Promoter and uses thereof

Abstract

Provided are a novel promoter and a method of producing a target product using the same.

Claims

1. A synthetic promoter comprising the nucleotide sequence of 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; 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.

8. A method of producing a target product, the method comprising: culturing the host cell of claim 5; and recovering the target product from the host cell or a culture medium including the cultured host cell.

Description

MODE FOR INVENTION

(1) 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

(2) (1) Preparation of a Recombinant Vector Comprising Po2 and a Transformed Strain Using the Same

(3) 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.

(4) 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.

(5) (1.1) Preparation of a Vector pECCG117-Po2-gfp and a Transformed Strain Using the Same

(6) (1.1.1) Preparation of a Vector

(7) 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. Afterwards, 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.

(8) 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.

(9) 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.

(10) (1.1.2) Preparation of a Transformed Strain Using the Vector

(11) 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.

(12) In addition, 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 Oct. 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.

(13) (1.2) Preparation of a Vector pECCG117-Po2-argJ and a Transformed Strain Using the Same

(14) (1.2.1) Preparation of a Vector

(15) 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.

(16) In detail, PCR was performed on a strain by using chromosomes of Corynebacterium glutamicum 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.

(17) (1.2.2) Preparation of a Transformed Strain Using the Vector

(18) 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.

(19) (1.3) Preparation of a Vector pECCG117-Po2-ilvE and a Transformed Strain Using the Same

(20) (1.3.1) Preparation of a Vector

(21) 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.

(22) In detail, PCR was performed on a strain by using chromosomes of Corynebacterium glutamicum 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.

(23) (1.3.2) Preparation of a Transformed Strain Using the Vector

(24) 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

(25) 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.

(26) 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.

(27) (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

(28) 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.

(29) 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.

(30) (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

(31) 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.

(32) 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.

(33) (3) Preparation of a Transformed Strain Having a Different Promoter from the Po2 in Terms of a Target Product Producibility Comparison with the Po2

(34) (3.1) Preparation of a Transformed Strain of Corynebacterium glutamicum, the Transformed Strain Having Arginine Producibility

(35) 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.

(36) 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.

(37) (3.2) Preparation of a Transformed Strain of Corynebacterium glutamicum, the Transformed Strain Having Valine Producibility

(38) 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.

(39) 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

(40) (1) Confirmation of a Po2 Activity for Inducing a Target Gene Expression in Corynebacterium glutamicum

(41) 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.

(42) 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? C. 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.

(43) [Seed Medium]

(44) 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 ?g of biotin, 1.5 mg of thiamine hydrochloride, 3 mg of calcium panthothenic acid, 3 mg of nicotinamide (based on 1 L of distilled water), and pH 7.2

(45) TABLE-US-00001 TABLE 1 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

(46) 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.

(47) (2) Confirmation of a Po2 Activity for Inducing a Target Gene Expression in E. coli

(48) 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 DH5?/pECCG117-Po2-gfp of (1.1.2) of Example 1 was compared with the measurement of GFP activity of the transformed strains each named DH5?/pECCG117-Ptrc-gfp, DH5?/pECCG117-Pcj1-gfp, and DH5?/pECCG117-Pcj4-gfp of (2) of Comparative Example 2.

(49) 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 Table 2.

(50) TABLE-US-00002 TABLE 2 Fluorescence intensity in E. coli Strain Fluorescence intensity DH5?/pECCG117-Ptrc-gfp 287.0 DH5?/pECCG117-Po2-gfp 248.9 DH5?/pECCG117-Pcj1-gfp 3041.9 DH5?/pECCG117-Pcj4-gfp 135.1

(51) 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 DH5?/pECCG117-Po2-gfp exhibited the fluorescence intensity at a similar level with that of the strain named DH5?/pECCG117-Ptrc-gfp, which is a known as a strong promoter and higher than that of the DH5?/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

(52) (1) Evaluation of Strains for Enhanced Production of Arginine

(53) 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.

(54) 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? C. 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.

(55) [Production Medium]

(56) 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

(57) TABLE-US-00003 TABLE 3 Production of arginine in KCCM10741P Concentration of arginine Strain OD (g/L) KCCM110741P 89 3.1 KCCM10741P/pECCG117-Pcj7-argJ 85 4.6 KCCM10741P/pECCG117-Po2-argJ 82 5.7

(58) 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.

(59) (2) Evaluation of Strains for Enhanced Production of L-Valine

(60) 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.

(61) 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? C. 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.

(62) [Production Medium]

(63) 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

(64) TABLE-US-00004 TABLE 4 Production of valine in KCCM11201P Concentration of valine Strain (g/L) KCCM11201P 2.8 KCCM11201P/pECCG117-Pcj7-ilvE 3.3 KCCM11201P/pECCG117-Po2-ilvE 3.7

(65) 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.

(66) [Accession Number]

(67) Accession institution: Korean Culture Center of Microorganisms (international)

(68) Accession number: KCCM11591P

(69) Accession date: Oct. 23, 2014

(70) 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.

(71) 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.

(72) 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.