KR20190055655A - Non-Genetically Modified Escherichia Strain for Expressing Membrane Protein and Method for Producing Membrane Protein using the Same - Google Patents

Abstract

The present invention relates to a non-genetically modified strain of colon bacillus for producing membrane proteins and a method of producing membrane proteins using the same. The present invention provides a modified strain of Escherichia sp. for producing membrane proteins, which comprises genes encoding the membrane proteins and has increased activity of diguanylate cyclase compared to a mother strain thereof. By using the non-genetically modified strain of colon bacillus according to an embodiment of the present invention, the membrane proteins may be efficiently produced.

Description

TECHNICAL FIELD [0001] The present invention relates to a non-recombinant Escherichia coli strain for membrane protein expression and a membrane protein production method using the same,

The present invention relates to a non-recombinant E. coli strain for membrane protein expression and a method for producing membrane protein using the same.

Membrane proteins account for about 25% to 30% of the biological proteome, and are various proteins involved in basic energy metabolism such as respiration and photosynthesis, communication between cells and cells, or external communication with cells, transport of substances, and lipid metabolism. Membrane proteins are an important target for drug development, and about 50% of commercially available drugs have been reported to act on G-protein coupled receptors (GPCRs), a type of membrane protein Lundstrom, K., Bioorg. Med. Chem. Lett., 15: 3654, 2005). Membrane proteins can be immobilized on various carriers unlike conventional water-soluble proteins, and thus can be safely developed with new medicines, foods, and bioconversion enzymes. However, even though it is an economically important protein, studies on the function or structure of the membrane protein are insufficient, because it is difficult to express and isolate the membrane protein as compared with the water-soluble protein.

Microorganisms that efficiently produce useful substances such as amino acids, antibodies, nucleic acids and organic acids which can be used for medical, industrial, etc., and methods using the microorganisms have been actively studied. However, when microorganisms are artificially manipulated to produce useful substances, safety problems due to recombination of genes may arise.

The present inventors completed the present invention by carrying out a study to develop a microorganism which can produce membrane protein safely and efficiently without genetic recombination.

1. Korean Patent No. 10-1197129

 1. Plant Physiol., 1987; 84: 25-30.  2. Biochemistry, 2009; 48: 10948-10955.

It is an object of the present invention to provide an Escherichia genus mutant having increased productivity of membrane protein versus parent strain.

Another object of the present invention is to provide a method for producing a membrane protein using Escherichia genus mutant strains with increased productivity of membrane protein versus parent strain.

In order to accomplish the above object, one aspect of the present invention is a method for producing a membrane protein, which comprises a gene encoding a membrane protein and which has an increased activity of diguanylate cyclase relative to the parent strain ( Escherichia ) ≪ / RTI >

As used herein, the term " mother strain " refers to a strain prior to the occurrence of a mutation, and is used interchangeably with an ancestral strain or a wildtype strain.

In one embodiment of the present invention, the parent strain may include a gene encoding a membrane protein.

As used herein, the term " membrane protein " refers to a protein or glycoprotein that is introduced into the lipid bilayer of the cell membrane. Membrane proteins may penetrate the lipid bilayer of the cell membrane, or may be located in the surface layer of the cell membrane, and may be fixed to the cell membrane by binding to other proteins.

As used herein, the term Escherichia mutant strain for membrane protein production refers to a strain capable of producing or secreting a membrane protein, which produces membrane protein in a larger or higher concentration than the parent strain, ancestor strain or wild type strain ≪ / RTI >

In one embodiment of the present invention, the Escherichia spp. Strain may be an evolutionary strain with increased productivity of a membrane protein selected through the evolution of parent strain or ancestor strain, and genetically modified organism (GMO ) Is a non-genetically modified strain that is not artificially modified.

In one embodiment of the present invention, the Escherichia spp. Mutant strain is characterized in that a locus- specific mutation occurs in the dgcQ gene (SEQ ID NO: 1) encoding guanylate cyclase to increase the activity of guanylate cyclase .

In one embodiment of the present invention, the Escherichia genus mutant strain may comprise a mutation in which the 1082nd nucleotide in the dgcQ gene (SEQ ID NO: 1) sequence is substituted with C to A.

In one embodiment of the present invention, the Escherichia spp. Strain may be Escherichia coli .

In one embodiment of the present invention, the Escherichia spp. Strain may be prepared by introducing into the parent strain a mutation with increased productivity of membrane protein, which occurs in the evolution process.

In one embodiment of the invention, the membrane protein is global to five bakteo (Gloeobacter) derived from the rhodopsin (rhodopsin), or atpF gene ATP encoded by (SEQ ID NO: 4) synthase (ATP synthase) F ₀ portion of b Subunit and may be a dicarboxylate transporter that is encoded by the dctA gene (SEQ ID NO: 5).

As used herein, the term ' Gloeobacter ' is a unicellular Cyanobacterium , which has a photosynthetic apparatus and respiratory system in the cell membrane instead of a thylakoid membrane ( Rippka Ret. Al., Arch Microbiol . 100: 419-436, 1974). Glow O bakteo genome a gene with a rhodopsin and the homology of microbial was found in the in the gene because the protein encoded by the gene to pump protons as proteosome rhodopsin (proteorhodopsin, PR) is GR (Gloeobacter Rhodopsin).

In one embodiment of the present invention, the Escherichia spp. Mutant strain comprises a GR gene sequence (SEQ ID NO: 2) that encodes rodeobacterium rhodopsin, and the gene includes a gene transfer And can be included in Escherichia spp.

Another aspect of the present invention relates to a method for producing Escherichia sp. Collecting the culture liquid; And obtaining a membrane protein from the recovered culture medium.

As used herein, the term " culture " means that the microorganism is grown under suitably controlled environmental conditions. The method of culturing the microorganism of the present invention can be carried out according to a suitable culture medium and culture conditions known in the art and can be easily adjusted by those skilled in the art depending on the selected strain. Examples of the culture method may include a batch culture, a continuous culture, and a fed-batch culture.

As used herein, the term " media " refers to a medium used for culturing a membrane protein producing strain, wherein the strain comprises a carbon source, a nitrogen source and inorganic salts so as to produce a membrane protein. The carbon source may be selected from the group consisting of starch, glucose, sucrose, galactose, fructose, glycerol, and mixtures thereof, for example, glycerol. The nitrogen source may be selected from the group consisting of ammonium sulfate, ammonium nitrate, sodium nitrate, glutamic acid, casamino acid, yeast extract, peptone, tryptone, soybean meal and mixtures thereof. The minerals include sodium chloride, potassium phosphate, magnesium sulfate And a mixture thereof can be used.

The step of obtaining the membrane protein may be carried out by a suitable method known in the art according to the culture method of the microorganism of the present invention, for example, batch, continuous, or fed-batch culture method, .

The membrane protein can be efficiently produced using the non-recombinant E. coli strain according to one embodiment of the present invention.

FIG. 1 shows the absorbance (OD ₆₀₀ ) of E. coli W3110 / pKJ606-GR strain cultured for 88 days in a chemostat fermenter. The upward arrow indicates the time point when a fresh medium was supplied to the storage tank .
FIG. 2 shows the result of observing the morphology of cells with a scanning electron microscope while culturing the E. coli W3110 / pKJ606-GR strain (ancestor strain) in a Kimostat fermenter for 88 days under light irradiation conditions: E. coli W3110 / pKJ606-GR strain (x10,000 magnification) observed first; b is an evolved E. coli W3110 / pKJ606-GR strain (x9,000 magnification) observed at 30 days of culture; And c is an evolved E. coli W3110 / pKJ606-GR strain (x10,000 magnification) observed at 88 days in culture.
Figure 3 shows the results of observing the cell morphology of the E. coli ET5 / pKJ606-GR strain by SEM before and after ash culture: a was the E. coli ET5 / pKJ606-GR strain (x10, 000 magnification); b is the E. coli ET5 / pKJ606-GR strain (x10,000 magnification) observed after ash culture; c was E. coli ET5 / pKJ606-GR strain (x80,000 magnification) observed before ash culture; And d is the E. coli ET5 / pKJ606-GR strain (x80,000 magnification) observed after ash culture.
Figure 4 shows the results of comparing the proton pumping ability of the E. coli W3110 / pKJ606-GR and E. coli ET5 / pKJ606-GR strains: The inset in the figure shows the proton pumping level The pH of the saline solution was measured.
FIG. 5 shows the results of confirming the number of gliobacteridopsis expressed in the E. coli W3110 / pKJ606-GR strain and the E. coli ET5 / pKJ606-GR strain.
FIG. 6 shows the results of comparing the b-subunit production ability of the ATP synthase F ₀ site in the E. coli W3110 / pTrc99a-atpF and E. coli ET5 / pTrc99a-atpF strains.
Figure 7 shows the results of comparing the ability of E. coli W3110 / pTrc99a- dctA and E. coli ET5 / pTrc99a-dctA to produce dicarboxylate transporters.

Hereinafter, one or more embodiments will be described in more detail by way of examples. However, these embodiments are intended to illustrate one or more embodiments, and the scope of the present invention is not limited to these embodiments.

Experimental Method

1. Microbial strains, medium and inducing adaptive evolution

J. Nanosci. Nanotechnol, 2011; 11:. Glow bakteo O (referred to Gloeobacter rhodopsin, hereinafter GR) derived from the rhodopsin disclosed 4261-4264 cryptographic pKJ606-GR plasmid (SEQ ID NO: 3), E. coli W3110 (Escherichia coli W3110) and used as an ancestral strain for adaptive evolution. Chemostat cultures of ancestor strains (hereinafter referred to as W3110 / pKJ606-GR strains) were performed by light transformation with some modification of M9 minimal medium.

The composition of M9 minimal medium used for the culture is as follows: 1 g / L _{glucose, 0.8 g / L NH 4 Cl} , 0.5 g / L NaCl, 7.5 g / L Na 2 HPO 4 · 2H 2 O, 3 g / L 5 μM all-trans-retinal (Cat. No. R-1) dissolved in KH ₄ PO ₄ , 0.2 g / L MgSO ₄ .7H ₂ O, 0.1 g / L CaCl ₂ , 50 μg / ml ampicillin, 0.1 mM IPTG, 2500, Sigma-Aldrich Co., USA), 1 mg / L thiamine HCl.

The culture medium was used to induce the evolution of the W3110 / pKJ606-GR strain (ancestor strain) as described below.

One single colony of W3110 / pKJ606-GR strain was taken and inoculated into 3 ml of M9 minimal medium and cultured at 37 DEG C for 16 hours with stirring at 200 rpm. 1 ml of the culture broth was collected and inoculated into a minichemostat fermenter jar (Hanil Inc., Korea) containing 100 ml of M9 minimal medium. In the fermenter, four 1W LED bulbs were installed at intervals of 1 cm and used as a light source. The fermenter was operated under conditions of 37 ° C, 200 rpm and ventilation (100 ml / min) and continuously irradiated with light. The 20 L medium reservoir was fed with fresh medium of the same composition as the initial culture medium whenever depleted. The reservoir was packed with aluminum foil to prevent the loss of light-sensitive retinal activity. The inlet and outlet tubes were controlled with a peristaltic pump at a rate of 10 ml / L, which corresponds to a dilution rate of 0.1 h ^-1 . 1 ml of the culture broth was collected from the effluent tube for 88 days to measure the absorbance (OD ₆₀₀ ).

2. Identification of microorganism type

The culture solution recovered from the outflow tube was confirmed (1,000X magnification) with a binocular microscope (BX41, Olympus, Japan) daily. Also, chymotrypsin stat culture in E.coli population (population) and the evolved descendants E. coli (evolved descendant E. coli) to the culture day 0, 30 and 88 primary linear scanning electron microscope (S-4800, Hitachi, Japan ) Respectively.

Cultures of chymostat on day 88 of culture were plated on M9 minimal medium agar plates (1.5% agar). A single colony was randomly selected to be named E. coli ET5 (5-㎛-long evolved type) after incubation at 37 ° C for 48 hours with light irradiation (using a 13 W bulb at 10 cm intervals) Respectively. E. Since the E. coli ET5 strain contains the pKJ606-GR plasmid in the same way as the W3110 / pKJ606-GR strain, the ET5 strain containing the pKJ606-GR plasmid will be described as the ET5 / pKJ606-GR strain hereinafter.

3. Photo nutrition  Growth Measurement ( 광학  growth measurement)

Cells were batch-cultured to determine the extent of photo-nutrient growth of W3110 / pKJ606-GR strain (ancestor strain) and ET5 / pKJ606-GR strain (evolutionary strain). Specifically, 50 ml of the medium was added to a 250 ml Erlenmeyer flask, and the strain was inoculated and cultured in a 37 ° C agitating incubator (13 W bulbs were installed at intervals of 20 cm) at 200 rpm for 24 hours. The same medium as the chymostat culture was used except that the glucose concentration was 2 g / L, and the degree of cell growth was determined by measuring the absorbance (OD ₆₀₀ ). For chemotrophic growth, the flasks were packed with aluminum foil and measured under light-shielded conditions. Photoperiodic growth was calculated from cell growth in light condition minus cell growth in dark condition. The degree of cell growth was measured three times.

4. Proton Pumping  Proton pumping measurement

W3110 / pKJ606-GR (ancestor strain) and ET5 / pKJ606-GR strain (an evolutionary strain) were cultured in order to confirm the level of proton pumping by GR. The cells in the log phase were collected by centrifugation (3,600 rpm, 10 min and room temperature) and the cell pellet was resuspended in saline (10 mM NaCl, 10 mM MgSO ₄ .7H ₂ O, and 0.1 mM CaCl ₂ ) Lt; / RTI > The washed cell pellet was placed in a spectrophotometric cuvette (path length 1 cm) and irradiated with light at an intensity of 100 W / m < 2 >. Protected CuSO ₄ filter _{(heat-protecting CuSO 4 filter)} -; (> 440 ㎚, Sigma Koki SCF50S-44Y, Japan short-wave cutoff filter), a focusing convex lens (focusing convex lens) and heat light irradiation, the short wavelength cut-off filter Respectively. Proton pumping levels were determined by measuring the pH for 1 min with an F-51 pH meter (Horiba Ltd., Kyoto, Japan) and repeated three or more times. After confirming the difference in the proton concentration, the proton concentration was calculated as pH = -log ₁₀ [H ⁺ ] by considering the correction by adding the acid.

5. Genome analysis

Genomic DNA and plasmid DNA of W3110 / pKJ606-GR strain and ET5 / pKJ606-GR strain (evolutionary strain) were purified using Wizard genomic DNA purification kit (Promega, USA) and Plasmid miniprep kit (Qiagen, Germany) Respectively, according to the protocol of the manufacturer. For the sequencing, the separated genomic DNA and plasmid DNA were mixed at a mass ratio of 100: 1, and the average insert size was 600 bp using an Illumina TruSeq Nano DNA kit (San Diego, Calif., USA) Sequencing library construction was constructed. Sequencing of the sequencing library constructs was carried out using the Illumina MiSeq platform (San Diego, Calif., USA) at the Korea Research Institute of Bioscience and Biotechnology, Korea, and a 300-cycle paired-end sequencing method Respectively. Read preprocessing was performed with a quality trim limit of 0.01, one uncertain nucleotide per reading, and a minimum read length of 100 bp. Reference mapping and fixed ploidy variant detection including insertion deletion (indel) and structural variant detection were all performed using CLC Genomics Workbench 8.0 (Insilicogen, Korea). E. The published complete genome sequence of the E. coli W3110 strain (RefSeq NC_007779.1) and the sequence of pKJ606-GR (SEQ ID NO: 3) were used as reference sequences.

6. Transduced E. coli  Strain production

6-1. HK769 strain ( KmR -tagged dgcQ ^C1082A  Strain) production

E. coli JW1912 strain [#CGSC (Coli Genetic Stock Center) 9590] was obtained from the Keio collection, which is an open reading frame of the amyA gene encoding alpha- amylase (kanamycin It has a resistance gene (kanamycin resistance gene; KmR). The kanamycin resistance gene is located 20 kb away from the dgcQ ^C1082A mutation in the chromosome. By introducing Δ amyA :: KmR transformants in ET5 / pKJ606 GR-strain was tested as described below to produce strain HK769.

6-1-1. P1 of JW1912 strain Melt ( lysate ) Produce

The strain JW1912 (donor strain) was inoculated overnight in 5 ml of LB liquid medium. On the next day, 50 μl of the above culture was added (1: 100 dilution) to 5 ml of LB liquid medium containing 50 μl of 20% glucose and 25 μl of 1M CaCl ₂ and incubated at 37 ° C for 30 minutes to 45 minutes. One drop of P1vir phage stock (10 ⁹ to 10 ¹⁰ pfu / ml; inventor's own) was added and further cultured for 2 to 4 hours until the cells were dissolved. When the cells were sufficiently dissolved, 50 μl of chloroform was added, vortexed for 30 seconds, and left for 15 minutes. The P1 lysate of strain JW1912 was transferred to a sterile centrifuge tube and stored at 4 < 0 > C.

6-1-2. Transduction

The ET5 / pKJ606-GR strain (donor strain) was inoculated in 5 ml of LB liquid medium overnight, and 1.5 ml of the culture broth was centrifuged at 14,000 rpm for 2 minutes to recover the cell pellet. The cell pellet P1 salt solution (10 mM CaCl, 5 mM MgSO 4) was suspended in 0.75 ㎖, captured in a test tube with a sterile suspension 100 ㎕. Three drops of the P1 lysate of the strain JW1912 prepared in Example 6-1-1 were added to the test tube and cultured at 30 DEG C for 30 minutes. Then, 1 ml of LB liquid medium and 200 占 퐇 of 1M sodium citrate were added to the test tubes and further cultured at 37 占 폚 for 1 hour. The culture broth was centrifuged (14,000 rpm, 2 minutes) to recover the cell pellet, and the pellet was resuspended in 100 μl of LB liquid medium. A portion of the suspension was plated on LB selective medium plates containing 5 mM sodium citrate and incubated overnight. One single colony was taken from the plate, streaked on selective medium plates containing 5 mM sodium citrate, and incubated overnight. The next day, a single colony was picked from the plate, streaked on selective medium plates, and cultured to obtain transformed strain HK769. Transduction was confirmed by DNA sequencing.

6-2. HK775 strain ( E. coli Δ amyA :: KmR , dgcQ ^C1082A Production

In the same manner as in 6-1 above, the strain HK774 ( E. coli Δ amyA :: KmR, dgcQ ^WT ) by introducing a point mutation of dgcQ ^C1082A into the homologous genotype HK775 strain (isogenic HK775 strain; E. coli ? AmyA :: KmR, dgcQ ^C1082A ). However, strain HK769 was used as a donor strain and strain HK774 was used as the strain. The strain HK774 is a strain produced by introducing Δ amyA :: KmR trait into W3110 strain.

7. Intracellular cdiGMP  Concentration measurement

Intracellular c-di-GMP concentrations were determined using methods known in the art (Methods Mol Biol. 2014; 1149: 271-279). (Strain E. coli W3110 dgcQ ^WT / pKJ606-GR) and strain ET5 / pKJ606-GR (genotype E. coli W3110 dgcQ ^C1082A / pKJ606-GR) trans-retinal and ampicillin (50 [mu] g / ml) in a minimal medium supplemented with M9 glucose.

When the OD ₆₀₀ value reached 0.5, the culture was centrifuged (6000 x g, 4 캜 and 10 minutes), the supernatant was discarded, the cells were quickly frozen with liquid nitrogen and stored at -80 ° C. After dissolving the frozen cells in the tube, 500 μl of an extraction buffer (methanol: acetonitrile: 0.1 N formic acid solution = 40: 40: 20) and an internal standard (3 ', 5'-cyclic xanthosine monophosphate, cXMP; Sigma -Aldrich, USA). The tube was sealed tightly and the cells were disrupted by freezing and thawing three times with liquid nitrogen and heating block (30 ° C). The tubes were then placed on ice for 30 minutes and then centrifuged (10,000xg, 4 ° C and 10 minutes). 400 μl of the supernatant was transferred to a new tube, concentrated by vacuum centrifuge, and resuspended by adding 100 μl of triple distilled water (TDW). The resuspended sample was centrifuged again (10,000xg, 4 < 0 > C and 30 minutes) to remove the solids and the supernatant was stored at -80 < 0 > C. The concentration of c-di-GMP contained in the supernatant was measured using a C18 column (Hypersil Gold column, 2.1 x 100 mm, 1.8 쨉 m; Thermo Fisher Scientific, USA) and a mass spectrometer (TSQ Quantum Access MAX Quadrupole MS, Thermo Fisher Scientific) And analyzed using an attached LC-MS / MS (Accela 1250 UPLC system, Thermo Fisher Scientific).

8. GR  Measurement of protein expression level

Ampicillin in 3 ㎖ LB liquid medium containing (50 ㎍ / ㎖) W3110 / pKJ606-GR strain and ET5 / pKJ606-GR respectively inoculated with stock (stock) 10 ㎕ of the strain at 37 ℃ until this OD ₆₀₀ 1 Lt; / RTI > 50 ml of M9 minimal medium was added to an Ellenmeyer flask, and the above culture was inoculated and further cultured. The composition of the M9 glucose minimal medium used is as follows: 1 g / L _{glucose, 1 g / L NH 4 Cl} , 0.5 g / L NaCl, 12.8 g / L Na 2 HPO 4 · 7H 2 O, 3 g / L KH _{2 PO 4, 2 mM MgSO 4} , 0.1 mM CaCl 2, 50 ㎍ / ㎖ ampicillin and dissolved in ethanol 5 μM all-trans-retinal ( Cat. Number R-2500, Sigma-Aldrich Co., USA) was added to 1 mg / L thiamine HCl.

To induce GR expression, IPTG (0.01 or 0.1 mM) was added and cultured until OD ₆₀₀ was 1. In the lighted incubator, the irradiated group was cultured as a flask, and the light non-irradiated group was cultured by packing the flask with aluminum foil. When the culture was completed, 1 ml of the culture solution was recovered and the OD ₆₀₀ was measured. The remaining culture was centrifuged to collect the pellet. The collected pellets were frozen in liquid nitrogen and stored at -80 ° C.

Cells were unfastened by adding unbuffered solution to the thawed pellet, and OD of 350 nm to 750 nm wavelength was measured with a UV-2450 UV visible spectrophotometer (Shimadzu, Japan). Using the OD measurement results, an exponential curve was created through the origin 61 program (OriginLab, USA) over the points corresponding to 350 ㎚ and 750 ㎚, and the difference from the OD measurement result was displayed in a graph. The value of the point indicated by the peak in the graph was obtained, and the number of GR was calculated according to the following equation (1). In addition, the cell number was measured with a hemocytometer, and the number of GRs expressed on the surface of one cell was calculated by dividing the estimated number of GRs by the number of cells.

9. The strain of ET5 Membrane protein  Capacity measurement

9-1. Transformation

The plasmids contained in the W3110 / pKJ606-GR strain and the ET5 / pKJ606-GR strain were cured. Specifically, the W3110 / pKJ606-GR strain and the ET5 / pKJ606-GR strain were cultured overnight in an LB liquid medium supplemented with 33 μM novobiocin, and then plated on an LB plate. Once colonies were formed, replica plating was performed on LB plates containing ampicillin (50 μg / ml) to further culture, and strains that did not grow in the ampicillin medium were selected. The plasmid-free W3110 / pKJ606-GR strain and the ET5 / pKJ606-GR strain are described as W3110 strain and ET5 strain, respectively.

(Encoding the b subunit of the F ₀ part in the ATP synthase, SEQ ID NO: 4) atpF encoding the endometrial protein gene and dctA (dicarboxylate transporter encryption, SEQ ID NO: 5) by inserting the gene in each pTrc99a vector pTrc99a-atpF vector and pTrc99a -dctA vector. GFP (green fluorescent protein) sequence was added to the end of the atpF gene to confirm the expression of endogenous protein by fluorescence microscopy. Each vector was added to the W3110 and ET5 strains selected above, and transformed by thermal shock at 42 DEG C for 1 minute. Transformants were selected by plating on selective media containing ampicillin (50 占 퐂 / ml): W3110 / pTrc99a-atpF strain, W3110 / pTrc99a-dctA strain, ET5 / pTrc99a-atpF strain and ET5 / pTrc99a-dctA strain.

9-2. atpF ATP synthase F ₀  Measurement of b subunit expression at the site

10 스톡 stocks of W3110 / pTrc99a-atpF and ET5 / pTrc99a-atpF selected in 9-1 were inoculated in 3 ml of LB liquid medium and cultured at 37 캜 and 230 rpm for 12 hours. The culture solution was inoculated in 50 ml of LB liquid medium supplemented with glucose (2 g / L), and the culture solution was inoculated such that the OD ₆₀₀ was 0.1. IPTG was added at a concentration of 0.1 mM and cultured at 37 캜 for 3 hours at 180 rpm. After 3 hours, fluorescence was measured three times by fluorescence spectrophotometer (Synergy MX, BioTek) under the conditions of excitation 489 nm and emission 530 nm.

9-3. dctA Encoded by a gene Dicarboxylate Transporter  Active measurement

10 占 퐇 of stocks of W3110 pTrc99a- dctA and ET5 pTrc99a- dctA selected in 9-1 were inoculated and cultured in 3 ml of LB liquid medium at 37 占 폚 and 230 rpm for 12 hours. The culture solution was inoculated in 50 ml of LB liquid medium supplemented with glucose (2 g / L), and the culture solution was inoculated such that the OD ₆₀₀ was 0.1. IPTG was added at a concentration of 0.1 mM and cultured at 37 캜 for 6 hours at 180 rpm. After the incubation, the cell pellet was recovered by centrifugation (4 ° C, 4,000 rpm) for 10 minutes, and the pellet was resuspended in 5 ml of succinate salt in PBS (potassium buffer system; 10 g / L). The suspension was vortexed at 2.5 ml each and vortexed for 1 minute at 25 ° C. This was to give the time for the dicarboxylate transporter encoded by dctA to transport the succinate into the cell. The supernatant was separated by centrifugation, and the separated supernatant was separated by HPLC (silica gel), and the supernatant was separated by centrifugation, followed by separation (4 ° C and 4,000 rpm) and washing the pellet twice with PBS. And the amount of succinate in the cells was measured.

Experiment result

One. GR  Expressed E. coli W3110 ( E. coli  W3110 / pKJ606 - GR ) Adaptive evolution of strains

For adaptive evolution, the W3110 / pKJ606-GR strain (ancestor strain) was cultured in a chymostat fermentor for 88 days using M9 minimal medium with a limited concentration (1 g / L) of D-glucose, And continued research.

As a result of measuring the absorbance of the culture broth in the course of culturing the chymostat, it was confirmed that when the new medium was supplied to the storage tank as shown in FIG. 1, the absorbance was increased and then gradually decreased. In addition, the OD ₆₀₀ remained at 0.1 to 0.7 levels during chymostatic culturing.

Observation of cell morphology by microscope revealed elongated cells in the 15th day of culture. The number of cells elongated during the growth of chymostats gradually increased, and most of the cells were observed to be elongated at 88 days of culture.

The morphological characteristics of E. coli on the 30th day of culture (corresponding to 100th generation) and the 88th day of culture (corresponding to 300th generation) were further confirmed by scanning electron microscopy. As a result, the W3110 / pKJ606-GR strain at day 0 of culture showed a typical rod-shaped shape having a long axis in the range of 1.5 to 2.2 mu m (see Fig. 2 (a)). On the 30th day of culture, an evolved form of E. coli with an unusual elongation compared to the strain W3110 / pKJ606-GR was identified (see Fig. 2b). The cells with the longest length in the culture medium had a major axis length of 9.3 ㎛ and a minor axis length of 0.5 ㎛. The number of cells showing atypical morphology at 88 days of culture increased compared with the 30th day of culture (c in Fig. 2). A single colony was randomly selected from E. coli evolved for 88 days and named ET5 / pKJ606-GR strain (evolutionary strain).

2. ET5 / pKJ606 - GR  Strain Photo nutrition  growth

Cultured in a minimal medium in a light irradiation condition (light state) or in a non-irradiated state (dark state), W3110 strain (negative control), W3110 / pKJ606-GR strain (parent strain) and ET5 / pKJ606- And the absorbance was measured to confirm the degree of growth.

As a result, the growth of W3110 was not influenced by light irradiation because there was no intracellular phototrophic machinery as shown in Table 1 below. On the other hand, W3110 / pKJ606 growth of GR-strain if it is not irradiated with light was OD ₆₀₀ is 1.52, the light irradiation by OD ₆₀₀ is slightly increased to 1.75 optical vegetative growth was 0.23 at OD _600. In the case of the ET5 / pKJ606-GR strain, the photo-nutritional growth was 0.51 at OD ₆₀₀ , which was 2.2 times higher than the W3110 / pKJ606-GR strain.

Bacterium Explanation Cell growth (OD ₆₀₀ ) ^a Photo nutrition growth
Δ [BA] (multiples) Cancer status (A) State (B) W3110 Negative control group 1.78 ± 0.05 1.78 + 0.04 0 ^b W3110 / pKJ606-GR Ancestral strain 1.52 ± 0.05 1.75 + 0.08 0.23 (1.0) ET5 / pKJ606-GR Evolutionary strain 1.52? 0.00 2.03 ± 0.08 0.51 (2.2)

^a means absorbance average of at least 3 repeated measurements ± standard deviation (SD)

^{b The} wild-type E. coli W3110 strain showed no difference in growth between dark (A) and light (B)

From the above experimental results, it can be seen that the ET5 / pKJ606-GR strain evolved from the W3110 / pKJ606-GR strain to grow effectively under the light irradiation condition during the kimostat culture.

3. ET5 / pKJ606 - GR  Type of strain and genome variation

As a result of the development of the W3110 / pKJ606-GR strain by the chymostat culturing, atypically elongated cells were observed. Therefore, the isolated strain ET5 / pKJ606-GR was asymmetrically cultured and observed under a scanning electron microscope.

Observation of the strain ET5 / pKJ606-GR before and after ash culture at a magnification of 10,000 magnifications revealed no elongated cells (see FIG. 3 a and FIG. 3 b). On the other hand, it was confirmed that the ET5 / pKJ606-GR strain (Fig. 3 (d)) obtained by ash culture as compared with the pre-culture ET5 / pKJ606-GR strain (c in Fig. 3) had a more corrugated surface structure I could confirm.

The genomic and plasmid sequences of W3110 / pKJ606-GR and ET5 / pKJ606-GR strains were analyzed using the genomic sequence (NC_007779.1) of E. coli W3110 and the sequence of pKJ606-GR (SEQ ID NO: Respectively.

As a result, it was confirmed that the genomic sequence of the W3110 / pKJ606-GR strain was partially different from the published reference sequence (NC_007779.1) as shown in Table 2 below. Cross between W3110 / 16 single nucleotide mutations identified in the genomic sequence of pKJ606-GR strains (single nucleotide variants, SNV), 4 of short insertion deletion (indel), and e14 15.2 kb deletion of Pro gripping portion (icd and icdC ) Was commonly present in all cultured E. coli cells, so that the candidate gene for the newly acquired trait (increased photoperiodic growth) of the ET5 / pKJ606-GR strain obtained by the evolution .

Reference Position Type Length Reference Allele Consequence 547694 SNV One A G 547832 Insertion One - G 556858 SNV One A T 696892 SNV One C T 987574 SNV One G T 1093686 SNV One T C 1197929 SNV One C A Icd ^D401E 1519222 SNV One G T 1669599 Deletion One C - 1979957 SNV One G T 2005401 SNV One C T 2245455 SNV One A G 2945541 SNV One G A 3268825 SNV One A G 3276046 SNV One G T 3746911 SNV One G A 3755017 SNV One T A 3958123 Deletion 32 AN ₃₀ T - 4371271 Deletion 2 AA - 4591729 SNV One C T

In addition, no plasmid mutation was found in the ET5 / pKJ606-GR strain, which means that the GR protein itself encoded by the pKJ606-GR plasmid is identical in the W3110 / pKJ606-GR strain and the ET5 / pKJ606-GR strain . For sequence analysis ET5 / pKJ606-GR strain W3110 / pKJ606-GR compared to the strain was confirmed that the dgcQ gene occurred a single nucleotide point mutations (C1082A), Due to this between Dhigu not rate encoded by dgcQ gene The amino acid sequence of the diguanylate cylase was changed (A361E).

The genomic DNA sequence analysis results of W3110 / pKJ606-GR and ET5 / pKJ606-GR strains are shown in Table 3 below.

Bacterium W3110 / pKJ606-GR
(Ancestral strain) ET5
(Evolutionary strain) DNA chromosome Plasmid chromosome Plasmid Mutation - - dgcQ ^C1081A - Average coverage
(Average coverage) 479 58,504 468 90,534 Read (after trim)
x10 ⁶ 13.2 14.2 Base (after trim)
x10 ⁶ 2754 2980 Average length (after trim)
x10 ⁶ 208 210 % Of read mapping
x10 ⁶ 99.41 99.50

The trim ratios based on the number of readings are 87.48% and 88.79%, respectively.

4. Photo-inducible proton Pumping (light-driven proton pumping)

The photo-induced growth rate of ET5 / pKJ606-GR strain was significantly better than that of W3110 / pKJ606-GR strain. Therefore, the degree of pH reduction of unbuffered saline solution was measured to confirm the photo-induced proton pumping efficiency of both strains.

As a result, as shown in FIG. 4 W3110 / pKJ606 GR-strain whereas the proton pumping level ^{0.38 (extracellular ΔH + x10 -7 /} min OD), ET5 / pKJ606-GR strain is 3.12 (extracellular ΔH ⁺ x10 ^-7 / min OD), indicating that the proton pumping level of ET5 / pKJ606-GR strain was increased about 8 times. 4 is a result of measuring the pH change of the saline solution, and it can be confirmed that when the light is irradiated, the pH of the saline containing the strain ET5 is greatly changed.

In addition, the mutation was introduced into strain HK774 to determine if the cause of the increase in the photo-induced proton pumping was due to dgcQ ^C1082A mutation.

As a result, as shown in Table 4, it was confirmed that the photo-nutritional growth of HK774 strain was 0.10 at OD ₆₀₀ , while the photo-nutritional growth of HK775 strain was 0.51. The proton pumping efficiencies of the two strains showed similar results, indicating that the proton pumping efficiency of the HK775 strain was doubled by the single point mutation compared to the HK774 strain.

Bacterium HK774 HK775 Genotype E. coli Δ amyA :: KmR, dgcQ ^WT E. coli ? AmyA :: KmR, dgcQ ^C1082A Cancer Status Growth (A) 0.98 + 0.02 1.13 + 0.04 State growth (B) 1.08 + 0.04 1.64 + 0.03 Photo nutrition growth
Δ [BA] ^a 0.10 0.51 Proton pumping
^{(ΔH + x10 -7 / min OD} ) 0.475 + 0.020 0.903 + - 0.107

^a Light nutrient growth is calculated from the difference in growth in light state (B) and growth in dark state (A), and the growth result means the mean absorbance ± standard deviation (SD) of at least three repeated measurements

5. c- di - GMP  Level comparison

To confirm the effect of single point mutation (C1082A) on enzyme activity, the metabolite of guanylate cyclase c-di-GMP levels were measured.

As shown in Table 5, the cdiGMP level of W3110 / pKJ606-GR (genotype E. coli W3110 dgcQ ^WT / pKJ606-GR) cultured in the dark state was less than 0.3 μM / OD, whereas ET5 / pKJ606-GR (Genotype E. coli W3110 dgcQ ^C1082A / pKJ606-GR) strain, cdiGMP level was significantly increased to 6.46 μM / OD. The cdiGMP level of the W3110 / pKJ606-GR strain was less than 0.02 μM / OD, while the cdiGMP level of the ET5 / pKJ606-GR strain was remarkably increased to 7.16 μM / OD Could know.

Bacterium Genotype c-di-GMP ([mu] M / OD) Cancer status State W3110 / pKJ606-GR E. coli W3110 dGCQ ^WT / pKJ606-GR <0.03 &Lt; 0.02 ET5 / pKJ606-GR E. coli W3110 dGCQ ^C1082A / pKJ606-GR 6.46 3.43 7.16 ± 2.40

6. Membrane protein  Comparison of expression

6-1. GR  Protein expression

The number of GR proteins expressed in the W3110 / pKJ606-GR strain and the ET5 / pKJ606-GR strain was calculated. As a result, the results shown in Table 6 and FIG. 5 were obtained.

Number of rhodopsin / cell Cancer status State IPTG (mM) 0.01 0.1 0.01 0.1 W3110 / pKJ606-GR 9.63 × 10 ² ± 3.88 × 10 ² 5.84x10 ⁴ ± 2.10x10 ³ 6.87 × 10 ² ± 8.55 × 10 ¹ 8.51 × 10 ⁴ ± 1.22 × 10 ⁴ ET5 / pKJ606-GR 6.05 x 10 ² ? 1.41 x 10 ² 1.12x10 ⁶ ± 1.55x10 ⁵ 5.03x10 ² ± 1.20x10 ² 7.05 x 10 ⁵ ? 1.38 x ^{10 5}

As a result, the number of expressed GR proteins was found to be larger in the strain ET5 / pKJ606-GR than in the strain W3110 / pKJ606-GR. Specifically, the number of GR proteins was found to be about 19 times in the cancerous state and about 8 times in the light state, and it was found that the proton pumping efficiency of the ET5 / pKJ606-GR strain was high.

6-2. ATP synthase F ₀  Site b-subunit and dicarboxylate transporter expression

As a result of confirming that the membrane protein production capacity of the ET5 strain was increased, the membrane protein production ability .

As shown in Figure 6 to determine that the fluorescent signal of the high strain ET5 atpF It was found that the b subunit at the F ₀ site of the ATP synthase, which is encoded by the gene, was highly expressed. In addition, as shown in FIG. 7, it was confirmed that the content of intracellular succinic acid salt of ET5 strain was high, so that a dicarboxylate transporter encoded by the dctA gene was also expressed to a large extent.

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

<110> CATHOLIC UNIVERSITY INDUSTRY-ACADEMIC COOPERATION FOUNDATION <120> Non-Genetically Modified Escherichia Strain for Expressing          Membrane Protein and Method for Producing Membrane Protein using          the Same <130> PN170253 <160> 5 <170> KoPatentin 3.0 <210> 1 <211> 1695 <212> DNA <213> Escherichia coli <220> <221> gene &Lt; 222 > (1) .. (1695) <223> dGCQ gene <400> 1 gtgcagcacg agacaaaaat ggaaaaccag agctggttga aaaaactcgc acgccgcctg 60 gggcctggtc atgtcgttaa tctctgcttt atcgtggtat tgcttttttc caccttgctc 120 acctggcgtg aagtggtggt gctggaagat gcctatatct ccagccagcg taatcatctg 180 gaaaacgttg ccaacgcgct cgataagcat ttgcagtata acgtcgacaa actgatcttt 240 ttacgtaatg gcatgcgcga agctctcgta gcgccactgg atttcacctc tctgcgtgat 300 gctgtaaccg agttcgaaca gcatcgcgac gagcacgcct ggaaaatcga actcaaccga 360 cgacgcactc tgccagttaa tggtgtgtcg gatgcattag tcagcgaggg gaatctcctg 420 tctcgcgaaa atgaaagcct cgacaatgaa attaccgctg cactggaagt tggttacttg 480 ctgcgactag cgcacaactc ctcgtcgatg gttgaacagg cgatgtatgt ctcgcgtgcc 540 ggattttacg tttcgacgca gccgaccttg tttacgcgca atgttccaac gcgttattac 600 ggctatgtca cccaaccctg gtttattggc cattcacaac gagaaaatcg tcaccgcgcg 660 gtacgctggt tcacttcgca accggaacac gccagcaata ctgaaccgca ggttaccgtc 720 agtgttccgg tagacagtaa taactactgg tatggcgtgc tggggatgag tattcccgtg 780 cgtactatgc agcaattttt aagaaacgcc atcgataaaa acctcgatgg tgagtatcag 840 ctctatgaca gtaagctgag atttttgacc tcttccaatc ctgaccatcc aacagggaat 900 atttttgatc ctcgtgaact ggccttgctg gcgcaggcaa tggaacatga cacgcggggc 960 ggcattcgta tggacagtcg ctatgttagc tgggaacgtc tggaccattt cgacggtgtg 1020 ctggtgcgtg tccatacgtt aagcgaaggc gtgcgcggcg atttcggcag tatcagcatt 1080 gcattaaccc tgctgtgggc gctctttacc accatgttac tcatctcctg gtatgtgatt 1140 cgccggatgg ttagcaacat gtatgttctg caaagctcgt tgcagtggca ggcgtggcac 1200 gacaccttaa cgcgtttata taaccgtggc gcactgttcg aaaaagcccg tccgctcgct 1260 aaattgtgtc agacgcacca acatcctttt tctgtcattc aggtcgatct tgaccatttt 1320 aaagcgatta atgaccgctt tggtcatcag gcgggcgacc gtgttctttc tcatgctgcc 1380 ggattaatta gcagttcctt gcgtgcgcag gacgttgccg ggcgggtcgg tggtgaggag 1440 ttttgtgtga ttctgccagg cgcgagtctg acggaggctg cggaggtcgc agaacgtatt 1500 cgactgaaat taaatgaaaa agagatgttg atcgccaaga gtacgacgat acgcatcagt 1560 gcctcgctgg gggtaagcag cagcgaggaa accggtgatt atgattttga acaactccag 1620 tcactggccg accgtcggct ttatctcgct aaacaggctg ggcgtaatcg ggtattcgcg 1680 agcgataacg cttaa 1695 <210> 2 <211> 714 <212> DNA <213> Gloeobacter violaceus <220> <221> gene <222> (1). (714) <223> Gloeobacter Rhodopsin <400> 2 atacatatga atttggagag tcttttacac tggatttatg ttgcgggaat gacaattggt 60 gcattgcact tttggtcact tagtcgaaat ccgcgcggtg ttccccagta cgaatacctt 120 gtggcgatgt ttattcccat ttggtcggga ctagcctata tggcaatggc aatagaccaa 180 ggtaaagttg aagcggctgg gcaaattgcc cactatgccc gttatattga ttggatggtg 240 acaacaccat tattactgct atctctttct tggacagcga tgcagtttat caaaaaagat 300 tggacactca ttggtttttt gatgagtacc cagatagttg taattacctc tgggttaatc 360 gcagatttat ctgagcgcga ttgggtgaga tatctatggt atatctgcgg ggtttgcgct 420 tttctcatta ttctttgggg tatttggaat ccattgcgcg ccaaaaccag aactcaaagt 480 tcagaactag cgaacttata cgataagctt gttacttatt ttacagtgct ttggataggc 540 tacccaatag tctggattat tggccctagt ggttttggct ggataaatca aactatagat 600 acatttttgt tttgtctact gccctttttc tccaaggttg gatttagttt tctggattta 660 cacggcttac gtaatctcaa tgattcccgc caacatcacc accatcatca ctag 714 <210> 3 <211> 4386 <212> DNA <213> Artificial Sequence <220> <223> recombinant vector <400> 3 gaattccgac accatcgaat ggcgcaaaac ctttcgcggt atggcatgat agcgcccgga 60 agagagtcaa ttcagggtgg tgaatgtgaa accagtaacg ttatacgatg tcgcagagta 120 tgccggtgtc tcttatcaga ccgtttcccg cgtggtgaac caggccagcc acgtttctgc 180 gaaaacgcgg gaaaaagtgg aagcggcgat ggcggagctg aattacattc ccaaccgcgt 240 ggcacaacaa ctggcgggca aacagtcgtt gctgattggc gttgccacct ccagtctggc 300 cctgcacgcg ccgtcgcaaa ttgtcgcggc gattaaatct cgcgccgatc aactgggtgc 360 cagcgtggtg gtgtcgatgg tagaacgaag cggcgtcgaa gcctgtaaag cggcggtgca 420 caatcttctc gcgcaacgcg tcagtgggct gatcattaac tatccgctgg atgaccagga 480 tgccattgct gtggaagctg cctgcactaa tgttccggcg ttatttcttg atgtctctga 540 ccagacaccc atcaacagta ttattttctc ccatgaagac ggtacgcgac tgggcgtgga 600 gcatctggtc gcattgggtc accagcaaat cgcgctgtta gcgggcccat taagttctgt 660 ctcggcgcgt ctgcgtctgg ctggctggca taaatatctc actcgcaatc aaattcagcc 720 gatagcggaa cgggaaggcg actggagtgc catgtccggt tttcaacaaa ccatgcaaat 780 gctgaatgag ggcatcgttc ccactgcgat gctggttgcc aacgatcaga tggcgctggg 840 cgcaatgcgc gccattaccg agtccgggct gcgcgttggt gcggatatct cggtagtggg 900 atacgacgat accgaagaca gctcatgtta tatcccgccg ttaaccacca tcaaacagga 960 ttttcgcctg ctggggcaaa ccagcgtgga ccgcttgctg caactctctc agggccaggc 1020 ggtgaagggc aatcagctgt tgcccgtctc actggtgaaa agaaaaacca ccctggcgcc 1080 caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca 1140 ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc 1200 attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga 1260 gcggataaca atttcacaca ggaaacagct atgaccatga ttacggattc actggccgtc 1320 gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca 1380 catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccggggg atcctctaga 1440 aataattttg tttaacttta agaaggagat atacatatga atttggagag tcttttacac 1500 tggatttatg ttgcgggaat gacaattggt gcattgcact tttggtcact tagtcgaaat 1560 ccgcgcggtg ttccccagta cgaatacctt gtggcgatgt ttattcccat ttggtcggga 1620 ctagcctata tggcaatggc aatagaccaa ggtaaagttg aagcggctgg gcaaattgcc 1680 gt; tggacagcga tgcagtttat caaaaaagat tggacactca ttggtttttt gatgagtacc 1800 cagatagttg taattacctc tgggttaatc gcagatttat ctgagcgcga ttgggtgaga 1860 tatctatggt atatctgcgg ggtttgcgct tttctcatta ttctttgggg tatttggaat 1920 ccattgcgcg ccaaaaccag aactcaaagt tcagaactag cgaacttata cgataagctt 1980 gttacttatt ttacagtgct ttggataggc tacccaatag tctggattat tggccctagt 2040 ggttttggct ggataaatca aactatagat acatttttgt tttgtctact gccctttttc 2100 tccaaggttg gatttagttt tctggattta cacggcttac gtaatctcaa tgattcccgc 2160 caacatcacc accatcatca ctaggcggcc gccctgaagc ttggcctttt tgcgtttcta 2220 caaactcttt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata 2280 accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg 2340 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac 2400 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact 2460 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat 2520 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtgttgacg ccgggcaaga 2580 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac 2640 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat 2700 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac 2760 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct 2820 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgcagcaa tggcaacaac 2880 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga 2940 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg 3000 gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact 3060 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac 3120 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta 3180 actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt 3240 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga 3300 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc 3360 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt 3420 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc 3480 gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc 3540 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg 3600 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg 3660 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga 3720 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc 3780 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg 3840 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg 3900 atttttgtga tgctcgtcaa gggggcggag cctatggaaa aacgccagca acgcggcctt 3960 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc 4020 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg 4080 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga tgcggtattt 4140 tctccttacg catctgtgcg gtatttcaca ccgcatatgc ggtgtgaaat accgcacaga 4200 tgcgtaagga gaaaataccg catcaggcgc cattcgccat tcaggctgcg caactgttgg 4260 gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct 4320 gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg taaaacgacg 4380 gccagt 4386 <210> 4 <211> 471 <212> DNA <213> Escherichia coli <220> <221> gene &Lt; 222 > (1) .. (471) <223> The atpF gene <400> 4 atgaatctta acgcaacaat cctcggccag gccatcgcgt ttgtcctgtt cgttctgttc 60 tgcatgaagt acgtatggcc gccattaatg gcagccatcg aaaaacgtca aaaagaaatt 120 gctgacggcc ttgcttccgc agaacgagca cataaggacc ttgaccttgc aaaggccagc 180 gcgaccgacc agctgaaaaa agcgaaagcg gaagcccagg taatcatcga gcaggcgaac 240 aaacgccgct cgcagattct ggacgaagcg aaagctgagg cagaacagga acgtactaaa 300 atcgtggccc aggcgcaggc ggaaattgaa gccgagcgta aacgtgcccg tgaagagctg 360 cgtaagcaag ttgctatcct ggctgttgct ggcgccgaga agatcatcga acgttccgtg 420 gatgaagctg ctaacagcga catcgtggat aaacttgtcg ctgaactgta a 471 <210> 5 <211> 1287 <212> DNA <213> Escherichia coli <220> <221> gene &Lt; 222 > (1) .. (1287) <223> dctA gene <400> 5 atgaaaacct ctctgtttaa aagcctttac tttcaggtcc tgacagcgat agccattggt 60 attctccttg gccatttcta tcctgaaata ggcgagcaaa tgaaaccgct tggcgacggc 120 ttcgttaagc tcattaagat gatcatcgct cctgtcatct tttgtaccgt cgtaacgggc 180 attgcgggca tggaaagcat gaaggcggtc ggtcgtaccg gcgcagtcgc actgctttac 240 tttgaaattg tcagtaccat cgcgctgatt attggtctta tcatcgttaa cgtcgtgcag 300 cctggtgccg gaatgaacgt cgatccggca acgcttgatg cgaaagcggt agcggtttac 360 gccgatcagg cgaaagacca gggcattgtc gccttcatta tggatgtcat cccggcgagc 420 gtcattggcg catttgccag cggtaacatt ctgcaggtgc tgctgtttgc cgtactgttt 480 ggttttgcgc tccaccgtct gggcagcaaa ggccaactga tttttaacgt catcgaaagt 540 ttctcgcagg tcatcttcgg catcatcaat atgatcatgc gtctggcacc tattggtgcg 600 ttcggggcaa tggcgtttac catcggtaaa tacggcgtcg gcacactggt gcaactgggg 660 cagctgatta tctgtttcta cattacctgt atcctgtttg tggtgctggt attgggttca 720 atcgctaaag cgactggttt cagtatcttc aaatttatcc gctacatccg tgaagaactg 780 ctgattgtac tggggacttc atcttccgag tcggcgctgc cgcgtatgct cgacaagatg 840 gagaaactcg gctgccgtaa atcggtggtg gggctggtca tcccgacagg ctactcgttt 900 aaccttgatg gcacatcgat atacctgaca atggcggcgg tgtttatcgc ccaggccact 960 aacagtcaga tggatatcgt ccaccaaatc acgctgttaa tcgtgttgct gctttcttct 1020 aaaggggcgg caggggtaac gggtagtggc tttatcgtgc tggcggcgac gctctctgcg 1080 gtgggccatt tgccggtagc gggtctggcg ctgatcctcg gtatcgaccg ctttatgtca 1140 gaagctcgtg cgctgactaa cctggtcggt aacggcgtag cgaccattgt cgttgctaag 1200 tgggtgaaag aactggacca caaaaaactg gacgatgtgc tgaataatcg tgcgccggat 1260 ggcaaaacgc acgaattatc ctcttaa 1287

Claims (7)

An Escherichia genus mutant strain for the production of a membrane protein containing a gene encoding a membrane protein and having increased diguanylate cyclase activity relative to the parent strain.
The mutant strain according to claim 1, wherein the mutant strain is a mutant strain wherein the 1082nd nucleotide in the dgcQ gene sequence of SEQ ID NO: 1 encoding guanylate cyclase is substituted with C to A.
The strain according to claim 2, wherein the Escherichia spp. Mutant strain is Escherichia coli .
The membrane protein according to claim 3, wherein the membrane protein is selected from the group consisting of Glooobacter rhodopsin, b subunit of ATP synthase F ₀ region encoded by the atpF gene of SEQ ID NO: 4, and a dicarboxylate transporter encoded by the dctA gene. &lt; RTI ID = 0.0 &gt; 11. &lt; / RTI &gt;
(a) culturing the Escherichia sp. strain according to claim 1 in a culture medium;
(b) recovering the culture medium; And
(c) obtaining a membrane protein from the recovered culture.
6. The production method of a membrane protein according to claim 5, wherein the Escherichia sp. Strain is Escherichia coli substituted with C at position 1082 in the dgcQ gene sequence of SEQ ID NO: 1 encoding guanylate cyclase .
6. The membrane protein according to claim 5, wherein the membrane protein is globobacter rhodopsin, a b subunit of the ATP synthase F ₀ region encoded by the atpF gene of SEQ ID NO: 4, and a dicarboxylate transporter encoded by the dctA gene.

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