JP2010284130A - Modified promoter derived from bacterium of genus bacillus - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a modified promoter functioning in a bacterium of the genus Bacillus and a use thereof. <P>SOLUTION: The modified promoter functioning in a bacterium of the genus Bacillus has improved promoter activity in comparison with a natural promoter by substitution with any sequence selected from the group consisting of sequence AAAAATAA, sequence AAAAAAATA, sequence AAAAATTA and sequence AAAAAATAA in a natural promoter of a bacterium of the genus Bacillus. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a modified promoter. In detail, it is related with the modified promoter constructed | assembled based on the promoter of Bacillus genus bacteria, and its use.

  Bacteria belonging to the genus Bacillus have long been used for fermented foods. It is also known to secrete many enzymes including protease and amylase. On the other hand, in gene recombination techniques currently widely used for the purpose of producing heterologous proteins or high protein production, E. coli is most frequently used as a host microorganism for gene recombination organisms. . When considering Bacillus bacteria from the viewpoint of host microorganisms for genetic recombination technology, unlike E. coli, pyrogens such as endotoxin are not produced and are not pathogenic, and as mentioned above, they have been used in the fermentation industry for a long time. It has many advantages, such as the fact that it has been developed, and its high ability to secrete substances, and is expected to be used in industry.

  There are very few examples of industrial use of Bacillus bacteria as host microorganisms compared to E. coli. Although several decades ago, an expression system for Bacillus subtilis has been developed, vectors have been discovered, transformation methods have been established, and many genes have been cloned. Is not progressing in the industry. One reason for this is that an expression system suitable for high production of the target protein, and thus an expression vector suitable for high production of the target protein has not been constructed. One promising strategy for overcoming the obstacles to the spread of this industry is to develop a high expression promoter, that is, to modify the promoter and increase its activity. In fact, there is a high demand for improvement of promoters, and various attempts have been made (for example, see Patent Documents 1 to 3).

Japanese Patent No. 3529774 JP 2002-272466 A JP-T-2002-504379

  An object of the present invention is to provide a modified promoter (promoter improved so as to be highly expressed) that functions in a Bacillus bacterium and use thereof in order to realize a high expression system using a Bacillus bacterium.

  In view of the above problems, the present inventors have repeatedly studied. First, by carrying out mutation treatment using Bacillus amyloliquefaciens as an α-amylase-producing bacterium, a mutant strain having improved α-amylase productivity was successfully obtained. When the α-amylase gene in the mutant strain and the base sequence of the promoter were identified, it was confirmed that a part of the base sequence of the promoter is different from the natural type (wild type) sequence. As a result of detailed examination, this modified site is 80 bp upstream from the promoter 3 ′ end, and the α-amylase promoter modified site in the previous reports (near the promoter 3 ′ end, mainly 30 bp upstream from the 3 ′ end) (See Patent Literatures 1 and 2). It should be noted that the modified site found this time is located upstream of the “-35 region” recognized by RNA polymerase, and at first glance, it is considered to be a site that affects the function of the promoter. There is no point. In other words, we succeeded in finding a new modified site that is not remarkable from the viewpoint of common technical knowledge.

  On the other hand, paying attention to the newly found modification site, the relationship between various modifications and the effect of improving promoter activity was investigated. As a result, a plurality of modified embodiments that lead to improved promoter activity were identified. In other words, it succeeded in constructing a plurality of high expression promoters. Moreover, it was confirmed that these high expression promoters are excellent in versatility.

Based on the above results, the following inventions are provided.
[1] A modified promoter that functions in a bacterium belonging to the genus Bacillus, wherein the sequence AAAAATAA is replaced with any sequence selected from the group consisting of the sequence AAAAAAATA, the sequence AAAAATTA, and the sequence AAAAAATAA in the natural promoter of the Bacillus bacterium A modified promoter characterized in that the promoter activity is improved as compared to a natural promoter.
[2] A modified promoter that functions in a bacterium belonging to the genus Bacillus, and the sequence AAAAATAA existing upstream and in the vicinity of the -35 region in the natural promoter of the genus Bacillus is selected from the group consisting of the sequence AAAAAAATA, the sequence AAAAATTA, and the sequence AAAAAATAA A modified promoter, characterized in that the modified promoter is substituted with any of the sequences described above.
[3] The modified promoter according to [2], comprising any one of SEQ ID NOs: 1 to 3 as a sequence including the -35 region.
[4] The modified promoter according to [2], comprising any one of SEQ ID NOs: 4 to 6 as a sequence including the -35 region and the -10 region.
[5] The modified promoter according to [2], comprising any one of SEQ ID NOs: 7 to 9.
[6] The modified promoter according to [2], comprising any of the following sequences (1) to (3):
(1) The sequence of SEQ ID NO: 10, or the region of bases 1 to 95, the region of bases 105 to 118, the region of bases 125 to 145, and the bases 152 to 181 of the sequence A sequence comprising one or more bases substituted, deleted, inserted and / or added in one or more regions selected from the group consisting of base regions and exhibiting higher activity than the natural promoter;
(2) The sequence of SEQ ID NO: 11, or the region of bases 1 to 95, the region of bases 104 to 117, the region of bases 124 to 144, and the base of 151 to 180 A sequence comprising one or more bases substituted, deleted, inserted and / or added in one or more regions selected from the group consisting of base regions and exhibiting higher activity than the natural promoter;
(3) the sequence of SEQ ID NO: 12, or the region of bases 1 to 95, the region of bases 105 to 118, the region of bases 125 to 145, and the region of bases 152 to 181 in the sequence A sequence comprising a sequence in which one or several bases are substituted, deleted, inserted and / or added in one or more regions selected from the group consisting of base regions, and exhibiting higher activity than the natural promoter.
[7] The modified promoter according to any one of [1] to [6], wherein the natural promoter is an α-amylase promoter of Bacillus amyloliquefaciens.
[8] The modified promoter according to [7], wherein the α-amylase promoter consists of the sequence of SEQ ID NO: 13.
[9] A modified promoter that functions in Bacillus bacteria, comprising the sequence of SEQ ID NO: 14 as a sequence containing the −35 region and the −10 region.
[10] The modified promoter according to [9], comprising the sequence of SEQ ID NO: 15 or 16.
[11] The modified promoter according to [9], comprising the sequence of SEQ ID NO: 17 or 18.
[12] An expression construct comprising the modified promoter according to any one of [1] to [11] and a base sequence encoding a polypeptide operably linked to the modified promoter.
[13] The expression construct according to [12], wherein the polypeptide is α-amylase, β-amylase, carboxyesterase or oxalate decarboxylase.
[14] An expression vector into which the expression construct according to [12] or [13] is inserted.
[15] An expression vector comprising the modified promoter according to any one of [1] to [11] and a cloning site disposed under the control of the modified promoter.
[16] A transformant obtained by transforming a host bacterium with the expression vector according to [14].
[17] The transformant according to [16], wherein the host bacterium is a genus Bacillus.
[18] a first step of culturing the transformant according to [16] or [17] and expressing the polypeptide;
And a second step of recovering the expressed polypeptide.
[19] The method for producing a polypeptide according to [18], wherein the first step comprises culturing and proliferating the transformant and inducing expression of the polypeptide.
[20] The sequence AAAAATAA existing upstream of and in the vicinity of the -35 region in the natural promoter of the genus Bacillus is any one selected from the group consisting of the sequence AAAAAAAATA, the sequence AAAAAAAAA, the sequence AAAAATTA, the sequence AAAAAATA, and the sequence AAAAAATAA A method for modifying a promoter, wherein the modification is performed so that the sequence is substituted.

The figure which shows the construction method of shuttle vector pUBC1. The figure which shows the arrangement | sequence of the natural alpha-amylase promoter of Bacillus amyloliquefaciens. The figure which compared the arrangement | sequence of the natural type | mold alpha-amylase promoter and the mutant | variant type | mold alpha-amylase promoter. Mutation sites are underlined. The figure which shows the variation | mutation site | part (underline) of the modified promoters 1-4 and the sequence | arrangement near it (sequence number 23-26 in order from the top). The figure which shows the construction method of genetically modified bacteria. The figure which shows the structure of a carboxyesterase expression plasmid vector. The figure which shows the structure of (beta) -amylase expression plasmid vector. The figure which shows the structure of the carboxyesterase expression plasmid vector containing a modified | denatured alpha-amylase promoter. The figure which shows the structure of the beta amylase expression plasmid vector containing a modified | denatured alpha-amylase promoter. Measurement results of carboxyesterase activity. The esterase relative activity (lower graph) was compared as the relative value (upper table) of the activity of each promoter. Measurement results of β-amylase activity. The relative activity of β-amylase (lower graph) was compared as the relative value of the activity of each promoter (upper table). The table | surface which shows the modified promoter and transgene which each recombinant bacterium has.

(Modified promoter)
The first aspect of the present invention relates to a modified promoter. “Modified promoter” refers to a promoter constructed by partially modifying a natural (wild-type) promoter. However, the preparation method is not particularly limited. Therefore, it is not limited to those obtained by directly modifying the natural promoter, and even those constructed using materials other than the natural promoter may fall under the “modified promoter”. Since the modified promoter is designed or prepared based on a natural promoter, it exhibits high sequence homology with the natural promoter from which it is derived. On the other hand, as a result of modification, the modified promoter is more active than the underlying promoter (natural promoter).

  One feature of the modified promoter of the present invention is that it functions in Bacillus bacteria. “Function in a Bacillus bacterium” means that it can exhibit a promoter activity when introduced into a Bacillus bacterium. In other words, it means a promoter that can be used to express a target polypeptide in an expression system using a Bacillus bacterium as a host. The Bacillus bacterium here is not particularly limited. Examples of Bacillus bacteria include Bacillus amyloliquefaciens, Bacillus subtilis, Bacillus licheniformis, Bacillus stearothermophilus, Bacillus stearothermophilus Ensis (Bacillus thuringiensis) can be mentioned. The modified promoter of the present invention functions in Bacillus bacteria, but does not prevent it from functioning in an expression system using another bacterium (for example, E. coli) as a host.

  The modified promoter of the present invention is derived from a natural promoter of Bacillus bacteria. That is, the modified promoter of the present invention corresponds to a modified natural promoter of a Bacillus bacterium. In the modified promoter of the present invention, the sequence AAAAATAA is replaced with any sequence selected from the group consisting of the sequence AAAAAAATA, the sequence AAAAATTA and the sequence AAAAAATAA in the natural promoter of the genus Bacillus. This feature improves the promoter activity compared to the natural promoter. The sequence AAAAAATAA in the native promoter is present upstream and in the vicinity of the -35 region. The -35 region is the site where RNA polymerase binds and is important for promoter activity. In fact, for the purpose of improving promoter activity, attempts have been made to modify the site to be targeted (for example, Patent Document 3). As described above, the present invention is notably different from the conventional improved technology in that the region in the upstream and the vicinity thereof is modified instead of the −35 region. The base promoter, that is, the natural promoter from which the modified promoter is derived is not particularly limited as long as it is possessed by bacteria belonging to the genus Bacillus. As an example, the sequence of Bacillus amyloliquefaciens α-amylase promoter is shown in SEQ ID NO: 13. In this sequence, the sequence AAAAATAA is present at the 96th base to the 103rd base upstream and in the vicinity of the -35 region.

  When comparing the modified region of the underlying natural promoter and the corresponding region of the modified promoter, there is a difference between them, and it can be seen that the modified region has been replaced with another sequence. As used herein, the modification according to the present invention is expressed as “substituted”. Actually, the modified promoter of the present invention is constructed by partial substitution (1-base or 2-base substitution), insertion, deletion, etc. in the modified region of the natural promoter.

  As described above, the present invention provides three types of modification (substitution from the sequence AAAAATAA to the sequence AAAAAAATA, substitution from the sequence AAAAATAA to the sequence AAAAATTA, and substitution from the sequence AAAAATAA to the sequence AAAAAATAA). The modified promoter in the case of the first modified embodiment (substitution of the sequence AAAAATAA to the sequence AAAAAAATA) preferably comprises the sequence of SEQ ID NO: 1. This sequence is derived from the α-amylase promoter of Bacillus amyloliquefaciens and includes a modified site, a −35 region, and a region linking both. A more preferred modified promoter includes a sequence (SEQ ID NO: 4) including the modified site, the -10 region, and a region (including the -35 region) linking both. An even more preferred modified promoter comprises a sequence (SEQ ID NO: 7) that includes the region up to the 3 ′ end region that continues to the −10 region.

  The second modified embodiment (substitution from the sequence AAAAAATAA to the sequence AAAAATTA) and the third modified embodiment (sequence AAAAAATAA to the sequence AAAAAATAA) are the same as in the first modified embodiment, that is, the second modification The modified promoter of the modified embodiment preferably comprises the sequence of SEQ ID NO: 2 (the modified site and the -35 region and a region linking both), and the modified promoter of the third modified embodiment is preferably the sequence of SEQ ID NO: 3. (Modified region and -35 region and a region connecting both). More preferably, in the second modified embodiment, it comprises the sequence of SEQ ID NO: 5 (sequence including the modified site and the -10 region and a region linking both), and in the third modified embodiment, SEQ ID NO: 6 (The sequence including the modification site and the -10 region and a region connecting both of them). Even more preferably, in the second modified embodiment, it comprises the sequence of SEQ ID NO: 8 (sequence including even the 3 ′ terminal region continuous with the −10 region), and in the third modified embodiment, the sequence of SEQ ID NO: 9 It comprises a sequence (sequence including even the 3 ′ terminal region connected to the −10 region).

  Examples of the sequence that defines the full length of the modified promoter of the present invention include the sequence of SEQ ID NO: 10, the sequence of SEQ ID NO: 11, and the sequence of SEQ ID NO: 12. The sequence of SEQ ID NO: 10 is a sequence obtained by modifying the sequence (SEQ ID NO: 13) including the full-length α-amylase promoter of Bacillus amyloliquefaciens according to the first modification mode defined above. Similarly, the sequence of SEQ ID NO: 11 is a sequence that has been similarly modified according to the second modification, and the sequence of SEQ ID NO: 12 is a sequence that has been similarly modified according to the third modification. is there.

Here, in general, even if a part of the base sequence is different, it may have an equivalent function. That is, the difference in the base sequence may not substantially affect the function. Based on the common general technical knowledge, the present invention comprises, as another aspect, a base sequence equivalent to a reference base sequence (sequence of SEQ ID NO: 10, sequence of SEQ ID NO: 11, sequence of SEQ ID NO: 12), which is essential for the present invention. Provided is a modified promoter having the characteristics, “being a promoter that functions in Bacillus bacteria”. In the modified promoter of this embodiment, one or several bases are substituted, deleted, inserted and / or added in the following region (any one or more) considered not to be important for promoter activity in the reference base sequence. ing.
(1) When the sequence of SEQ ID NO: 10 is a reference base sequence, the region of bases 1 to 95 of SEQ ID NO: 10, the region of bases 105 to 118, the region of bases 125 to 145, and 152 The region from the base No. to the base No. 181 (2) When the sequence of SEQ ID NO: 11 is the reference base sequence The region of the base No. 1 to the base No. 95 of SEQ ID No. 11, the region of the base No. 104 to the base No. 117, the base No. 124 144th base region and 151th to 180th base region (3) When the sequence of SEQ ID NO: 12 is a reference base sequence, the region of 1st base to 95th base of SEQ ID NO: 12, 105th base to 118th base Base region, region from base 125 to base 145, and region from base 152 to base 181

  The equivalent base sequence as described above can be obtained by a genetic engineering technique (for example, site-specific mutagenesis) so that a desired modification occurs. It can also be obtained by mutation treatment by ultraviolet irradiation or the like.

In addition to the various modified promoters listed above, the present inventors have succeeded in obtaining an effective modified promoter, as shown in the Examples described later. Based on the results, the present invention also provides the modified promoter defined below.
(1) A modified promoter comprising the sequence of SEQ ID NO: 14 as a sequence including the -35 region and the -10 region (2) In addition to the -35 region and the -10 region, up to the 3 'end region connected to the -10 region In addition to the modified promoter (3) -35 region and -10 region comprising a sequence (SEQ ID NO: 15) that also includes the sequence (SEQ ID NO: 15), the sequence (SEQ ID NO: 16) also includes the 3 'terminal region that continues to the -10 region (4) a sequence in which the sequence AAAAATAA in the sequence (SEQ ID NO: 13) containing the full length α-amylase promoter of Bacillus amyloliquefaciens is replaced by the sequence AAAAAAAA (sequence of SEQ ID NO: 17) (5) a sequence (SEQ ID NO: 1) in which the sequence AAAAATAA in the sequence (SEQ ID NO: 13) containing the full length α-amylase promoter of Bacillus amyloliquefaciens is replaced by the sequence AAAAAATA 8 modified promoter)

  The modified promoter of the present invention can be prepared by, for example, standard genetic engineering techniques (such as site-directed mutagenesis) with reference to the sequence information disclosed in this specification or the attached sequence listing. In Examples described later, a modified promoter was constructed by obtaining a Bacillus amyloliquefaciens α-amylase promoter and then mutating it. By the same method, it is possible to construct a desired modified promoter based on various promoters derived from Bacillus bacteria.

(Expression construct)
The second aspect of the present invention relates to the use of the modified promoter of the present invention, and provides an expression construct using the modified promoter. “Expression construct” refers to a structure capable of producing a polypeptide of interest in a host by introducing it into the host. The expression construct of the present invention comprises a modified promoter of the present invention and a base sequence encoding the polypeptide. The sequence encoding the polypeptide is operably linked to a modified promoter. Here, “operably linked” is synonymous with “arranged under control” and means that the sequence encoding the polypeptide is functionally linked to the modified promoter. To do. By achieving such ligation, transcription of the sequence encoding the polypeptide is controlled (regulated) by the modified promoter. Typically, a polypeptide-encoding sequence is directly linked to a modified promoter, but as long as the polypeptide-encoding sequence is transcriptionally controlled by the modified promoter, other sequences are interposed between the two. It may be.

  A poly A addition signal sequence or poly A sequence, an enhancer sequence, a selection marker sequence, a tag sequence, etc. can also be arranged in the expression construct of the present invention. The use of a poly A addition signal sequence or poly A sequence improves the stability of the mRNA resulting from the expression construct. The poly A addition signal sequence or poly A sequence is linked to the polynucleotide of the present invention on the downstream side. On the other hand, the use of an enhancer sequence can improve the expression efficiency. In addition, if an expression construct containing a selectable marker sequence is used, the presence or absence (and the extent) of the introduction of the expression construct can be confirmed using the selectable marker.

(Expression vector)
The third aspect of the present invention relates to an expression vector using a modified promoter. “Expression vector” refers to a vector capable of introducing a nucleic acid inserted therein into a target cell (host cell) and allowing expression in the cell. The modified promoter of the present invention is incorporated in the expression vector of the present invention.

  One embodiment of the expression vector of the present invention is an expression vector into which the expression construct of the present invention has been inserted. By using the expression vector, the target polypeptide can be forcibly expressed (produced) in bacteria belonging to the genus Bacillus. In another embodiment of the present invention, a cloning site is provided under the control of the modified promoter of the present invention. In the case of the expression vector, various genes (base sequences encoding polypeptides) can be inserted using a cloning site. In order to improve versatility and convenience, a multi-cloning site (MCS) may be adopted as a cloning site. There are no particular restrictions on the restriction enzyme sites (recognition sites and cleavage sites) that can be used as cloning sites. Examples of restriction enzyme sites are BamHI, XbaI, SmaI, XmaI, PstI, SacI, DraI, EcoNI and Bsa. In this embodiment, a poly A addition signal sequence, a poly A sequence, an enhancer sequence, a selectable marker sequence, a tag sequence, etc. may be arranged in the expression vector.

  For recombination operations (eg, insertion of genes, promoters, selection marker genes, etc. into vectors), standard recombinant DNA techniques (for example, Molecular Cloning, Third Edition, 1.84) such as methods using restriction enzymes and DNA ligases. , Cold Spring Harbor Laboratory Press, New York).

(Transformant)
The present invention further provides a transformant obtained by transforming a Bacillus genus bacterium with the expression vector of the present invention. As will be described later, the transformant is typically used for production of a target peptide. The expression vector can be introduced into a host bacterium by a known method. For transformation, for example, protoplast transformation method (see Chang, S. & Cohen, SN Mol. Gen. Genet. 168, 111-115 (1979), etc.) or competent cell method (Spizizen, J. Proc. Natl. Acad. Sci. .USA.44,1072-1078 (1958) etc.) can be used.

  Examples of host bacteria include Bacillus subtilis, Bacillus licheniformis, Bacillus megaterium, Bacillus brevis, Bacillus sphaericus, Bacillus sphaericus, polymyxa), Bacillus alcalophilus, and Geobacillus stearothermophilus. Of these, Bacillus subtilis is preferable, and Bacillus subtilis 168 strain is particularly preferable. When the expression vector of the present invention has a modified promoter that functions also in bacteria other than the genus Bacillus, bacteria other than the genus Bacillus, such as Escherichia coli, can be employed as host bacteria. Specific examples of E. coli include JM109 strain, MC1061 strain, DH5α strain, and BL21 strain. Host bacteria can be easily obtained from ATCC (American Type Culture Collection) or the like.

(Production method of polypeptide)
If the above transformant is used, the desired polypeptide can be produced inside or outside the Bacillus bacterium. Then, the further situation of this invention provides the manufacturing method of polypeptide using said transformant. In the production method of the present invention, the above transformant is cultured, and a step (first step) of expressing a sequence encoding the target polypeptide introduced into the transformant is performed. Culture conditions using a Bacillus bacterium as a host are known (for example, DNA cloning, Volume II, IRL Press and the like can be referred to), and those skilled in the art can easily set appropriate culture conditions.

  You may decide to implement the said 1st step in two steps, the step which culture | cultivates and transforms a transformant, and the step which induces | expresses the expression of the target polypeptide. According to this aspect, the manufacturing efficiency can be improved.

  The timing of inducing expression is not particularly limited, but it is preferably induced in the logarithmic growth phase or stationary phase from the viewpoint of increasing the production amount of the target polypeptide. In particular, it is preferably induced between the late phase of the logarithmic growth phase and the middle phase of the stationary phase. Expression may be induced at the start of culture. A person skilled in the art can set appropriate culture conditions by preliminary experiments.

  By sampling the culture solution over time and calculating the number of bacteria by measuring turbidity (or absorbance), the growth stage (such as logarithmic growth phase or stationary phase) can be determined. Of course, a growth curve may be created by preliminary culture, and the growth stage may be determined using the growth curve.

  Other culture conditions (medium, culture temperature, culture time, etc.) may be appropriately set according to the recombinant bacteria to be cultured. Appropriate culture conditions can be set by preliminary experiments.

  The composition of the medium is not particularly limited as long as the condition that the recombinant bacteria to be cultured can grow is satisfied. For example, maltose, sucrose, gentiobiose, soluble starch, glycerin, dextrin, molasses, organic acid and the like can be used as the carbon source of the medium. Further, as the nitrogen source, for example, ammonium sulfate, ammonium carbonate, ammonium phosphate, ammonium acetate, peptone, yeast extract, corn steep liquor, casein hydrolyzate, bran, meat extract and the like can be used. As the inorganic salt, potassium salt, magnesium salt, sodium salt, phosphate, manganese salt, iron salt, zinc salt and the like can be used. A medium supplemented with vitamins, amino acids and the like may be used to promote the growth of the recombinant bacteria.

  The pH of the medium is adjusted to, for example, about 3 to 8, preferably about 6 to 8, and the culture temperature is usually about 10 to 50 ° C., preferably about 27 to 37 ° C. for 1 to 15 days, preferably 1 to Incubate for about 3 days. Usually, it is cultured under aerobic conditions. As the culture method, for example, a shaking culture method, a rotary culture method, an aeration stirring culture method, or the like can be used.

  Following the culture step, a step of recovering the expressed polypeptide of interest is performed. The target polypeptide can be recovered from the culture broth or cells after culturing. That is, it can be recovered from the culture medium when it is produced outside the bacterial cell, and from the bacterial cell otherwise. When recovering from the culture solution, for example, the culture supernatant is filtered and centrifuged to remove insolubles, followed by salting out such as ammonium sulfate precipitation, dialysis, and various chromatography (ion exchange chromatography, hydrophobic chromatography, affinity chromatography). The target polypeptide can be obtained by performing separation and purification in combination with chromatography and the like. On the other hand, when recovering from the microbial cells, for example, the microbial cells are crushed by pressure treatment, ultrasonic treatment, etc., and then the target polypeptide can be obtained by separation and purification in the same manner as described above. In addition, after collect | recovering a microbial cell from a culture solution previously by filtration, a centrifugation process, etc., you may perform the said series of processes (crushing, isolation | separation, purification of a microbial cell).

The polypeptide produced by the production method of the present invention is not particularly limited. However, the production method of the present invention is useful for producing enzymes. For example, the production method of the present invention can be applied to the production of α-amylase, β-amylase, carboxyesterase, oxalate decarboxylase and the like.
Hereinafter, the present invention will be described in more detail with reference to examples.

1. Construction of shuttle vector pUBC1 (Figure 1)
When the genetic recombination technique was used with Bacillus subtilis 168 strain as a host microorganism, the pUB110 vector (ATCC (American Type Culture Collection)) generally used in the Bacillus genus bacterial expression system was used. Instead of using the pUB110 vector as it is, in order to improve the transformation efficiency when constructing a genetically modified microorganism, we first considered the construction of an expression plasmid in E. coli, and the shuttle vector of the pUC18 and pUB110 vectors for E. coli Built. Actually, pUC18 and pUB110 were treated with restriction enzymes AflIII and XbaI to obtain a fragment containing the replication origin of each other. Subsequently, the obtained fragments were ligated with ligase to obtain a shuttle vector pUBC1.

2. Acquisition of α-amylase promoter The α-amylase promoter was acquired from Bacillus amyloliquefaciens. First, chromosomal DNA was obtained by a conventional method from a wild strain of Bacillus amyloliquefaciens and a mutant strain in which α-amylase productivity was improved by mutation treatment. Primers that specifically amplify the α-amylase promoter region (SEQ ID NO: 13) were designed with reference to the sequence of the α-amylase gene (GenBank Accession No. J01542) on the database. The 5 'primer is added with a restriction enzyme site of XbaI. In addition, an α-amylase promoter 3 ′ terminal sequence and a carboxyesterase gene 5 ′ terminal were added to the 3 ′ primer.
5'-side primer (primer 1): 5'-CGGCTCTAGATGGATCGATTGTTTGAG-3 '(SEQ ID NO: 19)
3'-side primer (primer 2): 5'-CAGGGTTTCAATTTTAGACATGTTTCCTCTCCCTCTCA-3 '(SEQ ID NO: 20)

  The α-amylase promoter fragment was obtained by amplifying the α-amylase promoter using the obtained chromosomal DNA as a template with the 5′-side primer and the 3′-side primer. The sequence of the natural α-amylase promoter (SEQ ID NO: 13) is shown in FIG. In the α-amylase promoter obtained from Bacillus amyloliquefaciens whose α-amylase productivity has been improved by the mutation treatment (mutant type promoter. The full-length sequence is shown in SEQ ID NO: 10), the portion indicated by the underline in FIG. Mutations were observed. The mutation site and the sequence in the vicinity thereof were compared between the natural type (SEQ ID NO: 21) and the mutant type (SEQ ID NO: 22) (FIG. 3).

The mutation point in the mutant sequence was not one place, but substitution of 2 bases and insertion of 1 base occurred. In order to identify the mutation point that increases the promoter activity most effectively, four types of modified promoters (FIG. 4) having different promoter mutation sites were prepared and their promoter activity was confirmed (see below). 3 to 14). In order to confirm the influence of the mutation in the α-amylase promoter on the promoter activity, a β-amylase gene or a carboxyesterase gene was linked to the α-amylase promoter. The promoter activity of each modified promoter was estimated from the expression levels of these genes. In order to confirm the promoter activity when expressed and accumulated in the host microorganism cell, a carboxyesterase gene (SEQ ID NO: 39) derived from Bacillus thuringiensis NBRC 13866 (formerly IFO 13866) is used. On the other hand, a β-amylase gene (SEQ ID NO: 40) derived from Bacillus flexus APC9451 was used to confirm the promoter activity when secreted outside the host microorganism cells. The NBRC 13866 share can be easily obtained from the National Institute of Technology and Evaluation Technology by a prescribed procedure. In addition, the APC9451 strain is deposited with a predetermined depository as follows and can be easily obtained.
Depositary institution: NITE Biotechnology Headquarters Patent Microorganism Deposit Center (2-5-8 Kazusa Kamashiri, Kisarazu City, Chiba Prefecture 292-0818, Japan)
Deposit date: April 9, 2008 Deposit number: NITE BP-548

3. Acquisition of carboxyesterase gene The carboxyesterase gene was acquired from Bacillus sulindiensis. First, chromosomal DNA was obtained by a conventional method from a wild strain of Bacillus schlingiensis. Primers that specifically amplify the carboxyesterase gene region were designed with reference to the gene sequence on the database. In addition, a restriction enzyme site of XbaI is added to the 3 ′ primer.
5'-side primer (primer 3): 5'-TATAAAACTGCAGGATGTCTAAAATTGAAACCCCTGT-3 '(SEQ ID NO: 27)
3'-side primer (primer 4): 5'-ACTAGTCTAGATTATTTAATTTTCTTAAATGCAAGCG-3 '(SEQ ID NO: 28)

  Using the obtained chromosomal DNA as a template, the carboxyesterase gene was amplified with the 5′-side primer and the 3′-side primer to obtain a carboxyesterase gene fragment.

4). Linkage between α-amylase promoter and carboxyesterase gene
The α-amylase promoter and carboxyesterase gene to be inserted into pUBC1 were ligated by PCR. A natural α-amylase promoter obtained by PCR or a mutant α-amylase promoter and a carboxyesterase gene obtained by PCR are mixed, and PCR is performed using primers 1 and 4, immediately after the α-amylase promoter. A fragment to which a carboxyesterase gene was linked was obtained.

5). Construction of genetically modified bacteria No.1 and No.2 (Fig. 5)
Shuttle vector pUBC1 and α-amylase promoter (natural or mutant) carboxyesterase gene ligation fragment treated with XbaI and then ligated with ligase to carboxyesterase gene expression plasmid vectors pUBCkik3x (natural) and pUBCkik2x (mutant) Was obtained (FIG. 6). Subsequently, E. coli strain JM109 was transformed with the vector. A large amount of the vector was prepared from this recombinant Escherichia coli, and then Bacillus subtilis 168 strain was transformed with the vector by a protoplast-PEG-fusion method to obtain a recombinant carboxyesterase gene (natural type or mutant type). A recombinant bacterium having a natural α-amylase promoter was designated as a recombinant bacterium No. 1, and a recombinant bacterium having a mutant α-amylase promoter was designated as a recombinant bacterium No. 2.

6). Acquisition of β-amylase gene The β-amylase gene was acquired from Bacillus flexus. First, chromosomal DNA was obtained from a wild strain of Bacillus flexus by a conventional method. Primers that specifically amplify the β-amylase gene region were designed with reference to the completed base sequence of the β-amylase gene. The 5 ′ primer was added with a BspLU11I restriction enzyme site, and the 3 ′ primer was added with a PstI restriction enzyme site.
5'-side primer (primer 5): 5'-GGAATTACATGTACAAGCCAATTAAAAAG-3 '(SEQ ID NO: 29)
3'-side primer (primer 6): 5'-TGGCCCTCACTGCAGAACAGC-3 '(SEQ ID NO: 30)

  Using the obtained chromosomal DNA as a template, the β-amylase gene was amplified using the 5′-side primer and the 3′-side primer to obtain a β-amylase gene fragment.

7). Construction of genetically modified bacteria No.7 and No.8 (Figure 5)
The carboxyesterase gene expression plasmid pUBCkik3x or pUBCkik2x and the β-amylase gene fragment were treated with BspLU11I and PstI, and then ligated with ligase, thereby β-amylase gene expression plasmid vectors pUBCOBA-1 (natural type) and pUBCMBA-1 (mutant type) ) Was obtained (FIG. 7). Subsequently, E. coli strain JM109 was transformed with the vector. After preparing a large amount of the vector from this recombinant Escherichia coli, Bacillus subtilis 168 strain was transformed with the vector by the protoplast-PEG-fusion method to obtain a β-amylase gene recombinant bacterium (natural type or mutant type). . A recombinant bacterium having a natural α-amylase promoter was designated as a recombinant bacterium No. 7, and a recombinant bacterium having a mutant α-amylase promoter was designated as a recombinant bacterium No. 8.

8). Construction of an expression plasmid vector having a modified α-amylase promoter (FIG. 5)
Construction of the modified α-amylase promoter was performed using Stratagene's QuikChange II Site-Directed Mutagenesis Kit. Primers (primers 7 to 14) were prepared corresponding to each modification, and site-specific mutations were introduced using pUBCOBA-1 as a template. The full length sequence of the modified promoter 1 is SEQ ID NO: 17, the full length sequence of the modified promoter 2 is SEQ ID NO: 11, the full length sequence of the modified promoter 3 is SEQ ID NO: 18, and the full length of the modified promoter 4 Are shown in SEQ ID NO: 12, respectively.
5 ′ primer for modified promoter 1 (primer 7): 5′-GACTGTCCGCTGTGTAAAAAAAAGGAATAAAGGGGGGTTG-3 ′ (SEQ ID NO: 31)
3 ′ primer for modified promoter 1 (primer 8): 5′-CAACCCCCCTTTATTCCTTTTTTTTACACAGCGGACAGTC-3 ′ (SEQ ID NO: 32)
5 ′ primer for modified promoter 2 (primer 9): 5′-GACTGTCCGCTGTGTAAAAATTAGGAATAAAGGGGGGTTG-3 ′ (SEQ ID NO: 33)
Modified promoter 2 3 ′ primer (primer 10): 5′-CAACCCCCCTTTATTCCTAATTTTTACACAGCGGACAGTC-3 ′ (SEQ ID NO: 34)
5′-side primer for modified promoter 3 (primer 11): 5′-GACTGTCCGCTGTGTAAAAAATAGGAATAAAGGGGGGTTG-3 ′ (SEQ ID NO: 35)
3 ′ primer for modified promoter 3 (primer 12): 5′-CAACCCCCCTTTATTCCTATTTTTTACACAGCGGACAGTC-3 ′ (SEQ ID NO: 36)
5 ′ primer for modified promoter 4 (primer 13): 5′-GACTGTCCGCTGTGTAAAAAATAAGGAATAAAGGGGGGTTG-3 ′ (SEQ ID NO: 37)
3 ′ primer for modified promoter 4 (primer 14): 5′-CAACCCCCCTTTATTCCTTATTTTTTACACAGCGGACAGTC-3 ′ (SEQ ID NO: 38)

  Escherichia coli JM109 was transformed with an expression plasmid vector containing various modified α-amylase promoters introduced with mutations, and a large number of expression plasmid vectors containing the modified α-amylase promoter were prepared from recombinant E. coli. The expression plasmid vector having the modified promoter 1 is pUBCDBA-1 (-), the expression plasmid vector having the modified promoter 2 is pUBCDBA-2 (-), and the expression plasmid vector having the modified promoter 3 is pUBCDBA-3. (−), An expression plasmid vector having the modified promoter 4 was designated as pUBCDBA-4 (−).

9. Linkage of various modified α-amylase promoters and carboxyesterase genes (FIG. 5)
Various modified α-amylase promoters and carboxyesterase genes were linked by PCR. First, various modified α are obtained by performing PCR using primers 1 and 2 using pUBCDBA-1 (−), pUBCDBA-2 (−), pUBCDBA-3 (−), and pUBCDBA-4 (−) as templates, respectively. -The amylase promoter was obtained. Next, by mixing the obtained various modified α-amylase promoters and the carboxyesterase gene and performing PCR using the primers 1 and 4, fragments obtained by linking the carboxyesterase gene immediately after the various modified α-amylase promoters Acquired.

10. Construction of expression plasmid vector having modified α-amylase promoter Shuttle vector pUBC1 and various modified α-amylase promoters and carboxyesterase gene fragments were treated with XbaI, and then ligated with ligase to obtain a carboxyesterase gene expression plasmid vector. Obtained. Next, E. coli strain JM109 was transformed with the obtained plasmid vector, and the plasmid vector was prepared in large quantities from recombinant E. coli. The expression plasmid vector having the modified promoter 1 is pUBCkik4-1 (−), the expression plasmid vector having the modified promoter 2 is pUBCkik4-2 (−), and the expression plasmid vector having the modified promoter 3 is pUBCkik4-3. The expression plasmid vector carrying the modified promoter 4 (−) was designated pUBCkik4-4 (−).

11. Construction of genetically modified bacteria No.3, No.4, No.5, No.6
Since the carboxyesterase gene in pUBCkik4-1 (-), pUBCkik4-2 (-), pUBCkik4-3 (-), and pUBCkik4-4 (-) was amplified by PCR, an error was introduced by chance during PCR. there is a possibility. Therefore, in order to reduce the possibility of an error, the carboxyesterase gene in pUBCkik3x for which sequence confirmation was completed and pUBCkik4-1 (−), pUBCkik4-2 (−), pUBCkik4-3 (−), pUBCkik4-4 (− ) In each of the carboxyesterase genes. That is, pUBCkik3x, pUBCkik4-1 (−), pUBCkik4-2 (−), pUBCkik4-3 (−), pUBCkik4-4 (−) were each treated with BspLU11I, pUBCkik3x was treated with the carboxyesterase gene, pUBCkik4-1 ( For −), pUBCkik4-2 (−), pUBCkik4-3 (−), and pUBCkik4-4 (−), vector portions containing an α-amylase promoter were obtained. Subsequently, the carboxyesterase gene fragments obtained from pUBCkik3x were ligated to each of pUBCkik4-1 (−), pUBCkik4-2 (−), pUBCkik4-3 (−), and pUBCkik4-4 (−) treated with BspLU11I by ligase. Escherichia coli JM109 strain was transformed with these to prepare a large amount of plasmid vector. The obtained various expression plasmid vectors (FIG. 8) were designated as pUBCkik4-1, pUBCkik4-2, pUBCkik4-3, and pUBCkik4-4.

  Next, each of the obtained pUBCkik4-1, pUBCkik4-2, pUBCkik4-3, pUBCkik4-4 was transformed into Bacillus sacilli 168 strain using protoplast-PEG-fusion method, and genes having various plasmid vectors Recombinant bacteria were obtained. The obtained genetically modified bacteria were designated as No. 3 for pUBCkik4-1, No. 4 for pUBCkik4-2, No. 5 for pUBCkik4-3, and No. 6 for pUBCkik4-4.

12 Construction of genetically modified bacteria No.9, No.10, No.11, No.12 (Fig. 5)
pUBCOBA-1 was treated with BspLU11I and PstI to obtain a β-amylase gene. Further, each of pUBCkik4-1, pUBCkik4-2, pUBCkik4-3, and pUBCkik4-4 was treated with BspLU11I and PstI to obtain a vector portion containing an α-amylase promoter. Next, a β-amylase gene and a vector containing various modified α-amylase promoters were ligated with ligase to construct a β-amylase gene expression plasmid vector. Escherichia coli JM109 was transformed with the constructed expression plasmid vector, and each expression plasmid vector was prepared in large quantities. The obtained various expression plasmid vectors (FIG. 9) were designated as pUBCDBA-1, pUBCDBA-2, pUBCDBA-3, and pUBCDBA-4.

  Next, each of the obtained pUBCDBA-1, pUBCDBA-2, pUBCDBA-3, and pUBCDBA-4 was transformed into Bacillus sacilli 168 strain using protoplast-PEG-fusion method, and genes having various plasmid vectors Recombinant bacteria were obtained. The obtained genetically modified bacteria were designated as No. 9 for pUBCDBA-1, No. 10 for pUBCDBA-2, No. 11 for pUBCDBA-3, and No. 12 for pUBCDBA-4.

13. Cultivation and Activity Measurement of Carboxyesterase Recombinant Bacteria No. 1 to No. 6 The esterase productivity of all six obtained carboxyesterase gene recombinant bacteria No. 1 to No. 6 was confirmed. As a method for culturing the recombinant bacteria, pre-culture was performed overnight, and then main culture was performed for 2 nights. LB medium was used as the medium for the pre-medium and the main medium. In addition, kanamycin was added to the pre-culture medium so that the final concentration was 20 μg / mL. The preculture medium was inoculated from a colony of recombinant bacteria and cultured at 37 ° C. and 180 rpm for about 16 hours.

Pre-culture medium (Kanamycin added at a final concentration of 20 μg / mL)

  In addition, since the α-amylase promoter is induced by a saccharide of disaccharide or higher, maltose was added to this medium so that the final concentration was 1%. 500 μL of the preculture was inoculated into this medium and cultured at 37 ° C. and 200 rpm for about 48 hours.

This medium (with maltose added at a final concentration of 1%)

  After culturing, the culture solution was centrifuged to collect microbial cells. The collected cells are washed with 10 mM potassium phosphate buffer (pH 7.0), suspended in 10 mM potassium phosphate buffer (pH 7.0) buffer, an appropriate amount of glass beads are added, and a multi-bead shocker ( The cells were crushed by Yasui Kikai (600 seconds with a cycle of 60 seconds of operation and 30 seconds of stoppage, 2,000 rpm). After disrupting the cells, the mixture was centrifuged, and the supernatant was collected and used as a sample for measuring carboxyesterase activity.

The activity measurement was performed as follows. After 9 mL of 4% Triton X-100 was added to 2.9 mg of p-nitrophenol chrysanthemate (pNPC) and dissolved by heating, 1 mL of 1 mL of 1M Tris solution (pH 8.5) was added to obtain a substrate solution. 0.2 mL of the substrate solution and 0.2 mL of an activity measurement sample appropriately diluted with 10 mM potassium phosphate buffer (pH 7.0) were mixed and reacted at 40 ° C. for 30 minutes. The reaction was completed by adding 0.6 mL of ethanol. About the sample which stopped reaction, the light absorbency in absorption wavelength 405nm was measured, and the activity value was computed from the following formula. The amount of enzyme that liberates 1 μmol of Chrysanthemic acid per minute was defined as 1 u.

  The measurement result of carboxyesterase activity is shown in FIG. The activity value of the natural α-amylase promoter is shown as a relative value with 100%. The mutant and all modified promoters showed higher values than the native promoter. In particular, it was confirmed that the activity increased by a factor of 2 or more in the promoters of the mutant type, modified type 2 and modified type 3.

14 Cultivation and Activity Measurement of β-Amylase Recombinant Bacteria No. 7 to No. 12 The β-amylase productivity of all of the obtained β-amylase gene recombinants No. 7 to No. 12 was confirmed. As a method for culturing the recombinant bacteria, pre-culture was performed overnight, and then main culture was performed for 2 nights. LB medium was used as the medium for the pre-medium and the main medium. In addition, kanamycin was added to the pre-culture medium so that the final concentration was 20 μg / mL. The preculture medium was inoculated from a colony of recombinant bacteria and cultured at 37 ° C. and 180 rpm for about 16 hours.

Pre-culture medium (Kanamycin added at a final concentration of 20 μg / mL)

  In addition, since the α-amylase promoter is induced by a saccharide of disaccharide or higher, maltose was added to this medium so that the final concentration was 1%. 500 μL of the preculture was inoculated into this medium and cultured at 37 ° C. and 200 rpm for about 48 hours.

This medium (with maltose added at a final concentration of 1%)

After culturing, the culture solution was centrifuged and the supernatant was collected. This supernatant was used as a sample for measuring β-amylase activity. The activity was measured by improving a β-amylase measurement kit (manufactured by Megazyme). Specifically, for the measurement of activity using p-nitrophenyl-α-D-maltopentaoside (PNPG5) 0.475mg / mL and α-glucosidase 10μ / L as a substrate solution, diluted appropriately with 50μL of substrate solution and distilled water. After mixing 10 μL of the sample, the reaction was performed at 40 ° C. for 5 minutes. Moreover, the same reaction was performed with distilled water instead of the sample for activity measurement as a blank reaction liquid. Five minutes after the start of the reaction, 150 μL of 1.5% Tris solution (pH 8.5) was added as a reaction stop solution, and the reaction was stopped by stirring. About the sample which stopped reaction, the light absorbency in absorption wavelength 400nm was measured, and the activity value was computed from the following formula. The amount of enzyme that liberates 1 μmol of p-nitrophenol per minute was defined as 1 u.

  The results of measuring β-amylase activity are shown in FIG. The activity value of the natural α-amylase promoter is shown as a relative value with 100%. The mutant and all modified promoters showed higher values than the native promoter. In particular, an increase in activity of 1.5 times or more was confirmed with the promoters of mutant type, modified type 2 and modified type 4.

  The modified promoter of the present invention can be used in an expression system using a Bacillus bacterium as a host. By using the modified promoter of the present invention, a high expression system can be constructed. Preferably, the modified promoter of the present invention is used for enzyme production. Examples of the enzyme produced include β-amylase and carboxyesterase.

The present invention is not limited to the description of the embodiments and examples of the invention described above. Various modifications may be included in the present invention as long as those skilled in the art can easily conceive without departing from the description of the scope of claims.
The contents of papers, published patent gazettes, patent gazettes, and the like specified in this specification are incorporated by reference in their entirety.

SEQ ID NOs: 19, 20, 27-38: Description of artificial sequence: primer

Claims (20)

  A modified promoter that functions in a bacterium belonging to the genus Bacillus, wherein the sequence AAAAATAA is replaced with any sequence selected from the group consisting of the sequence AAAAAAAATA, the sequence AAAAATTA, and the sequence AAAAAATAA in the natural promoter of the genus Bacillus A modified promoter characterized in that the promoter activity is improved as compared with a natural promoter.   A modified promoter that functions in a bacterium belonging to the genus Bacillus, wherein the sequence AAAAATAA that is present upstream and in the vicinity of the -35 region in the natural promoter of the genus Bacillus is selected from the group consisting of the sequence AAAAAAATA, the sequence AAAAATTA, and the sequence AAAAAATAA A modified promoter, characterized by being substituted by any of the sequences.   The modified promoter according to claim 2, comprising any one of SEQ ID NOs: 1 to 3 as a sequence containing the -35 region.   The modified promoter according to claim 2, comprising the sequence of any one of SEQ ID NOs: 4 to 6 as a sequence including a -35 region and a -10 region.   The modified promoter according to claim 2, comprising any one of SEQ ID NOs: 7 to 9. The modified promoter according to claim 2, comprising any one of the following sequences (1) to (3):
(1) The sequence of SEQ ID NO: 10, or the region of bases 1 to 95, the region of bases 105 to 118, the region of bases 125 to 145, and the bases 152 to 181 of the sequence A sequence comprising one or more bases substituted, deleted, inserted and / or added in one or more regions selected from the group consisting of base regions and exhibiting higher activity than the natural promoter;
(2) The sequence of SEQ ID NO: 11, or the region of bases 1 to 95, the region of bases 104 to 117, the region of bases 124 to 144, and the base of 151 to 180 A sequence comprising one or more bases substituted, deleted, inserted and / or added in one or more regions selected from the group consisting of base regions and exhibiting higher activity than the natural promoter;
(3) the sequence of SEQ ID NO: 12, or the region of bases 1 to 95, the region of bases 105 to 118, the region of bases 125 to 145, and the region of bases 152 to 181 in the sequence A sequence comprising a sequence in which one or several bases are substituted, deleted, inserted and / or added in one or more regions selected from the group consisting of base regions, and exhibiting higher activity than the natural promoter.   The modified promoter according to any one of claims 1 to 6, wherein the natural promoter is an α-amylase promoter of Bacillus amyloliquefaciens.   The modified promoter according to claim 7, wherein the α-amylase promoter consists of the sequence of SEQ ID NO: 13.   A modified promoter that functions in a bacterium belonging to the genus Bacillus, comprising the sequence of SEQ ID NO: 14 as a sequence containing the -35 region and the -10 region.   The modified promoter according to claim 9, comprising the sequence of SEQ ID NO: 15 or 16.   The modified promoter according to claim 9, which consists of the sequence of SEQ ID NO: 17 or 18.   An expression construct comprising the modified promoter according to any one of claims 1 to 11 and a base sequence encoding a polypeptide operably linked to the modified promoter.   The expression construct according to claim 12, wherein the polypeptide is α-amylase, β-amylase, carboxyesterase or oxalate decarboxylase.   An expression vector into which the expression construct according to claim 12 or 13 is inserted.   An expression vector comprising the modified promoter according to any one of claims 1 to 11 and a cloning site arranged under the control of the modified promoter.   A transformant obtained by transforming a host bacterium with the expression vector according to claim 14.   The transformant according to claim 16, wherein the host bacterium is a genus Bacillus. A first step of culturing the transformant according to claim 16 or 17 and expressing the polypeptide;
And a second step of recovering the expressed polypeptide.   19. The method for producing a polypeptide according to claim 18, wherein the first step comprises culturing and growing the transformant, and inducing expression of the polypeptide.   The sequence AAAAATAA existing upstream and in the vicinity of the -35 region in the native promoter of the genus Bacillus is replaced with any sequence selected from the group consisting of the sequence AAAAAAATA, the sequence AAAAAAAA, the sequence AAAAATTA, the sequence AAAAAATA, and the sequence AAAAAATAA A method for modifying a promoter, characterized by being modified as described above.

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