Method for producing monoacylglycerol lipase

Abstract

To provide a method for producing monoacylglycerol lipase by a convenient procedure.SOLUTION: A method for producing monoacylglycerol lipase includes culturing Bacillus bacteria with a reduced activity of Bacillus subtilis AprE or protease corresponding thereto, for expressing of monoacylglycerol lipase.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a method for producing monoacylglycerol lipase.

Background Art

[0002] Monoacylglycerol (MAG) is a substance widely used in the fields of foods, pharmaceuticals, cosmetics, etc. as an emulsifier, antibacterial agent, etc. Furthermore, in recent years, it has been found that MAG has excellent physiological activities such as suppression of the increase in postprandial blood triglycerides, suppression of the increase in postprandial blood insulin, and suppression of postprandial GIP secretion. Chemically, MAG is produced by a method of esterifying a fatty acid and glycerin in the presence of an alkali catalyst under high-temperature conditions, a method of glycerolysis of fats and oils under high-temperature and high-pressure conditions, etc. However, these methods require high-temperature treatment and equipment for wastewater. On the other hand, a biochemical production method of MAG in which a fatty acid and glycerin are reacted in the presence of lipase has attracted attention as a method that can be used under milder conditions (for example, Patent Document 1). As the lipase used in this method, a lipase that recognizes a fatty acid monoglyceride or a fatty acid diglyceride such as monoglyceride lipase (MGLP) as a substrate is preferable.

[0003] Regarding MGLP, Patent Document 2 discloses H-257, which is MGLP derived from Bacillus sp. H-257. Also, MGLPs related to H-257 lipase such as EstGtA2 (Non-Patent Document 1), which is MGLP derived from Geobacillus thermodenitrificans, are known.

[0004] Microbial-derived MGLP from microorganisms such as Bacillus can be produced by culturing the microorganisms that produce it and then extracting MGLP from the culture. Furthermore, Patent Document 3 discloses that MGLP can be efficiently mass-produced by culturing an Escherichia coli strain into which the gene of H-257 lipase has been introduced and collecting MGLP from the culture. However, the conventional method has a complicated procedure because it requires a step of disrupting the cultured cells to recover the produced MGLP.

[0005] On the one hand, mutant Bacillus bacteria modified to highly produce a target protein are known. Patent Document 4 discloses a method for producing a protein by culturing a recombinant Bacillus subtilis transformed with a vector retaining a DNA fragment containing a promoter region and a secretion signal peptide region of an alkaline cellulase-producing gene derived from Bacillus bacteria and a gene encoding a target protein, wherein the aprE gene encoding extracellular protease AprE is deleted or inactivated. Patent Document 5 discloses a method for producing a protein using a recombinant microorganism in which a microorganism having a deleted or inactivated aprX gene of Bacillus subtilis and a gene selected from aprE, nprB, nprE, bpr, vpr, mpr, epr, and wprA of Bacillus subtilis as a host and into which a gene encoding a heterologous protein or polypeptide is introduced. Patent Document 6 discloses a method for producing BglC, which comprises using a Bacillus subtilis mutant strain in which eight or more protease genes selected from aprX, aprE, nprB, nprE, bpr, vpr, mpr, epr, and wprA are deleted from Bacillus subtilis strain 168, expressing the Bacillus subtilis endoglucanase gene bglC, and secreting full-length BglC outside Bacillus subtilis cells. Patent Document 7 discloses a method for producing SSI protein, which comprises culturing a recombinant Bacillus cell having inactivated aprE, nprE, epr, ispA, bpr, vpr, wprA, mpr-ybjF, and nprB genes and containing a nucleic acid encoding a fusion protein of a signal sequence and Streptomyces subtilisin inhibitor (SSI) protein. Patent Document 8 describes using a Bacillus host cell in which a gene encoding one or more proteases selected from the group consisting of AprE, Bpr, Epr, Mpr, NprE, Vpr, and WprA is disrupted as a host cell for producing foreign proteins such as glucosyltransferase, green fluorescent protein, and asparaginase.

Prior Art Documents

Patent Documents

[0006]

Patent Document 1

[0007] [Non-Patent Document 1] PLoS ONE, 2013, 8(10):e76675 [Summary of the Invention] [Problems to be Solved by the Invention]

[0008] The present invention provides a method for producing monoacylglycerol lipase. [Means for Solving the Problems]

[0009] The present inventors cultured Bacillus bacteria with reduced activity of secretory protease AprE in the production of monoacylglycerol lipase, which is not secreted naturally, to produce the monoacylglycerol lipase, and found that the produced monoacylglycerol lipase can be recovered from the culture supernatant without performing a cell disruption step.

[0010] Accordingly, the present invention provides a method for producing monoacylglycerol lipase, which includes culturing a Bacillus bacterium with reduced activity of Bacillus subtilis AprE or a protease corresponding thereto to express monoacylglycerol lipase. The present invention also provides a recombinant Bacillus bacterium in which the activity of Bacillus subtilis AprE or a protease corresponding thereto is reduced and a polynucleotide encoding monoacylglycerol lipase is introduced exogenously. The present invention also provides a method for producing a recombinant Bacillus bacterium, which includes incorporating a polynucleotide encoding monoacylglycerol lipase into a Bacillus bacterium with reduced activity of Bacillus subtilis AprE or a protease corresponding thereto.

Advantages of the Invention

[0011] According to the present invention, a naturally non-secretory monoacylglycerol lipase can be secreted and produced outside microbial cells, and can be easily recovered from the culture supernatant without performing a step of disrupting the cells after culture. According to the present invention, monoacylglycerol lipase can be produced by a simple procedure.

Brief Description of the Drawings

[0012]

Figure 1

Figure 2

Figure 3

Modes for Carrying Out the Invention

[0013] The names of the genes of Bacillus subtilis described in this specification are described based on the Bacillus subtilis genome data publicly available on the Internet ([bacillus.genome.ad.jp / ], updated on January 18, 2006) by JAFAN: Japan Functional Analysis Network for Bacillus subtilis (BSORF DB). The gene numbers of Bacillus subtilis described in this specification represent the gene numbers registered in BSORF DB.

[0014] In this specification, the identity of nucleotide sequences and amino acid sequences is calculated by the Lipman-Pearson method (Science, 1985, 227: 1435-1441). Specifically, it is calculated by performing an analysis using the homology analysis (Search homology) program of the genetic information processing software Genetyx-Win with Unit size to compare (ktup) set to 2.

[0015] In this specification, "at least 90% identity" with respect to an amino acid sequence or a nucleotide sequence means an identity of 90% or more, preferably 95% or more, more preferably 96% or more, still more preferably 97% or more, even more preferably 98% or more, and most preferably 99% or more.

[0016] Also in this specification, "operably linked" between a control region and a gene means that the gene and the control region are linked such that the gene can be expressed under the control of the control region. The procedure for "operably linking" a gene and a control region is well known to those skilled in the art.

[0017] In this specification, "upstream" and "downstream" with respect to a gene mean upstream and downstream in the transcription direction of the gene. For example, "a gene located downstream of a promoter" means that the gene is present on the 3'-side of the promoter in the DNA sense strand, and upstream of the gene means the region on the 5'-side of the gene in the DNA sense strand.

[0018] As used herein, the term "native" when used in relation to the function, properties, or traits of a cell is used to indicate that the function, properties, or traits originally exist in the cell. In contrast, the term "foreign" is used to indicate a function, property, or trait that is not originally present in the cell but is introduced from the outside. For example, a "foreign" gene or polynucleotide is a gene or polynucleotide introduced into a cell from the outside. The foreign gene or polynucleotide may be derived from the same species of organism as the cell into which it is introduced or from a different species of organism (i.e., a heterologous gene or polynucleotide).

[0019] The method for producing the monoacylglycerol lipase of the present invention (hereinafter also referred to as MGLP) includes culturing a microorganism with reduced activity of a specific secreted protease (also referred to as an extracellular protease) to express monoacylglycerol lipase.

[0020] Examples of the microorganism used for producing MGLP in the present invention include bacteria of the genus Bacillus such as Bacillus subtilis, bacteria of the genus Clostridium, and yeasts. Among them, bacteria of the genus Bacillus are preferred. Further, Bacillus subtilis or a mutant strain thereof is more preferred in view of the fact that the whole genome information has been clarified, the genome engineering technology has been established, and the ability to secrete and produce proteins outside the cells.

[0021] Examples of the secreted protease whose activity is reduced in the microorganism used in the present invention include Bacillus subtilis AprE (hereinafter simply referred to as AprE) and proteases corresponding thereto. AprE is a serine alkaline protease (subtilisin E) encoded by the Bacillus subtilis gene aprE (hereinafter simply referred to as aprE). aprE is a gene represented by the gene number BG10190 in the BSORF DB.

[0022] Examples of proteases corresponding to AprE include proteases having at least 90% identity with AprE in the amino acid sequence and having the same function as AprE. Preferably, a polypeptide having serine protease activity with an optimal pH on the alkaline side (i.e., serine alkaline protease) encoded by a gene corresponding to aprE is mentioned. In the present specification, a gene corresponding to aprE refers to a gene having at least 90% identity with aprE in the nucleotide sequence and encoding a polypeptide having serine protease activity with an optimal pH on the alkaline side. Specific examples of proteases corresponding to AprE include peptidase S8 of Bacillus mojavensis, peptidase S8 of Bacillus halotolerans, peptidase S8 of Bacillus tequilensis, and the like.

[0023] In addition to reducing the activity of AprE or a protease corresponding thereto, the activity of one or more other native proteases may also be reduced in the microorganism used in the present invention. Examples of such other native proteases include, for example, the Bacillus subtilis genes epr, wprA, mpr, nprB, bpr, nprE, vpr, aprX (hereinafter simply referred to as epr, wprA, mpr, nprB, bpr, nprE, vpr, aprX), and proteases encoded by genes corresponding thereto. By reducing the activities of these other native proteases together with AprE or a protease corresponding thereto, the release of MGLP extracellularly is promoted, and the yield of MGLP recovered from the culture supernatant can be improved. Examples of such microorganisms include those in which the aprE or a gene corresponding thereto is deleted or inactivated, and further at least one gene selected from the group consisting of epr, wprA, mpr, nprB, bpr, nprE, vpr, aprX, and genes corresponding thereto is deleted or inactivated. For epr, wprA, mpr, nprB, bpr, nprE, vpr, and aprX, or genes corresponding thereto, any one of them may be deleted, or any two or more of them may be deleted or inactivated in combination.

[0024] Table 1 shows the gene numbers of epr, wprA, mpr, nprB, bpr, nprE, vpr, and aprX and the functions of the proteins they encode. Examples of genes corresponding to epr, wprA, mpr, nprB, bpr, nprE, vpr, and aprX include epr, wprA, mpr, nprB, bpr, nprE, vpr, and aprX, respectively, and genes having at least 90% identity in nucleotide sequence and encoding proteins having the same function (described in Table 1).

[0025]

Table 1

[0026] Reduction of the activity of a target protein of a microorganism, such as AprE or a protease corresponding thereto or other native proteases, can be achieved by reducing the expression level of the target protein in the microorganism. For example, by deleting or inactivating the gene encoding the target protein in the microorganism (i.e., the target gene, such as aprE or a gene corresponding thereto), inactivating the mRNA transcribed from the target gene, or inhibiting the translation of the mRNA of the target gene into a protein, etc., the expression of the target protein can be suppressed, and thus the activity of the target protein can be reduced. Alternatively, the activity of the expressed target protein may be reduced.

[0027] Deletion or inactivation of a target gene in a cell can be achieved by removing part or all of the nucleotide sequence of the target gene from the genome or replacing it with another nucleotide sequence, inserting another polynucleotide fragment into the sequence of the target gene, introducing a mutation into the transcription or translation initiation region of the target gene, etc. Preferably, part or all of the nucleotide sequence of the target gene is deleted or inactivated. More specific examples include a method of specifically deleting or inactivating a target gene on the genome of a cell, and a method of randomly introducing deletion or inactivation mutations into a gene in a cell, followed by evaluating the expression level and activity of the target protein or performing gene analysis to select a cell having a desired mutation.

[0028] For specific deletion or inactivation of a target gene, methods such as homologous recombination can be used. That is, a DNA fragment of a target gene into which an inactivating mutation has been introduced by base substitution, base insertion, etc., or a DNA fragment that contains the outer region of the target gene but does not contain the target gene is constructed, and this is incorporated into a parental microbial cell to cause homologous recombination in the region containing the target gene of the parental microbial genome, thereby making it possible to delete or inactivate the target gene on the genome. Alternatively, a recombinant vector (such as a plasmid) having a DNA fragment containing a partial region of the target gene is incorporated into a parental microbial cell, and the partial region of the target gene of the parental microbial genome is disrupted by homologous recombination, thereby making it possible to inactivate the target gene. As a method for randomly deleting or inactivating genes in a cell, there are methods such as introducing a DNA fragment randomly cloned with a gene into which an inactivating mutation has been introduced into the cell and causing homologous recombination with the gene on the genome of the cell, and irradiating the cell with ultraviolet rays, γ-rays, etc. to induce mutations. An inactivating mutation of a gene means a mutation in which the function originally possessed by the target gene is lost due to a silent mutation, missense mutation, nonsense mutation, frameshift mutation, etc. For example, a gene into which an inactivating mutation has been introduced does not express a protein or expresses a protein with its original activity impaired.

[0029] As a method for preparing a DNA fragment containing a target gene into which an inactivating mutation has been introduced, site-directed mutagenesis can be mentioned. Site-directed mutagenesis can be carried out using a mutant primer containing the nucleotide mutation to be introduced. For example, by PCR using a target gene as a template with two sets of primers containing the nucleotide mutation to be introduced, DNA fragments amplified on the upstream side and the downstream side of the region containing the target gene are prepared respectively, and then these are ligated together by SOE-PCR (splicing by overlap extension PCR) (Gene, 1989, 77(1): p61-68), whereby a DNA fragment containing the desired mutation can be constructed. Alternatively, for site-directed mutagenesis, an inverse PCR method, an annealing method, etc. (edited by Muramatsu et al., "Revised 4th Edition New Genetic Engineering Handbook", Yodosha, p82-88), or commercially available site-directed mutagenesis kits such as Stratagene's QuickChange II Site-Directed Mutagenesis Kit and QuickChange Multi Site-Directed Mutagenesis Kit can also be used.

[0030] The mutant primer can be prepared by a well-known oligonucleotide synthesis method such as the phosphoramidite method (Nucleic Acids Research, 1989, 17: 7059-7071). The target gene used as a template may be prepared by a conventional method from the microorganism used for the production of MGLP described above, or may be chemically synthesized.

[0031] To introduce DNA fragments or vectors into microbial cells, well-known techniques such as the calcium phosphate method, electroporation method, lipofection method, particle gun method, PEG method, etc. can be applied. For example, methods applicable to Bacillus bacteria include the competent cell transformation method (J Bacteriol, 1967, 93: 1925 - 1937), electroporation method (FEMS Microbiol Lett, 1990, 55: 135 - 138), protoplast transformation method (Mol Gen Genet, 1979, 168: 111 - 115), Tris-PEG method (J Bacteriol, 1983, 156: 1130 - 1134), and the like.

[0032] Cells in which the target gene is deleted or inactivated can be selected by confirming their genomic sequences. Alternatively, cells in which the target gene is deleted or inactivated can be selected using the expression level or activity of the target protein as an indicator.

[0033] Examples of methods for inactivating the mRNA of the target gene in cells include target-specific mRNA inhibition using siRNA. Examples of methods for reducing the activity of the expressed target protein include using an inhibitor of the target protein.

[0034] The microorganism used for producing MGLP of the present invention may be a microorganism that originally has the ability to produce MGLP, or a microorganism modified to acquire or improve the MGLP-producing ability. Examples of methods for acquiring or improving the MGLP-producing ability of microorganisms include incorporating a polynucleotide encoding the target MGLP into the microorganism. In a preferred embodiment, the microorganism used for producing MGLP of the present invention is a recombinant microorganism into which a foreign polynucleotide encoding the target MGLP to be produced is incorporated.

[0035] The types of MGLP produced by the present invention are not particularly limited, but MGLP belonging to Bacterial Lypolytic Enzyme Family XV (Non-Patent Document 1) is mentioned as a preferred example. More preferred examples include non-secreted MGLP derived from Bacillus or Geobacillus bacteria belonging to the said Family XV. Preferred examples of the non-secreted MGLP include EstGtA2 (GenBank: AEN92268.1, SEQ ID NO: 1) which is MGLP derived from Geobacillus thermodenitrificans, and H-257 (PDB: 4KE8, SEQ ID NO: 2) which is MGLP derived from Bacillus sp. H-257. Other examples of the non-secreted MGLP include MGLP derived from Geobacillus stearothermophilus consisting of the amino acid sequence of SEQ ID NO: 3 (NCBI Reference Sequence: WP_053414863.1, hereinafter referred to as stLP), MGLP derived from Bacillus sp. FJAT-29814 consisting of the amino acid sequence of SEQ ID NO: 4 (NCBI Reference Sequence: WP_066306313.1, hereinafter referred to as CI220-6), MGLP derived from Bacillus sp. EB01 consisting of the amino acid sequence of SEQ ID NO: 5 (NCBI Reference Sequence: WP_04392037.1, hereinafter referred to as CI220-10), and MGLP derived from Burkholderia singularis consisting of the amino acid sequence of SEQ ID NO: 6 (GenBank: SMF98887.1, hereinafter referred to as CI220-12).

[0036] More preferred examples of the MGLP produced by the present invention include a polypeptide consisting of an amino acid sequence of any one of SEQ ID NOs: 1 to 6, and a polypeptide consisting of an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NOs: 1 to 6 and has monoacylglycerol lipase activity. Even more preferred examples of the MGLP produced by the present invention include a polypeptide consisting of an amino acid sequence of any one of SEQ ID NOs: 1 and 2, and a polypeptide consisting of an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NOs: 1 and 2 and has monoacylglycerol lipase activity.

[0037] In the present specification, the "monoglyceride lipase activity" refers to the activity of more selectively decomposing monoacylglycerol into glycerol and fatty acids as compared with the activity of decomposing triacylglycerol into diacylglycerol and fatty acids and the activity of decomposing diacylglycerol into monoacylglycerol and fatty acids, or refers to the activity of more selectively synthesizing monoacylglycerol as compared with triacylglycerol and diacylglycerol in the synthesis reaction of glycerol and fatty acids.

[0038] As described in Non-Patent Document 1, it has been proposed that the MGLP belonging to the above Bacterial Lypolytic Enzyme Family XV is equivalent in terms of sequence homology to Family abH11.03 in the Lipase Engineering Database ([www.led.uni-stuttgart.de / ]). Therefore, another preferred example of the type of MGLP produced by the present invention includes MGLP belonging to the Family abH11.03.

[0039] The polynucleotide encoding the target MGLP to be incorporated into the microorganism used in the present invention may be any polynucleotide that encodes the MGLP produced in the present invention described above. If necessary, the polynucleotide is codon-optimized according to the codon usage frequency in the cells into which it is incorporated and expressed. Information on codons used by various organisms is available from the Codon Usage Database ([www.kazusa.or.jp / codon / ]). For example, the polynucleotides encoding the amino acid sequences of SEQ ID NOs: 1 to 6 codon-optimized for Bacillus subtilis are polynucleotides consisting of the nucleotide sequences of SEQ ID NOs: 7 to 12, respectively.

[0040] Preferably, the polynucleotide encoding the MGLP is operably linked to one or more regions selected from the group consisting of a control region involved in gene transcription or translation. Examples of the control region include a transcription start control region and a translation start control region. Examples of the transcription start control region include a promoter and a transcription start point. Examples of the translation start region include a ribosome binding site and a start codon. Preferably, the polynucleotide encoding the MGLP is operably linked to one or more regions selected from the group consisting of a transcription start control region and a translation start control region. More preferably, the polynucleotide encoding the MGLP is operably linked to a transcription start control region and a translation start control region. On the other hand, the polynucleotide encoding the MGLP does not necessarily need to be linked to a region involved in protein secretion (e.g., a secretion signal peptide), and preferably, it is not linked to a region involved in the secretion of the protein.

[0041] Examples of such control regions include the control region of the α - amylase gene derived from Bacillus bacteria, the control region of the protease gene, the control region of the aprE gene, the control region of the spoVG gene, the control region of the cellulase gene of Bacillus sp. KSM - S237 strain (Japanese Patent Laid - Open No. 2000 - 210081), the control region of the cellulase gene of Bacillus sp. KSM - 64 strain (Japanese Patent Laid - Open No. 2011 - 103875), the control region of the kanamycin resistance gene derived from Staphylococcus aureus, the control region of the chloramphenicol resistance gene (both refer to Japanese Patent Laid - Open No. 2009 - 089708), etc., but are not particularly limited. More preferable examples of the control region include the control region of the cellulase gene of Bacillus sp. KSM - S237 strain (SEQ ID NO: 78) and the control region of the cellulase gene of Bacillus sp. KSM - 64 strain (SEQ ID NO: 79). Also, preferable control regions include nucleotide sequences having at least 90% identity with SEQ ID NO: 78 or 79 and having the function of controlling gene transcription and translation. The ligation of the polynucleotide encoding the MGLP and the control region can be carried out by the restriction enzyme method, SOE - PCR described above, etc.

[0042] For the incorporation of the polynucleotide encoding the MGLP into a microorganism, a DNA fragment or vector containing the polynucleotide encoding the MGLP and, if necessary, a further control region may be constructed and incorporated into the microorganism. The method for introducing the DNA fragment or vector into the microbial cell is as described above.

[0043] The vector can be constructed by inserting and ligating a polynucleotide encoding the MGLP and, if necessary, a control region into any vector by a conventional method. The type of the vector is not particularly limited and may be any vector such as a plasmid, phage, phagemid, cosmid, virus, YAC vector, shuttle vector, etc. Further, the vector is preferably a vector that can be amplified in the microorganism used in the present invention, and more preferably an expression vector. Examples of preferred vectors include, but are not limited to, pHA3040SP64, pHSP64R or pASP64 (Patent No. 3492935), pHY300PLK (an expression vector capable of transforming both Escherichia coli and Bacillus subtilis; Jpn J Genet, 1985, 60: 235-243), pAC3 (Nucleic Acids Res, 1988, 16: 8732) and other shuttle vectors; pUB110 (J Bacteriol, 1978, 134: 318-329), pTA10607 (Plasmid, 1987, 18: 8-15) and other plasmid vectors available for transformation of Bacillus bacteria, and the like. Also, plasmid vectors derived from Escherichia coli (for example, pET22b(+), pBR322, pBR325, pUC57, pUC118, pUC119, pUC18, pUC19, pBluescript, etc.) can be used.

[0044] When producing the microorganism used for the production of MGLP in the present invention, a polynucleotide encoding the target MGLP may be incorporated into a microorganism with reduced activity of AprE or a protease corresponding thereto, or a microorganism incorporated with a polynucleotide encoding the target MGLP may be modified to reduce the activity of AprE or a protease corresponding thereto. In any case, a recombinant microorganism of the present invention can be obtained in which the activity of AprE or a protease corresponding thereto is reduced and a foreign polynucleotide encoding monoacylglycerol lipase is incorporated.

[0045] By culturing the microorganism in which the activity of AprE obtained by the above procedure or a protease corresponding thereto is reduced to express MGLP, MGLP can be produced. The culturing of the microorganism for MGLP production can be carried out according to a general method in the art depending on the species and traits of the microorganism. For example, when the microorganism is a Bacillus bacterium, the medium for its culture contains a carbon source necessary for the growth of the Bacillus bacterium and an inorganic nitrogen source or an organic nitrogen source. Examples of the carbon source include glucose, dextran, soluble starch, sucrose, methanol, etc. Examples of the inorganic nitrogen source or the organic nitrogen source include ammonium salts, nitrates, amino acids, corn steep liquor, peptone, casein, meat extract, soybean meal, potato extract, etc. If necessary, the medium may contain other nutrients, such as inorganic salts (e.g., sodium chloride, calcium chloride, sodium dihydrogen phosphate, magnesium chloride), vitamins, antibiotics (e.g., tetracycline, neomycin, kanamycin, spectinomycin, erythromycin, etc.). The culture conditions, such as temperature, aeration and agitation conditions, pH of the medium, and culture time, etc., can be appropriately selected according to the species and traits of the microorganism, the culture scale, etc.

[0046] In the method for producing MGLP of the present invention, the MGLP expressed by the microorganism is released extracellularly. Therefore, the target MGLP can be recovered from the supernatant of the culture. For example, the culture supernatant is collected by centrifugation, filtration, etc., and then the target MGLP can be recovered from the culture supernatant by using a general method used for protein purification, such as ammonium sulfate precipitation, gel chromatography, ion exchange chromatography, affinity chromatography, etc. alone or in appropriate combination. Alternatively, the recovered or concentrated culture supernatant can be used as a crude enzyme solution as it is. In the method of the present invention, MGLP can be recovered without performing the step of disrupting the cultured cells.

[0047] The present invention also includes, as exemplary embodiments, the following substances, production methods, uses, methods, etc. However, the present invention is not limited to these embodiments.

[0048] 〔1〕A method for producing monoacylglycerol lipase, comprising culturing a Bacillus bacterium with reduced activity of AprE of Bacillus subtilis or a protease corresponding thereto to express monoacylglycerol lipase. 〔2〕The production method according to 〔1〕, further preferably comprising recovering the monoacylglycerol lipase from the supernatant of the culture. 〔3〕The production method according to 〔1〕 or 〔2〕, preferably wherein a polynucleotide encoding the monoacylglycerol lipase is introduced exogenously into the Bacillus bacterium. 〔4〕The polynucleotide encoding the monoacylglycerol lipase is preferably operably linked to a control region, preferably not linked to a region involved in protein secretion, more preferably operably linked to a transcription start control region and a translation start control region, even more preferably operably linked to a transcription start control region and a translation start control region and not linked to a region involved in protein secretion, The production method according to 〔3〕. 〔5〕The monoacylglycerol lipase is preferably a monoacylglycerol lipase belonging to Bacterial Lipolytic Enzyme Family XV, more preferably a non-secreted monoacylglycerol lipase derived from a Bacillus bacterium or a Geobacillus bacterium belonging to the Family XV, even more preferably a polypeptide consisting of an amino acid sequence of any one of SEQ ID NOs: 1 to 6, or a polypeptide encoding a polypeptide consisting of an amino acid sequence having at least 90% identity with the amino acid sequence of any one of SEQ ID NOs: 1 to 6 and having monoglyceride lipase activity, More preferably, it is a polypeptide consisting of the amino acid sequence of any one of SEQ ID NO: 1 and 2, or a polypeptide consisting of an amino acid sequence having at least 90% identity with the amino acid sequence of any one of SEQ ID NO: 1 and 2 and having monoglyceride lipase activity, or a polypeptide consisting of the amino acid sequence of any one of SEQ ID NO: 3 to 6, or a polypeptide consisting of an amino acid sequence having at least 90% identity with the amino acid sequence of any one of SEQ ID NO: 3 to 6 and having monoglyceride lipase activity, More preferably, it is a polypeptide consisting of the amino acid sequence of any one of SEQ ID NO: 1 and 2, or a polypeptide consisting of an amino acid sequence having at least 90% identity with the amino acid sequence of any one of SEQ ID NO: 1 and 2 and having monoglyceride lipase activity, The production method according to any one of [1] to [4]. 〔6〕Preferably, the Bacillus bacterium with reduced activity of the Bacillus subtilis AprE or a protease corresponding thereto is a Bacillus bacterium in which the Bacillus subtilis aprE or a gene corresponding thereto is deleted or inactivated, the production method according to any one of [1] to [5]. 〔7〕Preferably, the gene corresponding to aprE is a gene encoding a polypeptide having at least 90% identity with aprE (BG10190) in nucleotide sequence and having serine protease activity with an optimum pH on the alkaline side, the production method according to [6]. 〔8〕Preferably, the Bacillus bacterium is one in which at least one gene selected from the group consisting of Bacillus subtilis epr, wprA, mpr, nprB, bpr, nprE, vpr, aprX, and genes corresponding thereto is further deleted or inactivated, The production method according to [6] or [7]. 〔9〕Preferably, the genes corresponding to the Bacillus subtilis epr, wprA, mpr, nprB, bpr, nprE, vpr, and aprX are as follows, A gene corresponding to epr: a gene having at least 90% identity with epr (BG10561) in nucleotide sequence and encoding a minor extracellular serine protease; A gene corresponding to wprA: a gene having at least 90% identity with wprA (BG11846) in nucleotide sequence and encoding a cell wall-bound protease precursor; A gene corresponding to mpr: a gene having at least 90% identity with mpr (BG10690) in nucleotide sequence and encoding an extracellular metalloprotease; A gene corresponding to nprB: a gene having at least 90% identity with nprB (BG10691) in nucleotide sequence and encoding an extracellular neutral protease B; A gene corresponding to bpr: a gene having at least 90% identity with bpr (BG10233) in nucleotide sequence and encoding bacillopeptidase F; A gene corresponding to nprE: a gene having at least 90% identity with nprE (BG10448) in nucleotide sequence and encoding an extracellular neutral metalloprotease; A gene corresponding to vpr: a gene having at least 90% identity with vpr (BG10591) in nucleotide sequence and encoding a minor extracellular serine protease; A gene corresponding to aprX: a gene having at least 90% identity with aprX (BG12567) in nucleotide sequence and encoding an intracellular serine protease, The production method according to [8]. Preferably, the Bacillus bacterium is Bacillus subtilis or a mutant thereof. The production method according to any one of [1] to [9].

[0049] A recombinant Bacillus bacterium in which the activity of Bacillus subtilis AprE or a protease corresponding thereto is reduced and a polynucleotide encoding a monoacylglycerol lipase is introduced exogenously. The polynucleotide encoding the monoacylglycerol lipase is Preferably, it is operably linked to a control region, Preferably, it is not linked to a region involved in protein secretion, More preferably, it is operably linked to a transcription start control region and a translation start control region, Even more preferably, it is operably linked to a transcription start control region and a translation start control region and is not linked to a region involved in protein secretion, The recombinant Bacillus bacterium according to

[11] .

[13] The monoacylglycerol lipase is, Preferably, the monoacylglycerol lipase is a monoacylglycerol lipase belonging to Bacterial Lipolytic Enzyme Family XV, More preferably, it is a non-secreted monoacylglycerol lipase derived from a Bacillus bacterium or a Geobacillus bacterium belonging to the Family XV, Even more preferably, it is a polypeptide consisting of an amino acid sequence of any one of SEQ ID NOs: 1 to 6, or a polypeptide encoding a polypeptide consisting of an amino acid sequence having at least 90% identity with the amino acid sequence of any one of SEQ ID NOs: 1 to 6 and having monoglyceride lipase activity, Even more preferably, it is a polypeptide consisting of an amino acid sequence of any one of SEQ ID NOs: 1 and 2, or a polypeptide encoding a polypeptide consisting of an amino acid sequence having at least 90% identity with the amino acid sequence of any one of SEQ ID NOs: 1 and 2 and having monoglyceride lipase activity, or a polypeptide consisting of an amino acid sequence of any one of SEQ ID NOs: 3 to 6, or a polypeptide encoding a polypeptide consisting of an amino acid sequence having at least 90% identity with the amino acid sequence of any one of SEQ ID NOs: 3 to 6 and having monoglyceride lipase activity, Even more preferably, it is a polypeptide consisting of an amino acid sequence of any one of SEQ ID NOs: 1 and 2, or a polypeptide encoding a polypeptide consisting of an amino acid sequence having at least 90% identity with the amino acid sequence of any one of SEQ ID NOs: 1 and 2 and having monoglyceride lipase activity, The recombinant Bacillus bacterium according to

[11] or

[12] . 〔14〕Preferably, the recombinant Bacillus bacterium according to any one of

[11] to

[13] , wherein the Bacillus subtilis aprE or a gene corresponding thereto is deleted or inactivated. 〔15〕Preferably, the gene corresponding to aprE is a gene encoding a polypeptide having at least 90% identity in nucleotide sequence with aprE (BG10190) and having serine protease activity with an optimum pH on the alkaline side, and the recombinant Bacillus bacterium according to

[14] . 〔16〕Preferably, at least one gene selected from the group consisting of Bacillus subtilis epr, wprA, mpr, nprB, bpr, nprE, vpr, aprX, and genes corresponding thereto is further deleted or inactivated. The recombinant Bacillus bacterium according to

[14] or

[15] . 〔17〕Preferably, the genes corresponding to the Bacillus subtilis epr, wprA, mpr, nprB, bpr, nprE, vpr, and aprX are as follows. Gene corresponding to epr: a gene having at least 90% identity in nucleotide sequence with epr (BG10561) and encoding a minor extracellular serine protease. Gene corresponding to wprA: a gene having at least 90% identity in nucleotide sequence with wprA (BG11846) and encoding a cell wall-bound protease precursor. Gene corresponding to mpr: a gene having at least 90% identity in nucleotide sequence with mpr (BG10690) and encoding an extracellular metalloprotease. Gene corresponding to nprB: a gene having at least 90% identity in nucleotide sequence with nprB (BG10691) and encoding extracellular neutral protease B. Gene corresponding to bpr: a gene having at least 90% identity in nucleotide sequence with bpr (BG10233) and encoding bacillopeptidase F. A gene corresponding to nprE: a gene having at least 90% identity with nprE (BG10448) in nucleotide sequence and encoding an extracellular neutral metalloprotease; A gene corresponding to vpr: a gene having at least 90% identity with vpr (BG10591) in nucleotide sequence and encoding a minor extracellular serine protease; A gene corresponding to aprX: a gene having at least 90% identity with aprX (BG12567) in nucleotide sequence and encoding an intracellular serine protease, The recombinant Bacillus bacterium according to

[16] . The recombinant Bacillus bacterium according to any one of

[11] to

[17] , preferably, the Bacillus bacterium is Bacillus subtilis or a mutant thereof.

[0050] A method for producing a recombinant Bacillus bacterium, comprising incorporating a polynucleotide encoding a monoacylglycerol lipase into a Bacillus bacterium with reduced activity of Bacillus subtilis AprE or a protease corresponding thereto, or reducing the activity of Bacillus subtilis AprE or a protease corresponding thereto in a Bacillus bacterium into which a polynucleotide encoding a monoacylglycerol lipase has been incorporated. 〔20〕The polynucleotide encoding the monoacylglycerol lipase, preferably, is operably linked to a control region, preferably, is not linked to a region involved in protein secretion, more preferably, is operably linked to a transcription start control region and a translation start control region, even more preferably, is operably linked to a transcription start control region and a translation start control region and is not linked to a region involved in protein secretion, The method for producing a recombinant Bacillus bacterium according to

[19] . 〔21〕The monoacylglycerol lipase, Preferably, the monoacylglycerol lipase is a monoacylglycerol lipase belonging to Bacterial Lipolytic Enzyme Family XV, More preferably, it is a non-secreted monoacylglycerol lipase derived from a bacterium of the genus Bacillus or Geobacillus belonging to the Family XV, Even more preferably, it is a polypeptide consisting of the amino acid sequence of any one of SEQ ID NOs: 1 to 6, or a polypeptide consisting of an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NOs: 1 to 6 and has monoglyceride lipase activity, Even more preferably, it is a polypeptide consisting of the amino acid sequence of any one of SEQ ID NOs: 1 and 2, a polypeptide consisting of an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NOs: 1 and 2 and has monoglyceride lipase activity, a polypeptide consisting of the amino acid sequence of any one of SEQ ID NOs: 3 to 6, or a polypeptide consisting of an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NOs: 3 to 6 and has monoglyceride lipase activity, Even more preferably, it is a polypeptide consisting of the amino acid sequence of any one of SEQ ID NOs: 1 and 2, or a polypeptide consisting of an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NOs: 1 and 2 and has monoglyceride lipase activity, The method for producing a recombinant Bacillus bacterium according to

[19] or

[20] . Preferably, the method for producing a recombinant Bacillus bacterium according to any one of

[19] to

[21] , wherein the Bacillus subtilis aprE or a gene corresponding thereto is deleted or inactivated. Preferably, the gene corresponding to aprE is a gene encoding a polypeptide having serine protease activity with an optimal pH on the alkaline side and having at least 90% identity in nucleotide sequence with aprE (BG10190), the method for producing a recombinant Bacillus bacterium according to

[22] . [(24)] Preferably, at least one gene selected from the group consisting of Bacillus subtilis epr, wprA, mpr, nprB, bpr, nprE, vpr, aprX, and genes corresponding thereto is further deleted or inactivated. [(22)] or the method for producing a recombinant Bacillus bacterium according to [(23)]. [(25)] Preferably, the genes corresponding to the Bacillus subtilis epr, wprA, mpr, nprB, bpr, nprE, vpr, and aprX are as follows: Gene corresponding to epr: having at least 90% identity in nucleotide sequence with epr (BG10561) and encoding a minor extracellular serine protease; Gene corresponding to wprA: having at least 90% identity in nucleotide sequence with wprA (BG11846) and encoding a cell wall-bound protease precursor; Gene corresponding to mpr: having at least 90% identity in nucleotide sequence with mpr (BG10690) and encoding an extracellular metalloprotease; Gene corresponding to nprB: having at least 90% identity in nucleotide sequence with nprB (BG10691) and encoding an extracellular neutral protease B; Gene corresponding to bpr: having at least 90% identity in nucleotide sequence with bpr (BG10233) and encoding bacillopeptidase F; Gene corresponding to nprE: having at least 90% identity in nucleotide sequence with nprE (BG10448) and encoding an extracellular neutral metalloprotease; Gene corresponding to vpr: having at least 90% identity in nucleotide sequence with vpr (BG10591) and encoding a minor extracellular serine protease; Gene corresponding to aprX: having at least 90% identity in nucleotide sequence with aprX (BG12567) and encoding an intracellular serine protease, The method for producing a recombinant Bacillus bacterium described in

[24] . The method for producing a recombinant Bacillus bacterium according to any one of

[19] to

[25] , preferably, the Bacillus bacterium is Bacillus subtilis or a mutant strain thereof.

Examples

[0051] Hereinafter, the present invention will be described more specifically using examples. However, the technical scope of the present invention is not limited to these examples.

[0052] In the following examples, PCR was performed using PrimeSTAR Max Premix (Takara Bio). The In-fusion method was performed using the In-Fusion HD Cloning Kit (Takara Bio). Introduction of the plasmid into the host microorganism was performed by the protoplast method (Mol. Gen. Genet, 1979, 168: 111-115). For culturing recombinant microorganisms for protein production, LB medium (1% tryptone, 0.5% yeast extract, 1% NaCl), or 2×L-maltose medium (2% tryptone, 1% yeast extract, 1% NaCl, 7.5% maltose, 7.5 ppm manganese sulfate tetrahydrate) was used. Measurement of lipase activity in the culture supernatant was performed according to the following procedure.

[0053] (Method for measuring lipase activity) 5 μL of appropriately diluted culture supernatant was dispensed into each well of a 96-well assay plate (IWAKI). To each well, 183 μL of deionized water, 10 μL of 1 M Tris-HCl (pH 8.0), and 2 μL of substrate solution were further dispensed to initiate the reaction. As the substrate solution, a dimethyl sulfoxide (DMSO) solution containing 100 mM of 4-Nitrophenyl octanoate (SIGMA-ALDRICH) was used. The reaction was carried out at 40 °C for 15 minutes, and the absorbance at 405 nm of the reaction solution was measured using a microplate reader (TECAN). 1 U was defined as the amount of enzyme that liberates 1 μmol of pNA per minute at 40 °C.

[0054] Table 2 to 7 show the names and nucleotide sequences of the primers used in the following examples.

[0055]

Table 2

[0056]

Table 3

[0057]

Table 4

[0058]

Table 5

[0059]

Table 6

[0060]

Table 7

[0061] Example 1 Construction of a gene deletion Bacillus subtilis strain (1) Construction of an aprE deletion strain (Construction of a plasmid for gene deletion) Using the genomic DNA extracted from Bacillus subtilis strain 168 as a template, 0.6 kb fragment (A) adjacent to the upstream of the aprE gene on the genome and 0.5 kb fragment (B) adjacent to the downstream were respectively prepared using each primer set of aprEfw1 and aprEUp r (SEQ ID NOs: 27 and 28), and aprEDNf and aprErv-repU (SEQ ID NOs: 29 and 30) shown in Table 3. Separately, a 1.2 kb fragment (C) was prepared by ligating the promoter region of the repU gene (Nucleic Acids Res, 1989, 17:4410) derived from plasmid pUB110 (Plasmid, 1986, 15:93-103) upstream of the chloramphenicol resistance gene derived from plasmid pC194 (J. Bacteriol, 150(2), 815, 1982). In the preparation of fragment (C), first, a 0.4 kb fragment (D) containing the repU gene promoter region was prepared using the primer set of repUfw and repUr-Cm (SEQ ID NOs: 32 and 33, Table 3) and plasmid pUB110 as a template. Further, an 0.8 kb fragment (E) containing the chloramphenicol resistance gene was prepared using the primer set of CmUf-rep and Cmrv1 (SEQ ID NOs: 34 and 35, Table 3) and plasmid pC194 as a template. Then, fragment (C) was prepared by SOE-PCR using the mixture of fragments (D) and (E) as a template and primers repUfw and Cmrv1. Next, using the mixture of the obtained fragments (A), (B) and (C) as a template, the fragments were ligated in the order of (A)(B)(C) by SOE-PCR using primers aprEfw2 and Cmrv2 (SEQ ID NOs: 31 and 36, Table 3) to obtain a 2.2 kb DNA fragment. The ends of this DNA fragment were blunted and 5'-phosphorylated, and inserted into the SmaI restriction enzyme site of plasmid pUC118 (Methods Enzymol, 1987, 153:3-11) to construct the plasmid pUC118-CmrΔaprE for aprE gene deletion.

[0062] (Construction of gene deletion strain) The constructed plasmid pUC118-CmrΔaprE was introduced into the Bacillus subtilis mutant trpC2 (trpC2 strain) by the competent cell transformation method (J Bacteriol, 1967, 93, 1925-1937). A transformant in which the plasmid and genomic DNA were fused by single crossover homologous recombination between the corresponding regions of the upstream or downstream region of the aprE gene was obtained using chloramphenicol resistance as an indicator. The obtained transformant was inoculated into LB medium and cultured at 37°C for 2 hours, and then the competence induction operation was performed again to induce intragenomic homologous recombination between the upstream or downstream region of the aprE gene that overlapped on the introduced plasmid and the genome. When homologous recombination occurred in a region different from that at the time of plasmid introduction, the aprE gene and the chloramphenicol resistance gene derived from the plasmid were deleted.

[0063] Next, in order to increase the proportion of chloramphenicol-sensitive strains, the ampicillin concentration enrichment operation was performed as follows. The culture solution after competent cell induction was inoculated into 1 mL of LB medium containing chloramphenicol at a final concentration of 5 ppm and sodium ampicillin at a final concentration of 100 ppm so that the turbidity (OD600) at 600 nm was 0.003. After culturing at 37°C for 5 hours, 10 μL of a 10,000 ppm aqueous solution of sodium ampicillin was added and further cultured for 3 hours. After the culture was completed, the cells were centrifuged and washed with a 2% sodium chloride aqueous solution, then suspended in 1 mL of a 2% sodium chloride aqueous solution, and 100 μL of the suspension was spread on an LB agar medium. After incubation at 37°C for about 15 hours, among the grown strains, those that became chloramphenicol-sensitive due to the deletion of the plasmid region were selected. Using the genomic DNA of the selected strain as a template, PCR was performed using the primers aprEfw2 and aprErv-repU (SEQ ID NOs: 31 and 30, Table 3) to confirm the deletion of the aprE gene, and the aprE gene deletion strain trpC2 / ΔaprE was obtained.

[0064] (2) Construction of epr, wprA, mpr, nprB, bpr, nprE or vpr deletion strains By repeating the same operation as (1), gene deletion strains trpC2 / Δepr, trpC2 / ΔwprA, trpC2 / Δmpr, trpC2 / ΔnprB, trpC2 / Δbpr, trpC2 / ΔnprE, and trpC2 / Δvpr in which each gene of epr, wprA, mpr, nprB, bpr, nprE, and vpr was deleted from the trpC2 strain were obtained. The sequences of the primers used for the preparation of each deletion strain are shown in Tables 4 to 5, and the correspondence between each primer and the aprE gene deletion primer used in (1) is shown in Table 6.

[0065] (3) Construction of aprX deletion strain An aprX gene deletion strain in which the aprX gene was replaced with a spectinomycin resistance gene was constructed by the double crossover method. Using the genomic DNA extracted from Bacillus subtilis strain 168 as a template, with each primer set of aprX+5F and aprX+563R (SEQ ID NOs: 72 and 73), and aprX+775F and aprX+1320R (SEQ ID NOs: 74 and 75) shown in Table 7, a 558 bp fragment (F) containing the upstream of the aprX gene on the genome at the 5'-terminal side, and a 545 bp fragment (G) at the 3'-terminal side were respectively prepared. Separately, the spectinomycin resistance gene region was excised from the BamHI and XhoI restriction enzyme cleavage sites of plasmid pDG1727 (Gene, 167, 335, 1995) and inserted into the BamHI and XhoI restriction enzyme cleavage sites of pBluescript II SK(+) (Stratagene) to construct plasmid pBlueSPR. Using the DNA of pBlueSPR as a template, the spectinomycin resistance gene region (H) was amplified with the primer set of PB-M13-20 and PB-M13Rev (SEQ ID NOs: 76 and 77, Table 7). Using the mixture of (F), (G) and (H) fragments as a template, a DNA fragment ligated in the order of (E)(H)(G) was prepared by the SOE-PCR method using the primer set of aprX+5F and aprX+1320R (SEQ ID NOs: 72 and 75, Table 7). Using the prepared DNA fragment, transformation of the trpC2 strain was carried out by the competent cell transformation method, and then colonies grown on an LB agar medium containing spectinomycin (100 μg / mL) were separated as transformants. The genome of the obtained transformant was extracted, and it was confirmed by PCR that the aprX gene was deleted and replaced with the spectinomycin resistance gene, and the aprX gene deletion strain trpC2 / ΔaprX was obtained.

[0066] Example 2 Comparison of MGLP Productivity of Various Gene Deletion Strains (1) Construction of EstGtA2-Expressing Recombinant Bacillus subtilis Strains A recombinant Bacillus subtilis strain was constructed by introducing the gene encoding monoacylglycerol lipase EstGtA2 (SEQ ID NO: 1) into the gene deletion strain prepared in Example 1. A plasmid (EstGtA2-pUC57) in which the gene encoding codon-optimized EstGtA2 for Bacillus subtilis (SEQ ID NO: 7) was inserted into plasmid pUC57 was prepared using the artificial gene synthesis service of GenScript. Using this as a template, PCR was performed using the primer set of primer EstGtA2-F and primer EstGtA2-R (SEQ ID NOs: 13 and 14, Table 2). Similarly, using plasmid pHY-S237 described in Example 7 of WO2006 / 068148A1 as a template, a PCR reaction was performed using primers pHY-S237-F and pHY-S237-R (SEQ ID NOs: 15 and 16, Table 2). Each PCR product was treated with DpnI (New England Biolabs). An appropriate amount of the two obtained fragments was mixed, and a plasmid was synthesized using the In-fusion method (pHY-EstGtA2). At this time, it was designed so that the control region of cellulase S237 (a region containing a transcription start control region and a translation start control region, SEQ ID NO: 78) and the gene of EstGtA2 were operably linked. The obtained plasmid was introduced into the trpC2 strain (control) and the gene deletion strains trpC2 / ΔaprE, trpC2 / Δepr, trpC2 / ΔwprA, trpC2 / Δmpr, trpC2 / ΔnprB, trpC2 / Δbpr, trpC2 / ΔnprE, trpC2 / Δvpr, and trpC2 / ΔaprX prepared in Example 1 by the protoplast transformation method.

[0067] (2) Comparison of MGLP productivity (1) The recombinant Bacillus subtilis strain obtained in (1) was cultured with shaking at 30 °C for 15 hours at 250 rpm in 3 mL of LB medium in a large test tube (φ18×180 mm). 0.02 mL of the obtained culture solution was inoculated into 20 mL of 2×L - maltose medium in a 500 mL Erlenmeyer flask with baffles (Biot), and cultured with shaking at 30 °C for 96 hours at 210 rpm. After culturing, the culture solution was centrifuged (8000 rpm × 30 minutes), and the cells were removed from the obtained supernatant using a filter (0.45 μm, PVDF, manufactured by Merck Millipore), and the lipase activity of the remaining culture supernatant was measured. Also, the accumulation of EstGtA2 in the culture supernatant was confirmed by SDS - PAGE. In SDS - PAGE, protein bands were confirmed using Bio - Safe CBB G - 250 stain (Bio - Rad).

[0068] Figure 1 shows the lipase activity in the culture supernatant obtained after culturing each recombinant Bacillus subtilis strain for 96 hours. In the figure, the horizontal axis shows the hosts of the recombinant Bacillus subtilis strains (trpC2, trpC2 / Δepr, trpC2 / ΔwprA, trpC2 / Δmpr, trpC2 / ΔnprB, trpC2 / Δbpr, trpC2 / ΔnprE, trpC2 / Δvpr, trpC2 / ΔaprE, trpC2 / ΔaprX), and the vertical axis shows the lipase activity in the culture supernatant for each strain. The lipase activity values are shown as relative activities with the activity of the tprC2 strain set as 100%. The lipase activity in the culture supernatant was significantly improved in the aprE - deficient strain. Figure 2 shows the results of SDS - PAGE in the culture supernatant after culturing each recombinant Bacillus subtilis strain for 96 hours. Each lane is indicated by the deleted gene of the recombinant Bacillus subtilis strain. EstGtA2 was accumulated in the culture supernatant in the aprE - deficient strain, while the accumulation of EstGtA2 in the culture supernatant could not be confirmed in other deficient strains. From the above results, it was found that by using a strain in which the aprE gene or a gene corresponding thereto was deleted or inactivated, the monoacylglycerol lipase EstGtA2, which is originally an intracellular enzyme, can be secreted extracellularly and recovered from the culture supernatant.

[0069] Example 3 Productivity of various MGLPs by aprE - deficient strain (1) Construction of MGLP - expressing recombinant Bacillus subtilis strain Recombinant Bacillus subtilis strains expressing MGLP H-257, stLP, CI220-6, CI220-10, and CI220-12 (SEQ ID NOs: 2-6) belonging to the Bacterial Lipolytic Enzyme Family XV were constructed. Genes encoding each MGLP codon-optimized for Bacillus subtilis (SEQ ID NOs: 8-12) were inserted into plasmid pUC57 to produce plasmids (H-257-pUC57, stLP-pUC57, CI220-6-pUC57, CI220-10-pUC57, and CI220-12-pUC57) using GenScript's artificial gene synthesis service. Using the prepared plasmids as templates, PCR was performed using the primer pairs H257-F / R (SEQ ID NOs: 17 and 18), stLP-F / R (SEQ ID NOs: 19 and 20), CI220-6-F / R (SEQ ID NOs: 21 and 22), CI220-10-F / R (SEQ ID NOs: 23 and 24), and CI220-12-F / R (SEQ ID NOs: 25 and 26) shown in Table 2, respectively. Similarly, using plasmid pHY-S237 as a template, a PCR reaction was performed using primers pHY-S237-F and pHY-S237-R (SEQ ID NOs: 15 and 16). Similar to Example 2(1), the PCR products were treated with DpnI, and plasmids were synthesized using the In-fusion method (pHY-H-257, pHY-stLP, pHY-CI220-6, pHY-CI220-10, and pHY-CI220-12), and the obtained plasmids were introduced into the trpC2 strain and the trpC2 / ΔaprE strain by the protoplast transformation method.

[0070] (2) MGLP productivity of aprE-deleted recombinant Bacillus subtilis strains (1) The recombinant Bacillus subtilis strains obtained were shake-cultured overnight at 30 °C in 1 mL of LB medium. 0.003 mL of the obtained culture solution was inoculated into 3 mL of 2×L-maltose medium and shake-cultured at 30 °C for 72 hours. After culturing, the culture supernatant was separated by the same procedure as in Example 2(2), and the amount of MGLP produced extracellularly was confirmed by SDS-PAGE. Protein bands were confirmed using a Mini-PROTEAN TGX Stain-Free gel (Bio-Rad).

[0071] Figure 3 shows the results of SDS-PAGE in the culture supernatant after 72-hour culture for each recombinant Bacillus subtilis strain. Each lane is labeled with the deleted gene of the recombinant Bacillus subtilis strain and the type of MGLP expressed. Any of the MGLPs to be expressed (H-257, stLP, CI220-6, CI220-10, and CI220-12) accumulated in the culture supernatant of the aprE deletion strain, while no accumulation of any MGLP was confirmed in the culture supernatant of the trpC2 strain. From the above results, it was found that by using a strain in which the aprE gene or a gene corresponding thereto was deleted or inactivated, various MGLPs, which are intracellular enzymes, can be secreted extracellularly and recovered from the culture supernatant.

[0072] As described above, the embodiments of the present invention have been described, but it should be understood that these are not intended to limit the present invention to the specific embodiments described. Various other changes and modifications within the scope of the present invention will be apparent to those skilled in the art. The documents and patent applications cited in this specification are incorporated by reference as if fully set forth herein.

Claims

1. A method for producing monoacylglycerol lipase, comprising culturing a Bacillus bacterium with reduced activity of Bacillus subtilis AprE or a protease corresponding thereto to express monoacylglycerol lipase, wherein the Bacillus subtilis AprE is subtilisin E, a serine alkaline protease encoded by the Bacillus subtilis gene aprE, the protease corresponding to the Bacillus subtilis AprE is a polypeptide having at least 90% identity in amino acid sequence with the Bacillus subtilis AprE and having serine protease activity with an optimal pH on the alkaline side, and the monoacylglycerol lipase is a polypeptide consisting of the amino acid sequence of any one of SEQ ID NOs: 1 and 2, or a polypeptide consisting of an amino acid sequence having at least 90% identity with the amino acid sequence of any one of SEQ ID NOs: 1 and 2. Method.

2. The production method according to claim 1, further comprising recovering the monoacylglycerol lipase from the supernatant of the culture.

3. The production method according to claim 1 or 2, wherein a polynucleotide encoding the monoacylglycerol lipase is introduced exogenously into the Bacillus bacterium.

4. The production method according to claim 3, wherein a polynucleotide encoding the monoacylglycerol lipase is operably linked to a control region.

5. The production method according to claim 3 or 4, wherein a polynucleotide encoding the monoacylglycerol lipase is not linked to a polynucleotide region encoding a secretion signal peptide.

6. The Bacillus bacterium with reduced activity of the Bacillus subtilis AprE or a protease corresponding thereto is a Bacillus bacterium in which the Bacillus subtilis gene aprE or a gene corresponding thereto is deleted or inactivated, and the gene corresponding to aprE is a gene encoding a polypeptide having at least 90% identity in nucleotide sequence with the aprE and having serine protease activity with an optimal pH on the alkaline side. The production method according to any one of claims 1 to 5.

7. The production method according to claim 6, wherein the Bacillus bacterium has at least one gene selected from the group consisting of Bacillus subtilis epr, wprA, mpr, nprB, bpr, nprE, vpr, aprX, and genes corresponding thereto further deleted or inactivated.

8. The production method according to any one of claims 1 to 7, wherein the Bacillus bacterium is Bacillus subtilis or a mutant strain thereof.

9. A recombinant Bacillus bacterium in which the activity of Bacillus subtilis AprE or a protease corresponding thereto is reduced, and a polynucleotide encoding monoacylglycerol lipase is introduced exogenously, wherein the Bacillus subtilis AprE is subtilisin E, a serine alkaline protease encoded by the Bacillus subtilis gene aprE, the protease corresponding to the Bacillus subtilis AprE is a polypeptide having at least 90% identity in amino acid sequence with the Bacillus subtilis AprE and having serine protease activity with an optimum pH on the alkaline side, the monoacylglycerol lipase is a polypeptide consisting of the amino acid sequence of any one of SEQ ID NOs: 1 and 2, or a polypeptide consisting of an amino acid sequence having at least 90% identity with the amino acid sequence of any one of SEQ ID NOs: 1 and 2, a recombinant Bacillus bacterium.

10. The recombinant Bacillus bacterium according to claim 9, wherein the polynucleotide encoding the monoacylglycerol lipase is operably linked to a control region.

11. The recombinant Bacillus bacterium according to claim 9 or 10, wherein the polynucleotide encoding the monoacylglycerol lipase is not linked to a polynucleotide region encoding a secretion signal peptide.

12. The Bacillus subtilis gene aprE or a gene corresponding thereto is deleted or inactivated, and the gene corresponding to the aprE is a gene encoding a polypeptide having at least 90% identity in nucleotide sequence with the aprE and having serine protease activity with an optimum pH on the alkaline side, the recombinant Bacillus bacterium according to any one of claims 9 to 11.

13. The recombinant Bacillus bacterium according to claim 12, wherein at least one gene selected from the group consisting of Bacillus subtilis epr, wprA, mpr, nprB, bpr, nprE, vpr, aprX, and genes corresponding thereto is further deleted or inactivated.

14. The recombinant Bacillus bacterium according to any one of claims 9 to 13, wherein the Bacillus bacterium is Bacillus subtilis or a mutant strain thereof.

15. A method for producing a recombinant Bacillus bacterium, comprising incorporating a polynucleotide encoding a monoacylglycerol lipase into a Bacillus bacterium in which the activity of Bacillus subtilis AprE or a protease corresponding thereto is reduced, or reducing the activity of Bacillus subtilis AprE or a protease corresponding thereto in a Bacillus bacterium incorporated with a polynucleotide encoding a monoacylglycerol lipase, wherein the Bacillus subtilis AprE is subtilisin E, a serine alkaline protease encoded by the Bacillus subtilis gene aprE, the protease corresponding to the Bacillus subtilis AprE is a polypeptide having at least 90% identity with the Bacillus subtilis AprE in the amino acid sequence and having a serine protease activity with an optimum pH on the alkaline side, and the monoacylglycerol lipase is a polypeptide consisting of the amino acid sequence of any one of SEQ ID NOs: 1 and 2, or a polypeptide consisting of an amino acid sequence having at least 90% identity with the amino acid sequence of any one of SEQ ID NOs: 1 and 2, method.

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