# Patent

## Title
Modified promoter

## Abstract

The present invention provides a modified promoter, an expression vector and a transformant each containing the promoter, and a method for producing a gene product of interest using the transformant. The invention provides a modified promoter, including a nucleotide sequence of a promoter derived from bacterium belonging to the genus Bacillus in which at least one nucleotide sequence selected from the following has been modified: a nucleotide sequence represented by SEQ ID NO: 1; a nucleotide sequence equivalent to the nucleotide sequence represented by SEQ ID NO: 1, except that one or a plurality of bases therein are substituted, deleted, added or inserted; and a nucleotide sequence having a sequence identity of 70% or more with respect to the nucleotide sequence represented by SEQ ID NO: 1.

## Background
REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY
The content of the electronically submitted substitute sequence listing, file name 2537—0650002SEQListing.txt; size 53,861 bytes; and date of creation Feb. 28, 2012, filed herewith, is incorporated herein by reference in its entirety.
FIELD THE INVENTION
The present invention relates to a modified promoter, to an expression vector and a transformant each containing the promoter, and to a method for producing a gene product of interest using the transformant.
BACKGROUND OF THE INVENTION
Through use of microorganisms, a variety of useful substances have been produced on an industrial scale, including foods such as alcoholic beverages, miso, and shoyu (soy source); and other substances such as amino acids, organic acids, nucleic-acid-related substances, antibiotics, sugars, lipids, and proteins. Such substances are useful in a wide range of fields such as foods, pharmaceuticals, and daily-use articles such as detergents and cosmetics, as well as raw materials for chemical products.
In such industrial production of useful substances by the mediation of microorganisms, enhancement in productivity is an important issue. One approach which has been employed for enhancing productivity is breeding, through a genetic technique such as mutation, a bacterium which produces a substance of interest. In recent years, accompanied with the development of microbial genetics and biotechnology, more effective methods based on a recombinant technique or the like have been focused for producing useful substances.
A number of studies have been conducted on promoters which are required for the transcription of genes. Regarding Bacillus subtilis, a promoter domain of an alkaline cellulase gene derived from Bacillus sp. KSM-64 strain (FERM BP-2886) (see, for example, Non-Patent Document 1), a promoter domain present on the upstream side of an alkaline cellulase gene derived from Bacillus sp. KSM-S237 strain (FERM BP-7875) (see, for example, Patent Document 1 and Non-Patent Document 2), and other promoter domains are employed as promoter domains which actively transcript a gene encoding a heterologous protein or polypeptide.
However, in order to reduce production cost in industrial production, there is demand for a promoter or a microorganism which realizes higher productivity.
Generally, a promoter domain has various sites relating to regulation of transcription. These sites play a role in promoting or suppressing expression of a gene by the mediation of external signals. For example, when a culture medium for microorganisms contains large amounts of catabolites such as glucose, the microorganisms utilize mainly the catabolites as carbon sources, and the expression of an enzyme for degrading higher-molecule sugar is suppressed. This phenomenon is called “catabolite repression,” which has been observed in E. coil, Bacillus, etc. Catabolite repression in Bacillus subtilis is known to occur at the transcription level. The domain which is involved in catabolite repression in Bacillus is called “catabolite responsive element (cre).”
Non-Patent Document 3 discloses that catabolite repression may be increased or reduced by a point mutation in a catabolite repression operator domain in the Bacillus subtilis-derived α-amylase gene. Meanwhile, in a promoter in the Bacillus subtilis-derived endo-glucanase, there is a nucleotide sequence similar to that of the catabolite repression domain of α-amylase, which is called a “catabolite repression operator-like sequence” (Non-Patent Document 2). However, the actual role of the sequence has not been elucidated.

    [Patent Document 1] JP-A-2000-210081
    [Non-Patent Document 1] Sumitomo et al., Biosci. Biotech. Biochem., 59: 2172-2175, 1995
    [Non-Patent Document 2] Hakamada et al., Biosci. Biotech. Biochem. 64: 2281-2289, 2000
    [Non-Patent Document 3] Weickert & Chambliss, Proc. Natl. Acad. Sci. USA, 87: 6238-6242, 1990


DISCLOSURE OF THE INVENTION
The present invention is directed to a modified promoter, including a nucleotide sequence of a promoter derived from bacterium belonging to the genus Bacillus in which at least one nucleotide sequence selected from the following has been modified: a nucleotide sequence represented by SEQ ID NO: 1; a nucleotide sequence equivalent to the nucleotide sequence represented by SEQ ID NO: 1, except that one or a plurality of bases therein are substituted, deleted, added or inserted; and a nucleotide sequence having a sequence identity of 700 or more with respect to the nucleotide sequence represented by SEQ ID NO: 1.
The present invention is also directed to a method for producing a modified promoter, the method including modifying, in a promoter derived from bacterium belonging to the genus Bacillus, at least one nucleotide sequence selected from the following: a nucleotide sequence represented by SEQ ID NO: 1; a nucleotide sequence equivalent to the nucleotide sequence represented by SEQ ID NO: 1, except that one or a plurality of bases therein are substituted, deleted, added or inserted; and a nucleotide sequence having a sequence identity of 70% or more with respect to the nucleotide sequence represented by SEQ ID NO: 1.
The present invention is also directed to the above-mentioned modified promoter or a modified promoter produced by the above-mentioned method, wherein one or a plurality of bases therein are further substituted, deleted, added or inserted.
The present invention is also directed to an expression vector including the above-mentioned modified promoter or the modified promoter produced by the above-mentioned method.
The present invention is also directed to a transformant including the above-mentioned modified promoter or the modified promoter produced by the above-mentioned method.
The present invention is also directed to a method for producing a gene product of interest, the method includes employing the transformant.
In one embodiment, the nucleotide sequence having a sequence identity of 70% or more with respect to the nucleotide sequence represented by SEQ ID NO: 1 is a nucleotide sequence represented by SEQ ID NO: 2.
In another embodiment, the promoter derived from a bacterium belonging to the genus Bacillus is a promoter from Bacillus subtilis or a mutant strain of Bacillus subtilis. 
In another embodiment, the promoter derived from a bacterium belonging to the genus Bacillus is: a promoter of an alkaline cellulase gene from Bacillus sp. KSM-64 strain (FERN BP-2886); a promoter of an alkaline cellulase gene from Bacillus sp. KSM-5237 strain (FERN BP-7875); or a promoter containing a nucleotide sequence which has a sequence identity of 700 or more with respect to the nucleotide sequence of any one of said promoters and having a cre-like sequence.
In another embodiment, the modification of the nucleotide sequences is deletion, substitution or addition of a base in the nucleotide sequences.
In another embodiment, the modified promoter is operably linked to the upstream of a gene encoding a gene product of interest.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 A procedure for producing a mutated promoter having a deletion through the SOE-PCR method.
FIG. 2 A procedure for preparing a pDL2-S237 plasmid.
FIG. 3 β-galactosidase activity of the transformant of the present invention and that of a wild type. A: Activity in glucose-containing medium. B: Activity in maltose-containing medium. C: Activity in sorbitol-containing medium. D: Activity in model saccharified solution-containing medium.



DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to provision of a modified promoter which enhances the gene transcription, an expression vector and a transformant each containing the promoter, and a method for producing a gene product of interest by use of the transformant.
The present inventors have found that a plurality of sequences which are similar to catabolite repression sequences in the promoter domain of a cellulase gene in a microorganism belonging to the genus Bacillus. The present inventors have also found that when the thus-found sequences were mutated, expression of the gene regulated by the promoter was considerably increased and productivity of the gene product encoded by the gene was enhanced.
The modified promoter of the present invention remarkably enhances the transcription amount of the gene which is linked to the downstream of the promoter. Thus, according to the present invention, a target gene product is yielded more efficiently.
In the present invention, sequence identity between amino acid sequences and that between nucleotide sequences are both determined through the Lipman-Pearson method (Science, 227, 1435 (1985)). Specifically, sequence identity is calculated through analysis by use of a homology analysis (search homology) program of genetic information processing software Genetyx-Win (Software Development Co., Ltd.), with the parameter ktup (unit size to compare) being set to 2.
Unless otherwise specified, the nucleotide sequence and base Nos. of Bacillus subtilis DNA used in the present specification are based on the Bacillus subtilis transcriptional factor retrieval site (http://dbtbs.hgc.jp/).
The modified promoter of the present invention is produced by modifying a nucleotide sequence similar to that of the catabolite responsive element (hereinafter referred to as “cre-like sequence”) present in a polynucleotide encoding a promoter derived from a gene in a microorganism belonging to the genus Bacillus having the cre-like sequence. Examples of the cre-like sequence include a nucleotide sequence represented by SEQ ID NO: 1, and a nucleotide sequence equivalent to the nucleotide sequence represented by SEQ ID NO: 1, except that one or a plurality of bases therein are substituted, deleted, added or inserted. Examples of the cre-like sequence further include a nucleotide sequence having a sequence identity of 700 or more, preferably 800 or more, more preferably 900 or more with respect to the nucleotide sequence represented by SEQ ID NO: 1. As used herein, the term “a plurality of” refers to 1 to 6, preferably 1 to 3.
The method of substituting, deleting, adding or inserting a base is disclosed in, for example, Dieffenbach et al. (Cold Spring Harbor Laboratory Press, New York, 581-621, 1995). Through employment of this technique, one or a plurality of bases can be substituted, deleted, added, or inserted. Whether or not the sequence of interest is a cre-like sequence may be confirmed, for example, by observing enhanced expression of a target gene in the presence of a catabolite such as glucose when mutation is introduced in the sequence in a manner similar to that of the Examples described hereinbelow. Examples of the nucleotide sequence having a sequence identity of 70% or more with respect to the nucleotide sequence represented by SEQ ID NO: 1 include a nucleotide sequence represented by SEQ ID NO: 2. The nucleotide sequence represented by SEQ ID NO: 2 is equivalent to the nucleotide sequence represented by SEQ ID NO: 1, except that three bases therein have been substituted. Therefore, the nucleotide sequence represented by SEQ ID NO: has a homology of 76.9% with respect to the nucleotide sequence represented by SEQ ID NO: 1.
No particular limitation is imposed on the promoter derived from a bacterium belonging to the genus Bacillus from which the modified promoter of the present invention is produced, i.e., the parent promoter corresponding to the modified promoter of the present invention, so long as the parent promoter is a promoter which is derived from a bacterium belonging to the genus Bacillus and which has a cre-like sequence. The parent promoter is preferably derived from Bacillus subtilis or a mutant strain of Bacillus subtilis. Examples of preferred parent promoter include a promoter of an alkaline cellulase gene derived from Bacillus sp. KSM-64 strain (FERM BP-2886) (base Nos. 1 to 609 in SEQ ID NO: 3) and a promoter of an alkaline cellulase gene derived from Bacillus sp. KSM-S237 strain (FERM BP-7875) (base Nos. 1 to 572 in SEQ ID NO: 4). The nucleotide sequences represented by SEQ ID NO: 1 and 2 correspond to the nucleotide sequences of base Nos. 399 to 412 and 264 to 276, respectively, in the promoter of an alkaline cellulase gene from the KSM-64 strain which is represented by base Nos. 1 to 609 in SEQ ID NO: 3, and correspond to the nucleotide sequences of base Nos. 359 to 372 and 223 to 235, respectively, in the promoter of an alkaline cellulase gene from the KSM-5237 strain which is represented by base Nos. 1 to 572 in SEQ ID NO: 4.
Other examples of the parent promoter in the present invention include a promoter derived from a bacterium belonging to the genus Bacillus and having a cre-like sequence, which has: a nucleotide sequence equivalent to the nucleotide sequence of the promoter of the alkaline cellulase gene represented by SEQ ID NO: 3 or 4, except that one or a plurality of bases therein are substituted, deleted, added or inserted; or a nucleotide sequence having a sequence identity of 70% or more, preferably 80% or more, more preferably 90% or more, even more preferably 95% or more, yet more preferably 98% or more, with respect to the nucleotide sequence of the promoter of the alkaline cellulase gene represented by SEQ ID NO: 3 or 4. Whether or not the promoter has a cre-like sequence may be confirmed by determining the presence of a cre-like sequence in the nucleotide sequence of the promoter through sequencing, or by comparing the sequence of the promoter with that of the promoter of the alkaline cellulase gene represented by SEQ ID NO: 3 or 4 to identify the region corresponding to the cre-like sequence. The method of substituting, deleting, adding or inserting a base is disclosed in, for example, Dieffenbach et al. (Cold Spring Harbor Laboratory Press, New York, 581-621, 1995). Through employment of this technique, one or a plurality of bases can be substituted, deleted, added or inserted. The promoter sequence may be identified, for example, by observing expression of β-galactosidase when a LacZ gene has been introduced in a manner similar to that of the Examples described hereinbelow.
In the case where the parent promoter includes two or more of cre-like sequences, at least one nucleotide sequence of the cre-like sequences present in the parent promoter may be modified. In other words, one, two or more, or all of the cre-like sequences present in the parent promoter may be modified. For example, when the parent prompter has two cre-like sequences represented by SEQ ID NOs: 1 and 2, only the nucleotide sequence represented by SEQ ID NO: 1 or the nucleotide sequence represented by SEQ ID NO: 2 may be modified, or both of the two nucleotide sequences represented by SEQ ID NO: 1 and SEQ ID NO: 2 may be modified. Preferably, the two nucleotide sequences represented by SEQ ID NO: 1 and SEQ ID NO: 2 are modified.
In one embodiment, the modified promoter of the present invention is produced by modifying at least one cre-like sequence present in a promoter from Bacillus subtilis or a mutant strain of Bacillus subtilis. 
In another embodiment, the modified promoter of the present invention is produced by modifying at least one of the cre-like sequence represented by base Nos. 264 to 276 and the cre-like sequence represented by base Nos. 399 to 412, in the promoter of an alkaline cellulase gene derived from KSM-64 strain (FERM BP-2886) (base Nos. 1 to 609 in SEQ ID NO: 3). Preferably, both the cre-like sequence represented by base Nos. 264 to 276 and the cre-like sequence represented by base Nos. 399 to 412 are modified.
In another embodiment, the modified promoter of the present invention is produced by modifying at least one of the cre-like sequence represented by base Nos. 223 to 235 and the cre-like sequence represented by base Nos. 359 to 372, in the promoter of an alkaline cellulase gene derived from Bacillus sp. KSM-S237 strain (FERM BP-7875) (base Nos. 1 to 572 in SEQ ID NO: 4). Preferably, both the cre-like sequence represented by base Nos. 223 to 235 and the cre-like sequence represented by base Nos. 359 to 372 are modified.
Examples of the modification of the nucleotide sequence of the cre-like sequence include deletion of a part of or the entirety of the bases in the nucleotide sequence, substitution of a part of or the entirety of the bases in the nucleotide sequence, and addition of a base at any position in the nucleotide sequence. As used herein the term “a part of the bases” refers to one or more bases, preferably three or more bases, more preferably 6 or more bases, even more preferably 10 or more bases. Deletion, substitution, or addition of a base may be performed through a method generally employed in the art, such as site-directed mutagenesis. The site-directed mutagenesis may be performed through the Kunkel method (Proc. Natl. Acad. Sci. USA., 82, 488, 1985), PCR, or the like.
Alternatively, the modified promoter of the present invention may be produced through the SOE (splicing by overlap extension)-PCR method (Gene, 77, 51, 1989), in which a base is deleted, substituted, or added in the nucleotide sequence of the cre-like sequence present in the promoter.
According to the SOE-PCR method, an upstream DNA fragment having a nucleotide sequence modification site at the downstream end, and a downstream DNA fragment having a nucleotide sequence modification site at the upstream end are prepared through the first PCR. In the case where a fragment in which a cre-like sequence has been deleted is prepared, a primer is designed such that the downstream side of the upstream DNA fragment and the upstream side of the downstream DNA fragment are mutually annealed, and a cre-like-sequence-deleted nucleotide sequence is provided (FIG. 1).
Subsequently, the second PCR is performed by use of the two DNA fragments prepared by the first PCR as templates, and the upstream primer of the upstream DNA fragment and the downstream primer of the DNA fragment. As a result, annealing occurs between the downstream end of the upstream DNA fragment and the upstream end of the downstream DNA fragment, whereby the two DNA fragments are linked to each other. Thus, a DNA fragment containing a promoter sequence in which a cre-like sequence has been deleted is produced (FIG. 1). In this case, when the primers employed in PCR are designed to have a restriction sequence, a promoter DNA fragment including restriction sequences at the ends is produced, and incorporated into a vector through the restriction enzyme method.
Similarly, when a primer is designed such that the downstream side of the upstream DNA fragment and the upstream side of the downstream DNA fragment are mutually annealed and that a nucleotide sequence including a cre-like sequence in which at least one base is substituted or added is prepared, there can be prepared a DNA fragment containing a promoter sequence in which at least one base of the cre-like sequence has been substituted or added.
In the modified promoter of the present invention, one or a plurality of bases may be further substituted, deleted, added or inserted, so long as the function of the promoter and the effect of enhancing expression of the gene of interest are maintained. The method of substituting, deleting, adding or inserting a base is disclosed in, for example, Dieffenbach et al. (Cold Spring Harbor Laboratory Press, New York, 581-621, 1995). Through employment of this technique, one or a plurality of bases can be substituted, deleted, added or inserted. Whether or not the modified promoter which has been further modified has promoter function and exhibits an effect of enhancing expression of a gene of interest may be confirmed, for example, by observing an effect of enhancing expression of a target gene in the presence of a catabolite such as glucose in a manner similar to that of the Examples described hereinbelow.
Through incorporation of the thus-prepared modified promoter of the present invention into an appropriate vector, the expression vector of the present invention can be produced. No particular limitation is imposed on the vector into which the modified promoter is incorporated, and vectors such as pDL2 and pMUTIN are preferred. Incorporation of a modified promoter into a vector may be performed in accordance with any methods generally employed in the art. For example, a vector is cleaved by means of restriction enzymes, and combined with a promoter having the above-produced restriction sequences at the ends, whereby the promoter is incorporated into the vector (restriction enzyme method).
In the expression vector of the present invention, the modified promoter of the present invention is operably linked to the upstream of the gene encoding a gene product of interest. Examples of the gene product of interest include protein, polypeptide and non-coding RNA. No particular limitation is imposed on the species of the protein and polypeptide, and the protein or the polypeptide includes enzymes for industrial uses such as detergents, foods, fiber, livestock feed, chemicals, medical care, and diagnosis, as well as physiologically active peptides. Examples of the enzyme for industrial uses include oxidoreductase, transferase, hydrolase, lyase, isomerase and ligase/synthetase. Genes of hydrolases such as cellulase, amylase and protease are preferred. Specific examples of the hydrolase include cellulase belonging to the family 5 as defined by the classification of polysaccharide hydrolases (Biochem. J., 280, 309, 1991). Among them, cellulases derived from microorganisms; for example, a microorganism belonging to the genus Bacillus, are preferred. More specific examples include an alkaline cellulase derived from Bacillus sp. KSM-64 strain (FERM BP-2886) consisting of an amino acid sequence represented by SEQ ID NO: 3, an alkaline cellulase derived from Bacillus sp. KSM-S237 strain (FERM BP-7875) consisting of an amino acid sequence represented by SEQ ID NO: 4, and cellulases consisting of an amino acid sequence having a sequence identity of 70% or more, preferably 80% or more, more preferably 90% or more, even more preferably 95% or more, yet more preferably 98% or more, with respect to the amino acid sequence represented by SEQ ID NO: 3 or 4.
Specific examples of the α-amylase include α-amylases derived from microorganisms. A liquefying α-amylase derived from a microorganism belonging to the genus Bacillus is preferred. More specific examples include an alkaline amylase derived from Bacillus sp. KSM-K38 strain (FERM BP-6946) consisting of an amino acid sequence represented by SEQ ID NO: 5 and amylases consisting of an amino acid sequence having a sequence identity of 70% or more, preferably 80% or more, more preferably 90% or more, even more preferably 95% or more, yet more preferably 98% or more, with respect to the amino acid sequence represented by SEQ ID NO: 5. Specific examples of the protease include serine protease and metalloprotease derived from a microorganism such as a microorganism belonging to the genus Bacillus. 
Preferably, the aforementioned protein gene or polypeptide gene is operably linked to the region of the modified promoter of the present invention and to a secretion signal peptide region. For example, a structural gene of the target protein or polypeptide may be operably linked to a secretion signal peptide region of a cellulase gene derived from KSM-64 strain (FERM BP-2886) or KSM-S237 strain (FERM BP-7875). More specifically, a structural gene of the target protein or polypeptide may be operably linked to a nucleotide sequence represented by base Nos. 610 to 696 of SEQ ID NO: 3; a nucleotide sequence represented by base Nos. 573 to 659 of SEQ ID NO: 4; a DNA fragment consisting of a nucleotide sequence having a sequence identity of 70% or more, preferably 80% or more, more preferably 90% or more, even more preferably 95% or more, yet more preferably 98% or more, with respect to any of the above nucleotide sequences; or a DNA fragment equivalent to any of the nucleotide sequences, except that a part of a base or bases are deleted.
The non-coding RNA is an RNA which is not translated to protein after transcription from the corresponding DNA. Examples of the non-coding RNA include a non-translated region including a regulatory sequence, tRNA, rRNA, mRNA-type ncRNA (mRNA-like non-coding RNA), snRNA (small nuclear RNA), snoRNA (small nucleolar RNA), miRNA (microRNA), stRNA (small temporal RNA), and siRNA (short-interfering RNA). These non-coding RNAs are involved in various mechanisms of biological activity including regulation of cell expression, development, differentiation and others, and may be utilized in research, medical care, diagnosis, drug development, agriculture (e.g., breeding or pesticide production), fisheries, the livestock industry, and chemical production.
The modified promoter of the present invention and a gene encoding a gene product of interest or a secretion signal peptide region may be linked before incorporating them into a vector. Alternatively, the modified promoter of the present invention may be incorporated into a vector already containing a gene encoding a gene product of interest or the vector further containing a secretion signal peptide region.
As used herein, the term “operable linking” between the promoter and a gene, or between the promoter, a gene and a secretion signal refers to such a linkage that the promoter can induce transcription of the gene, or such a linkage that the protein encoded by the gene is secreted by the secretion signal. The procedure of “operable linking” between the promoter and a gene, or between the promoter, a gene and a secretion signal, is known to those skilled in the art.
Through incorporation of the produced expression vector of the present invention into a host microorganism by an ordinary transformation method, the transformant of the present invention is yielded. Alternatively, the transformant of the present invention may also be produced by constituting a DNA fragment in which an appropriate region homologous to the genome of the host microorganism is combined with a fragment prepared by linking the modified promoter of the present invention to a gene encoding a gene product of interest and optionally to a secretion signal peptide region; and directly incorporating the DNA fragment into the genome of the host microorganism through homologous recombination.
The transformant of the present invention may also be provided by incorporating the expression vector of the present invention into a host microorganism with an ordinary recombination technique.
A gene product of interest may be produced by use of the transformant of the present invention through the following procedure: inoculating the transformant strain to a medium containing an assimilable carbon source, nitrogen source and other essential ingredients; culturing the transformants through an ordinary culturing method; and subsequently collecting or purifying a gene product of interest. As described in the Examples hereinbelow, the transformant of the present invention exhibits higher productivity of a gene product of interest, as compared with a microorganism in which the cre-like sequence has not been modified.
EXAMPLES
The present invention will next be described in more detail by way of examples.
Example 1
Preparation of Modified Promoter Derived from Alkaline Cellulase Gene
A promoter DNA fragment derived from KSM-S237 cellulase (Hakamada et al., Biosci. Biotechnol. Biochem. 64, 2281-2289, 2000) was prepared by PCR. A fragment of promoter region in an alkaline cellulase gene DNA derived from Bacillus sp. KSM-S237 (base Nos. 1 to 572 of SEQ ID NO: 4), which has been incorporated in a shuttle vector pHY300PLK for E. coli-Bacillus subtilis, was used as a template. EcoRI_PS237 FW (SEQ ID NO: 6) and BamHI_PS237 RV (SEQ ID NO: 7) shown in Table 1 was used as primers. A promoter DNA fragment having an EcoRI-recognition site on the upstream side and a BamHI-recognition site on the downstream side was prepared by PCR using the template and the primers described above. Then, for preparing a mutant promoter DNA fragment in which a target sequence in the promoter DNA fragment has been deleted, PCRs were conducted using the fragment prepared through the above PCR as a template, and a primer set of Δcre1 FW (SEQ ID NO: 8) and BamHI_PS237 RV or a primer set of EcoRI_PS237 FW and Δcre1 RV (SEQ ID NO: 9), each shown in Table 1, thereby preparing fragments having, on the upstream side and the downstream side respectively, a region in which a target sequence has been deleted. The thus-obtained two fragments were combined to be used as a mixed template. PCR was conducted with the mixed template and a EcoRI_PS237 FW primer and a BamHI_PS237 RV primer to prepare a promoter sequence DNA fragment in which base residues 223 to 235 in the nucleotide sequence represented by SEQ ID NO: 4 have been deleted (Δcre1) (FIG. 1). In a similar manner, a promoter domain sequence DNA fragment in which base residues 359 to 372 in the nucleotide sequence represented by SEQ ID NO: 4 have been deleted (Δcre2) was prepared, by use of Δcre2 FW (SEQ ID NO: 10) instead of Δcre1 FW, and Δcre2 RV (SEQ ID NO: 11) instead of Δcre1 RV. Further PCRs were conducted using Δcre1 as a template, and a primer set of Δcre2 FW and BamHI_PS237 RV or a primer set of EcoRI_PS237 FW and Δcre2 RV. Using the two fragments thus-obtained as a mixed template, PCR was conducted with primers EcoRI_PS237 FW and BamHI_PS237 RV, thereby preparing a promoter sequence DNA fragment in which base residues 223 to 235 and 359 to 372 in the nucleotide sequence represented by SEQ ID NO: 4 have been deleted (Δcre1.2).
For amplifying a DNA fragment, PCR was conducted by means of a GeneAmp PCR System (product of Applied Biosystems) and by use of Pyrobest DNA Polymerase (Product of Takara Bio) and reagents attached thereto. The PCR solution (50 μL) was prepared so as to combine an appropriately diluted template DNA fragment (1 μL), each of a sense primer and an anti-sense primer (10 pmol each), and Pyrobest DNA Polymerase (2.5 U). PCR included 30 cycles of the 3-step thermal treatment (98° C. for 10 sec, 55° C. for 30 sec, and 72° C. for 1 to 5 min (about 1 min/kb, with adjusting in accordance with the target product) and reaction at 72° C. for 5 min.









TABLE 1







SEQ




ID


Primer
Nucleotide sequence 5′→3′
NO:







EcoRI_PS237 FW
AAAGAATTCGCTTATATTTAGAGGAAATTTC
 6


 


BamHI_PS237 RV
TTTGGATCCATTACCTCCTAAATATTTTTAAAG
 7


 


Δcre1 FW
GAAAATACTGTTTACTATAAAACCTTATATTC
 8


 


Δcre1 RV
GAATATAAGGTTTTATAGTAAACAGTATTTTC
 9


 


Δcre2 FW
CTTTTTTTACGATATACCTTGTGCTATATG
10


 


Δcre2 RV
CATATAGCACAAGGTATATCGTAAAAAAAG
11









Example 2
Preparation of Expression Vectors
Each of the promoter sequence DNA fragments prepared in Example 1 was cleaved by restriction enzymes EcoRI and BamHI and ligated to pDL2 plasmid (Fukuchi et al., Microbiology, 146, 1573-1583, 2000) cleaved out by EcoRI and BamHI. Cells of E. coli were transformed by use of the thus-obtained plasmid through a routine method, and the plasmid was recovered from the obtained transformant cells (see FIG. 2). Sequencing of the promoter sequence inserted into the obtained plasmid was performed by means of a 3100DNA sequencer (Applied Biosystems). A plasmid containing each promoter sequence DNA fragment prepared in Example 1 was recovered as an expression vector.
Example 3
Production of Transformants
The expression vector prepared in Example 2 was cleaved by a restriction enzyme ScaI to form fragments, and incorporated into cells of Bacillus subtilis 168 strain through the competent method for transformation. The thus-obtained cells were cultured in an LB agar medium containing ampicillin (1 μg/mL). From the grown colonies, a transformant in which the promoter region and a LacZ gene were incorporated into an amyE gene were recovered.
Example 4
Determination of β-Galactosidase Activity
Each transformant obtained in Example 3 was cultured in media containing a variety of carbon sources. The transcription efficiency of each promoter was evaluated by β-galactosidase activity as an index. As a carbon source, there was used a glucose, a maltose, sorbitol, or a model saccharified solution (carbohydrate solution imitating a biomass-derived carbon source: 4% glucose, 0.5% xylose, and 0.5% cellobiose). Each transformant was cultured in an LB medium (1 mL) at 30° C. for 12 hours, and the thus-obtained culture solution was inoculated to an LB medium (1 mL) containing a carbon source at a final concentration of 0.5% in such an amount that the final cell concentration was adjusted to 1%. The cells were cultured at 37° C. for 8 hours, and OD(600) was measured. A culture solution having a OD(600) of 1.0 was centrifuged to recover the cells. The cells were re-suspended in 25 mM Tris-HCl (pH: 7.4) (0.5 mL) to completely remove the culture medium, and the cells were re-collected through centrifugation. The thus-collected cells were suspended in Z buffer (60 mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCl, 1 mM MgSO4/7H2O, and 2 mM DTT) (0.64 mL), and 2.5-mg/mL solution (0.16 mL) of lysozyme in Z buffer was added to the suspension. The mixture was incubated at 37° C. for 5 minutes to remove cell walls, whereby protoplast cells were prepared. To the protoplast cells, 10% Triton-X100 (8 μL) was added, and the thus-obtained admixture was employed as a lyzed cell solution. A control lyzed cell solution was prepared through the similar procedure from a wild-type strain into which lacZ was incorporated.
The β-galactosidase activity of each of the obtained lyzed cell solutions was determined through the ONPG method. Specifically, the lyzed cell solution was preliminarily incubated at 30° C. for three minutes, and a 4-mg/mL solution (0.2 mL) of ONPG in Z buffer was added thereto. From start of addition of the ONPG solution (0 sec), when the solution was yellowed by β-galactosidase activity, the reaction was terminated by adding 1M Na2CO3 (0.4 mL) to the solution. Insoluble fractions were removed through centrifugation, and OD(420) of each reaction mixture was measured, and β-galactosidase activity was calculated by the following equation:

β-Galactosidase activity (Unit)=1000×OD(420)/reaction time (min).

The results are shown in Table 2 and FIGS. 3A to 3D. In the case of culturing in a medium containing glucose, a transformant containing a Δcre1 promoter exhibited a β-galactosidase activity about 1.9 times that of the wild-type cell, and a transformant containing a Δcre2 promoter exhibited a β-galactosidase activity about 6.2 times that of the wild-type cell. A transformant containing a Δcre1.2 promoter (in which both Δcre1 and Δcre2 were deleted) exhibited a β-galactosidase activity about 7.8 times that of the wild-type cell, which is the highest transcription efficiency.
In the same experiment performed by use of maltose or sorbitol as a carbon source, the transcription efficiency of each promoter in the corresponding transformant was remarkably enhanced, as compared with the wild-type cell. In the experiment using the culture medium containing a carbon source imitating a carbohydrate solution of a biomass such as a plant (model saccharified solution), a remarkably enhanced transcription efficiency was also observed.










TABLE 2








β-Galactosidase


Carbon source
Strain
Transducing vector
activity (Unit)


















Glucose
Wild type
PS237-lacZ
135.2



Δcre1
PΔcre1-lacZ
256.8



Δcre2
PΔcre2-lacZ
840.5



Δcre1.2
PΔcre1.2-lacZ
1050.0


Maltose
Wild type
PS237-lacZ
354.0



Δcre1
PΔcre1-lacZ
462.9



Δcre2
PΔcre2-lacZ
850.0



Δcre1.2
PΔcre1.2-lacZ
723.8


Sorbitol
Wild type
PS237-lacZ
140.0



Δcre1
PΔcre1-lacZ
308.7



Δcre2
PΔcre2-lacZ
624.3



Δcre1.2
PΔcre1.2-lacZ
846.8


Model
Wild type
PS237-lacZ
290.7


saccharified
Δcre1
PΔcre1-lacZ
706.4


solution
Δcre2
PΔcre2-lacZ
1359.0



Δcre1.2
PΔcre1.2-lacZ
1604.8

## Claims

1. A modified promoter, comprising a nucleotide sequence of a promoter derived from a bacterium belonging to the genus Bacillus in which at least one nucleotide sequence selected from the group consisting of a nucleotide sequence represented by SEQ ID NO: 1 and a nucleotide sequence represented by SEQ ID NO: 2 has been deleted,
wherein the promoter derived from a bacterium belonging to the genus Bacillus is: a promoter of an alkaline cellulase gene from Bacillus sp. KSM-64 strain (FERM BP-2886); a promoter of an alkaline cellulase gene from Bacillus sp. KSM-S237 strain (FERM BP-7875); or a promoter containing a nucleotide sequence that has a sequence identity of 90% or more with respect to the nucleotide sequence of any one of said promoters and that has a nucleotide sequence represented by SEQ ID NO:1 or 2, or nucleotide sequences represented by SEQ ID NOs: 1 and 2.

2. A method for producing a modified promoter, the method comprising deleting, from a promoter derived from a bacterium belonging to the genus Bacillus, at least one nucleotide sequence selected from the group consisting of a nucleotide sequence represented by SEQ ID NO: 1 and a nucleotide sequence represented by SEQ ID NO: 2,
wherein the promoter derived from a bacterium belonging to the genus Bacillus is: a promoter of an alkaline cellulase gene from Bacillus sp. KSM-64 strain (FERN BP-2886); a promoter of an alkaline cellulase gene from Bacillus sp. KSM-S237 strain (FERN BP-7875); or a promoter containing a nucleotide sequence that has a sequence identity of 90% or more with respect to the nucleotide sequence of any one of said promoters and that has a nucleotide sequence represented by SEQ ID NO:1 or 2, or nucleotide sequences represented by SEQ ID NOs: 1 and 2.

3. An expression vector comprising a modified promoter according to claim 1.

4. The expression vector according to claim 3, wherein the modified promoter is operably linked to the upstream of a gene encoding a gene product of interest.

5. A transformant comprising a modified promoter according to claim 1, wherein the modified promoter is operably linked to the upstream of a gene encoding a gene product of interest.

6. A method for producing a gene product of interest, wherein the method comprises employing a transformant according to claim 5 to produce said gene product of interest.

7. An expression vector comprising a modified promoter produced by the method according to claim 2.

8. The expression vector according to claim 7, wherein the modified promoter is operably linked to the upstream of a gene encoding a gene product of interest.

9. A transformant comprising a modified promoter produced by the method according to claim 2, wherein the modified promoter is operably linked to the upstream of a gene encoding a gene product of interest.

10. A method for producing a gene product of interest, wherein the method comprises employing the transformant according to claim 9 to produce said gene product of interest.