JP5901964B2 - Recombinant microorganism - Google Patents

Description

  The present invention relates to a recombinant microorganism.

Cellulase is a general term for enzymes that hydrolyze the β-1,4-glucoside bond of cellulose, and is a typical hydrolase along with amylase, protease, and lipase. Cellulose, which is a substrate for cellulase, is biomass that is produced by more than 100 billion tons per year as the main component of plant cell walls, and is effectively used for clothing, paper, building materials, and the like. In addition, since cellulose is a macromolecule in which glucose is linearly bonded, it can be converted into a fuel substance or a higher value-added metabolite by decomposition. Therefore, research on efficient degradation of cellulose and effective utilization of the degradation products has been widely conducted. When cellulose is converted into a high value-added substance, the cellulose raw material is usually converted into highly reactive cellulose by pretreatment and then decomposed into low molecular sugars such as glucose. The obtained low molecular weight sugar is converted into a high value-added substance through a chemical reaction such as fermentation by a microorganism. In recent years, in the cellulose decomposition step of this series of processes, enzymatic decomposition by cellulase is generally used as a more economical method.
However, at present, the rate of cellulose degradation by cellulase is not sufficient, and in order to achieve the necessary degradation rate, a large amount of enzyme must be used or a mixture of many types of enzymes must be used. This inevitably results in significant costs for the enzyme. Accordingly, there is a need for a cellulase gene screening method that efficiently selects cellulase genes that encode highly active cellulases that efficiently produce cellulose degradation products.

As a conventional screening method for cellulase genes, a microbial source such as soil is cultured on a solid medium supplemented with carboxymethylcellulose (CMC), microcrystalline cellulose or the like, and halo accompanying the degradation of CMC using a pigment such as Congo Red. Examples include a method of isolating a cellulase-producing bacterium using the formation and formation of clear halo accompanying microcrystalline cellulose decomposition as an index, and obtaining a cellulase gene from the microorganism. In recent years, it is possible to obtain a cellulase gene from a transformant that forms a halo in the same manner as above by degrading DNA with an appropriate restriction enzyme or obtaining cDNA from RNA and performing transformation using these DNAs. It has become. However, in these methods, it is necessary to find a microorganism having a small number of cellulase genes from microorganisms not having an infinite number of cellulase genes, and a great deal of trial and error is required.
Therefore, as a more efficient screening method for cellulase, a screening method using a microorganism having a glucomannan utilization (utilization) operon is conceivable.
On the genome of Bacillus subtilis etc., there exists a glucomannan utilization operon composed of a gene encoding a protein involved in utilization of glucomannan. In Bacillus subtilis, the glucomannan utilization operon consists of eight ORFs (open reading frames).
The mechanism of action of the glucomannan utilization operon of Bacillus subtilis is described in detail in Non-Patent Document 1, etc., and its outline will be described based on FIG. The glucomannan utilization operon of Bacillus subtilis is composed of gmuB gene, gmuA gene, gmuC gene, gmuD gene, gmuR gene, gmuE gene, gmuF gene, and gmuG gene in this order. Of these, β-1,4-mannanase encoded by the gmuG gene and secreted extracellularly (hereinafter, β-1,4-mannanase is also referred to as “GmuG” in this specification) is a part of glucomannan. It is decomposed to produce oligoglucomannan (indicated as “Glucomannan ^* ” in FIG. 2). This oligoglucomannan is taken into cells by PTS (phosphotransferase system) composed of GmuB, GmuA, and GmuC encoded by the gmuB gene, gmuA gene, and gmuC gene, respectively. The glucomannan degradation products such as cellobiose and mannobiose (indicated as “Glucomannan ^**- P” in FIG. 2) incorporated into the cell function as an inducer of the glucomannan-utilizing operon. Induces operon expression. On the other hand, the expression of the glucomannan-utilizing operon is controlled by an internal repressor encoded by the gmuR gene (hereinafter also referred to as “GmuR” in this specification), a carbon catabolite repressing factor (CcpA), and the like.

  When such a microorganism is used to efficiently screen the cellulase gene using the expression of the glucomannan-utilizing operon, a selection marker such as a drug resistance gene or an auxotrophic gene is introduced into the host microorganism (parent microorganism). It is necessary to be able to select a transformant based on the expression of a selection marker accompanying the expression of a glucomannan-utilizing operon. However, to date, it has not been elucidated how expression of a selectable marker using a glucomannan-utilized operon is induced in a microorganism having a glucomannan-utilized operon.

Sadaie Y. et al., FEMS Microbiol. Lett., 2008, vol. 279, no. 1, p. 103-109

The present invention utilizes the fact that a glucomannan degradation product such as cellobiose controls the expression of a glucomannan-utilizing operon as an inducer in a microorganism having a glucomannan-utilizing operon, and the target gene inserted into the genome of the host microorganism It is an object to provide a recombinant microorganism whose expression is controlled by cellobiose.
Another object of the present invention is to provide a method for producing a recombinant microorganism for efficiently producing the recombinant microorganism.
Furthermore, an object of the present invention is to provide a method for screening a cellulase gene using the recombinant microorganism.

In view of the above-mentioned problems, the present inventors, in a microorganism having a glucomannan-utilizing operon on the genome, expresses a target gene such as a selection marker inserted into the genome using the operon. We intensively studied the mechanism of expression. As a result, by introducing the target gene downstream of the gmuG gene or the gene corresponding to the gmuG gene among the eight ORFs constituting the glucomannan utilization operon, the expression of the target gene is caused by a glucomannan degradation product such as cellobiose. I found out that it becomes controllable. Furthermore, since the expression of the target gene is controlled by cellobiose, it has been found that such a recombinant microorganism can be suitably used for screening a gene encoding cellulase that produces cellobiose using cellulose as a substrate. The present invention has been completed based on these findings.

The present invention relates to a glucomannan utilization operon comprising a gmuB gene, a gmuA gene, a gmuC gene, a gmuD gene, a gmuR gene, a gmuE gene, a gmuF gene and a gmuG gene of Bacillus subtilis or a gene corresponding to the gene. The present invention relates to a recombinant microorganism in which a target gene is operably linked downstream of the gmuG gene or a gene corresponding to the gmuG gene in a microorganism having an operon functioning as an operon on the genome.

The present invention also relates to a glucomannan-utilizing operon comprising a gmuB gene, a gmuA gene, a gmuC gene, a gmuD gene, a gmuR gene, a gmuE gene, a gmuF gene and a gmuG gene of Bacillus subtilis or a gene corresponding to each of the above genes. The present invention relates to a method for producing the recombinant microorganism, comprising a step of operably linking a target gene downstream of the gmuG gene or a gene corresponding to the gmuG gene in a microorganism having an operon functioning as a mannan-utilizing operon on the genome.

  Furthermore, the present invention provides a cellulase gene screening method using the above-mentioned recombinant microorganism, wherein the cellulase gene is selected by selecting the gene encoding cellulase using the expression of the target gene introduced into the recombinant microorganism as an index. Regarding the method.

In the recombinant microorganism of the present invention, since the target gene is operably linked to the glucomannan-utilizing operon, the expression of the target gene is controlled by a glucomannan degradation product such as cellobiose which is an inducer of the glucomannan-utilizing operon. Is done.
Moreover, the method for producing a recombinant microorganism of the present invention can produce the recombinant microorganism efficiently.
Further, the cellulase gene screening method using the recombinant microorganism of the present invention is suitable for cellulase gene screening.

It is a figure which shows typically the target gene insertion site | part in the recombinant microorganism of this invention. It is a figure which shows typically the action mechanism of the glucomannan utilization operon of Bacillus subtilis. It is a figure which shows the base sequence of the chloramphenicol resistance gene derived from Staphylococcus aureus . It is a schematic diagram which shows the introduction | transduction procedure of the target gene by the double crossing method using a SOE-PCR fragment. It is a figure which shows typically the insertion position of the chloramphenicol resistance gene ( cat gene) on the genome of Bacillus subtilis son1 strain, Bacillus subtilis son2 strain, Bacillus subtilis son3 strain, Bacillus subtilis son4 strain, and Bacillus subtilis son5 strain. FIG. 5 (a) is a diagram showing Bacillus subtilis son5 strain cultured on cellobiose and chloramphenicol-containing LB agar medium, and FIG. 5 (b) is a Bacillus subtilis son5 strain cultured on chloramphenicol-containing LB agar medium. FIG. It is a figure which shows the growth degree in the presence of cellobiose of Bacillus subtilis son5 strain. It is a figure which shows the growth degree of Bacillus subtilis son5 strain | stump | stock in the presence of cellobiose, cellotriose, cellotetraose, cellopentaose or cellohexaose. It is a figure which shows the screening result of the cellulase gene using Bacillus subtilis son5 strain. It is a figure which shows typically the action mechanism of the glucomannan utilization operon of Bacillus subtilis son5 strain | stump | stock in the screening of the cellulase gene performed in the Example. It is a schematic diagram which shows the deletion procedure of the ccpA gene by the double crossing method using a SOE-PCR fragment. It is a figure which shows the growth degree in the presence of cellobiose and / or glucose of Bacillus subtilis son5A strain.

  As used herein, “upstream of a gene” refers to a region that is not located from the replication start point but continues to the 5 ′ end of the start codon of the gene of interest. Further, “downstream of the gene” refers to a region continuing to the 3 ′ end side of the stop codon of the target gene.

  The name and arrangement of each gene of Bacillus subtilis described in the present specification, and the name and function of the protein encoded by each gene are described in FEMS Microbiol. Lett., 2008, vol. 279, no. 1, p. 103-109, genome data are BSORF; Bacillus subtilis Genome Database (http://bacillus.genome.jp/bsorf.html) and NCBI Database (http://www.ncbi.nlm.nih.gov/sites / entrez? db = nucleotide), but in the present invention, it is described based on the genome data of Bacillus subtilis of NCBI Database.

  In the present invention, the identity of amino acid sequences and base sequences is calculated by the Lipman-Pearson method (Science, 227, 1435, (1985)). Specifically, it is calculated by performing analysis with the parameter Unit size to compare (ktup) set to 2 using the homology analysis (Search homology) program of genetic information processing software Genetyx-Win (software development).

In the present invention, the glucomannan utilization operon of Bacillus subtilis consists of gmuB gene, gmuA gene, gmuC gene, gmuD gene, gmuR gene, gmuE gene, gmuF gene and gmuG gene, or genes corresponding to the respective genes, It is a mannanase-like protein that encodes an enzyme related to the degradation and utilization of glucomannan. Hereinafter, these genes will be described in detail.

In the present invention, (also referred ydhM gene) GmuB gene, (also referred ydhO gene) GmuA gene (also referred ydhN gene) and gmuC gene is a gene encoding GmuB, the GmuA and GmuC respectively . GmuB, GmuA and GmuC are proteins constituting PTS, and a glucomannan degradation product existing outside the cell of Bacillus subtilis is taken into the cell via this PTS. The glucomannan degradation product such as cellobiose and mannobiose thus taken up into the cell functions as an inducer of the glucomannan-utilizing operon and induces the expression of the glucomannan-utilizing operon.
In the present specification, “a gene corresponding to the gmuB gene”, “a gene corresponding to the gmuA gene” and “a gene corresponding to the gmuC gene” are genes ( gmuB or ydhM ) encoding Bacillus subtilis GmuB described in the NCBI Database. , genes encoding Bacillus subtilis GmuA (gmuA or YdhN) or genes encoding Bacillus subtilis GmuC (gmuC or YdhO) to a nucleotide sequence of 90% or more, more preferably 95% or more, particularly preferably 98% or more The gene which codes the protein which has the base sequence which has the identity of these, and comprises PTS similarly to GmuB, GmuA, or GmuC. Alternatively, GmuB gene, a gene having gmuA gene or gmuC gene homology (homology) or similarity to (Similarity) also, "gene corresponding to GmuB gene" corresponds to and "gmuC gene" gene corresponding to gmuA gene " Included in "gene".

In the present invention, the gmuD gene (also referred to as ydhP gene) is a gene encoding a protein GmuD similar to phospho-β-glucosidase. The gmuE gene (also referred to as ydhR gene) is a gene encoding a protein GmuE having homology with fructokinase. The gmuF gene (also referred to as ydhS gene) is a gene encoding a protein GmuF having homology with mannose-6-phosphate isomerase. GmuD, GmuE, and GmuF all metabolize the glucomannan degradation product taken into the cell by the PTS.
In the present specification, “a gene corresponding to the gmuD gene”, “a gene corresponding to the gmuE gene” and “a gene corresponding to the gmuF gene” are genes ( gmuD or ydhP ) encoding Bacillus subtilis GmuD described in the NCBI Database. , genes encoding Bacillus subtilis GmuE (gmuE or YdhR) or genes encoding Bacillus subtilis GmuF (gmuF or YdhS) to a nucleotide sequence of 90% or more, more preferably 95% or more, particularly preferably 98% or more And a gene that encodes a protein that metabolizes a glucomannan degradation product incorporated into cells in the same manner as GmuD, GmuE, or GmuF. Alternatively, included in gmuD genes, genes with gmuE gene or gmuF gene homology or similarity also, "gene corresponding to gmuD gene", "gene corresponds to gmuE gene" and "gene corresponding to gmuF gene" .

In the present invention, the gmuR gene (also referred to as ydhQ gene) is a gene encoding a DNA-binding protein GmuR belonging to the GntR family, which is a kind of transcription regulatory factor. GmuR suppresses the expression of the glucomannan utilization operon.
By "gene corresponding to GmuR gene" as used herein, a gene encoding a Bacillus subtilis GmuR according to NCBI Database (gmuR or YdhQ) to a nucleotide sequence of 90% or more, more preferably 95% or more, Particularly preferably, it refers to a gene encoding a protein having a base sequence having an identity of 98% or more and suppressing the expression of a glucomannan-utilizing operon in the same manner as GmuR. Alternatively, a gene having gmuR gene homology or similarity is also included in the "gene corresponding to gmuR gene".

In the present invention, the gmuG gene (also referred to as ydhT gene) is a gene encoding β-1,4-mannanase GmuG having a signal sequence on the N-terminal side. When GmuG encoded by the gmuG gene is secreted extracellularly, glucomannan is decomposed into cellobiose, mannobiose and the like.
By "gene corresponding to GmuG gene" as used herein, a gene encoding a Bacillus subtilis GmuG according to NCBI Database (gmuG or YdhT) to a nucleotide sequence of 90% or more, more preferably 95% or more, particularly preferably refers to a gene encoding a 98 percent or more, of having a nucleotide sequence identity with, and GmuG as well as protein degrades when secreted extracellularly glucomannan cellobiose and mannobiose like. Alternatively, a gene having gmuG gene homology or similarity is also included in the "gene corresponding to gmuG gene".

Regarding the glucomannan-utilizing operon of the recombinant microorganism of the present invention and the base sequence of each gene constituting the operon, the base sequence of each gene constituting the glucomannan-utilizing operon of Bacillus subtilis 168 strain shown in SEQ ID NO: 1 Based on However, the present invention is not limited to this.
969-1280 th region of the nucleotide sequence the nucleotide sequence is shown in SEQ ID NO: 1 of gmuB gene, 1280-1612 th region of the nucleotide sequence the nucleotide sequence is shown in SEQ ID NO: 1 of gmuA gene, nucleotide sequence of gmuC gene SEQ ID NO: The 1631 to 2959th region of the base sequence shown in Fig. 1, the base sequence of the gmuD gene is the 2977 to 4374th region of the base sequence shown in SEQ ID NO: 1, and the base sequence of the gmuR gene is 4517 to the base sequence shown in SEQ ID NO: 1. The 5230th region, the gmuE gene base sequence is the 5259-6158th region of the base sequence shown in SEQ ID NO: 1, the gmuF gene base sequence is the 6155-7102th region of the base sequence shown in SEQ ID NO: 1, the gmuG gene The base sequence corresponds to the 7121st to 8209th region of the base sequence shown in SEQ ID NO: 1.

a gene corresponding to the gmuB gene, a gene corresponding to the gmuA gene, a gene corresponding to the gmuC gene, a gene corresponding to the gmuD gene, a gene corresponding to the gmuR gene, a gene corresponding to the gmuE gene, a gene corresponding to the gmuF gene, and gmuG Examples of the gene corresponding to the gene include homologous genes described in Table 1 below. Table 1 lists representative strains of each bacterial species and homologous genes corresponding to the respective genes. The same homologous genes as those in Table 1 or different homologous genes possessed by different strains of the same species as those in Table 1 are also included in the genes corresponding to the respective genes in the present invention.

Regarding the gmuB gene, gmuA gene, gmuC gene, gmuD gene, gmuR gene, gmuE gene, gmuF gene and gmuG gene in the glucomannan utilization operon of the recombinant microorganism of the present invention, or the sequence of the gene corresponding to each gene There are no particular restrictions as long as the operons constituted by these genes function as glucomannan-utilizing operons. For example, GmuB gene or a homologous gene thereof, GmuA gene or a homologous gene thereof, GmuC gene or a homologous gene thereof, GmuD gene or a homologous gene thereof, GmuR gene or a homologous gene thereof, GmuE gene or a homologous gene thereof, GmuF gene or a homologous gene thereof , And the gmuG gene or homologous genes thereof are preferably linked in this order on the genome to constitute a glucomannan-utilizing operon, gmuB gene, gmuA gene, gmuC gene, gmuD gene, gmuR gene, gmuE gene, gmuF gene More preferably, the gmuG gene is linked in this order on the genome to constitute a glucomannan-utilizing operon. Or as long as the gene corresponding to each said gene functions as a glucomannan utilization operon, each gene may exist in the distant position on a genome, respectively. For example, in Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) DSM7 strain, GmuR gene is present in a position away from the other genes constituting the glucomannan assimilation operon, there is glucomannan hydrolysates as the inducer In a state where the glucomannan is not used, expression of the glucomannan utilization operon is suppressed. Therefore, as long as these genes function as a glucomannan-utilizing operon, even in the case of microorganisms in which genes corresponding to the respective genes are present at distant positions on the genome, such as the Bacillus amyloliquefaciens DSM7 strain, , To be included in the host microorganism used in the present invention.

In the recombinant microorganism of the present invention, the target gene is operably linked downstream of the gmuG gene or a gene corresponding to the gmuG gene. Control of the inducer of the glucomannan-utilizing operon, which is an expression-inducible promoter, by operably linking the target gene downstream of the gmuG gene or a gene corresponding to the gmuG gene and upstream of the terminator of the operon The target gene can be expressed below. Here, the expression inducible promoter means a promoter having a function of inducing gene expression only under specific conditions. In the present invention, “specific conditions” means that a degradation product of glucomannan such as cellobiose is present in the expression system. Further, “operably linking the target gene” means that the target gene is linked so that transcription of the gene can be induced with the expression of the glucomannan-utilizing operon.

The target gene to be operably linked downstream of the gmuG gene or the gene corresponding to the gmuG gene is not particularly limited, and is a contraction protein, transport protein, signal protein, biological defense protein, receptor protein, structural protein, gene regulatory protein, Examples include genes that encode proteins such as enzymes and storage proteins, drug resistance genes, auxotrophic genes, and non-coding RNAs such as functional RNAs. When the microorganism of the present invention is used for screening a foreign gene such as a cellulase gene, it serves as a selection marker for a drug resistance gene, an auxotrophic gene, a reporter gene, etc. from the viewpoint of selecting a foreign gene using the expression of the target gene as an index. The gene obtained is preferred.
The drug resistance gene is not particularly limited, and is a chloramphenicol resistance gene ( cat gene), tetracycline resistance gene ( tet gene), neomycin resistance gene ( nem gene), erythromycin resistance gene ( erm gene), spectinomycin resistance Gene ( spm gene), kanamycin resistance gene and the like.
There is no restriction | limiting in particular as an auxotrophic gene, Nucleic acid requirement genes, such as uracil and adenine, Amino acid requirement genes, such as methionine and tryptophan, etc. are mentioned.
Other target genes include reporter genes such as green fluorescent protein (GFP), β-glucuronidase (GUS), and β-galactosidase (β-Gal).
These target genes preferably do not have a promoter sequence.

As an example of the target gene used in the present invention, a chloramphenicol resistance gene ( cat gene) will be described.
The nucleotide sequence (including promoter sequence) of the chloramphenicol resistance gene derived from Staphylococcus aureus is shown in SEQ ID NO: 2 and FIG. 3, and the amino acid sequence encoded by the chloramphenicol resistance gene comprising the nucleotide sequence is SEQ ID NO: 3. Shown in FIG. 3 also shows the promoter sequence and Shine-Dalgarno sequence (also referred to as SD sequence in this specification) in the chloramphenicol resistance gene derived from Staphylococcus aureus.
Eubacterial promoters consist of two short sequences found about 10 bases upstream and about 35 bases upstream from the start of transcription. A sequence about 10 bases upstream from the transcription start point is also called “-10 region”, and a common sequence of 6 bases “5′-TATAAT-3 ′” is found in the -10 region of many genes. . This -10 region is essential for the initiation of transcription in prokaryotes. The sequence about 35 bases upstream from the transcription start point is also called “-35 region”, and is a 6-base consensus sequence of 5′-TTGACA-3 ′. This -35 region has the function of increasing the transfer strength. If the -10 region and -35 region are not included upstream of the transcription start site of the target gene, transcription by itself is impossible. The SD sequence is a ribosome binding site and is an essential sequence for protein translation.
In the present invention, the target gene linked downstream of the gmuG gene or the gene corresponding to the gmuG gene preferably does not have a promoter sequence (does not have the -10 region and -35 region). That is, a sequence upstream of the translation start point (or SD sequence) of the target gene is not included, and the transcription start point (or SD sequence) of the target gene and a region downstream thereof are downstream of the gene corresponding to the gmuG gene or gmuG gene. And preferably operatively connected upstream of the operon terminator. With such a configuration, the target gene can be expressed only under the control of the inducer of the glucomannan-utilizing operon.

  The gmuG gene or a gene corresponding to the gmuG gene and the target gene (marker gene) can be linked by a method usually used in the art. For example, as shown in FIG. 4, a SOE-PCR fragment for introducing a target gene is prepared by SOE (splicing by overlap extension) -PCR method (see Gene, 1989, vol. 77 (1), p. 61-68). The recombinant microorganism of the present invention can be produced by introducing the SOE-PCR fragment into the host microorganism cell and directly integrating the marker gene into the genome of the host microorganism by homologous recombination by the double crossover method.

Hereinafter, the method for producing the recombinant microorganism of the present invention will be described with specific examples. However, the present invention is not limited to this.
First, an upstream fragment (hereinafter also referred to as a fragment (A)) and a downstream fragment (hereinafter also referred to as a fragment (B)) of a target gene insertion site using a genomic DNA of a host microorganism into which the target gene is introduced or a cDNA thereof as a template, A target gene fragment (hereinafter also referred to as fragment (C)) is prepared by PCR using a plasmid or the like in which the base sequence of the target gene is incorporated as a template. PCR is performed using a primer designed to add about 10 to 30 base pairs on the 5 ′ end side of the target gene to the 3 ′ end of the fragment (A). Further, PCR is performed using a primer designed to add about 10 to 30 base pairs on the 3 ′ end side of the target gene to the 5 ′ end of the fragment (B). Here, the size of the fragment (A) and the fragment (B) is not particularly limited, but is preferably 0.1 kb to 3 kb, more preferably 0.5 kb to 3 kb, and even more preferably 0.5 kb to 2 kb.
Purpose of addition to the 3 ′ end of the fragment (A) and the 5 ′ end of the fragment (B) by performing PCR using the three types of PCR fragments of the fragment (A), the fragment (B) and the fragment (C) as a template An annealing occurs between the partial sequence of the gene and the fragment (C), and the SOE-PCR fragment for introducing the target gene (fragment (D) with the fragment (C) inserted between the fragment (A) and the fragment (B) )) Is obtained.
When the fragment (D) thus obtained is introduced into a host microorganism cell by a conventional method and the fragment (D) and the genome of the host microorganism are brought close to each other under conditions where homologous recombination occurs, upstream of the target gene insertion site. In the homologous region downstream, double crossover homologous recombination occurs in the cell, and the target gene is inserted into the target gene insertion site on the genome of the host microorganism.

Here, the conditions under which homologous recombination occurs means a state in which the function of an enzyme involved in the recombination reaction inherently possessed by the host microorganism has not been lost. The recombination method between the fragment (D) and the genome of the host microorganism is not particularly limited, and a general competent cell transformation method (for example, J. Bacterial., 1967, vol. 93, p. 1925, etc.) Protoplast transformation method (see, for example, Mol. Gen. Genet., 1979, vol. 168, p. 111, etc.), electroporation method (see, for example, FEMS Microbiol. Lett., 1990, vol. 55, p. 135), LP transformation methods (for example, Archives of Microbiology, 1987, vol. 146, p.353-357; Bioscience, Biotechnology, and Biochemistry, 2001, vol. 65, No. 4, p. 823-). 829), calcium chloride method, calcium phosphate method, lipofection method, Agrobacterium method, particle gun method, PEG method, spheroplast method, lithium acetate method and the like. For example, when the host microorganism is Bacillus subtilis, transformation can be performed as follows according to the competent cell transformation method.
First, the strain of the host microorganism was changed to SPI medium (0.20% (w / v) ammonium sulfate, 1.40% (w / v) dipotassium hydrogen phosphate, 0.60% (w / v) dihydrogen phosphate. Potassium, 0.10% (w / v) trisodium citrate dihydrate, 0.50% (w / v) glucose, 0.02% (w / v) casamino acid (Difco), 5 mM sulfuric acid (Magnesium, 0.25 μM manganese chloride, 50 μg / mL tryptophan) at 37 ° C., for example, with shaking until the degree of growth (OD ₆₀₀ ) is about 1. After shaking culture, a portion of the culture broth was added to 9 times the amount of SPII medium (0.20% (w / v) ammonium sulfate, 1.40% (w / v) dipotassium hydrogen phosphate, 0.60% (w / v). v) Potassium dihydrogen phosphate, 0.10% (w / v) trisodium citrate dihydrate, 0.50% (w / v) glucose, 0.01% (w / v) casamino acid (Difco (Commercially available), 5 mM magnesium sulfate, 0.40 μM manganese chloride, 5 μg / mL tryptophan), and further cultured by shaking until, for example, the degree of growth (OD ₆₀₀ ) is about 0.4. To prepare. Next, the DNA fragment is added to a competent cell of the host microorganism strain and cultured for 1 to 2 hours. The amount of the DNA fragment to be added is not particularly limited, but is preferably 100 pg to 100 μg, more preferably 1 ng to 10 μg. Moreover, although there is no restriction | limiting in particular in the usage-amount of a competent cell, 10 < ⁵ > -10 < ⁹ > CFU is preferable and 10 < ⁶ > -10 < ⁸ > CFU is more preferable.

Confirmation of insertion of the target gene can be performed by a usual method. For example, PCR may be performed using genomic DNA extracted from the transformant as a template to confirm that the target gene has been inserted.
In addition, when the target gene is a selection marker such as a drug resistance gene or an auxotrophic gene, a transformant that has undergone homologous recombination can be isolated using the expression of the selection marker as an index. For example, when the target gene is a drug resistance gene, the expression of the drug resistance gene is controlled by the glucomannan-utilized operon. Therefore, drug resistance is the first in the presence of cellobiose or mannobiose that functions as an inducer of the glucomannan-utilized operon. What is necessary is just to select the microorganism which acquires.

As a host microorganism for producing the recombinant microorganism of the present invention, a glucomannan-utilizing operon consisting of the Bacillus subtilis gmuB gene, gmuA gene, gmuC gene, gmuD gene, gmuR gene, gmuE gene, gmuF gene and gmuG gene or There is no particular limitation as long as it has an operon consisting of genes corresponding to the above genes and functioning as a glucomannan-utilizing operon on the genome, and these may be wild type or mutant strains. Specifically, Bacillus licheniformis (Bacillus licheniformis), Bacillus megaterium (Bacillus megaterium), Bacillus Anshirashisu (Bacillus anthracis), Bacillus halodurans (Bacillus halodurans), Bacillus such as Bacillus subtilis (Bacillus) bacterium, Clostridium (Clostridium) bacteria, Corynebacterium (Corynebacterium) bacteria, Pseudomonas (Pseudomonas) bacteria, E. coli (Escherichia coli), include yeasts such as, among others Bacillus bacterium is preferable. In particular, since the whole genome information has been clarified, genetic engineering and genomic engineering techniques have been established, it has been recognized as safe and excellent as a host microorganism, and it has the ability to secrete and produce proteins outside the bacterial cell. Bacteria are more preferred. Furthermore, as the host microorganism to be selected, either a wild type strain or a mutant strain in which a predetermined mutation is introduced into the wild type strain can be used.

In the recombinant microorganism of the present invention, when the expression of the glucomannan-utilizing operon is suppressed by a carbon catabolite repressing factor (CcpA), the ccpA gene encoding CcpA or a gene corresponding thereto may be deleted or inactivated. preferable. By deleting or inactivating the ccpA gene or a gene corresponding thereto, the expression of the target gene can be confirmed by cellobiose even in the presence of a sugar that is easily metabolized such as glucose. The ccpA gene is not limited as long as it is a gene encoding a carbon catabolite repressor, but is preferably the Bacillus subtilis ccpA gene.
The base sequence of the Bacillus subtilis ccpA gene will be described based on SEQ ID NO: 40. The base sequence shown in SEQ ID NO: 40 shows the base sequence of the Bacillus subtilis ccpA gene (the 1087-2091st region of the base sequence shown in SEQ ID NO: 40), and the upstream and downstream base sequences. The gene corresponding to ccpA gene, the nucleotide sequence to be 90% or more, more preferably 95% or more, particularly preferably has a nucleotide sequence having identity of 98% or more, and encoding CcpA A gene.

There is no restriction | limiting in particular as a deletion or inactivation method of a ccpA gene or a gene corresponding to it, It can carry out by a normal method. Examples thereof include a double crossover method using a linear DNA fragment prepared by SOE-PCR, a single crossover method using a circular plasmid, and a mutation method using UV irradiation or a mutagen.
Specifically, by the method using the double crossover method using the DNA fragment for deletion prepared by the SOE-PCR method (see Gene, 1989, vol. 77 (1), p. 61-68), ccpA A gene can be deleted from the genome. The deletion DNA fragment used in the present method comprises an about 0.1 to 3 kb fragment adjacent to the upstream of the ccpA gene (referred to as upstream fragment), a drug resistance marker gene fragment, and an about 0.1 to 3 kb adjacent to the downstream of the ccpA gene. A DNA fragment in which three fragments (referred to as downstream fragments) are linked.

First, an upstream fragment and a downstream fragment of a deletion target gene ( ccpA gene) and three fragments of a drug resistance marker gene fragment are prepared by the first PCR. At this time, a primer to which a sequence of 10 to 30 base pairs at the end of the DNA fragment to be bound is added is designed. For example, a sequence corresponding to 10 to 30 bases upstream of the drug resistance marker gene fragment is added to the 5 ′ end of the primer located at the downstream end (annealing) of the upstream fragment, and located at the upstream end of the downstream fragment. A sequence corresponding to 10 to 30 bases downstream of the drug resistance marker gene fragment is added to the 5 ′ end of the (annealing) primer. When the upstream fragment and the downstream fragment are amplified using the primer set designed in this way, a region corresponding to the upstream side of the drug resistance marker gene fragment is added to the downstream side of the amplified upstream fragment. A region corresponding to the downstream side of the drug resistance marker gene fragment is added to the upstream side of the downstream fragment.
Next, the first prepared upstream fragment, drug resistance marker gene fragment and downstream fragment are mixed to form a template, which is located upstream of the upstream fragment (annealed) and located downstream of the downstream fragment (annealed). SOE-PCR is performed using a pair of primers. By this second PCR, a deletion DNA fragment in which an upstream fragment, a drug resistance marker gene fragment and a downstream fragment are combined in this order can be amplified.

  Next, transformation of the host bacterium with the deletion DNA fragment is performed by a competent cell transformation method (see, for example, J. Bacterial., 1967, vol. 93, p. 1925, etc.) A transformant in which the drug resistance marker gene is replaced with the ccpA gene on the host fungal genome is obtained by double crossover homologous recombination between homologous regions on the genome. For selection of transformants, drug resistance by a drug resistance marker gene inserted into the deletion DNA fragment may be used as an index. Genomic DNA may be extracted from these strains and the deletion of the target gene may be confirmed by PCR or the like.

  Since the target gene is operably linked to the glucomannan-utilizing operon in the recombinant microorganism of the present invention, the expression of the target gene is controlled by a glucomannan degradation product such as cellobiose which is an inducer of the glucomannan-utilizing operon. The Therefore, by using the recombinant microorganism of the present invention, a cellulase that produces cellobiose by hydrolyzing the β-1,4-glucoside bond of cellulose using the expression of the target gene introduced into the recombinant microorganism as an index. Screening of the gene to be encoded becomes possible.

  Hereinafter, embodiments of the cellulase gene screening method of the present invention will be described. However, the present invention is not limited to this.

When the gene to be screened is transformed and introduced into the recombinant microorganism of the present invention, the gene introduced into the recombinant microorganism of the present invention is expressed in the presence of cellulose, and the gene is a cellulase gene. Cellobiose is produced using cellulose as a substrate. When this cellobiose is taken into the cells of the recombinant microorganism, it functions as an inducer of the glucomannan utilization operon of the recombinant microorganism, and the target gene linked to the downstream of the gene corresponding to the gmuG gene or gmuG gene is expressed. Therefore, the cellulase gene can be screened by using the expressed target gene as an index. For example, when a drug resistance gene is used as the target gene of the recombinant microorganism of the present invention, the expression of a test gene introduced into the recombinant microorganism is induced, the recombinant microorganism is cultured in the presence of cellulose, When drug resistance is acquired, a test gene introduced into the recombinant microorganism may be selected as a cellulase gene.
When screening a cellulase gene from a metagenome, the DNA can be treated with an appropriate restriction enzyme to provide a gene for screening. When RNA is used, it can be used as a gene for screening by reverse transcription of RNA and synthesis of cDNA.

In the screening of the cellulase gene of the present invention, the medium for culturing the recombinant microorganism is not particularly limited as long as cellulose capable of hydrolyzing cellulase is present, and glycerol, fructose, maltose, xylose, arabinose, various organic acids For the cultivation of host microorganisms of the recombinant microorganisms, including carbon sources such as various amino acids, polypeptone, tryptone, nitrogen sources such as ammonium sulfate and urea, inorganic salts such as sodium salts and other necessary nutrients, trace metal salts, etc. Any medium that is normally used may be used. In addition, a medium using a sugar as a carbon source such as glucose can be used by deleting or inactivating the ccpA gene of the recombinant microorganism of the present invention.
Further, the cellulose used in the present invention is not particularly limited, and milled cellulose, CMC, Avicel PH101 (trade name, manufactured by Fluka), Cellulose Powder (manufactured by Fluka), pulp, phosphoric acid expanded cellulose, KC floc Etc. Of these, milled cellulose and CMC are preferred.

In the recombinant microorganism of the present invention, the target gene is operably linked downstream of the gmuG gene or a gene corresponding to the gmuG gene. The inducers of the glucomannan-utilizing operon include cellobiose and mannobiose. Therefore, the expression of the target gene is controlled by cellobiose, mannobiose, or the like.
Furthermore, by using the target gene whose expression is controlled by cellobiose as an index, the recombinant microorganism of the present invention can be suitably used for screening for a gene encoding cellulase that produces cellobiose using cellulose as a substrate.

EXAMPLES Hereinafter, although this invention is demonstrated further in detail based on an Example, the technical scope of this invention is not limited to a following example.
In addition, the name of the primer used in the Example, its sequence | arrangement, and sequence number are shown in Table 2 and 3.

  For the polymerase chain reaction (PCR) for DNA fragment amplification in the following examples, GeneAmp PCR System (Applied Biosystems) is used, Pyrobest DNA Polymerase (Takara Bio) and attached reagents are used. DNA amplification was performed. The PCR reaction solution composition was 1 μL of appropriately diluted template DNA, 20 pmol each of sense and antisense primers, and 2.5 U of Pyrobest DNA Polymerase, so that the total reaction solution volume was 50 μL. PCR reaction conditions were 98 ° C. for 10 seconds, 55 ° C. for 30 seconds and 72 ° C. for 1 to 5 minutes (adjusted according to the target amplification product. The standard is 1 minute per 1 kb). After repeating, the reaction was carried out at 72 ° C. for 5 minutes.

Further, Bacillus subtilis was transformed as follows.
The Bacillus subtilis strain was cultured with shaking in an SPI medium at 37 ° C. until the degree of growth (OD ₆₀₀ ) reached about 1. After shaking culture, a portion of the culture solution is inoculated into 9 times the amount of SPII medium, and further cultured by shaking until the degree of growth (OD ₆₀₀ ) is about 0.4. Prepared. Next, 5 μL of a solution containing various DNA fragments (SOE-PCR reaction solution, etc.) is added to 100 μL of the prepared competent cell suspension (culture medium in SPII medium), shaken at 37 ° C. for 1 hour, The whole amount was smeared on LB agar medium (1% tryptone, 0.5% yeast extract, 1% NaCl, 1.5% agar) containing the drug. After stationary culture at 37 ° C., the grown colonies were isolated as transformants. The genome of the obtained transformant was extracted, and it was confirmed that the intended genomic structure was altered by PCR using this as a template.

Example 1 Production of Recombinant Microorganism As shown in FIG. 5, a Bacillus subtilis son5 strain in which a drug resistance gene without a promoter was inserted downstream of the gmuG gene of Bacillus subtilis 168 strain was produced by the method shown in the schematic diagram of FIG. did. Details will be described below.

Genomic DNA extracted from Bacillus subtilis 168 strain as a template and using a primer set consisting of an oligonucleotide represented by the nucleotide sequence of son5f1 shown in Table 2 (SEQ ID NO: 22) and son5 / catr (SEQ ID NO: 24), cat A 1.0 kb fragment (A) having a 5 ′ terminal homologous sequence of the gene on the 3 ′ terminal side was prepared by PCR. Similarly, the genomic DNA as a template, using son5 / catf primer set consisting of an oligonucleotide represented by the nucleotide sequence of (SEQ ID NO: 25) and Son5r1 (SEQ ID NO: 27) shown in Table 2, 3 of the cat gene A 1.0 kb fragment (B) having a 'terminal homologous sequence at the 5' end was prepared by PCR. Furthermore, using the DNA of plasmid pC194 (see J. Bacteriol., Vol. 150, No. 2, p. 815, 1982) as a template, catf2 (SEQ ID NO: 4) and catr2 (SEQ ID NO: 5) shown in Table 2 A 0.7 kb fragment (C) containing a chloramphenicol resistance gene ( cat gene) was prepared by PCR using a primer set consisting of an oligonucleotide represented by the nucleotide sequence.

  Next, 3 fragments of the obtained 1.0 kb fragment (A), 1.0 kb fragment (B) and 0.7 kb fragment (C) were mixed and used as a template to form son5f2 (SEQ ID NO: 23) shown in Table 2. ) And son5r2 (SEQ ID NO: 26) are subjected to SOE-PCR using a primer set consisting of oligonucleotides, and the three fragments are in the order of fragment (A), fragment (C) and fragment (B). Thus, a 2.7 kb DNA fragment (D) was obtained.

Furthermore, Bacillus subtilis 168 strain was transformed by the competent cell transformation method using the obtained DNA fragment (D). After transformation, colonies grown on LB agar medium containing cellobiose (0.1%) and chloramphenicol (10 ppm) were isolated as transformants. Genomic DNA of the obtained transformant was extracted, and it was confirmed by PCR that the cat gene was inserted downstream of the gmuG gene. The strain thus constructed is designated as Bacillus subtilis son5 strain.

Similar to Bacillus subtilis son5 strain except that the primers used for the preparation of fragment (A), fragment (B), fragment (C) and fragment (D) were as shown in Table 4, Bacillus subtilis son1 strain, Bacillus subtilis Bacteria son2 strain, Bacillus subtilis son3 strain and Bacillus subtilis son4 strain were prepared, respectively. FIG. 5 shows the insertion position of the cat gene on the genome of Bacillus subtilis son1, strain Bacillus subtilis son2, strain Bacillus subtilis son3, Bacillus subtilis son4, and Bacillus subtilis son5.

Test example 1
Each transformant prepared in Example 1 was planted on an LB + Cm plate (chloramphenicol: 10 ppm) and an LB + Cm + cellobiose plate (chloramphenicol: 10 ppm, cellobiose: 0.1% (w / v)). After culturing and culturing at 30 ° C. for 2 days, the growth status (presence or absence of colonies) was confirmed. The results are shown in Table 5. FIG. 6 shows an LB + Cm plate and an LB + Cm + cellobiose plate in which Bacillus subtilis son5 strain was cultured.

As shown in Table 5, Bacillus subtilis son1 strain, Bacillus subtilis son2 strain, Bacillus subtilis son3 strain, and Bacillus subtilis son4 strain in which a chloramphenicol resistance gene was inserted at a site other than the downstream of the gmuG gene were tested for the presence or absence of cellobiose. Nevertheless, it was able to grow. That is, in these recombinant microorganisms, the expression of the chloramphenicol resistance gene could not be controlled by cellobiose. Since Bacillus subtilis son1 strain introduced the cat gene into the cre sequence of the promoter of the glucomannan-utilizing operon, it affected the binding of the internal repressor GmuR encoded by the gmuR gene, and cellobiose, the inducer of the glucomannan-utilizing operon. It is thought that it was able to grow up with or without. Bacillus subtilis son3 strain and son4 strain, since the introduction of the cat gene upstream or gmuR gene of gmuR gene affects expression of GmuR that gmuR gene encodes, cat gene constitutively expressed, in the presence of cellobiose It is thought that it was able to grow regardless.
On the other hand, as shown in Table 5 and FIG. 6, Bacillus subtilis son5 strain cannot grow in a medium containing chloramphenicol in the absence of cellobiose, but acquires drug resistance in the presence of cellobiose, and chloram It is also possible to grow on a medium containing phenicol.

Test example 2
The culture solution of Bacillus subtilis son5 strain cultured overnight at 30 ° C. in 5 mL of LB medium containing 10 ppm chloramphenicol and 0.1% (w / v) cellobiose is adjusted to an OD ₆₀₀ = 0.03. The inoculum was inoculated and cultured at 37 ° C. with shaking at 180 rpm, and the growth degree (OD ₆₀₀ ) of the strain was measured. For comparison, the same culture was performed except that cellobiose was not contained, and the growth degree of Bacillus subtilis son5 strain was measured. The result is shown in FIG.
As shown in FIG. 7, the Bacillus subtilis son5 strain could not grow in the absence of cellobiose, whereas the Bacillus subtilis son5 strain could grow in the presence of cellobiose. This is because cellobiose functions as an inducer of the glucomannan- utilized operon, induces the expression of the glucomannan- utilized operon, and a drug resistance gene operably linked downstream of the gmuG gene of the glucomannan- utilized operon This is because Bacillus subtilis son5 strain was expressed and acquired drug resistance.

Moreover, it culture | cultivated similarly except having used cellotriose, cellotetraose, cellopentaose, or cellohexaose instead of cellobiose, and the growth degree of Bacillus subtilis son5 stock | strain was measured. The result is shown in FIG.
As shown in FIG. 8, Bacillus subtilis son5 strain could not grow in the presence of cellotriose, cellotetraose, cellopentaose or cellohexaose. Therefore, it is suggested that the expression of the target gene operably linked downstream of the gmuG gene of the glucomannan utilization operon of the recombinant microorganism of the present invention is controlled by cellobiose.

Example 2 Cellulase Gene Screening (1) Construction of Bacillus subtilis cellulase gene bglC overexpression plasmid
According to the protocol of In-Fusion ^™ Advantage PCR Cloning Kit (manufactured by Clontech), an alkaline cellulase gene derived from Bacillus sp. KSM-S237 strain (FERM BP-7875) (see JP 2000-210081) Table 3 shows a recombinant plasmid pHYS237 (see Biosci. Biotechnol. Biochem., 2000, vol. 64 (11), p. 2281-2289) into which a DNA fragment encoding a promoter and a signal sequence is inserted as a template. The DNA fragment was amplified using a primer set consisting of oligonucleotides represented by the base sequences of PSF_infu (EcI) (SEQ ID NO: 28) and PS237R (SEQ ID NO: 29). Further, a primer set comprising oligonucleotides represented by the base sequences of bglC-sig / PSF (SEQ ID NO: 30) and bglC_infu (SaI) (SEQ ID NO: 31) shown in Table 3 using the genomic DNA of Bacillus subtilis 168 strain as a template Was used to amplify the Bacillus subtilis cellulase gene bglC . These DNA fragments, the shuttle vector pHY300PLK treated with restriction enzymes Eco RI and Sal I and linearized, and In-Fusion enzyme were mixed in appropriate amounts and reacted for 30 minutes. Escherichia coli HB101 competent cell (manufactured by TaKaRa) was transformed with the thus prepared plasmid. Using an LB agar medium containing CMC and trypan blue, transformants that acquired tetracycline resistance were selected. In this way, to produce a promoter sequence and signal sequence of the S237 cellulase ligated with the structural gene of BglC with plasmid pHPS-bglC.
As Bacillus subtilis, 168 strains used were the same strains registered as MGNA-A001 at the National Institute of Genetics and access number 1A1 at Bacillus Genetic Stock Center (BGSC) in the United States.

(2) Preparation of transformant using bglC overexpression plasmid According to the protoplast transformation method (Mol. Gen. Genet., 1979, vol. 168, p. 111), the plasmid pHPS-bglC Introduced. The transformant was DM3 agar medium containing tetracycline (Tc) (1% CMC, 0.5% casamino acid, 0.5% yeast extract (Difco), 0.01% methionine, 0.01% lysine, 0.01% tryptophan, 8.1% sodium succinate. 6H ₂ O, 0.35% dipotassium hydrogen phosphate, 0.15% monopotassium hydrogen phosphate, 0.5% glucose, 20 mM magnesium chloride, 0.01% trypan blue, 0.01% BSA, 50 ppm tetracycline).

(3) Cellulase gene screening
Bacillus subtilis son5 strain introduced with pHPS-bglC and Bacillus subtilis son5 strain introduced with pHY300PLK as a control were grown by culturing on an LB agar medium containing chloramphenicol 10 ppm and milled cellulose or CMC 0.1%. Single colonies were inoculated with a platinum loop on SPI + Cm agar medium and LB + Cm agar medium containing a cellulose source (milled cellulose or CMC) or no cellulose source, and cultured at 30 ° C. for 3 days. Thereafter, the growth of the strain on the plate was confirmed. The result is shown in FIG.
The mill-pulverized cellulose is a powdered pulp (trade name: W-400G, manufactured by Nippon Paper Chemical Co., Ltd., 400 mesh pass 90 or more, cellulose content 99% by mass, moisture content 1% by mass). 500 g is put into “MA1D-X” manufactured by Mitsui Mining Co., Ltd., total capacity of container: 5.5 L, and 11 kg of zirconia balls (diameter: 10 mm, material: zirconia) is filled in the attritor as a medium (filling rate 59%) ) And pulverizing for 2 hours under the condition of a rotation speed of 307 rpm.

As shown in FIG. 9, Bacillus subtilis son5 strain into which a plasmid not containing a cellulase gene has been introduced cannot produce cellulase. Therefore, cellobiose as an inducer does not exist, and as a result, the chloramphenicol resistance gene inserted into the genome of the host microorganism is not expressed and cannot grow in the presence of chloramphenicol. In contrast, Bacillus subtilis son5 strain into which the bglC high expression plasmid pHPS-bglC has been introduced produces cellulase by expressing the bglC gene, and cellulose is decomposed by cellulase to produce cellobiose. Since this cellobiose functions as an inducer of the glucomannan-utilizing operon, the cat gene introduced into Bacillus subtilis son5 strain is expressed to acquire chloramphenicol resistance, and Bacillus subtilis son5 strain is in the presence of chloramphenicol. But it can grow.

  FIG. 10 schematically shows the mechanism of action of the glucomannan-utilizing operon of Bacillus subtilis son5 strain in cellulase gene screening. When a cellulase gene encoding cellulase is introduced into the recombinant microorganism of the present invention and the cellulase gene is expressed, cellobiose is generated from cellulose contained in the medium, and cellobiose induces the expression of the glucomannan-utilizing operon. Since the drug resistance gene is operably linked to the glucomannan-utilized operon, drug resistance is acquired by inducing the expression of the glucomannan-utilized operon. Therefore, the cellulase gene can be screened by using the drug resistance gene as an index.

  From the above results, according to the present invention, it is possible to create a recombinant microorganism in which the expression of the target gene is controlled by the inducer of the glucomannan-utilizing operon. Furthermore, by using the recombinant microorganism of the present invention, cellulase gene screening can be performed using the expression of the target gene as an index.

Example 3
As shown in FIG. 11, a strain son5A strain in which the ccpA gene was deleted from Bacillus subtilis son5 strain was prepared using a neomycin resistance gene ( neo gene) as a marker. Details will be described below.

Using a genomic DNA extracted from Bacillus subtilis 168 as a template, and using a primer set consisting of oligonucleotides represented by the base sequences of ccpAFW (SEQ ID NO: 32) and ccpA / NeR (SEQ ID NO: 33) shown in Table 3, neo A 1.0 kb fragment (E) having the 5 ′ end homologous sequence of the gene on the 3 ′ end side was prepared by PCR. Similarly, using the above-described genomic DNA as a template, a primer set consisting of oligonucleotides represented by the base sequences of ccpA / NeF (SEQ ID NO: 34) and ccpARV (SEQ ID NO: 35) shown in Table 3 is used. A 1.0 kb fragment (F) having a 'terminal homologous sequence at the 5' end was prepared by PCR. Furthermore, oligonucleotides represented by the nucleotide sequences of rneof (SEQ ID NO: 38) and neor (SEQ ID NO: 39) shown in Table 3 using plasmid pUB110 DNA (Plasmid, vol. 15, p. 93, 1986) as a template. A 1.3 kb fragment (G) containing the neo gene was prepared by PCR using a primer set consisting of

  Next, 3 fragments of the obtained 1.0 kb fragment (E), 1.0 kb fragment (F) and 1.3 kb fragment (G) were mixed and used as a template to obtain ccpAFW2 (SEQ ID NO: 36) shown in Table 3. ) And ccpARV2 (SEQ ID NO: 37) are subjected to SOE-PCR using a primer set consisting of oligonucleotides, and the three fragments are in the order of fragment (E), fragment (G) and fragment (F). Thus, a 3.3 kb DNA fragment (H) was obtained.

Furthermore, Bacillus subtilis son5 strain was transformed using the obtained DNA fragment (H) by competent cell transformation method. After transformation, colonies grown on LB agar medium containing neomycin (20 ppm) were isolated as transformants. The genomic DNA of the obtained transformant was extracted, and it was confirmed by PCR that the ccpA gene was replaced with the neo gene. The strain constructed in this way is designated as son5A strain.

Test example 3
10 ppm chloramphenicol and (i) cellobiose 0.1% (w / v), (ii) cellobiose 0.1% (w / v) and glucose 0.1% (w / v), or (iii) glucose 0.1% (w / v) in 5 mL of LB medium, inoculated with a culture solution of Bacillus subtilis son5A strain cultured overnight at 30 ° C. so that the cell concentration is OD ₆₀₀ = 0.03, and at 180 ° C. at 180 rpm. The strain was grown with shaking and the growth degree (OD ₆₀₀ ) of the strain was measured. For comparison, culture was performed in the same manner except that cellobiose and glucose were not contained, and the degree of growth of Bacillus subtilis son5A strain was measured. The result is shown in FIG.
As shown in FIG. 12, the Bacillus subtilis son5A strain in which the ccpA gene or a corresponding gene was deleted or inactivated had acquired resistance to chloramphenicol regardless of the presence or absence of glucose. This is because cellobiose functions as an inducer of the glucomannan- utilized operon, induces the expression of the glucomannan- utilized operon, and a drug resistance gene operably linked downstream of the gmuG gene of the glucomannan- utilized operon This is because Bacillus subtilis son5A strain was expressed and acquired drug resistance. Therefore, by deleting or inactivating the ccpA gene of the recombinant microorganism of the present invention or a gene corresponding thereto, the expression of the target gene can be confirmed by cellobiose even in the presence of a sugar that is easily metabolized such as glucose. Is possible.

Claims (10)

Glucmannan assimilation operon consisting of gmuB gene, gmuA gene, gmuC gene, gmuD gene, gmuR gene, gmuE gene, gmuF gene and gmuG gene of Bacillus subtilis or a gene corresponding to the above gene A microorganism having an operon functioning as an operon on the genome, wherein a target gene is operably linked downstream of the gmuG gene or a gene corresponding to the gmuG gene. The recombination according to claim 1, wherein the gmuB gene, the gmuA gene, the gmuC gene, the gmuD gene, the gmuR gene, the gmuE gene, the gmuF gene, and the gmuG gene are linked in this order on the genome to constitute a glucomannan-utilizing operon. Microorganisms. The recombinant microorganism according to claim 1 or 2, wherein the Bacillus subtilis ccpA gene or a gene corresponding thereto is deleted or inactivated.   The recombinant microorganism according to any one of claims 1 to 3, wherein the expression of the target gene is controlled by cellobiose.   The recombinant microorganism according to any one of claims 1 to 4, wherein the target gene is a drug resistance gene.   The recombinant microorganism according to claim 5, wherein the drug resistance gene is a chloramphenicol resistance gene. The recombinant microorganism according to any one of claims 1 to 6, wherein the recombinant microorganism is a bacterium belonging to the genus Bacillus . The recombinant microorganism according to claim 7, wherein the Bacillus bacterium is Bacillus subtilis . A glucomannan-utilizing operon consisting of the Bacillus subtilis gmuB gene, gmuA gene, gmuC gene, gmuD gene, gmuR gene, gmuE gene, gmuF gene and gmuG gene, or a gene corresponding to each of the above genes. The microorganism according to claim 1, further comprising a step of operably linking a target gene downstream of the gmuG gene or a gene corresponding to the gmuG gene in a microorganism having an operon functioning as an operon on the genome. A method for producing a replacement microorganism.   A cellulase gene screening method using the recombinant microorganism according to any one of claims 1 to 8, wherein a cellulase-encoding gene is selected using the expression of the target gene introduced into the recombinant microorganism as an index. Gene screening method.