novel promoter and use thereof
Technical field
[1]
The present application relates to a novel promoter and a method of manufacturing a target product using the same.
[2]
Background
[3]
As the understanding of intracellular mechanisms increases with the development of technology, methods for producing various target products by regulating the metabolism of cells are being developed. Currently, microorganisms such as the genus Escherichia or the genus Corynebacterium are most commonly used, and are directly used for the production of various small molecules, high molecular compounds, pharmaceutical proteins, such as amino acids, polyphenols, flavonoids, antibodies, and natural rubber.
[4]
[5]
Among various methods of regulating metabolism, a highly efficient gene expression system for inducing overexpression of a target gene has been mainly studied. For example, as it is well known that the most important factor in gene expression is a promoter, studies using several promoters (Ptac, Ptrc, Plac) derived from E. coli are actively progressing. In addition, the present applicant has discovered a cj1 promoter derived from Corynebacterium ammoniagenes, which exhibits a strong activity of 296% compared to the tac promoter of Escherichia coli through prior research (Korean Patent Publication No. 10-2006-0068505). However, there is still a need for research on how to efficiently produce the desired product with high yield.
[6]
Detailed description of the invention
Technical challenge
[7]
As a result of intensive research to develop a method that can increase the expression of the target gene and eventually produce the target product, the present inventors mutated the sequence of the existing cj1 promoter, which is known to have high activity, to create a new promoter (cj2.2) It was prepared, and this application was completed by confirming that it exhibits about three times higher activity than the cj1 promoter.
[8]
Means of solving the task
[9]
One object of the present application is to provide a polynucleotide having promoter activity, including the polynucleotide sequence of SEQ ID NO: 1.
[10]
Another object of the present application is to provide a vector containing a polynucleotide having the promoter activity.
[11]
Another object of the present application is to provide a host cell into which the vector has been introduced.
[12]
Another object of the present application is the step of culturing the host cell; And it is to provide a method for producing a target material comprising the step of isolating the target material from the host cell or the culture medium.
[13]
Effects of the Invention
[14]
Since the novel promoter of the present application can be introduced into a microorganism to increase the expression and activity of genes linked thereto, it can be effectively used to efficiently produce the target product affected by the promoter and the gene.
[15]
Brief description of the drawing
[16]
1 is a graph showing the results of analyzing the fluorescence intensity of green fluorescent protein expressed in individual clones of a recombinant vector mutant library prepared according to an embodiment of the present application. cj1 refers to a clone containing the cj1 promoter, and M1 to M20 refers to each individual clone of the mutant library.
[17]
2 is a cleavage map of a vector including the cj2.2 promoter and metZ gene prepared in an embodiment of the present application.
[18]
3 is a cleavage map of a vector including a cj2.2 promoter and opss gene prepared in an embodiment of the present application.
[19]
Best mode for carrying out the invention
[20]
This will be described in detail as follows. Meanwhile, each description and embodiment disclosed in the present application may be applied to each other description and embodiment. That is, all combinations of various elements disclosed in the present application belong to the scope of the present application. In addition, it cannot be seen that the scope of the present application is limited by the specific description described below.
[21]
[22]
One aspect of the present application provides a polynucleotide having promoter activity, including the polynucleotide sequence of SEQ ID NO: 1.
[23]
[24]
In the present application, the term "promoter" includes a binding site for a polymerase and has a transcription initiation activity of a promoter gene of interest into an mRNA, a non-translated nucleotide sequence upstream of the coding region, that is, a polymerase It refers to a DNA region that binds to initiate transcription of a gene. The promoter may be located at the 5'site of the mRNA transcription initiation site.
[25]
[26]
In the present application, a polynucleotide having promoter activity including the polynucleotide sequence of SEQ ID NO: 1 may be referred to as "polynucleotide" or "cj2.2 promoter", and all of the terms described above are used herein. Can be used.
[27]
[28]
For the purposes of the present application, the polynucleotide may have an increased promoter activity compared to a conventional promoter. In addition, it is possible to increase the expression of the target gene operably linked to the polynucleotide in the host cell of interest, and increase the expression and activity of the protein encoded by the target gene as well as the expression of the target gene.
[29]
In addition, the polynucleotide can be used as a universal promoter.
[30]
[31]
In this case, the term "target gene" refers to a gene encoding a protein of interest to increase expression, and may be a gene involved in amino acid production, but is not limited thereto.
[32]
Specifically, the gene may encode a protein involved in the production of amino acids such as methionine or cysteine, and more specifically, a methionine converting enzyme involved in the production of methionine or a cysteine ​​converting enzyme involved in the production of cysteine. More specifically, O-acetylhomoserine sulfhydrylase (O-acetylhomoserine sulfhydrylase) involved in the production of methionine or O-phosphoserine sulfhydrylase (OPSS) involved in the production of cysteine ​​was used. It may be coding, and most specifically, a metZ gene or an opss gene, but is not limited thereto. The sequence of each gene can be readily obtained by those skilled in the art through a known database such as GenBank of the National Institutes of Health.
[33]
[34]
In the present application, the polynucleotide may be composed of the nucleotide sequence of SEQ ID NO: 1.
[35]
In addition, the nucleotide sequence of the present application may be modified by conventionally known mutagenesis methods, for example, direct evolution and site-directed mutagenesis.
[36]
Thus, the polynucleotide is at least 60% or more, specifically 70% or more, more specifically 80% or more, even more specifically 83% or more, 84% or more, 88% of the nucleotide sequence of SEQ ID NO: 1 It may include a polynucleotide comprising a nucleotide sequence having homology of at least 90%, at least 93%, at least 95%, or at least 97%. As a sequence having homology with the above sequence, if a polynucleotide sequence having a biological activity substantially identical to or corresponding to the nucleotide sequence of SEQ ID NO: 1, some sequences have a deleted, modified, substituted or added polynucleotide sequence. It is obvious that it is included in the scope of this application.
[37]
[38]
In the present application, the term "homology" means the degree to which it matches a given nucleotide sequence and may be expressed as a percentage. In the present specification, its homologous sequence having the same or similar activity as a given nucleotide sequence is indicated as "% homology". Homology to the nucleotide sequence can be determined, for example, by the literature algorithm BLAST (Karlin and Altschul, Pro. Natl. Acad. Sci. USA, 90, 5873, 1993) or FASTA by Pearson (Method Enzymol. , 183, 63, 1990). Based on this algorithm BLAST, a program called BLASTN or BLASTX has been developed (see: http://www.ncbi.nlm.nih.gov).
[39]
The “stringent conditions” refer to conditions that allow specific hybridization between polynucleotides. These conditions are specifically described in known literature. For example, among genes with high homology, genes with homology of 60% or more, specifically 90% or more, more specifically 95% or more, more specifically 97% or more, particularly 99% or more Under conditions that hybridize to each other and do not hybridize to genes with lower homology, or to wash conditions for general Southern hybridization, 60°C, 1×SSC, 0.1% SDS, specifically 60°C, 0.1×SSC, 0.1 At a salt concentration and temperature corresponding to% SDS, more specifically 68° C., 0.1×SSC, and 0.1% SDS, the conditions for washing once, specifically two to three times, can be enumerated. Hybridization requires that two nucleotides have a complementary sequence, although a mismatch between bases is possible depending on the stringency of the hybridization. The term “complementary” is used to describe the relationship between nucleotide bases capable of hybridizing to each other. For example, with respect to DNA, adenosine is complementary to thymine and cytosine is complementary to guanine. Thus, the present application may also include substantially similar polynucleotide sequences as well as isolated polynucleotide fragments that are complementary to the entire sequence.
[40]
Specifically, polynucleotides having homology can be detected using hybridization conditions including a hybridization step at a Tm value of 55° C. and using the above-described conditions. In addition, the Tm value may be 60°C, 63°C, or 65°C, but is not limited thereto and may be appropriately adjusted by a person skilled in the art according to the purpose. The appropriate stringency to hybridize a polynucleotide depends on the length and degree of complementarity of the polynucleotide, and the parameters are well known in the art (see Sambrook et al., supra, 9.50-9.51, 11.7-11.8).
[41]
[42]
In particular, the expression consisting of the nucleotide sequence of SEQ ID NO: 1 in the above refers to the addition of nucleotides that may occur during the process of linking to the target gene, such as the use of restriction enzymes, when the polynucleotide is linked to the target gene as a promoter and used. Or it does not exclude cases of deletion, and/or mutation.
[43]
In addition, if the polynucleotide having promoter activity consisting of the nucleotide sequence of SEQ ID NO: 1 is a nucleotide sequence having the promoter activity of the present application by hydride under stringent conditions with a complementary sequence to all or part of the nucleotide sequence of SEQ ID NO: 1 It can be included without limitation.
[44]
[45]
Another aspect of the present application provides a vector comprising a polynucleotide having the promoter activity.
[46]
In this case, the description of the "promoter" is as described above.
[47]
[48]
In the present application, the term "vector" refers to an artificial DNA molecule containing a genetic material so as to express a gene of interest in a suitable host cell, and includes a suitable gene expression control sequence; And it means a DNA preparation comprising the nucleotide sequence of the gene of interest operably linked thereto.
[49]
[50]
Specifically, the term “gene expression control sequence” refers to a sequence capable of expressing a gene of interest that includes a polynucleotide having the promoter activity and is operably linked thereto.
[51]
[52]
Specifically, the gene expression control sequence includes an arbitrary operator sequence for controlling transcription, a sequence encoding a suitable mRNA ribosome binding site, and a DNA controlling the termination of transcription and translation in addition to a promoter for carrying out transcription of the gene. However, it is not limited thereto. In addition, a promoter and a ribosome binding site may be included as a regulatory sequence suitable for prokaryotic organisms, but are not limited thereto. The polynucleotide having the promoter activity of the present application may constitute a sequence for regulating gene expression as described above as needed by a person skilled in the art.
[53]
[54]
In addition, the term "operatively linked" means that the polynucleotide having the promoter activity is functionally linked with the nucleotide sequence of the target gene to initiate and mediate transcription of the target gene. The operable linkage may be prepared using a gene recombination technique known in the art, and site-specific DNA cleavage and linkage may be prepared using a cleavage and linkage enzyme in the art, but is not limited thereto.
[55]
[56]
The vector used in the present application is not particularly limited as long as it can be expressed in the host cell, and any vector known in the art may be used to transform the host cell. Examples of commonly used vectors include natural or recombinant plasmids, cosmids, viruses and bacteriophages.
[57]
For example, pWE15, M13, λLB3, λBL4, λIXII, λASHII, λAPII, λt10, λt11, Charon4A, and Charon21A can be used as a phage vector or a cosmid vector, and pBR-based, pUC-based, pBluescriptII-based plasmid vector , pGEM system, pTZ system, pCL system, pET system, etc. can be used.
[58]
In addition, the endogenous promoter in the chromosome may be replaced with a polynucleotide having the promoter activity of the present application through the vector for chromosome insertion into the host cell. For example, pECCG117, pDZ, pACYC177, pACYC184, pCL, pUC19, pBR322, pMW118, pCC1BAC, pCES208, pXMJ19 vectors, etc. may be used, but are not limited thereto.
[59]
In addition, the insertion of the polynucleotide into the chromosome may be performed by any method known in the art, for example, by homologous recombination.
[60]
Since the vector of the present application can be inserted into a chromosome by causing homologous recombination, it may additionally include a selection marker to confirm whether the chromosome is inserted. The selection marker is for selecting cells transformed with a vector, i.e., to confirm whether a polynucleotide is inserted, and confers a selectable phenotype such as drug resistance, nutritional demand, resistance to cytotoxic agents, or expression of surface proteins Markers can be used. In an environment treated with a selective agent, only cells expressing the selection marker survive or exhibit other phenotypic traits, and thus transformed cells can be selected.
[61]
[62]
Another aspect of the present application provides a host cell into which the vector has been introduced.
[63]
In this case, the description of the "vector" is as described above.
[64]
[65]
In the present application, the term "host cell" refers to a transformant transformed by the vector containing the polynucleotide.
[66]
At this time, the term "transformation" means introducing the vector, including the polynucleotide having promoter activity, into a host cell so that the target gene under the control of the polynucleotide can be expressed in the host cell. .
[67]
Specifically, the host cell may be included without limitation as long as the polynucleotide is introduced and can act as a promoter, and thus the expression of the target gene under the control of the polynucleotide is increased. As an example, it may be a microorganism, a plant cell, an animal cell, etc., specifically, may be a microorganism such as Escherichia genus or Corynebacterium genus, and more specifically, may be Escherichia coli, Not limited.
[68]
[69]
The method of transformation includes all methods of introducing the vector into a host cell, and can be performed by selecting an appropriate standard technique as known in the art according to the host cell. For example, electroporation, calcium phosphate (CaPO 4 ) precipitation, calcium chloride (CaCl 2 ) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, and Lithium acetate-DMSO method, and the like, but is not limited thereto.
[70]
[71]
Another aspect of the present application is culturing the host cell; And it provides a method for producing a target material comprising the step of isolating the target material from the host cell or a culture medium in which the same.
[72]
At this time, the description of the "host cell" is as described above.
[73]
[74]
In the present application, the target substance may be an amino acid. Specifically, the amino acid may be an L-form amino acid unless otherwise stated, and glycine, alanine, valine, leucine, isoleucine, threonine, serine, cysteine, glutamine, methionine, aspartic acid, asparagine, glutamic acid, lysine , Arginine, histidine, phenylalanine, tyrosine, tryptophan, proline, and may be selected from the group consisting of a combination thereof, but is not limited thereto. More specifically, the amino acid may be methionine or cysteine, but is not limited thereto.
[75]
[76]
In the present application, the term "culture" means growing a host cell in an appropriately artificially controlled environmental condition. In the present application, a method of producing a target substance using a host cell containing the polynucleotide may be performed using a method widely known in the art. Specifically, the culture may be continuously cultured in a batch process, an injection batch or a repeated fed batch process, but is not limited thereto. The medium used for cultivation must meet the requirements of the specific strain in an appropriate manner.
[77]
Sugar sources that can be used in the medium include sugars and carbohydrates such as glucose, saccharose, lactose, fructose, maltose, starch, cellulose, oils and fats such as soybean oil, sunflower oil, castor oil, coconut oil, palmitic acid, stearic acid. , Fatty acids such as linoleic acid, alcohols such as glycerol and ethanol, and organic acids such as acetic acid. These materials may be used individually or as a mixture, but are not limited thereto.
[78]
Nitrogen sources that can be used include peptone, yeast extract, broth, malt extract, corn steep liquor, soybean meal and urea or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate. The nitrogen source may also be used individually or as a mixture, but is not limited thereto.
[79]
Personnel that may be used may include potassium dihydrogen phosphate or dipotassium hydrogen phosphate or salts containing the corresponding sodium. In addition, the culture medium may contain a metal salt such as magnesium sulfate or iron sulfate required for growth. Finally, essential growth substances such as amino acids and vitamins can be used in addition to the above substances. In addition, precursors suitable for the culture medium may be used. The above-described raw materials may be added batchwise or continuously in a manner suitable to the culture during the cultivation process.
[80]
In the culture of the host cell, a basic compound such as sodium hydroxide, potassium hydroxide, ammonia or an acid compound such as phosphoric acid or sulfuric acid may be used in an appropriate manner to adjust the pH of the culture. In addition, foaming can be suppressed by using an antifoaming agent such as fatty acid polyglycol ester. Oxygen or an oxygen-containing gas (eg, air) may be injected into the culture to maintain an aerobic condition.
[81]
The temperature of the culture (medium) may be usually 20°C to 45°C, specifically 25°C to 40°C. The cultivation time may be continued until a desired amount of the target substance is obtained, but specifically, may be 10 to 160 hours.
[82]
[83]
Recovery of the target material from the culture (medium) may be separated and recovered by a conventional method known in the art. In this separation method, methods such as centrifugation, filtration, chromatography, and crystallization may be used. For example, the culture medium may be centrifuged at low speed to remove biomass, and the resulting supernatant may be separated through ion exchange chromatography, but is not limited thereto.
[84]
The recovery step may further include a purification process.
[85]
Mode for carrying out the invention
[86]
Hereinafter, the present application will be described in more detail through examples. However, these examples are for illustrative purposes only, and the scope of the present application is not limited to these examples.
[87]
[88]
Example 1: Construction of a mutant library of a recombinant vector containing the cj1 promoter sequence
[89]
In order to discover a promoter capable of strongly inducing gene expression in a microbial strain, a mutation library was prepared based on the existing cj1 promoter (Korea Patent Publication No. 10-2006-0068505), which is known to exhibit strong activity.
[90]
Specifically, a mutant library was prepared using the recombinant vector pCL-cj1-gfp containing the sequence of the cj1 promoter as a template. The library was constructed using an error-prone PCR kit (clontech Diversify® PCR Random Mutagenesis Kit). Under conditions in which mutation may occur, PCR was performed using the primers of SEQ ID NOs: 2 and 3. Conditions in which one mutation occurs per 1000 bp is as follows: dGTP is added without adding MnSO 4 (8 mM), 40 μM (final rxn), and pre-heating at 94° C. for 30 seconds, and then 94° C. At 30 seconds, the process of 1 minute at 68° C. was repeated 25 times. The PCR product obtained at this time was subjected to denaturation at 95°C for 50 seconds, annealing at 60°C for 50 seconds, and extension for 12 minutes at 68°C using a mega primer (500-125 ng). After repeating 25 times, a library was prepared by transforming into E. coli DH5α strain through a DpnI treatment process.
[91]
[92]
Example 2: Green fluorescent protein ( GFP ) activity intensity evaluation of the recombinant vector
[93]
In order to evaluate the activity of the recombinant vector variant library prepared in Example 1, its GFP activity was compared.
[94]
Specifically, a library of about 10,000 weeks or more was inoculated into M9 medium and cultured at 37° C. and 900 rpm for about 24 hours. After the cultured E. coli was centrifuged at 4000 rpm for 10 minutes to obtain cells, the cells were suspended in phosphate buffered saline (PBS), and a mutant strain having strong green fluorescent protein intensity was selected through a fluorescence cytometer.
[95]
As a primary screening, 20 mutant strains having a GFP activity of about 1.1 to 5 times higher than that of the cj1 promoter were selected (Fig. 1). Among them, the mutant promoter M11 having the highest GFP activity was named the cj2.2 promoter (SEQ ID NO: 1), and the sequence was analyzed.
[96]
[97]
Example 3: Construction of expression vector of cj2.2 promoter
[98]
In Example 2, it was confirmed that the cj2.2 promoter had the highest activity among the recombinant vector libraries, and site-directed mutagenesis was performed using the pCL_Pcj1 vector to introduce it into the microorganism strain.
[99]
Specifically, PCR was performed using the primers of SEQ ID NOs: 4 and 5. The PCR includes denaturation at 94° C. for 30 seconds, annealing at 55° C. for 30 seconds, and extension at 68° C. for 12 minutes, and the processes were repeated 18 times.
[100]
The product obtained through the above process was treated with DpnI, and then transformed into a DH5α strain to prepare a pCL_Pcj2.2 vector containing the cj2.2 promoter.
[101]
[102]
Example 4: Measurement of activity of cj2 .2 promoter
[103]
Example 4-1: methionine-converting enzyme (O- acetyl homoserine sulfinyl deurilra the expression level measurement)
[104]
In order to confirm the activity of the cj2.2 promoter and the ability to induce gene expression accordingly, a methionine converting enzyme (O-acetylhomoserine sulfhydrylase) was ligated to the cj2.2 promoter and its expression level was confirmed.
[105]
Specifically, the metZ gene (Korean Patent Publication No. 10-1250651) encoding O-acetylhomoserine sulfhydrylase was cloned into the pCL_Pcj2.2 vector prepared in Example 3. PCR was performed using the chromosome of Rhodobacter spheroid as a template, using primers of SEQ ID NOs: 6 and 7, and 30 seconds of denaturation at 94°C, annealing at 55°C for 30 seconds, and an extension of 2 minutes at 72°C. It was carried out repeatedly. The DNA fragment obtained by the PCR reaction was digested with BamHI/SalI, and then cloned into a pCL_P2.2 vector digested with the same restriction enzyme. The vector produced by the above method was named pCL-Pcj2.2-metZ, and a schematic diagram of the vector is shown in FIG. 2.
[106]
The pCL-Pcj2.2-metZ vector was transformed into an Escherichia coli K12 strain, cultured in an LB plate medium containing 50 μg/L spectinomycin, and colonies were selected. The selected colonies were inoculated into 3 ml of LB medium containing 50 μg/L spectinomycin, and cultured for 16 hours at 37° C. and 200 rpm. This was re-inoculated into a new 25 ml LB liquid medium (250 ml flask) and grown under the same culture conditions so that an OD 600 of 0.5 to 0.6 (2 to 3 hours), 500 ml of LB medium with 4% glucose added 1L container) was incubated until the glucose was depleted. After the culture was completed, 1 ml of the culture solution was collected and the supernatant was removed by a centrifuge to obtain cells. The cells were washed with 0.1M potassium phosphate buffer (pH7.5), and then suspended in 1 ml of potassium phosphate buffer, and the cells were disrupted using ultrasonic waves.
[107]
Then, the activity of the methionine converting enzyme (O-acetylhomoserine sulfhydrylase) in the lysate was measured. To measure the enzyme activity, in 1 ml of 80 g/L O-acetylhomoserine, 0.01 ml of sodium methylmercaptan (15%, w/v), 0.01 ml of O-acetylhomoserine sulfhydrylase, and pyridoxalphosphate (pyridoxal 5'-phosphate) To a substrate solution mixed with 0.1 mM, 5 µl of the lysate was added, followed by enzymatic reaction at 33° C. under stirring at 800 rpm. After the reaction was completed, HPLC was used to analyze the concentrations of methionine and O-acetylhomoserine, and protein activity was calculated through a conventional method using the results analyzed by HPLC. The results are shown in Table 1 below.
[108]
[109]
[Table 1]
vector Active (Unit/ml) Relative activity
pCL-Pcj1-metZ 9.3 1.0
pCL-Pcj2.2-metZ 29.2 3.1
[110]
[111]
As can be seen in Table 1, compared to the cj1 promoter, which is known to strongly induce gene expression in a microbial strain in the past, the cj2.2 promoter of the present application is more active than that of the gene (O-acetylhomoserine sulfhydrylase) by about 3.1. It was confirmed that it increased more times.
[112]
Accordingly, it was confirmed that the cj2.2 promoter has a stronger activity than that of a conventional promoter, and accordingly, the activity of the target enzyme encoded by the gene linked thereto may be further increased.
[113]
[114]
Example 4-2: Measurement of the expression level of cysteine ​​converting enzyme (O -phosphoserine sulfhydrylase )
[115]
In order to confirm the activity of the cj2.2 promoter and the ability to induce gene expression accordingly, a cysteine ​​converting enzyme (O-phosphoserine sulfhydrylase) was ligated to the cj2.2 promoter, and its expression level was confirmed.
[116]
Specifically, O-phosphoserine sulfhydrylase (hereinafter referred to as “OPSS”, Republic of Korea Patent Publication 10-1208267) was cloned into the vector prepared in Example 3. PCR was performed using the chromosome of Mycobacterium smegmatics as a template and primers of SEQ ID NOs: 8 and 9, and a vector was constructed in a similar manner to Example 4-1. The prepared vector was named pCL-Pcj2.2-opss, and a schematic diagram of the vector is shown in FIG. 3.
[117]
The pCL-Pcj2.2-opss vector was transformed into an Escherichia coli K12 strain, cultured on an LB plate containing 50 μg/L spectinomycin, and colonies were selected. The selected colonies were inoculated into 5 ml of LB medium containing 50 μg/L spectinomycin, and cultured for 16 hours at 37° C. and 200 rpm. This was re-inoculated into a new 25 ㎖ LB liquid medium (250 ㎖ flask) and cultivated under the same culture conditions so that an OD 600 of 0.5-0.6 (2-3 hours), LB medium 500 with 4% glucose added. Ml (1L container) was incubated until the glucose was depleted. Xylene 2% (v/v) was added to the culture solution and crushed, which was used for activity evaluation.
[118]
Thereafter, conditions and methods for evaluating the activity of the OPSS enzyme prepared using the pCL-Pcj2.2-opss vector are described in the existing literature (Mino K and Ishikawa K, FEBS letters, 551: 133-138, 2003; BurnsKE et al., J Am. Chem. Soc., 127: 11602-11603, 2005; Westrop GD et al., J. Biol. Chem., 281: 25062-25075, 2006). The addition unit of the substrate is ml, and conditions for measuring the enzyme activity are shown in Table 2 below.
[119]
[120]
[Table 2]
Blank OPSS reaction solution
enzyme - 40 ml of lysate (or 50 mg of crude protein)
1M HEPES (pH7.4) 100 ㎖ 100 ㎖
0.5M Na2S 20 ㎖ 20 ㎖
10mM PLP 20 ㎖ 20 ㎖
100mM OPS 0 50 ㎖
DW 790 ㎖ 750 ㎖
Total 1000 ㎖ 1000 ㎖
[121]
[122]
After pre-incubating the mixture excluding the enzyme in Table 2 at 37°C for 5 minutes, 50 mg of OPSS enzyme was added to react at 37°C, and then 0.1 ml of 33.2% TCA was added to the reaction solution to stop the reaction. , Cysteine ​​and cystine were analyzed through LC analysis by diluting in 0.1N HCl. The measured activity results are shown in Table 3 below.
[123]
[124]
[Table 3]
vector Active (Unit/ml) Relative activity
pCL-Pcj1-opss 6.0 1.0
pCL-Pcj2.2-opss 12.0 2.0
[125]
[126]
As can be seen in Table 3, compared to the cj1 promoter known to strongly induce gene expression in conventional microbial strains, the cj2.2 promoter of the present application has about twice the activity of the gene (O-phosphoserine sulfhydrylase). It was confirmed to increase further.
[127]
Accordingly, it was confirmed that the cj2.2 promoter has a stronger activity than that of a conventional promoter, and accordingly, the activity of the target enzyme encoded by the gene linked thereto may be further increased.
[128]
[129]
Example 5: Production of the target product using an enzyme produced by the cj2.2 promoter
[130]
Example 5-1: Production of methionine
[131]
It was confirmed through Example 4-1 that the use of the cj2.2 promoter can increase the activity of the methionine converting enzyme (O-acetylhomoserine sulfhydrylase), and the target product, methionine, was produced using the enzyme. By doing so, its activity and the efficiency of methionine production in the reactor system were confirmed.
[132]
On the other hand, O-acetylhomoserine sulfhydrylase converts O-acetylhomoserine into methionine.
[133]
Specifically, first, O-acetylhomoserine, a substrate for enzymatic reaction, was produced by fermenting the microbial strain described in the existing patent (Korean Patent No. 10-1250651). After removing the cells, 500 ml of the culture solution having a concentration of about 70 g/L was used as a substrate. 50 ml of the lysate (enzyme solution) produced in Example 4-1 was used as the enzyme O-acetylhomoserine sulfhydrylase. About 50 ml of sodium methylmercaptan was used for the enzymatic reaction, and pyridoxal phosphate (pyridoxal: 5'-phosphate, Sigma, USA) was added at a concentration of 0.1 mM. The reaction was set to pH 7.0, 33° C., and the stirring was set to 700 rpm. The reaction time was carried out for 3 hours while supplying sodium methyl mercaptan. Analysis of the concentration of O-acetylhomoserine and L-methionine over time was performed using HPLC, and the results are shown in Table 4 below.
[134]
[135]
[Table 4]
Hours [minutes] Cj1 Cj2.2
O-acetylhomoserine [g/L] L-methionine [g/L] O-acetylhomoserine [g/L] L-methionine [g/L]
0 66.1 0 66.1 0
30 20.6 24.1 9.2 35.5
90 9.5 42.0 2.8 47.7
120 3.1 47.2 0 49.4
180 0 49.5 - -
[136]
[137]
As can be seen in Table 4, compared to the conventional cj1 promoter, which is known to strongly induce gene expression in microbial strains, when the cj2.2 promoter of the present application is used, the rate at which O-acetylhomoserine is converted to L-methionine is more It was confirmed that it is fast, and thus L-methionine is produced more rapidly.
[138]
This was found to be due to an increase in the activity of the target product encoded by the gene, that is, methionine converting enzyme (O-acetylhomoserine sulfhydrylase) when the gene is expressed with the cj2.2 promoter of the present application.
[139]
[140]
Example 5-2: Production of cysteine
[141]
It was confirmed through Example 4-2 that the use of the cj2.2 promoter can increase the activity of the cysteine ​​converting enzyme (O-phosphoserine sulfhydrylase). By using the enzyme, the target product, cysteine, is produced. Its activity and the efficiency of cysteine ​​production in the reactor system were confirmed.
[142]
On the other hand, O-phosphoserine sulfhydrylase converts OPS into cysteine ​​(or its oxide, cystine).
[143]
Specifically, the cysteine ​​concentration in the conversion reaction solution was quantified by Gaitonde method and LC analysis. In addition, cystine produced by oxidation of cysteine ​​was quantified by LC analysis. First, prior to the conversion reaction in the 1L container scale, centrifugation (10000rpm, 10 minutes, 4°C) was performed on the OPS fermentation broth obtained through the 1L container scale fermentation to obtain a supernatant, which was permeated through the membrane (0.45 μm). The cells were removed. 72 g Na 2 S was added to the OPS fermentation broth (19.317 g/L), and the resulting precipitate was removed using Whatman filter paper (6 μm). Thereafter, 10 mM PLP (pyridoxal 5'-phosphate) was added, pre-incubated at 37° C., 200 rpm, for 5 minutes, and finally 50 mg OPS sulfhydrylase was added to perform a conversion reaction in a 1 L vessel scale. Conditions for measuring the enzyme activity are shown in Table 5 below.
[144]
[145]
[Table 5]
Reaction solution composition Concentration (or weight) in the reaction solution Mixed quantity (weight or volume)
OPS sulfhydrylase (2.59 µg/µl) 50 ㎎ 19 ㎖
Na 2 S 300 mM 72g
10 mM PLP 0.1 mM 10 ㎖
OPS fermentation broth from which cells were removed (19.317 g/ℓ) 104.42 mM 1000 ㎖
Total - 1029 ㎖
[146]
[147]
Thereafter, 100 µl of the reaction solution was taken for each time period at 0, 10, 30, 60, and 120 minutes of reaction, and the reaction solution was mixed with 100 µl of 33.2% TCA to stop the reaction. Cysteine ​​and cystine were analyzed. In addition, cysteine ​​and cystine were quantified through the Gaitonde method. The analyzed results are shown in Table 6 below.
[148]
[149]
[Table 6]
Hours [minutes] Cysteine ​​and cystine [g/L]
Cj1 Cj2.2
0 1.36 2.5
10 3.58 4.76
30 6.45 7.65
60 8.57 8.98
120 9.07 -
[150]
[151]
As can be seen from Table 6, it was confirmed that cysteine ​​and cystine were produced more rapidly when the cj2.2 promoter of the present application was used, compared to the cj1 promoter, which is known to strongly induce gene expression in conventional microbial strains.
[152]
This was found to be due to an increase in the activity of the target product encoded by the gene, that is, cysteine ​​converting enzyme (O-phosphoserine sulfhydrylase) when the gene is expressed with the cj2.2 promoter of the present application.
[153]
[154]
From the above description, those skilled in the art to which the present application belongs will understand that the present application may be implemented in other specific forms without changing the technical spirit or essential features thereof. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not limiting. The scope of the present application should be construed as including all changes or modified forms derived from the meaning and scope of the claims to be described later rather than the above detailed description, and equivalent concepts thereof.
Claims
[Claim 1]
A polynucleotide having promoter activity, comprising the polynucleotide sequence of SEQ ID NO: 1.
[Claim 2]
A vector comprising a polynucleotide having the promoter activity of claim 1.
[Claim 3]
The vector according to claim 2, wherein the vector contains a gene encoding a protein of interest.
[Claim 4]
A host cell into which the vector of claim 2 has been introduced.
[Claim 5]
The host cell according to claim 4, wherein the host cell is a bacterial cell belonging to the genus Corynebacterium or genus Escherichia.
[Claim 6]
Culturing the host cell of claim 4; And separating the target material from the host cell or a culture medium in which it is cultured.