BACTERIUM MODIFIED TO EXPRESS HETEROLOGOUS Tat PROTEIN

Abstract

Provided is a technology whereby a heterologous protein can be expressed in a bacterium which does not have an endogenous Tat system secretion machine. The present specification discloses a bacterium that is selected from the group consisting of bifidobacteria, lactococci, and staphylococci and has been modified to express a heterologous Tat protein. The bacterium has the ability to produce and secrete a heterologous protein.

Description

Bacteria engineered to express heterologous Tat-based proteins

[0001] The present invention relates to bacteria engineered to express heterologous Tat system proteins, and more particularly to bacteria selected from the group consisting of Bifidobacterium, Lactococcus, Lactobacillus, Limosilactobacillus, and Staphylococcus engineered to express heterologous Tat system proteins.

[0002] A common method for producing and secreting heterologous proteins is known to utilize a pathway known as the Sec system, which is widely present in prokaryotes and eukaryotes. In the Sec system pathway, produced proteins are secreted outside the cell in an unfolded (unstructured) state. Recently, a protein secretion pathway completely different from the Sec system has been discovered in the thylakoid membrane of plant chloroplasts (Non-Patent Document 1). Because the signal sequences of proteins secreted through this pathway share an arginine-arginine sequence, it has come to be called the Tat system (Twin-Arginine Translocation system). As mentioned above, in the Sec system pathway, proteins are secreted in a state prior to the formation of higher-order structures, whereas in the Tat system pathway, proteins are secreted through the cell membrane after forming higher-order structures within the cell (Non-Patent Document 2).

[0003] As a method for expressing a heterologous protein using the Tat system, Patent Document 1 describes a method in which a construct containing the tatABC genes derived from Streptomyces coelicolor, a nucleic acid sequence encoding the Tat signal peptide derived from S. scabies, and a nucleic acid sequence encoding an agarase derived from Streptomyces coelicolor is introduced into Bacillus subtilis to express the agarase derived from Streptomyces coelicolor in Bacillus subtilis. Patent Document 2 describes a method in which a gene construct containing a nucleic acid sequence encoding a Tat system-dependent signal peptide and a nucleic acid sequence encoding a heterologous protein is introduced into a coryneform bacterium (Corynebacterium glutamicum) having a Tat secretion apparatus, thereby producing and secreting a heterologous protein in the coryneform bacterium. Non-Patent Document 3 describes the expression of Tat derived from Pseudomonas syringae, Streptomyces coelicolor, and Aquifex aeolicus in E. coli. Non-Patent Document 4 describes the expression of Tat derived from Bacillus subtilis in E. coli.

[0004] Patent Document 1: WO2011 / 135370 Patent Document 2: WO2005 / 103278 Non-Patent Document 1: Chaddock AM, Mant A, Karnauchov I, Brink S, Herrmann RG, Klosgen RB, Robinson C, A new type of signal peptide: central role of a twin-arginine motif in transfer signals for the delta pH-dependent thylakoidal protein translocase, EMBO Journal, (1995), 14(12):2715-2722 Non-Patent Document 2: Hynds PJ, Robinson D, Robinson C. The sec-independent twin-arginine translocation system can transport both tightly folded and malfolded proteins across the thylakoid membrane. J Biol Chem. (1998), 25; 273(52):34868-74 Non-Patent Document 3: Matthew G Hicks et al., Formation of functional Tat translocases from heterologous components. BMC Microbiology (2006), 6:64 Non-patent document 4: Anna M Albiniak et al., High-level secretion of a recombinant protein to the culture medium with a Bacillus subtilis twin-arginine translocation system in Escherichia coli. FEBS J. (2013), 280(16):3810-21.

[0005] However, the microorganisms used in Patent Documents 1 and 2 and Non-Patent Documents 3 and 4 (Bacillus subtilis, coryneform bacteria, and E. coli) all have an endogenous Tat-system secretion system (i.e., have endogenous genes encoding Tat-system proteins that constitute the Tat-system secretion system) (Protein J. 2019; 38(4): 377-388., Proc Natl Acad Sci USA. 2006 Nov 21; 103(47): 17927-32., Crit Rev Biotechnol. 2017 Jun; 37(4): 541-551.), and they do not focus on expressing heterologous proteins using microorganisms that do not have an endogenous Tat-system secretion system.

[0006] As a result of extensive research, the present inventors have found that even microorganisms lacking an endogenous Tat-system secretion system can secrete heterologous proteins using heterologous Tat-system proteins. This makes it possible to secrete larger amounts of protein even in microorganisms lacking an endogenous Tat-system secretion system. Specifically, the present invention encompasses the following aspects: [1] A bacterium selected from the group consisting of the genera Bifidobacterium, Lactococcus, Lactobacillus, Limosilactobacillus, and Staphylococcus, which has been modified to express a heterologous Tat-system protein and has the ability to produce and secrete heterologous proteins. [2] The bacterium according to [1], wherein the bacterium is a bacterial strain lacking an endogenous Tat-system secretion system. [3] The bacterium according to [1] or [2], which has the ability to extracellularly secrete a heterologous protein via a heterologous Tat system protein. [4] The bacterium according to any one of [1] to [3], which is a bacterium selected from the group consisting of Lactococcus lactis, Bifidobacterium longum, Lactobacillus reuteri (also known as Limosilactobacillus reuteri), and Staphylococcus epidermidis. [5] The bacterium according to any one of [1] to [4], which comprises a gene construct comprising, in the 5' to 3' direction, a nucleic acid sequence encoding a Tat system-dependent signal peptide and a nucleic acid sequence encoding a heterologous protein. [6] The bacterium according to [5], wherein the gene construct further comprises a nucleic acid sequence encoding a heterologous Tat-system protein.[7] The bacterium according to any one of [1] to [6], wherein the heterologous Tat system protein is a Tat system protein derived from a bacterium selected from the genera Corynebacterium, Bacillus, and Bifidobacterium. [8] The bacterium according to any one of [1] to [7], wherein the heterologous Tat system protein comprises at least one selected from TatA, TatB, and TatC. [9] The bacterium according to any one of [1] to [8], wherein the heterologous Tat system protein comprises TatA, TatB, and TatC.

[10] The bacterium according to any one of [1] to [9], wherein the heterologous protein comprises at least one selected from FGF2 and FGF1.

[11] The bacterium according to any one of [5] to

[10] , wherein the gene construct further comprises a promoter derived from a bacterium selected from the group consisting of the genera Bifidobacterium, Lactococcus, Lactobacillus, Limosilactobacillus, and Staphylococcus.

[12] The bacterium according to any one of [5] to

[11] , wherein the gene construct further comprises a nucleic acid sequence encoding a signal peptidase.

[13] The bacterium according to any one of [5] to

[12] , comprising a recombinant vector into which the gene construct has been incorporated.

[14] A method for producing a heterologous protein, comprising the step of culturing the bacterium according to any one of [1] to

[13] and producing and secreting the heterologous protein.

[15] A pharmaceutical composition comprising the bacterium according to any one of [1] to

[13] .

[16] The pharmaceutical composition according to

[15] , for treating or preventing a disease or condition selected from cancer, lower limb ischemic disease, rash or oral mucositis associated with cancer treatment, ichthyosis, dental caries, irritable bowel syndrome, obesity, trauma, and surgical scars.

[0007] According to the present invention, heterologous proteins can be expressed using heterologous Tat proteins even in microorganisms that do not have an endogenous Tat secretion system, which has the advantage that proteins that are not secreted by the endogenous Sec system of the microorganism can be produced and secreted, thereby broadening the range of applications of the microorganism.

[0008] Figure 1 shows whether the heterologous protein FGF2 was produced and secreted when Lactococcus lactis / pNZ9148 (lane 4), Lactococcus lactis / pNZ9148-CgtatABC-torAss-fgf2 (lane 5), Lactococcus lactis / pNZ9148-CgtatABC (lane 6), or Lactococcus lactis / pNZ9148-torAss-fgf2 (lane 7) were cultured. Of lanes 4 to 7, production and secretion of FGF2 was confirmed only in lane 5. Figure 2 shows whether the TorA signal peptide attached to the heterologous protein FGF2 was cleaved and mature FGF2 was produced and secreted when Lactococcus lactis / pNZ9148-CgtatABC-torAss-fgf2 (lane 3), Lactococcus lactis / pNZ9148-CgtatABC-torAss-fgf2-LllepB (lanes 7, 8, and 9), or Lactococcus lactis / pNZ9148-CgtatABC-torAss-fgf2-CglepB (lanes 10, 11, and 12) were cultured. Increased production and secretion of cleaved FGF2 was confirmed in lanes 7 to 9 and lanes 10, 11, and 12. Figure 3 shows whether the heterologous protein Pro-PGHisx6 was produced and secreted when Lactococcus lactis / pNZ9148-torAss-ppghis-LllepB (lane 1) and Lactococcus lactis / pNZ9148-Pusp45-CgtatABC-torAss-ppghis-LllepB (lane 2) were cultured. Of the two strains, only lane 2 confirmed the production and secretion of Pro-PGHisx6.Figure 4 shows whether the heterologous protein FGF2 was produced and secreted when Lactobacillus reuteri / pNZ9148 (lane 3), Lactobacillus reuteri / pNZ9148-CgtatABC (lane 4), Lactobacillus reuteri / pNZ9148-torAss-fgf2 (lane 5), or Lactobacillus reuteri / pNZ9148-CgtatABC-torAss-fgf2 (lane 6) were cultured. Of lanes 3 to 6, production and secretion of FGF2 was confirmed only in lane 6.

[0009] <<Bacteria>> A first aspect of the present invention is a bacterium selected from the group consisting of Bifidobacterium, Lactococcus, Lactobacillus, Limosilactobacillus, and Staphylococcus, which has been modified to express a heterologous Tat-system protein and has the ability to produce and secrete the heterologous protein. Each component is explained below. In this specification and claims, "endogenous" proteins or genes of a microorganism refer to proteins or genes derived from the microorganism, and are used to distinguish them from proteins or genes derived from a "heterologous" organism that is not derived from the microorganism. For example, the endogenous Tat system secretion system or endogenous Tat system protein of a certain microorganism means a Tat system secretion system or Tat system protein that the microorganism naturally possesses, and does not include a Tat system secretion system or Tat system protein that is obtained when a gene encoding a Tat system secretion system or Tat system protein derived from another organism is incorporated into the microorganism and expressed.

[0010] The bacteria used in the present invention are selected from the group consisting of the genera Bifidobacterium, Lactococcus, Lactobacillus, Limosilactobacillus, and Staphylococcus. Examples of bacteria in the genus Bifidobacterium include Bifidobacterium longum, Bifidobacterium breve, and Bifidobacterium bifidum. Preferred examples of bacteria of the genus Bifidobacterium include Bifidobacterium longum strain 105-A, Bifidobacterium longum JCM 1217 (ATCC 15707 (Z. sun, et al., Front. Microbiol., 10, 796 (2009))), Bifidobacterium breve UCC2003 (AN Shkoporov, et al., FEMS Microbiol. Lett., 362, 12 (2015)), and Bifidobacterium bifidum S17 (Z. sun, et al., Bioengineered, 5:6, 371-377 (2014)). Examples of bacteria of the genus Lactococcus include Lactococcus lactis and Lactococcus raffinolactis. Among these, Lactococcus lactis is preferred, and Lactococcus lactis subsp. cremoris is more preferred.Preferred examples of bacteria of the genus Lactococcus include bacterial strains such as Lactococcus lactis subsp. cremoris MG1363, Lactococcus lactis subsp. cremoris SK11, and Lactococcus lactis subsp. lactis IL1403 (see, for example, US008759088B2 and N. Noreen, et al., Microb. Cell. Fact., 10, 28 (2011)). Examples of bacteria of the genus Lactobacillus and / or Limosilactobacillus include Lactobacillus plantarum, Lactobacillus reuteri (also known as Limosilactobacillus reuteri), etc. Among these, Lactobacillus reuteri is preferred. Preferred examples of bacteria of the Lactobacillus and / or Limosilactobacillus genus include bacterial strains such as Lactobacillus reuteri DSM20016 strain, R2LC strain, ATCC PTA 6475 strain, and ATCC PTA 5290 strain (see EP3735979A1, EP1868622A1, EP1765975A1, etc.). In recent years, the genus classification of Lactobacillus reuteri has been reevaluated at the genome level and it has been reclassified as Limosilactobacillus reuteri (Int J Syst Evol Microbiol. 2020 Apr; 70(4): 2782-2858), but it is still referred to as Lactobacillus reuteri in many papers.In this specification and claims, Lactobacillus reuteri and Limosilactobacillus reuteri are interchangeably used.In addition, in this specification and claims, the bacteria of the genus Lactobacillus are not limited to the bacteria classified into the genus Lactobacillus at the time of filing of this application, but also refer to the bacteria classified into the genus Lactobacillus at least at any time before, at the time of, or after filing of this application.If further limitation is required, the bacteria of the genus Lactobacillus may be the bacteria classified into the genus Lactobacillus at the time of filing of this application. Examples of bacteria of the genus Staphylococcus include Staphylococcus epidermidis, Staphylococcus aureus, Staphylococcus saprophyticus, etc. Preferred examples of bacteria of the genus Staphylococcus include bacterial strains such as Staphylococcus epidermidis ATCC 12228, Staphylococcus epidermidis NRRL B-4268, and Staphylococcus epidermidis ATCC 14990 (see, for example, US20210162030). The bacterium of the present invention is preferably a bacterial strain that does not have an endogenous Tat system secretion apparatus (or does not have an endogenous Tat system protein), i.e., the bacterium of the present invention is preferably a bacterial strain that does not have an endogenous gene encoding a Tat system protein that constitutes the Tat system secretion apparatus.Bacterial strains that do not have an endogenous Tat system secretion apparatus include Bifidobacterium longum (especially strain 105-A), Lactococcus lactis, Staphylococcus epidermidis, Bifidobacterium breve (especially strains S-17, UCC2003, and LMG 13208), Bifidobacterium breve (especially strain S-27), Lactobacillus plantarum (especially strain WCSF1), and Lactobacillus reuteri (especially strain DSM20016) (Appl Environ Microbiol. 2006). Dec;72(12):7626-33., Appl Environ Microbiol. 2018 Aug 1;84(16):e00796-18., J Bacteriol. 2009 Oct; 191(19): 5921-5929., Proc Natl Acad Sci US A. 2003 Feb 18; 100(4): 1990-1995., Appl Environ Microbiol. 2003 Dec;69(12):6994-7001, PLoS One. 2015 Jun; 10(6): e0128802, FEMS Microbiol Rev. 2010 Mar;34(2):199-230.).The bacteria used in the present invention are preferably selected from the group consisting of Lactococcus lactis, Bifidobacterium longum, Staphylococcus epidermidis, Bifidobacterium breve, Bifidobacterium bifidum, Lactobacillus plantarum, and Lactobacillus reuteri, and the bacteria used in the present invention are preferably selected from the group consisting of Lactococcus lactis, Bifidobacterium longum, Lactobacillus reuteri, and the bacteria used in the present invention are preferably selected from the group consisting of Lactococcus lactis, Bifidobacterium longum, Lactobacillus reuteri, and Staphylococcus epidermidis. More preferred is a bacterium selected from the group consisting of Lactococcus epidermidis, and even more preferred is Lactococcus lactis. Hereinafter, the bacterium or bacterial strain used in the present invention may be referred to as the "host".

[0011] <Ability to Produce and Secrete Heterologous Proteins> The bacteria used in the present invention have the ability to produce and secrete heterologous proteins. Furthermore, the bacteria used in the present invention preferably have the ability to extracellularly secrete heterologous proteins via heterologous Tat-system proteins. The ability to produce and secrete heterologous proteins can be obtained, for example, by having a genetic construct for producing and secreting heterologous proteins in the bacteria used in the present invention. In the present invention, the term "protein" encompasses not only proteins but also oligopeptides and polypeptides. In this specification, "secretion" of a protein or peptide refers to the transport of protein or peptide molecules outside the bacterial cell (extracellularly), and includes cases where the protein or peptide molecules are ultimately left completely free in the culture medium, as well as cases where only a portion of the protein or peptide is present outside the bacterial cell or on the bacterial cell surface.

[0012] In the present invention, having the ability to "produce and secrete" a heterologous protein means that when the bacterium of the present invention is cultured in a medium, the bacterium can produce the heterologous protein and secrete it outside the bacterial cell.

[0013] <Heterologous Protein> In the present invention, the term "heterologous protein" refers to a protein foreign to the bacterium used in the present invention. The heterologous protein is preferably a protein derived from an organism other than bacteria selected from the group consisting of Bifidobacterium, Lactococcus, Lactobacillus, Limosilactobacillus, and Staphylococcus. The heterologous protein may be an animal-derived protein, a plant-derived protein, a bacterial-derived protein, a fungal-derived protein, or a viral-derived protein. Among these, the heterologous protein is more preferably an animal-derived protein or a bacterial-derived protein, even more preferably an animal-derived protein, and particularly preferably a mammal-derived protein. Furthermore, the mammal-derived protein is preferably a human-derived protein. The heterologous protein may be a protein having the same amino acid sequence as a protein endogenous to an organism other than a bacterium selected from the group consisting of Bifidobacterium, Lactococcus, Lactobacillus, Limosilactobacillus, and Staphylococcus, or may be a protein obtained by modifying the amino acid sequence of a protein endogenous to the aforementioned organism. Examples of proteins obtained by modifying the amino acid sequence of a protein endogenous to the aforementioned organism include proteins with improved enzymatic activity.

[0014] The heterologous protein produced and secreted by the bacterium of the present invention is not particularly limited and may be any desired heterologous protein. Specific examples of heterologous proteins include human-derived fibroblast growth factors (FGFs) such as human-derived basic fibroblast growth factor (bFGF or FGF2) and human-derived acidic fibroblast growth factor (aFGF or FGF1), protein glutaminase, isomaltodextranase, and transglutaminase. Among these, FGF is preferred, and at least one selected from FGF2 and FGF1 is preferred, with at least one selected from FGF2 and FGF1 being more preferred, and FGF2 being even more preferred. The heterologous protein may have a presequence (signal sequence) and / or prosequence that it may have in its naturally occurring state, or may have such a presequence and / or prosequence removed. Furthermore, the heterologous protein may also be one to which a sequence not native to the protein has been added, for example, a desired functional sequence such as a signal sequence or tag sequence.

[0015] <Tat System-Dependent Signal Peptide (Signal Sequence)> In the present invention, a Tat system-dependent signal peptide refers to a signal peptide recognized by the Tat system. A Tat system-dependent signal peptide has an arginine-arginine sequence, and when a Tat system protein (or the Tat secretion apparatus) recognizes this sequence, it can secrete a protein having the signal peptide at its N-terminus extracellularly (extracellularly). A heterologous protein may or may not have a Tat system-dependent signal peptide linked to its N-terminus. When a heterologous protein has a Tat system-dependent signal peptide at its N-terminus, the Tat system recognizes the arginine-arginine sequence in the signal peptide and secretes the heterologous protein from within the bacterial cell to outside the cell. However, the Tat system-dependent signal peptide can be cleaved from the heterologous protein simultaneously with or after secretion. Therefore, a Tat system-dependent signal peptide may or may not be linked to a heterologous protein to be produced and secreted. Preferably, a Tat system-dependent signal peptide is not linked to a heterologous protein to be produced and secreted. Examples of Tat-dependent signal peptides include the signal peptide of Escherichia coli trimethylamine N-oxidoreductase (TorA) (SEQ ID NO: 49), the signal peptide of Escherichia coli Sufl (suppressor of ftsl) (SEQ ID NO: 50), the signal peptide of PhoD (phosphodiesterase) derived from Bacillus subtilis (SEQ ID NO: 51), the signal peptide of XlnC (xylanase) derived from Streptomyces lividans (SEQ ID NO: 52), the signal peptide of isomaltodextranase (IMD) derived from Arthrobacter globiformis (SEQ ID NO: 53), and the signal peptide of FAD-dependent oxidoreductase derived from Bifidobacterium longum (SEQ ID NO: 72).Among these, the signal peptide of trimethylamine N-oxidoreductase derived from Escherichia coli (TorA signal peptide, TorAss) and the signal peptide of FAD-dependent oxidoreductase derived from Bifidobacterium longum are preferred as Tat-dependent signal peptides, with the signal peptide of trimethylamine N-oxidoreductase derived from Escherichia coli (TorA signal peptide, TorAss) being more preferred. Examples of the amino acid sequence of the signal peptide include amino acid sequences that have 70% or more (preferably 80% or more, more preferably 90% or more, even more preferably 95% or more, even more preferably 98% or more, even more preferably 99% or more, and most preferably 100%) sequence identity to any of the amino acid sequences of SEQ ID NOs: 49 to 53 and 72 (preferably the amino acid sequence of SEQ ID NO: 49, which is the amino acid sequence of the signal peptide of trimethylamine N-oxidoreductase derived from Escherichia coli, or the amino acid sequence of SEQ ID NO: 72, which is the amino acid sequence of the signal peptide of FAD-dependent oxidoreductase derived from Bifidobacterium longum).

[0016] As described above, when a heterologous protein is secreted outside the bacterial cell via the Tat system secretion apparatus (Tat system, Tat pathway), the signal peptide may or may not be partially or completely removed by an endogenous or heterologous signal peptidase possessed by the host. Therefore, the heterologous protein produced and secreted by the bacterium of the present invention may be a heterologous protein from which the signal peptide has been completely removed, i.e., which retains no signal peptide at all (a fully-cleaved heterologous protein), a heterologous protein from which a portion of the signal peptide has been removed (a partially-cleaved heterologous protein), or a heterologous protein that retains a signal peptide (an uncleaved heterologous protein). A heterologous protein from which the signal peptide has been completely removed is preferred.

[0017] <Tat System Proteins> In the present invention, the term "Tat system" refers to a pathway also known as the "twin-arginine translocation pathway," which refers to a mechanism or pathway that recognizes a conserved arginine-arginine region in a signal peptide to secrete proteins. Examples of Tat system proteins include TatA, TatB, and TatC. In the present invention, the term "Tat system secretion apparatus" refers to a membrane protein composed of one or more of TatA, TatB, and TatC. TatA, TatB, and TatC are transmembrane proteins present in the cell membrane and are thought to form pores in the cell membrane through which proteins pass. In the present invention, the term "Tat system protein" refers to a membrane-permeable protein used in the Tat system. Examples of Tat system proteins include TatA, TatB, and TatC. TatA is the most abundant component of the Tat complex and is thought to be most involved in the formation of protein transport channels. TatB is known to bind to a Tat-dependent signal peptide and then bind to the mature protein. TatC is known to assist in binding to the protein to be transported (Protein J. 2019; 38(4): 377-388). Depending on the organism, the Tat secretion apparatus is either composed of three proteins (TatA, TatB, and TatC) or two proteins (TatA and TatC). In the latter case, TatA is known to also function as TatB. In the latter case, for example, in Bacillus subtilis, TatAd and TatAy are known to be equivalent to TatA, and TatCd and TatCy are known to be equivalent to TatC. In the present invention, "TatA" refers not only to TatA in various microorganisms but also to TatAd and TatAy in Bacillus subtilis. Similarly, "TatC" is a concept that includes not only TatC of various microorganisms but also TatCd and TatCy of Bacillus subtilis.The heterologous Tat system protein expressed by the bacterium of the present invention preferably comprises at least one selected from TatA, TatB and TatC, more preferably TatA and TatC, and particularly preferably TatA, TatB and TatC.

[0018] In the present invention, the term "heterologous Tat system protein" refers to a Tat system protein foreign to the bacterium used in the present invention. The heterologous protein may be a plant-derived Tat system protein or a bacterial-derived Tat system protein, but is preferably a bacterial-derived Tat system protein, more preferably a Tat system protein derived from a bacterium selected from the genera Corynebacterium, Bacillus, and Bifidobacterium, and even more preferably a Tat system protein derived from a bacterium of the genus Corynebacterium or Bacillus. Examples of bacteria belonging to the genus Corynebacterium include Corynebacterium glutamicum, Corynebacterium ammoniagenes, and Corynebacterium casei. Among these, Corynebacterium glutamicum is preferred, with C. glutamicum strain 2256 being more preferred. Examples of bacteria belonging to the genus Bacillus include Bacillus subtilis and Bacillus thuringiensis. Among these, Bacillus subtilis is preferred, with Bacillus subtilis strain 168 being more preferred. Among these, Tat proteins derived from bacteria of the genus Corynebacterium are preferred, and Tat proteins derived from Corynebacterium glutamicum are more preferred.

[0019] The heterologous Tat system protein may be a Tat system protein having the same amino acid sequence as a natural Tat system protein present in the aforementioned organism, or may be a Tat system protein whose amino acid sequence has been modified as long as the function of the Tat system protein present in the aforementioned organism is not impaired; however, a Tat system protein having the same amino acid sequence as a natural Tat system protein present in the aforementioned organism is preferred.

[0020] The TatA, TatB, and TatC proteins may be those derived from the organisms described above. Among these, the TatA, TatB, and TatC proteins derived from Corynebacterium glutamicum, the TatAd, TatCd, TatAy, and TatCy proteins derived from Bacillus subtilis, and the TatA, TatB, and TatC proteins derived from Bifidobacterium longum (preferably Bifidobacterium longum strain E-18) are preferred. The TatA, TatB, and TatC proteins derived from Corynebacterium glutamicum have the amino acid sequences of SEQ ID NOs: 24, 25, and 26, respectively. The TatAd, TatCd, TatAy, and TatCy proteins derived from Bacillus subtilis have the amino acid sequences of SEQ ID NOs: 39, 40, 41, and 42, respectively. The TatA, TatB, and TatC proteins derived from Bifidobacterium longum strain E-18 have the amino acid sequences of SEQ ID NOs: 46, 47, and 48, respectively.

[0021] The heterologous TatA used in the present invention is not particularly limited in its amino acid sequence, as long as it retains the membrane-permeabilizing function of TatA having the amino acid sequence of SEQ ID NO: 24, 39, 41, or 46. However, it is preferable for the heterologous TatA to have an amino acid sequence that has 70% or more sequence identity with the amino acid sequence of SEQ ID NO: 24, 39, 41, or 46, more preferably an amino acid sequence that has 80% or more sequence identity, even more preferably an amino acid sequence that has 90% or more sequence identity, even more preferably an amino acid sequence that has 95% or more sequence identity, even more preferably an amino acid sequence that has 98% or more sequence identity, even more preferably an amino acid sequence that has 99% or more sequence identity, and even more preferably an amino acid sequence that has 100% sequence identity. The heterologous TatB used in the present invention is not particularly limited in its amino acid sequence, as long as it retains the membrane-permeabilizing function of TatB having the amino acid sequence of SEQ ID NO: 25 or 47. However, it preferably has an amino acid sequence with 70% or more sequence identity to the amino acid sequence of SEQ ID NO: 25 or 47, more preferably has an amino acid sequence with 80% or more sequence identity, even more preferably has an amino acid sequence with 90% or more sequence identity, even more preferably has an amino acid sequence with 95% or more sequence identity, even more preferably has an amino acid sequence with 98% or more sequence identity, even more preferably has an amino acid sequence with 99% or more sequence identity, and even more preferably has an amino acid sequence with 100% sequence identity.The heterologous TatC used in the present invention is not particularly limited in its amino acid sequence, as long as it retains the membrane-permeabilizing function of TatC having the amino acid sequence of SEQ ID NO: 26, 40, 42, or 48. However, it is preferable for the heterologous TatC to have an amino acid sequence that has 70% or more sequence identity with the amino acid sequence of SEQ ID NO: 26, 40, 42, or 48, more preferably an amino acid sequence that has 80% or more sequence identity, even more preferably an amino acid sequence that has 90% or more sequence identity, even more preferably an amino acid sequence that has 95% or more sequence identity, even more preferably an amino acid sequence that has 98% or more sequence identity, even more preferably an amino acid sequence that has 99% or more sequence identity, and even more preferably an amino acid sequence that has 100% sequence identity.

[0022] <Signal peptidase> In the present invention, the term "signal peptidase" refers to a protein (enzyme) having the activity of removing part or all of a Tat-dependent signal peptide from a protein having such a signal peptide. The bacterium of the present invention preferably expresses a signal peptidase that removes part or all (preferably all) of a Tat-dependent signal peptide from a protein having such a signal peptide. The signal peptidase is not particularly limited as long as it has the above-mentioned activity. However, a signal peptidase having four conserved regions, Box B, Box C, Box D, and Box E, in the amino acid sequence is preferred (Pharmacol. Ther. 2000 87:27-49). Examples of such signal peptidases include type I signal peptidase derived from Escherichia coli (EcLepB; Mol. Gen. Genet. 1991 227;40-44), type I signal peptidase derived from Bacillus subtilis (SipS, SipT; Genes Dev. 1998 12;2318-2331), type I signal peptidase derived from Corynebacterium glutamicum 2256 strain (CgLepB; WO 2020 / 085511), and type I signal peptidase derived from Lactococcus lactis (Lactococcus lactis). More preferred are the type I signal peptidase (LlLepB) derived from the MG1363 strain, the type I signal peptidase (LrLepB) derived from Lactobacillus reuteri (L. reuteri), the type I signal peptidase (BlLepB) derived from Bifidobacterium longum (B. longum), and the type I signal peptidase (SeLepB) derived from Staphylococcus epidermidis (S. epidermidis), and the type I signal peptidase (CgLepB; WO 2020 / 085511) derived from the Corynebacterium glutamicum (C. glutamicum) 2256 strain and the type I signal peptidase (LlLepB) derived from the Lactococcus lactis (L. lactis) MG1363 strain. The amino acid sequences of the above signal peptidases are as follows:Amino acid sequence of EcLepB: SEQ ID NO: 30 Amino acid sequence of SipS: SEQ ID NO: 31 Amino acid sequence of SipT: SEQ ID NO: 32 Amino acid sequence of CgLepB: SEQ ID NO: 33 Amino acid sequence of LlLepB: SEQ ID NO: 34 Amino acid sequence of LrLepB: SEQ ID NO: 74 Amino acid sequence of BlLepB: SEQ ID NO: 75 Amino acid sequence of SeLepB: SEQ ID NO: 78 As the amino acid sequence of the signal peptidase, an amino acid sequence having 70% or more sequence identity (preferably 80% or more, more preferably 90% or more, even more preferably 95% or more, even more preferably 98% or more, even more preferably 99% or more, and most preferably 100%) to the amino acid sequence of SEQ ID NO: 30, 31, 32, 33, 34, 74, 75 or 78 is preferred.

[0023] <Other Configurations> The bacterium of the present invention may be deficient in an endogenous protease gene or have a reduced expression level. By deficient in a protease gene or having a reduced expression level, degradation of the protein produced and secreted can be prevented.

[0024] <Gene Construct> In the present invention, a "gene construct" refers to a construct containing a promoter, a sequence encoding an appropriate signal peptide, a nucleic acid fragment encoding a target protein, and control sequences (such as operators and terminators) necessary for expressing the target protein gene, all at appropriate positions so that they can function. The bacterium of the present invention preferably has a gene construct containing a nucleic acid sequence encoding a heterologous protein. Furthermore, the gene construct preferably contains, from 5' to 3', a nucleic acid sequence encoding a Tat-dependent signal peptide and a nucleic acid sequence encoding the heterologous protein. In this case, there is no stop codon between the nucleic acid sequence encoding the Tat-dependent signal peptide and the nucleic acid sequence encoding the heterologous protein. This allows the production of a heterologous protein with a Tat-dependent signal peptide linked to its N-terminus in the host bacterium. Furthermore, the gene construct preferably contains a nucleic acid sequence encoding a heterologous Tat-system protein. The nucleic acid sequence encoding the heterologous Tat-system protein may be located either at the 5' end of the nucleic acid sequence encoding the Tat-dependent signal peptide or at the 3' end of the nucleic acid sequence encoding the heterologous protein. Furthermore, the nucleic acid sequence encoding the heterologous Tat-system protein and the nucleic acid sequence encoding the heterologous protein may be present in the same gene construct, or may be present in different gene constructs.

[0025] <Vector> The bacterium used in the present invention preferably contains a recombinant vector incorporating a gene construct. Examples of vectors used to incorporate a gene construct include plasmids, transposons, and phages. There are no particular limitations on the type of plasmid used, as long as it can incorporate the gene construct in an appropriate location, and various plasmids can be used. Specific examples of plasmids that can be used in the present invention include pNZ8148 vector (GoldBio, SEQ ID NO: 62), pBS423 (Appl. Environ. Microbiol. 2012 Jun;78(14):4984-4944., SEQ ID NO: 63), and pRMC2 (Plasmid. 2009 Mar;61(2):126-9.). There are no particular limitations on the method for incorporating a gene construct or each gene and promoter sequence contained in a gene construct into a vector. For example, a gene construct can be incorporated into a vector by ligating it to a cleavage site obtained by digesting a specific sequence of the vector with a restriction enzyme.

[0026] <Nucleic acid sequence encoding a heterologous Tat system protein> The gene construct or vector preferably contains a nucleic acid sequence encoding a heterologous Tat system protein. In the present invention, the term "nucleic acid sequence encoding a heterologous Tat system protein" refers to a nucleic acid sequence encoding a Tat system protein foreign to the bacterium used in the present invention. Examples of heterologous Tat system proteins include those described above. The nucleic acid sequence encoding a heterologous Tat system protein preferably contains a nucleic acid sequence encoding at least one Tat system protein selected from TatA, TatB, and TatC, more preferably contains a nucleic acid sequence encoding TatA (tatA) and a nucleic acid sequence encoding TatC (tatC), and even more preferably contains a nucleic acid sequence encoding TatA (tatA), a nucleic acid sequence encoding TatB (tatB), and a nucleic acid sequence encoding TatC (tatC).

[0027] The nucleic acid sequences encoding the TatA, TatB, and TatC proteins may be those derived from the organisms described above. Among these, nucleic acid sequences encoding the TatA, TatB, and TatC proteins derived from Corynebacterium glutamicum, the TatAd, TatCd, TatAy, and TatCy proteins derived from Bacillus subtilis, or the TatA, TatB, and TatC proteins derived from Bifidobacterium longum (preferably Bifidobacterium longum strain E-18) are preferred. The nucleic acid sequences encoding the TatA, TatB, and TatC proteins derived from Corynebacterium glutamicum have the nucleic acid sequences of SEQ ID NOs: 4, 5, and 6, respectively. Nucleic acid sequences encoding the TatAd, TatCd, TatAy, and TatCy proteins derived from Bacillus subtilis have the nucleic acid sequences of SEQ ID NOs: 35, 36, 37, and 38, respectively. Nucleic acid sequences encoding the TatA, TatB, and TatC proteins derived from Bifidobacterium longum E-18 have the nucleic acid sequences of SEQ ID NOs: 43, 44, and 45, respectively. The nucleic acid sequences encoding the TatA, TatB, and TatC proteins used in the present invention preferably have 70% or more (preferably 80% or more, more preferably 90% or more, even more preferably 95% or more, even more preferably 98% or more, even more preferably 99% or more, and most preferably 100%) sequence identity to the above nucleic acid sequences.

[0028] Furthermore, nucleic acid sequences encoding heterologous TatA, TatB, and TatC may be optimized for the host bacterium (it is preferable to optimize the nucleic acid sequences encoding them without changing the amino acid sequences of TatA, TatB, and TatC). For example, gene translation efficiency can be improved by replacing rare codons present in the gene with more frequently used synonymous codons. That is, the gene to be introduced may be modified to have optimal codons depending on the codon usage frequency of the host used. Codon substitution can be performed, for example, by site-directed mutagenesis, which introduces the desired mutation into the target site in DNA. Alternatively, a gene fragment with substituted codons may be totally synthesized. Codon usage frequencies in various organisms are disclosed in the "Codon Usage Database" (http: / / www.kazusa.or.jp / codon; Nakamura, Y. et al., Nucl. Acids Res., 28, 292 (2000)). Preferred are nucleic acid sequences having 70% or more (preferably 80% or more, more preferably 90% or more, even more preferably 95% or more, even more preferably 98% or more, even more preferably 99% or more, and most preferably 100%) sequence identity to SEQ ID NOs: 15, 16, and 17, which are sequences optimized for Lactococcus lactis and Lactobacillus reuteri, SEQ ID NOs: 18, 19, and 20, which are sequences optimized for Bifidobacterium longum, and SEQ ID NOs: 21, 22, and 23, which are sequences optimized for Staphylococcus epidermidis.

[0029] Furthermore, the nucleic acid sequences encoding heterologous TatA, TatB and TatC used in the present invention preferably have nucleic acid sequences corresponding to the amino acid sequences described above in the section "Heterologous Tat-based proteins."

[0030] In a gene construct or vector, the nucleic acid sequence encoding a heterologous Tat system protein may be located 5' to the nucleic acid sequence encoding a Tat system-dependent signal peptide (described below) or 3' to the nucleic acid sequence encoding the heterologous protein, but is preferably located on the 5' side. Also, in a gene construct or vector, the nucleic acid sequence encoding a heterologous Tat system protein is preferably located 3' to the promoter sequence (described below). Most preferably, in the gene construct or vector, the promoter sequence, the nucleic acid sequence encoding the heterologous Tat system protein, the nucleic acid sequence encoding the Tat system-dependent signal peptide, and the nucleic acid sequence encoding the heterologous protein are arranged in this order from 5' to 3' in the gene construct or vector.

[0031] <Nucleic Acid Sequence Encoding a Tat System-Dependent Signal Peptide> The gene construct or vector preferably further comprises a nucleic acid sequence encoding a Tat system-dependent signal peptide. The nucleic acid sequence encoding the Tat system-dependent signal peptide preferably has a nucleic acid sequence encoding a heterologous protein at its 3'-end. That is, the gene construct preferably comprises, from 5' to 3', a nucleic acid sequence encoding a Tat system-dependent signal peptide and a nucleic acid sequence encoding a heterologous protein. Furthermore, the nucleic acid sequence encoding the Tat system-dependent signal peptide and the nucleic acid sequence encoding the heterologous protein are preferably configured such that, upon expression, the Tat system-dependent signal peptide is directly linked to the N-terminus of the heterologous protein. Examples of Tat system-dependent signal peptides include those described above in the section "Signal Peptides (Signal Sequences)." Furthermore, the nucleic acid sequence encoding the signal peptide may be optimized for the host. Optimization methods include, for example, methods similar to those described in the section "Nucleic Acid Sequences Encoding Heterologous Tat System Proteins." As a nucleic acid sequence encoding a Tat system-dependent signal peptide, a nucleic acid sequence having 70% or more (preferably 80% or more, more preferably 90% or more, even more preferably 95% or more, even more preferably 98% or more, even more preferably 99% or more, and most preferably 100%) sequence identity to the nucleic acid sequence encoding the signal peptide of trimethylamine N-oxidoreductase derived from Escherichia coli (SEQ ID NO: 10) or the nucleic acid sequence encoding the signal peptide of FAD-dependent oxidoreductase derived from Bifidobacterium longum (SEQ ID NO: 73) is preferred.Also preferred are nucleic acid sequences that have 70% or more (preferably 80% or more, more preferably 90% or more, even more preferably 95% or more, even more preferably 98% or more, even more preferably 99% or more, and most preferably 100%) sequence identity to the nucleic acid sequence of SEQ ID NO: 27, which is a nucleic acid sequence encoding the signal peptide of trimethylamine N-oxide reductase derived from Escherichia coli optimized for Lactococcus lactis and Lactobacillus reuteri, SEQ ID NO: 28, which is optimized for Bifidobacterium longum, or SEQ ID NO: 29, which is optimized for Staphylococcus epidermidis.

[0032] <Nucleic Acid Sequence Encoding Signal Peptidase> In the present invention, "signal peptidase" refers to a protein (enzyme) that has the activity of removing part or all of a signal peptide from a protein having a Tat-dependent signal peptide. The gene construct or vector may further contain a nucleic acid sequence encoding a signal peptidase. By including a nucleic acid sequence encoding a signal peptidase, part or all of the Tat-dependent signal peptide is more easily removed from a heterologous protein having a Tat-dependent signal peptide at its N-terminus after it is secreted extracellularly via the Tat system. Examples of signal peptidases include those described above in the "Signal Peptidase" section. The nucleic acid sequences encoding EclepB, SipS, SipT, CglepB, LllepB, LrlepB, and BllepB described in the signal peptidase section are as follows: Nucleic acid sequence encoding EcLepB: SEQ ID NO: 80 Nucleic acid sequence encoding SipS: SEQ ID NO: 81 Nucleic acid sequence encoding SipT: SEQ ID NO: 82 Nucleic acid sequence encoding CgLepB: SEQ ID NO: 83 Nucleic acid sequence encoding LlLepB: SEQ ID NO: 84 Nucleic acid sequence encoding LrLepB: SEQ ID NO: 76 Nucleic acid sequence encoding BlLepB: SEQ ID NO: 77 Nucleic acid sequence encoding SeLepB: SEQ ID NO: 79

[0033] The nucleic acid sequence encoding the signal peptidase may or may not be optimized for the host bacterium. Methods for optimizing the nucleic acid sequence for the host bacterium include, for example, the same methods as those described in the section "Nucleic acid sequences encoding heterologous Tat-system proteins." The nucleic acid sequence encoding the signal peptidase preferably has 70% or more (preferably 80% or more, more preferably 90% or more, even more preferably 95% or more, even more preferably 98% or more, even more preferably 99% or more, and most preferably 100%) sequence identity with the nucleic acid sequence of SEQ ID NO: 80, 81, 82, 83, 84, 76, 77, or 79. Also preferred is a nucleic acid sequence having 70% or more (preferably 80% or more, more preferably 90% or more, even more preferably 95% or more, even more preferably 98% or more, even more preferably 99% or more, and most preferably 100%) sequence identity with the nucleic acid sequence of SEQ ID NO: 64, which is a nucleic acid sequence encoding signal peptidase I derived from Corynebacterium glutamicum optimized for Lactococcus lactis and Lactobacillus reuteri.

[0034] <Nucleic acid sequence encoding a heterologous protein>

[0035] The nucleic acid sequence encoding the heterologous protein is preferably a nucleic acid sequence encoding a protein derived from an organism other than a bacterium selected from the group consisting of Bifidobacterium, Lactococcus, Lactobacillus, Limosilactobacillus, and Staphylococcus. Examples of heterologous proteins include those described above. The nucleic acid sequence encoding the heterologous protein may or may not be optimized for the host bacterium. Methods for optimizing a nucleic acid sequence for the host bacterium include, for example, methods similar to those described in the section "Nucleic Acid Sequence Encoding a Heterologous Tat-System Protein." The nucleic acid sequence encoding the heterologous protein preferably has a nucleic acid sequence encoding a Tat-system-dependent signal peptide at its 5'-end. That is, the gene construct preferably comprises, from 5' to 3', a nucleic acid sequence encoding a Tat-system-dependent signal peptide and a nucleic acid sequence encoding the heterologous protein. Furthermore, it is preferable that the nucleic acid sequence encoding the Tat-dependent signal peptide and the nucleic acid sequence encoding the heterologous protein are configured so that there is no stop codon between them and the heterologous protein is expressed with the signal peptide linked to its N-terminus.

[0036] <Promoter> The gene construct or vector preferably further comprises a promoter. The promoter is preferably located on the 5'-end side of the nucleic acid sequence encoding the Tat-dependent signal peptide and the nucleic acid sequence encoding the heterologous protein. There are no limitations on the promoter as long as it functions as a promoter in the host bacterium, but it is preferably a promoter derived from bacteria selected from the group consisting of Bifidobacterium, Lactococcus, Lactobacillus, Limosilactobacillus, and Staphylococcus. Examples of such promoters include the promoter of the pepN gene from Lactococcus lactis (SEQ ID NO: 1), the promoter of the dnaJ gene from Lactococcus lactis (SEQ ID NO: 55), the promoter of the tuf gene from Lactococcus lactis (SEQ ID NO: 56), the promoter of the pfkA gene from Lactococcus lactis (SEQ ID NO: 57), the promoter of the usp45 gene from Lactococcus lactis (SEQ ID NO: 58), the promoter of the cbaH gene from Bifidobacterium longum (SEQ ID NO: 59), the promoter of the gapA gene from Bifidobacterium longum (SEQ ID NO: 60), the promoter of the tuf gene from Lactococcus lactis (SEQ ID NO: 61), the promoter of the pfkA gene from Lactococcus lactis (SEQ ID NO: 62), the promoter of the usp45 gene from Lactococcus lactis (SEQ ID NO: 63), the promoter of the cbaH gene from Bifidobacterium longum (SEQ ID NO: 64), the promoter of the gapA gene from Bifidobacterium longum (SEQ ID NO: 65), the promoter of the gapA gene from Bifidobacterium longum (SEQ ID NO: 66), the promoter of the tuf gene from Lactococcus lactis (SEQ ID NO: 67), the promoter of the pfkA gene from Lactococcus lactis (SEQ ID NO: 68), the promoter of the tuf gene from Lactococcus lactis (SEQ ID NO: 69), the promoter of the tuf gene from Lactococcus lactis (SEQ ID NO: 70), the promoter of the tuf gene from Lactococcus lactis (SEQ ID NO: 71), the promoter of the tuf gene from Lactococcus lactis (SEQ ID NO: 72 Examples of such promoters include the promoter of the tuf gene from Bacillus longum (SEQ ID NO: 61), the Pxyl / tetO promoter from Bacillus subtilis (SEQ ID NO: 54), and the promoter of the tuf gene from Lactobacillus reuteri (SEQ ID NO: 71).Among these, the pepN gene promoter (PpepN), the cbaH gene promoter (PcbaH), Pxyl / tetO, and the like are preferred. The promoter may be a naturally occurring promoter derived from the aforementioned bacteria, or a modified naturally occurring promoter. Examples of modifications to naturally occurring promoters include approximating the -35 and -10 regions within the promoter region to a consensus sequence (WO00 / 18935) and randomly modifying non-consensus sequences (Microbiology 2006 Dec;152:1011-1019).

[0037] The promoter sequence is not particularly limited as long as it functions as a promoter in the host bacterium (has the function of regulating the transcription of a nucleic acid sequence encoding a heterologous protein), but it is preferable that the promoter sequence be a nucleic acid sequence that has 70% or more sequence identity (preferably 80% or more, more preferably 90% or more, even more preferably 95% or more, even more preferably 98% or more, and most preferably 100%) with the nucleic acid sequence of the above-mentioned SEQ ID NO.

[0038] Furthermore, the gene construct or vector of the present invention preferably has a Shine-Dalgarno sequence (SD sequence) in addition to the promoter sequence. The Shine-Dalgarno sequence (SD sequence) preferably contains the nucleic acid sequence AGGAG.

[0039] <Others> A terminator for terminating transcription can be placed in the gene construct or vector. The terminator is preferably placed downstream of the nucleic acid sequence encoding the heterologous protein and / or signal peptidase and / or Tat system protein. The terminator is not particularly limited as long as it functions in the bacterium of the present invention. The terminator may be a terminator derived from the host or a terminator derived from a heterologous species. The terminator may be a terminator inherent to the gene to be introduced or a terminator of another gene.

[0040] Vectors, promoters, and terminators that can be used in various microorganisms are described in detail in, for example, "Basic Microbiology Lectures 8: Genetic Engineering, Kyoritsu Shuppan, 1987," and they can be used.

[0041] Furthermore, when two or more genes are introduced, it is sufficient that each gene is carried in an expressible manner in the bacterium of the present invention. For example, all of the genes may be carried on a single expression vector, or all may be carried on a chromosome. Alternatively, each gene may be carried separately on multiple expression vectors, or may be carried separately on a single or multiple expression vectors and on a chromosome. Alternatively, two or more genes may be introduced as an operon.

[0042] <Introduction Method> The method for introducing a gene construct or recombinant vector that can be used in the present invention into a host bacterium is not particularly limited, and any conventionally known method can be used. For example, a method for treating recipient cells with calcium chloride to increase DNA permeability, as reported for Escherichia coli K-12 (Mandel, M. and Higa, A., J. Mol. Biol. 1970, 53, 159-162), or a method for preparing competent cells from cells in the growth stage and introducing DNA, as reported for Bacillus subtilis (Duncan, CH, Wilson, GA and Young, FE, 1977. Gene 1: 153-167), can be used. Alternatively, a method known for Bacillus subtilis, actinomycetes, and yeasts can be used in which the recipient cells are transformed into protoplasts or spheroplasts, which readily incorporate recombinant DNA, and then the recombinant DNA is introduced into the recipient cells (Chang, S. and Choen, SN, 1979, Mol. Gen. Genet. 168: 111-115; Bibb, MJ, Ward, JM, and Hopwood, OA, 1978, Nature 274: 398-400; Hinnen, A., Hicks, JB, and Fink, GR, 1978, Proc. Natl. Acad. Sci. USA 75: 1929-1933). Alternatively, an electric pulse method, as reported for coryneform bacteria (JP 2-207791), can be used.

[0043] <Production Method> A second aspect of the present invention is a method for producing a heterologous protein, comprising the step of culturing the above-described bacterium to produce and secrete the heterologous protein. The method for culturing the bacterium is not particularly limited, and known culture methods can be used. The production method of the present invention may include a step of removing the bacterial cells after culturing the bacterium to produce and secrete the heterologous protein. Examples of the removal method include centrifugation and filtration. The method may also include a step of purifying the heterologous protein after recovery. Examples of the purification method include salting out, ethanol precipitation, ultrafiltration, gel filtration chromatography, ion exchange column chromatography, affinity chromatography, medium- to high-pressure liquid chromatography, reversed-phase chromatography, and hydrophobic chromatography. The method may also include a step of adding a signal peptidase to the bacterial cells or a culture medium containing the bacterial cells during or after culturing the bacteria.

[0044] <Pharmaceutical Composition> A third aspect of the present invention is a pharmaceutical composition comprising the bacteria described above. In addition to the bacteria, the pharmaceutical composition of the present invention may also contain a pharmaceutically acceptable carrier or diluent. Examples of the carrier or diluent include water (water for injection, saline, etc.), non-volatile oil, polyethylene glycol, glycerin, propylene glycol, antioxidants (ascorbic acid, sodium bisulfite, etc.), chelating agents (ethylenediaminetetraacetic acid, etc.), buffers (acetates, citrates, phosphates, etc.), and isotonic agents (sodium chloride, glucose, etc.). Examples of dosage forms of the pharmaceutical composition include liquids, solid preparations, creams, gels, patches, and sprays (including inhalants).

[0045] In the case of a liquid preparation, for example, the bacterium of the present invention can be placed in physiological saline or the like, and pharmaceutical additives can be added as needed, such as antioxidants (ascorbic acid, sodium bisulfite, etc.), chelating agents (ethylenediaminetetraacetic acid, etc.), buffers (acetate, citrate, phosphate, etc.), and isotonic agents (sodium chloride, glucose, etc.).

[0046] In the case of solid formulations, for example, physiological saline or the above-mentioned liquid formulation containing the bacterium of the present invention can be freeze-dried to produce a freeze-dried formulation. Alternatively, physiological saline or the above-mentioned liquid formulation containing the bacterium of the present invention can be placed in a container and frozen to produce a frozen formulation. Pharmaceutical additives can be added to freeze-dried and frozen formulations as needed. Pharmaceutical additives include excipients, binders, disintegrants, lubricants, tonicity agents, stabilizers, preservatives, etc. Furthermore, a mixture of the bacterium of the present invention with pharmaceutical additives can also be used as a solid formulation. In this case, it is preferable to refrigerate the mixture of the bacterium of the present invention and pharmaceutical additives until use. Pharmaceutical additives include excipients, binders, disintegrants, lubricants, tonicity agents, stabilizers, preservatives, etc. Furthermore, the pharmaceutical composition of the present invention may be in the form of other solid formulations, such as tablets, pills, granules, powders, oral tablets (troches, sublingual tablets, buccal tablets, adhesive tablets, gums), and capsules.

[0047] In the case of creams, for example, the bacteria of the present invention can be used as a cream by mixing it with an oil phase component consisting of a solid oil component made of white petrolatum and higher alcohols and a liquid oil component made of squalane, an aqueous phase component, and a surfactant. In addition to the above-mentioned white petrolatum, higher alcohols, and squalane, other solid oils and liquid oils may also be added to the oil phase components of creams. In the case of gels, for example, the bacteria of the present invention can be dissolved in a solvent such as ethanol or a polyhydric alcohol together with a solubilizer, and mixed with a gel phase swollen with water and a thickener to form a gel. In the case of patches, for example, the bacteria of the present invention can be used as a patch by combining it with a (solid) carrier or matrix such as a bandage, band-aid, or tape. In the case of sprays (including inhalants), for example, the bacteria of the present invention can be used as a spray by delivering it in the form of an aerosol spray from a pressurized container or dispenser containing an appropriate propellant, for example, a gas such as carbon dioxide, or a nebulizer.

[0048] The method of administration of the pharmaceutical composition of the present invention is not limited, and may be oral or parenteral. Oral administration includes oral ingestion by drinking or eating the pharmaceutical composition of the present invention, as well as application of the pharmaceutical composition to the oral cavity. Parenteral administration includes intravenous injection, transdermal administration, intradermal administration, subcutaneous administration, nasal administration (nasal inhalation), intraocular administration, intramuscular administration, and intratumoral administration. Among these, oral administration, intravenous injection, transdermal administration, nasal administration, and intratumoral administration are preferred, with intravenous injection, transdermal administration, and intratumoral administration being more preferred. Furthermore, there are no particular limitations on the dosage and administration interval; appropriate dosage and administration interval may be selected depending on the intended use and patient. The pharmaceutical composition of the present invention can be administered to mammals, with humans being preferred. The pharmaceutical composition of the present invention can be used to treat and / or prevent various types of diseases or conditions depending on the type of heterologous protein produced and secreted. Examples of the disease or condition include central nervous system diseases (e.g., Alzheimer's disease, Parkinson's disease, ischemic neuropathy), inflammatory diseases (e.g., allergic diseases, asthma, rheumatism, osteoarthritis), cardiovascular diseases (e.g., heart failure, cardiac hypertrophy, angina pectoris, arteriosclerosis), cancer (e.g., non-small cell lung cancer, ovarian cancer, prostate cancer, gastric cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer), diabetes, immune system diseases (e.g., autoimmune diseases, immune diseases, atopic dermatitis, allergic diseases, immunodeficiency, asthma, rheumatoid arthritis, psoriasis, arteriosclerosis, diabetes, Alzheimer's disease), contusion, liver and gallbladder diseases (e.g., cirrhosis, hepatitis, liver failure, cholestasis, stones), digestive system diseases (e.g., ulcers, enteritis, dyspepsia, irritable bowel disease, ulcerative colitis, diarrhea, ileus), burns, bone fractures, alopecia, acne, acneiform rash, stomatitis, etc. Among these, central nervous system diseases, inflammatory diseases, cancer, immune system diseases, burns, alopecia, acne, acneiform rash, and stomatitis are preferred, and cancer, immune system diseases, burns, alopecia, acne, acneiform rash, and stomatitis are particularly preferred. Furthermore, the pharmaceutical composition of the present invention can be suitably used for treating or preventing a disease or condition selected from cancer, lower limb ischemic disease, rash and oral mucositis associated with cancer treatment, ichthyosis, dental caries, irritable bowel syndrome, obesity, trauma, and surgical scars. Particularly preferably, the pharmaceutical composition of the present invention can be suitably used for treating or preventing lower limb ischemic disease.

[0049] A fourth aspect of the present invention is the use of the bacterium described above in the manufacture of a medicine. Examples of the medicine and its uses include those described in the section on pharmaceutical compositions above.

[0050] The present invention will be described in more detail below with reference to examples, although the present invention is not limited to the examples shown below.

[0051] <1> Secretion of human basic fibroblast growth factor (bFGF or FGF2) using the signal peptide (TorAss) of Escherichia coli trimethylamine N-oxide reductase by Lactococcus lactis subsp. cremoris MG1363 (LMBP 3019) heterologously expressing the Tat-based secretion apparatus (CgTatABC) from Corynebacterium glutamicum 2256 (ATCC 13869).

[0052] <1-1> Construction of a Plasmid Vector (pNZ9148) Carrying a Constitutive Expression Promoter, PpepN The promoter region and SD sequence of the pepN gene from Lactococcus lactis subsp. cremoris MG1363 strain (SEQ ID NO: 1; hereafter referred to as PpepN; IJ van Alen-Boerrigter et al., Appl. Environ. Microbiol., 57(9), 2555-2561 (1991)) were obtained by artificial gene synthesis (Genscript). This sequence was amplified by PCR using the synthetic DNAs of SEQ ID NOs: 2 and 3 as primers. The resulting PCR product was ligated to the pNZ8148 vector (GoldBio) sequence digested with BglII and PaeI (Thermo Fisher Scientific) using In-Fusion Snap Assembly Master Mix (Clontech). The resulting DNA was used to transform Escherichia coli MC1061F. -Competent cells (Lucigen) of the strain were transformed and plated on LB agar medium (tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 5 g / L, agar 15 g / L) containing 10 μg / mL of chloramphenicol and cultured overnight at 37°C. Plasmids were extracted from the resulting transformants, and one containing the desired PCR product was designated pNZ9148.

[0053] <1-2> Construction of Corynebacterium glutamicum-derived TatABC expression vector (pNZ-PpepN-CgtatAB) The tatA, tatB, and tatC genes from C. glutamicum strain 2256 (SEQ ID NOS: 4, 5, and 6; Y. Kikuchi et al., Appl. Environ. Microbial., 72(11), 7138-7192 (2006)) were each codon-optimized for L. lactis strain MG1363, and the resulting sequence (SEQ ID NOS: 7; hereafter referred to as CgtatABC) containing these three genes was obtained by artificial gene synthesis (Twist Bioscience). This sequence was amplified by PCR using the synthetic DNAs SEQ ID NOS: 8 and 9 as primers. The resulting PCR product was ligated to pNZ9148 vector digested with SpeI and HindIII (Takara) using In-Fusion Snap Assembly Master Mix (Clontech). The obtained DNA was used to transform Escherichia coli MC1061F - Competent cells (Lucigen) of the strain were transformed, plated on LB agar medium containing 10 μg / mL chloramphenicol, and cultured overnight at 37°C. Plasmids were extracted from the resulting transformants, and the one containing the desired PCR product was designated pNZ9148-CgtatABC.

[0054] <1-3> Construction of the TorAss-FGF2 Fusion Protein Expression Vector (pNZ9148-torAss-fgf2) To express the Escherichia coli TorA signal peptide and human FGF2 as a fusion protein, the nucleotide sequences encoding their respective amino acid sequences (SEQ ID NOS: 27 and 11) were ligated. The ORF sequence (SEQ ID NOS: 12; hereafter referred to as "torAss-fgf2") was optimized for the Lactococcus lactis subsp. cremoris MG1363 strain codons and synthesized by artificial gene synthesis (Twist Bioscience). This sequence was amplified by PCR using the synthetic DNA sequences SEQ ID NOS: 13 and 14 as primers. The resulting PCR product was ligated with pNZ9148 vector digested with SpeI and HindIII (Takara) using In-Fusion Snap Assembly Master Mix (Clontech). The resulting DNA was used to transform Escherichia coli MC1061F - Competent cells (Lucigen) of the strain were transformed, plated on LB agar medium containing 10 μg / mL chloramphenicol, and cultured overnight at 37°C. Plasmids were extracted from the resulting transformants, and the one containing the desired PCR product was designated pNZ9148-torAss-fgf2.

[0055] <1-4> Construction of the CgTatABC and TorAss-FGF2 fusion protein expression vector (pNZ9148-CgtatAB-torAss-fgf2). The CgtatABC sequence, the pepN promoter sequence, and the downstream torA-fgf sequence (CgtatABC-PpepN-torA-fgf2) were synthesized by artificial gene synthesis (Twist Bioscience). This sequence was amplified by PCR using the synthetic DNAs of SEQ ID NOs: 8 and 14 as primers. The resulting PCR product was ligated with pNZ9148 vector digested with SpeI and HindIII (Takara) using In-Fusion Snap Assembly Master Mix (Clontech). The resulting DNA was transformed into E. coli MC1061F. -Competent cells (Lucigen) of the strain were transformed, plated on LB agar medium containing 10 μg / mL chloramphenicol, and cultured overnight at 37°C. Plasmids were extracted from the resulting transformants, and the one containing the desired PCR product was designated pNZ9148-CgtatABC-torAss-fgf2.

[0056] <1-5> Construction of Lactococcus lactis expressing CgTatABC or TorAss-FGF2 pNZ9148, pNZ9148-CgtatABC, pNZ9148-torAss-fgf2, and pNZ9148-CgtatABC-torAss-fgf2 were each introduced into competent cells of Lactococcus lactis subsp. cremoris MG1363 (GoldBio) by electroporation, and the cells were plated on M17 agar medium (BD Difco) containing 10 μg / mL chloramphenicol and 5 g / L glucose and cultured overnight at 30°C. The resulting transformants were designated Lactococcus lactis MG1363 / pNZ9148, Lactococcus lactis MG1363 / pNZ9148-CgtatABC, Lactococcus lactis MG1363 / pNZ9148-torAss-fgf2, and Lactococcus lactis MG1363 / pNZ9148-CgtatABC-torAss-fgf2, respectively. These strains were inoculated into 5 mL of M17 medium containing 10 μg / mL chloramphenicol and 5 g / L glucose and cultured overnight at 30°C. The resulting cultures were mixed with an equal volume of 30% (v / v) glycerol solution and stored at -80°C.

[0057] <1-6> FGF2 production and secretion by Lactococcus lactis MG1363 / pNZ9148-CgtatABC-torAss-fgf2 strain. Stock solutions of Lactococcus lactis MG1363 / pNZ9148, Lactococcus lactis MG1363 / pNZ9148-CgtatABC, Lactococcus lactis MG1363 / pNZ9148-torAss-fgf2, and Lactococcus lactis MG1363 / pNZ9148-CgtatABC-torAss-fgf2 strains were scraped with a 10-μL syringe and inoculated into 5 mL of M17 (G5M17) medium containing 10 μg / mL chloramphenicol and 5 g / L glucose. The cultures were incubated for 30 minutes under anaerobic conditions using Anaeropack (registered trademark) (Mitsubishi Gas Chemical Company, Ltd.). The culture was then cultured at 30°C for 20 hours with shaking. 250 μL of the resulting culture was then inoculated into 5 mL of M17 (G10M17) medium containing 10 μg / mL chloramphenicol, 10 mg / L catalase (derived from bovine liver), 40 g / L calcium carbonate, and 10 g / L glucose. The culture was then cultured at 30°C for 48 hours with shaking under anaerobic conditions using Anaeropack® (Mitsubishi Gas Chemical Company). The resulting culture was then centrifuged at 7500 × g for 10 minutes at 4°C. The supernatant was filtered, and the filtrate was concentrated using an ultrafiltration filter (Amicon® Ultra 3K, Merck) to obtain a culture supernatant concentrate. The resulting culture supernatant concentrate was subjected to reducing SDS-PAGE using NuPAGE 10% Bit-Tris Protein Gels (Thermo Fisher Scientific) and NuPAGE MOPS-SDS Running buffer (Thermo Fisher Scientific). The samples were then transferred to a PVDF membrane using iBlot Gel Transfer Stacks PVDF, Mini (Thermo Fisher Scientific) and iBlot 2 Dry Blotting System (Thermo Fisher Scientific).The transferred PVDF membrane was blotted with anti-FGF2 monoclonal antibody (Thermo Fisher Scientific) and goat anti-mouse IgG (H+L)-HRP conjugate (Thermo Fisher Scientific) using iBind Western Systems (Thermo Fisher Scientific) and the I Bind Flex Solution Kit (Thermo Fisher Scientific). After blotting, the PVDF membrane was washed with ultrapure water and immersed in ECL Prime Western Blotting Detection Reagent (Cytiva) at room temperature for 5 minutes, followed by detection using a ChemiDoc (Bio-Rad).

[0058] The results are shown in Figure 1. Surprisingly, an FGF2 band was detected only in the Lactococcus lactis MG1363 / pNZ9148-CgtatABC-torAss-fgf2 strain, demonstrating that although Lactococcus lactis MG1363 does not have an endogenous Tat system secretion apparatus, heterologous expression of the Tat system secretion apparatus derived from Corynebacterium glutamicum enables the expression and secretion of a desired protein via the Tat system.

[0059] <2> Secretion of FGF2 using the E. coli TorA signal peptide by the L. lactis MG1363 strain (LMBP 3019) heterologously expressing the Tat secretion apparatus (CgTatABC) derived from C. glutamicum 2256 and the type I signal peptidase (CgLepB; WO 2020 / 085511) derived from C. glutamicum 2256 or the type I signal peptidase homolog (LllepB) derived from L. lactis MG1363.

[0060] <2-1> Construction of the CgTatABC, TorAss-FGF2, and CgLepB Expression Vector (pNZ9148-CgtatABC-torAss-fgf2-CglepB) The type I signal peptidase gene CglepB (SEQ ID NO: 64) from C. glutamicum 2256, codon-optimized for L. lactis MG1363, was obtained by artificial gene synthesis (Twist Bioscience). Using this sequence as a template, a sequence containing CglepB was amplified by PCR using synthetic DNAs SEQ ID NOs: 65 and 66 as primers. The resulting PCR product was ligated to the pNZ9148-CgtatABC-torAss-fgf2 vector, which had been linearized by inverse PCR using synthetic DNAs SEQ ID NOs: 67 and 68 as primers, using In-Fusion Snap Assembly Master Mix (Clontech). The resulting DNA was used to transform E. coli MC1061F - Competent cells (Lucigen) of the strain were transformed, plated on LB agar medium containing 10 μg / mL chloramphenicol, and cultured overnight at 37°C. Plasmids were extracted from the resulting transformants, and the one containing the desired PCR product was designated pNZ9148-CgtatABC-torAss-fgf2-CglepB.

[0061] <2-2> Construction of TorAss-FGF2 and CgLepB Expression Vector (pNZ9148-torAss-fgf2-CglepB) As in <2-1>, a sequence containing CglepB was amplified by PCR using the CglepB sequence obtained by gene synthesis as a template and synthetic DNAs of SEQ ID NOs: 65 and 66 as primers. The resulting PCR product and the pNZ9148-torAss-fgf2 vector, which had been linearized by inverse PCR using synthetic DNAs of SEQ ID NOs: 67 and 68 as primers, were ligated using In-Fusion Snap Assembly Master Mix (Clontech). The resulting DNA was used to transform E. coli MC1061F -Competent cells (Lucigen) of the strain were transformed, plated on LB agar medium containing 10 μg / mL chloramphenicol, and cultured overnight at 37°C. Plasmids were extracted from the resulting transformants, and the one containing the desired PCR product was designated pNZ9148-torAss-fgf2-CglepB.

[0062] <2-3> Construction of the CgTatABC, TorAss-FGF2, and LllepB Expression Vector (pNZ9148-CgtatABC-torAss-fgf2-LllepB) Using the genome of L. lactis MG1363 as a template, a sequence containing LllepB was amplified by PCR using synthetic DNA primers SEQ ID NOs: 69 and 70. The resulting PCR product was ligated to the pNZ9148-CgtatABC-torAss-fgf2 vector, which had been linearized by inverse PCR using synthetic DNA primers SEQ ID NOs: 67 and 68, using In-Fusion Snap Assembly Master Mix (Clontech). The resulting DNA was transformed into E. coli MC1061F. - Competent cells (Lucigen) of the strain were transformed, plated on LB agar medium containing 10 μg / mL chloramphenicol, and cultured overnight at 37°C. Plasmids were extracted from the resulting transformants, and the one containing the desired PCR product was designated pNZ9148-CgtatABC-torAss-fgf2-LllepB.

[0063] <2-4> Construction of TorAss-FGF2 and LllepB Expression Vector (pNZ9148-torAss-fgf2-LllepB) As in <2-3>, a sequence containing LllepB was amplified by PCR using the genome of L. lactis MG1363 as a template and the synthetic DNAs of SEQ ID NOs: 69 and 70 as primers. The resulting PCR product and pNZ9148-torAss-fgf2, which had been linearized by PCR using SEQ ID NOs: 67 and 68, were ligated using In-Fusion Snap Assembly Master Mix (Clontech). The resulting DNA was used to transform E. coli MC1061F -Competent cells (Lucigen) of the strain were transformed, plated on LB agar medium containing 10 μg / mL chloramphenicol, and cultured overnight at 37°C. Plasmids were extracted from the resulting transformants, and the one containing the desired PCR product was designated pNZ9148-torAss-fgf2-LllepB.

[0064] <2-5> Construction of Lactococcus lactis expressing CgTatABC, TorAss-FGF2, and CglepB or LllepB. pNZ9148-CgtatABC-torAss-fgf2-CglepB, pNZ9148-torAss-fgf2-CglepB, pNZ9148-CgtatABC-torAss-fgf2-LllepB, and pNZ9148-torAss-fgf2-LllepB were each introduced into competent cells of Lactococcus lactis subsp. cremoris MG1363 (GoldBio) by electroporation. The cells were then plated on M17 agar medium (BD Difco) containing 10 μg / mL chloramphenicol and 5 g / L glucose and cultured overnight at 30°C. The resulting transformants were designated Lactococcus lactis MG1363 / pNZ9148-CgtatABC-torAss-fgf2-CglelpB, Lactococcus lactis MG1363 / pNZ9148-torAss-fgf2-CglepB, Lactococcus lactis MG1363 / pNZ9148-CgtatABC-torAss-fgf2-LllepB, and Lactococcus lactis MG1363 / pNZ9148-torAss-fgf2-LllepB, respectively. These strains were inoculated into 5 mL of M17 medium containing 10 μg / mL chloramphenicol and 5 g / L glucose and cultured overnight at 30°C. The resulting culture medium was mixed with an equal volume of 30% (v / v) glycerol solution and stored at -80°C.

[0065] <2-6> Improvement of production and secretion of mature FGF2 by Lactococcus lactis MG1363 / pNZ9148-CgtatABC-torAss-fgf2-CglepB strain and Lactococcus lactis MG1363 / pNZ9148-CgtatABC-torAss-fgf2-LllepB strain Lactococcus lactis MG1363 / pNZ9148 strain, Lactococcus lactis MG1363 / pNZ9148-CgtatABC strain, Lactococcus lactis MG1363 / pNZ9148-torAss-fgf2 strain, Lactococcus lactis MG1363 / pNZ9148-CgtatABC-torAss-fgf2 strain, Lactococcus lactis The MG1363 / pNZ9148-CgtatABC-torAss-fgf2-CglelpB strain, Lactococcus lactis MG1363 / pNZ9148-torAss-fgf2-CglepB strain, Lactococcus lactis MG1363 / pNZ9148-CgtatABC-torAss-fgf2-LllepB strain, and Lactococcus lactis MG1363 / pNZ9148-torAss-fgf2-LllepB strain were scraped with a 10-µL syringe and inoculated into 5 mL of M17 (G5M17) medium containing 10 µg / mL chloramphenicol and 5 g / L glucose. The medium was then cultured at 30°C with shaking under anaerobic conditions for 20 hours using Anaeropack (registered trademark, Mitsubishi Gas Chemical Company). Subsequently, 250 μL of the resulting culture was inoculated into 5 mL of M17 (G10M17) medium containing 10 μg / mL chloramphenicol, 10 mg / L catalase (derived from bovine liver), 40 g / L calcium carbonate, and 10 g / L glucose. The medium was then cultured for 48 hours under anaerobic conditions at 30°C with shaking using Anaeropack® (Mitsubishi Gas Chemical Company). The resulting culture was then centrifuged at 7500 × g for 10 minutes at 4°C. The supernatant was filtered, and the resulting filtrate was concentrated using an ultrafiltration filter (Amicon® Ultra 3K, Merck) to obtain a concentrated culture supernatant.The protein concentration in the resulting culture supernatant concentrate was quantified using the Quick Start Bradford Protein Assay Kit (Bio-Rad). The culture supernatant was diluted to 12 μg total protein and subjected to reducing SDS-PAGE using NuPAGE 10% Bit-Tris Protein Gels (Thermo Fisher Scientific) and NuPAGE MOPS-SDS Running buffer (Thermo Fisher Scientific). The protein was then transferred to a PVDF membrane using iBlot Gel Transfer Stacks PVDF, Mini (Thermo Fisher Scientific) and the iBlot 2 Dry Blotting System (Thermo Fisher Scientific). The transferred PVDF membrane was blotted with anti-FGF2 monoclonal antibody (Thermo Fisher Scientific) and goat anti-mouse IgG (H+L)-HRP conjugate (Thermo Fisher Scientific) using iBind Western Systems (Thermo Fisher Scientific) and the I Bind Flex Solution Kit (Thermo Fisher Scientific). After blotting, the PVDF membrane was washed with ultrapure water and immersed in ECL Prime Western Blotting Detection Reagent (Cytiva) for 5 minutes at room temperature. Bands were detected using ChemiDoc (Bio-Rad). Bands were quantified using Quantity One software (Bio-Rad).

[0066] The results are shown in Figure 2. The secretion levels of mature FGF2 with its signal sequence cleaved were increased on average by 1.9-fold and 1.5-fold, respectively, in the Lactococcus lactis MG1363 / pNZ9148-CgtatABC-torAss-fgf2-LllepB and Lactococcus lactis MG1363 / pNZ9148-CgtatABC-torAss-fgf2 strains compared to Lactococcus lactis MG1363 / pNZ9148-CgtatABC-torAss-fgf2. Therefore, it was demonstrated that heterologous expression of Corynebacterium glutamicum signal peptidase I or enhancement of Lactococcus lactis signal peptidase I improved the ability of Lactococcus lactis MG1363, which heterologously expresses the Corynebacterium glutamicum Tat secretion system, to secrete mature proteins.

[0067] <3> Secretion of Pro-PGHisx6 using the E. coli TorA signal peptide by L. lactis MG1363 (LMBP 3019), which heterologously expresses the Tat secretion apparatus (CgTatABC) from C. glutamicum 2256 and the type I signal peptidase homolog (LllepB) from L. lactis MG1363.

[0068] <3-1> Construction of the TorAss-Pro-PGHisx6 and LllepB Expression Vector (pNZ9148-torAss-ppghis-LllepB) The gene sequence for Chryseobacterium proteolyticum protein glutaminase (Pro-PGHisx6), which contains an N-terminal pro-sequence and a C-terminal polyhistidine tag, was codon-optimized for Lactococcus lactis MG1363 (SEQ ID NO: 85) and obtained by artificial gene synthesis (Twist Bioscience). The obtained sequence was amplified by PCR using synthetic DNAs represented by SEQ ID NOs: 86 and 87 as primers. The obtained sequence and the pNZ9148-torAss-fgf2-LllepB vector, which had been linearized by PCR to remove the fgf2 sequence using synthetic DNAs represented by SEQ ID NOs: 88 and 89 as primers, were ligated using In-Fusion Snap Assembly Master Mix (Clontech). The resulting DNA was used to transform E. coli MC1061F - Competent cells (Lucigen) of the strain were transformed, plated on LB agar medium containing 10 μg / mL chloramphenicol, and cultured overnight at 37°C. Plasmids were extracted from the resulting transformants, and the one containing the desired PCR product was designated pNZ9148-torAss-ppghis-LllepB.

[0069] <3-2> Construction of CgTatABC, TorAss-Pro-PGHisx6, and LlLepB Expression Vector (pNZ9148-Pusp45-CgtatABC-torAss-ppghis-LllepB) As in <3-1>, the Pro-PG gene sequence was amplified by PCR using the synthetic DNAs of SEQ ID NOs: 86 and 87 as primers, and the pNZ9148-CgtatABC-torAss-fgf2-LllepB vector was linearized by PCR using the synthetic DNAs of SEQ ID NOs: 88 and 89 to remove the fgf2 sequence. The resulting DNA was ligated using In-Fusion Snap Assembly Master Mix (Clontech). E. coli MC1061F -Competent cells of the strain (Lucigen) were transformed, plated on LB agar medium containing 10 μg / mL chloramphenicol, and cultured overnight at 37°C. Plasmids were extracted from the resulting transformants, and one containing the desired PCR product was designated pNZ9148-CgtatABC-torAss-ppghis-LllepB. To modify the CgtatABC expression promoter, a sequence containing the usp45 primer (SEQ ID NO: 58) was amplified by PCR using the genome of L. lactis MG1363 as a template and synthetic DNA primers SEQ ID NOs: 90 and 91. This and the pNZ9148-CgtatABC-torAss-ppghis-LllepB vector, which had been linearized by PCR to remove the pepN promoter sequence using synthetic DNA primers SEQ ID NOs: 92 and 93, were then ligated using In-Fusion Snap Assembly Master Mix (Clontech). The resulting DNA was used to transform E. coli MC1061F - Competent cells (Lucigen) of the strain were transformed, plated on LB agar medium containing 10 μg / mL chloramphenicol, and cultured overnight at 37°C. Plasmids were extracted from the resulting transformants, and the one containing the desired PCR product was designated pNZ-Pusp45-CgtatABC-torAss-ppghis-LllepB.

[0070] <3-3> Construction of Lactococcus lactis MG1363 strain expressing CgtatABC, TorAss-pro-PG, and LllepB. pNZ9148-torAss-ppg-LllepB and pNZ9148-Pusp45-CgtatABC-torAss-ppghis-LllepB were introduced into competent cells of Lactococcus lactis subsp. cremoris MG1363 (GoldBio) by electroporation. The cells were then plated on M17 agar medium (BD Difco) containing 10 μg / mL chloramphenicol and 5 g / L glucose, and cultured overnight at 30°C. The resulting transformants were designated Lactococcus lactis MG1363 / pNZ9148-torAss-ppghis-LllepB and Lactococcus lactis MG1363 / pNZ9148-Pusp45-CgtatABC-torAss-ppghis-LllepB, respectively. These strains were inoculated into 5 mL of M17 medium containing 10 μg / mL chloramphenicol and 5 g / L glucose, and cultured overnight at 30°C. The resulting cultures were mixed with an equal volume of 30% (v / v) glycerol solution and stored at -80°C.

[0071] <3-4> Improvement of production and secretion of mature Pro-PGHisx6 by Lactococcus lactis MG1363 / pNZ9148-torAss-ppghis-LllepB strain The Lactococcus lactis MG1363 / pNZ9148-torAss-ppghis-LllepB strain and the Lactococcus lactis MG1363 / pNZ9148-Pusp45-CgtatABC-torAss-ppghis-LllepB strain were scraped with a 1-µL volume of enzyme and inoculated into 5 mL of M17 (G5M17) medium containing 10 µg / mL of chloramphenicol and 5 g / L of glucose. The medium was then shake-cultured at 30°C under anaerobic conditions for 20 hours using Anaeropack (registered trademark) (manufactured by Mitsubishi Gas Chemical Company). Subsequently, 100 μL of the resulting culture was inoculated into 5 mL of M17 (G10M17) medium containing 10 μg / mL chloramphenicol, 10 mg / L catalase (derived from bovine liver), 40 g / L calcium carbonate, and 10 g / L glucose. The culture was then cultured for 48 hours under anaerobic conditions at 30°C with shaking using Anaeropack® (Mitsubishi Gas Chemical Company). The resulting culture was then centrifuged at 7500 × g for 10 minutes at 4°C. The supernatant was filtered, and the filtrate was concentrated using an ultrafiltration filter (Amicon® Ultra 3K, Merck) to obtain a culture supernatant concentrate. The protein concentration in the resulting culture supernatant concentrate was quantified using a Quick Start Bradford Protein Assay Kit (Bio-Rad). The culture supernatant was diluted to a total protein content of 18 μg and subjected to reducing SDS-PAGE as described in <2-6>. Next, the fragments were transferred to a PVDF membrane using the same method as in <2-6> and blotted with a 6x-His tag monoclonal antibody (His.H8) and goat anti-mouse IgG (H+L)-HRP conjugate (Thermo Fisher Scientific). After blotting, the PVDF membrane was washed with ultrapure water and immersed in ECL Prime Western Blotting Detection Reagent (Cytiva) at room temperature for 5 minutes, followed by detection using a ChemiDoc (Bio-Rad).The molecular weight was estimated using Quantity One software (Bio-Rad).

[0072] The results are shown in Figure 3. Only in the Lactococcus lactis MG1363 / pNZ9148-Pusp45-CgtatABC-torAss-ppghis-LllepB strain was a band detected at approximately 33.5 kDa, the predicted molecular mass of Pro-PGHisx6. In addition, no band was detected at approximately 43.3 kDa, the molecular mass of the induced protein TorAss-Pro-PGHisx6 with an intact signal sequence. These results demonstrate that enhanced Lactococcus lactis signal peptidase I and heterologous expression of the Corynebacterium glutamicum Tat secretion system result in the secretion of signal-sequence-truncated Pro-PGHisx6.

[0073] <4> Secretory expression of FGF2 using the E. coli TorA signal peptide or the Bifidobacterium longum BIOML-A18 FAD-dependent oxidoreductase signal peptide by Bifidobacterium longum 105-A (JCM 31944) strain heterologously expressing the Tat-based secretion system from C. glutamicum 2256 (CgTatABC) or the Tat-based secretion system from Bifidobacterium longum E-18 (BlTatABC).

[0074] <5> Secretion of FGF2 using the E. coli TorA signal peptide by Staphylococcus epidermidis ATCC 12228 strain heterologously expressing the Tat secretion system (CgTatABC) derived from C. glutamicum 2256 or the Tat secretion system (BsTatAdCd) derived from Bacillus subtilis 168.

[0075] <6> Secretory expression of FGF2 using the E. coli TorA signal peptide by Lactobacillus reuteri DSM20016 strain heterologously expressing the Tat-based secretion apparatus (CgTatABC) derived from C. glutamicum strain 2256.

[0076] <6-1> Construction of Lactobacillus reuteri DSM20016 strain expressing CgTatABC or TorAss-FGF2. Competent cells of Lactobacillus reuteri DSM20016 were prepared according to a previously published method (Holo, H. and Nes, IN, 1995. Mehotds. Mol. Biol. 47: 195-199). A 1-μL sample of a glycerol stock of Lactobacillus reuteri DSM20016 was spread onto MRS (BD Difco) (MRSCS) agar medium containing 0.22% (w / v) L-cysteine ​​hydrochloride monohydrate and 3.4% (w / v) sodium L-ascorbate. The cells were then cultured overnight at 37°C under anaerobic conditions using Anaeropack® (Mitsubishi Gas Chemical Company). Approximately 0.5 cups of the resulting bacterial cells were scraped and inoculated into 40 mL of MRSCS medium containing 0.5 M sucrose and 2% (w / v) glycine. The culture was grown statically until the OD660 reached 0.5-0.6. The resulting culture was centrifuged at 7500 × g for 10 minutes at 4°C in a large centrifuge, and the supernatant was discarded. The resulting bacterial pellet was washed three times with 20 mL of ice-cold washing solution (0.5 M sucrose, 10% (v / v) glycerol), suspended in 1.8 mL of ice-cold 10% (v / v) glycerol, and centrifuged at 16,200 × g for 10 minutes at 4°C in a small centrifuge. The supernatant was discarded, and the resulting cell pellet was resuspended in 1.8 mL of 10% (v / v) glycerol, dispensed into 60 μL aliquots, and stored at -80°C.

[0077] The plasmid vectors pNZ9148, pNZ9149-CgtatABC, pNZ9148-torAss-fgf2, and pNZ9148-CgtatABC-torAss-fgf2 constructed in <1-1>, <1-2>, <1-3>, and <1-4> were each introduced into competent cells of Lactobacillus reuteri DSM20016 by the electric pulse method. The cells were then plated onto MRSCS agar medium containing 10 μg / mL of chloramphenicol and cultured overnight at 37°C under anaerobic conditions using Anaeropack (registered trademark) (Mitsubishi Gas Chemical Company). The resulting transformants were designated Lactobacillus reuteri DSM20016 / pNZ9148, Lactobacillus reuteri DSM20016 / pNZ9148-CgtatABC, Lactobacillus reuteri DSM20016 / pNZ9148-torAss-fgf2, and Lactobacillus reuteri DSM20016 / pNZ9148-CgtatABC-torAss-fgf2, respectively. These strains were inoculated into 5 mL of MRSCS medium containing 10 μg / mL chloramphenicol and cultured overnight at 37°C under anaerobic conditions using Anaeropack® (Mitsubishi Gas Chemical Company). The resulting cultures were mixed with an equal volume of 30% (v / v) glycerol solution and stored at -80°C.

[0078] <6-2> FGF2 production and secretion by Lactobacillus reuteri DSM20016 / pNZ9148-CgtatABC-torAss-fgf2 strain. Glycerol stocks of Lactobacillus reuteri DSM20016 / pNZ9148, Lactobacillus reuteri DSM20016 / pNZ9148-CgtatABC, Lactobacillus reuteri DSM20016 / pNZ9148-torAss-fgf2, and Lactobacillus reuteri DSM20016 / pNZ9148-CgtatABC-torAss-fgf2 strains were scraped into 10-μL volumes and cultured in MRSCS medium containing 10 μg / mL chloramphenicol. 1 mL of the culture medium was inoculated and cultured at 37°C under anaerobic conditions using Anaeropack® (Mitsubishi Gas Chemical Company) for 20 hours with shaking. Then, 100 μL of the resulting culture medium was inoculated into 5 mL of MRSCS medium containing 10 μg / mL chloramphenicol, 40 g / L calcium carbonate, and 10 mg / L catalase. Culture was then cultured at 37°C under anaerobic conditions using Anaeropack® (Mitsubishi Gas Chemical Company) for 48 hours with shaking. The resulting culture medium was then centrifuged at 7500 × g at 4°C for 10 minutes. The supernatant was filtered, and the resulting filtrate was concentrated using an ultrafiltration filter (Amicon® Ultra 3K, Merck) to obtain a culture supernatant concentrate. The resulting culture supernatant concentrate was subjected to Western blotting using the same method as in <1-6>.

[0079] The results are shown in Figure 4. An FGF2 band was detected only in the Lactobacillus reuteri DSM20016 / pNZ9148-CgtatABC-torAss-fgf2 strain, indicating that although the Lactobacillus reuteri DSM20016 strain does not have an endogenous Tat secretion system, heterologous expression of the Tat secretion system derived from Corynebacterium glutamicum enables the expression and secretion of a desired protein via the Tat system.

[0080]

[0081] According to the present invention, the Tat system can be utilized to express heterologous proteins even in bacteria that do not have an endogenous Tat secretion system, making the present invention extremely useful industrially.

Claims

1. 1. A bacterium selected from the group consisting of the genera Bifidobacterium, Lactococcus, Lactobacillus, Limosilactobacillus and Staphylococcus, which has been modified to express a heterologous Tat system protein, The bacterium described above, which has the ability to produce and secrete a heterologous protein.

2. The bacterium according to claim 1 , wherein the bacterium is a bacterial strain that does not have an endogenous Tat-based secretion apparatus.

3. The bacterium according to claim 1 , which has the ability to secrete the heterologous protein extracellularly via a heterologous Tat system protein.

4. 2. The bacterium of claim 1, wherein the bacterium is selected from the group consisting of Lactococcus lactis, Bifidobacterium longum, Lactobacillus reuteri (also known as Limosilactobacillus reuteri), and Staphylococcus epidermidis.

5. The bacterium of claim 1, comprising a genetic construct comprising, in the 5' to 3' direction, a nucleic acid sequence encoding a Tat system-dependent signal peptide and a nucleic acid sequence encoding a heterologous protein.

6. The bacterium of claim 5 , wherein the genetic construct further comprises a nucleic acid sequence encoding a heterologous Tat-system protein.

7. 2. The bacterium according to claim 1, wherein the heterologous Tat system protein is a Tat system protein derived from a bacterium selected from the genera Corynebacterium, Bacillus and Bifidobacterium.

8. The bacterium according to claim 1, wherein the heterologous Tat system protein comprises at least one selected from TatA, TatB and TatC.

9. The bacterium of claim 1 , wherein the heterologous Tat system proteins include TatA, TatB, and TatC.

10. The bacterium according to claim 1 , wherein the heterologous protein comprises at least one selected from FGF2 and FGF1.

11. 6. The bacterium of claim 5, wherein the genetic construct further comprises a promoter derived from a bacterium selected from the group consisting of Bifidobacterium, Lactococcus, Lactobacillus, Limosilactobacillus, and Staphylococcus.

12. The bacterium of claim 5 , wherein the genetic construct further comprises a nucleic acid sequence encoding a signal peptidase.

13. The bacterium of claim 5 , comprising a recombinant vector incorporating a gene construct.

14. A method for producing a heterologous protein, comprising the step of culturing the bacterium according to any one of claims 1 to 13 to produce and secrete the heterologous protein.

15. A pharmaceutical composition comprising the bacterium according to any one of claims 1 to 13.

16. The pharmaceutical composition according to claim 15, for treating or preventing a disease or condition selected from cancer, lower limb ischemic disease, rash or oral mucositis associated with cancer treatment, ichthyosis, tooth decay, irritable bowel syndrome, obesity, trauma, and surgical scars.