KR100485614B1 - A method of high cell-density culture of Escherichia coli by overproducing Mlc protein - Google Patents

Abstract

The present invention relates to a method for overexpressing Mlc protein, a regulator of carbohydrate metabolism of Escherichia coli. Specifically, the present invention provides a method of overexpressing Mlc protein by inducing mutations in the -10 site and the ribosome binding site of the transcription control site of the Escherichia coli mlc gene, and the Mlc overexpressing mutant strain produced by the above method. Due to the overexpression of the Mlc protein by the present invention, E. coli can be cultured at high concentration by inhibiting the production of acetate, a toxic metabolite byproduct of glucose metabolism, by decreasing the glucose metabolism rate and the non-growth rate of E. coli in the medium to which glucose is added. Mlc overexpressing mutant strains produced by the present invention can be used as foreign gene expression host cells to increase the yield of recombinant protein.

Description

Method of culturing Escherichia coli at high concentration by overexpressing Mlc protein {A method of high cell-density culture of Escherichia coli by overproducing Mlc protein}

The present invention relates to a novel E. coli mutant strain that improves the productivity of recombinant protein by regulating metabolic pathways to reduce the accumulation of toxic metabolites, acetate, in the medium to which glucose is added, and a method for overexpressing recombinant protein in said strain.

Of the host cell strains that express many useful proteins, Escherichia coli is the most well known and the chromosomal base sequence is fully revealed, and the strain that has been studied the most, and has a high growth rate even in cheap nutrient medium. In addition, its genetic properties are well known, and many cloning and expression vector systems have been developed. For this reason, E. coli is widely used as a host cell in high-cell-density aerobic fermentation to produce recombinant proteins.

On the other hand, research on overexpression of recombinant proteins began with the development of gene cloning technology, and the focus has been on the development of strains that produce useful substances from humans, animals, plants and bacteria. The study of overexpression of recombinant protein can be divided into two categories: first, increase in specific productivity in one cell per unit time, and second, increase in cell productivity per unit time. In order to increase the production of protein per unit time and cell per cell, a system that expresses genes encoding generally useful proteins under a high copy number strong promoter is used, and pET vector is a highly efficient expression vector among commercial expression vectors. It is used a lot in. In addition, as research on the molecular physiology of cells continues, methods for increasing the expression of foreign genes at the level of transcription and translation have been proposed.

However, high expression levels of exogenous mRNA cause problems such as ribosomal destruction, cell death, decreased expression rate, instability of plasmids or expression patterns, and also improper folding and the formation of intracellular inclusion bodies in the expression of proteins in cells. There is a problem to facilitate. In addition, the vector is maintained through the selection marker in the host cell, there is a problem that requires the treatment of expensive antibiotics or inducers.

To date, high cell density culture (HCDC) technology for Escherichia coli has evolved toward lower culture volume, less waste, lower costs, and lower equipment costs. However, in high density culture of Escherichia coli, there are barriers to be solved such as inhibition by substrate, limited oxygen transfer, generation of byproducts that inhibit growth, and limited heat transfer. Among these, by-products produced during fermentation and inhibiting the growth of Escherichia coli, there are various kinds of organic acids, and acetate is a representative toxic metabolite.

For high concentration growth of host cells, significant amounts of glucose are added to the growth medium because glucose is relatively inexpensive and readily available as a carbon and energy source. The biggest problem of high density aerobic cell culture is that organic acid byproducts are produced by fermentation, which inhibits cell growth, among which acetate production is the biggest problem. The production of acids as by-products, in particular the production of acetate, leads to a decrease in pH due to the accumulation of acetate in the medium and stops the growth of host cells before the glucose is completely consumed in the medium, resulting in high density cell growth as well as recombinant proteins. It acts as a limiting factor in production (Han et al. , Biotechnol. Bioeng . 39: 663, 1992; Luli et al. , Appl. Environ. Microbio. 56: 1004, 1990). Under aerobic conditions, in the glucose metabolism of Escherichia coli, the carbon flow exceeding the TCA circuit capacity is converted to acetate and released out of the cell (Majewski and Donmach, Biotechnol. Bioeng. , 35: 732, 1990). Inhibits growth and production of the desired recombinant protein. Synthesis of acetate is acetyl coenzyme A (acetyl coenzyme A) and phospho-trans-acetyl-raised (phosphotransacetylase; pts) and acetate kinase; mutant of acetate synthase makin consists of a series of reactions according to (acetate kinase ack) (pts and ack genes Enzyme activity is reduced, but still produces a significant amount of acetate. Mutations also result in higher levels of lactic and pyruvic acid accumulation than parental strains (Diaz-Ricci et al., Biotechnol. Bioeng. , 38: 1318, 1991). These results show that there are alternative pathways for acetate synthesis and that inactivating known acetate synthesis pathways converts the carbon stream to other organic acids.

In order to solve the above problems, fed-batch techniques (Fieschko et al., Chem. Eng. Commun. 45: 2875, 1986; Ohta et al., J. Fermet. Bioeng. 75: 155 , 1993; Yang, J. Biotechnol. 23: 271, 1992), methods for removing organic acids from cultures (Landwall et al., J. Gen. Microbiol. 103: 345, 1997; Meyer et al. , Proceedings of the 3rd European Congress on Biotechnology, 1984; MacDonald, Appl. Environ.Microbiol. 56: 640, 1990) or methods of changing the medium composition (Holmes, Curr. Topics Cell.Regul . 28:69, 1986; Han et al., Biotechnol, Bioeng. , 41: 316, 1993; Reihng, J. Biotechnol. 2: 191, 1985; Mor et al., J. Ferment. Technol. , 50: 519, 1972) and glycerol instead of glucose A method of lowering the fermentation temperature (37 ° C. → 25-30 ° C.) to slow the growth rate has been used. Recently, methods have also been developed to measure acetate concentrations online and to reduce nutrient supply rates with acetate accumulation.

However, these methods often have low productivity and low productivity of recombinant proteins due to inferior metabolic activity, resulting in a complicated and error-prone method of supplying culture medium (Chot et al., Biotechnol. Bioeng. , 44: 952, 1994). In addition, the addition of sodium phosphate buffer solution to the medium for pH control can reuse the accumulated acetate in the medium, but if glucose remains in the medium, such reuse does not occur, and the salts added to the medium for pH control are frequently used. There is a problem that it can inhibit the growth and / or production of host cells (Jensen et al., Biotechnol. Bioeng. , 36: 1, 1990).

Thus, there has been a need for a search for a new host cell in which the organic acid production function is reduced as a whole and the productivity of a desired recombinant protein is improved.

Mlc protein is a newly discovered regulatory factor in carbohydrate metabolism and has been cloned due to its ability to increase glucose availability in rich media, slowing the growth of E. coli and reducing acetate accumulation when expressed on plasmids, thus forming larger colonies. It is known to have the ability to make (mlc = making large colonies) (Hosono et al., Biosci Biotech Biochem 59: 256-261, 1995). mlc gene, encoding the 44 kDa protein and the protein is ⅡCB ptsG encoding the ^Glc, manno Oz (mannose) malT and expression of mlc itself encoding the activator of the manXYZ, maltose control group coding for the enzyme Ⅱ of PTS It is known to regulate (Soon-Young Kim et al., J. Biol. Chem. 274 (36): 25398-25402). As such, Mlc is known to be a negative regulator of genes involved in the glycolysis system and, among other things, PTS genes involved in glucose transport when E. coli grows in glucose-containing media. (Katja D. et al., Mol. Microbiology 27 (2): 381-390, 1998; Jacqueline P. Mol. Microbiology 29 (4): 1053-1063, 1998).

The expression of the mlc gene in cells differs greatly from the nucleotide sequence of the transcriptional regulatory region of the mlc gene and the common nucleotide sequence of the transcriptional regulatory region of most genes, and unlike the general ATG initiation codon, the GTG initiation codon is used. Due to autoregulation by Mlc proteins, they are quite limited in cells. Therefore, the present invention attempts to overexpress the Mlc protein by inducing mutations in the mlc gene transcriptional regulatory region. Specifically, it was confirmed that the mlc gene was overexpressed when mutation was induced in the -10 region and the SD sequence of the transcription control region of the mlc gene by PCR, and the glucose was released to the medium due to the slowed glucose consumption rate in the Mlc overexpressed mutant. The present invention was completed by confirming that an increase in the growth of E. coli and an increase in the expression rate of external genes occurred due to a decrease in the concentration of acetate accumulation, a toxic by-product of the use.

In the present invention, "Mlc overexpression mutant" or "mutant" refers to a mutant strain in which a mutation occurs in the mlc wild-type gene transcription control site on the E. coli chromosome, resulting in overexpression of the mlc gene.

It is an object of the present invention to provide a method of overexpressing Mlc protein by inducing mutations in the transcriptional regulatory region of the mlc gene, which is a negative regulator of glycolysis system. Specifically, an object of the present invention is to overexpress the Mlc protein by inducing mutations in the -10 site and the ribosome binding site in the transcription control site of the E. coli mlc gene.

It is another object of the present invention to provide an E. coli mutant that overexpresses the Mlc protein due to mutation in the transcriptional regulatory region of the mlc gene.

In addition, using the E. coli mutant as a host cell provides a method for increasing the cell productivity and foreign protein productivity.

In order to achieve the above object, the present invention provides a method of overexpressing the mlc gene by inducing mutations in transcriptional and translational regulatory sites.

In another aspect, the present invention provides a mutant in which a mutation occurs in the transcriptional regulatory region and the mlc gene is overexpressed.

In addition, the present invention provides a method for culturing Escherichia coli at high concentration using the mutant as a host cell, and a method for mass production of external genes.

Hereinafter, the present invention will be described in detail.

The present invention provides a method of overexpressing the Mlc protein by inducing mutations in the -10 region of the transcriptional regulatory region of the mlc gene and the SD sequence which is the ribosomal binding site.

In the embodiment of the present invention the method for producing a mutant overexpressed mlc gene is

1) preparing and PCR a site-specific PCR primer to mutate the transcriptional regulatory region of the mlc gene (see FIG. 2);

2) Recombinant vector prepared by cloning the PCR product into a suicide vector (pKO3) having a temperature sensitive replication origin (rep ^ts ), a selective antibiotic resistance gene (cat) and a reverse selective marker gene (sac) (see FIG. 5A). Transforming the host cell so that recombination occurs in the chromosome (see FIG. 5B); and

3) selecting the mlc mutant in which recombination has occurred.

The wild-type mlc gene transcription control region of E. coli has a configuration as shown in FIG. The mlc gene has a cAMP-CRP complex binding site of the ATGTGCTGTTAATCACAT base sequence about 60 bp upstream from the start codon, indicating that the mlc gene is positively controlled by the cAMP-CRP complex. On the other hand, genes with particularly high transcriptional efficiency in E. coli have strong promoters, which are known to have conserved two sites, the -10 site sequence (or Pribnow box; TATAAT) and the -35 site sequence (TTGACAT). It is known that the frequency of transcription of genes with different sequences decreases. The -10 site and -35 site, which are transcription control sites of the mlc gene, are considered to be weak promoters because they differ from the conserved sequences of the strong promoters. Therefore, the inventors of the present invention induce mutations in the transcriptional control sites and convert them into powerful promoters, thus the mlc gene. We assumed that we could overexpress.

The position where the mutation is induced in the transcriptional regulatory region of the mlc gene may be selected from 100 bp upstream nucleotide sequences from the initiation codon of the mlc gene, including the CRP binding site, -35 site, -10 site, Mlc binding site and SD sequence . A method of inducing mutations in the base sequence of the mlc gene transcription control site may be induced by irradiating chemicals, X-rays, UV, etc., but preferably, a mutation induction method using PCR capable of inducing base-specific mutations. Can be used. In the embodiment of the present invention, the left, relative to the Mlc binding site in order to induce a mutation to change the -10 site into a conserved sequence, a mutation of the Mlc binding site, a mutation of the SD sequence or a mutation to change the start codon from GTG of wild type to ATG PCR primers with mutant sequences capable of obtaining 500 bp DNA fragments on each of the right sides were prepared (see FIG. 2). As a result of the first PCR, PCR was performed using primers of SEQ ID NOs: 3 and 5, 3, and 8 , respectively, to obtain a DNA fragment left of the Mlc binding site having a mutated wild type sequence and a -10 site, respectively, SEQ ID NOs: 4 , 6, 4, and 7, 4 and 9 were PCR-produced with primers to obtain DNA fragments on the right side of the Mlc binding site having the changed start codon, the changed Mlc binding site and the changed start codon, the Mlc binding site and the changed start codon (see FIG. 5). ).

The PCR was performed using a linker obtained by linking two Mlc binding site left DNA fragments and three Mlc binding site right DNA fragments as a template, and using SEQ ID NOs: 3 and 4 as a primer. Three DNA fragments with mlc gene transcription control site sequences were obtained (see FIG. 6). Inserting the DNA fragment into the pUC vector and sequencing the nucleotide sequence in which only the start codon is mutated; A base sequence in which a start codon and a Mlc binding site are mutated; After identifying the nucleotide sequence in which the initiation codon, the Mlc binding site, the -10 site, and the SD sequence were mutated, the DNA fragment was cut with a restriction enzyme and introduced into the suicide vector pKO3, named pKO-L1, pKO-L2, and pKO-L3, respectively. It was.

E. coli MC4100 was transformed with a recombinant suicide vector having the mutation-derived mlc gene transcription control site fragment, and the transcription control site fragment sequence was introduced onto the chromosome by homologous recombination to obtain mutated cells.

In a preferred embodiment of the present invention, suicide vectors used for gene-substituting mutations are susceptible to specific substances with genes and counter-selectable makers that allow them to exhibit antibiotic resistance at the point of temperature-sensitive replication origin and selection markers. Contains genes to be assigned. The vector, which has a temperature-sensitive replication origin (rep ^ts ), can replicate itself at low temperatures (30 ° C) but not at high temperatures (42 ° C), so that the vector is inserted onto the chromosome in order to survive. Can only survive with replication. Therefore, when a recombinant vector having a temperature-sensitive replication origin such as the vector is transformed into a host cell and cultured at high temperature (42 ° C.), it is possible to insert the vector onto a chromosome by a single homologous recombination. . The transformant resulting from the recombination can be found by selecting a transformant that shows resistance to the antibiotic chloroamphenicol as a selection marker and simultaneously shows sensitivity to a specific substance as a reverse selection marker. As the specific material, a carbon source or a nitrogen source is used, and sucrose is preferably used. In a preferred embodiment of the present invention, a gene-substituted mutant vector including the sacB gene was used as a gene to confer susceptibility to sucrose. The sacB gene encodes levansucrase and the enzyme catalyzes levan synthesis, a toxic substance from sucrose, so that the host cell transformed with the plasmid containing the sacB gene exhibits sucrose sensitivity.

When the plasmid-recombinant host cell is cultured at low temperature on a rich medium free of chloroamphenicol antibiotics, a second recombination occurs, whereby the recombinant vector in which the gene is substituted is separated from the chromosome, and as a result, the chromosome of the host cell The recombinant vector can be removed. The host cell in which the vector is deleted on the chromosome can be obtained by selecting a transformant which simultaneously exhibits sensitivity to selection markers and resistance to reverse selection markers. In a preferred embodiment of the present invention, a transformant is selected that exhibits both susceptibility to chloroamphenicol antibiotics and susceptibility to sucrose to select a cell in which gene substitution occurs and the vector is deleted (see FIG. 4).

In an embodiment of the present invention, E. coli mutants SR752, which were transformed with pKO-L1 and then mutated to the initiation codon by homologous recombination, were mutated to the initiation codon and Mlc binding site by homologous recombination. Escherichia coli mutants SR753 and pKO-L3 were transformed with homologous recombination to obtain coliform mutants SR754 mutated at the start codon, Mlc binding site, -10 site and SD sequence. Confirmed

Western blots were performed to confirm that the mlc gene was overexpressed with mutations that occurred at the transcriptional regulatory sites in each mutant (see FIG. 7). As a result, all of the mutants except SR754 showed similar expression pattern to wild type. In the present invention, mutations induced at the start codon and Mlc binding site did not affect the transcription of the mlc gene, whereas Induced mutations suggest that increased expression of the mlc gene.

The Mlc overexpressing mutants of the present invention increase cell growth in culture compared to wild type and also show higher productivity compared to wild type when used as host cells for foreign protein production (see FIGS. 8 to 11 and 12). Since the Mlc protein acts as a negative regulator of sugar metabolism-related genes, when the protein is overexpressed, the glucose utilization rate is lowered and the acetate excretion rate is slowed, so that cell growth continues even after logarithmic growth. In the embodiment of the present invention, the cell growth was measured in the minimal medium and the rich medium to which glucose was added, and it was confirmed that Mlc overexpressing mutants continued cell growth after the logarithmic growth period compared to the wild type (see FIGS. 9 and 10). In another embodiment of the present invention, it was confirmed that the productivity of the genes was increased in the Mlc overexpression mutant transformed with a recombinant vector in which a gene encoding β -galactosidase and a GFPuv protein was introduced as an external gene (FIG. 10 and FIG. 12). These results suggest that the Mlc overexpressing mutants of the present invention can be effectively used as host cells for foreign protein production.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

<Example 1> mlc Construction of a PCR primer for mutation induction of transcriptional regulatory regions of genes and mutation-induced mlc Construction of Recombinant Vectors Containing Genes

A site-specific mutated PCR primer was constructed to induce mutations in the transcriptional regulatory region of the mlc gene.

Mutations were induced at the −10 conserved site, Mlc binding site, SD sequence or initiation codon, and the base sequence at the 5 ′ end of each primer had a mutant base sequence different from that of the template. Part of the 5 'side of the outer primer was constructed to have a Bam HI cleavage site. The first PCR was performed using one of the 5 'end and 3' end outer primers and one of the three mutation-induced inner primers as the PCR primers, so that two left PCR products and three right PCRs based on the Mlc binding site were performed. After obtaining the product, ligation of the five PCR products obtained in the first PCR, and then performing a second PCR using the primers of SEQ ID NOs: 3 and 4 to connect the left PCR product and the right PCR product mutation PCR products of about 1 kb in size containing the derived mlc gene transcription site were obtained.

In the first PCR, the PCR conditions were determined by using a thermocycler, using the DNA sequence of SEQ ID NO: 1 as a template in Vent DNA (PCR buffer solution containing 1 unit of polymerase (10 mM dNTP, 1.5 mM MgCl ₂ , 20 mM Tris-Cl, 50 mM KCl). Denature the E. coli chromosomal DNA containing the wild-type mlc gene and the above primer for 5 minutes at 95 ° C, then perform 30 cycles at 95 ° C for 30 seconds, annealing temperature for 30 seconds, and 72 ° C for 1 minute. And finally reacted for 10 minutes at 72 ° C. The heating and cooling temperatures when using SEQ ID NOs: 3 and 5 , SEQ ID NOs: 4 and 7, or SEQ ID NOs: 3 and 8 as primers were 56 ° C., SEQ ID NOs: 4 and 9 The heating cooling temperature at the time of use was 57 ° C., and the heating cooling temperature was 60 ° C. when using SEQ ID NOs: 4 and 6. Agarose gel electrophoresis on the PCR product confirmed that the fragment of about 500 bp was amplified. PCR product separation key (Boehringer Mannheim. USA) was used for pure separation and kinase reaction to add phosphate groups at both ends of each PCR fragment, followed by ligation, followed by ligation reaction. A ligation reaction was carried out by reacting a total of 20 µl of the reaction solution including 5 µl each of PCR products, 2 Weiss units of T4 DNA ligation enzyme, and 1 µl of T4 DNA ligation buffer solution at room temperature for 15 minutes, followed by 10 minutes at 65 ° C. .

The PCR product conjugate obtained in the ligation reaction was used as a template for the second PCR, SEQ ID NOs: 3 and 4 as primers, and a vent DNA polymerase as a DNA polymerase, and PCR was performed at a heat and cooling temperature of 63 ° C. The second PCR product of about 1 kb in length obtained as a result of PCR was cut and purified with restriction enzyme Bam HI and inserted into the Bam HI position of the pUC19 vector. The recombinant pUC19 vector was digested with Bam HI restriction enzyme to insert the insert at the enzyme site of the suicide vector pKO3 (Harvard medical school, USA) to prepare recombinant vectors pKO-L1, pKO-L2, pKO-L3. The recombinant vector was transformed into E. coli MC 4100 to introduce a mlc gene containing a mutation-induced transcriptional regulatory site onto the chromosome.

The transformants were incubated for 12 hours at 42 ° C. on a medium containing 20 μg / ml of chloroamphenicol to introduce a recombinant suicide vector having a temperature-sensitive replication origin onto a chromosome by a single homologous recombination. It was induced to obtain a transconjugate in which a recombinant vector was introduced on a chromosome. In order to remove the vector portion from the trans-conjugate, it was incubated at 30 ° C. for 12 hours on a medium containing 5% sucrose and no chloroamphenicol. Transformants exhibiting sucrose resistance and chloroamphenicol sensitivity were found by screening using primers of SEQ ID NOs: 11 and 12 . Through the above procedure, the selected transformant was subjected to sequencing to confirm that gene substitution occurred at a desired position (see FIG. 3).

In the present invention, three kinds of mutants were obtained in which mutations were made in the transcriptional regulatory region of the mlc gene by mutation induction using PCR. As shown in FIG. 3, the sequencing of the SR752 strain at the start codon, the SR753 strain at the Mlc binding site and the start codon, and the SR754 strain at the -10 site, the Mlc binding site, the SD sequence, and the start codon were performed. I could check through.

<Example 2> mlc  Increase in growth due to Mlc overexpression of mutant strains

Western blot analysis was performed to determine whether the Mlc protein was overexpressed in the mlc mutant strain. The protein extracted from the wild type strain (MC4100) and mutant strain (SR752, SR753, SR754) protein samples and the Mlc protein, mlc structural gene purified by the positive control transfection with a recombinant vector pOH106 introduced into the expression vector pUC19 conversion strains derived from Samples were electrophoresed and then transferred to nitrocellulose membranes. The transfer of the protein was performed using a blot transition cell kit (Amersham Pharmacia Biotech), 100 volts using a transition buffer (20% methanol, 0.05% SDS, 25 mM Tris-Cl, 192 mM glycine) in a cold room maintained at 4 ° C. Vt) for 1 hour. Then, the mixture was reacted with phosphate buffered saline (PBS) containing 1% skim milk at room temperature for 1 hour and washed three times with PBST solution (1% Triton x-100 in PBS). As the primary antibody, the anti-Mlc antibody (Seoul National University Microbiology and Microbial Physiology Laboratory) was diluted 100-fold with PBS solution and reacted with the protein bound to the membrane. After completion of the reaction, the membrane was washed three times with PBS solution at room temperature, and then reacted by diluting an anti-mouse antibody as a secondary antibody (ECL. USA). The excess antibody that did not participate in the reaction was washed and then detected using ECL chemiluminescence detection reagent and the membrane was photosensed on X-ray film for 15 seconds to 1 minute. It was observed whether the overexpression of Mlc protein through Western blot as described above (Fig. 7). As a result, only SR754 mutants mutated to -10 site, Mlc binding site, and initiation codon among mutants SR752, SR753, SR754 overexpressed Mlc protein compared to wild type, and SR752, SR753 where Mlc binding site or initiation codon was mutated. It was confirmed that the mutant showed a similar degree of expression as the wild type strain. These results suggest that changes in the nucleotide sequence at the -10 site are important for activating the transcription of the mlc gene and inducing overexpression of the Mlc protein.

<Example 3> The effect of overexpression of Mlc protein on the growth of host cells

The effect of overexpression of Mlc protein on the growth of host cells was investigated by measuring growth curves by measuring OD every hour while culturing cells. As a result of incubation by adding glucose to TB (trypton broth 1%, NaCl 0.8%) as a minimum medium, there was no difference between wild-type strains and overexpressing mutants in the medium without glucose (Fig. 8A). The higher the mutant compared to the wild type strain in the growth curve after the logarithmic growth period, and the mutant was confirmed that the growth continues for a long time (Fig. 8B, C).

In addition, as a result of the culture experiment in L-broth (rich broth), there was no difference between wild-type strains and over-expressing mutants in medium without glucose (FIG. 9A). Was different in the growth curve after the logarithmic growth period, and the mutants were able to confirm that the growth continued for a long time (Figs. 9B to E).

Example 4 Increased expression of foreign genes in Mlc overexpressed mutants

It was confirmed that the expression of foreign genes in Mlc overexpressing mutant host cells increased compared to wild type host cells. As a foreign gene, a gene encoding β -galactosidase and a gene encoding GFPuv (green fluorescence protein variant) were used. In addition, the number of viable cells was measured to determine the proportion of the OD value for the growth curve and the actual number of viable cells.

4-1) Exogenous Genes in Mlc Overexpression β Investigation of the expression level of galactosides

In order to investigate the expression level when a gene encoding β -galactosidase was used as a foreign gene, pGAL, a plasmid expressing β -galactosidase, was transformed into a wild-type strain and an Mlc overexpressing mutant, respectively. It was. The pGAL plasmid was prepared by introducing the lacZ gene in pRS415 to be functionally linked downstream of the lac promoter of the pUC19 vector. The pGAL plasmid was transformed into the wild type strain and Mlc overexpressing mutant SR754, and cultured by adding 0.3% glucose to 37 ° C, 230 rpm, and L-broth. To prevent plasmid deletion from host cells, 50 μg / ml of antibiotics empicillin was added to the medium, and IPTG 1 mM was added at different times to induce β -galactosidase. As a result, the growth of the bacteria and the expression of β -galactosidase gene measured by induction time are shown in FIG. 10. In terms of cell growth, the wild type enters the plateau during 4 hours after incubation, while the growth of Mlc overexpressing mutants continues continuously until 14 hours after incubation, and the cell concentration was higher than that of the wild type. In addition, it was confirmed that the enzyme activity was 5 to 9 times higher when the Mlc overexpression mutant was used as the host cell than when the wild type was used as the host cell of the β -galactosidase gene. On the other hand, when comparing the degree of the growth of foreign genes with the strain growth according to the induction period, it was confirmed that a higher expression amount can be obtained when induced at the end of the logarithmic growth stage.

4-2) Measurement of Viable Cell Number

In order to check the difference between the OD value and the actual number of live cells when the wild-type strain and the mutant SR754 were grown, the viable cell number was measured by measuring the sampling time with time difference, and the results are shown in FIG. 11. In viable count, the wild type strain and the mutant SR754 grew similarly up to 5 hours after incubation, but after 5 hours, the wild type strains showed a sharp decrease in growth. On the other hand, the mutant SR754 continued to grow after 5 hours of culture and was found to enter the plateau at about 8 hours after the culture. These results confirm that the increase in the total number of cells in Mlc overexpressing mutants is due to the increase in the number of living cells.

4-3) Exogenous Genes in Mlc Overexpression gfpuv Of expression level

When the gene encoding GFPuv was used as a foreign gene, pGFPuv, a plasmid expressing GFPuv, was transformed into a wild-type strain and an Mlc overexpressing mutant, respectively, to examine the expression level. The pGFPuv plasmid was prepared by introducing the gfpuv gene to be functionally linked downstream of the lac promoter of the pUC19 vector (clontech. USA). The pGFPuv plasmid was transformed into the wild type strain and Mlc overexpressing mutant SR754 and cultured under the same conditions as in Example 4-1). To prevent plasmid deletion from host cells, 50 μg / ml of antibiotics empicillin was added to the medium, and IPTG 1 mM was added during the logarithmic growth phase to induce gfpuv gene. As a result, the growth of bacteria and the expression of gfpuv gene measured by time are shown in FIG. 12. Similar to β -galactosidase gene expression, Mlc overexpressing mutants were found to have higher values than wild type in cell growth or enzyme activity.

The Mlc overexpressing mutants of the present invention are inhibited from glucose metabolism, and the accumulation and release of acetate, a toxic byproduct of glucose metabolism, is reduced, resulting in long-lasting growth compared to wild-type strains, resulting in increased cell production and protein production when foreign genes are introduced. As a result, it can be used as a host cell having higher productivity and efficiency than a host cell for gene expression.

Figure 1 shows the transcriptional regulation site of mlc (making large colonies) in E. coli MC4100 strain,

2 is a schematic diagram showing the position of a PCR primer having a mutant sequence for producing a mutant of mlc gene using PCR,

Figure 3 shows the wild-type and mutated base sequence of the gene transcription control site in the chromosome of E. coli wild type strain (MC 4100) and mlc mutants (SR752, SR753, SR754),

CRP (cyclic AMP receptor protein) site: CRP binding site

Mlc spot: Mlc protein binding position

SD (Shine-Dalgarno) sequence: ribosomal binding site

4A is a cleavage map showing a vector configuration of a suicide vector to which a mlc gene containing a transcriptional regulatory region having a mutant sequence has been introduced,

4b schematically shows a process of inducing mutations on a chromosome of a host cell by homologous recombination in response to the suicide vector,

5 is a result of PCR using a PCR primer having a mutant sequence,

M: size cover

Lane 1: wild-type Mlc binding site left PCR product

Lane 2: PCR product of the right Mlc binding site mutated start codon

Lane 3: PCR product of the Mlc binding site right mutated Mlc binding site and the start codon

Lane 4: Mlc binding site left PCR product with the -10 site mutated

Lane 5: Mlc binding site right PCR product mutated Mlc binding site, SD sequence and initiation codon

FIG. 6 shows the results of PCR using a template of the conjugate of the left product and the right product of the Mlc binding site of the first PCR,

M: size cover

Lane 1: PCR was performed using a concatenation of the left PCR product of the wild type Mlc binding site and the right PCR product of the Mlc binding site mutated with the start codon.

Lane 2: PCR of the left PCR product of the wild type Mlc binding site and the linkage of the Mlc binding site right PCR product mutated with the start codon

Lane 3: PCR was performed using a concatenation of the PCR product on the left side of the Mlc binding site where the -10 site was mutated and the PCR product on the right side of the Mlc binding site, the SD sequence and the right side of the Mlc binding site where the start codon was mutated.

7 is a result of Western blot analysis for overexpression of Mlc protein in E. coli wild type strain and mlc mutant.

Lane 1: 125ng of purified Mlc protein

Lane 2: sample of Mlc expression in wild type strain

Lane 3: sample of Mlc expression in mutant SR752

Lane 4: sample of Mlc expression in mutant SR753

Lane 5: Mlc expression sample in mutant SR754

Lane 6: Mlc expression sample from wild type strain transformed with recombinant vector pOH106 into which mlc structural gene was introduced

8 shows the effect of Mlc overexpression on host cell growth by measuring growth curves in minimal medium supplemented with glucose,

(A) shows growth curve in medium without glucose, (B), (C) shows growth curve in medium with 0.1% and 1.0% glucose, respectively

9 is a result confirming the effect of Mlc overexpression on the growth of host cells by measuring the growth curve in the rich medium,

(A) is a growth curve in the medium without glucose, (B), (C), (D) is a growth curve in the medium added with 0.3%, 0.5%, 1% glucose, respectively

FIG. 10 shows that there is a difference in the expression of foreign genes in wild-type strains and Mlc overexpressing mutants, which are induced by transformation of a recombinant vector having a β -galactosidase gene introduced into each strain. ), (B) and (C) are the results of induction of expression by addition of IPTG at the beginning of logarithmic growth (OD ₆₀₀ value 0.5), medium (OD ₆₀₀ value 2.3), and late (OD ₆₀₀ value 4.3), respectively,

FIG. 11 shows the results of measuring live cells to investigate the difference between OD values and viable cells in growth of wild-type strains and mlc mutant SR754.

12 shows whether there is a difference in the expression of foreign genes in wild-type strains and Mlc overexpressing mutants (A) after preparing a recombinant vector in which gfpuv (variant of green fluorescent protein) gene is introduced, (B) the recombinant vector To transform the wild-type strains and mutants to induce expression.

<110> BIPIDO INC. <120> A method of high cell-density culture of Escherichia coli by over producing Mlc protein <130> NPF1631 <160> 12 <170> KOPATIN 1.5 <210> 1 <211> 1355 <212> DNA <213> Escherichia coli <400> 1 cgaaaaatgt gctgttaatc acatgcctaa gtaaaaattt gacgacacgt attgaagtgc 60 tttataatag cctacaaggc cgggcaatgt taatccgtaa ggaggagtat gcgatggttg 120 cgtaaaacca gcctgtggtt gctgaaaacc agcctgggca cattgatcaa ataaagcaga 180 ccaacgcggg cgcggtttat cgcctgattg atcagcttgg tccagtctcg cgtatcgatc 240 tttcccgtct ggcgcaactg gctcctgcca gtatcactaa aattgtccgt gagatgctcg 300 aagcacacct ggtgcaagag ctggaaatca aagaagcggg gaaccgtggc cgtccggcgg 360 tggggctggt ggttgaaact gaagcctggc actatctttc tctgcgcatt agtcgcgggg 420 agattttcct tgctctgcgc gatctgagca gcaaactggt ggtggaagag tcgcaggaac 480 tggcgttaaa agatgacttg ccattgctgg atcgtattat ttcccatatc gatcagtttt 540 ttatccgcca ccagaaaaaa cttgagcgtc taacttcgat tgccataacc ttgccgggaa 600 ttattgatac ggaaaatggt attgtacatc gcatgccgtt ctacgaggat gtaaaagaga 660 tgccgctcgg cgaggcgctg gagcagcata ccggcgttcc ggtttatatt cagcatgata 720 tcagcgcatg gacgatggca gaggccttgt ttggtgcctc acgcggggcg cgcgatgtga 780 ttcaggtggt tatcgatcac aacgtggggg cgggcgtcat taccgatggt catctgctac 840 acgcaggcag cagtagtctc gtggaaatag gccacacaca ggtcgacccg tatgggaaac 900 gctgttattg cgggaatcac ggctgcctcg aaaccatcgc cagcgtggac agtattcttg 960 agctggcaca gctgcgtctt aatcaatcca tgagctcgat gttacatgga caaccgttaa 1020 ccgtggactc attgtgtcag gcggcattgc gcggcgatct actggcaaaa gacatcatta 1080 ccggggtggg cgcgcatgtc gggcgcattc ttgccatcat ggtgaattta tttaacccac 1140 aaaaaatact gattggctca ccgttaagta aagcggcaga tatcctcttc ccggtcatct 1200 cagacagcat ccgtcagcag gcccttcctg cgtatagtca gcacatcagc gttgagagta 1260 ctcagttttc taaccagggc acgatggcag gcgctgcact ggtaaaagac gcgatgtata 1320 acggttcttt gttgattcgt ctgttgcagg gttaa 1355 <210> 2 <211> 53 <212> DNA <213> Mutated sequence between -13 region and start codon in mlc gene <400> 2 tataatagcc tacaggccgg gcaatgttaa tccgtaagga ggagtatgcg atg 53 <210> 3 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> upstream outer primer <400> 3 cttatggcga ggatcccgca cgatcatccc 30 <210> 4 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Down stream outer primer <400> 4 caatggatcc cggcaaggtt atggcaatcg 30 <210> 5 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Mlc binding site primer <400> 5 attttcgcgc tccgaaataa tctg 24 <210> 6 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Mlc binding site primer <400> 6 atagggagta tgcgatggtt gctg 24 <210> 7 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Mlc binding site primer <400> 7 ttatacccag cgcgggagta tgcgatggtt gctg 34 <210> 8 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Mlc binding site primer <400> 8 cattgcccgc ctgtaggcta ttataaagca cttcaa 36 <210> 9 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> Mlc binding site primer <400> 9 ttaatccgta aggaggagta tgcgatggtt gctgaaa 37 <210> 10 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Sequencing primer <400> 10 cccgcgttgg tctgcagtat ttgatc 26 <210> 11 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Screening forward primer <400> 11 agggcagggt cgttaaatag c 21 <210> 12 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Screening reverse primer <400> 12 ttaatgcgcc gctacagggc g 21

Claims (17)

Characterized in that the nucleotide sequence from the -13 position to the translation initiation codon in the transcription control region of the wild type E. coli mlc (making large colonies) gene shown in Figure 1 characterized in that the mutant Method of overexpressing Mlc protein. delete delete delete delete delete delete High concentration culture of E. coli, characterized in that the nucleotide sequence from the -13 position to the translation initiation codon of the transcription control region of the wild type E. coli mlc gene shown in Figure 1 is mutated to the nucleotide sequence represented by SEQ ID NO: 2 Way. delete delete The transcriptional regulation according to claim 8, wherein the nucleotide sequence from the -13 position to the translation initiation codon in the transcription control region of the wild-type Escherichia coli mlc gene shown in FIG. 1 is mutated to the nucleotide sequence represented by SEQ ID NO: 2. Transforming Escherichia coli with a vector comprising a site and a mlc structural gene linked thereto , and inserting the gene into the chromosome of Escherichia coli through homologous recombination. DNA fragment mutated to the nucleotide sequence shown in SEQ ID NO: 2 from the base sequence at the -13 position to the translation start codon in the transcription control region of the wild-type E. coli mlc gene shown in FIG. The DNA fragment of claim 12 linked to the mlc structural gene DNA. The DNA according to claim 13, which is represented by SEQ ID NO: 1. Mutant strain SR754 comprising the DNA of claim 14 (Accession No. KFCC 11244). A method for increasing cell productivity using the mutant strain of claim 15 as a host cell. A method for overproducing Mlc protein using the mutant strain of claim 15 as a host cell.