were named DH5 ⁇ /pECCG117-Po2-gfp and assigned as a deposit designation "CA01-2290".
  • CA01-2290 was deposited at the Korean Culture Center of Microorganisms (KCCM) on October 23, 2014, under the accession number of KCCM11591P, in accordance with the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.
  • a main biosynthetic gene e.g., an argJ gene (Ncgl1341, SEQ ID NO: 7) encoding bifunctional ornithine acetyltransferase/N-acetylglutamate synthase
  • the recombinant vector pECCG117-Po2-gfp was used to prepare a vector pECCG117-Po2-argJ.
  • PCR was performed on a strain by using chromosomes of Corynebacterium glutamicuum ATCC13869 as a template and a primer set of SEQ ID NO: 8 and SEQ ID NO: 9, thereby ensuring DNA fragments including the argJ genes.
  • the PCR was performed according to cycles of denaturation at a temperature of 94°C for 1 minute, annealing at a temperature of 58°C for 30 seconds, and polymerization at a temperature of 72°C for 2 minutes by using a Pfu polymerase, wherein the cycles were performed 30 times. Consequently, a fragment having a length of about 1,201 bp and including EcoRV and 3' PstI restriction enzyme sites on a 5' end was amplified.
  • the amplified fragments generated by the PCR were purified, mixed with the vector pECCG117-Po2-gfp to which EcoRV and PstI restriction enzymes were treated, and then, joined together using the In-fusion Cloning Kit, thereby preparing a recombinant vector, which was named pECCG117-Po2-argJ.
  • Corynebacterium glutamicum KCCM10741P which is an arginine producing strain, was transformed with the recombinant vector pECCG117-Po2-argJ by an electric pulse method (see KR 10-0791659), and then, transformed strains were obtained from a selective medium containing 25 mg/L of kanamycin. The obtained strain was named KCCM10741P/pECCG117-Po2-argJ.
  • a main biosynthetic gene e.g., an ilvE gene (Ncgl2123, SEQ ID NO: 10) encoding a branched-chain amino acid aminotransferase, for enhanced production of valine is expressed by the Po2
  • the recombinant vector pECCG117-Po2-gfp was used to prepare a vector pECCG117-Po2-ilvE.
  • PCR was performed on a strain by using chromosomes of Corynebacterium glutamicuum ATCC14067 as a template and a primer set of SEQ ID NO: 11 and SEQ ID NO: 12, thereby ensuring DNA fragments including the ilvE genes.
  • the PCR performed according to cycles of denaturation at a temperature of 94°C for 1 minute, annealing at a temperature of 58°C for 30 seconds, and polymerization at a temperature of 72°C for 2 minutes by using a Pfu polymerase, wherein the cycles were performed 30 times. Consequently, a fragment having a length of about 1,201 bp and including EcoRV and 3' PstI restriction enzyme sites on a 5' end was amplified.
  • the amplified fragments generated by the PCR were purified, mixed with the vector pECCG117-Po2-gfp to which EcoRV and PstI restriction enzymes were treated, and then, joined together using the In-fusion Cloning Kit, thereby preparing a recombinant vector, which was named pECCG117-Po2-ilvE.
  • Corynebacterium glutamicum KCCM111201P which is a valine producing strain, was transformed with the recombinant vector pECCG117-Po2-ilvE by an electric pulse method (see KR 10-1117022), and then, transformed strains were obtained from a selective medium containing 25 mg/L of kanamycin.
  • the obtained strains were named KCCM11201P/pECCG117-Po2-ilvE.
  • Comparative Example 1 Preparation of a recombinant vector including a control promoter and a transformed strain using the vector
  • the GFP gene was used to operatively link a known strong promoter (e.g., Ptrc, Pcj1, Pcj4, or Pcj7 (see KR 10-0620092)) or a wild-type promoter (e.g., aceEP (WT)) to the ORF of the GFP gene to prepare a recombinant vector. Then, each of strains of Corynebacteria and E. coli was transformed with the recombinant vector, thereby preparing each of the transformed strains of Corynebacteria and E. coli.
  • a known strong promoter e.g., Ptrc, Pcj1, Pcj4, or Pcj7 (see KR 10-0620092
  • WT wild-type promoter
  • a transformed strain including a main biosynthetic gene for enhanced production of arginine or a transformed strain including a main biosynthetic gene for enhanced production of valine were each prepared.
  • aceEP(WT) Base sequences of aceEP(WT) were ensured based on the U.S. National Institute of Health (NIH Genbank), and PCR was performed thereon by using chromosomes of wild-type Corynebacterium glutamicum ATCC13032 as a template and a primer set of SEQ ID NO: 13 and SEQ ID NO: 14 including Kpn I/EcoR V restriction sites. The PCR was performed in the same manner as in (1.1) of Example 1, thereby preparing a recombinant vector named pECCG117-aceEP(WT)-gfp.
  • Strains of E. coli were each of the prepared vector pECCG117-Ptrc-gfp and vectors pECCG117-Pcj1-gfp and pECCG117-Pcj4-gfp, in the same manner as in (1.1) of Example 1, thereby preparing transformed strains that were each named DH5 ⁇ /pECCG117-Ptrc-gfp, DH5 ⁇ /pECCG117-Pcj1-gfp, and DH5 ⁇ /pECCG117-Pcj4-gfp.
  • the vector pECCG117-Pcj7-gfp was used to prepare a vector named pECCG117-Pcj7-argJ in the same manner as in (1.2.1) of Example 1, except that PCR was performed by using a primer set of SEQ ID NO: 9 and SEQ ID NO: 17.
  • a transformed strain was prepared by using the vector pECCG117-Pcj7-argJ in the same manner as in (1.2.2) of Example 1, and was named KCCM10741P/pECCG117-Pcj7-argJ.
  • the vector pECCG117-Pcj7-gfp vector was used to prepare a vector named pECCG117-Pcj7-ilvE in the same manner as in (1.3.1) of Example 1, except that PCR was performed by using a primer set of SEQ ID NO: 12 and SEQ ID NO: 18.
  • a transformed strain was prepared by using the vector pECCG117-Pcj7-ilvE in the same manner as in (1.3.2) of Example 1, and was named KCCM11201P/pECCG117-Pcj7-ilvE.
  • Example 2 Confirmation of a Po2 activity for inducing a target gene expression
  • the measurement of green fluorescence protein (GFP) activity of the transformed strain named ATCC13032/pECCG117-Po2-gfp of (1.1.2) of Example 1 was compared with the measurement of GFP activity of the transformed strains each named ATCC13032/pECCG117 of (1.1.2) of Example 1 and ATCC13032/ pECCG117-aceEP(WT)-gfp, ATCC13032/pECCG117-Pcj1-gfp, ATCC13032/pECCG117-Pcj4-gfp, and ATCC13032/pECCG117-Pcj7-gfp of (1) of Comparative Example 1.
  • GFP green fluorescence protein
  • the cells were disrupted for 6 minutes by using a bead beater after glass beads were added at 1.25 g/1.5 ml of the suspension.
  • a supernatant containing a cell extract was collected by centrifugation (at a speed of about 15,000 rpm for about 20 minutes), and then, quantitatively measured in terms of a protein concentration therein according to the Bradford method (see Bradford, M.M 1976. Anal. Biochem. 72:248-254).
  • the strain named ATCC13032/pECCG117-Po2-gfp exhibited fluorescence intensity at a level that is much higher than that of the strains each named ATCC13032/pECCG117-Pcj1-gfp, ATCC13032/ pECCG117-Pcj4-gfp, and ATCC13032/pECCG117-Pcj7-gfp using known strong promoters (e.g., Pcj1, Pcj4, and Pcj7). Consequently, it was confirmed that the Po2 served as a strong promoter to express a target gene.
  • known strong promoters e.g., Pcj1, Pcj4, and Pcj7
  • the cells were collected by centrifugation (at a speed of about 5,000 rpm for about 15 minutes), washed twice with a 0.1% Tris.HCl (pH 8.0) buffer solution, suspended in the same buffer solution, disrupted by sonication, and then, subjected to centrifugation (at a speed of about 15,000 rpm for about 20 minutes) to obtain a supernatant containing a cell extract.
  • centrifugation at a speed of about 5,000 rpm for about 15 minutes
  • a 0.1% Tris.HCl (pH 8.0) buffer solution suspended in the same buffer solution, disrupted by sonication, and then, subjected to centrifugation (at a speed of about 15,000 rpm for about 20 minutes) to obtain a supernatant containing a cell extract.
  • the supernatant was quantitatively measured in terms of a protein concentration therein according to the Bradford method. Then, the same amount of the cell extract was irradiated with light at an excitation wavelength of 488 nm using the method of Laure Gory or the like, and light at an emission wavelength of 511 nm was measured by using the LS-50B spectrophotometer (Perkin-Elmer), thereby determining the expression of the GFP gene, The results of measuring GFP activity in each of the strains are shown in Table2.
  • the strain named KCCM10741P/pECCG117-Po2-argJ of (1.2.2) of Example 1 which was used as the strain for enhanced production of the argJ gene, was compared with the non-transformed strain named KCCM10741P (having no transformed arginine producibility) and the strain named KCCM10741P/pECCG117-Pcj7-argJ of (3.1) of Comparative Example 1 in terms of the arginine producibility.
  • glucose 6% ammonium sulfate 3%, monopotassium phosphate 0.1%, magnesium sulfate heptahydrate 0.2%, corn steep liquor (CSL) 1.5%, NaCl 1%, yeast extract 0.5%, biotin 100 ⁇ g/L, and pH7.2
  • the strain named KCCM11201P/pECCG117-Po2-ilvE of (1.3.2) of Example 1 which was used as the strain for enhanced production of the ilvE gene, was compared with the non-transformed strain named KCCM11201P (having no transformed L-valine producibility) and the strain named KCCM11201P/pECCG117-Pcj7-ilvE of (3.2) of Comparative Example 1 in terms of L-valine producibility.
  • glucose 5% ammonium sulfate 2%, monopotassium phosphate 0.1%, magnesium sulfate heptahydrate 0.05%, CSL 2.0%, biotin 200 ⁇ g/L, and pH 7.2
  • valine Production of valine in KCCM11201P Strain Concentration of valine (g/L) KCCM11201P 2.8 KCCM11201P/pECCG117-Pcj7-ilvE 3.3 KCCM11201P/pECCG117- Po2-ilvE 3.7
  • the Po2 resulted in improved production of valine in Corynebacterium glutamicum in which the expression of the ilvE gene was enhanced.
  • the valine production in the strain named KCCM11201P/pECCG117-Po2-ilvE was significantly increased by about 32% compared to that in the control strain, and was increased by about 17% compared to that in the strain named KCCM10741P/pECCG117-Pcj7-ilvE. Consequently, it was confirmed that the Po2 influenced the enhanced expression of the ilvE gene.
  • a novel promoter may have various activities according to a microorganism used to induce expression of a target gene.
  • the novel promoter may be used in a case where activity of a target gene needs to be controlled during the production of the target product, resulting in efficient production of the target product.

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Abstract

Provided are a novel promoter and a method of producing a target product using the same, wherein the novel promoter is synthesized to be expressed in a variety of different microorganism and to exhibit much stronger expression effects.

Description

NOVEL PROMOTER AND USES THEREOF
CROSS-REFERENCE TO RELATED APPLICATION
This application claims the benefit of Korean Patent Application No. 10-2015-0014587, filed on January 29, 2015, in the Korean Intellectual Property Office, the disclosure of which is incorporated herein in its entirety by reference.
One or more exemplary embodiments relate to a novel promoter and a method of producing a target product using the same.
To produce target products, e.g., L-amino acids, organic acids, or nucleic acid materials, with high yield by using microorganisms, there is a need to selectively control the expression of genes related to a number of metabolic processes in the microorganisms. In particular, there is a need to enhance the expression of target genes involved in a biosynthetic pathway of the target products, and for example, a modification of an expression regulatory sequence may be performed. Such a modification of the expression regulatory sequence may include, for example, a substitution a native promoter for a strong promoter, a modification of a native promoter, or a modification of a Shine-Dalgarno (SD) sequence. The substitution a native promoter for a strong promoter has been used the most, and in this regard, it is considered essential to develop a useful promoter.
However, strong promoters known in the art are limited, and may be expressed only in a limited microorganism. In some cases, the strong promoters may fail to exhibit strong expression effects at various intensity levels as desired.
A tac promoter derived from Escherichia coli is widely known as a strong promoter in the art. In the case of the genus Corynebacterium, a new promoter has been developed by modification of a native promoter (see Gene, 102, 93-98, 1991; Microbiology, 142, 1297-1309, 1996). For example, it was reported that a promoter derived from Corynebacterium ammoniagenesis had about 10% higher activity than that of the tac promoter derived from E. coli (see Biotechnol. Lett. 25, 1311-1316, 2003). In addition, as a strong promoter derived from Corynebacterium ammoniagenesis, promoters of Pcj1 to Pcj7 with various intensity levels have been developed and had strong promoter activity that is at least 10 times as large as that of the tac promoter (see KR 10-0620092). In addition, a promoter derived from Brevibacterium flavum MJ-233 (FERM BP-1497) was reported to have a stronger activity than that of the tac promoter, but had a difficulty in its expression in a microorganism other than a microorganism belonging to the genus Brevibacterium (see US 5,593,781).
In this regard, when the inventors of the present inventive concept explore a region including a promoter sequence that can have various intensity levels in a variety of different microorganisms, a novel promoter synthesized according to the present inventive concept is found to be expressed in a variety of different microorganisms and to exhibit much stronger expression effects than those of the existing promoters known in the art, thereby completing the present inventive concept.
One or more exemplary embodiments include, a novel promoter, an expression regulatory sequence comprising the novel promoter, a vector including the novel promoter, a host cell including the vector, and a method of producing a target product using the host cell.
According to one or more exemplary embodiments, there is provided a promoter comprising a nucleotide sequence represented by SEQ ID NO: 1. In the present inventive concept, the promoter is named "an o2 promoter (hereinafter, referred to as "Po2")".
The term "promoter" as used herein refers to a DNA region to which an RNA polymerase is combined to allow initiation of transcription of a gene that is operatively linked to the promoter, and may be positioned at a 5'-end of an initiation site for transcription of an mRNA.
A polynucleotide having promoter activity as used herein may be modified to some extents according to recent studies of several techniques, such as a directed evolution technique or a site-directed mutagenesis technique. For example, a promoter having a homology of 70% or more, 80% or more, 90% or more, or 95% or more to the nucleotide sequence represented by SEQ ID NO: 1 is also included within the scope of the present inventive concept.
The term "homology" as used herein refers to the degree (represented in percentage) of sequence identity between polynucleotide sequences. In the present specification, a homology of a sequence identical to a given polynucleotide sequence or a sequence having similar activity with that of a given polynucleotide sequence is represented in terms of "% homology". For example, the homology of polynucleotide sequences may be determined by using standard software, e.g., BLAST 2.0, to calculate parameters such as score, identity, and similarity. Alternatively, the homology of polynucleotide sequences may be identified by comparing sequences according to a hybridization method performed under defined stringent conditions. The defined and appropriate conditions for the hybridization method may be determined in consideration of methods that are well known to one of person skilled in the art (see Infra in Sambrook et al., 1989).
According to one or more exemplary embodiments, there is provided an expression regulatory sequence, which controls expression of a target gene, comprising the nucleotide sequence represented by SEQ ID NO: 1.
The term "expression regulatory sequence" as used herein refers to a DNA sequence used for expression of a coding sequence that is operatively linked to the DNA sequence in a host organism. Such an expression regulatory sequence may include, a promoter required for initiating transcription, an operator sequence for regulating transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence for regulating termination of transcription and translation. For example, an expression regulatory sequence suitable for prokaryotes may include a promoter, an operator sequence, and a ribosome binding site, but is not limited thereto. An expression regulatory sequence includes Po2 of the present inventive concept may constitute, if necessary, one of person skilled in the art may constitute an expression regulatory sequence as described above.
According to one or more exemplary embodiments, there is provided a vector including the expression regulatory sequence and a target gene operatively linked to the expression regulatory sequence.
The term "operatively linked" as used herein refers to a linking between an expression regulatory sequence which controls a target gene (e.g., a promoter) and other nucleotide sequences. In this regard, the expression regulatory sequence may be able to control transcription and/or translation of the other nucleotide sequences.
The term "vector" as used herein refers to a DNA product including base sequences of a polynucleotide to encode a target protein that is operatively linked to an appropriate expression regulatory sequence to express the target protein. In addition, a plurality of nucleotide sequences may be bonded to or recombined with the vector so that a DNA sequence of a selected gene with an appropriate 3'-untranslated sequence, and a promoter may be introduced into a cell. The vector may be used for transformation in an appropriate host cell, and then, may be replicated regardless of the genome of the host cell. Alternatively, the vector may be integrated into the genome of the host cell. In addition, the vector may include the promoter or a variant thereof and the target gene, and furthermore, may include a replication origin, a promoter regulatory site, a ribosome binding site, a transcription termination site, a selective marker, or a combination thereof.
The vector used in the present inventive concept is not particularly limited so long as it can be replicated in a host cell, and any vector available in the art may be used. Examples of the vector typically used in the art include a natural or recombinant plasmid vector, a cosmid vector, a viral vector, and a bacteriophage vector. Examples of the bacteriophage and cosmid vectors include pWE15, M13, λMBL3, λMBL4, λIXII, λASHII, λAPII, λt10, λt11, Charon4A, and Charon21A, and examples of the plasmid vectors include pBR-based, pUC-based, pBluescriptII-based, pGEM-based, pTZ-based, pCL-based, and pET-based vectors, but the vectors are not limited thereto.
The target gene refers to a gene encoding a product to be expression in an excess amount. For example, the target gene may be a gene involved in the production of a product selected from the group consisting of amino acids (e.g., L-amino acid), organic acids, and a combination thereof. In detail, the target gene may be a gene encoding an enzyme involved in biosynthesis of amino acids, a gene encoding an enzyme involved in biosynthesis of organic acids, or a gene encoding a protein involved in exporting a target product, but is not limited thereto.
According to one or more exemplary embodiments, there is provided a host cell including a vector, wherein the vector includes an expression regulatory sequence which controls a target gene and includes a promoter having a nucleotide sequence represented by SEQ ID NO: 1 and a target gene that is operatively linked to the expression regulatory sequence.
The host cell is not particularly limited so long as a microorganism used as the host cell is capable of introducing the vector including the expression regulatory sequence which controls a target gene and includes the promoter having the nucleotide sequence represented by SEQ ID NO: 1 and the target gene that is operatively linked to the expression regulatory sequence. The host cell may be both a prokaryotic cell and a eukaryotic cell, but in an exemplary embodiment, may be a prokaryotic cell. For example, the host cell may include a strain of a microorganism belonging to the genus Escherichia, the genus Erwinia, the genus Serratia, the genus Providencia, the genus Corynebacterium, and the genus Brevibacterium. For example, the host cell may be a strain of a microorganism belonging to the genus Corynebacterium or the genes Escherichia. For example, the host cell may be a strain of a microorganism belonging to Escherichia coli, Corynebacterium glutamicum, Corynebacterium thermoaminogenes, Brevibacterium flavum, or Brevibacterium lactofermentum.
The introduction of the vector may be performed by, as described in the art, selecting a suitable technique according to the host cell. The introduction of the vector may be performed by, for example, electroporation, heat-shock, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG), a DEAE-dextran method, a cationic liposome method, a lithium acetate-DMSO method, or a combination thereof, but is not limited thereto.
According to one or more exemplary embodiments, there is provided a method of producing a target product, the method including: culturing a host cell in a medium, wherein the host cell comprise a vector that includes an expression regulatory sequence comprising a promoter having a nucleotide sequence represented by SEQ ID NO: 1 and a target gene that is operatively linked to the expression regulatory sequence; and recovering the target product from the host cell or the medium including the cultured host cell.
The target product may be selected from the group consisting of amino acids (e.g., L-amino acid), organic acids, and a combination thereof. The term "amino acid" or "L-amino acid" as used herein generally refers to a basic unit of a protein constituting a living body, in which an amino acid group and a carboxyl group are linked to the same carbon atom. The amino acid may be selected from the group consisting of glycine, alanine, valine, leucine, isoleucine, threonine, serine, cysteine, cystine, methionine, aspartic acid, asparagine, glutamic acid, diiodotyrosine, lysine, arginine, histidine, phenylalanine, tyrosine, tryptophan, proline, oxyproline, and a combination thereof, but is not limited thereto. The term "organic acid" as used herein refers to an organic compound having acidity, and for example, may include an organic compound having a carboxyl group and a sulfonic group. The organic acid may include, for example, lactic acid, acetic acid, succinic acid, butyric acid, palmitic acid, oxalic acid, tartaric acid, propionic acid, hexenoic acid, capric acid, caprylic acid, valeric acid, or citric acid, but is not limited thereto.
The culture of the host cell may be performed according to typical methods known in the art. The medium used for the culturing of the host cell may include, as a source of sugar, sugar and carbohydrates, such as glucose, saccharose, lactose, fructose, maltose, starch, and cellulose; oils and fats, such as soybean oil, sunflower oil, castor oil, and coconut oil; fatty acids, such as palmitic acid, stearic acid, and linoleic acid; alcohols, such as glycerol and ethanol; and organic acids, such as acetic acid, individually or as a mixture, but is not limited thereto. The medium used for the culturing of the host cell may include, as a source of nitrogen, peptones, yeast extract, meat extract, malt extract, maize steep liquor, soybean meal, and urea, or inorganic compounds, such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, or ammonium nitrate, individually or as a mixture, but is not limited thereto. The medium used for the culturing of the host cell may include, as a source of phosphorus, potassium dihydrogen phosphate or dipotassium hydrogen phosphate, or its corresponding sodium-containing salts, but is not limited thereto. The medium used for the culturing of the host cell may include, salts of metals, such as magnesium sulfate or iron sulfate, which are needed for growth, but the salts of metals are not limited thereto. In addition, during the culturing of the host cell, essential growth substances, such as amino acids and vitamins, or suitable precursors may be added to the culture medium. Such materials may be added to the culture medium during the culturing of the host cell in an appropriate manner, and for example, may be added in a batch or continuous manner.
Furthermore, during the culturing of the host cell, compounds, such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid, may be added to the culture medium in a suitable manner to regulate the pH of the culture medium. In addition, during the culturing of the host cell, an anti-forming agent, such as fatty acid polyglycol ester, may be used to prevent the production of foams. To maintain aerobic conditions of the culture medium, oxygen or oxygen-containing gas (e.g., air) may be introduced into the culture medium. Here, a temperature of the culture medium may be in a range of about 20℃ to about 45℃, for example, about 25℃ to about 40℃. In addition, the culturing of the host cell may be continued until a desired amount of a target product is achieved, and in this regard, the culturing of the host cell may be continues for about 10 hours to about 160 hours.
The recovering of the target product from the host cell or the culture medium including the cultured host cell may be performed (i.e., separated or recovered) through an appropriate reaction known in the art. For example, an appropriate reaction may be made according to a treatment using protein precipitants (i.e., a saltingout method), centrifugation, extract, sonication, ultrafiltration, dialysis, various chromatography techniques, such as molecular sieve chromatography (i.e., gel filtration), adsorption chromatography, ion-exchange chromatography, affinity chromatography, and a combination thereof, but is not limited thereto. In this regard, the produced target product may be collected, recovered, or separated from the host cell or the medium including the cultured host cell.
Hereinafter, the present inventive concept will be described in further detail with reference to the following examples. However, these examples are for illustrative purpose only and are not intended to limit the scope of the present inventive concept.
Example 1 : Preparation of a recombinant vector comprising a novel promoter and a transformed strain using the same
(1) Preparation of a recombinant vector comprising Po2 and a transformed strain using the same
In terms of synthesizing a promoter that induces expression of a target gene, sequences of various promoters derived from a microorganism belonging to the genus Corynebacterium and a microorganism belonging to the genus Escherichia were analyzed, thereby synthesizing a promoter having a nucleotide sequence of SEQ ID NO: 1. The synthesized promoter was referred to as an o2 promoter (hereinafter, named "Po2"). To measure a Po2 activity for inducing a target gene expression , the Po2 was operatively linked to an open reading frame (ORF) of a GFP gene so that a recombinant vector was prepared. Then, each of strains of Corynebacteria and E. coli was transformed with the recombinant vector, thereby preparing each of the transformed strains of Corynebacteria and E. coli.
In addition, to prepare a strain having an enhanced ability of producing L-amino acids, e.g., L-arginine, or branched amino acids, such as L-valine, as an example of a target product,, the Po2 was used to enhance the expression of biosynthetic gene for arginine or valine.
(1.1) Preparation of a vector pECCG117 - Po2 - gfp and a transformed strain using the same
(1.1.1) Preparation of a vector
PCR was performed by using the synthesized Po2 as a template and a primer set of SEQ ID NO: 2 and SEQ ID NO: 3 including Kpn I/EcoR V restriction sites. The PCR was performed according to cycles of denaturation at a temperature of 94℃ for 5 minutes, denaturation at a temperature of 94℃ for 30 seconds, annealing at a temperature of 60℃ for 30 seconds, and polymerization at a temperature of 72℃ for 30 seconds, wherein the cycles were performed 30 times. Afterwards, polymerization was performed again on the strains at a temperature of 72℃ for 7 minutes, thereby consequently obtaining Po2 having a length of about 100 bp.
PCR was performed by using a pGFPuv vector (manufactured by Clontech, USA) as a template and a primer set of SEQ ID NO: 4 and SEQ ID NO: 5 including PstI/EcoR V restriction sites. PCR was performed according to cycles of denaturation at a temperature of 94℃ for 5 minutes, denaturation at a temperature of 94℃ for 30 seconds, annealing at a temperature of 55℃ for 30 seconds, and polymerization at a temperature of 72℃ for 1 minute, wherein the cycles were performed 30 times. Afterwards, polymerization was performed again at a temperature of 72℃ for 7 minutes, thereby consequently obtaining SEQ ID NO: 6 including the ORF of the GFP gene.
The Po2 was treated with restriction enzymes PstI and EcoR V and the ORF of the GFP gene was treated with restriction enzymes Kpn I and EcoR V, at PstI and Kpn I sites of a shuttle vector pECCG117 (see Biotechnology letters vol 13, No. 10, p. 721-726 (1991)) that can be expressed in E. coli and Corynebacteria. Then, the treated Po2 and ORF of the GFP gene were operatively linked to each other by using a DNA conjugating enzyme, thereby manufacturing a recombinant vector in which the Po2 and the GFP gene were linked to each other. Here, the recombinant vector was named pECCG117-Po2-gfp.
(1.1.2) Preparation of a transformed strain using the vector
Corynebacterium glutamicum ATCC13032 was transformed with each of a vector pECCG117 and the recombinant vector pECCG117-Po2-gfp by an electric pulse method, and then, transformed strains were obtained from a selective medium containing 25 mg/L of kanamycin. The obtained strains that were transformed with the vector pECCG117 and the recombinant vector pECCG117-Po2-gfp were each named ATCC13032/pECCG117 and ATCC13032/pECCG117-Po2-gfp.
In addition, E. coli DH5α was transformed with the recombinant vector pECCG117-Po2-gfp by a heat shock method, and then, transformed strains were obtained from a Luria-Bertani (LB) agar medium containing 25 mg/L of kanamycin. The obtained strains were named DH5α/pECCG117-Po2-gfp and assigned as a deposit designation "CA01-2290". CA01-2290 was deposited at the Korean Culture Center of Microorganisms (KCCM) on October 23, 2014, under the accession number of KCCM11591P, in accordance with the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.
(1.2) Preparation of a vector pECCG117 - Po2 - argJ and a transformed strain using the same
(1.2.1) Preparation of a vector
In terms of synthesizing a vector in which a main biosynthetic gene, e.g., an argJ gene (Ncgl1341, SEQ ID NO: 7) encoding bifunctional ornithine acetyltransferase/N-acetylglutamate synthase, for enhanced production of arginine is expressed by the Po2, the recombinant vector pECCG117-Po2-gfp was used to prepare a vector pECCG117-Po2-argJ.
In detail, PCR was performed on a strain by using chromosomes of Corynebacterium glutamicuum ATCC13869 as a template and a primer set of SEQ ID NO: 8 and SEQ ID NO: 9, thereby ensuring DNA fragments including the argJ genes. The PCR was performed according to cycles of denaturation at a temperature of 94℃ for 1 minute, annealing at a temperature of 58℃ for 30 seconds, and polymerization at a temperature of 72℃ for 2 minutes by using a Pfu polymerase, wherein the cycles were performed 30 times. Consequently, a fragment having a length of about 1,201 bp and including EcoRV and 3' PstI restriction enzyme sites on a 5' end was amplified. The amplified fragments generated by the PCR were purified, mixed with the vector pECCG117-Po2-gfp to which EcoRV and PstI restriction enzymes were treated, and then, joined together using the In-fusion Cloning Kit, thereby preparing a recombinant vector, which was named pECCG117-Po2-argJ.
(1.2.2) Preparation of a transformed strain using the vector
Corynebacterium glutamicum KCCM10741P, which is an arginine producing strain, was transformed with the recombinant vector pECCG117-Po2-argJ by an electric pulse method (see KR 10-0791659), and then, transformed strains were obtained from a selective medium containing 25 mg/L of kanamycin. The obtained strain was named KCCM10741P/pECCG117-Po2-argJ.
(1.3) Preparation of a vector pECCG117 - Po2 - ilvE and a transformed strain using the same
(1.3.1) Preparation of a vector
In terms of synthesizing a vector in which a main biosynthetic gene, e.g., an ilvE gene (Ncgl2123, SEQ ID NO: 10) encoding a branched-chain amino acid aminotransferase, for enhanced production of valine is expressed by the Po2, the recombinant vector pECCG117-Po2-gfp was used to prepare a vector pECCG117-Po2-ilvE.
In detail, PCR was performed on a strain by using chromosomes of Corynebacterium glutamicuum ATCC14067 as a template and a primer set of SEQ ID NO: 11 and SEQ ID NO: 12, thereby ensuring DNA fragments including the ilvE genes. The PCR performed according to cycles of denaturation at a temperature of 94℃ for 1 minute, annealing at a temperature of 58℃ for 30 seconds, and polymerization at a temperature of 72℃ for 2 minutes by using a Pfu polymerase, wherein the cycles were performed 30 times. Consequently, a fragment having a length of about 1,201 bp and including EcoRV and 3' PstI restriction enzyme sites on a 5' end was amplified. The amplified fragments generated by the PCR were purified, mixed with the vector pECCG117-Po2-gfp to which EcoRV and PstI restriction enzymes were treated, and then, joined together using the In-fusion Cloning Kit, thereby preparing a recombinant vector, which was named pECCG117-Po2-ilvE.
(1.3.2) Preparation of a transformed strain using the vector
Corynebacterium glutamicum KCCM111201P, which is a valine producing strain, was transformed with the recombinant vector pECCG117-Po2-ilvE by an electric pulse method (see KR 10-1117022), and then, transformed strains were obtained from a selective medium containing 25 mg/L of kanamycin. The obtained strains were named KCCM11201P/pECCG117-Po2-ilvE.
Comparative Example 1: Preparation of a recombinant vector including a control promoter and a transformed strain using the vector
To measure a Po2 activity for inducing a target gene expression, the GFP gene was used to operatively link a known strong promoter (e.g., Ptrc, Pcj1, Pcj4, or Pcj7 (see KR 10-0620092)) or a wild-type promoter (e.g., aceEP (WT)) to the ORF of the GFP gene to prepare a recombinant vector. Then, each of strains of Corynebacteria and E. coli was transformed with the recombinant vector, thereby preparing each of the transformed strains of Corynebacteria and E. coli.
In addition, to evaluate the transformed strains including a main biosynthetic gene using the Po2 for enhanced production of a target gene, a transformed strain including a main biosynthetic gene for enhanced production of arginine or a transformed strain including a main biosynthetic gene for enhanced production of valine were each prepared.
(1) Preparation of a gfp expression vector having a different promoter from the Po2 in terms of activity comparison with the Po2 and a transformed strain of Corynebacterium glutamicum
Base sequences of aceEP(WT) were ensured based on the U.S. National Institute of Health (NIH Genbank), and PCR was performed thereon by using chromosomes of wild-type Corynebacterium glutamicum ATCC13032 as a template and a primer set of SEQ ID NO: 13 and SEQ ID NO: 14 including Kpn I/EcoR V restriction sites. The PCR was performed in the same manner as in (1.1) of Example 1, thereby preparing a recombinant vector named pECCG117-aceEP(WT)-gfp.
Strains of Corynebacterium glutamicum were transformed with each of the prepared vector pECCG117-aceEP(WT)-gfp and vectors pECCG117-Pcj1-gfp, pECCG117-Pcj4-gfp, and pECCG117-Pcj7-gfp (see KR 10-0620092), in the same manner as in (1.1) of Example 1, thereby preparing transformed strains that were each named ATCC13032/pECCG117-aceEP(WT)-gfp, ATCC13032/pECCG117-Pcj1-gfp, ATCC13032/pECCG117-Pcj4-gfp, and ATCC13032/pECCG117-Pcj7-gfp.
(2) Preparation of a gfp expression vector having a different promoter from the Po2 in terms of activity comparison with the Po2 and a transformed strain of E. coli
Base sequences of Ptrc were ensured based on the U.S. National Institute of Health (NIH Genbank), and PCR was performed thereon by using pTrc99A (NCBI GenBank, M22744) as a template and a primer set of SEQ ID NO: 15 and SEQ ID NO: 16 including Kpn/EcoR restriction sites. The PCR was performed in the same manner as in (1.1) of Example 1, thereby preparing a recombinant vector named pECCG117-Ptrc-gfp.
Strains of E. coli were each of the prepared vector pECCG117-Ptrc-gfp and vectors pECCG117-Pcj1-gfp and pECCG117-Pcj4-gfp, in the same manner as in (1.1) of Example 1, thereby preparing transformed strains that were each named DH5α/pECCG117-Ptrc-gfp, DH5α/pECCG117-Pcj1-gfp, and DH5α/pECCG117-Pcj4-gfp.
(3) Preparation of a transformed strain having a different promoter from the Po2 in terms of a target product producibility comparison with the Po2
(3.1) Preparation of a transformed strain of Corynebacterium glutamicuum , the transformed strain having arginine producibility
The vector pECCG117-Pcj7-gfp was used to prepare a vector named pECCG117-Pcj7-argJ in the same manner as in (1.2.1) of Example 1, except that PCR was performed by using a primer set of SEQ ID NO: 9 and SEQ ID NO: 17.
Then, a transformed strain was prepared by using the vector pECCG117-Pcj7-argJ in the same manner as in (1.2.2) of Example 1, and was named KCCM10741P/pECCG117-Pcj7-argJ.
(3.2) Preparation of a transformed strain of Corynebacterium glutamicuum , the transformed strain having valine producibility
The vector pECCG117-Pcj7-gfp vector was used to prepare a vector named pECCG117-Pcj7-ilvE in the same manner as in (1.3.1) of Example 1, except that PCR was performed by using a primer set of SEQ ID NO: 12 and SEQ ID NO: 18.
Then, a transformed strain was prepared by using the vector pECCG117-Pcj7-ilvE in the same manner as in (1.3.2) of Example 1, and was named KCCM11201P/pECCG117-Pcj7-ilvE.
Example 2: Confirmation of a Po2 activity for inducing a target gene expression
(1) Confirmation of a Po2 activity for inducing a target gene expression in Corynebacterium glutamicum
To measure a Po2 activity for inducing a target gene expression in the transformed strains of Corynebacterium glutamicum, the measurement of green fluorescence protein (GFP) activity of the transformed strain named ATCC13032/pECCG117-Po2-gfp of (1.1.2) of Example 1 was compared with the measurement of GFP activity of the transformed strains each named ATCC13032/pECCG117 of (1.1.2) of Example 1 and ATCC13032/ pECCG117-aceEP(WT)-gfp, ATCC13032/pECCG117-Pcj1-gfp, ATCC13032/pECCG117-Pcj4-gfp, and ATCC13032/pECCG117-Pcj7-gfp of (1) of Comparative Example 1.
Each of the transformed strains of Corynebacterium glutamicum above was inoculated in a 250-ml corner bottle containing 25 ml of a seed medium by a volume ratio of 1: 20, and then, shake-cultured (at a speed of about 200 rpm) at a temperature of 30℃ until the strains were grown in a culture metaphase phase (OD600=10.0). After completion of the culture, the cells were collected by centrifugation (at a speed of about 5,000 rpm for about 15 minutes). The collected cells were washed twice with a 0.1% Tris.HCl (pH 8.0) buffer solution, and then, suspended in the same buffer solution for a turbidity at 610 nm of about 160. The cells were disrupted for 6 minutes by using a bead beater after glass beads were added at 1.25 g/1.5 ml of the suspension. A supernatant containing a cell extract was collected by centrifugation (at a speed of about 15,000 rpm for about 20 minutes), and then, quantitatively measured in terms of a protein concentration therein according to the Bradford method (see Bradford, M.M 1976. Anal. Biochem. 72:248-254). Then, the same amount of the cell extract was irradiated with light at an excitation wavelength of 488 nm using the method of Laure Gory or the like, and light at an emission wavelength of 511 nm was measured by using the LS-50B spectrophotometer (Perkin-Elmer), thereby determining the expression of the GFP gene, The results of measuring GFP activity in each of the strains are shown in Table 1.
[Seed medium]
20 g of glucose, 5 g of ammonium sulfate, 5 g of yeast extract, 1.5 g of urea, 4 g of KH2PO4, 8 g of K2HPO4, 0.5 g of MgSO47H2O, 150㎍ of biotin, 1.5 mg of thiamine hydrochloride, 3 mg of calcium panthothenic acid, 3 mg of nicotinamide (based on 1L of distilled water), and pH 7.2
Fluorescence intensity in Corynebacterium glutamicum
Strain Fluorescence intensity
ATCC13032/pECCG117 0.0
ATCC13032/ pECCG117-Po2-gfp 2339.5
ATCC13032/ pECCG117-aceEP(WT)-gfp 170.7
ATCC13032/ pECCG117-Pcj1-gfp 589.6
ATCC13032/ pECCG117-Pcj4-gfp 920.5
ATCC13032/ pECCG117-Pcj7-gfp 270.4
As shown in the results of Table 1, it was confirmed that the Po2 had a promoter activity in Corynebacterium glutamicum, and that the strain named ATCC13032/pECCG117-Po2-gfp exhibited fluorescence intensity that is at least 13 times as large as that of the wild-type ATCC13032/pECCG117-aceEP(WT)-gfp strain. In addition, it was confirmed that the strain named ATCC13032/pECCG117-Po2-gfp exhibited fluorescence intensity at a level that is much higher than that of the strains each named ATCC13032/pECCG117-Pcj1-gfp, ATCC13032/ pECCG117-Pcj4-gfp, and ATCC13032/pECCG117-Pcj7-gfp using known strong promoters (e.g., Pcj1, Pcj4, and Pcj7). Consequently, it was confirmed that the Po2 served as a strong promoter to express a target gene.
(2) Confirmation of a Po2 activity for inducing a target gene expression in E. coli
To measure a Po2 activity for inducing a target gene expression in the transformed strains of E. coli, measurement of GFP activity of the transformed strain named DH5α/pECCG117-Po2-gfp of (1.1.2) of Example 1 was compared with the measurement of GFP activity of the transformed strains each named DH5α/pECCG117-Ptrc-gfp, DH5α/pECCG117-Pcj1-gfp, and DH5α/pECCG117-Pcj4-gfp of (2) of Comparative Example 2.
Each of the transformed strains of E. coli above was inoculated in a 250-ml corner bottle containing 25 ml of a kanamycin-containing LB medium a volume ratio of 1: 20, and then, shake-cultured (at a speed of about 200 rpm) at a temperature of until the strains were grown in a culture metaphase phase (OD600=3.0). After completion of the culture, the cells were collected by centrifugation (at a speed of about 5,000 rpm for about 15 minutes), washed twice with a 0.1% Tris.HCl (pH 8.0) buffer solution, suspended in the same buffer solution, disrupted by sonication, and then, subjected to centrifugation (at a speed of about 15,000 rpm for about 20 minutes) to obtain a supernatant containing a cell extract. The supernatant was quantitatively measured in terms of a protein concentration therein according to the Bradford method, Then, the same amount of the cell extract was irradiated with light at an excitation wavelength of 488 nm using the method of Laure Gory or the like, and light at an emission wavelength of 511 nm was measured by using the LS-50B spectrophotometer (Perkin-Elmer), thereby determining the expression of the GFP gene, The results of measuring GFP activity in each of the strains are shown in Table2.
Fluorescence intensity in E. coli
Strain Fluorescence intensity
DH5α/pECCG117-Ptrc-gfp 287.0
DH5α/pECCG117-Po2-gfp 248.9
DH5α/pECCG117-Pcj1-gfp 3041.9
DH5α/pECCG117-Pcj4-gfp 135.1
As shown in the results of Table 2, it was confirmed that the Po2 had a promoter activity in E. coli, and that the strain named DH5α/pECCG117-Po2-gfp exhibited the fluorescence intensity at a similar level with that of the strain named DH5α/pECCG117-Ptrc-gfp, which is a known as a strong promoter and higher than that of the DH5α/pECCG117-Pcj4-gfp strain. Consequently, it was confirmed that the Po2 served as a strong promoter to express a target gene in E. coli.
Example 3: Evaluation of strains for enhanced target product producibility
(1) Evaluation of strains for enhanced production of arginine
In terms of evaluating factors influencing the production of arginine, when the main biosynthetic gene for arginine, i.e., the argJ gene, was expressed by using the Po2, the strain named KCCM10741P/pECCG117-Po2-argJ of (1.2.2) of Example 1, which was used as the strain for enhanced production of the argJ gene, was compared with the non-transformed strain named KCCM10741P (having no transformed arginine producibility) and the strain named KCCM10741P/pECCG117-Pcj7-argJ of (3.1) of Comparative Example 1 in terms of the arginine producibility.
1 loop of each of the transformed strains above was inoculated in a 250-ml corner bottle containing 25 ml of a production medium, and then, shake-cultured at a speed of about 200 rpm at a temperature of 30℃ for 48 hours. After completion of the culture, the production of L-arginine was measured by HPLC. The results of measuring the production of L-arginine are shown in Table 3.
[Production medium]
glucose 6%, ammonium sulfate 3%, monopotassium phosphate 0.1%, magnesium sulfate heptahydrate 0.2%, corn steep liquor (CSL) 1.5%, NaCl 1%, yeast extract 0.5%, biotin 100㎍/L, and pH7.2
Production of arginine in KCCM10741P
Strain OD Concentration of arginine (g/L)
KCCM110741P 89 3.1
KCCM10741P/pECCG117-Pcj7-argJ 85 4.6
KCCM10741P/pECCG117- Po2-argJ 82 5.7
As shown in the results of Table 3, it was confirmed that the Po2 resulted in improved production of arginine in Corynebacterium glutamicum in which the expression of the argJ gene was enhanced. In particular, the arginine production in Corynebacterium glutamicum was increased by about 84% compared to that in the control strain, and was increased by about 23% compared to that in the strain named KCCM10741P/pECCG117-Pcj7-argJ. Consequently, it was confirmed that the Po2 influenced the enhanced expression of the argJ gene.
(2) Evaluation of strains for enhanced production of L- valine
In terms of evaluating factors influencing the production of valine, when the main biosynthetic gene for valine, i.e., the ilE gene, was expressed by using the Po2, the strain named KCCM11201P/pECCG117-Po2-ilvE of (1.3.2) of Example 1, which was used as the strain for enhanced production of the ilvE gene, was compared with the non-transformed strain named KCCM11201P (having no transformed L-valine producibility) and the strain named KCCM11201P/pECCG117-Pcj7-ilvE of (3.2) of Comparative Example 1 in terms of L-valine producibility.
1 loop of each of the transformed strains above was inoculated in a 250-ml corner bottle containing 25 ml of a production medium, and then, shake-cultured at a speed of about 200 rpm at a temperature of 30℃ for 72 hours. After completion of the culture, the production of L-valine was measured by HPLC. The results of measuring the production of L-valine are shown in Table 4.
[Production medium]
glucose 5%, ammonium sulfate 2%, monopotassium phosphate 0.1%, magnesium sulfate heptahydrate 0.05%, CSL 2.0%, biotin 200 ㎍/L, and pH 7.2
Production of valine in KCCM11201P
Strain Concentration of valine (g/L)
KCCM11201P 2.8
KCCM11201P/pECCG117-Pcj7-ilvE 3.3
KCCM11201P/pECCG117- Po2-ilvE 3.7
As shown in the results of Table 4, it was confirmed that the Po2 resulted in improved production of valine in Corynebacterium glutamicum in which the expression of the ilvE gene was enhanced. In particular, the valine production in the strain named KCCM11201P/pECCG117-Po2-ilvE was significantly increased by about 32% compared to that in the control strain, and was increased by about 17% compared to that in the strain named KCCM10741P/pECCG117-Pcj7-ilvE. Consequently, it was confirmed that the Po2 influenced the enhanced expression of the ilvE gene.
[Accession number]
Accession institution : Korean Culture Center of Microorganisms (international)
Accession number : KCCM11591P
Accession date : October 23, 2014
According to the one or more of the exemplary embodiments above, a novel promoter may have various activities according to a microorganism used to induce expression of a target gene. In this regard, the novel promoter may be used in a case where activity of a target gene needs to be controlled during the production of the target product, resulting in efficient production of the target product.
It should be understood that exemplary embodiments described herein should be considered in a descriptive sense only and not for purposes of limitation. Descriptions of features or aspects within each exemplary embodiment should typically be considered as available for other similar features or aspects in other exemplary embodiments.
While one or more exemplary embodiments have been described, it will be understood by those of ordinary skill in the art that various changes in form and details may be made therein without departing from the spirit and scope of the inventive concept as defined by the following claims.
Figure PCTKR2016000444-appb-I000001

Claims (7)

  1. A promoter comprising a nucleotide sequence represented by SEQ ID NO: 1.
  2. An expression regulatory sequence, comprising the promoter of claim 1.
  3. A vector comprising the expression regulatory sequence of claim 2 and a target gene that is operatively linked to the expression regulatory sequence.
  4. A host cell comprising the vector of claim 3.
  5. The host cell according to claim 4, wherein the host cell is a bacterial cell belonging to the genus Corynebacterium or the genus Escherichia.
  6. A method of producing a target product, the method comprising:
    culturing the host cell of claim 4 or claim 5; and
    recovering the target product from the host cell or a culture medium including the cultured host cell.
  7. The method of claim 6, wherein the target product is an amino acid.
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