DESCRIPTION RECOMBINANT MICROORGANISM

Field of the Invention [0001]

The present invention relates to a recombinant microorganism used in the production of a useful protein or polypeptide, and a method for producing the protein or polypeptide .

Background of the Invention

[0002]

Industrial production of useful materials by use of microorganisms is carried out for an extensive range of materials , the types including foodstuffs such as alcoholic beverages, soybean paste and soybean sauce, as well as amino acids, organic acids, nucleic acid-related substances, antibiotic substances, carbohydrates, lipids, proteins, and the like. Applications of these substances are also being expanded over a broad range of fields, including foods, pharmaceuticals, detergents, products for dailyuse suchas cosmetics, andavarietyof chemical raw materials.

[0003]

With regard to such industrial production of useful materials by microorganisms, one of important challenges is to improve productivity, and as a measure therefor, breeding of

productive microorganisms through genetic techniques such as mutation has been conducted. Recently, in particular, advances in microbial genetics and biotechnology have allowed more efficient breeding of productive microorganisms using genetic recombination technology and the like. In addition, the rapid advancement of genome analysis technology in recent years has resulted in attempts to interpret the genomic data of microorganisms of interest and industriallyutilize the obtained information more actively. Examples of industrially useful host microorganisms whose genomic data have been disclosed include Bacillus subtilis Marburg No. 168 (Non-Patent Document 1) , Escherichia coli K-12 MG1655 (Non-Patent Document 2) , CorynebacteriumglutamicumATCC132032 , and the like, and further improved microbial strains have been developed using these genomic data. However, regardless of such efforts, the production efficiencies are not necessarily satisfactory. [0004]

For certain types of microorganisms, strains in which a gene associated with the early stage of sporulation has been deleted or inactivated, have been recently constructed, and thereby an effect of improving the productivity for proteins or polypeptides is being obtained. For example, it has been reported that the productivity for the secretion of cellulase and the like is increased by using a host strain in which sigE gene, sigFgene, spoIIEgene, spoIISBgene or sigGgene of Bacillus subtilis, or a group of genes included in a region extending

from spoIVCB gene to spoIIIC gene, has been deleted (Patent Document 1) .

Furthermore, the functions for operating the protein translocation system (Sec route) in Bacillus subtilis are shared by SecA which serves as a motor for expelling secreted proteins to the outside of cells, and the three proteins, SecY, SecE and SecG, constituting the main part of the translocation channel through which the secreted proteins pass , as well as SecDF which is a Co-factor for the translocationchannel , andthe like . Inter alia, there have been reports on an expression vector capable of overexpressing secG gene, which encodes SecG protein (Patent Document 2) , or a Gram-positive bacterium in which expression of secG gene has been changed by altering the ribosome binding site of SecG gene (Patent Document 3) . Thus, it is shown that the breeding of a Bacillus subtilis species having SecG gene destroyed is inhibited during the production of heterologous proteins. It is also reported that in Escherichia coli, variation in secY gene inhibits the breeding at low temperatures or the secretion of proteins (Non-Patent Document 3). [0005]

However, a microorganism which overexpresses secY gene, andalsohas a sporulation-associatedgene deletedor inactivated, is not known hitherto.

[Patent Document 1] JP-A-2003 -47490

[Patent Document 2] JW-A-2001-510046

[Patent Document 3] US-A-2003/0157642

[Non-Patent Document 1] Nature, 390, 249, 1997 [Non-Patent Document 2] Science, 277, 1453, 1997 [Non-Patent Document 3] Cell, 32, 789, 1983.

Disclosure of the Invention [0006]

The present invention has the following aspects. (I)A recombinant microorganism obtained by transfecting a gene encoding a desired protein or polypeptide into a microorganism strain which is obtained by genetically constructing to overexpress secY gene of Bacillus subtilis or a gene corresponding to the secY gene, and deleting or inactivating one or more genes selected from sporulation-associated genes and genes corresponding to the sporuation-associated genes from the genome.

(2) A method of producing a recombinant microorganism as set forth in claim 1, which includes, in a microorganism, introducing a transcription initiation control region or a site of transcription initiation control region-ribosome binding, having a function in a microorganism, to the upstream on the genome of secY gene of Bacillus subtilis or a gene corresponding to the secY gene, or to the upstream of the leader gene of an operon on the genome containing secY gene of Bacillus subtilis or a corresponding gene, or obtained by introducing a gene fragment in which a transcription initiation control region or a site of transcription initiation control

region-ribosome binding, having a function in a microorganism, is linked to the upstream of secY gene of Bacillus subtilis or a gene corresponding to the secY gene; deleting or inactivating one or more genes selected from sporulation-associated genes and genes corresponding to the sporulation-associated genes; and transfecting a gene encoding a desired protein or polypeptide into a microorganism strain.

(3 ) Amethod forproducing a desiredprotein or polypeptide using the recombinant microorganism.

Brief Description of the Drawings [0007]

Fig. 1 is a schematic diagram showing gene transfection using a bound nucleic acid fragment prepared by SOE-PCR method.

Fig. 2 is a diagram illustrating the sporulation signal transduction in Bacillus subtilis .

Fig .3 is a schematic diagram showing a method forpreparing a DNA fragment for the production of a strain with enhanced secY expression, through SOE-PCR.

Fig. 4 is a schematic diagram showing the deletion of a target gene through a double crossover method using SOE-PCR fragment .

Best Mode for Carrying Out the Invention [0008]

The present invention relates to a microorganism having improved productivity for a protein or polypeptide , and a method for producing a desired protein or polypeptide using the microorganism . [0009]

The inventors of the present invention have searched for a gene which affects the production of a useful protein or polypeptide among a variety of genes encoded on the genome of microorganism, and found that when a gene encoding a desired proteinorpolypeptide is transfected intoamicroorganismstrain in which secY gene of Bacillus subtilis has been enhanced to overexpress SecY of Bacillus subtilis, as well as sporulation-associated genes are regulated, the productivity for the desired protein or polypeptide is improved compared to the productivity before the alteration. [0010]

The recombinant microorganism of the present invention is amicroorganism having highproductivity fora desiredprotein or a desired polypeptide. Therefore, when production of a desired protein or a desired polypeptide is performed using this recombinant microorganism, the time or costs required for the production of the material can be reduced. [0011]

The identity of amino acid sequences and base sequences in the present invention is calculated by the Lipman-Pearson method (Science, 227, 1435 (1985)) . Specifically, the identity

is calculatedbyperforming an analysis using a homologyanalysis program (Search Homology) of genetic data processing software, Genetyx-Win (Software Development Co. , Ltd. ) , with the unit size to compare (ktup) being set to 2. [0012]

In the present specification, the transcription initiation control region is a region including a promoter and a transcription initiation point , and the ribosome binding site is a site corresponding to the Shine-Dalgarno (SD) sequence which forms a translation initiation control region together with an initiation codon (Proc. Natl. Acad. Sci. USA, 71, 1342 (1974)) . [0013]

According to the present invention, the terms upstream and downstream of a gene refer not to the locations with respect to the replication initiation point, but the upstream indicates a region following the 5' -end of a gene or region of interest, while the downstream indicates a region following the 3 ' -end of a gene or region of interest . [0014]

Parent microorganisms for constructing the microorganism of the present invention may be any microorganisms having secY gene of Bacillus subtilis or a gene corresponding thereto, and these may be wild-type microorganisms as well as mutated microorganisms. Specifically, bacteria of the genus Bacillus, bacteria of the genus Clostridium, yeast or the like may be mentioned, and among them, bacteria of the genus Bacillus are

preferred. Furthermore, Bacillus subti1is is even more preferred fromtheviewpoints that the entire genomic information for the microorganism has been revealed, and thus relevant technologies of genetic engineering and genomic engineering are constructed, and that the microorganismhas an ability to secrete and produce a protein outside the bacterial cell.

The names of various genes and gene regions of Bacillus subtilis described the present specification are described on the basis of the Bacillus subtilis genome data reported inNature, 390, 249-256 (1997), and published in the Internet on the website of JAFAN: JapanFunctionalAnalysisNetwork forBacillus subtilis (BSORFDB) (http://bacillus.genome.ad.jp/, renewedonMarch 10 , 2004) . [0015]

The secY gene of Bacillus subtilis of the present invention refers to a gene having the base sequence set forth in SEQ ID NO : 1. The gene corresponding to secY gene of Bacillus subtilis refers to a gene having the substantially same function as that of the secY gene of Bacillus subtilis, and for example, the respective secY genes identified mainly by genome analysis and genes encoding SecY protein in Bacillus licheniformis, Bacillus anthracis, Bacillus cereus, Bacillus thuringiensis , Oceanobacillus iheyensis and the like, may be mentioned. Furthermore, there are cases where two kinds of the corresponding gene are identified in a microorganism, such as in Bacillus anthracis . As the genes corresponding to the secY gene of

Bacillus subtilis, any of the following genes of (1) to (4) may be mentioned. [0016]

(1) A gene comprising a DNA which has abase sequence having at least 90%, preferably at least 95%, and more preferably at least 99%, identity to the base sequence set forth in SEQ ID NO: 1, and encodes a protein which is functionally equivalent to a protein having the amino acid sequence set forth in SEQ ID NO : 2.

[0017]

(2) A gene comprising a DNA which is hybridized with a DNAcomprising abase sequence complementary to the base sequence set forth in SEQ ID NO: 1 under stringent conditions, and encodes a protein which is functionally equivalent to a protein having the amino acid sequence set forth in SEQ ID NO: 2.

Additionally, as for the "stringent conditions" as used herein, there maybe mentioned, forexample, the method described in Molecular Cloning - A LABORATORY MANUAL THIRD EDITION [Joseph Sambrook, DavidW. Russell, ColdSpringHarborLaboratoryPress] , and for example, conditions for hybridizing by thermostatically maintaining at 65°C for 8 to 16 hours, together with a probe in a solution containing 6 x SSC (composition of I x SSC: 0.15 M sodium chloride, 0.015 M sodium citrate, pH 7.0), 0.5% SDS, 5 xDenhardts andl . OOmg/mLof herringspermDNA, maybementioned. [0018]

(3) A gene comprising a DNAwhich has an amino acid sequence

having at least 90%, preferably at least 95%, and more preferably at least 99%, identity to the amino acid sequence set forth in SEQ ID NO: 2, and encodes a protein which is functionally equivalent to a protein having the amino acid sequence set forth in SEQ ID NO : 2. [0019]

(4) A gene comprising a DNAwhich has an amino acid sequence having one or two or more amino acids deleted, substituted or added in the amino acid sequence set forth in SEQ ID NO: 2, and encodes a protein which is functionally equivalent to a protein having the amino acid sequence set forth in SEQ ID NO: 2.

Additionally, the amino acid sequence having one or two or more amino acids deleted, substituted or added in the amino acid sequence set forth in SEQ ID NO: 2 as used herein includes amino acid sequences having one or several, preferably 1 to 10, amino acids deleted, substituted or added, and the addition includes addition of one to several amino acids to both terminals of an amino acid sequence. [0020]

In addition, the protein which is functionally equivalent to a protein having the amino acid sequence set forth in SEQ ID NO: 2 refers to a protein which has the substantially same function as that of a protein encoded by secY gene , and is capable of constituting an important part of a translocation channel through which secreted proteins pass. [0021]

The term overexpression of secY gene of Bacillus subtilis or a gene corresponding to the secYgene indicates that expression in a host of secYgene of Bacillus subtilis or a gene corresponding to the secY gene in an amount exceeding the usual amount of expression is observed. Examples of the means for overexpressing the secY gene of Bacillus subtilis or the gene corresponding to the secY gene include introduction of a transcription initiation control region or a site of transcription initiation control region-ribosome binding, having a function in a microorganism, to the upstream of the secY gene of Bacillus subtilis or the gene corresponding to the secY gene in the genome or to the upstream of the leader gene of an operon on the genome containing secY gene of Bacillus subtilis or a gene corresponding to the secY gene , or introduction of a gene fragment in which a transcription initiation control region or a site of transcription initiation control region-ribosome binding, having a function in a microorganism, is linked to the upstream of the secY gene of Bacillus subtilis or a gene corresponding to the secY gene . [0022]

Here, the transcription initiation control region or the site of transcription initiation control region-ribosome binding, having a function in a microorganism, is not particularly limited as long as the region is a transcription initiation control region or a site of transcription initiation control region-ribosome binding, having a function in the

microorganism which serves as a host, but for example, a transcription initiation control region or a site of transcription initiation control region-ribosome binding located upstream to spoVG gene or aprE gene of Bacillus subtilis, or a gene corresponding to any of these genes, is preferred, and a transcription initiation control region or a site of transcription initiation control region-ribosome binding located upstream to spoVG gene of Bacillus subtilis, or a gene corresponding to the spoVG gene is more preferred. [0023]

As the transcription initiation control regionof the spoVG gene of Bacillus subtilis, there may be mentioned a region for controlling the transcription of spoVG gene, which is a gene disclosed as Gene No. BG10112 in the Internet website (http://bacillus.genome.ad.jp/, renewed on march 10, 2004) by JAFAN: Japan Functional Analysis Network for Bacillus subtilis (BSORF DB) . More specifically, a DNA having a base sequence from base No. 38 to base No. 210 of the base sequence set forth in SEQ ID NO: 9 , or a DNA comprising a base sequence homologous to the aforementioned base sequence and having a function as the transcription initiation control region of spoVG gene of Bacillus subtilis, may be mentioned. Furthermore, as the site of transcription initiation control region-ribosome binding of spoVG gene of Bacillus subtilis, there may be mentioned a DNA comprising a base sequence from base No. 38 to base No. 230 of the base sequence set forth in SEQ ID NO: 9, or a DNA comprising

a base sequence homologous to the aforementioned base sequence and having a function as the site of transcription initiation control region-ribosome binding of spoVG gene of Bacillus subtilis . [0024]

Examples of the base sequence homologous to the base sequence from base No. 38 to base No. 210 or the base sequence from base No. 38 to base No. 230 of the base sequence set forth in SEQ ID NO: 9, include (A) a base sequence comprising a DNA which is hybridized with a DNA comprising a base sequence complementary to the base sequence from base No. 38 to base No. 210 or from base No. 38 to base No. 230 of the base sequence set forth in SEQ ID NO: 9, under stringent conditions; (B) a base sequence having at least 90%, preferably at least 95%, and more preferably at least 99%, identity to the base sequence from base No. 38 to base No. 210 or from base No. 38 to base No. 230 of the base sequence set forth in SEQ ID NO: 9; and the like.

Additionally, the term "stringent conditions" as used herein may be exemplified by the same conditions as described above . [0025]

The phrase "having a function as the transcription initiation control region of spoVG gene of Bacillus subtilis, or as the site of transcription initiation control region-ribosome binding" implies that when the DNA having the function is introduced to the upstream of secY gene of Bacillus

subtilis or a gene corresponding to the secY gene, or to the upstream of the leader gene of an operon (rpsJ gene for Bacillus subtilis) on the genome containing secY gene of Bacillus subtilis or a corresponding gene, the secY gene or the gene corresponding to the secY gene is overexpressed, thereby resulting in an improvement of the productivity for a desired protein or polypeptide, and also the extent of improvement is equal to the improvement obtained in the case where the transcription initiation control region of spoVG gene of Bacillus subtilis or the site of transcription initiation control region-ribosome binding is introduced to the upstream of the secY gene of Bacillus subtilis or a gene corresponding to the secY gene, or to the upstream of the leader gene of an operon (rpsJ gene for Bacillus subtilis) on the genome containing secY gene of Bacillus subtilis or a corresponding gene . [0026]

The introduction of the transcription initiation control region or the site of transcription initiation control region-ribosome binding, to the upstream of the secY gene or a gene corresponding to the secY gene on the genome, or to the upstream of the leader gene of an operon (rpsJ gene for Bacillus subtilis) on the genome containing secY gene of Bacillus subtilis or a corresponding gene, includes partial or entire substitution of the original transcription initiation control region of the secY gene of Bacillus subtilis or a gene corresponding to the secY gene, or of an operon on the genome containing secY gene

of Bacillus subtilis or a corresponding gene, as well as insertion while retaining the original transcription initiation control region or the site of transcription initiation control region-ribosome binding. [0027]

The substitution of the transcription initiation control region or the site of transcription initiation control region-ribosome binding can be performed, for example, using a known method involving homologous recombination. That is, first, to the upstream of a DNA fragment containing such transcription initiation control region or the site of transcription initiation control region-ribosome binding, a DNA fragment containing the upstream region of the original transcription initiation control region of an operon containing secY gene, and a drug resistance gene fragment are linked, while to the downstream of the former DNA fragment, a DNA fragment containing a part or the entirety of the translation initiation control region and the structural gene region of rpsJ gene , which is the leader gene of an operon containing secY gene, or a part or the entirety of the structural gene region of rpsJ gene, is linked, by a known method such as the SOE (splicing by overlap extension) -PCR method (Gene, 77, 61, 1989). In this manner, a DNA fragment is obtained, in which the DNA fragment containing the upstream region of the original transcription initiation control region of an operon containing secY gene, a drug resistance gene fragment, a DNA fragment containing the subject

transcription initiation control region or the subject site of ribosome biding to the transcription initiation control region, and a DNA fragment containing a part or the entirety of the translation initiation control region and the structural gene region of rpsJ gene, or a part or the entirety of the structural gene region of rpsJ gene, are linked in this order. [0028]

Next, when such DNA fragment is transfected into the cells of a parent microorganism by a known method, double crossover homologous recombination occurs at two spots of the genome of the parent microorganism, such as the upstream region of the original transcription initiation control region of the operon containing secY gene, and the region including a part or the entirety of the translation initiation control region and the structural gene region of rpsJ gene, or a part or the entirety of the structural gene region of rpsJ gene. As a result, a transformant in which the original transcription initiation control region or the site of transcription initiation control region-ribosome binding is substituted by the subject transcription initiation control region or the subject site of transcription initiation control region-ribosome binding, can be isolated using the drug resistance gene as a marker. In this manner, the transcription initiation control region or the site of transcription initiation control region-ribosome binding introduced to the upstream of the operon containing secY gene onthe genome, maybe genetically stablymaintained. Inaddition,

as the known method for transfecting a host microorganism with a DNA fragment for transfection, specifically there may be mentioned a competent cell transformation method (J. Bacteriol . 93, 1925 (1967)), a protoplast transformation method (MoI. Gen. Genet. 168, 111 (1979)), an electroporation method (FEMS Microbiol. Lett. 55, 135 (1990)), or the like, and a competent cell transformation method in particular is preferred.

In particular, in the case of using Bacillus subtilis as the hostmicroorganismof thepresent invention, the substitution through homologous recombination from the original transcription initiation control region of the operon containing secY gene or the site of transcription initiation control region-ribosome binding, to the subject transcription initiation control region or the subject site of transcription initiation control region-ribosome binding, can be performed using the method described in MoI . Gen. Genet., 223, 268 (1990) or the like. [0029]

The insertion of the subject transcription initiation control region or the subject site of transcription initiation control region-ribosome binding can be performed by the same method as the aforementioned method of substitution, if the sequences of the DNA fragments to be added to the two terminals of the transcription initiation control region or the site of transcription initiation control region-ribosome binding desired for insertion, are appropriately selected. For example,

to the upstream of such transcription initiation control region, a DNA fragment containing the upstream region of the original transcription initiation control region of an operon containing secY gene, and a drug resistance gene fragment are linked, while to the downstreamof suchtranscription initiationcontrol region, a DNA fragment containing a part or the entirety of the original transcription initiation control region is linked. In this manner, there is obtained a DNA fragment in which a DNA fragment containing the upstream region of the original transcription initiation control region of an operon containing secY gene, a drug resistance gene fragment, the subject transcription initiation control region, and a DNA fragment containing a part or the entirety of the original transcription initiation control region, are linked in this order. Subsequently, such DNA fragment is inserted into a host microorganism, and then the transformant can be isolated using the drug resistance gene as a marker. On the genome of the transformant thus isolated, the original transcription initiation control region of the operon containing secY gene and the subject transcription initiation control region may be stably maintained, while being adjacent to each otherwithout anygap therebetween. Alternatively, when a DNA fragment in which a DNA fragment containing the upstream region of secY gene and a drug resistance gene fragment are linked upstreamto the subject transcription initiationcontrol region, while a DNA fragment containing a part or the entirety of secY gene is linkeddownstreamtothe subject transcription initiation

control region, is prepared and used, the subject transcription initiation control region may be maintained stably, while being introduced immediately upstream to the secY gene. [0030]

According to the present invention, the upstream on the genome to which a transcription initiation control region or a site of transcription initiation control region-ribosome binding, having a function in a microorganism, is introduced includes secY gene. The upstream is not particularly limited if the region is the upstream side of the initiation codon of rpsJ gene which is the operon leader gene, or the upstream side of the initiation codon of secY gene, but a region including 2000 adjacent base pairs is preferred, a region including 500 base pairs is more preferred, a region including 100 base pairs is even more preferred, and a region including 50 base pairs is even more preferred. [0031]

According to the present invention, the introduction of a gene fragment in which the subject transcription initiation control region or the site of transcription initiation control region-ribosome binding is linked upstream to secY gene or a gene corresponding to the secY gene, can be performed using a gene fragment obtained by linking a fragment of the subject transcription initiation control region or the site of transcription initiation control region-ribosome binding, to a fragment of the secY gene or a gene corresponding to the secY

gene, which fragments have beenobtainedbya known cloningmethod, such as a PCR method, with the genome of a microorganism other than Bacillus subtilis used as the template, through a known method such as a restriction enzyme method or an SOE (splicing by overlap extension) -PCR method (Gene, 77, 61 (1989)). Such fragments can be introduced to the chromosome by allowing homologous recombination between a nucleic acid fragment introduced into the cell and the chromosome, according to a known transformation method. [0032]

The base sequence of the secY gene or a gene corresponding to the secY gene to be introduced, may not coincide with the base sequence of the secY gene originally possessed by the microorganism or a gene corresponding to the secY gene, as long as it is the base sequence of secY gene or a gene corresponding to the secY gene. Furthermore, the base sequence of the transcription initiation control region or the site of transcription initiation control region-ribosome binding, having a function in a microorganism, such as the transcription initiationcontrol regionorthe site of transcription initiation control region-ribosome binding of spoVG gene of Bacillus subtilis tobe introduced, maynot coincide with the base sequence possessed by the microorganism, as long as it is the subject base sequence . Themethodof introducinganucleic acid fragment into a host maybe exemplified by a competent cell transformation method, a protoplast transformation method, an electroporation

method or the like, and a competent cell transformation method in particular is preferred.

Furthermore, such fragment can also be introduced into the cytoplasm by a vector such as a plasmid. Additionally, as shown in the Examples that will be described later, since such fragment exhibits sufficient effects on the production of a desired protein or polypeptide by introducing one copy per one bacterial cell, when introduced by means of a plasmid, the fragment is hardly affected even if some of the plasmids are dropped out during the production and culture. [0033]

In addition, as for the region of chromosome allowing the introduction in a host, the inner part of a non-essential gene, or the inner part of a non-gene region upstream to a non-essential gene is preferred. For example , there may be mentioned the inner parts of aprE gene, sacB gene, nprE gene, amyE gene and ybxG gene, or the inner parts of non-gene regions upstream to these genes, but the inner part of amyE gene, or the inner part of a non-gene region upstream to ybxG gene is preferred.

The term "non-essential gene" as used herein means a gene with which the host can still survive at least under certain conditions even when the gene is destroyed. Also, even if the introduction is accompaniedby deletion of a non-essential gene, or of a part or the entirety of a non-gene region upstream of a non-essential gene, there is no problem resulting therefrom. [0034]

According to the present invention, overexpression of the secY gene of Bacillus subtilis or a gene corresponding to the secY gene, as well as overexpression of another gene associated with the Sec route of Bacillus subtilis, for example, secE gene or the like, may be carried out within the scope of not affecting the improvement of productivity for a desired protein or polypeptide, and inactivation or deletion of one or two or more genes may also be implemented in parallel. Additionally, the inactivation or deletion of a gene includes substitution and deletion of a part or all of the bases in the gene, as well as insertion of bases into the gene. [0035]

Hereinafter, the method of introducing a gene fragment in which the transcription initiation control region or the site of transcription initiation control region-ribosome binding of spoVG gene is linked to the upstream of secY gene, to the genome of a host by means of double crossover, using a DNA fragment preparedaccordingtotheSOE (splicingbyoverlap extension) -PCR method (Gene, 77, 61 (1989)), will be described in more detail . However, the method of introducing according to the present invention is not intended to be limited to the following.

The DNA fragment for introduction used in the present method is aDNAfragment inwhichbetweena fragment (hereinafter, fragment (I)) of about 0.1 to 3 kb in size, preferably 0.4 to 3 kb, adjacent to the upstream of the site of introduction on the genome of the host, and a fragment (hereinafter, fragment

(2) ) of about 0.1 to 3 kb in size, preferably 0.4 to 3 kb, adjacent to the downstream of the site of introduction, there are inserted a fragment containing the transcription initiation control region or the site of transcription initiation control region-ribosome binding of the sp _j oVGgene (hereinafter, fragment

(3) ) , a secY gene fragment (hereinafter, fragment (4) ) , and a fragment of a drug resistance marker gene such as chloramphenicol resistance gene (hereinafter, fragment (5)), in this order. First, five fragments of fragment (1) to fragment (5) areprepared in the first round of PCR. At this time, a primer designed such that, for example, a sequence of 10 to 30 base pairs on the upstream side of the fragment (3) is added to the downstream end of the fragment (1) , a sequence of 10 to 30 base pairs on the downstream side of the fragment (3) is added to the upstream end of the fragment (4) , a sequence of 10 to 30 base pairs on the upstream side of the fragment (5) is added to the downstream end of the fragment (4) , and a sequence of 10 to 30 base pairs on the downstream side of the fragment (5) is added upstream to the fragment (2) , is used (Fig. 1) .

Subsequently, the five kinds of PCR fragments prepared in the first round are used as templates, and the second round of PCR is performed using the primer located upstream to the fragment (1) and the primer located downstream to the fragment (2) . Thereby, annealing occurs with the fragment (3) in the sequence of the fragment (3) added to the downstream end of the fragment (1), annealing occurs with the fragment (3) in the

sequence of the fragment (3) added to the upstream end of the fragment (4), annealing occurs with the fragment (5) in the sequence of the fragment (5) added to the downstream end of the fragment (4) , and annealing occurs with the fragment (5) in the sequence of the fragment (5) added upstream to the fragment (2) . Thus, as a result of PCR amplification, a DNA fragment in which the five fragments of fragment (1) to fragment (5) are bound in the order of (1), (3), (4), (5) and (2), can be obtained (Fig. 1) . [0036]

The PCR reaction performed herein may be favorably- performed under conventional conditions described in literatures (PCR Protocols. Current methods and Applications, Edited by B.A. White, Humana Press, pp. 251, 1993; Gene, 77, 61 (1989) ) , using the primer set indicated in Table 1 and using an enzyme kit for general PCR, such as Pyrobest DNA polymerase (Takara Shuzo Co., Ltd.) .

When the DNA fragment for transfection thus obtained is transfected into cells by a competent method or the like, gene recombination occurs within the cells , in the homologous regions upstreamanddownstream to the site of introduction on the genome, where identity exists, and cells transfectedwith a gene fragment in which the transcription initiation control region or the site of transcription initiation control region-ribosome binding of the spoVG gene is linked to the upstream of the secY gene, can be isolated through selectionbased on the drug resistance marker.

The selectionbasedonthedrugresistancemarkermaybe favorably- performed by isolating colonies which grow on an agar medium containing chloramphenicol, and then selecting the cells for which introduction on the genome is confirmed by a PCR method using the genome as the template, or the like.

In addition, the drug resistance marker gene is not particularly limited as long as it can be used in the selection using a generally used antibiotic substance, but in addition to the chloramphenicol resistance gene, there may be mentioned drug resistance marker genes such as erythromycin resistance gene, neomycin resistance gene, spectinomycin, tetracycline resistance gene and blasticidin S resistance gene. [0037]

In the recombinant microorganismof the present invention, in addition to the overexpression of SecY resulting from the enhancement of secY gene, deletion or inactivation of one or more of sporulation-associated genes and genes corresponding thereto from the genome is achieved. The deletion or inactivation of the gene is an alteration to suppress sporulation, as shown in Examples 2 and 3.

According to the present invention, examples of the sporulation-associated genes include groups of genes accelerating the formation of spores, thus deletion or inactivation of each of those genes resulting in substantial inhibition of the process of sporulation, such as the group of genes encoding sigma factors specific to the stages of

sporulation, or the group of genes associatedwith the expression of the sigma factor genes and the activation of sigma factors. Furthermore, the group of genes that are transcribed by the corresponding sigma factors , andare involved inthe acceleration of sporulation, is also included.

For Bacillus subtilis, among the bacteria of the genus Bacillus, 17 sigma factors have been identified, and starting from SigA which is a major sigma factor involved in the transcription of the genes essential for growth in the nutrition growth stage (housekeeping sigma factor) , SigH, SigF, SigE, SigG and SigK which are sigma factors for controlling the process of sporulation, SigD which is a sigma factor for controlling the formation of flagella or cell wall digestion, SigL which is a sigma factor for controlling the metabolism of certain types of aminoacidor sugar, SigBwhichisa sigma factor forcontrolling the response to environmental changes, sigma factors called ECF sigma factors, and the like are known to exist (Bacillus subtilis and Its Closest Relatives: From Genes to Cells, Edited by A. L. Sonenshein, American Society for Microbiology, pp.289 (2002) ) . [0038]

Among them, the sigma factors for controlling the process of sporulation are known to be sequentially expressed and activated according to the progress of the sporulation process, as shown in Fig .2. In other words , when Bacillus subtilis enters a nutrient starvation state, there occurs first phosphorylation of SpoOA, which is a spoluration initiation control factor, via

a multi-stage phosphate transduction system involving a plurality of proteins, called the phosphate relay system (Cell, 64, 545 (1991)). More specifically, auto-phosphorylation of KinA present in the cytoplasm, and of KinB and KinC present in the cell membrane, occurs as a result of nutrient starvation, and the phosphoryl group is transferred to SpoOA via SpoOF and SpoOB, thereby phosphorylated SpoOA (Phosphorylated SpoOA) beingproduced. Furthermore, KapB is required in the activation of the process of sporulation involving KinB (MoI. Microbiol. , 26, 1097 (1997)), while KipA binds to Kipl, which is an auto-phosphorylation inhibitor of KinA, to avoid the sporulation suppressive action thereof (Genes Dev. , 11, 2569 (1997)) . Also, PhrA inhibits the function of RapA which is a dephosphorylation enzyme for phosphorylated SpoOF (Proc. Natl. Acad. Sci. USA, 94, 8612 (1997)) . KinA, KinB, KinC, SpoOF, SpoOB, SpoOA, KapB, Kipl, KipA, RapA and PhrA are encoded by the genes kinA, kinB, kinC, spoOF, spoOB, spoOA, kapB, kipl, kipA, rapA and phrA, respectively. [0039]

Accompanied by an increase in the concentration of Phosphorylated SpoOA, induction of repressor AbrB which suppresses the expression of a structural gene of SigH (sigH) is suppressed, and as a result, transcription of sigH is induced in a SigA-dependent manner (J. Bacteriol., 173, 521 (1991)). Additionally, while the Phosphorylated SpoOA transcription regulatory function is inhibited by Soj which is involved in

the chromosome separation, SpoOJ which is also involved in the chromosome separation suppresses such action of Soj (J. Bacteriol., 182, 3446, 2000). Soj and SpoOJ are encoded by soj gene and spoOJgene , respectively. After the activationof SigH, the cytoplasm of Bacillus subtilis is partitioned by the formation of an asymmetric septum, into the mother cell side and the daughter cell side. Subsequently, on the daughter cell side, Phosphorylated SpoOA and SigH are conjugated to induce the transcription of an operon (spolIAA-spolIAB-sigF) containing the structural gene of SigF (sigF) (Gene, 101, 113 (1991) ) , and on the mother cell side, Phosphorylated SpoOA and SigA are conjugated to induce the transcription of an operon (spoIIGA-sigE) containing the structural gene of SigE precursor (sicfE) (J. Bacteriol., 169, 3329 (1987)). There exists two-stage suppression in which SigF is functionally suppressed by an anti-sigma factor, SpoIIAB, and an anti-anti-sigma factor, SpoIIAA, suppresses the action of SpoIIAB. That is, while the functional deletion of SpoIIAA leads to the inhibition of sporulation as in the case of the functional deletion of SigF, the functional deletion of SpoIIAB is also known to inhibit sporulation (Proc. Natl. Acad. Sci. USA, 87, 9221 (1990); J. Bacteriol., 173, 6678 (1991)). Furthermore, the activation is controlled by SpoIIE which is a dephosphorylation enzyme for SpoIIAA (Genes Cells, 1, 881 (1996)), and activated SigF induces the transcription of the structural gene of SpoIIR, which is a signal transduction protein. It is conceived that SpoIIR

secreted from the daughter cell side activates SpoIIGA which is a SigE precursor activating protease localized in the asymmetric septum on the mother cell side, and therebyactivation of SigE takes place (Proc. Natl. Acad. Sci . USA, 92, 2012 (1995) ) . SpoIIAA, SpoIIAB, SpoIIE, SpoIIR and SpoIIGA are encoded by spoIIAA, spoIIAB, spoIIE, spoIIR and spoIIGAgenes , respectively. Further, on the daughter cell side, SigF induces the transcription of the structural gene of SigG (sigG) , and on the mother cell side, SigE induces the transcription of the structural genes of SigK (spoIIIC gene and spoIVCB gene) . However, the activation of SigG on the daughter cell side occurs after the activation of SigE on the mother cell side, and the activation of SigK occurs thereafter on the mother cell side (MoI. Microbiol., 31, 1285 (1999)). [0040]

Among the genes described above, the genes which inhibit the process of sporulation when deleted or inactivated are, as obvious from Fig.2, kinA, kinB, kinC, spoOF, spoOB, spoOA, kapB, kipA, phrA, spoOJ, sigH, sigF, sigE, spoIIAA, spoIIAB, spoIIE, spoIIR, spoIIGA, sigG, spoIIIC and spoIVCB genes. The sporulation-associated genes to be deleted or inactivated in the present invention are preferably selected from these genes belonging to Bacillus subtilis, and more preferably selected from sigF gene, sigE gene and phrA gene of Bacillus subtilis .

The sigF gene is a gene for encoding a sigma factor which is responsible for the expression of genes occurring in the

daughter cell side from stage II on, where the asymmetric septum is formed in the Bacillus subtilis cells, during the sporulation stages, while the sigE gene is a gene for encoding a sigma factor which is responsible for the expression of genes occurring in the mother cell side from stage II on, where the asymmetric septum is formed in the Bacillus subtilis cells, during the sporulation stages.

Furthermore, the phrA gene is one of the genes involved in the mechanism of intercellular information transmission needed in the case of sensing changes in the external growth environment and responding thereto in various ways, and the gene product is secreted outside the cell for the moment. It is reported that the gene is, after being processed outside the cell, ingested into the cell as a pentapeptide, and is bound to RapA protein which controls phosphorylation of SpoOF in the phosphor-relay system for transmitting signals for the initiation of sporulation, thereby participating in the signal transduction for the initiationof sporulation (Proc. Natl. Acad. Sci. USA, 94, 8612 (1997)).

The gene number and function of each of the above-mentioned genes are summarized in Table 6. [0041]

A gene corresponding to the sporulation-associated genes such as kinA, kinB, kinC, spoOF, spoOB, spoOA, kapB, kipA, phrA, spoOJ, sigH, sigF, sigE, spoIIAA, spoIIAB, spoIIE, spoIIR, spoIIGA, sigG, spoIIIC and spoIVCB, refers to a gene having the

substantially same function as that of anyone of the above genes, and for example, there maybementioneda gene derived fromanother microorganism, preferably one derived from a bacterium of the genus Bacillus, having a base sequence at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95%, and still more preferably at least 98%, identical to the base sequence of any one of those genes. Additionally, the identity of base sequences here is calculated according to the Lipman-Pearson method (Science, 227, 1435 (1985)). [0042]

Deletion or inactivation of such genes may be deletion or inactivationof an individual among the aforementionedvarious genes, or may be deletion or inactivation of a combination of two or more thereof. Further, enhancement, or deletion or inactivation of a gene other than the subject gene may also be performed in parallel. Additionally, the deletion or inactivation of a gene includes substitution or deletion of a part or all of bases in the gene, as well as insertion of bases into the gene . [0043]

As for the order of the deletion or inactivation of a group of genes ora gene, theremaybementionedamethodof intentionally deleting or inactivating the subject gene (target gene) , as well as a method of mutating a random gene through deletion or inactivation, and then performing evaluation of protein productivity and genetic analysis by appropriate methods.

[ 0044 ]

To delete or inactivate a target gene, for example, amethod involving homologous recombination may be used. That is, a cyclic recombinant plasmid obtained by cloning a DNA fragment containing a part of the target gene in an appropriate plasmid can be transfected into the cells of a parent microorganism, so that the target gene on the genome of the parent microorganism is split by homologous recombination in some region of the target gene, thereby inactivating the target gene. Alternatively, it is also possible to substitute a target gene on the genome with a deleted or inactivated gene fragment, by constructing a target gene inactivated through mutation such as base substitution or base insertion, or a linear DNA fragment containing the upstream and downstream regions of the target gene but not containing the target gene, as shown in Fig. 3, according to a PCR method or the like, and transfecting the resultant into the cells of a parent microorganism, to thereby cause double crossover homologous recombination at two sites exterior to the mutation site within the target gene on the genome of the parent microorganism, or on the upstream side and downstream side of the target gene .

[0045]

Particularly, in the case of using Bacillus subtilis as the parent microorganism for constructing the microorganism of the present invention, several reports are already available on the method of deleting or inactivating a target gene by

homologous recombination (MoI. Gen. Genet., 223, 268 (1990) and the like) , and thus the host microorganism of the present invention can be obtained by repeating such methods. [0046]

The deletion or inactivation of random genes can also be performedby a method of inducing homologous recombination, such as the above-described method using a randomly cloned DNA fragment, or by irradiating a parent microorganism with radiation. [0047]

Hereinafter, the method of deletion by double crossover using a DNA fragment for deletion which is prepared according to the SOE (splicing by overlap extension) -PCR method (Gene, 77, 61 (1989) ) will bemore specificallyexplained, but themethod of gene deletion in the present invention is not intended to be limited to the following. [0048]

The DNA fragment for deletion used in the present method is a fragment prepared by inserting a drug resistance marker gene fragment between a fragment of about 0.1 to 3 kb in size, preferably 0.4 to 3 kb, which is adjacent to the upstream of the gene to be deleted, and a fragment of about 0.1 to 3 kb in size, preferably 0.4 to 3 kb, which is adjacent to the downstream of the gene to be deleted. First, three fragments of an upstream fragment and a downstream fragment of the gene to be deleted, and a drug resistance marker gene fragment are prepared by the

first round of PCR, and at this time, a primer designed such that, for example, a sequence of 10 to 30 base pairs on the upstream side of the drug resistance marker gene is added to the downstream end of the upstream fragment, and conversely, a sequence of 10 to 30 base pairs on the downstream side of the drug resistance marker gene is addedto the upstreamendof the downstream fragment, is used (Fig. 4) . [0049]

Subsequently, the three kinds of PCR fragments prepared in the first round are used as templates, and the second round of PCR is performedusing the upstream side primer of the upstream fragment and the downstream primer of the downstream fragment . Thereby, annealing occurs with the drug resistance marker gene fragment in the drug resistance marker gene sequences added to the downstream end of the upstream fragment and the upstream end of the downstream fragment, and as a result of PCR amplification, a DNA fragment having a drug resistance marker gene inserted between an upstream side fragment and a downstream side fragment, can be obtained (Fig. 4) . [0050]

In the case of using a chloramphenicol resistance gene as the drug resistance marker gene, DNA fragments for deletion of various genes are obtained by performing SOE-PCR under conventional conditions that are shown in literatures (PCR Protocols. CurrentMethods andApplications, EditedbyB.A. White, Humana Press, p.251, 1993; Gene, 77, 61 (1989) ) , using the primer

set indicated in Table 1 and using an enzyme kit for general PCR, such as Pyrobest DNA polymerase (Takara Shuzo Co. , Ltd. ) . [0051]

When the DNA fragment for deletion thus obtained is transfected into cells by a competent method or the like, gene recombination occurs within the cells , in the homologous regions upstream and downstream to the gene to be deleted, where identity exists, and cells in which the desired gene has been substituted with the drug resistance gene, can be isolated through selection basedon the drug resistance marker . That is, when a DNA fragment for deletion prepared using the primer set indicated in Table 1 is transfected, colonies growing on an agar medium containing chloramphenicol maybe isolated and confirmed on that the desired gene on the genome is substituted with the chloramphenicol resistance gene by a PCR method using the genome as the template, or the like. [0052]

The microorganism of the present invention can be obtained by transfecting a gene, which encodes a desired protein or a desired polypeptide, into the microorganism thus produced. Here, the term "desired protein or polypeptide" means a protein or polypeptide for which one of the purposes is production or purification. Also, with regard to the "microorganism having a gene which encodes a desired protein or polypeptide, " the gene is meant to include the genes originally possessed by the microorganism, as well as genes not originally possessed by the

microorganism, that is, foreign genes. [0053]

The desired protein or desired polypeptide is not particularly limited, and examples thereof include various industrial enzymes or physiologically active peptides used in detergents, foods, fabrics, feedstuff, chemicals, medicine, diagnosis and the like, while industrial enzymes are preferred. Also, in terms of function of the industrial enzymes, oxidoreductases, transferases, hydrolases, lyases, isomerases, ligases/synthetases, and the like are included, and there may be favorablymentioned hydrolases such as cellulases, γ-amylases and proteases.

As for the proteases, proteases derived from microorganisms, preferably those derived from bacteria of the genus Bacillus, more preferably those derived from Bacillus clausii strain KSM-K16 (FERM BP-3376) , may be mentioned. More specific examples of alkali proteases derived from Bacillus clausii strain KSM-K16 include an alkali protease derived from a bacterium of the genus Bacillus, comprising an amino acid sequence from amino acid No. 1 to amino acid No. 380 of the amino acid sequence set forth in SEQ ID NO : 4 , or a protease comprising an amino acid sequence having 70%, preferably 80%, more preferably at least 90%, even more preferably 95%, and still more preferably at least 98%, identity to the above amino acid sequence .

As for the cellulase, there may be mentioned cellulases

belonging to Family 5 among the class of polysaccharide hydrolases (Biochem. J., 280, 309 (1991)), and among them, cellulases derived from microorganisms, particularly those derived from bacteria of the genus Bacillus, may be mentioned. Forexample, cellulases derived fromBacillus sp . strainKSM-S237 (FERM BP-7875) and Bacillus sp. strain KSM-64 (FERM BP-2886) maybe mentioned, and suitable examples thereof include an alkali cellulase derived from a bacterium of the genus Bacillus, comprising an amino acid sequence from amino acid No. 1 to amino acid No. 795 of the amino acid sequence set forth in SEQ ID NO: 6, or an alkali cellulase derived from a bacterium of the genus Bacillus, comprising an amino acid sequence from amino acid No. 1 to amino acid No. 793 of the amino acid sequence set forth in SEQ ID NO: 8, or a cellulase comprising an amino acid sequence having 70%, preferably 80%, more preferably at least 90%, even more preferably at least 95%, and still more preferably at least 98%, identity to the above amino acid sequence.

Furthermore, as for the γ-amylase, there may be mentioned γ-amylases derived from microorganisms, preferably those derived from bacteria of the genus Bacillus, and more preferably those derived from Bacillus sp . strain KSM-K38. [0054]

It is desirable that the gene of the desired protein or desired polypeptide to be transfected into the microorganism of the present invention is coupled with one or more regions selected from the control regions associatedwith transcription,

translation and secretion of the gene, namely, a transcription initiation control region containing a promoter and a transcription initiation point, a translation initiation control region containing a ribosome binding site and an initiation codon, and a secretion signal peptide region, in the upstream in an adequate form. Inparticular, a gene having three bound regions, including a transcription initiation control region, a translation initiation control region and a secretion signal peptide region, is preferred, and also, it is desirable that the secretion signal peptide region is one derived from a cellulase gene of a bacterium of the genus Bacillus, while the transcription initiation control region and translation initiation control region are regions of 0.6 to 1 kb in size upstream to the cellulase gene, and these regions are adequately- coupled with the gene of the desired protein or desired polypeptide. For example, it is desirable that the cellulase gene derived from a bacterium of the genus Bacillus, that is, strain KSM-S237 (FERM BP-7875) or strain KSM-64 (FERM BP-2886) , and the transcription initiation control region, translation initiation control region and secretion signal peptide region of the cellulase gene are adequately coupled with the structural gene of a desired protein or desired polypeptide. More specifically, it is desirable that a DNA fragment comprising abase sequence from base No.1 to base No.659 of the base sequence set forth in SEQ ID NO: 5, or a base sequence from base No. 1 to base No. 696 of the base sequence set forth in SEQ ID NO:

7, or a base sequence having at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95%, and still more preferably at least 98%, identity to the above base sequence , or a DNA fragment comprising a base sequence resulting from deletion, substitution or addition of a part of any of the above base sequences, is adequately coupled with the structural gene of a desired protein or desired polypeptide. Additionally, the DNA fragment comprising a base sequence resulting from deletion, substitution or addition of a part of the above base sequence as used herein means a DNA fragment which has a part of the above base sequence deleted, substituted or added, but maintains the function associated with the transcription, translation and secretion of a gene. [0055]

The introduction of such gene encoding a desired protein or desired polypeptide can be performed, for example, through (1) introduction by a vector, or (2) insertion into the genome . Inthe case of the (1) introductionbyavector, avector containing a gene which encodes a desired protein or desired polypeptide, and is coupled in the upstream with one or more regions selected from the control regions associated with transcription, translation and secretion of the gene, namely, a transcription initiation control region containing a promoter and a transcription initiation point, a translation initiation control region containing a ribosome binding site and an initiation codon, and a secretion signal peptide region, in an

adequate form, maybe introducedbyanappropriate transformation method such as a competent cell transformation method, a protoplast transformation method or an electroporation method. Here, the vector is not particularly limited as long as it is an appropriate carrier nucleic acid molecule for transfecting a desired gene into a host so as to propagate and express the gene, and there maybe mentioned plasmids, and also, for example, artificial chromosomes such as YAC and BAC, vectors using transposon, cosmids and the like. Examples of the plasmid include pUBHO and pHY300PLK. [0056]

Furthermore, the (2) insertion into the genome may be performed, for example, using a method involving homologous recombination. That is, a DNA fragment having a part of a chromosome region for inducing introduction bound to a gene encoding a desired protein or desired polypeptide, can be incorporated into the genome by transfecting the DNA fragment into the cells of a microorganism, and inducing homologous recombination in some part of the chromosome region. Here, the chromosome region for inducing introduction is not particularly limited, but a non-essential gene region or a non-gene region upstream to a non-essential gene region is preferred. [0057]

The production of a desired protein or polypeptide using the recombinant microorganism of the present invention may be performed by inoculating the bacterial strain into a medium

containing assimilable carbon sources, nitrogen sources and other essential components, culturing the strain by a conventional microorganism culturing method, and after completion of the culture, collecting and purifying the protein or polypeptide . As described in the Examples that are described later, an improvement in the productivity for a desired protein or polypeptide is achieved as compared with the case of using a microorganism that has not been altered in the gene. [0058]

Hereinafter, a method for constructing the recombination microorganism of the present invention, and a method for producing a cellulase and an amylase using the recombinant microorganism will be described in detail.

EXAMPLES [0059]

In the polymerase chain reaction (PCR) for the amplification of DNA fragments in- the following Examples, DNA amplification was performed using a GeneAmp PCR System (Applied Biosystems, Inc.) and using Pyrobest DNA Polymerase (TakaraBio, Inc.) and accessory reagents. The composition of the PCR reaction solution was prepared by adding 1 μL of appropriately diluted template DNA, 20 pmol each of a sense primer and an antisense primer, and 2.5 U of Pyrobest DNA Polymerase, and adjusting the total amount of the reaction solution to 50 μL. PCR was performed under the reaction conditions of repeating

30 rounds of three-stage temperature change of 10 seconds at 98°C, 30 seconds at 55°C, and 1 to 5 minutes at 72°C (adjusted in accordance with the desired amplification product. A rough standard is 1 minute per 1 kb) , and then allowing the reaction to proceed for 5 minutes at 72°C.

Furthermore, in the following Examples, the upstream and downstream of a gene refer not to the locations from the replication initiationpoint , but theupstream indicates a region following the 5 ' -end of a gene or region of interest in various operations andprocesses , while the downstream indicates a region following the 3 ' -end of a gene or region of interest in various operations and processes.

Furthermore, the names of the respective genes and gene regions in the following Examples are described on the basis of the genome data of Bacillus subtilis reported in Nature, 390, 249-256 (1997) and published in the Internet at the website of JAFAN: Japan Functional Analysis Network for Bacillus subtilis (BSORFDB) (http://bacillus.genome.ad.jp/, renewed onMarch 10 , 2004) . [0060]

Transformation of Bacillus subtilis was performed in the following manner. Specifically, a strain of Bacillus subtilis was cultured with shaking in SPI medium (0.20% ammonium sulfate, 1.40% dipotassium hydrogen phosphate, 0.60% potassium dihydrogen phosphate, 0.10% trisodium citrate dihydrate, 0.50% glucose, 0.02% casamino acid (Difco Laboratories, Inc.), 5 mM

magnesium sulfate, 0.25 μM manganese chloride, and 50 μg/mL tryptophan) at 37°C, until the value of degree of growth (OD600) reaches about 1. After the culturing with shaking, some of the culture solution was inoculated into a 9-fold amount of SPII medium (0.20% ammonium sulfate, 1.40% dipotassium hydrogen phosphate, 0.60% potassium dihydrogen phosphate, 0.10% trisodium citrate dihydrate, 0.50% glucose, 0.01% casamino acid (Difco Laboratories, Inc.), 5 mM magnesium sulfate, 0.40 μM manganese chloride, and 5 μg/mL tryptophan) , and the cells were further cultured with shaking until the value of degree of growth (OD600) reached about 0.4. Thus, competent cells of Bacillus subtilis were prepared.

Subsequently, to 100 μL of the competent cell suspension thus prepared (culture solution in SPII medium) , 5 μL of a solution containing various DNA fragments (SOE-PCR reaction solution or the like) was added, the mixture was incubated with shaking for 1 hour at 37°C, and the total amount was smeared onto LB agar medium (1% tryptophan, 0.5% yeast extract, 1% NaCl, and 1.5% agar) containing an appropriate drug . After stationary culture at 37°C, grown colonies were isolated as a transformant . The genome of the obtained transformant was extracted, and it was confirmed by PCR using the genome as the template, that a desired alteration in the genome structure was achieved. [0061]

The transfection of a gene encoding a desired protein or polypeptide into a host microorganism was performed following

any of the competent cell transformation method (J. Bacteriol . , 93, 1925 (1967)), the electroporation method (FEMS Microbiol . Lett., 55, 135 (1990)) , and the protoplast transformation method (MoI. Gen. Genet., 168, 111 (1979)).

As for the culture for protein production by the recombinant microorganism, LB medium (1% tryptophan, 0.5% yeast extract, and 1% NaCl) , 2xYT medium (1.6% tryptophan, 1% yeast extract, and 0.5% NaCl) , 2xL-maltose medium (2% tryptophan, 1% yeast extract, 1% NaCl, 7.5% maltose, and 7.5 ppm manganese sulfate tetra- or pentahydrate) , or CSL fermentation medium (2% yeast extract, 0.5% corn steep liquor (CSL), 0.05% magnesium chloride heptahydrate , 0.6% urea, 0.2% L-tryptophan, 10% glucose, 0.15% sodium dihydrogen phosphate, and 0.35% disodium hydrogen phosphate, pH 7.2) were used. [0062]

EXAMPLE 1 Construction of strain overexpressing secY gene

Construction of a variant overexpressing secY gene was performed as follows (see Fig. 3) . A 0.2 kb fragment (A) containing the transcription initiation control region and the ribosome binding site of spoVG gene, and a 1.3 kb fragment (B) containing spoVG gene were amplified by PCR using the genome DNA extracted from Bacillus subtilis strain 168 as the template, and using primer sets of PVG-FW and PVG-R, and secY/PVG-F and secY/Cm-R. Furthermore, a 0.9 kb fragment (C) containing chloramphenicol (Cm) resistance gene was amplified by PCR using

plasmidpC194 (J. Bacteriol . , 150(2) , 815 (1982) ) as the template, and using the primer set of catf and catr shown in Table 1.

Next, a DNA fragment (D) of 2.2 kb in size, in which the three fragments of (A) , (B) and (C) were coupled in this order, the transcription initiation control region and the ribosome binding site of spoVG gene were linked to the upstream of secY gene (linked suchthat the initiation codonof secY gene is located at the location of the initiation codon of spoVG gene) , and a Cm resistance gene was bound downstream thereto, was obtained by using a mixture of the obtained three fragments of (A) , (B) and (C) as a template, and performing SOE-PCR using the primer set of PVG-FW2 and catr2 shown in Table 1. Subsequently, a 1.0 kb fragment (E) containing the region on the 5 ' -end of amyE gene, and a 1.0 kb fragment (F) containing the region on the 3 ' -end of amyε genewere amplifiedby PCR, using the genome data extracted from Bacillus subtilis strain 168 as the template, and using the primer sets of aτnyEfw2 and amyE/PVG2-R and amyE/Cm2-F and amyErv2 shown in Table 1.

Subsequently, aDNAfragment (G) having a total base length of 4.2 kb, in which the three fragments of (E) , (D) and (F) were bound in this order, secY gene was linked to the downstream of the transcription initiation control region and the ribosome binding site of spoVG gene, and a DNA fragment of 2.2 kb in size coupled with a chloramphenicol resistance gene in the downstream thereto was inserted in the center of amyE gene, was obtained by using a mixture of the obtained three fragments of (E) , (F)

and (D) as a template, and performing SOE-PCR using the primer set of amyEfwl and amyErvl shown in Table 1.

Using the obtained DNA fragment (G) of 4.2 kb, Bacillus subtilis strain 168 was transformedby the competent cell method, and colonies growing on LB agar medium containing (10 μg/mL) were isolated as a transformant . Amplification of DNA fragments of 2.5 kb and 3.1 kb in size, respectively, was confirmed by performing PCR using the genome DNA extracted from the obtained transformant as a template, and using the primer sets of amyEfw2 and secY/Cm-R, and secY/PVG-F and amyErv2 shown in Table 1, and it was confirmed that a DNA fragment in which secY gene was linked downstream to the transcription initiation control region and the ribosome binding site of spoVG gene, was inserted at the amyE gene site on the genome of Bacillus subtilis strain 168. The strain thus obtained was designated as strain secY-K.

[0063]

[Table 1-1]

[0064]

[Table 1-2]

[0065]

[Table 1-3]

[0066]

EXAMPLE 2 Substitution of sigF gene in the genome with drug resistance gene

A method for substituting sigE gene in the genome with a drug resistance gene will be described on the basis of Fig. 4.

The genome DNA extracted from Bacillus subtilis strain

168 was used as a template, a 1.0 kb fragment (A) adjacent to the upstream of sigE gene in the genome was amplified by PCR using the primer set of sigF-FW and sigF/Sp-R shown in Table 1. Also, the above-mentioned genome DNA was used as a template, and a 1.0 kb fragment (B) adjacent to the downstream of sigF gene in the genome was amplified by PCR using the primer set of sigF/sp-F and sigF-RV.

Furthermore, a spectinomycin (Sp) resistance gene region (C) of 1.2 kb in size was prepared by PCR using plasmid pDG1727 (Gene, 167, 335 (1995)) DNA as a template, and using the primer set of spf and spr shown in Table 1.

Subsequently, as shown in Fig.4 , a mixture of the obtained three fragments of 1.0 kb fragment (A) , 1.0kb fragment (B) and Sp resistance gene region (C) was used as a template, and a DNA fragment (D) of 2.8 kb in which the three fragments were contained in the order of the 1.0kb fragment (A) , the Sp resistance gene region (C) and the 1.0kb fragment (B) , was obtained according to the SOE-PCRmethodusingtheprimer set of sigF-FW2 andsigF-RV2 shown in Table 1.

Then, transformation of the strain 168 was performedusing the obtained DNA fragment (D) , according to the competent cell transformation method. After the transformation, colonies growing on an LB agar medium containing spectinomycin (100 μg/mL) were isolated as a transformant .

The genome DNA of the obtained transformant was extracted, and it was confirmed that sigF gene was substituted with the

Sp resistance gene by PCR. As such, a sigF gene deleted strain (strain δsigF) was constructed. Furthermore, a strain in which sigF gene was substituted with the Sp resistance gene in the genome of strain secY-K (strain secYKδsigF) was constructedusing the strain secY-K that was constructed in Example 1 by changing the strain to Bacillus subtilis strain 168 in the transformation. [0067]

EXAMPLE 3 Substitution of sigE gene and phrA gene in the genome with drug resistance gene

Substitution of sigE gene and phrA gene in the genome of strain 168 with a spectinomycin resistance gene was performed in the same manner as in the substitution of sigE gene with a drug resistance gene as shown in Example 2, to thus construct a sigE gene deleted strain (strain δsigE) and a phrA gene deleted strain (strain δphrA) . For the construction of the respective strains, the primers shown in Table 1 were used, and the correspondence of the respective primers to the primers used in the construction of strain δsigF is shown in Table 2. [0068]

[ 0069]

By substituting sigE gene or phrA gene on the genome of strain secY-K constructed in Example 1, with a spectinomycin resistance gene, strain secYKδsigE and strain secYKδphrA were constructed.

[0070]

EXAMPLE 4 Evaluation of secretion and production of alkali protease

Evaluation of the productivity for heterologous proteins of the strain secY-K, strain secYKδsigF, strain secYKδsigE and strain secYKδphrA obtained in Examples 1 to 3 was performed as follows, using the productivity for an alkali protease derived from a bacterium of the genus Bacillus , whichprotease comprising the amino acid sequence set forth in SEQ ID NO: 4, as an index. As control , evaluation was performed also for Bacillus subtilis strain 168, strain δsigF, strain δsigE and strain δphrA. That is, a DNA fragment (W) of 1.3 kb in size which encodes an alkali protease having the amino acid sequence set forth in SEQ ID NO: 3 (Appl. Microbiol. Biotechnol . , 43, 473 (1995)), was amplified by performing PCR using the genome DNA extracted from Bacillus clausii strain KSM-K16 (FERM BP-3376) as a template, and using the primer set of S237pKAPpp-F and KAPter-R (BgIII) shown in Table 1. Also, a DNA fragment (X) of 0.6 kb in size containing the promoter region of an alkali cellulase gene (JP-A No. 2000-210081) , was amplified by performing PCR using the genome DNA extracted from Bacillus sp. strain KSM-S237 (FERM BP-7875)

as a template, and using the primer set of S237ppp-F2 (BamHI) and S237pKAPpp-R shown in Table 1.

Subsequently, a DNA fragment (Y) of 1.8 kb in size, in which alkali protease gene is linked downstream to the promoter region of alkali cellulase gene, was obtained by performing SOE-PCR using a mixture of the obtained two fragments of (W) and (X) as a template, and using the primer set of S237ppp-F2 (Bam HI) and KAPter-R (BgIII) and the like shown in Table 1. The resulting DNA fragment (Y) of 1.8 kb was inserted at the BamHI-Bglll restrictionenzyme cleavage site of a shuttlevector, pHY300PLK (Yakult HonshaCo. , Ltd.) , to construct plasmidpHYKAP (S237p) for the evaluation of alkali protease production.

The constructed plasmid pHYKAP (S237p) was transfected into various bacterial strains according to the protoplast transformation method. Each of the obtained recombinant strains was subjected to shaking culture overnight at 37°C in 10 mL of LB medium, and 0.05 mL of this culture solution was inoculated into 50 mL of 2xL-maltose medium (2% tryptone, 1% yeast extract, 1% NaCl, 7.5% maltose, 7.5 ppm manganese sulfate tetra- or pentahydrate , and 15 ppm tetracycline) , and was subjectedto shakingculture for 3 days at 30°C . Afterthe culture, the supernatant of culture solution from which bacterial cells were removed by centrifugation, was measured for the alkali protease activity to determine the amount of the alkali protease secreted and produced outside the bacterial cells during the culture. The determination of the protease activity in the

culture supernatant was performed as follows. Specifically, to 50 μL of the culture supernatant appropriately diluted with a 2 mM CaCl ₂ solution, 100 μL of a 75 mM boric acid-KCl buffer solution (pH 10.5) containing 7.5 mM of

Succinyl-L-Alanyl-L-Alanyl-L-Alanine p-Nitroanilide (STANA, Peptide Institute, Inc.) as a substrate, was added and mixed. Quantification of the amount of p-nitroaniline detached when a reaction was performed at 30°C, was carried out by measuring changes in the absorbance at 420 nm (OD420nm) . The amount of enzyme for detaching 1 μ-mol of p-nitroaniline in one minute was taken as 1 U. [0071]

As a result of the measurement of alkali protease activity, as shown in Table 3, when strain secY-K was used as the host, the productivity for alkali protease was equal to that of the control, strain 168 (wild type), and in particular, no improvement of productivity was recognized. On the other hand, significant improvement of productivitywas observedwith strain secYKδsigF, strain secYKδsigE and strain secYKδphrA, in which deletions of sporulation-associated genes were combined. An improvement of productivity exceeding the improvement for strain 168 was also observed for strain δsigF, strain δsigE and strain δphrA, which were not subject to the introduction of enhancement in the expression of secY gene,- however, the manifestation of the productivity improving effects resulting from deletion of these genes clearly was obviously increasing in the combination

with the enhancement in the expression of secY gene. In other words, it was conceived that when the amount of SecY protein, which is the device for secretion, is first increased, and then deletion of the sporulation-associated genes such as sigF, sigE and phrA is provided, a synergistic effect is obtained in the improvement of productivity for heterologous proteins. [0072]

[Table 3]

[0073]

EXAMPLE 5 Evaluation of secretion and production of alkali cellulase

An evaluation of productivity for other heterologous proteins was performed as follows, using the productivity for analkali cellulase derived fromabacteriumof the genus Bacillus , comprising the amino acid sequence set forth in SEQ ID NO: 6, as an index. Specifically, a fragment (3.1 kb) of alkali cellulase gene (JP-ANo.2000-210081) derived from Bacillus sp. strain KSM-S237 (FERM BP-7875) was amplified using the primer

set of 237UBl and 237DBl shown in Table 1, and then was treated withBamHI restriction enzyme to insert the fragment at the BatnHI restriction enzyme cleavage site of shuttle vector pHY300PLK. Recombinant plasmidpHY-S237 thus obtained was transfected into various bacterial strains according to the protoplast transformation method. Each of the recombinant strains thus obtained was subjected to shaking culture for 3 days under the same conditions as in Example 4. The supernatant of culture solution from which bacterial cells were removed by centrifugation, was measured for the alkali cellulase activity to determine the amount of the alkali cellulase secreted and produced outside the bacterial cells during the culture.

For the determination of the cellulase activity, 50 μL of 0.4 mM p-nitrophenyl-β-D-cellotrioside (Seikagaku Corporation) was added to 50 μLof a sample solution appropriately diluted with a 1/7.5 M phosphate buffer solution (pH 7.4, Wako Pure Chemical Industries, Ltd.) and mixed, and quantification of the amount of p-nitrophenol detached when a reaction was performed at 30 ⁰ C, was carried out by measuring changes in the absorbance at 420 nm (OD420nm) . The amount of enzyme for detaching 1 μmol of p-nitrophenol for one minute was taken as 1 U. [0074]

As a result of the measurement of alkali cellulase activity, as shown in Table 4, when strain secY-K was used as the host, higher secretion and production of alkali cellulase were

recognized as compared to the case of the control, strain 168 (wild type) . Also, further significant improvement of productivity was recognized with strain secYKδsigF, strain secYKδsigE and strain secYKδphrA, in which deletions of sporulation-associated genes were combined. An improvement of productivity exceeding the improvement for strain 168 was also observed for strain δsigF, strain δsigE and strain δphrA, which were not subject to the introduction of enhancement in the expression of secY gene; however, the manifestation of the productivity improving effects resulting from deletion of these genes clearly was obviously increasing in the combination with the enhancement in the expression of secY gene . In other words , it was conceived that when the amount of SecY protein, which is the device for secretion, is first increased, and then deletion of the sporulation-associated genes such as sigF, sigE and phrA is provided, a synergistic effect is obtained in the improvement of productivity. [0075]

[Table 4]

[ 0076 ]

COMPARATIVE EXAMPLE 1 Substitution of rsiX gene with drug resistance gene

Substitution of rsiX gene on the genome of Bacillus subtilis strain 168 with a chloramphenicol resistance gene was performed in the same manner as in the substitution of sigF gene with a spectinomycin resistance gene shown in Example 2, to construct strain δrsiX. The primers used are shown in Table 1 , and the correspondence of the respective primers to the primers used in the construction of strain δsigF is shown in Table 2. Strain δsigFδrsiX was also constructed by substituting rsiX gene on the genome of the strain δsigF constructed in Example 2, with a chloramphenicol resistance gene in the same manner. Additionally, the rsiX gene is a gene encoding an anti-sigma factor (anti-SigX) that suppresses the function of SigX, which is one of sigma factors belonging to ECF (extracytoplasmic function) family of Bacillus subtilis . SigX is activated when the environment around the cells changes under thermal stress or the like, and has a function of coping with the environmental change by inducing the transcription of a gene having a promoter for recognizing the activation, or of an operon (J. Bacteriol . , 179, 2915 (1997) ) . [0077]

COMPARATIVE EXAMPLE 2 Evaluation of secretion and production of alkali protease

Evaluation of secretion productivity for alkali protease

of the strain δsigFδrsiX constructed in Comparative Example 1 was performed in the same manner as in Example 4. As a control, the same evaluation was performed for Bacillus subtilis strain 168, strain δrsiX and the strain δsigF constructed in Example 1.

As a result, as shown in Table 5 , although the productivity for protease of the strain δrsiX with deleted rsiX gene was higher than that of the wild type strain 168 , the productivity of strain δsigFδrsiX in which deletion of sigF gene was combined was rather lower than that of strainδsigF. In otherwords, it was confirmed that manifestation of a synergistic effect for the production of heterologous proteins when deletions of sporulation-associated gene are combined, is not recognized for the deletion of rsiX gene. [0078]

[Table 5]

[0079]

COMPARATIVE EXAMPLE 3 Substitution of yacP gene, yvdE gene, yurK gene, yhdQ gene and glcT gene with drug resistance gene

Substitution of yacP gene, yvdE gene, yurK gene, yhdQ gene

and glcT gene on the genome of Bacillus subtilis strain 168 with a chloramphenicol resistance gene performed in the same manner as in the substitution of sigF gene with a spectinomycin resistance gene shown in Example 2, to construct strain δyacP, strain δyvdE, strain δyurK, strain δyhdQ and strain δglcT, respectively. The primers used are shown in Table 1, and the correspondence of the respective primers used to the primers used in the construction of strain δsigF is shown in Table 2. Also, by substituting yacP gene, yvdE gene , yurK gene , yhdQ gene and glcT gene on the genome of strain δsigF constructed in Example 2, with a chloramphenicol resistance gene, strain δsigFδyacP, strain δsigFδyvdE, strain δsigFδyurK, strain δsigFδyhdQ and strain δsigFδglcT were respectively constructed.

With regard to the genes related to the present invention, the gene No. and function thereof are presented in Table 6.

[0080]

[Table 6]

[ 0081]

COMPARATIVE EXAMPLE 4 Evaluation of secretion and production of alkali cellulase

Evaluation of the secretion productivity for alkali cellulase of the strainδyacP, strainδyvdE, strainδyurK, strain δyhdQ and strain δglcT constructed in Comparative Example 3 was performed in the same manner as in Example 5. As a control, evaluation was performed also for Bacillus subtilis strain 168. As a result, as shown in Table 7, the a productivity higher than that of the wild type strain was recognized with strain δyacP, strain δyvdE, strain δyurK, and strain δglcT, while the productivity was slightly decreased with strain δyhdQ. Furthermore, evaluation of the secretionproductivity for alkali cellulase of the strain δsigFδyacP, strain δsigFδyvdE, strain δsigFδyurK, strainδsigFδyhdQ and strainδsigFδglcT constructed in Comparative Example 3 was performed in the same manner as in Example 5. As a control, the evaluation was also performed for Bacillus subtilis strain 168 and the strain δsigF constructed in Example 1. As a result, as shown in Table 8, with any of the constructed strains , theproductivityfor cellulasewas lower than that of strain δsigF. In other words, it was strongly suggested that manifestation of a synergistic effect for the production of heterologous proteins when deletions of sporulation-associated genes are combined, is a feature for the combination with enhancement of the expression of secY gene.

[0082]

[Table 7]

[0083]

[Table 8]