JP7702230B2 - Method for producing monoacylglycerol lipase - Google Patents

Description

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

Monoacylglycerol (MAG) is a substance that is widely used in the fields of food, medicine, cosmetics, etc. as an emulsifier, antibacterial agent, etc. Furthermore, in recent years, MAG has been found to have excellent physiological activities such as suppressing the rise in blood triglycerides after meals, suppressing the rise in blood insulin after meals, and suppressing postprandial GIP secretion. MAG is chemically produced by a method of esterifying fatty acids and glycerin in the presence of an alkaline catalyst under high temperature conditions, or a method of glycerolysis of oils and fats under high temperature and high pressure conditions. However, these methods require equipment for high temperature treatment and drainage. On the other hand, a biochemical method of producing MAG by reacting fatty acids and glycerin 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 fatty acid monoglycerides or fatty acid diglycerides as a substrate, such as monoglyceride lipase (MGLP), is preferable.

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

MGLP derived 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 mass-produced efficiently by culturing an Escherichia coli strain into which the H-257 lipase gene has been introduced and collecting MGLP from the culture. However, conventional methods require a step of disrupting the cultured cells in order to recover the produced MGLP, making the procedure complicated.

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

JP 2008-220236 A JP 2010-183873 A Japanese Patent Application Publication No. 5-168468 JP 2004-313169 A JP 2006-174707 A JP 2012-115219 A Special Publication No. 2011-508610 Special Publication No. 2016-521579

PLoS ONE, 2013, 8(10):e76675

The present invention provides a method for producing monoacylglycerol lipase.

The present inventors have found that, in the production of monoacylglycerol lipase, which is naturally non-secreted, by culturing a Bacillus bacterium in which the activity of the secretory protease AprE is reduced to produce the monoacylglycerol lipase, the produced monoacylglycerol lipase can be recovered from the culture supernatant without a cell disruption step.

Therefore, the present invention provides a method for producing monoacylglycerol lipase, which comprises culturing a Bacillus bacterium having reduced activity of Bacillus subtilis AprE or a protease equivalent thereto, and expressing monoacylglycerol lipase.
The present invention also provides a recombinant Bacillus bacterium in which the activity of Bacillus subtilis AprE or a protease equivalent thereto has been reduced, and into which a polynucleotide encoding monoacylglycerol lipase has been exogenously introduced.
The present invention also provides a method for producing a recombinant Bacillus bacterium, which comprises incorporating a polynucleotide encoding monoacylglycerol lipase into a Bacillus bacterium having reduced activity of Bacillus subtilis AprE or a protease equivalent thereto.

According to the present invention, monoacylglycerol lipase, which is naturally non-secreted, can be secreted outside the microbial cells and easily recovered from the culture supernatant without a process of disrupting the cells after culture. According to the present invention, monoacylglycerol lipase can be produced by a simple procedure.

Relative lipase activity in culture supernatants obtained after 96 hours of culture of various gene-deleted recombinant Bacillus subtilis strains. SDS-PAGE results of the culture supernatant obtained after culturing various gene-deleted recombinant Bacillus subtilis strains for 96 hours. The arrow indicates the EstGtA2 band. SDS-PAGE results of the culture supernatant of aprE-deleted recombinant Bacillus subtilis strains expressing various MGLPs. Arrows indicate, from the left, the bands of H-257, stLP, CI220-6, CI220-10, and CI220-12.

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

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 Search homology program of the genetic information processing software Genetyx-Win with a unit size to compare (ktup) of 2.

As used herein, "at least 90% identity" with respect to an amino acid sequence or a nucleotide sequence means identity of 90% or more, preferably 95% or more, more preferably 96% or more, even more preferably 97% or more, even more preferably 98% or more, and even more preferably 99% or more.

In addition, in this specification, "operably linked" between a regulatory region and a gene means that the gene and regulatory region are linked in such a way that the gene can be expressed under the control of the regulatory region. The procedure for "operably linked" between a gene and a regulatory region is well known to those skilled in the art.

In this specification, "upstream" and "downstream" in relation to a gene refer to the 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 a gene" means the region on the 5' side of the gene in the DNA sense strand.

As used herein, the term "native" when referring to a function, property, or trait of a cell is used to indicate that the function, property, or trait is inherently present in the cell. In contrast, the term "exogenous" is used to indicate a function, property, or trait that is not inherently present in the cell, but is introduced from outside. For example, a "exogenous" gene or polynucleotide is a gene or polynucleotide that is introduced into a cell from outside. An exogenous gene or polynucleotide may be from the same organism as the cell into which it is introduced, or from a different organism (i.e., a heterologous gene or polynucleotide).

The method for producing monoacylglycerol lipase (hereinafter also referred to as MGLP) of the present invention includes culturing a microorganism in which the activity of a specific secretory protease (also referred to as an extracellular protease) is reduced, and expressing monoacylglycerol lipase.

The microorganisms used in the production of MGLP in the present invention include Bacillus bacteria such as Bacillus subtilis, Clostridium bacteria, yeast, etc., among which Bacillus bacteria are preferred. Furthermore, Bacillus subtilis or its mutant strains are more preferred because the complete genome information has been revealed, genome engineering technology has been established, and it has the ability to produce and secrete proteins outside the bacterial cell.

Examples of secreted proteases whose activity is reduced in the microorganisms 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 designated by gene number BG10190 in the BSORF DB.

Proteases corresponding to AprE include proteases that have at least 90% identity to AprE in amino acid sequence and have the same function as AprE, and preferably include a polypeptide having serine protease activity with an optimal pH on the alkaline side (i.e., serine alkaline protease) that is encoded by a gene corresponding to aprE. In this specification, a gene corresponding to aprE refers to a gene that has at least 90% identity to aprE in nucleotide sequence and encodes a polypeptide having serine protease activity with an optimal pH on the alkaline side. Specific examples of proteases equivalent to AprE include peptidase S8 from Bacillus mojavensis, peptidase S8 from Bacillus halotolerans, and peptidase S8 from Bacillus tequilensis.

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

The gene numbers of epr, wprA, mpr, nprB, bpr, nprE, vpr, and aprX and the functions of the proteins they encode are shown in Table 1. Genes corresponding to epr, wprA, mpr, nprB, bpr, nprE, vpr, and aprX include genes that have at least 90% identity in nucleotide sequence with epr, wprA, mpr, nprB, bpr, nprE, vpr, and aprX, respectively, and encode proteins with the same functions (described in Table 1).

The activity of a target protein of a microorganism, such as AprE or an equivalent protease or other native proteases, can be reduced by reducing the expression level of the target protein in the microorganism. For example, the activity of the target protein can be reduced by suppressing the expression of the target protein by deleting or inactivating a gene encoding the target protein in the microorganism (i.e., a target gene, e.g., aprE or an equivalent gene), inactivating the mRNA transcribed from the target gene, or inhibiting the translation of the mRNA of the target gene into protein. Alternatively, the activity of the expressed target protein may be reduced.

The 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, or mutating the transcription or translation initiation region of the target gene. 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 deleting or inactivating a gene in a cell, and then evaluating the expression level or activity of the target protein, or performing genetic analysis to select cells having the desired mutation.

For example, a method using homologous recombination may be used to specifically delete or inactivate a target gene. That is, a DNA fragment of a target gene into which an inactivating mutation has been introduced by base substitution or base insertion, or a DNA fragment containing an outer region of the target gene but not the target gene, is constructed, and the DNA fragment is introduced into a parent microbial cell to cause homologous recombination in a region of the parent microbial genome containing the target gene, thereby deleting or inactivating the target gene on the genome. Alternatively, a recombinant vector (plasmid, etc.) having a DNA fragment containing a partial region of the target gene can be introduced into a parent microbial cell, and a partial region of the target gene in the parent microbial genome can be disrupted by homologous recombination, thereby inactivating the target gene. Methods for randomly deleting or inactivating genes in cells include a method in which a DNA fragment in which an inactivating mutation has been introduced is randomly cloned into a cell, and homologous recombination occurs between the gene and the genome of the cell, and a method in which a cell is irradiated with ultraviolet light, gamma rays, etc. to induce mutations. An inactivating mutation in a gene refers to a mutation that causes the target gene to lose its original function due to a silence 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 whose original activity is impaired.

A method for producing a DNA fragment containing a target gene into which an inactivating mutation has been introduced includes site-specific mutagenesis. Site-specific mutagenesis can be carried out using a mutation primer containing the nucleotide mutation to be introduced. For example, DNA fragments are produced by amplifying the upstream and downstream sides of a region containing the target gene by PCR using two pairs of primers containing the nucleotide mutation to be introduced as a template, and then these are linked together by SOE-PCR (splicing by overlap extension PCR) (Gene, 1989, 77(1): p61-68), thereby constructing a DNA fragment containing the desired mutation. Alternatively, site-specific mutagenesis can be performed using inverse PCR or annealing methods (Muramatsu et al., eds., "Revised 4th Edition New Genetic Engineering Handbook," Yodosha, pp. 82-88), or commercially available site-specific mutagenesis kits such as Stratagene's QuickChange II Site-Directed Mutagenesis Kit and QuickChange Multi Site-Directed Mutagenesis Kit.

The mutation 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.

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

Cells in which the target gene has been deleted or inactivated can be selected by confirming their genomic sequence. Alternatively, cells in which the target gene has been deleted or inactivated can be selected using the expression level or activity of the target protein as an indicator.

Methods for inactivating the mRNA of a target gene in a cell include target-specific mRNA inhibition using siRNA. Methods for reducing the activity of an expressed target protein include the use of an inhibitor of the target protein.

The microorganism used in the MGLP production of the present invention may be a microorganism that originally has the ability to produce MGLP, or may be a microorganism that has been modified to acquire or improve the ability to produce MGLP. A method for acquiring or improving the MGLP production ability of a microorganism includes a method of incorporating a polynucleotide encoding the MGLP of interest into the microorganism. In a preferred embodiment, the microorganism used in the MGLP production of the present invention is a recombinant microorganism into which an exogenous polynucleotide encoding the MGLP of interest to be produced has been incorporated.

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

More preferred examples of MGLP produced by the present invention include a polypeptide consisting of the 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. More preferred examples of MGLP produced by the present invention include a polypeptide consisting of the 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.

In this specification, "monoglyceride lipase activity" refers to the activity of more selectively decomposing monoacylglycerol into glycerin and fatty acids compared to the activity of decomposing triacylglycerol into diacylglycerol and fatty acids and the activity of decomposing diacylglycerol into monoacylglycerol and fatty acids, or the activity of more selectively synthesizing monoacylglycerol compared to triacylglycerol and diacylglycerol in the synthetic reaction between glycerin and fatty acids.

As described in Non-Patent Document 1, it has been proposed that the MGLP belonging to the 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 is MGLP belonging to the Family abH11.03.

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

Preferably, the polynucleotide encoding the MGLP is operably linked to a control region involved in the transcription or translation of a gene. Examples of the control region include a transcription initiation control region and a translation initiation control region. Examples of the transcription initiation control region include a promoter and a transcription start point. Examples of the translation initiation 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 initiation control region and a translation initiation control region. More preferably, the polynucleotide encoding the MGLP is operably linked to a transcription initiation control region and a translation initiation control region. On the other hand, the polynucleotide encoding the MGLP does not need to be linked to a region involved in the secretion of a protein (e.g., a secretory signal peptide), and is preferably not linked to a region involved in the secretion of the protein.

Examples of the control region include, but are not limited to, the control region of the α-amylase gene derived from a bacterium of the genus Bacillus, the control region of a 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 (JP 2000-210081 A), the control region of the cellulase gene of Bacillus sp. KSM-64 strain (JP 2011-103875 A), the control region of the kanamycin resistance gene derived from Staphylococcus aureus, and the control region of the chloramphenicol resistance gene (for all, see JP 2009-089708 A). More preferred 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). In addition, preferred control regions include nucleotide sequences that have at least 90% identity with SEQ ID NO: 78 or 79 and have the function of controlling gene transcription and translation. The polynucleotide encoding MGLP can be linked to the control region by the restriction enzyme method, the above-mentioned SOE-PCR, or the like.

To introduce the polynucleotide encoding the MGLP into a microorganism, a DNA fragment or vector containing the polynucleotide encoding the MGLP and, if necessary, a control region may be constructed and introduced into the microorganism. The method for introducing the DNA fragment or vector into the microbial cell is as described above.

The vector can be constructed by inserting and linking the 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, a phage, a phagemid, a cosmid, a virus, a YAC vector, or a shuttle vector. The vector is preferably a vector that can be amplified in the microorganism used in the present invention, and more preferably an expression vector. Preferred examples of the vector include, but are not limited to, shuttle vectors such as 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), and pAC3 (Nucleic Acids Res, 1988, 16:8732); and plasmid vectors that can be used for transformation of bacteria of the genus Bacillus, such as pUB110 (J Bacteriol, 1978, 134:318-329) and pTA10607 (Plasmid, 1987, 18:8-15). Plasmid vectors derived from Escherichia coli (e.g., pET22b(+), pBR322, pBR325, pUC57, pUC118, pUC119, pUC18, pUC19, pBluescript, etc.) can also be used.

When producing a microorganism used to produce MGLP in the present invention, a polynucleotide encoding the MGLP of interest may be incorporated into a microorganism in which the activity of AprE or a protease equivalent thereto is reduced, or a microorganism in which a polynucleotide encoding the MGLP of interest has been incorporated may be modified to reduce the activity of AprE or a protease equivalent thereto. In either case, a recombinant microorganism of the present invention can be obtained in which the activity of AprE or a protease equivalent thereto is reduced and an exogenous polynucleotide encoding monoacylglycerol lipase has been incorporated.

MGLP can be produced by culturing the microorganisms with reduced activity of AprE or a protease corresponding thereto obtained by the above procedure and expressing MGLP. The microorganisms for producing MGLP can be cultured according to a general method in the field depending on the species and characteristics of the microorganisms. For example, when the microorganism is a Bacillus genus bacterium, the medium for culturing the microorganism contains a carbon source necessary for the growth of the Bacillus genus bacterium, and an inorganic or organic nitrogen source. Examples of carbon sources include glucose, dextran, soluble starch, sucrose, and methanol. Examples of inorganic or organic nitrogen sources include ammonium salts, nitrates, amino acids, corn steep liquor, peptone, casein, meat extract, soybean meal, and potato extract. 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.), etc. Culture conditions, such as temperature, aeration and agitation conditions, pH of the medium, and culture time, can be appropriately selected depending on the species and characteristics of the microorganism, the culture scale, etc.

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

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

[1] A method for producing monoacylglycerol lipase, comprising culturing a Bacillus bacterium in which Bacillus subtilis AprE or a protease activity equivalent thereto has been reduced, and expressing monoacylglycerol lipase.
[2] The method according to [1], further comprising recovering the monoacylglycerol lipase from the culture supernatant.
[3] The production method according to [1] or [2], wherein a polynucleotide encoding the monoacylglycerol lipase is exogenously introduced into the Bacillus bacterium.
[4] 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 the secretion of a protein,
More preferably, it is operably linked to a transcription initiation control region and a translation initiation control region,
More preferably, it is operably linked to a transcription initiation control region and a translation initiation control region, and is not linked to a region involved in the secretion of a protein.
The manufacturing method described in [3].
[5] The monoacylglycerol lipase,
Preferably, the monoacylglycerol lipase is a monoacylglycerol lipase belonging to Bacterial Lipolytic Enzyme Family XV,
More preferably, the non-secretory monoacylglycerol lipase is derived from a bacterium of the genus Bacillus or Geobacillus belonging to Family XV,
More preferably, it is a polypeptide consisting of any one of the amino acid sequences of SEQ ID NOs: 1 to 6, or a polypeptide consisting of an amino acid sequence that is at least 90% identical to any one of the amino acid sequences of SEQ ID NOs: 1 to 6 and encoding a polypeptide having monoglyceride lipase activity,
More preferably, the polypeptide 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 at least 90% identical to the amino acid sequence of any one of SEQ ID NOs: 1 and 2 and encoding a polypeptide having monoglyceride lipase activity, a polypeptide consisting of an amino acid sequence of any one of SEQ ID NOs: 3 to 6, or a polypeptide consisting of an amino acid sequence at least 90% identical to the amino acid sequence of any one of SEQ ID NOs: 3 to 6 and encoding a polypeptide having monoglyceride lipase activity,
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 encoding a polypeptide having monoglyceride lipase activity.
The method according to any one of [1] to [4].
[6] The production method according to any one of [1] to [5], wherein the Bacillus bacterium in which the activity of Bacillus subtilis AprE or a protease corresponding thereto is reduced is a Bacillus bacterium in which Bacillus subtilis aprE or a gene corresponding thereto has been deleted or inactivated.
[7] The production method according to [6], wherein the gene corresponding to aprE preferably has at least 90% identity in nucleotide sequence to aprE (BG10190) and encodes a polypeptide having serine protease activity with an optimal pH on the alkaline side.
[8] The Bacillus bacterium is preferably 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 has been further deleted or inactivated.
The manufacturing method according to [6] or [7].
[9] Preferably, the genes corresponding to 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;
A 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;
A gene corresponding to mpr: a gene having at least 90% identity in nucleotide sequence with mpr (BG10690) and encoding an extracellular metalloprotease;
A gene corresponding to nprB: a gene having at least 90% identity in nucleotide sequence with nprB (BG10691) and encoding extracellular neutral protease B;
A 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 in nucleotide sequence with nprE (BG10448) and encoding an extracellular neutral metalloprotease;
Gene corresponding to vpr: a gene having at least 90% identity in nucleotide sequence with vpr (BG10591) and encoding a minor extracellular serine protease;
A gene corresponding to aprX: a gene having at least 90% identity in nucleotide sequence with aprX (BG12567) and encoding an intracellular serine protease;
The manufacturing method described in [8].
[10] The method according to any one of [1] to [9], wherein the Bacillus bacterium is Bacillus subtilis or a mutant thereof.

[11] A recombinant Bacillus bacterium, in which the activity of Bacillus subtilis AprE or a protease equivalent thereto has been reduced, and a polynucleotide encoding monoacylglycerol lipase has been exogenously introduced.
[12] 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 the secretion of a protein,
More preferably, it is operably linked to a transcription initiation control region and a translation initiation control region,
More preferably, it is operably linked to a transcription initiation control region and a translation initiation control region, and is not linked to a region involved in the secretion of a protein.
The recombinant Bacillus bacterium described in [11].
[13] The monoacylglycerol lipase,
Preferably, the monoacylglycerol lipase is a monoacylglycerol lipase belonging to Bacterial Lipolytic Enzyme Family XV,
More preferably, the non-secretory monoacylglycerol lipase is derived from a bacterium of the genus Bacillus or Geobacillus belonging to Family XV,
More preferably, it is a polypeptide consisting of any one of the amino acid sequences of SEQ ID NOs: 1 to 6, or a polypeptide consisting of an amino acid sequence that is at least 90% identical to any one of the amino acid sequences of SEQ ID NOs: 1 to 6 and encoding a polypeptide having monoglyceride lipase activity,
More preferably, the polypeptide 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 at least 90% identical to the amino acid sequence of any one of SEQ ID NOs: 1 and 2 and encoding a polypeptide having monoglyceride lipase activity, a polypeptide consisting of an amino acid sequence of any one of SEQ ID NOs: 3 to 6, or a polypeptide consisting of an amino acid sequence at least 90% identical to the amino acid sequence of any one of SEQ ID NOs: 3 to 6 and encoding a polypeptide having monoglyceride lipase activity,
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 encoding a polypeptide having monoglyceride lipase activity.
The recombinant Bacillus bacterium according to [11] or [12].
[14] The recombinant Bacillus bacterium according to any one of [11] to [13], preferably in which Bacillus subtilis aprE or a gene corresponding thereto is deleted or inactivated.
[15] The recombinant Bacillus bacterium according to [14], wherein the gene corresponding to aprE preferably has at least 90% identity in nucleotide sequence to aprE (BG10190) and encodes a polypeptide having serine protease activity with an optimal pH on the alkaline side.
[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 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;
A 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;
A gene corresponding to mpr: a gene having at least 90% identity in nucleotide sequence with mpr (BG10690) and encoding an extracellular metalloprotease;
A gene corresponding to nprB: a gene having at least 90% identity in nucleotide sequence with nprB (BG10691) and encoding extracellular neutral protease B;
A 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 in nucleotide sequence with nprE (BG10448) and encoding an extracellular neutral metalloprotease;
Gene corresponding to vpr: a gene having at least 90% identity in nucleotide sequence with vpr (BG10591) and encoding a minor extracellular serine protease;
A gene corresponding to aprX: a gene having at least 90% identity in nucleotide sequence with aprX (BG12567) and encoding an intracellular serine protease;
The recombinant Bacillus bacterium described in [16].
[18] The recombinant Bacillus bacterium according to any one of [11] to [17], wherein the Bacillus bacterium is Bacillus subtilis or a mutant thereof.

[19] A method for producing a recombinant Bacillus bacterium, comprising incorporating a polynucleotide encoding monoacylglycerol lipase into a Bacillus bacterium having reduced activity of Bacillus subtilis AprE or a protease equivalent thereto, or comprising reducing the activity of Bacillus subtilis AprE or a protease equivalent thereto in a Bacillus bacterium having incorporated a polynucleotide encoding monoacylglycerol lipase.
[20] 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 the secretion of a protein,
More preferably, it is operably linked to a transcription initiation control region and a translation initiation control region,
More preferably, it is operably linked to a transcription initiation control region and a translation initiation control region, and is not linked to a region involved in the secretion of a protein.
A method for producing the recombinant Bacillus bacterium described in [19].
[21] The monoacylglycerol lipase,
Preferably, the monoacylglycerol lipase is a monoacylglycerol lipase belonging to Bacterial Lipolytic Enzyme Family XV,
More preferably, the non-secretory monoacylglycerol lipase is derived from a bacterium of the genus Bacillus or Geobacillus belonging to Family XV,
More preferably, it is a polypeptide consisting of any one of the amino acid sequences of SEQ ID NOs: 1 to 6, or a polypeptide consisting of an amino acid sequence that is at least 90% identical to any one of the amino acid sequences of SEQ ID NOs: 1 to 6 and encoding a polypeptide having monoglyceride lipase activity,
More preferably, the polypeptide 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 at least 90% identical to the amino acid sequence of any one of SEQ ID NOs: 1 and 2 and encoding a polypeptide having monoglyceride lipase activity, a polypeptide consisting of an amino acid sequence of any one of SEQ ID NOs: 3 to 6, or a polypeptide consisting of an amino acid sequence at least 90% identical to the amino acid sequence of any one of SEQ ID NOs: 3 to 6 and encoding a polypeptide having monoglyceride lipase activity,
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 encoding a polypeptide having monoglyceride lipase activity.
The method for producing the recombinant Bacillus bacterium according to [19] or [20].
[22] The method for producing a recombinant Bacillus bacterium according to any one of [19] to [21], preferably wherein Bacillus subtilis aprE or a gene corresponding thereto is deleted or inactivated.
[23] The method for producing a recombinant Bacillus bacterium according to [22], wherein the gene corresponding to aprE preferably has at least 90% identity in nucleotide sequence to aprE (BG10190) and encodes a polypeptide having serine protease activity with an optimal pH on the alkaline side.
[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.
The method for producing the recombinant Bacillus bacterium according to [22] or [23].
[25] Preferably, the genes corresponding to 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;
A 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;
A gene corresponding to mpr: a gene having at least 90% identity in nucleotide sequence with mpr (BG10690) and encoding an extracellular metalloprotease;
A gene corresponding to nprB: a gene having at least 90% identity in nucleotide sequence with nprB (BG10691) and encoding extracellular neutral protease B;
A 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 in nucleotide sequence with nprE (BG10448) and encoding an extracellular neutral metalloprotease;
Gene corresponding to vpr: a gene having at least 90% identity in nucleotide sequence with vpr (BG10591) and encoding a minor extracellular serine protease;
A gene corresponding to aprX: a gene having at least 90% identity in nucleotide sequence with aprX (BG12567) and encoding an intracellular serine protease;
A method for producing the recombinant Bacillus bacterium described in [24].
[26] The method for producing a recombinant Bacillus bacterium according to any one of [19] to [25], wherein the Bacillus bacterium is Bacillus subtilis or a mutant thereof.

The present invention will be described in more detail below using examples. However, the technical scope of the present invention is not limited to these examples.

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). Plasmids were introduced into host microorganisms by the protoplast method (Mol. Gen. Genet, 1979, 168: 111-115). Recombinant microorganisms for protein production were cultured in LB medium (1% tryptone, 0.5% yeast extract, 1% NaCl) or 2x L-maltose medium (2% tryptone, 1% yeast extract, 1% NaCl, 7.5% maltose, 7.5 ppm manganese sulfate 4-5 hydrate). Lipase activity in the culture supernatant was measured as follows.

(Lipase activity measurement method)
5 μL of appropriately diluted culture supernatant was dispensed into each well of a 96-well assay plate (IWAKI). 183 μL of deionized water, 10 μL of 1M Tris-HCl (pH 8.0), and 2 μL of substrate solution were further dispensed into each well to initiate the reaction. A dimethyl sulfoxide (DMSO) solution containing 100 mM 4-nitrophenyl octanoate (SIGMA-ALDRICH) was used as the substrate solution. The reaction was carried out at 40°C for 15 minutes, and the absorbance of the reaction solution at 405 nm 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.

Tables 2 to 7 show the names and nucleotide sequences of the primers used in the following examples.

Example 1 Construction of a gene-deleted Bacillus subtilis strain (1) Construction of an aprE-deleted strain (Construction of a plasmid for gene deletion)
A 0.6 kb fragment (A) adjacent to the upstream side of the aprE gene on the genome and a 0.5 kb fragment (B) adjacent to the downstream side of the aprE gene on the genome were prepared using the genomic DNA extracted from Bacillus subtilis strain 168 as a template and the primer sets of aprEfw1 and aprEUpr (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 linking the promoter region (Nucleic Acids Res, 1989, 17: 4410) of the repU gene derived from plasmid pUB110 (Plasmid, 1986, 15: 93-103) to the upstream side of the chloramphenicol resistance gene derived from plasmid pC194 (J. Bacteriol, 150 (2), 815, 1982). In preparing fragment (C), first, a 0.4 kb fragment (D) containing the repU gene promoter region was prepared using a primer set of repUfw and repUr-Cm (SEQ ID NOs: 32 and 33, Table 3) and plasmid pUB110 as a template. Further, a 0.8 kb fragment (E) containing a chloramphenicol resistance gene was prepared using a primer set of CmUf-rep and Cmrv1 (SEQ ID NOs: 34 and 35, Table 3) and plasmid pC194 as a template. Next, fragment (C) was prepared by SOE-PCR using primers repUfw and Cmrv1 with a mixture of fragments (D) and (E) as a template. Next, the resulting mixture of fragments (A), (B) and (C) was used as a template, and the fragments were ligated in the order of (A), (B) and (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 the fragment was inserted into the SmaI restriction enzyme site of plasmid pUC118 (Methods Enzymol, 1987, 153: 3-11) to construct a plasmid for deleting the aprE gene, pUC118-CmrΔaprE.

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

Next, in order to increase the proportion of chloramphenicol-sensitive strains, ampicillin concentration 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 ampicillin sodium at a final concentration of 100 ppm so that the turbidity at 600 nm (OD600) was 0.003. After 5 hours of cultivation at 37°C, 10 μL of 10,000 ppm ampicillin sodium aqueous solution was added and cultivated for another 3 hours. After the cultivation 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. The cells were incubated at 37°C for about 15 hours, and among the grown strains, those that became chloramphenicol-sensitive due to the loss of the plasmid region were selected. The genomic DNA of the selected strain was used as a template to perform PCR using primers aprEfw2 and aprErv-repU (sequence numbers 31 and 30, Table 3) to confirm the deletion of the aprE gene, and the aprE gene-deleted strain trpC2/ΔaprE was obtained.

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

(3) Construction of aprX deletion strain An aprX deletion strain in which the aprX gene was replaced with a spectinomycin resistance gene was constructed by double crossover. Using genomic DNA extracted from Bacillus subtilis 168 strain as a template, a 558 bp fragment (F) on the 5'-end side including the upstream of the aprX gene on the genome and a 545 bp fragment (G) on the 3'-end side were prepared using the primer sets 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. Separately, a spectinomycin resistance gene region was excised from the BamHI and XhoI restriction enzyme cleavage site of plasmid pDG1727 (Gene, 167, 335, 1995) and inserted into the BamHI and XhoI restriction enzyme cleavage site of pBluescript II SK(+) (Stratagene) to construct plasmid pBlueSPR. Using pBlueSPR DNA as a template, a spectinomycin resistance gene region (H) was amplified using a primer set of PB-M13-20 and PB-M13Rev (SEQ ID NOs: 76 and 77, Table 7). A DNA fragment in which the fragments (E), (H) and (G) were linked in this order was prepared by SOE-PCR using a primer set of aprX+5F and aprX+1320R (SEQ ID NOs: 72 and 75, Table 7) with a mixture of the fragments (F), (G) and (H) as a template. The prepared DNA fragment was used to transform the trpC2 strain by competent cell transformation, and colonies grown on LB agar medium containing spectinomycin (100 μg/mL) were isolated as transformants. The genome of the obtained transformant was extracted, and it was confirmed by PCR that the aprX gene had been deleted and replaced with a spectinomycin resistance gene, to obtain an aprX gene-deleted strain trpC2/ΔaprX.

Example 2 Comparison of MGLP productivity of various gene deletion strains (1) Construction of EstGtA2-expressing recombinant Bacillus subtilis strain A recombinant Bacillus subtilis strain was constructed by introducing a gene encoding monoacylglycerol lipase EstGtA2 (SEQ ID NO: 1) into the gene deletion strain prepared in Example 1. A plasmid (EstGtA2-pUC57) was prepared by inserting a gene (SEQ ID NO: 7) encoding EstGtA2 codon-optimized for Bacillus subtilis into plasmid pUC57, using the artificial gene synthesis service of GenScript Inc. Using this as a template, PCR was performed using a primer set of primer EstGtA2-F and primer EstGtA2-R (SEQ ID NOs: 13 and 14, Table 2). Similarly, a PCR reaction was carried out using the plasmid pHY-S237 described in Example 7 of WO2006/068148A1 as a template and 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). Appropriate amounts of the two obtained fragments were mixed, and a plasmid was synthesized using the in-fusion method (pHY-EstGtA2). At this time, the control region of cellulase S237 (a region including a transcription initiation control region and a translation initiation control region, SEQ ID NO: 78) and the EstGtA2 gene were designed to be 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 protoplast transformation.

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

Figure 1 shows lipase activity in the culture supernatant obtained after 96 hours of culture of each recombinant Bacillus subtilis strain. In the figure, the horizontal axis shows the host of the recombinant Bacillus subtilis strain (trpC2, trpC2/Δepr, trpC2/ΔwprA, trpC2/Δmpr, trpC2/ΔnprB, trpC2/Δbpr, trpC2/ΔnprE, trpC2/Δvpr, trpC2/ΔaprE, trpC2/ΔaprX), and the vertical axis shows lipase activity in the culture supernatant for each strain. Lipase activity values are shown as relative activities, with the activity for the tprC2 strain taken as 100%. Lipase activity in the culture supernatant was significantly improved with the aprE deletion strain. FIG. 2 shows the results of SDS-PAGE of the culture supernatant after 96 hours of culture for each recombinant Bacillus subtilis strain. Each lane indicates the deleted gene of the recombinant Bacillus subtilis strain. While EstGtA2 accumulated in the culture supernatant of the aprE deleted strain, accumulation of EstGtA2 in the culture supernatant of the other deleted strains was not confirmed. From these results, it was found that by using a strain in which the aprE gene or a gene equivalent thereto is deleted or inactivated, monoacylglycerol lipase EstGtA2, which is originally an intracellular enzyme, can be secreted outside the cells and recovered from the culture supernatant.

Example 3 Productivity of various MGLPs by aprE-deficient strains (1) Construction of recombinant Bacillus subtilis strains expressing MGLPs A recombinant Bacillus subtilis strain was constructed that expresses H-257, stLP, CI220-6, CI220-10, and CI220-12 (SEQ ID NOs: 2 to 6), which are MGLPs belonging to Bacterial Lipolytic Enzyme Family XV. Plasmids (H-257-pUC57, stLP-pUC57, CI220-6-pUC57, CI220-10-pUC57, and CI220-12-pUC57) in which genes (SEQ ID NOs: 8 to 12) encoding each MGLP that had been codon-optimized for Bacillus subtilis were inserted into plasmid pUC57 were prepared using the artificial gene synthesis service provided by GenScript. Using the prepared plasmid as a template, 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. Similarly, PCR reaction was performed using the plasmid pHY-S237 as a template and the primers pHY-S237-F and pHY-S237-R (SEQ ID NOs: 15 and 16). As in Example 2(1), the PCR products were treated with DpnI and plasmids were synthesized by 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 protoplast transformation method.

(2) MGLP productivity of aprE-deleted recombinant Bacillus subtilis strain The recombinant Bacillus subtilis strain obtained in (1) was cultured overnight at 30°C with shaking in 1 mL of LB medium. 0.003 mL of the resulting culture was inoculated into 3 mL of 2xL-maltose medium and cultured at 30°C for 72 hours with shaking. After the culture, the culture supernatant was separated in the same manner as in Example 2(2), and the amount of MGLP produced outside the cells was confirmed by SDS-PAGE. Protein bands were confirmed by Mini-Protean TGX Stain-Free Gel (Bio-Rad).

Figure 3 shows the results of SDS-PAGE of the culture supernatant after 72 hours of culture for each recombinant Bacillus subtilis strain. Each lane indicates the deleted gene of the recombinant Bacillus subtilis strain and the type of MGLP expressed. All of the expressed MGLPs (H-257, stLP, CI220-6, CI220-10, and CI220-12) accumulated in the culture supernatant of the aprE deleted strain, whereas no accumulation of any MGLP was confirmed in the culture supernatant of the trpC2 strain. These results demonstrate that by using a strain in which the aprE gene or a gene equivalent thereto is deleted or inactivated, various MGLPs, which are intracellular enzymes, can be secreted outside the cells and recovered from the culture supernatant.

Although embodiments of the present invention have been described above, it should be understood that they 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.
All publications and patent applications cited herein are incorporated by reference as if fully set forth herein.

Claims (15)

1. A method for producing monoacylglycerol lipase, comprising culturing a Bacillus bacterium having reduced activity of Bacillus subtilis AprE or a protease corresponding thereto, and expressing monoacylglycerol lipase,
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 to the Bacillus subtilis AprE and having serine protease activity with an optimal 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 that is at least 90% identical to the amino acid sequence of any one of SEQ ID NOs: 1 and 2 .
method . The method of claim 1, further comprising recovering the monoacylglycerol lipase from the culture supernatant. The method according to claim 1 or 2, wherein a polynucleotide encoding the monoacylglycerol lipase is exogenously introduced into the Bacillus bacterium. The method according to claim 3, wherein the polynucleotide encoding the monoacylglycerol lipase is operably linked to a control region. The method according to claim 3 or 4, wherein the polynucleotide encoding the monoacylglycerol lipase is not linked to a polynucleotide region encoding a secretory signal peptide. the Bacillus bacterium having reduced activity of Bacillus subtilis AprE or a protease corresponding thereto is a Bacillus bacterium in which the Bacillus subtilis gene aprE or a gene corresponding thereto has been deleted or inactivated;
The gene corresponding to aprE has at least 90% identity in nucleotide sequence with aprE and encodes a polypeptide having serine protease activity with an optimal pH on the alkaline side.
The method according to any one of claims 1 to 5 . 7. The method according to claim 6, wherein 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 has been further deleted or inactivated. The method according to any one of claims 1 to 7 , wherein the Bacillus bacterium is Bacillus subtilis or a mutant thereof. A recombinant Bacillus bacterium having reduced activity of Bacillus subtilis AprE or a protease equivalent thereto, and having an exogenously introduced polynucleotide encoding monoacylglycerol lipase,
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 to the Bacillus subtilis AprE and having serine protease activity with an optimal 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 that is at least 90% identical to the amino acid sequence of any one of SEQ ID NOs: 1 and 2 .
Recombinant Bacillus spp. The recombinant Bacillus of claim 9 , wherein the polynucleotide encoding the monoacylglycerol lipase is operably linked to a regulatory region. 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 secretory signal peptide. the Bacillus subtilis gene aprE or a corresponding gene is deleted or inactivated,
The gene corresponding to aprE has at least 90% identity in nucleotide sequence with aprE and encodes a polypeptide having serine protease activity with an optimal pH on the alkaline side.
The recombinant Bacillus bacterium according to any one of claims 9 to 11 . 13. The recombinant Bacillus bacterium of 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. The recombinant Bacillus bacterium according to any one of claims 9 to 13 , wherein the Bacillus bacterium is Bacillus subtilis or a mutant thereof. A method for producing a recombinant Bacillus bacterium, comprising: incorporating a polynucleotide encoding monoacylglycerol lipase into a Bacillus bacterium having reduced activity of Bacillus subtilis AprE or a protease equivalent thereto; or reducing activity of Bacillus subtilis AprE or a protease equivalent thereto in a Bacillus bacterium having incorporated a polynucleotide encoding monoacylglycerol lipase,
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 to the Bacillus subtilis AprE and having serine protease activity with an optimal 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 that is at least 90% identical to the amino acid sequence of any one of SEQ ID NOs: 1 and 2 .
method.