KR100629773B1 - Vectors containing guaA gene expressible in Escherichia coli Escherichia cells harboring the same and processes for accumulating 5'-guanylic acid synthetase in a medium using them - Google Patents

Abstract

The present invention relates to a vector comprising a guaA gene expressible in Escherichia coli, an Escherichia spp. Including the same, and a method of accumulating 5'-guanylic acid synthase in a medium using the microorganism. According to the present invention, 5'-guanylic acid synthase can be supplied in a stable and large amount in the 5'-guanylic acid production process.

PL promoter, Shindalgano sequence, 5'-guanylic acid synthase, guaA gene, Escherichia

Description

A vector comprising a g uAA gene expressible in Escherichia coli, an Escherichia microorganism comprising the same, and a method of accumulating ＇-guanylic acid synthase in a medium using the microorganism. harboring the same, and processes for accumulating 5'-guanylic acid synthetase in a medium using them}

1 shows the construction of the recombinant plasmid pUPSG according to the present invention.

2 shows the construction of the recombinant plasmid pUC-guaA :: loxPKm and the construction of the DNA fragment guaA :: loxPKm therefrom according to the present invention.

The present invention relates to a vector comprising a guaA gene expressible in Escherichia coli, an Escherichia spp. Including the same, and a method of accumulating 5'-guanylic acid synthase in a medium using the microorganism.

5'-guanylic acid (GMP) is a method of digesting or chemically degrading yeast RNA using microbial enzymes, culturing microorganism mutants in a medium containing sugar, nitrogen and phosphate sources, and directly nucleotides. The production method, fermentation method to produce the intermediate of nucleotide synthesis and then chemically or enzymatically converted to nucleotides, the current economically advantageous production method is a complex production method by fermentation and chemical synthesis or fermentation and enzyme conversion. It is widely used industrially.

On the other hand, the complex production method by enzymatic conversion consists of a fermentation process that produces 5'-xanthyl acid (XMP) by direct fermentation and a reaction process enzymatically converts the fermentation products to 5'-guanylic acid (GMP) Two kinds of microorganisms are used. In the first step (fermentation process), mutant strains bred as XMP producing bacteria are used, and in the second step (reaction step), microorganisms overexpressing the gene encoding 5'-guanylic acid synthase (GMP synthetase) are used. .

In the second step, a high-production plasmid made by combining the guaA gene encoding the enzyme with the PL promoter of bacteriophage lambda, a powerful promoter, is expressed and expressed in an Escherichia spp. The process cannot be considered to have optimized efficiency due to the deletion of ribosomal attachment sequence of Escherichia spp. (EP0263716A2). In addition, the plasmid stability of the overexpressed microorganisms in this process is drastically reduced in the late stage of the culturing process, which is known that 5'-guanylic acid synthase is toxic to the parent strain during overproduction. It is known to inhibit microbial growth. Therefore, it is necessary to increase the productivity by increasing the efficiency of the high-production plasmids currently used, and in addition, it is required in the art to increase the plasmid stability in microorganisms to produce even enzymes throughout the culture process.

It is an object of the present invention to provide a vector comprising a guaA gene that can be highly expressed in E. coli.

Another object of the invention to provide a microorganism of the genus Escherichia (Escherichia) comprising the vector.

Still another object of the present invention is to provide a method of culturing the microorganism and accumulating 5'-guanylic acid synthase at a high concentration in a medium.

In order to achieve the object of the present invention, the present invention

A vector is provided in which a polynucleotide encoding a bacteriophage lambda-derived PL promoter, a ribosomal binding sequence functional in Escherichia, and a 5′-guanylic acid synthetase is operably linked.

A high-production plasmid is obtained by combining a guaA structural gene encoding a 5'-guanylic acid synthase with a PL promoter of bacteriophage lambda and a base sequence optimized for ribosomal attachment sequence of Escherichia coli. The PL promoter of the present invention is derived from bacteriophage lambda, a virus that infects Escherichia spp., And is known to be a very powerful promoter. In particular, the PL promoter is widely used for academic and industrial purposes with a cI inhibitor, which is a transcriptional regulator. The PL promoter is able to inhibit the transcription process because the site where the cI inhibitor is attached overlaps with the site where the RNA polymerase is attached. In particular, the temperature-sensitive mutation factor with reduced thermal stability among the cI inhibitors can regulate transcription according to temperature. It is widely used. In addition, it is known that the PL promoter is promoted by IHF (integration host factor), a comprehensive transcriptional regulator of Escherichia spp. Therefore, the present invention aims to bind the PL promoter region including all of the IHF attachment sequences to the guaA gene, and in this case, the technique of optimizing and inserting the shineAlgano sequence, which is essential for the translation process, is missing. To provide.

The ribosome attachment sequence of Escherichia coli of the present invention is known as a shine-dalgarno sequence (SD sequence), and is expressed as 5'-AGGAGG-3 'within 10 base pairs from AUG, which is a codon initiation process. Known consensus sequences are located. This is a part that complementarily binds to the 3'-UCCUCC-5 'region of 16S rRNA, a constituent of ribosomes responsible for the detoxification of Escherichia spp., Even if the consensus sequence is missing. It is well known that the efficiency decreases. Another study shows that the 5'-TAAGGAG-3 'sequences are strongly conserved and are optimal when their positions are located at the 13th base pair from the first AUG start codon of the structural gene. Therefore, in the present invention, the artificially combined Shindalgano sequencing was combined with the guaA gene so that the mRNA of the guaA structural gene transcribed from the strong PL promoter can be translated with high efficiency.

The PL promoter was obtained by polymerase chain reaction (PCR) using bacteriophage lambda as a template, and the guaA structural gene was obtained by PCR using genomic DNA of Escherichia sp. K12 as a template. Ribosome attachment sequences of Escherichia spp. Strains can consist of up to about 20 base pairs, which are located between the PL promoter and the guaA structural gene. Therefore, each PCR product is manufactured by sharing the ribosome attachment sequence of artificially combining 3'-terminal one of the primers used to obtain the PL promoter and 5'-terminal one of the primers used to obtain the guaA structural gene. Complementary binding between the ends was possible based on the ribosome attachment sequence, and the final PCR product of the PL promoter, the ribosome attachment sequence, and the guaA structural gene was combined in the secondary PCR using the remaining primers as a template. In addition, the primers used to obtain the PL promoter and guaA structural gene were prepared by inserting the restriction enzyme cleavage sequence. Therefore, the final PCR product was complemented to the pUC18 vector digested with the same restriction enzyme after the restriction enzyme treatment. It is easily introduced through integration. In order to overcome the low cloning efficiency caused by high mortality of the parent strain following 5'-guanylic acid synthase overexpression, Escherichia spp POP2136 strain containing the cI inhibitor of bacteriophage lambda on the genome was used to prepare the plasmid. It was.

In one embodiment of the present invention, as a variant of Escherichia sp. K12, there is provided an Escherichia spp microorganism, wherein the vector of the present invention is transformed and the endogenous guaA gene is inactivated. The Escherichia spp. Microorganism of the present invention is a strain in which the endogenous guaA gene is inactivated, thereby improving the stability in the microorganism of the vector of the present invention. In addition, the production of 5'-guanylic acid synthase was increased along with improved stability of the overexpression vector.

Inactivation of the guaA gene can be accomplished by using various knockout mutations. Inactivation mutations include, but are not necessarily in vitro, convenient foreign fragments of DNA that provide dominant selectable markers inserted into specific protein coding regions of native chromosomal DNA to inactivate their sequences. Inactivation mutations within the protein coding region inhibit the expression of the wild type protein or lose the function provided by that protein.

The inactivation process can be performed by mixing the inactivated gene cassette with the culture of the strain. Naturally, strains can compete for DNA influx and mold conversion, but it is desirable to make the strain competent for DNA influx in advance by appropriate methods. The inactivated gene cassette refers to a fragment of native chromosomal DNA into which a foreign DNA fragment is inserted that can provide a selection marker, introduces the foreign DNA fragment into the fragment of genomic DNA and replaces the wild-type chromosomal copy of this sequence with the inactivation cassette. It is formed by. As an aspect of the invention the inactivation protocol involves cloning a foreign DNA fragment into the target DNA such that a "tail" containing the target site DNA remains at the 5 'and 3' ends of the inactivation cassette. The tail is preferably at least 50 base pairs, more preferably 200 to 500 base pairs for efficient recombination and / or gene conversion. Conveniently, foreign DNA cloned into the target DNA provides a selection marker, for example an antibiotic resistance gene. When the target DNA is inactivated by the antibiotic resistance gene, selection of the transformants is carried out on an agar plate containing the appropriate antibiotic. After transformation, the cell fraction into which the inactivation cassette is introduced undergoes homologous recombination or gene conversion along the genomic DNA tail of the cassette and eventually the wild type genomic sequence is replaced with the inactivation cassette. Whether inactivation recombination has been performed can be easily confirmed, for example, by Southern blotting hybrid experiments, and more conveniently, by PCR. Those skilled in the art will appreciate that inactivation cassettes and DNA fragments of the present invention can be prepared by conventional cloning methods. PCR includes genomic DNA, suitable enzymes, primers and buffers and is conveniently performed by Peltier Thermal Cycler (MJ research). Positive PCR results can be determined, for example, by detecting DNA fragments of appropriate size by agarose gel electrophoresis.

In order to increase stability by removing foreign gene information from genomic DNA, foreign DNA cloned into target DNA, such as antibiotic resistance genes, must be removed. In order to remove the antibiotic resistance gene from the genomic DNA of the transformant in which the target gene is inactivated, a plasmid expressing a gene encoding an enzyme that recognizes a part of the nucleotide sequence present in the inactivation cassette and removes the antibiotic resistance gene is transformed. Switch Selection of the transformants is carried out on agar plate medium containing ampicillin. The elimination of antibiotic genes on the target DNA is confirmed by the formation of colonies via tupis on agar plates containing appropriate antibiotics.

Therefore, the microorganism of the present invention transforms Escherichia sp. K12 with a recombinant vector in which an antibiotic marker gene fragment is inserted into the guaA gene, selects a strain in which the guaA gene is inactivated, and removes the antibiotic resistance gene from the strain. The obtained microorganism is preferred.

Specifically, as can be seen in the following examples, microorganisms of the genus Escherichia produced by the manufacturing method including the following process is preferable. That is, genomic DNA is isolated from Escherichia sp. K12, and the guaA gene is amplified by PCR reaction using the template as a template. Transformation of the recombinant vector (e.g., recombinant plasmid pUC-guaA) formed by cloning the obtained gene product into a plasmid or other vector (e.g., natural or recombinant plasmid, cosmid, virus, bacteriophage, etc.) Is introduced into a host cell such as E. coli. After culturing the transformant, the recombinant vector having the guaA gene was extracted from the recombinant vector, and the antibiotic vector gene fragment containing the loxP sequence was inserted into the guaA gene in the extracted recombinant vector to inactivate the guaA gene. For example, pUC-guaA :: loxPKm) is constructed, and then the recombinant vector is introduced into the host cell by transformation as described above and cultured. The recombinant vector propagated from the resulting transformant is isolated and an inactivated cassette fragment (eg, guaA :: loxPKm) of the guaA gene is obtained by appropriate restriction enzyme treatment or PCR reaction. These fragments were introduced into Escherichia sp. K12 by a technique such as an electroshock method, and then the fragments of the guaA gene containing antibiotic markers were recombined with the wild guaA gene on the chromosome to continue to inactivate the guaA gene. Isolate strains that are antibiotic resistant. To remove the antibiotic marker on the chromosome of the selected strain, a recombinant vector expressing a gene encoding an enzyme that removes the antibiotic resistance gene by recognizing the loxP sequence in the antibiotic resistance gene is transformed. This vector is introduced into host cells by transformation into Escherichia sp. K12, and the selection of the transformants is carried out on agar plate medium containing ampicillin using the antibiotic resistance of the vector. The elimination of antibiotic genes on the target DNA is determined by colony formation through two-pic on agar plates containing appropriate antibiotics, and fragments of the guaA gene containing some of the antibiotic markers may be combined with wild guaA genes on the chromosome. Even after recombination and growth, strains having inactivation characteristics of the guaA gene (for example, Escherichia sp. K12ΔguaA) are isolated.

Therefore, the microorganism of the present invention is preferably a microorganism obtained by transforming Escherichia sp. K12 with a recombinant vector pUC-guaA :: loxPKm and selecting a strain in which the guaA gene is inactivated, and Escherichia sp. desirable.

The present invention also provides a method of culturing the microorganism of the present invention and accumulating 5'-guanylic acid synthase (GMP synthetase) in a medium at a high concentration.

The microorganism of the present invention can be cultured while controlling the temperature, pH and the like in a conventional medium containing a suitable carbon source, nitrogen source, amino acids, vitamins and the like.

As the carbon source, carbohydrates such as glucose, fructose, sterilized pretreated molasses (i.e., molasses converted to reducing sugars) and the like can be used. As nitrogen sources, various inorganic nitrogen sources such as ammonia, ammonium chloride and ammonium sulfate and peptone, NZ-amine Organic nitrogen sources may be used, such as meat extracts, enzyme extracts, corn steep liquors, casein hydrolysates, fish or degradation products thereof, skim soy cakes, or degradation products thereof. As the inorganic compound, potassium monohydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, etc. may be used, and vitamins and nutrient-constituting bases may be added as necessary.

The culture can be carried out under aerobic conditions, for example, by shaking culture or aeration agitation culture, preferably at a temperature of 30-40 ° C. The pH of the medium is preferably maintained near neutral during the cultivation, and the culture may be performed for 1 to 2 days, and microorganisms containing the activity of 5'-guanylic acid synthase are accumulated in the medium.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrating the present invention, and the present invention is not limited by these examples.

Example 1 Construction of Overexpression Vector pUPSG Overproducing 5′-Guanylic Acid Synthase

The PL promoter 245bp region was amplified by polymerase chain reaction (PCR) using bacteriophage lambda 1316 (bacteriophage lambda 1316) as a template. The primers used were 5'-GCT GAATTC TTGCCTCACGATCGCCCCCAAAAC-3 '(SEQ ID NO: 1) and 5'-ATGATC' towards 3'-end (SEQ ID NO: 2), and the underscore of SEQ ID NO: 1 was limited. Recognition sequence of the enzyme EcoRI is shown, and the double underline of SEQ ID NO: 2 shows the part including the artificially combined Shindalgano sequence (SD sequence). The reaction conditions were performed for 3 minutes at 95 ° C for the first denaturation step, then 30 seconds at 95 ° C for denaturation step, 30 seconds at 50 ° C for annealing step, and 30 seconds at 72 ° C for extension step. After repeating 30 cycles, the final extension step was carried out for 5 minutes at 72 ℃.

Primers used to obtain 1.6 kb guaA gene from PCR reactions based on genomic DNA of Escherichia sp. K12 were 5'-end toward 5'-ACCATGATAAGGAGGATCATATGACGGAAAACATTCATAAG-3 '(SEQ ID NO: 3) and 3'-end. 5'-GCC AAGCTT TCATTCCCACTCAATGGTAG-3 '(SEQ ID NO: 4), and the double underline of SEQ ID NO: 3 contains the artificially combined Shindalgalano sequencing, which is complementarily identical to that of SEQ ID NO: 2 in the future for two PCRs. It acts as a site to which the product can bind, and the underline of SEQ ID NO: 4 indicates the recognition sequence of restriction enzyme HindIII. The reaction conditions were the same as above except that the extension step was carried out to 1 minute 30 seconds and the last extension step to 10 minutes.

Each PCR result was electrophoresed on 1.0% agarose gel, and then the specific bands were eluted and their relative amounts were confirmed by electrophoresis.They were mixed in the same mole ratio and used as a template for the second PCR. The reaction proceeded. In this reaction, the primers of SEQ ID NO: 1 and SEQ ID NO: 4 were used, and the reaction conditions were the same as above except that the extension step was performed by increasing the time to 2 minutes. Secondary PCR products were also electrophoresed on agarose gel, followed by elution of 1.9 kb of band, followed by pure separation with the restriction enzymes EcoRI and HindIII, incubated at 37 ° C. overnight with appropriate buffer, and the pUC18 vector. . After recovering only the DNA from each reaction, and mixed in an appropriate ratio, the reaction was carried out for 4 hours at 16 ℃ by adding a DNA ligase ligase, part of which was transformed into a strain of Escherichia spp. The plate was plated and incubated overnight at 37 ° C.

Colonies were buried in platinum and inoculated in 3 ml of ampicillin-containing liquid medium and incubated at 37 ° C. overnight, and then plasmid DNA was extracted using miniprep kit (Intron). Plasmid DNA was treated with restriction enzymes EcoRI and HindIII to generate 1.9 kb fragments of PL-SD-guaA. The completed plasmid was confirmed by sequencing analysis that no error occurred during PCR reaction (Bacteriophage lambda-derived PL promoter (SEQ ID NO: 5) and guaA gene sequence (NCBI accession no. M10101)), which was named pUPSG ( 1).

Example 2 Preparation of a Novel Microorganism Inactivated guaA Gene of Escherichia sp. K12 Strain

Genomic DNA was extracted from Escherichia sp. K12 using a genomic-tip system (QIAGEN), and the guaA structural gene was amplified by PCR using the template. The primers used were 5'-ATGACGGAAAACATTCATAAGC-3 '(SEQ ID NO: 7) and 5'-TCATTCCCACTCAATGGTAGC-3' (SEQ ID NO: 8) at the 5'-end, and the reaction conditions were subjected to the initial denaturation step. After 3 minutes at 95 ° C, 30 seconds at the denaturation step 95 ° C, 30 seconds at the annealing step 50 ° C, 1 minute 30 seconds at the extension step 72 ° C, and 25 cycles were repeated. The extension step was carried out at 72 ° C. for 10 minutes.

The PCR product was electrophoresed on 1.0% agarose gel and eluted with a band of 1.6 kb to connect overnight to the HincII site of the pUC18 cloning vector. The recombinant plasmid pUC-guaA thus formed was transformed into E. coli POP2136 and plated in agar plate medium containing ampicillin and incubated overnight at 37 ° C.

Colonies were buried in platinum and inoculated in 3 ml of ampicillin-containing liquid medium and incubated at 37 ° C. overnight, and then plasmid DNA was extracted using miniprep kit (Intron). Plasmid DNA was treated with restriction enzymes EcoRI and HindIII to generate 1.6 kb fragments. The guaA structural gene was cloned. The identified plasmid pUC-guaA was treated with restriction enzyme HincII, followed by eluting a band of about 4.3 kb in a 1.0% agarose gel. The kanamycin resistance gene fragment (about 1.7 kb) containing the loxP moiety obtained by treatment of the plasmid pUG6 with restriction enzymes EcoRV and HincII was blunt-ended to obtain a recombinant plasmid pUC-guaA :: loxPKm (FIG. 2).

This recombinant plasmid pUC-guaA :: loxPKm was transformed into POP2136, plated in solid medium containing ampicillin and kanamycin and incubated overnight at 37 ° C. Colonies were buried in platinum and inoculated in 3 ml of liquid medium containing ampicillin and kanamycin, followed by incubation overnight, and plasmid DNA was extracted using a miniprep kit. PCR reaction using the plasmid DNA as a template amplified a DNA fragment (guaA :: loxPKm) containing a guaA structural gene and a loxPKm region, and a fragment of about 2.3 kb in size. The primers used were SEQ ID NO: 7 and SEQ ID NO: 8, and the reaction conditions were performed in the initial denaturation step at 95 ° C. for 3 minutes and then again at 30 ° C. at the denaturation step at 95 ° C., and at 30 ° C. at the annealing step at 50 ° C. , Extension step (2 minutes 30 seconds) was performed at 72 ℃ and 30 cycles were repeated after the final extension step was carried out for 10 minutes at 72 ℃. The DNA fragment guaA :: loxPKm was introduced into an Escherichia spp. K12 strain by electroshock treatment, plated on agar plate medium containing kanamycin, and colonies were selected.

To remove antibiotic markers of the selected colonies, pCP20 plasmids were transformed into selected colonies, plated in agar plate medium containing ampicillin and incubated overnight at 30 ° C. Colonies were buried in platinum and two-spiced in agar plate media without ampicillin and kanamycin, and then incubated overnight at 43 ° C. After overnight incubation, colonies formed only in antibiotic-free medium were obtained, and they were named K12ΔguaA after reconfirming that they could not grow on agar plate medium containing kanamycin.

Example 3 Measurement of Stability of pUPSG Plasmid and Production of 5′-Guanylic Acid Synthase in Selected Strain Escherichia sp. K12ΔguaA Strain

Escherichia sp. K12 strain and K12ΔguaA strain were transformed with pUPSG and plasmid stability and 5'-guanylic acid synthase production in Erlenmeyer flasks were investigated using 5'-guanylic acid titer (Table 1). Transgenic strains were plated on an agar plate medium containing antibiotics ampicillin and incubated at 37 ° C for 10 hours, inoculated into 3ml titer medium containing ampicillin, and cultured at 37 ° C for 250 rpm for 16 hours. 1% of 50ml titer was inoculated and incubated for 10 hours at 200 rpm at 30 ℃ and 37 ℃. Production of 5'-guanylic acid synthase was measured using the cultured strains, and some strains were plated at appropriate dilution concentrations in agar plate medium containing no antibiotics and agar plate medium containing antibiotics at 30 ° C. The number of colonies generated by incubating for 16 hours was counted to determine the stability of the pUPSG plasmid. As a result, as shown in Table 2, the plasmid stability was improved from K12 ΔguaA strain to 2 to as many as 100 times according to the temperature of K12 strain, and the yield of 5'-guanylic acid synthase increased more than 3 times. It can be seen.

5'-guanylic acid titer Composition Concentration (g / L) Bakto peptone 16 NaCl 5 Yeast extract 10 pH 7.0

Comparison of 5'-Guanylic Acid Synthase Productivity and pUPSG Plasmid Stability in Transgenic Strains K12 [pUPSG] K12 △ guaA [pUPSG] Enzyme titer Number of colonies stability Enzyme titer Number of colonies stability 30 ℃ Antibiotic free medium - 5 × 10 ⁸ 40% - 4 × 10 ⁸ 75% Antibiotic medium 2U / ml 2 × 10 ⁸ 4U / ml 3 × 10 ⁸ 37 ℃ Antibiotic free medium - 7 × 10 ⁹ 0.09% - 8 × 10 ⁹ 8.8% Antibiotic medium 6U / ml 6 × 10 ⁶ 21U / ml 7 × 10 ⁸

Stability is the percentage of colonies grown on antibiotic-free media divided by the number of colonies grown on antibiotic-free media.

A K12ΔguaA strain transformed with pUPSG was entrusted with an accession number of KCCM-10629 (accession date: November 30, 2004).

The novel vector pUPSG, which always overexpresses the guaA gene by optimizing the PL promoter and Shindallagan base sequence according to the present invention, and the Escherichia sp. K12ΔguaA strain having increased plasmid stability, are the The production of 5'-guanylic acid synthase was significantly higher than that of K12 wild strain. Therefore, a stable and large amount of 5'-guanylic acid synthase in the 5'-guanylic acid production process is proposed.

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Claims (15)

delete delete delete delete delete An endogenous transformed with a vector of a bacteriophage lambda-derived PL promoter, a ribosome binding sequence functional in Escherichia, and a polynucleotide encoding a 5'-guanylic acid synthetase. A microorganism of the genus Escherichia in which the guaA gene is inactivated. The microorganism of claim 6, wherein the microorganism is E. coli K12 mutant strain. The microorganism according to claim 7, which is E. coli K12 mutant transformed with the pUPSG vector described in FIG. 1 (Accession No. KCCM-10629). 8. The microorganism according to claim 7, wherein the E. coli K12 mutant strain is inactivated by guaA gene substitution by nucleic acid by homologous recombination. A method of accumulating 5'-guanylic acid synthase at a high concentration in a medium by culturing the microorganism according to any one of claims 6 to 9. The method of claim 10, wherein the ribosomal binding sequence is a polynucleotide of SEQ ID NO: 6. delete The microorganism of claim 6, wherein the bacteriophage lambda derived PL promoter is a polynucleotide of SEQ ID NO: 5. 8. The microorganism of claim 6, wherein the ribosomal binding sequence is a polynucleotide of SEQ ID NO: 6. 8. 7. The microorganism of claim 6, wherein the polynucleotide encoding the 5'-guanylic acid synthase has an NCBI accession number of M10101.

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