KR101123070B1 - High energy charged cell and use thereof - Google Patents

Abstract

The present invention relates to cells produced by recombinant methods or mutations and uses thereof having increased intracellular ATP concentrations relative to parent cells.

Phosphoenolpyruvate carboxykinase (PCK), overexpression, promoter exchange, cells.

Description

Cells with high intracellular energy concentrations and uses thereof

The present invention relates to cells produced by recombinant methods or mutations and uses thereof having increased intracellular ATP concentrations relative to parent cells.

Microbes are being used as cell factories to produce useful biomaterials such as proteins, drugs, fine chemicals, stereospecific chemicals, and the like. By combining genetic and biological engineering techniques with existing fermentation engineering techniques, it is possible to modify the metabolic pathways of bacteria, yeasts, fungi or flora and fauna cells, or to overexpress or inhibit or eliminate specific genes to efficiently produce useful target substances. Methods for improving microorganisms have been widely studied and used.

Energy is required to biosynthesize substances such as proteins, nucleic acids and the like in vivo. Most of the energy used in vivo is preserved and used in the form of NADH, NADPH and ATP. In particular, ATP is an energy carrier that transfers the chemical energy produced during metabolism to the various activities of organisms. ATP is produced through photosynthesis, fermentation and respiration processes and is consumed in in vivo activities such as biosynthesis, exercise, signaling and cell division. As such, since ATP regulates many intracellular processes such as growth, ribosomal RNA synthesis, and cellular metabolic pathways, intracellular ATP concentration is one of the most important factors for the physiological activity of cells. Thus, the production and maintenance of sufficient energy is required for microorganisms to be used as efficient plants for producing useful materials.

For the production of useful target substances using microorganisms, target substance specific approaches have been mainly employed, such as increasing the expression of genes encoding enzymes involved in the production of specific target substances or removing unnecessary genes. For example, a number of useful strains have been developed, including Escherichia coli, which can produce high yields of L-amino acids. However, each strain developed as described above can be used only for the production of a specific target material, and cells that can be used universally as a cell plant for high yield production of the target material, regardless of the type of the target material, are still Not developed. Therefore, the present inventors conducted studies to increase the ATP production yield and intracellular ATP level of cells, and overexpression of phosphoenolpyruvate carboxykinase involved in gluconegenesis, or the corresponding process. The present invention was completed by finding that expression in increases the intracellular ATP production and levels.

It is an object of the present invention to provide cells with high intracellular ATP production yields or concentrations.

It is still another object of the present invention to provide a method for producing a target substance using cells having a high intracellular ATP production yield or concentration.

In order to achieve the above object, the present invention uses a cell having an increased intracellular ATP production yield or concentration compared to the parental cell by the expression change of the gene encoding phosphoenolpyruvate carboxykinase (PCK) To provide a method for producing the target substance.

The method comprises culturing cells having an increased intracellular ATP concentration relative to the parent cell under conditions suitable for producing a target substance, and

It may include the step of recovering the target material from the culture.

Phosphoenolpyruvate carboxykinase (PCK) is an enzyme that catalyzes the reaction of phosphoenolpyruvate (PEP) from oxaloacetic acid (OAA) in gluconeogenesis in vivo. Since the reaction catalyzed by the enzyme is reversible, in the presence of CO ₂ , PCK produces one molecule of ATP while catalyzing the reaction to produce OAA from PEP. Thus, unlike phosphoenolpyruvate carboxylase (ppc), which catalyzes the same reaction in the glycolysis pathway, increased reactions by PCK can increase intracellular ATP production and concentration. ATP production by PCK by increasing the expression of a gene encoding PCK in a cell, or by altering the regulatory sequence so that the gene encoding the PCK can be expressed in that pathway, or by increasing the activity of PCK Can be increased.

The method for producing a target substance according to an embodiment of the present invention includes culturing cells having an increased intracellular ATP concentration compared to the parent cell under conditions suitable for producing the target substance.

The step of culturing the cells can be carried out under suitable culture conditions with suitable media known in the art. Conditions suitable for producing the target substance can be easily determined according to the target substance and cells by those skilled in the art. For example, when induction is required for expression of a gene encoding a target substance introduced from the outside, culturing the cell may further include inducing expression of the gene. Cultures include, but are not limited to, batch, continuous and fed-batch cultures.

Cells with high intracellular ATP concentrations may be suitable for producing foreign proteins because they may increase protein synthesis by increasing the expression of genes involved in biosynthesis such as amino acids and ribosomal concentrations. In addition, since ATP is used for active substance transport into and out of cells, when the concentration of intracellular ATP is high, cells can actively transport the substance using ATP, thereby producing a desired metabolite in higher yield. . That is, the generated metabolite may be transported to the outside of the cell, and the required carbon source may be transported into the cell to activate the metabolic pathway for generation of the target substance while excluding feedback regulation.

In one embodiment of the invention, the target material may be a protein, amino acid, nucleic acid, antibiotic, or metabolite.

In one embodiment of the present invention, the expression of the gene encoding the phosphoenolpyruvate carboxykinase (PCK) in the cell having an increased intracellular ATP concentration compared to the parent cell is overexpressed compared to the parent cell or in the corresponding pathway It may be altered to be expressed to adjust to increase the intracellular ATP concentration.

In one embodiment of the present invention, cells having an increased intracellular ATP concentration compared to the parent cells increase the number of copies of the gene encoding phosphoenolpyruvate carboxykinase in the parent cell or the expression control sequence of the gene. Or by modifying the coding sequence.

In one embodiment of the invention, the gene encoding phosphoenolpyruvate carboxykinase can be derived from bacteria, yeast, plant cells or animal cells, including E. coli.

In one embodiment of the present invention, an increase in the number of copies of the gene encoding the phosphoenolpyruvate carboxykinase in a cell having an increased intracellular ATP concentration compared to the parent cell results in a phosphoenolpyruvate carboxykinase. By the introduction of a foreign gene encoding.

Introduction of the foreign gene includes, but is not limited to, transformation or transfection, and may be performed by conventional methods known to those skilled in the art.

In one embodiment of the invention, the modification of the expression control sequence of the gene encoding the phosphoenolpyruvate carboxykinase in a cell having an increased intracellular ATP concentration compared to the parent cell is made by exchange of a promoter or enhancer Can be.

In one embodiment of the invention, the promoter exchanged for overexpressing the gene encoding the phosphoenolpyruvate carboxykinase can be selected from the group consisting of trc promoter, tac promoter, T7 promoter, lac promoter and trp promoter. have.

In one embodiment of the present invention, the promoter of the gene (pck) encoding the phosphoenolpyruvate carboxykinase to the gene (ppc) encoding the phosphoenol pyruvate carboxylase is expressed in glycolysis It may be exchanged by. When the promoter of the pck gene is exchanged with the promoter of the ppc gene expressed in the pathway in the cell, the cell expresses pck in the pathway, through which PCK converts phosphoenolpyruvate to oxaloacetate. As ATP can be produced, it has an increased intracellular ATP concentration compared to parental cells. That is, a cell having a pck gene expressed under the control of the ppc promoter may spontaneously select a pck gene in the process by exchanging a regulatory sequence of the pck gene present on the genome, in particular, a promoter, without introducing an additional pck gene from outside. Expression to increase intracellular ATP production.

In one embodiment of the present invention, a cell having an increased intracellular ATP concentration compared to the parent cell is increased by expression of a gene encoding phosphoenolpyruvate carboxykinase or activity of the enzyme compared to the parent cell by mutation. It may have been.

In one embodiment of the invention, the cells may be selected from the group consisting of bacteria, yeast, fungi, plant cells and animal cells.

In one embodiment of the present invention, the cell having an increased intracellular ATP concentration compared to the parent cell may be E. coli transformed by a recombinant vector comprising a gene encoding a phosphoenolpyruvate carboxykinase. The gene encoding the phosphoenolpyruvate carboxykinase in the E. coli may be operably linked to a trc promoter.

The method for producing a target substance according to the present invention is to introduce a gene encoding the target substance from the outside into a cell having an increased intracellular ATP concentration compared to the parent cell, or to genetically modulate the metabolic pathway of the cell to produce the target substance. It may further comprise the step of modifying. Cells with increased intracellular ATP concentrations compared to the parental cells may be introduced with the necessary genes from outside to be suitable for producing the target substance, or the cells may be expressed so that expression of the gene on the genome in the cell may be suitable for production of the target substance. It can be transformed entirely.

In one embodiment of the present invention, the target substance may be a protein, and the target substance may be produced by introducing and expressing a gene encoding the protein in the cell. Introduction of a gene encoding a target substance into the cell from the outside may include, but is not limited to, transformation or transfection, and may be performed by conventional methods known to those skilled in the art.

In one embodiment of the present invention, the target substance may be a metabolite such as succinic acid, and increases, inhibits or increases the expression or activity of a gene encoding the enzyme of the step involved in the production of succinic acid in the metabolic pathway of the cell. The removal can be genetically modified to produce high yields of succinic acid.

In one embodiment of the invention, the target substance may be an amino acid, and the cell increases the amino acid by increasing, inhibiting or eliminating the expression or activity of a gene encoding an enzyme involved in the biosynthetic pathway of the amino acid. It can be genetically modified to produce in high yields.

As used herein, a "parent cell" is modified by molecular biology techniques such as mutation or recombination methods such that the expression of a gene encoding phosphoenolpyruvate carboxykinase (PCK) is increased relative to the parent cell or is By bacteria, yeast, fungi, plant cells or animal cells before the gene is altered to be expressed in that pathway, thereby increasing the yield or concentration of intracellular ATP production.

As used herein, "change in expression of a gene encoding phosphoenolpyruvate carboxykinase (PCK)" means that a gene encoding phosphoenolpyruvate carboxykinase relative to a parent cell is introduced from outside or a modification of an expression control sequence. It is to be understood to include those that are overexpressed by or altered to modulate such genes to be expressed in that pathway or to increase the activity of the resulting PCK.

As used herein, "energy cell" refers to a cell having a high intracellular energy level, especially ATP concentration.

As used herein, a "cell" should be understood to include bacteria, yeasts, fungi, animal cells or plant cells.

As used herein, "target material" means a material produced by a method comprising the step of culturing a cell according to one embodiment of the invention, a protein, L-amino acid, nucleic acid, antibiotic, vitamin, bioactive material Metabolites, including but not limited to.

As used herein, "genetically modified to produce a target substance" refers to the introduction of a gene necessary for biosynthesis of a target substance into a cell from outside or expression of a gene involved in the generation of a target substance present on the genome of a cell. It is meant that the cells are transformed into an advantageous state for producing the target substance by increasing the or inhibiting or eliminating the expression of a gene that interferes with the production of the target substance or is involved in the degradation of the target substance.

The method for producing a target substance according to the present invention includes the step of recovering the target substance from the culture of the cells. Recovering the desired material from the culture can be carried out by conventional methods known in the art. Centrifugation, filtration, chromatography, crystallization, and the like may be used to separate the target substance, but is not limited thereto.

The present invention also provides cells with increased intracellular ATP production yield or concentration compared to parental cells, by expression change of the gene encoding phosphoenolpyruvate carboxykinase (PCK).

In one embodiment of the invention, the expression change of the gene encoding the phosphoenolpyruvate carboxykinase increases the number of copies of the gene encoding the phosphoenolpyruvate carboxykinase in the cell or It may be made by modifying the expression control sequence of the gene.

In one embodiment of the present invention, the promoter of the gene encoding the phosphoenolpyruvate carboxykinase may be exchanged with the promoter of the gene encoding the phosphoenolpyruvate carboxylase.

Since the cell according to one embodiment of the present invention has a high intracellular ATP concentration, it can be usefully used as a cell factory for producing a target substance.

In one embodiment of the present invention, a cell having an increased intracellular ATP concentration compared to the parent cell may be a cell into which a foreign gene encoding a target substance is introduced or a metabolic pathway is genetically modified for production of the target substance. have.

In one embodiment of the present invention, the target substance may be a protein, and the target substance may be produced by introducing and expressing a gene encoding the protein in the cell. Introduction of a gene encoding a target substance into the cell from the outside may include, but is not limited to, transformation or transfection, and may be performed by conventional methods known to those skilled in the art.

In one embodiment of the present invention, the target substance may be a metabolite such as succinic acid, and increases or inhibits the expression or activity of the gene encoding the enzyme of the step involved in the production of succinic acid in the metabolic pathway of the cell. Or upon removal the cells can be genetically modified to produce high yields of succinic acid.

In one embodiment of the invention, the cells may be bacteria, yeast, fungi, plant cells or animal cells.

Hereinafter, an Example demonstrates this invention in more detail. The following examples are intended to illustrate the invention in more detail and should not be construed as limiting the scope of the invention.

Cells having an increased intracellular ATP concentration compared to the parent cell according to the present invention, and using the production method of the target substance using the same, high intracellular ATP concentration enhances gene expression, biosynthesis, substance transport, etc. It can efficiently produce useful target materials, including.

Example 1 Preparation of PCK Overexpressing Strains

In this example, a strain overexpressing a gene encoding E. coli phosphoenolpyruvate carboxykinase was prepared.

(1) Preparation of pEcPCK

A plasmid having a gene encoding the PCK of Escherichia coli (pck) was prepared. PCR was performed using genomic DNA of Escherichia coli W3110 (Korena Collection of Type Culture, KCTC 2223) as a template and primers of SEQ ID NOs: 1 and 2 having EcoRI and PstI recognition sites, respectively (denatured for 40 seconds at 95 ° C, Pck was amplified with 30 cycles consisting of annealing for 40 seconds at 68.5 ° C., and elongation for 40 seconds at 72 ° C.). The 1.6 kb PCR product corresponding to the pck gene was cloned into pGEM-T easy vector (Promega, Madison, WI, USA) (PCR cloning vector, Ap ^R ). The pck gene obtained was then subcloned into the EcoRI-PstI site of the pTrc99A expression vector (AP Biotech, Uppsala, Sweden) to obtain pEcPCK.

(2) Preparation of Escherichia Coli W3110 (DE3) / pEcPCK

E. coli W3110 (DE3) was prepared from E. coli W3110 (KCTC 2223) using the DE3 Lysogenation kit (Novagen, Madison, Wis., USA) according to the manufacturer's instructions. Escherichia coli W3110 (E3) which transformed pEcPck prepared in (1) to E. coli W3110 (DE3) using electroporation (Gene Pulser, Bio-Rad, Hercules, CA, USA) to introduce pck gene to overexpress pck ( DE3) / pEcPCK was prepared. 1 shows the reaction catalyzed by phosphoenolpyruvate carboxykinase (PCK) and phosphoenolpyruvate carboxylase (PPC) in E. coli W3110 (DE3) and E. coli W3110 (DE3) / pEcPCK. . In wild-type Escherichia coli W3110 (DE3), PPC is involved in the conversion of PEP to OAA in glycolysis, and PCK is involved in the same reaction. However, in Escherichia coli W3110 (DE3) / pEcPCK transformed to overexpress PCK, both PPC on the chromosome and PCK present on the introduced plasmid work in the course of glycolysis.

(3) Preparation of Escherichia coli BL21 (DE3) / pEcPCK

The pEcPck prepared in the above (1) to () E. coli gal dcm ompT hsdS B (r _B ^{- -} m _B) cell physiology research purposes in addition to the wild type E. coli W3110 E. coli strain BL21 is used mainly (DE3) for recombinant protein synthesis E. coli BL21 (DE3) / pEcPCK was prepared by transformation by electroporation and introducing the pck gene to overexpress pck.

Example 2 Physiological Characteristics of PCK Overexpressing Strains

In order to determine the physiological characteristics of PCK overexpressing Escherichia coli W3110 (DE3) / pEcPCK prepared in Example 1, biomass, intracellular ATP concentration and ribosome concentration were measured relative to the parent strain (E. coli W3110 (DE)).

(1) biomass

The culture was carried out at 37 ° C., 250 rpm in a 50-mL test tube containing 6 mL of medium (9 g glucose, 10 g NaHCO3, IPTG 1 mM, 50 μg / ml antibiotics (ampicillin and kanamycin) and minerals / L). It was carried out for hours. The amount of biomass converted from optical density (OD) measured at 600 nm was 0.42 ± 0.02 (g-cell / L) and 0.16 ± 0.01 (g-) for E. coli W3110 (DE) and E. coli W3110 (DE3) / pEcPCK, respectively. cell / L).

(2) intracellular ATP concentration

Cultured under the same conditions as in (1) above, when the initial exponential phase of cell growth was active, that is, when the OD at 600 nm reached about 0.3, cells were harvested to measure intracellular ATP concentration. Intracellular ATP concentrations were measured using ATP determination kit (FL-AA; Sigma Chemical, St. Louis, MO) and luminometer (20 / 20n Luminometer System, Turner Biosystems, Sunnyvale, CA) and ATP standards. It was. The amount of intracellular ATP was 0.58 ± 0.02 and 1.45 ± 0.03 (nmol / g-cell) for E. coli W3110 (DE) and E. coli W3110 (DE3) / pEcPCK, respectively. Escherichia coli W3110 (DE3) / pEcPCK overexpressing PCK had an intracellular ATP concentration about three times higher than the control group.

(3) ribosomal concentration

Intracellular ribosomal concentrations were measured to determine if the microorganisms overexpressing PCK had the increased concentration of ribosomes required for protein synthesis.

0.5)을 채취하고, 15 ml의 완충액 A(50 mM Tris-HCl, 10 mM MgCl₂, 100 mM NH₄Cl, 6 mM 2-머캅토에탄올, 0.5 mM EDTA, pH 7.6)에 현탁시켰다. 상기 세포들을 얼음 상에서 1초 간격으로 3분 동안 30 W로 설정된 초음파 처리기에 의해 파쇄시켰다. 10 ㎕의 DNase I(Sigma, St. Louis, MO, U.S.A.)을 첨가한 후, 상층액을 얻기 위해 용해물을 4℃, 15,000 rpm에서 15분간 원심분리하였다. 상기 상층액에 0.5M NH₄Cl 용액의 첨가 후에, 초원심분리(4℃, 100,000 g, 5시간)에 의해 리보솜을 회수하였다. 상층 액을 버린 후에, 펠렛을 완충액 B(50 mM Tris-HCl, 6 mM MgCl₂, 100 mM NH₄Cl, 6 mM 2-머캅토에탄올, 0.5 mM EDTA, pH 7.6)에 용해시켰다. 12.5% 폴리아크릴아미드 겔에서 전기영동에 의해 단백질을 분리시키고 니트로셀룰로오스 막으로 옮겼다. L4-리보솜에 대한 제1 항체(한국 외국어대학교 환경학과 이규호 교수로부터 공여됨) 및 제2 항체(항-랫트 IgG AP-conjugated, Sigma)를 순차적으로 상기 니트로셀룰로오스 막에 결합시켰다. AP 완충액(100 mM Tris-HCl, 100 mM NaCl, 5 mM MgCl₂, pH 9.5)을 이용하여 리보솜을 확인하였다. 이미지 분석기(GelDoc1000/2000, Bio-Rad Lab, Inc., Hercules, CA. U.S.A)에 의한 강도의 분석을 통해 추정된 리보솜의 농도(임의 단위)는 대장균 W3110(DE)와 대장균 W3110(DE3)/pEcPCK에 대해 각각 342.5 및 605.9였다. 대장균 W3110(DE3)/pEcPCK는 대조군인 대장균 W3110(DE)에 비해 약 1.8배 더 높은 리보솜 농도를 보였고, 이는 높은 세포내 ATP 농도를 갖는 세포들이 보다 많은 단백질 합성 기구를 갖는다는 것을 보여준다. Actively growing cells cultured under the same conditions as in (1) (OD)

0.5) was collected and suspended in 15 ml of Buffer A (50 mM Tris-HCl, 10 mM MgCl ₂ , 100 mM NH ₄ Cl, 6 mM 2-mercaptoethanol, 0.5 mM EDTA, pH 7.6). The cells were disrupted by sonication set at 30 W for 3 minutes at 1 second intervals on ice. After addition of 10 μl of DNase I (Sigma, St. Louis, Mo., USA), the lysates were centrifuged at 4 ° C., 15,000 rpm for 15 minutes to obtain supernatant. After addition of 0.5M NH ₄ Cl solution to the supernatant, ribosomes were recovered by ultracentrifugation (4 ° C., 100,000 g, 5 hours). After the supernatant was discarded, the pellet was dissolved in buffer B (50 mM Tris-HCl, 6 mM MgCl ₂ , 100 mM NH ₄ Cl, 6 mM 2-mercaptoethanol, 0.5 mM EDTA, pH 7.6). Proteins were separated by electrophoresis on 12.5% polyacrylamide gels and transferred to nitrocellulose membranes. A first antibody against L4-ribosome (donated by Professor Kyu-ho Lee, Department of Environmental Studies, Hankuk University of Foreign Studies) and a second antibody (anti-rat IgG AP-conjugated, Sigma) were sequentially bound to the nitrocellulose membrane. Ribosomes were identified using AP buffer (100 mM Tris-HCl, 100 mM NaCl, 5 mM MgCl ₂ , pH 9.5). Ribosomal concentrations (arbitrary units) estimated by intensity analysis by an image analyzer (GelDoc1000 / 2000, Bio-Rad Lab, Inc., Hercules, CA.USA) are E. coli W3110 (DE) and E. coli W3110 (DE3) / 342.5 and 605.9 for pEcPCK, respectively. E. coli W3110 (DE3) / pEcPCK showed about 1.8 times higher ribosome concentrations than control E. coli W3110 (DE), indicating that cells with high intracellular ATP concentrations had more protein synthesis machinery.

The results showed that E. coli W3110 (DE3) / pEcPCK overexpressing PCK was about 3 times and 2 times higher in intracellular ATP and ribosomal concentrations than the control, but the cell growth rate was slower than the control. Because of the high intracellular ATP concentration, it is thought that cell growth is restricted by the inhibition of glycolysis and that energy for growth can be used for biosynthesis.

Example 3. Preparation of Foreign Protein by PCK Overexpressing Strains

3-1. Preparation of Foreign Protein eGFP

(1) Construction of eGFP Expression Vector

PCR was performed using genomic DNA of E. coli W3110 (Korena Collection of Type Culture, KCTC 2223) as a template and primers of SEQ ID NOs: 3 and 4 having EcoRI and XhoI recognition sites, respectively (denatured at 95 ° C for 1 minute, 25 cycles consisting of 1 minute anneal at 68 ° C. and 1 minute elongation at 72 ° C. were performed) eGFP was amplified. PCR products corresponding to the eGFP gene were cloned into pGEM-T easy vector (Promega, Madison, Wis., USA) and confirmed by DNA sequencing (solgent, Daejeon, Korea). Thereafter, the obtained eGFP gene was subcloned into the XhoI-EcoRI site of pET41a (Novagen) to obtain pEGFP (eGFP expression vector based on pET41a, p _T7 , Km ^R ).

(2) transformation and expression

The pEGFP prepared in (1) was transformed into E. coli W3110 (DE3) / pEcPCK, which is a PCK overexpressing strain prepared in Example 1, by electroporation. Transformed cell W3110 (DE3) / pEGFP / pEcPCK and control cell W3110 (DE3) / pEcPCK were treated with glucose-minimum medium (liter, glucose (9 g / L) containing IPTG (isopropyl-β-D-thiogalactopyranoside) and antibiotics. ), NaHCO ₃ (10 g), IPTG (1 mM), antibiotics (ampicillin and kanamycin, 25 μg / mL each, kanamycin 50 μg / mL for control, and minerals) and LB-glucose medium (per liter, peptone (5 g), tryptone (10 g), NaCl (10 g), glucose (9 g / L), NaHCO ₃ (10 g), IPTG (1 mM), and antibiotics (ampicillin and kanamycin, 25 μg / mL, respectively, For the control group, kanamycin 50 μg / mL)) was incubated at 37 ° C. and 250 rpm for 24 hours. To estimate the expression level of eGFP, cultures (700 microliters) were placed in a fluorescence spectrometer (RF-5301PC, Shimadzu, Kyoto, Japan) under excitation at 395 nm and emission conditions at 509 nm. Cells were observed using a phase contrast microscope and a fluorescence microscope (LSM 510, Carl Zeiss). 2 and 3 show the results of E. coli W3110 (DE3) / pEcPCK and E. coli W3110 (DE3) / pEGFP / pEcPCK cultured in glucose-minimum medium and LB-glucose medium, respectively. The volumetric expression (fluorescence intensity, RF) was 367.1 ± 0.02 and 643.6 ± 0.03, respectively, and the specific expression (fluorescence intensity / g-cell) was 869.5 and 3984.6.

In addition, the pEGFP prepared in the above (1) was transformed into the PCK overexpressing Escherichia coli BL21 (DE) pECK prepared in Example 1 by electroporation to prepare Escherichia coli BL21 (DE3) / pEGFP / pEcPCK. Transformed Escherichia coli pEGFP prepared in (1) was transformed by electroporation. Transformed Escherichia coli BL21 (DE3) / pEGFP / pEcPCK and Control Escherichia coli BL21 (DE3) / pEcPCK were transformed into LB-glucose medium containing IPTG and antibiotics (per liter, peptone (5 g), tryptone (10 g), NaCl (10 g). ), Glucose (9 g / L), NaHCO ₃ (10 g), IPTG (1 mM), and antibiotics (ampicillin and kanamycin, 25 μg / mL, respectively, kanamycin 50 μg / mL for control) at 37 ° C. Incubated for 12 hours under conditions of 250 rpm. The expression level of eGFP was measured in the same manner as in the case of E. coli W3110 (DE3) / pEGFP / pEcPCK. The expression levels of E. coli BL21 (DE3) / pEGFP and BL21 (DE3) / pEcPCK / pEGFP in glucose-LB medium were 463.3 and 873.3, respectively, and the specific expression (RF / mg-cell) was 923.9 ± 5.1 and 1440.8 ± 104.9. . These results showed that the BL21 (DE3) strain, which co-expressed PCK and eGFP, increased about 56% exoprotein eGFP expression and 89% overall exoprotein eGFP expression per cell.

3-2. Preparation of Foreign Protein Beta-galactosidase (LacZ)

(1) Preparation of lacZ expression vector

Using genomic DNA of E. coli W3110 (Korena Collection of Type Culture, KCTC 2223) as a template and PCR using primers of SEQ ID NOs: 5 and 6 (denatured at 95 ° C for 40 seconds, and annealed at 67.5 ° C for 40 seconds) And 25 cycles of 1 min elongation at 72 ° C.) lacZ was amplified. PCR products corresponding to the lacZ gene were cloned into pGEM-T easy vector (Promega, Madison, Wis., USA) and confirmed by DNA sequencing (solgent, Daejeon, Korea). Thereafter, the obtained lacZ gene was subcloned into the XhoI-EcoRI site of pET41a (Novagen) to obtain pLacZ (LacZ expression vector based on pET41a, p _T7 , Km ^R ).

(2) transformation and expression

Carried out using the same method as in Example 1, E. coli BL21 (DE3) (F ^- the gal dcm (DE3) (by transforming the pEcPCK by electroporation in ^{_{Novagen Inc.) pck - ompT hsdS B}} (r B - m B E. coli BL21 (DE3) / pEcPCK, which is an overexpressed strain, was prepared, and pLacZ prepared in (1) was transformed by electroporation into the strain and control Escherichia coli BL21 (DE3), respectively. ) pEcPCK / pLacZ and BL21 (DE3) / pLacZ were added to glucose-minimum medium (liter, glucose (9 g / L), NaHCO ₃ (10 g), IPTG (1 mM), antibiotics (ampicillin and kanamycin, 25 μg / each). mL), and minerals) and LB-glucose medium (liter, peptone (5 g), tryptone (10 g), NaCl (10 g), glucose (9 g / L), NaHCO ₃ (10 g), IPTG (1 mM) ), And antibiotics (ampicillin and kanamycin, 25 μg / mL each) for 24 hours under conditions of 250 ° C. at 37 ° C. After 24 hours of anaerobic culture, cells were harvested and disrupted by sonication. -neat Beta-galacto of the lysate using phenyl-β-D-galactoside (ONPG) (Miller, J. 1972. Experiments in Molecular Genetics, p. 352-355. Cold Spring Harbor Laboratory, NY.) As substrate. Let the dehydratase activity was measured. When the yellow color development was stirred for 2 minutes at 30 ℃ ONPG was added to the cell lysate to complete the reaction by the addition of Na ₂ CO ₃ 500㎕ and measuring the activity in the OD _420nm and the OD _550nm respectively It was.

Figure 4 shows the activity of the foreign protein, beta galactosidase produced by the culture of cells (BL21 (DE3) pEcPCK / pLacZ) and control cells (BL21 (DE3) / pLacZ) according to an embodiment of the present invention do. The beta-galactosidase activity of BL21 (DE3) pEcPCK / pLacZ was about three times higher than BL21 (DE3) / pLacZ, which was similar to the expression of eGFP. That is, overexpression of PCK shows that cells with high intracellular ATP concentrations can be usefully used for the production of foreign proteins.

Example 4 Preparation of Metabolites by PCK Overexpressing Strains

Expression of foreign proteins by the strains overexpressing PCK and the subsequent production of metabolites were performed.

(1) Preparation of ygjE Expression Vector

The genomic DNA of E. coli W3110 (Korena Collection of Type Culture, KCTC 2223) is based on the gene ygjE, which encodes the active transporter, export intracellular succinate outward (YgjE) of E. coli, and the primers of SEQ ID NOs: 7 and 8, respectively. PCR was performed using (a cycle of 1 minute denaturation at 95 ° C., 1 minute annealing at 63 ° C., and 1 minute elongation at 72 ° C.) was amplified. PCR products corresponding to the ygjE gene were cloned into pGEM-T easy vector (Promega, Madison, Wis., USA) and confirmed by DNA sequencing (solgent, Daejeon, Korea). Thereafter, the obtained ygjE gene was subcloned into the EcoRI-XhoI site of pET41a (Novagen) to obtain pYgjE (YgjE expression vector based on pET41a, p _T7 , Km ^R ).

(2) transformation and expression

Carried out using the same method as in Example 1, E. coli BL21 (DE3) (F ^- the gal dcm (DE3) (by transforming the pEcPCK by electroporation in ^{_{Novagen Inc.) pck - ompT hsdS B}} (r B - m B E. coli BL21 (DE3) / pEcPCK, which is an overexpressing strain, was prepared, and pYgjE prepared in (1) was transformed by electroporation into the strain and control Escherichia coli BL21 (DE3), respectively. As a membrane-integrated transporter, transformed cells BL21 (DE3) /ygjE.pck and BL21 (DE3) / ygjE were subjected to anaerobic conditions with minimal glucose (liter, glucose (9 g / L), NaHCO ₃ (10 g)). , IPTG (50 μM), antibiotics (ampicillin and kanamycin, 25 μg / mL each, and minerals) were incubated for 24 hours at 37 ° C., 250 rpm.After 24 hours anaerobic culture, the activity of YgjE It confirmed by measuring the succinate concentration inside and outside.

5 shows the amount of succinate produced by anaerobic culture of cells (BL21 (DE3) /ygjE.pck) and control cells (BL21 (DE3) / ygjE) according to one embodiment of the invention.

Production of succinate, a culture product under anaerobic conditions, was increased in Escherichia coli BL21 (DE3) /ygjE.pck, which overexpresses PCK than control Escherichia coli BL21 (DE3) / ygjE. This result shows that cells with high intracellular ATP concentration according to the present invention can be usefully used for the production of foreign proteins and metabolites.

Example 5 Preparation of Spontaneous PCK Expression Strains and Preparation of Foreign Proteins Using the Same

(1) Preparation of Spontaneous PCK Expression Strains

In the genome of E. coli, the pck promoter regulated in the native condition is exchanged with the ppc promoter regulated in the glycolytic condition, thereby allowing pck to be expressed by the ppc promoter regulation in the relevant condition, thereby spontaneously without overexpression by the introduction of the plasmid vector. Cells were produced that produce higher energy.

First, PCR was performed using the genomic DNA of Escherichia coli W3110 as a template and using SEQ ID NOs: 9 and 10, respectively. Repeated cycles), PCR using SEQ ID NOs: 11 and 12 (25 cycles consisting of 1 minute denaturation at 95 ° C., 1 minute annealing at 61.5 ° C., and 1 minute elongation at 72 ° C.) and SEQ ID NO: PCR was performed using primers 13 and 14 (25 cycles of 1 minute denaturation at 95 ° C., 1 minute annealing at 69 ° C., and 1 minute elongation at 72 ° C.) to fragment I (pck upstream). , 1.5 kb), fragment II (ppc promoter region, 0.5 kb), and fragment III (pck structural region 1.6 kb) were amplified. The amplification products of fragments I, II, and III obtained above were cloned into T vectors (T & A cloning vector, RBC Co., Taiwan) to confirm the sequence through DNA sequencing, and then SmaI and XbaI, XbaI and PstI, and After treatment with PstI and HindIII, they were sequentially cloned into pTrc99A expression vector (AP Biotech, Uppsala, Sweden). After cloning, digestion with each restriction enzyme confirmed the DNA size. PTrc99A-I-II-III obtained by the cloning using the primers of SEQ ID NOs: 15 and 16 as a template, a total of 3.6 kb of fragment I-II-III in total (denatured for 40 seconds at 95 ° C, 25 cycles consisting of annealing for 40 seconds at 67.5 ° C. and 1 minute elongation at 72 ° C. were amplified and finally a temperature-sensitive suicide vector (Ts) for promoter exchange. suicide vector) subcloned into the NotI insert of pKOV (J. Bacteriology 171: 4617-4622 [1989]; donated by Harvard Medical School, Department of Genetics, Prof. George M. Church). The vector was then transformed into E. coli W3110 (DE3) using electroporation. Immediately after transformation, cells were cultured at 30 ° C. in LB liquid medium for 1 hour and cultured in LB solid medium containing chloramphenicol (50 ug / mL) at 42 ° C. to generate first crossover of colonies. To choose. The colonies were inoculated again in 1 mL LB liquid medium and incubated for about 5 hours with stirring at 200 rpm at 30 ° C. Immediately, LB solid medium containing 5% sucrose and 5% sucrose and chloramphenicol (85 ug / mL) were added. After smearing in an LB solid medium containing the culture was selected at 30 ℃ strains that lost resistance to chloramphenicol to select the cells having a double crossover (double crossover). 6 schematically shows homologous recombination exchanging the promoter of the pck gene with the promoter of the ppk gene on the genome of E. coli K12. Using the genome of the selected strain as a template, PCR was performed using primers of SEQ ID NOs: 15 and 16 (denature for 40 seconds at 95 ° C, annealing for 40 seconds at 67.5 ° C, and extension for 1 minute at 72 ° C). 25 cycles of the constructed cycle were performed.) The E. coli strain whose promoter of pck gene was exchanged with the ppc promoter was identified and named as HEC01. FIG. 7A shows a schematic diagram of the spontaneous energy cell genome in which the promoter of pck prepared in this example was exchanged with the ppc promoter, and FIG. 7B shows the biochemical reaction occurring in the cells. Finally, the cells were cultured using the method described in (2) of Example 2 and the intracellular ATP concentration was measured. Intracellular ATP concentrations were determined by measuring HEC01 strains grown actively in glucose minimal media, and the concentration of 35.81 ± 0.08 nmol / g-cell was about 50% higher than the 24.73 ± 1.26 nmol / g-cell of W3110 (DE3). It was confirmed that the above improvement. In addition, this concentration was confirmed to show an intracellular ATP concentration similar to 37.41 ± 1.88 nmol / g-cell of the W3110 (DE3) / pEcPck strain overexpressed pck by IPTG induction.

(2) Preparation of transformed and expressed foreign protein (eGFP)

PEGFP prepared in Example 3-1 was transformed by HEC01, which is a spontaneous PCK expressing energy cell prepared in (1), and E. coli W3110 (DE3), which is a control group, respectively, by electroporation. Transformed cells HEC01 / pEGFP and W3110 (DE3) / pEGFP were converted to glucose-minimum medium (liter, glucose (9 g / L), NaHCO ₃ (10 g) containing IPTG (isopropyl-β-D-thiogalactopyranoside) and antibiotics. ), IPTG (1 mM), antibiotics (ampicillin and kanamycin, 25 μg / mL each, kanamycin 50 μg / mL for control, and minerals) and LB-glucose medium (per liter, peptone (5 g), tryptone ( 10 g), NaCl (10 g), glucose (9 g / L), NaHCO ₃ (10 g), IPTG (1 mM), and antibiotics (ampicillin and kanamycin, 25 μg / mL, respectively, 50 μg of kanamycin for control) / mL)) was incubated for 12 hours under conditions of 37 ℃, 250 rpm. To estimate the expression level of eGFP, cultures (700 microliters) were placed in a fluorescence spectrometer (RF-5301PC, Shimadzu, Kyoto, Japan) under excitation at 395 nm and emission conditions at 509 nm. Cells were observed using a phase contrast microscope and a fluorescence microscope (LSM 510, Carl Zeiss). The expression amount (fluorescence intensity) of E. coli HEC01 / pEGFP was 194.45 ± 9.27, which was about 2 times higher than that of 100.4 ± 9.35 of E. coli W3110 (DE3) / pEGFP.

1 shows the reaction catalyzed by phosphoenolpyruvate carboxykinase (PCK) and phosphoenolpyruvate carboxylase (PPC) in E. coli. P and c in the subscripts indicate that the promoter and chromosomal genes, respectively.

Figure 2 shows the results of observation under phase contrast and fluorescence microscopy of foreign protein eGFP (enhanced Green Fluorescent Protein) produced by culturing cells in glucose-minimal media according to one embodiment of the invention. do.

3 shows the results of observation under phase contrast and fluorescence microscopy of foreign protein eGFP produced by culturing cells in LB (Luria Bertani) -glucose medium in accordance with one embodiment of the present invention.

Figure 4 shows the activity of the foreign protein, beta galactosidase produced by the culture of cells (BL21 (DE3) pEcPCK / pLacZ) and control cells (BL21 (DE3) / pLacZ) according to an embodiment of the present invention do.

5 shows the amount of succinate produced by anaerobic culture of cells (BL21 (DE3) /ygjE.pck) and control cells (BL21 (DE3) / ygjE) according to one embodiment of the invention.

FIG. 6 schematically shows homologous recombination in which a promoter of the pck gene is exchanged for a promoter of the ppk gene on the genome of E. coli K12 to prepare a cell (HECO1) according to an embodiment of the present invention.

7 shows the genomic structure of E. coli HEC01, a spontaneous pck expressing cell, and E. coli W3110 (DE3), a control cell, according to an embodiment of the present invention, B is a biochemical that occurs in the pathway by PCK expression in E. coli HEC01. Indicates a reaction.

<110> Industry-Academic Cooperation Foundation of the Catholic University of Korea <120> High energy charged cell and use <160> 16 <170> KopatentIn 1.71 <210> 1 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> forward primer for pck <400> 1 gaattcatgc gcgttaacaa tggtttgacc cc 32 <210> 2 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> backward primer for pck <400> 2 ctgcagttac agtttcggac cagccgctac 30 <210> 3 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> forward primer for eGFP <400> 3 gaattcatgg tgagcaaggg cgagga 26 <210> 4 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for eGFP <400> 4 ctcgagcttg tacagctcgt ccatgcc 27 <210> 5 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> forward primer for lacZ <400> 5 gaattcatgg tgagcaaggg cgagga 26 <210> 6 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for lacZ <400> 6 ctcgagcttg tacagctcgt ccatgcc 27 <210> 7 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> forward primer for ygjE <400> 7 gaattcatga aaccttccac tgaatggt 28 <210> 8 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for ygjE <400> 8 ctcagattaa agcaacacca cgggcat 27 <210> 9 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> forward primer for fragment I <400> 9 cccgggggct tcgtccccag ttttccatcc t 31 <210> 10 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for fragment I <400> 10 tctagatggc tggcgctagt gtttattgcc 30 <210> 11 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> forward primer for fragment II <400> 11 tctagaacct acagtgactc aaacgatgc 29 <210> 12 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for fragment II <400> 12 ctgcagatta ccccagacac cccatcttat 30 <210> 13 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> forward primer for fragment III <400> 13 ctgcagatgc gcgttaacaa tggtttgac 29 <210> 14 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for fragment III <400> 14 aagcttttac agtttcggac cagccgcta 29 <210> 15 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> forward primer for fragment I-II-III <400> 15 gcggccgcgg cttcgtcccc agttttccat cc 32 <210> 16 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for fragment I-II-III <400> 16 gcggccgctt acagtttcgg accagccgct 30  

Claims (17)

Introducing a gene encoding a target substance from the outside into a cell having an increased intracellular ATP concentration compared to a parent cell, Culturing the cells under conditions suitable for producing the target material, and Recovering the target material from the culture; Cells with increased intracellular ATP concentration compared to the parental cells are exchanged for the promoter of the gene encoding phosphoenolpyruvate carboxykinase by the promoter of the gene encoding phosphoenolpyruvate carboxylase. The expression of a gene encoding phosphoenolpyruvate carboxykinase (PCK) relative to the parental cell is E. coli, which is changed to increase intracellular ATP concentration. How to produce a substance. delete The method of claim 1, wherein the target substance is a protein, amino acid, nucleic acid, antibiotic, or metabolite. delete delete delete delete delete delete delete delete As a cell having an increased intracellular ATP concentration compared to the parental cell by a change in the expression of a gene encoding phosphoenolpyruvate carboxykinase (PCK), the expression change of the gene is the phosphoenolpyruvate carboxykinase A cell which is caused by being exchanged with a promoter of a gene encoding a phosphoenolpyruvate carboxylase and is Escherichia coli. delete delete The cell according to claim 12, wherein the cell has introduced a foreign gene encoding a target substance. The cell of claim 15, wherein the target substance is a protein, amino acid, nucleic acid, antibiotic, or metabolite. delete

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