JP2009065837A - Promoter and activation method thereof - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for increasing the expression amount of protein in Actinomycetes. <P>SOLUTION: A method for activating a promoter comprises a step of culturing a recombinant microorganism incorporated with a promoter-inserted vector in a medium including a metal salt selected from the group consisting of magnesium, calcium and manganese. The activation of the promoter is to increase the expression amount of protein in a recombinant microorganism by the promoter. In this method, a promoter comprises a specific base sequence having a sequence of SEQ ID NO: 1 to 427, or a base sequence in which one or several bases are deleted, substituted or added in the base sequence and which can exhibit equal activity to or higher activity than that of the base sequence. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a promoter derived from actinomycetes and a method for activating the promoter. More specifically, the present invention relates to a method for increasing the protein expression level by activating a promoter.

  Streptomyces actinomycetes are very important microorganisms in the production of useful substances such as antibiotics and pesticides. These microorganisms are also extensively studied for enzymes and their production to produce various industrially useful enzymes.

  For example, aminopeptidases are catalysts for reactions during the production of seasonings (Patent Documents 1 to 3), molecular tools in protein sequencing, additives in a two-stage assay for endopeptidases (Non-Patent Document 1), recombinants It is used as a curing reagent (Non-Patent Document 2) and optically active amino acid production (Non-Patent Document 3) in the processing of proteins and fusion products. However, conventionally used aminopeptidases have limited use conditions in terms of stability, reactivity, cofactor requirements, substrate specificity, and the like. In contrast, the Streptomyces septatus strain TH-2 has a sufficiently high thermal stability and optimal reaction temperature, and appropriate reactivity, cofactor requirements, and substrate specificity. It was found to produce a novel aminopeptidase (SSAP) that exhibits

  On the other hand, the Streptomyces septatas TH-2 strain has also been found to secrete a large amount of metalloendopeptidase (SSMP) in the presence of inorganic phosphate and a carbon source (Patent Document 5). That is, Streptomyces ceptatas TH-2 secretes SSMP at a high concentration of 0.3 g / l in a medium rich in inorganic phosphate and glucose.

By the way, a large-scale expression system in actinomycetes is required. For example, it has been found that a DNA construct containing an H-nitrile hydratase gene cluster derived from Rhodococcus rhodochrous functions in microorganisms belonging to the genus Streptomyces (Patent Document 6). It is also known that an expression vector containing an L11 protein gene promoter derived from S. griseius highly expresses a heterologous gene during the actinomycetes growth phase (Patent Document 7).
JP 2002-355047 A JP 2000-325090 A Japanese Patent Application Laid-Open No. 2000-232828 JP 2005-218319 A JP 2006-197802 A JP 2005-237223 A JP-A-2005-21031 Orowski, M. et al., Biochemistry, 18, 4942-4950 (1981). Ben-Bassat, A. et al., Journal of Bacteriology, 169, 751-757 (1987). YA Yamaskov et al., Enzyme Microb. Technol., 3, 137-140 (1981)

  An object of this invention is to provide the method of increasing the expression level of protein in actinomycetes.

The present invention provides a method for activating a promoter, wherein the promoter has a base sequence from position 1 to position 427 of SEQ ID NO: 1 in the sequence listing, or one or several bases in the base sequence Consisting of a base sequence that is deleted, substituted or added and can exhibit an activity equal to or higher than that of the base sequence, the method comprising:
Culturing a recombinant microorganism into which a vector into which the promoter has been inserted is incorporated in a medium containing a metal salt selected from the group consisting of magnesium, calcium, and manganese, wherein the promoter is a protein gene A process coupled to the upstream side of
The activation of the promoter means that the expression level of the protein in the recombinant microorganism is increased by the promoter.

The present invention also provides a method of increasing the expression level of a protein in a recombinant microorganism, the method comprising culturing the recombinant microorganism in a medium,
In the recombinant microorganism, a vector into which a promoter linked to the upstream side of the protein gene is inserted is incorporated,
The promoter has a base sequence from position 1 to position 427 of SEQ ID NO: 1 in the sequence listing, or one or several bases are deleted, substituted or added in the base sequence, and is equivalent to the base sequence or It consists of a base sequence that can exert further activity, and the medium contains a metal salt selected from the group consisting of magnesium, calcium, and manganese.

The present invention further provides a method for producing a protein, the method comprising:
Culturing in a medium a recombinant microorganism in which a vector having a promoter linked upstream of the protein gene is inserted, and recovering the protein from the obtained culture,
The promoter has a base sequence from position 1 to position 427 of SEQ ID NO: 1 in the sequence listing, or one or several bases are deleted, substituted or added in the base sequence, and is equivalent to the base sequence or It consists of a base sequence that can exert further activity, and the medium contains a metal salt selected from the group consisting of magnesium, calcium, and manganese.

  In the above method, in one embodiment, the vector includes a palindromic sequence downstream of the protein gene, and the palindromic sequence includes a base sequence from position 1 to position 537 of SEQ ID NO: 2 in the sequence listing. Or a base sequence in which one or several bases are deleted, substituted or added and can take a palindromic structure.

  In one embodiment of the above method, the recombinant microorganism is actinomycetes.

  In a further embodiment of the above method, the actinomycetes is Streptomyces lividans.

  The present invention also has a base sequence from position 1 to position 427 of SEQ ID NO: 1 in the sequence listing, or one or several bases are deleted, substituted or added in the base sequence, and is equivalent to the base sequence Or providing a promoter comprising a base sequence capable of exhibiting an activity higher than that, and exhibiting an activity of increasing the expression level of the protein in the host cell body by binding to the upstream side of the protein gene. Can do.

  In one embodiment, the host fungus is cultured in a medium comprising a metal salt selected from the group consisting of magnesium, calcium, and manganese.

  The present invention further provides a vector comprising the above promoter.

  In one embodiment, the vector has a base sequence from position 1 to position 537 of SEQ ID NO: 2 in the sequence listing on the downstream side of the promoter, or one or several bases are deleted in the base sequence. A palindromic sequence consisting of a base sequence which can be substituted or added and can take a palindromic structure.

  In one embodiment, the vector has a protein gene inserted downstream of the promoter.

  The present invention further provides a recombinant microorganism in which the above vector is incorporated into a host cell.

  In one embodiment, the host bacterium is actinomycetes.

  In a further embodiment, the actinomycetes is Streptomyces lividans.

  According to the present invention, a promoter useful in host bacteria, particularly actinomycetes, is provided. By activating this promoter, the expression level of the protein can be increased. Activation of the promoter can be performed by a very simple means of culturing a cell having the promoter in a medium containing a metal salt selected from the group consisting of magnesium, calcium, and manganese. Therefore, a desired protein can be expressed in a large amount by using the promoter of the present invention.

  The promoter of the present invention can exhibit the activity of increasing the expression level of protein in the host cell. This promoter is preferably derived from the promoter of the metalloendopeptidase (SSMP) gene of S. septatus (hereinafter sometimes referred to as SSMP promoter). Specifically, it has the base sequence from position 1 to position 427 of SEQ ID NO: 1 in the sequence listing, or one or several bases are deleted, substituted or added in the base sequence and is equivalent to the SSMP promoter or It consists of a base sequence that can exhibit further promoter activity.

  In the present specification, the “deleted, substituted or added” base sequence may be a base sequence having a naturally-occurring mutation or a base sequence into which an artificial mutation has been introduced. “Substitution, deletion, addition or insertion of a base sequence” can be caused to a nucleic acid comprising a base sequence by a site-directed mutagenesis method ordinarily used in the art. It is only necessary that the obtained base sequence exhibits an activity equal to or higher than that of the original base sequence.

  The promoter of the present invention can be excised from, for example, bacterial cells carrying the SSMP promoter using a restriction enzyme. In this case, a DNA fragment of this promoter region can be obtained by amplifying a desired promoter region by PCR using a primer provided with an appropriate restriction enzyme recognition site. Alternatively, a desired promoter region can be chemically synthesized based on the base sequence information shown in SEQ ID NO: 1.

  The promoter of the present invention is activated by culturing a host bacterium in which this promoter is incorporated in a medium containing a metal salt selected from the group consisting of magnesium, calcium and manganese, and downstream of this promoter. The expression level of a protein from a base sequence (protein gene) encoding a certain protein increases.

  In the present invention, “promoter activation” means that the expression level of a protein in a host cell is increased by a promoter linked to the upstream side of a protein gene. In other words, it means that transcription of a protein gene on the downstream side of the promoter is further promoted. On the other hand, “promoter activity” refers to an activity in which a transcription factor binds to a promoter region to induce transcription. The downstream side means the 3 'side of the transcribed polynucleotide chain, and the upstream side means the 5' side of the transcribed polynucleotide chain.

  The promoter is preferably inserted into a vector suitable for introduction into a host bacterium. As such a vector, an appropriate vector may be selected according to the purpose of use of the recombinant DNA, and examples thereof include a plasmid vector and a phage vector. A vector that can be amplified by both actinomycetes and E. coli can be used as an E. coli-actinomycetes shuttle vector.

  For example, vectors suitable for actinomycetes include an origin of replication (ori) for amplification with actinomycetes. Furthermore, it may have an origin of replication for amplification in E. coli. The origin of replication (ori) for amplification with actinomycetes may be low copy or multicopy. A low copy plasmid refers to a plasmid that causes plasmid amplification of about 1 to 10 copies in actinomycetes, and a multi-copy plasmid results in a copy number of more, specifically, 40 copies to 400 copies or more. It refers to a plasmid. Examples of low copy ori include SCP2 * ori (Lydiate, D.J. et al., Gene, Vol. 35, No. 3, pp. 223-235, 1985). Examples of the high copy ori include pIJ101 ori (Katz, E. et al., J. Gen. Microbiol., 129, 2703-2714, 1983).

  Examples of vectors suitable for actinomycetes include pIJ486 (Mol. Gen. Genet., 203, 468-478, 1986), pKC1064 (Gene, 103, 97-99, 1991), pUWL- KS (Gene, 165, 149-150, 1995), pIJ702 (Katz, E. et al., J. Gen. Microbiol., 129, 2703-2714, 1983), and pIJ8600 (Sun, J. Et al., Microbiology, 145, 2221-2227, 1999). Suitable vectors for E. coli include, for example, pET22b, pTrc99A, pQE32, pIJ702 (Molnar et al., J. Ferment. Bioeng., 72, 368-372, 1991).

  Such a vector may be a genome integration type vector. A genome-integrated vector is a vector that can be incorporated into the actinomycetes genome and expressed from the genome when introduced into actinomycetes, for example. This vector can also be prepared in the form of a plasmid prior to introduction into actinomycetes. In preparing a genome integration type vector, a recombination sequence required for integration of phage DNA into a host genome can be used. Specifically, for example, an int gene of φ31 phage and attP may be inserted into a vector (Bierman, M. et al., Gene, 116, 43-49, 1992). In order to keep the plasmid in the host cell, it is usually necessary to continue selection based on the drug corresponding to the drug resistance gene incorporated in the plasmid. However, since the genome-integrated expression vector is stable, Is superior in that there is no need for drug selection.

  In addition to the above promoter, the vector may appropriately have a transcription element such as an inducible promoter, a selection marker gene, and a terminator.

  As the terminator, a terminator derived from any gene may be used as long as it can exhibit activity in the host fungus. The terminator usually has a palindromic sequence for stopping the translation of the protein to be expressed. For example, TH-3 coll terminator, PLD terminator, fd phage terminator (fd-ter), T4 terminator (T4-ter) and the like can be used. In the present invention, the palindromic sequence preferably has a base sequence from position 1 to position 537 of SEQ ID NO: 2 in the sequence listing, or one or several bases are deleted, substituted or substituted in the base sequence. It consists of a base sequence that can be added and take a palindromic structure.

  In addition, the vector preferably contains an appropriate drug resistance gene or a marker gene for selection such as a metabolic enzyme so that a host holding the vector can be selected. Such selectable marker genes include, for example, ampicillin resistance gene, kanamycin resistance gene, thiostrepton resistance gene, hygromycin resistance gene, apramycin resistance gene, etc., all of which are well known to those skilled in the art. .

  A multicloning site (MCS) can be introduced into the vector of the present invention. MCS is a DNA fragment having a plurality of types of restriction enzyme sites, and is used for linking a target gene for expression to MCS and inserting it into a vector. For example, MCS has restriction enzyme sites (at least one) such as NdeI, EcoRI, XbaI, HindIII, BglII, BamHI.

  A base sequence (protein gene) encoding a protein to be expressed is inserted downstream of the promoter in the vector. In the present invention, the protein is not particularly limited as long as it is a protein that can be expressed in a host bacterium. The protein may be a desired enzyme, a structural protein, a regulatory factor, or the like, and may be a natural protein, a mutant protein, an artificially created protein, or the like. For example, if a secreted protein is expressed using the vector of the present invention, this protein can be secreted in a large amount into the culture medium. Even a protein that is not secreted naturally can be secreted outside the cell by expressing it as a fusion protein to which a secretion signal is added. The vector of the present invention may previously encode a secretory signal sequence in the vector so that the protein encoded by the protein gene to be inserted is secreted.

  Examples of the bacterium used for the preparation of such a vector include a host bacterium commonly used in the technical field, and specific examples include actinomycetes and E. coli. More specifically, actinomycetes include Streptomyces lividans 1326, Streptomyces lividans TK-23 and the like, and Escherichia coli include BL21 (DE3), JM109 and the like. Among these host bacteria, Escherichia coli BL21 (DE3) and JM109 are desirable from the viewpoints of easy purification, mass preparation, safety and the like.

  A vector having a desired protein gene is introduced into an appropriate host bacterium, and the host bacterium is transformed to produce a recombinant microorganism. As the transformation method, for example, methods well known to those skilled in the art such as calcium phosphate method, DEAE-dextran method, electroporation method and the like are employed.

  Examples of host bacteria used in the present invention include actinomycetes, Escherichia coli, Bacillus, Pseudomonas, Thermus, and Agrobacterium. Among these, actinomycetes are preferably used in the present invention.

  In the present invention, actinomycetes refer to bacteria belonging to the order Actinomycetals. Actinomycetes are a taxonomic group belonging to Gram-positive bacteria and mainly inhabit soil. Although it is a prokaryotic organism, many actinomycetes show filamentous growth with branching and take various forms. In addition, there are species that generally form spores, including sporangia and motile spores. In addition, various antibiotics and other biologically important compounds have been discovered from actinomycetes. From the order of the Actinomycetes, the family Franciaceae, Micromonosporaceae, Propionibacterium cteraceae, Psudonocardiaceae, Streptomyces spp. (Streptosporangaceae), Thermomonosporaaceae, Corynebacteriumaceae, Mycobacterium faecae, and Nocdiaceae are included. Actinomycetes in the present invention are preferably bacteria belonging to the family Streptomyces, more preferably belonging to the genus Streptomyces. Examples of the bacteria belonging to the genus Streptomyces include, for example, Streptomyces septus, S. lividans, Streptomyces griseus, Streptomyces lavendulae. (S. lavendulae), Streptomyces virginia (S. virginia), and St. mycocolor (S. coelicolor). In the present invention, Streptomyces lividans is particularly preferably used as the host cell. Such actinomycetes belonging to the genus Streptomyces and other actinomycetes are described in various catalogs and can be easily obtained by those skilled in the art.

  The recombinant microorganism of the present invention transformed with the SSMP promoter and a vector having a base sequence encoding a desired protein is cultured in a medium containing a metal salt selected from the group consisting of magnesium, calcium, and manganese. The Such a medium is a medium containing an appropriate concentration of a metal salt in a medium usually used in the art.

  Commonly used media include carbon sources, nitrogen sources, inorganic salts, and the like that can be assimilated by the host bacteria, and natural media and synthetic media as long as the transformants can be cultured efficiently. Any of these may be used. Liquid or solid media can be used. Examples of the carbon source include carbohydrates such as glucose, galactose, fructose, sucrose, raffinose and starch, organic acids such as acetic acid and propionic acid, and alcohols such as ethanol and propanol. Examples of the nitrogen source include ammonia, ammonium salts of inorganic acids or organic acids such as ammonium chloride, ammonium sulfate, ammonium acetate, and ammonium phosphate, or other nitrogen-containing compounds. In addition, it may contain peptone, meat extract, corn steep liquor, various amino acids and the like. In addition, the inorganic salt may contain a trace amount of phosphates such as potassium, sodium, and iron, hydrochlorides, nitrates, acetates, and the like. Moreover, you may add antifoamers, such as vegetable oil, surfactant, and silicone, as needed.

  Examples of the metal salt selected from the group consisting of magnesium, calcium, and manganese contained in such a medium include magnesium phosphate, magnesium sulfate, manganese sulfate, and calcium carbonate. The concentrations of these metal salts vary depending on the type and concentration of the host fungus, the type of medium, and the like. These metal salts are usually included in the medium at least 0.005 w / v%, preferably at least 0.01 w / v%, more preferably at least 0.02 w / v%, usually at most 2.0 w. / V%, preferably at most 1.0 w / v%, more preferably at most 0.5 w / v%.

  The culture conditions may be appropriately selected depending on the type of culture medium, the culture method, and the like, and are not particularly limited as long as the host bacteria can grow and produce the target protein. Usually, it is carried out at 10 ° C. to 40 ° C., preferably at 28 ° C. for 12 to 120 hours under aerobic conditions such as shaking culture or aeration stirring culture in a liquid medium. The pH is adjusted to 4 to 10, preferably 6 to 8. The pH is adjusted using an inorganic or organic acid, an alkaline solution, or the like.

  Thus, by culturing the recombinant microorganism of the present invention, the incorporated protein gene is expressed, and the protein can be obtained from the culture. In particular, by culturing the recombinant microorganism in a medium containing a metal salt selected from the group consisting of magnesium, calcium, and manganese, the SSMP promoter is activated and promotes transcription of the protein gene. Compared with the case of culturing in a medium not containing magnesium, calcium, or manganese, the expression level of the protein is further increased. Therefore, the target protein can be produced efficiently.

  If the protein accumulates in the host cell, the transformed cells are collected by centrifugation after completion of the culture, and the resulting cell is disrupted by sonication or the like, and then the cell-free extract is obtained by centrifugation or the like. obtain. Using this as a starting material, it can be purified by a general protein purification method such as salting-out, various types of chromatography such as ion exchange chromatography, gel filtration chromatography, hydrophobic chromatography, and affinity chromatography. When the expressed protein is secreted extracellularly, it can be similarly purified from the culture supernatant.

  EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not limited by an Example. Unless otherwise stated, various manipulations were performed according to the Molecular Cloning A Laboratory Manual, Second Edition (Sambrook, J. et al., 1989). In Examples,% represents w / v% unless otherwise specified.

Example 1: Construction of expression vector pTONA5
Using pBluescript II KS (-) (TOYOBO) as a template, the ori (0.6 kb) portion of E. coli JM109 was amplified by PCR using primers 1 and 2 (SEQ ID NOs: 3 and 4). The PCR conditions were 94 ° C. for 2 minutes, 94 ° C. for 15 seconds, 55 ° C. for 30 seconds, and 68 ° C. for 1 minute for 30 cycles, and then cooled to 4 ° C. For PCR, KOD plus DNA polymerase (TOYOBO) was used. Subsequently, pTYM18 (Onaka, H. et al., J. Antibiotics, 56, 950-956, 2003) was used as a template, aphII (E. coli-actinomycetes kanamycin resistance gene: 1.3 kb), primer 3 And 4 (SEQ ID NOS: 5 and 6) were amplified by PCR as described above.

  The obtained two fragments were treated with restriction enzymes SspI and PstI, respectively, and the two fragments were ligated to each other and then introduced into E. coli. The plasmid pBSaphII containing the ligated fragment was obtained by using E. coli in LB medium (1% NaCl, 1% polypeptone, and 0.5% yeast extract) containing 50 μg / mL kanamycin at 37 ° C. It was. pIJ702 (Molnar et al., J. Ferment. Bioeng., 72, 368-372, 1991) and the resulting pBSaphII were each treated with the restriction enzyme PstI, and the two fragments were ligated and then transferred to E. coli. Introduced. Plasmid pIJE702 was obtained using kanamycin as a selection marker.

  Next, oriT (0.8 kb) was amplified by PCR in the same manner as described above using pTYM18 as a template and primers 5 and 6 (SEQ ID NOs: 7 and 8). The resulting fragment was treated with restriction enzymes HincII and SmaI, and pIJE702 was treated with the restriction enzyme SspI. The two fragments obtained were ligated and then introduced into E. coli. Plasmid pIJEC702 was obtained using kanamycin as a selection marker. The NdeI site of pIJEC702 was then deleted by silent mutation using QuikChange II Kit (Stratagene) and primers 7 and 8 (SEQ ID NOs: 9 and 10), resulting in pIJEC'702.

  Next, S. septatus TH-2 genomic DNA is designated as S. septatus TH-2 [named and displayed as Streptomyces septatus TH-2, accession number: FERM P-17329 (date of deposit: March 24, 1999) , Institute of Biotechnology, Institute of International Trade and Industry, Ministry of International Trade and Industry, Patent Microorganism Depositary Center (currently National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center) (1-1 1-1 East Higashi Tsukuba, Ibaraki, Japan) ] By the Saito Miura method (Saito, H. et al., Biochem. Biophys. Acta., 72, 619-629, 1963). The SSMP promoter (0.4 kb) was amplified by PCR in the same manner as described above using primers 9 and 10 (SEQ ID NOs: 11 and 12) using S. septatus TH-2 genomic DNA as a template. In addition, S. aureofaciens TH-3 genomic DNA was independently designated as S. aureofaciens TH-3 [named and displayed as Streptomyces aureofaciens TH-3, accession number: FERM P-21343 (deposit date: August 17, 2007). It was prepared by the same method as described above from the National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center (deposited at 1-1 1-1 Higashi 1-1, Tsukuba, Ibaraki, Japan). A TH-3 coll terminator (0.5 kb) was amplified by PCR in the same manner as described above using primers 11 and 12 (SEQ ID NOs: 13 and 14, respectively) using S. aureofaciens TH-3 genomic DNA as a template. The SSMP promoter fragment was treated with AseI and EcoRI, the terminator fragment was treated with EcoRI and BglII, and pIJEC'702 was treated with the restriction enzymes AseI and BglII. The three fragments obtained were ligated and then introduced into E. coli JM109. Plasmid pTONA5 was obtained using kanamycin as a selection marker. The structure of plasmid pTONA5 (restriction enzyme map) is shown in FIG. This plasmid pTONA5 contains an SSMP promoter sequence (SEQ ID NO: 1) and a TH-3 coll terminator (SEQ ID NO: 2).

Example 2: Construction of SSAP expression strain (S. lividans) The SSAP gene was obtained by PCR in the same manner as described above using primers S13 and 14 (SEQ ID NOS: 15 and 16) using the S. septatus TH-2 genomic DNA as a template. Amplified. pTONA5 was treated with restriction enzymes NdeI and HindIII, the SSAP gene fragment was treated with NdeI and HindIII, and the resulting two fragments were ligated and then introduced into E. coli JM109. Plasmid pTONA5-SSAP was obtained using kanamycin as a selection marker. In this plasmid pTONA5-SSAP, the SSAP gene (SEQ ID NO: 17) is arranged downstream of the SSMP promoter so that it can be expressed.

  pTONA5-SSAP was introduced into conjugative E. coli S17-1, and S. lividans 1326 strain was transformed from the transformed E. coli by conjugative transformation. Regarding the conjugation transformation method, “Genetic Manupulation of Streptomyces” (Hopwood, DA et al., 1985, Genetic Manupulation of Streptomyces: a Laboratory Manual, the John Innes Foundation, Norwich). In detail, it carried out as follows.

1) The conjugated Escherichia coli (S17-1) carrying pTONA5-SSAP was cultured overnight at 37 ° C. in 2-5 ml of LB medium containing 50 μg / ml of kanamycin;
2) After 1 ml of culture broth was centrifuged (8,000 rpm, 5 minutes, 4 ° C.), the supernatant was discarded by decantation;
3) After the cell pellet was suspended in 1 ml of LB medium, the cell was washed in the same manner as in the above operation 2). This operation was repeated twice;
4) 500 μl of LB medium was added to the washed cell pellet and gently suspended with a pipette;
5) 100 μl of the Escherichia coli suspension obtained in the above operation 4 was collected and mixed with 10 μl of a spore suspension (1010 cells / ml) of actinomycetes S. lividans 1326 by pipetting;
6) All the mixture was spread on ISP4 medium plate medium (Difco ^™ ISP Medium 4: 3.7%) and incubated for 18 hours at 30 ° C; and 7) Kanamycin 50 μg / ml and Nalidixic acid sodium 67 μg / ml NB soft agar containing 3 ml of NB soft agar (Difco ^™ Nutrient broth: 0.8%, agar: 0.5%) was overlaid and cultured at 30 ° C. for 5 days to obtain a transformant.

  The obtained transformant (SSAP expression strain) has the SSAP gene sequence shown in SEQ ID NO: 17 downstream of the SSMP promoter.

Example 3: Culture experiment using SSAP expression strain (S. lividans) (Influence of divalent metal salt on SSMP promoter)
In the culture experiment, 25 ml of various media were dispensed into 500-ml baffled Erlenmeyer flasks and steam-sterilized at 121 ° C. for 20 minutes (kanamycin was added after sterilization). The composition of the basic medium was as follows: glucose 2%, K ₂ HPO ₄ 0.8%, polypeptone (manufactured by Nippon Pharmaceutical) 0.5%, yeast extract (manufactured by Difco) 0.5%, kanamycin 50 μg / mL, pH 7.0. The SSAP-expressing strain was cultured with shaking at 28 ° C. and 160 rpm for 72 hours. Absorbance at 660 nm was measured as a measure of the growth amount of the SSAP-expressing strain.

The activity of SSAP expressed by culture was measured as follows: First, 0.059 g of L-Leucyl-p-nitroanilide which is a substrate of SSAP was accurately weighed and 2 mM CaCl ₂ · 2H _2. A substrate solution was prepared by dissolving in 0.1 M Tris-HCl (pH 8.0) containing O and making up to 200 ml. Next, 2 ml of the substrate solution was heated at 37 ± 0.5 ° C. for 5 minutes, and then 0.05 ml of the sample solution (culture solution diluted with demineralized water) was added to initiate the reaction. After exactly 5 minutes, 0.3 ml of reaction stop solution (5% trichloroacetic acid solution) was added. Absorbance _AT at a wavelength of 405 nm was measured using water as a control. As a blank, the absorbance was measured A _B using the same method by adding demineralized water instead of the sample solution. Separately, a calibration curve for p-nitroaniline was prepared in advance. The A _T and _A respectively corresponding p- nitroaniline concentration _B obtained from p- nitroaniline calibration curve, respectively of p- nitroaniline concentration (μmol / ml) and _{N T} and _{N B.} The enzyme activity was determined from the following formula.

Enzyme activity (U / g or U / ml) = (N _T −N _B ) × (2.35 / 0.05) × (1/5) × (1 / W)
N _T -N _B : Concentration of produced p-nitroaniline (μmol / ml)
0.05: Amount of sample solution used for reaction (ml)
2.35: Final liquid volume (ml)
5: Reaction time (minutes)
W: Amount of sample in 1 ml of sample solution (g or ml) = dilution ratio Here, the unit of enzyme activity (U) is the amount of enzyme that produces 1 μmol of p-nitroaniline per minute under the measurement conditions. To express.

  Table 1 shows the activity of SSAP and the absorbance at 660 nm after culturing the SSAP-expressing strain in various media for 72 hours.

  Among divalent metal ions, when cultured in a medium containing magnesium (Mg), calcium (Ca), or manganese (Mn), the SSAP activity in the culture is lower than when cultured in a basic medium. It was very expensive. Even in the case of a divalent metal ion, a medium containing iron (Fe), copper (Cu), zinc (Zn), or cobalt (Co) has a lower SSAP activity than when cultured in a basic medium. Or no activity at all. However, it is clear from the measured value of OD660 that the growth of the SSAP-expressing strain itself is not inhibited by these metals. On the other hand, even when the SSAP activity was high (that is, when cultured in a medium containing Mg, Ca, or Mn), the amount of growth of the SSAP-expressing strain was the same as or slightly higher than that in the case of using the basic medium. It can be seen that the high SSAP activity in the culture solution does not depend on the number of cells.

  It is known that the activity (enzyme activity) of SSAP is not affected by metal ions (Patent Document 4). Therefore, it is considered that the high SSAP activity shown in the above results is due to the fact that the SSAP concentration in the culture medium is high, that is, a large amount of SSAP is expressed and secreted. Therefore, it can be seen that the SSAP expression level was increased by culturing the SSAP-expressing strain in a medium containing Mg, Ca, or Mn. This indicates that a large amount of SSAP linked under the control of the SSMP promoter was expressed. This is thought to be due to the fact that the transcription of the protein gene linked under the control of the SSMP promoter is further promoted, in other words, the SSMP promoter is activated, thereby increasing the expression level of the protein.

  According to the present invention, a promoter useful in a host, particularly actinomycetes, is provided. By activating this promoter, the expression level of the protein can be increased. Activation of the promoter can be performed by a very simple means of culturing a cell having the promoter in a medium containing a metal salt selected from the group consisting of magnesium, calcium, and manganese. Therefore, a desired protein can be expressed in a large amount by using the promoter of the present invention. In particular, actinomycetes are very important microorganisms in the production of useful enzymes, and thus the method of the present invention can provide proteins such as enzymes more efficiently.

It is a schematic diagram which shows the structure of plasmid pTONA5.

Claims (20)

A method of activating a promoter, wherein the promoter has a base sequence from position 1 to position 427 of SEQ ID NO: 1 in the sequence listing, or one or several bases are deleted or replaced in the base sequence Or consisting of a base sequence that is added and can exhibit an activity equal to or higher than the base sequence,
Culturing a recombinant microorganism into which a vector into which the promoter has been inserted is incorporated in a medium containing a metal salt selected from the group consisting of magnesium, calcium, and manganese, wherein the promoter is a protein gene A process coupled to the upstream side of
The method wherein the activation of the promoter is that the expression level of the protein in the recombinant microorganism is increased by the promoter.   The vector includes a palindromic sequence downstream of the protein gene, the palindromic sequence has a base sequence from position 1 to position 537 of SEQ ID NO: 2 in the sequence listing, or 1 or 2 in the base sequence The method according to claim 1, comprising a base sequence in which several bases are deleted, substituted or added and can take a palindromic structure.   The method according to claim 1 or 2, wherein the recombinant microorganism is actinomycetes.   The method according to claim 3, wherein the actinomycetes is Streptomyces lividans. A method for increasing the expression level of a protein in a recombinant microorganism,
Culturing the recombinant microorganism in a medium,
In the recombinant microorganism, a vector into which a promoter linked to the upstream side of the protein gene is inserted is incorporated,
The promoter has a base sequence from position 1 to position 427 of SEQ ID NO: 1 in the sequence listing, or one or several bases are deleted, substituted or added in the base sequence, and is equivalent to the base sequence or A method comprising a base sequence capable of exerting further activity, and wherein the medium comprises a metal salt selected from the group consisting of magnesium, calcium, and manganese.   The vector includes a palindromic sequence downstream of the protein gene, the palindromic sequence has a base sequence from position 1 to position 537 of SEQ ID NO: 2 in the sequence listing, or 1 or 2 in the base sequence 6. The method according to claim 5, comprising a base sequence in which several bases are deleted, substituted or added and can take a palindromic structure.   The method according to claim 5 or 6, wherein the recombinant microorganism is actinomycetes.   The method according to claim 7, wherein the actinomycetes is Streptomyces lividans. A method for producing a protein comprising:
Culturing in a medium a recombinant microorganism in which a vector having a promoter linked upstream of the protein gene is inserted, and recovering the protein from the obtained culture,
The promoter has a base sequence from position 1 to position 427 of SEQ ID NO: 1 in the sequence listing, or one or several bases are deleted, substituted or added in the base sequence, and is equivalent to the base sequence or A method comprising a base sequence capable of exerting further activity, and wherein the medium comprises a metal salt selected from the group consisting of magnesium, calcium, and manganese.   The vector includes a palindromic sequence downstream of the protein gene, the palindromic sequence has a base sequence from position 1 to position 537 of SEQ ID NO: 2 in the sequence listing, or 1 or 2 in the base sequence The method according to claim 9, comprising a base sequence in which several bases are deleted, substituted or added and can take a palindromic structure.   The method according to claim 9 or 10, wherein the recombinant microorganism is actinomycetes.   The method according to claim 11, wherein the actinomycetes is Streptomyces lividans. It has a base sequence from position 1 to position 427 of SEQ ID NO: 1 in the sequence listing, or one or several bases are deleted, substituted or added in the base sequence, and an activity equivalent to or higher than the base sequence A promoter consisting of a base sequence capable of exhibiting
A promoter capable of exerting an activity of increasing the expression level of the protein in the host cell body by being bound to the upstream side of the protein gene.   The promoter according to claim 13, wherein the host bacterium is cultured in a medium containing a metal salt selected from the group consisting of magnesium, calcium, and manganese.   A vector comprising the promoter according to claim 13 or 14.   It has a base sequence from position 1 to position 537 of SEQ ID NO: 2 in the sequence listing on the downstream side of the promoter, or one or several bases are deleted, substituted or added in the base sequence, and a palindromic structure The vector according to claim 15, comprising a palindromic sequence consisting of a base sequence capable of taking   The vector according to claim 15 or 16, wherein a protein gene is inserted downstream of the promoter.   A recombinant microorganism in which the vector according to claim 17 is incorporated into a host cell.   The recombinant microorganism according to claim 18, wherein the host bacterium is an actinomycete.   The recombinant microorganism according to claim 19, wherein the actinomycetes is Streptomyces lividans.

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