Method for producing target proteins

Abstract

To provide methods for producing target proteins.SOLUTION: Disclosed is a method for producing a protein encoded by a target gene comprising a step of expressing a target gene in a bacterium of the genus Burkholderia where the bacterium does not have one or more genes selected from the group consisting of BSFP_068740, BSFP_068730 and BSFP_068720, or the expression of the gene(s) or the expression of the protein(s) encoded by the gene(s) is inhibited.SELECTED DRAWING: Figure 4

Description

Technical Field

[0001] The present invention relates to a method for producing a target protein in bacteria of the genus Burkholderia, etc.

Background Art

[0002] Bacteria of the genus Burkholderia are Gram-negative bacteria that secrete and produce useful lipases (Patent Document 1), and can express and secrete extracellular secretory lipases, etc., for which it is difficult to express the active form in Escherichia coli. Therefore, their use as a platform for a useful protein expression system has been reported (see Patent Documents 2 and 3).

[0003] However, since the amount of recombinant protein produced using bacteria of the genus Burkholderia is small compared to host-vector systems such as Escherichia coli, yeast, and filamentous fungi, there are few examples of use, and for producing lipases derived from the genus Burkholderia, there are also examples where a host-vector system using Escherichia coli rather than bacteria of the genus Burkholderia is used (Patent Document 4 and Non-Patent Document 1).

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Non-Patent Documents

[0005]

Non-Patent Document 1

Summary of the Invention

[0006] The object of the present invention is to provide a novel method for expressing recombinant proteins in large quantities in Burkholderia bacteria, which have few examples of use as a platform for useful protein expression systems. [Means for solving the problem]

[0007] The inventors have discovered that by inhibiting the function of a gene located on a specific chromosome in Burkholderia bacteria, the expression vector becomes stable in Burkholderia bacteria, and desirable properties are conferred to the bacteria through stable protein production. A novel promoter capable of increasing the expression level of the target protein was also discovered.

[0008] In other words, this application encompasses the following inventions. [1] A method for producing a protein encoded by a target gene, A method comprising the step of expressing a target gene in a Burkholderia bacterium that does not have one or more genes selected from the group consisting of BSFP_068740, BSFP_068730, and BSFP_068720, or in which the expression of such genes or the expression of the protein encoded by such genes is inhibited. [2] The gene BSFP_068740 contains one of the following DNAs (i) to (viii): (i) DNA consisting of the base sequence of Sequence ID No. 1; (ii) DNA that can hybridize under stringent conditions with DNA having a nucleotide sequence complementary to the nucleotide sequence of Sequence ID No. 1, wherein the protein encoded thereby has the function of reducing the copy number of the expression construct in Burkholderia bacteria or reducing its stability; (iii) DNA comprising a nucleotide sequence in which one or more nucleotides are deleted, substituted, or added in the nucleotide sequence of Sequence ID No. 1, wherein the protein encoded thereby has the function of reducing the copy number of the expression construct in Burkholderia bacteria or reducing its stability; (iv) DNA comprising a nucleotide sequence having at least 80% sequence identity with Sequence ID No. 1, wherein the protein encoded thereby has the function of reducing the copy number of an expression construct in Burkholderia bacteria or reducing its stability; (v) DNA consisting of a base sequence that codes for a protein consisting of the amino acid sequence of Sequence ID No. 2; (vi) DNA that can hybridize under stringent conditions with DNA comprising a base sequence encoding a protein comprising the amino acid sequence of Sequence ID No. 2, wherein the protein encoded thereby has the function of reducing the copy number of an expression construct in Burkholderia bacteria or reducing its stability; (vii) DNA comprising a protein encoding a protein having an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence of Sequence ID No. 2, and which has the function of reducing the copy number of the expression construct in Burkholderia bacteria or reducing its stability; or (viii) A protein comprising an amino acid sequence having at least 80% sequence identity with Sequence ID No. 2, and having the function of reducing the copy number of the expression construct in Burkholderia bacteria or reducing its stability, comprising a DNA sequence that encodes a protein. The method described in [1], including the method described in [1]. [3] The gene BSFP_068730 contains one of the following DNAs (i) to (viii): (i) DNA consisting of the base sequence of Sequence ID No. 3; (ii) DNA that can hybridize under stringent conditions with DNA having a nucleotide sequence complementary to the nucleotide sequence of Sequence ID No. 3, wherein the protein encoded thereby has the function of reducing the copy number of the expression construct in Burkholderia bacteria or reducing its stability; (iii) DNA comprising a nucleotide sequence in which one or more nucleotides are deleted, substituted, or added in the nucleotide sequence of Sequence ID No. 3, wherein the protein encoded thereby has the function of reducing the copy number of the expression construct in Burkholderia bacteria or reducing its stability; (iv) DNA comprising a nucleotide sequence having at least 80% sequence identity with Sequence ID No. 3, wherein the protein encoded thereby has the function of reducing the copy number of the expression construct in Burkholderia bacteria or reducing its stability; (v) DNA consisting of a base sequence that codes for a protein consisting of the amino acid sequence of Sequence ID No. 4; (vi) DNA that can hybridize under stringent conditions with DNA comprising a base sequence encoding a protein comprising the amino acid sequence of Sequence ID No. 4, wherein the protein encoded thereby has the function of reducing the copy number of an expression construct in Burkholderia bacteria or reducing its stability; (vii) DNA comprising a protein encoding a protein having an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence of SEQ ID NO: 4, and which has the function of reducing the copy number of the expression construct in Burkholderia bacteria or reducing its stability; or (viii) A protein comprising an amino acid sequence having at least 80% sequence identity with Sequence ID No. 4, and having the function of reducing the copy number of the expression construct in Burkholderia bacteria or reducing its stability, comprising a DNA sequence that encodes a protein. The method described in [1] or [2], including the method described in [1] or [2]. [4] The gene BSFP_068720 contains one of the following DNAs (i) to (viii): (i) DNA consisting of the base sequence of Sequence ID No. 5; (ii) DNA that can hybridize under stringent conditions with DNA having a nucleotide sequence complementary to the nucleotide sequence of Sequence ID No. 5, wherein the protein encoded thereby has the function of reducing the copy number of the expression construct in Burkholderia bacteria or reducing its stability; (iii) DNA comprising a nucleotide sequence in which one or more nucleotides are deleted, substituted, or added in the nucleotide sequence of Sequence ID No. 5, wherein the protein encoded thereby has the function of reducing the copy number of the expression construct in Burkholderia bacteria or reducing its stability; (iv) DNA comprising a nucleotide sequence having at least 80% sequence identity with Sequence ID No. 5, wherein the protein encoded thereby has the function of reducing the copy number of the expression construct in Burkholderia bacteria or reducing its stability; (v) DNA consisting of a base sequence that codes for a protein consisting of the amino acid sequence of Sequence ID No. 6; (vi) DNA that can hybridize under stringent conditions with DNA comprising a base sequence encoding a protein comprising the amino acid sequence of Sequence ID No. 6, wherein the protein encoded thereby has the function of reducing the copy number of an expression construct in Burkholderia bacteria or reducing its stability; (vii) DNA comprising a protein encoding a protein having an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence of Sequence ID No. 6, and which has the function of reducing the copy number of the expression construct in Burkholderia bacteria or reducing its stability; or (viii) A protein consisting of an amino acid sequence having at least 80% or more sequence identity with SEQ ID NO: 6, which has a function of reducing the copy number of an expression construct in bacteria of the genus Burkholderia or reducing its stability, a DNA consisting of a nucleotide sequence encoding the protein, The method according to any one of [1] to [3], comprising [5] The method according to any one of [1] to [4], wherein the target gene is linked to the expression construct so as to be capable of expression. [6] The method according to any one of [2] to [5], wherein the expression construct is an expression vector. [7] The method according to [6], wherein the expression construct is a plasmid replicable in bacteria of the genus Burkholderia. [8] The method according to [7], wherein the plasmid is a broad-host vector. [9] The method according to [8], wherein the broad-host vector is pBBR122, pSa or RK2.

[10] The method according to any one of [2] to [9], wherein the expression construct contains a promoter that controls the expression of the target gene.

[11] The method according to

[10] , wherein the promoter is derived from bacteria of the genus Burkholderia.

[12] The promoter is any one of the following DNAs (i) to (iv): (i) A DNA consisting of a nucleotide sequence of more than 80 consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 7 and having promoter activity; (ii) A DNA that can hybridize under stringent conditions with a DNA consisting of a complementary nucleotide sequence of more than 80 consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 7 and having promoter activity; (iii) A DNA consisting of a nucleotide sequence in which one or several nucleotides of a nucleotide sequence of more than 80 consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 7 are deleted, substituted or added and having promoter activity; or (iv) DNA having promoter activity, consisting of more than 80 consecutive base sequences and base sequences having at least 80% sequence identity with the sequence sequence of Sequence ID No. 7, A method according to any of [1] to

[11] , including the method described above.

[13] The promoter is one of the following DNAs (i) to (iv): (i) DNA consisting of the base sequence of Sequence ID No. 8; (ii) DNA that can hybridize under stringent conditions with DNA having a nucleotide sequence complementary to the nucleotide sequence of Sequence ID No. 8 and possesses promoter activity; (iii) DNA having promoter activity, consisting of a nucleotide sequence in which one or more nucleotides are deleted, substituted, or added in the nucleotide sequence of Sequence ID No. 8; (iv) A DNA having a nucleotide sequence that is at least 80% identical to sequence number 8 and possesses promoter activity, The method described in

[12] , including the method described in

[12] .

[14] The promoter is one of the following DNAs (i) to (iv): (i) DNA having promoter activity, consisting of more than 100 consecutive base pairs in the base sequence of Sequence ID No. 9; (ii) DNA that can hybridize under stringent conditions with DNA consisting of more than 100 consecutive complementary base sequences in the base sequence of Sequence ID No. 9, and that possesses promoter activity; (iii) DNA having promoter activity, consisting of a sequence in which one or more bases in a sequence of more than 100 consecutive bases of sequence number 9 are deleted, substituted, or added; or (iv) DNA having promoter activity, consisting of more than 100 consecutive base sequences and base sequences having at least 80% sequence identity with respect to sequence sequence 9, A method according to any of [1] to

[13] , including the method described above.

[15] The promoter is one of the following DNAs (i) to (iv): (i) DNA consisting of the base sequence of Sequence ID No. 10; (ii) DNA that can hybridize under stringent conditions with DNA having a nucleotide sequence complementary to the nucleotide sequence of Sequence ID No. 10 and that possesses promoter activity; (iii) DNA having promoter activity, consisting of a nucleotide sequence in which one or more nucleotides are deleted, substituted, or added in the nucleotide sequence of Sequence ID No. 10; (iv) DNA having a nucleotide sequence that is at least 80% identical to sequence number 10 and possessing promoter activity, The method described in

[14] , including the method described in

[14] .

[16] The method described in any of [1] to

[15] , wherein the target gene encodes an esterase.

[17] The gene encoding the esterase is one of the following DNAs (i) to (iv): (i) DNA consisting of one of the base sequences from sequence numbers 11 to 13; (ii) DNA that can hybridize under stringent conditions with DNA consisting of a base sequence complementary to any one of the base sequences of sequence numbers 11 to 13, wherein the protein encoded thereby has esterase activity; (iii) DNA consisting of a nucleotide sequence in which one or more nucleotides are deleted, substituted, or added in any one of the nucleotide sequences of sequence numbers 11 to 13, wherein the protein encoded thereby has esterase activity; or (iv) DNA comprising a base sequence having at least 80% sequence identity with any one of sequence numbers 11 to 13, wherein the protein encoded thereby has esterase activity, The method described in

[16] , including the method described in

[16] .

[18] The method according to any one of [1] to

[17] , wherein the expression construct comprises DNA encoding a foldase.

[19] The DNA encoding foldase is one of the following (i) to (iv): (i) DNA consisting of one of the base sequences from sequence numbers 14 to 16; (ii) DNA that can hybridize under stringent conditions with DNA consisting of a base sequence complementary to any one of the base sequences of sequence numbers 14-16, wherein the protein encoded thereby has foldase activity; (iii) DNA consisting of a nucleotide sequence in which one or more nucleotides are deleted, substituted, or added in any one of the nucleotide sequences of sequence numbers 14 to 16, wherein the protein encoded thereby has foldase activity; or (iv) DNA comprising a base sequence having at least 80% sequence identity with any one of the base sequences of sequence numbers 14 to 16, wherein the protein encoded thereby has foldase activity, The method described in

[18] , including the method described in

[18] .

[20] The method described in any of [1] to

[19] , wherein the target gene further encodes a signal sequence. [twenty one] The method described in any of [1] to

[20] , wherein the bacterium of the genus Burkholderia is Burkholderia stabilis. [twenty two] One of the following DNA samples (i) to (iv): (i) DNA that has promoter activity and consists of more than 80 consecutive base pairs in the base sequence of Sequence ID No. 7; (ii) DNA that can hybridize under stringent conditions with DNA consisting of more than 80 consecutive complementary base sequences in the base sequence of Sequence ID No. 7, and that possesses promoter activity; (iii) DNA having promoter activity, consisting of a sequence in which one or more bases in the sequence of Sequence ID No. 7 are deleted, substituted, or added to more than 80 consecutive bases; or (iv) DNA having promoter activity, consisting of more than 80 consecutive base sequences and base sequences having at least 80% sequence identity with the sequence sequence of Sequence ID No. 7, A promoter consisting of [something]. [twenty three] The promoter is one of the following DNAs (i) to (iv): (i) DNA consisting of the base sequence of Sequence ID No. 8; (ii) DNA that can hybridize under stringent conditions with DNA having a nucleotide sequence complementary to the nucleotide sequence of Sequence ID No. 8 and possesses promoter activity; (iii) DNA having promoter activity, consisting of a nucleotide sequence in which one or more nucleotides are deleted, substituted, or added in the nucleotide sequence of Sequence ID No. 8; (iv) A DNA having a nucleotide sequence that is at least 80% identical to sequence number 8 and possesses promoter activity, The promoter described in

[22] , including the promoter described in

[22] . [twenty four] One of the following DNA samples (i) to (iv): (i) DNA having promoter activity, consisting of more than 100 consecutive base pairs in the base sequence of Sequence ID No. 9; (ii) DNA that can hybridize under stringent conditions with DNA consisting of more than 100 consecutive complementary base sequences in the base sequence of Sequence ID No. 9, and that possesses promoter activity; (iii) DNA having promoter activity, consisting of a sequence in which one or more bases in a sequence of more than 100 consecutive bases of sequence number 9 are deleted, substituted, or added; or (iv) DNA having promoter activity, consisting of more than 100 consecutive base sequences and base sequences having at least 80% sequence identity with respect to sequence sequence 9, A promoter consisting of [something]. [twenty five] The promoter is one of the following DNAs (i) to (iv): (i) DNA consisting of the base sequence of Sequence ID No. 10; (ii) DNA that can hybridize under stringent conditions with DNA having a nucleotide sequence complementary to the nucleotide sequence of Sequence ID No. 10 and that possesses promoter activity; (iii) DNA having promoter activity, consisting of a nucleotide sequence in which one or more nucleotides are deleted, substituted, or added in the nucleotide sequence of Sequence ID No. 10; (iv) DNA having a nucleotide sequence that is at least 80% identical to sequence number 10 and possessing promoter activity, The promoter described in

[24] , including the promoter described in

[24] .

[26] A mutant strain of the genus Burkholderia, modified to inhibit the expression of one or more genes selected from the group consisting of BSFP_068740, BSFP_068730, and BSFP_068720, or the expression of the protein encoded by said genes.

[27] The mutant strain according to

[26] , wherein the mutant strain comprises an expression construct, and the expression construct comprises a promoter that controls the expression of a target gene.

[28] The mutant strain described in

[27] , wherein the promoter is one of the promoters described in

[22] to

[25] .

[29] A method for maintaining expression constructs in bacteria of the genus Burkholderia, A method comprising the step of culturing a Burkholderia bacterium that does not have one or more genes selected from the group consisting of BSFP_068740, BSFP_068730, and BSFP_068720, or in which the expression of said genes or the expression of the protein encoded by said genes is inhibited, the Burkholderia bacterium containing an expression construct.

[30] The method according to

[29] , wherein the copy number of the expression construct is increased or its stability is increased in cultured Burkholderia bacteria. [Effects of the Invention]

[0009] According to the present invention, compared to Burkholderia bacteria in which the function of genes on specific chromosomes is not inhibited, high production of target proteins becomes possible, and the stability of expression vectors in Burkholderia bacteria can be increased. [Brief explanation of the drawing]

[0010] [Figure 1] Figure 1 shows the process of creating a gene disruption vector. [Figure 2] Figure 2 shows the electrophoresis results (bottom) of PCR products of the wild-type strain (1) and the disrupted gene region (2) of the gene knockout strain, and schematic diagrams (top) of the wild-type gene and the disrupted gene. [Figure 3] Figure 3 shows the cholesterol esterase expression vector. [Figure 4] Figure 4 shows the number of pBBR122 copies per bacterium extracted from wild-type strains, BSFP_068720 knockout strains, BSFP_068730 knockout strains, BSFP_068740 knockout strains, and chromosome 3 deletion strains into which pBBR122 was introduced. [Figure 5] Figure 5 shows the electrophoretic results of pBBR122 extracted from wild-type strains, BSFP_068720 knockout strains, BSFP_068730 knockout strains, BSFP_068740 knockout strains, and chromosome 3 deletion strains into which pBBR122 was introduced. [Figure 6] Figure 6 shows the cholesterol esterase activity in wild-type strains, BSFP_068720 knockout strains, BSFP_068730 knockout strains, BSFP_068740 knockout strains, and chromosome 3 knockout strains, all of which were introduced into pBBR122 without the cholesterol esterase gene or pBBR122-182 containing the cholesterol esterase gene. [Figure 7] Figure 7 shows the electrophoretic results of pSa extracted from wild-type strains, BSFP_068720 knockout strains, BSFP_068730 knockout strains, BSFP_068740 knockout strains, and chromosome 3 deletion strains into which pSa was introduced. [Figure 8]Figure 8 shows the cholesterol esterase activity in the wild-type strain, BSFP_068720 knockout strain, BSFP_068730 knockout strain, BSFP_068740 knockout strain, and chromosome 3 deletion strain, all of which were introduced with pRK2-182. [Figure 9] Figure 9 shows the lipase activity in wild-type strains, BSFP_068720 knockout strains, BSFP_068730 knockout strains, BSFP_068740 knockout strains, and chromosome 3 deletion strains, all of which were introduced with a vector containing lipase derived from Burkholderia cepacia. [Figure 10] Figure 10 shows the lipase activity in wild-type strains, BSFP_068720 knockout strains, BSFP_068730 knockout strains, BSFP_068740 knockout strains, and chromosome 3 deletion strains that were introduced with a vector containing lipase derived from Burkholderia plantarii. [Figure 11] Figure 11 shows the expression levels of cholesterol esterase when the 14020 promoter and the 48230 promoter are shortened. [Figure 12] Figure 12 shows the electrophoresis results of amplification products obtained by PCR using primers SEQ ID NOs. 98 and 99, with total DNA of Burkholderia silva tranthica and total DNA of Burkholderia stabilis as templates. [Modes for carrying out the invention]

[0011] In a first embodiment, a method is provided for producing a protein encoded by a target gene, comprising the step of expressing the target gene in a Burkholderia bacterium that does not have one or more genes selected from the group consisting of BSFP_068740, BSFP_068730, and BSFP_068720, or in which the expression of such genes or the expression of the protein encoded by such genes is inhibited.

[0012] The nucleotide sequence encoding the protein is not particularly limited, but for example, it may be a nucleotide sequence obtained by modifying the nucleotide sequence of SEQ ID NOs: 1, 3, or 5 to match the codon usage frequency of a host, such as Burkholderia bacteria such as Burkholderia stabilis. Furthermore, in the case of the above proteins, any nucleotide sequence encoding an amino acid sequence substantially equivalent to that of each protein may be used, as long as it is a protein that has the function of reducing the copy number of the expression construct or reducing its stability. For example, it may be a nucleotide sequence encoding an equivalent of an amino acid sequence in which some amino acids not involved in the above function of each protein have been mutated, for example, one or more amino acids have been deleted, substituted, or added. Preferably, it is particularly preferable that all or part of the nucleotide sequence of SEQ ID NOs: 1, 3, or 5 is included.

[0013] Burkholderia bacteria, which are Gram-negative aerobic rods, are sometimes used as platforms for useful protein expression systems, and more than 60 species, such as Burkholderia stabilis, are registered. Among Burkholderia bacteria, for example, Burkholderia stabilis FERMP-21014 (Genome Announcements Volume 5 Issue 29 e00636-17) has three chromosomes: chromosome 1 (3.6 Mbp), chromosome 2 (3.2 Mbp), and chromosome 3 (0.9 Mbp), with the smallest chromosome 3 containing 849 genes. By deleting a specific chromosome in Burkholderia bacteria, the production of target proteins may be increased compared to the same bacteria without the deletion. While not intended to be constrained by theory, it is thought that inhibiting the function of genes located on the specific chromosomes mentioned above that have a function that disrupts the stability of expression vectors such as plasmids increases the stability of the expression vectors, and as a result, increases protein production.

[0014] As used herein, "Burkholderia bacteria" means any bacterium of the genus Burkholderia. Burkholderia bacteria may be naturally occurring or artificially created, as long as the function of genes that have a function that causes the expression vector such as a plasmid to lose stability is inhibited. Among Burkholderia bacteria, Burkholderia stabilis and Burkholderia silva trantica are preferred.

[0015] The genome sequence of Burkholderia stabilis FERMP-21014 is publicly known, ( https: / / www.ncbi.nlm.nih.gov / genome / 45559?genome _ assembly _ id=331877 For example, the gene sequences that make up chromosome 3 are also publicly known (https: / / www.ncbi.nlm.nih.gov / nuccore / AP018113.1). Examples of genes that have a function that disrupts the stability of expression vectors such as plasmids include BSFP_068740, BSFP_068730, or BSFP_068720 of Burkholderia stabilis FERMP-21014, which are located on chromosome 3. BSFP_068740, BSFP_068730, and BSFP_068720 may have different or similar functions, but inhibiting one or more functions of each gene can improve the stability of the expression vector. The BSFP number represents the locus tag according to NCBI (National Center for Biotechnology Information).

[0016] In a preferred embodiment, the gene BSFP_068740 is one of the following DNAs (i) to (viii): (i) DNA consisting of the base sequence of Sequence ID No. 1; (ii) DNA that can hybridize under stringent conditions with DNA having a nucleotide sequence complementary to the nucleotide sequence of Sequence ID No. 1, wherein the protein encoded thereby has the function of reducing the copy number of the expression construct in Burkholderia bacteria or reducing its stability; (iii) DNA comprising a nucleotide sequence in which one or more, preferably one or more, nucleotides are deleted, substituted, or added in the nucleotide sequence of Sequence ID No. 1, wherein the protein encoded thereby has the function of reducing the copy number of the expression construct in Burkholderia bacteria or reducing its stability; (iv) DNA comprising a nucleotide sequence having at least 80%, preferably 90%, sequence homology, preferably sequence identity, with SEQ ID NO: 1, wherein the protein encoded thereby has the function of reducing the copy number of the expression construct in Burkholderia bacteria or reducing its stability; (v) DNA consisting of a base sequence that codes for a protein consisting of the amino acid sequence of Sequence ID No. 2; (vi) DNA that can hybridize under stringent conditions with DNA comprising a base sequence encoding a protein comprising the amino acid sequence of Sequence ID No. 2, wherein the protein encoded thereby has the function of reducing the copy number of an expression construct in Burkholderia bacteria or reducing its stability; (vii) DNA comprising a protein encoding a protein having an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence of Sequence ID No. 2, and which has the function of reducing the copy number of the expression construct in Burkholderia bacteria or reducing its stability; or (viii) A protein comprising an amino acid sequence having at least 80% or more, preferably 90% or more sequence homology, preferably sequence identity, with SEQ ID NO: 2, and having the function of reducing the copy number of the expression construct in Burkholderia bacteria or reducing its stability, comprising a DNA base sequence encoding a protein. It may include.

[0017] In a preferred embodiment, the gene BSFP_068730 is one of the following DNAs (i) to (viii): (i) DNA consisting of the base sequence of Sequence ID No. 3; (ii) DNA that can hybridize under stringent conditions with DNA having a nucleotide sequence complementary to the nucleotide sequence of Sequence ID No. 3, wherein the protein encoded thereby has the function of reducing the copy number of the expression construct in Burkholderia bacteria or reducing its stability; (iii) DNA comprising a nucleotide sequence in which one or more, preferably one or more, nucleotides are deleted, substituted, or added in the nucleotide sequence of Sequence ID No. 3, wherein the protein encoded thereby has the function of reducing the copy number of the expression construct in Burkholderia bacteria or reducing its stability; (iv) DNA comprising a base sequence having at least 80%, preferably 90%, sequence homology, preferably sequence identity, with SEQ ID NO: 3, wherein the protein encoded thereby has the function of reducing the copy number of the expression construct in Burkholderia bacteria or reducing its stability; (v) DNA consisting of a base sequence that codes for a protein consisting of the amino acid sequence of Sequence ID No. 4; (vi) DNA that can hybridize under stringent conditions with DNA comprising a base sequence encoding a protein comprising the amino acid sequence of Sequence ID No. 4, wherein the protein encoded thereby has the function of reducing the copy number of an expression construct in Burkholderia bacteria or reducing its stability; (vii) DNA comprising a protein encoding a protein having an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence of SEQ ID NO: 4, and which has the function of reducing the copy number of the expression construct in Burkholderia bacteria or reducing its stability; or (viii) A protein comprising an amino acid sequence having at least 80% or more, preferably 90% or more sequence homology, preferably sequence identity, with SEQ ID NO: 4, and having the function of reducing the copy number of the expression construct in Burkholderia bacteria or reducing its stability, comprising a DNA base sequence encoding a protein. It may include.

[0018] In a preferred embodiment, the gene BSFP_068720 is one of the following DNAs (i) to (viii): (i) DNA consisting of the base sequence of Sequence ID No. 5; (ii) DNA that can hybridize under stringent conditions with DNA having a nucleotide sequence complementary to the nucleotide sequence of Sequence ID No. 5, wherein the protein encoded thereby has the function of reducing the copy number of the expression construct in Burkholderia bacteria or reducing its stability; (iii) DNA comprising a nucleotide sequence in which one or more, preferably one or more, nucleotides are deleted, substituted, or added in the nucleotide sequence of Sequence ID No. 5, wherein the protein encoded thereby has the function of reducing the copy number of the expression construct in Burkholderia bacteria or reducing its stability; (iv) DNA comprising a nucleotide sequence having at least 80%, preferably 90%, sequence homology, preferably sequence identity, with SEQ ID NO: 5, wherein the protein encoded thereby has the function of reducing the copy number of the expression construct in Burkholderia bacteria or reducing its stability; (v) DNA consisting of a base sequence that codes for a protein consisting of the amino acid sequence of Sequence ID No. 6; (vi) DNA that can hybridize under stringent conditions with DNA comprising a base sequence encoding a protein comprising the amino acid sequence of Sequence ID No. 6, wherein the protein encoded thereby has the function of reducing the copy number of an expression construct in Burkholderia bacteria or reducing its stability; (vii) DNA comprising a protein encoding a protein having an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence of Sequence ID No. 6, and which has the function of reducing the copy number of the expression construct in Burkholderia bacteria or reducing its stability; or (viii) A protein comprising an amino acid sequence having at least 80% or more, preferably 90% or more sequence homology, preferably sequence identity, with SEQ ID NO: 6, and having the function of reducing the copy number of the expression construct in Burkholderia bacteria or reducing its stability, comprising a DNA base sequence encoding a protein. It may include.

[0019] In this specification, "stringent conditions" refer to conditions such as "1xSSC, 0.1% SDS, 37°C," more stringent conditions such as "0.5xSSC, 0.1% SDS, 42°C," and even more stringent conditions such as "0.2xSSC, 0.1% SDS, 65°C." The more stringent the hybridization conditions, the higher the expected isolation of DNA with high homology, preferably high identity, to the probe sequence. However, the above combinations of SSC, SDS, and temperature are illustrative examples, and those skilled in the art can achieve similar stringency by appropriately combining the above or other factors that determine hybridization stringency (e.g., probe concentration, probe length, hybridization reaction time, etc.). DNA hybridized under such stringent conditions generally exhibits high sequence identity. High sequence identity means having at least 60%, preferably 70%, more preferably 80%, particularly preferably 90%, and most preferably 95% or more homology.

[0020] As used herein, "one or more bases" means that the number of bases varies depending on the total length of the reference sequence to which deletion, substitution, or addition is made, but for example, for a base sequence consisting of 100 bases, it means 1 to 30 bases, preferably 1 to 20 bases, and more preferably 1 to 10 bases. The same applies to "one or several bases," which means, for example, for a base sequence consisting of 100 bases, it means 1 to 10 bases, preferably 1 to 5 bases, and more preferably 4, 3, 2, or 1 base.

[0021] As used herein, "one or more amino acids" means that the number of amino acids varies depending on the total length of the reference sequence to which deletion, substitution, or addition is made, but for example, for an amino acid sequence consisting of 100 amino acids, it means 1 to 30, preferably 1 to 20, and more preferably 1 to 10 amino acids. The same applies to "one or several amino acids," which means, for example, for an amino acid sequence consisting of 100 amino acids, it means 1 to 10, preferably 1 to 5, and more preferably 4, 3, 2, or 1 amino acid.

[0022] Sequence homology or sequence identity, when calculated using BLAST or similar tools (for example, using default, i.e., initial parameters), is at least 80%, preferably 90%, more preferably 95%, and even more preferably 98% for a given sequence.

[0023] A protein encoded by a target gene, intended to be produced in bacteria of the genus Burkholderia, is also called a "target protein." The target protein is not particularly limited as long as it is a protein that can be produced in bacteria of the genus Burkholderia. Preferably, the target protein is an extracellular secretion type protein that is difficult to express in conventionally known host vector systems such as Escherichia coli, yeast, or filamentous fungi. Enzymes are examples of such proteins, including esterases, lipases, amylases, glucoamylases, α-galactosidases, β-galactosidases, α-glucosidases, β-glucosidases, mannosidases, isomerases, invertases, transferases, ribonucleases, deoxyribonucleases, chitinases, catalases, laccases, phenol oxidases, oxidases, oxidoreductases, cellulases, xylanases, peroxidases, hydrolases, cutinases, proteases, phytases, lyases, pectinases, aminopeptidases, and carboxypeptidases. Among these enzymes, esterases classified as EC 3.1.1 Carboxylic-ester hydrolases are preferred, esterases classified as EC 3.1.1.1 carboxylesterases are more preferred, sterol esterases classified as EC 3.1.1.13 - sterol esterases and triacylglycerol lipases (lipases) classified as EC 3.1.1.3 are even more preferred, and cholesterol esterases are particularly preferred.

[0024] Examples of cholesterol esterases include sterol ester hydrolase from Burkholderia stabilis (EC3.1.1.13). Examples of lipases include lipase from Burkholderia grumae (LG Frenken et al., Mol Microbiol. 9(3), 591-9 (1993)). The target protein may or may not contain a signal peptide. Preferably, the target protein further contains a signal peptide. For example, a signal peptide from E. coli can be used. Signal peptides reported in papers, etc. (Tullman-Ercek et al., J Bio Chem. 16, 282(11), 8309-16 (2007)) or signal peptides obtained by searching the amino acid sequence in the protein database Uniprot (http: / / www.uniprot.org / ) can also be used. Using the latter method, for example, sequence information can be obtained for the signal peptides of the E. coli protein BsmA (https: / / www.uniprot.org / uniprot / P39297), the YafY protein (https: / / www.uniprot.org / uniprot / P77365), and the YnjE protein (https: / / www.uniprot.org / uniprot / P78067).

[0025] The base sequence encoding the esterase is not particularly limited, but for example, it may be a base sequence obtained by modifying one of the base sequences of SEQ ID NOs: 11 to 13 to match the codon usage frequency of the host, such as Burkholderia stabilis or other Burkholderia bacteria. Alternatively, any base sequence encoding a substantially identical amino acid sequence of a protein having esterase activity may be used, for example, a base sequence encoding an equivalent of an amino acid sequence in which some amino acids not involved in esterase activity have been mutated, such as one or more amino acids being deleted, substituted, or added.

[0026] In a preferred embodiment, the gene encoding the esterase is, for example, one of the following DNAs (i) to (iv): (i) DNA consisting of one of the base sequences from sequence numbers 11 to 13; (ii) DNA that can hybridize under stringent conditions with DNA consisting of a base sequence complementary to any one of the base sequences of sequence numbers 11 to 13, wherein the protein encoded thereby has esterase activity; (iii) DNA comprising a base sequence in which one or more, preferably one or more, bases are deleted, substituted, or added in any one of the base sequences of sequence numbers 11 to 13, wherein the protein encoded thereby has esterase activity; or (iv) DNA comprising a base sequence having at least 80% sequence identity with any one of sequence numbers 11 to 13, wherein the protein encoded thereby has esterase activity, It may include.

[0027] The target gene encoding the target protein is preferably ligated to an expression construct such as a plasmid so that it can be expressed in Burkholderia bacteria. The expression construct only needs to be replicable in Burkholderia bacteria, and examples include expression vectors, preferably autonomously replicating plasmid vectors. Examples of expression vectors include plasmid vectors that can replicate in Burkholderia bacteria, such as broad-range host vectors like pBBR122, pSa, and pRK2.

[0028] The expression construct preferably includes a promoter that controls the expression of the target gene. The promoter may be derived from a bacterium of the genus Burkholderia. By selecting a promoter with appropriate promoter activity, the transcription of the target gene increases, and the amount of protein produced also increases. Examples of such promoters include the promoter of the BSFP_015180 gene, which encodes glycerol-3-phosphate dehydrogenase, having promoter activity in a continuous region of approximately 80 bp or more upstream of the start codon of the gene (hereinafter also referred to as the "14020 promoter"), and the promoter of the BSFP_052240 gene, which encodes a protein of unknown function, having promoter activity in a continuous region of approximately 100 bp or more upstream of the start codon of the gene (hereinafter also referred to as the "48230 promoter").

[0029] Expression constructs can be constructed by those skilled in the art, for example, by incorporating a promoter at any location in a plasmid vector that autonomously replicates in Burkholderia bacteria, and by placing a multi-cloning site downstream of the promoter sequence to facilitate the introduction of the target gene and a terminator to terminate transcription.

[0030] Expression constructs are replicated within the host from their origin of replication. Therefore, it is preferable to use expression constructs that have an origin of replication that can be replicated in the host. In Gram-negative bacteria such as Burkholderia, expression constructs with an origin of replication that can be replicated in Gram-negative bacteria are preferred. The pBBR122 vector is an example of a plasmid vector that has an origin of replication that can be replicated in Gram-negative bacteria. Additionally, plasmids with replication origins different from pBBR122, such as pSa (Datta, American Society for Microbiology, Washington, DC, pp9-15 (1975), Gorai et al., Plasmid 2 489-492 (1979), Ward and Grinsted, Plasmid 7 239-250 (1982), Tait et al., Mol. Gen. Genet. 186 10-15 (1982)), and plasmids containing RK2ori (TJ Schmidhauser et al., Plasmid. 9(3) 325-30 (1983)), can also be used.

[0031] The expression construct may further include a selection marker and a conjugation site. The introduction of the expression construct into Burkholderia bacteria can be carried out by means known to those skilled in the art. Examples include conjugation and electroporation (Kim et al., Applied Biochemistry and Biotechnology, Vol. 73, 81-88 (1998)).

[0032] By transforming host cells with a plasmid vector containing the target gene introduced downstream of the promoter of the constructed expression construct, the target protein is expected to be constitutively expressed in the transformants selected according to the selection marker of the expression construct. The shuttle vector, multi-cloning site, and terminator used are not particularly limited. Expression constructs also include those into which such target genes have been introduced.

[0033] The target gene may be located downstream of the promoter sequence, under the control of the promoter sequence, for example, by 30 bases or less, preferably 15 bases or less, more preferably 6 bases or less, and particularly preferably directly linked to the promoter sequence. The target gene may have a signal sequence, for example, if the protein it encodes is secreted.

[0034] A terminator sequence is not necessarily required to be placed downstream of the target gene, but it is preferable to do so. A terminator sequence is a sequence that dissociates RNA polymerase from DNA and terminates transcription, and is usually placed downstream of the gene. Various known terminator sequences can be used without particular limitations. If the target gene to be expressed encodes cholesterol esterase, it is also convenient and preferable to use the natural terminator sequence that is already attached to that gene.

[0035] Depending on the target protein, foldases and chaperones may be expressed simultaneously with the target protein for expression or activation. This leads to the formation of polypeptide chains with the correct three-dimensional structure, and ultimately to the production of proteins with the correct function. When foldases and chaperones are required for protein production, it is preferable to express the protein and the foldases and chaperones simultaneously.

[0036] The nucleotide sequence encoding the chaperone is not particularly limited, but for example, it may be a known nucleotide sequence modified to match the codon usage frequency of a host, such as Burkholderia stabilis or other Burkholderia bacteria. Alternatively, any nucleotide sequence encoding a substantially identical amino acid sequence is acceptable, as long as it is a protein that functions as a chaperone. For example, it may be a nucleotide sequence encoding an equivalent of an amino acid sequence in which some amino acids not involved in chaperone function have been mutated, such as one or more amino acids being deleted, substituted, or added.

[0037] If foldases or chaperones are to be expressed simultaneously with the target protein, they may be located at distant locations in the sequence, and it is preferable that they be located downstream of the promoter necessary to initiate transcription.

[0038] To express foldases or chaperones simultaneously with the target gene, the foldase sequence or chaperone sequence may be inserted between the target gene and the terminator, and the foldase or chaperone may be expressed at the promoter of the target gene.

[0039] When foldases or chaperones function at a specific location, it is preferable that the encoding sequence includes a signal sequence.

[0040] In a preferred embodiment, the DNA encoding the foldase is one of the following DNAs (i) to (iv): (i) DNA consisting of one of the base sequences from sequence numbers 14 to 16; (ii) DNA that can hybridize under stringent conditions with DNA consisting of a base sequence complementary to any one of the base sequences of sequence numbers 14-16, wherein the protein encoded thereby has foldase activity; (iii) DNA comprising a base sequence in which one or more, preferably one or more, bases are deleted, substituted, or added in any one of the base sequences of sequence numbers 14 to 16, wherein the protein encoded thereby has foldase activity; or (iv) DNA comprising a base sequence having at least 80% sequence homology, preferably 90% or more, and preferably sequence identity with any one of the base sequences of sequence numbers 14 to 16, wherein the protein encoded thereby has foldase activity, It may include.

[0041] The target protein can be produced by expressing the target gene in Burkholderia bacteria. Preferably, the target protein can be produced by culturing Burkholderia bacteria (transformed organisms) transformed with an expression construct containing the gene encoding the target gene, and then isolating and purifying the protein encoded by the target gene from the culture. Here, the culture refers to a mixture of bacterial cells obtained by culturing bacteria and a solid or liquid culture medium. The transformed organisms can be cultured using a medium suitable for the host used, by methods such as static culture or roller bottle culture. Culturing can be carried out by known methods, and the medium and culture conditions can be appropriately determined depending on the type of Burkholderia bacterial strain used.

[0042] If the target protein is produced within the bacterial cell, the protein can be collected by disrupting the Burkholderia bacteria. If the target protein is produced outside the bacterial cell, the culture medium can be used as is, or the host cells can be removed by centrifugation or other means. Subsequently, the target protein can be isolated and purified from the culture by using various general biochemical methods employing chromatography, either alone or in appropriate combinations.

[0043] In a second embodiment, a 14020 promoter or a 48230 promoter is provided.

[0044] The 14020 promoter or 48230 promoter can be widely used to express target genes in any Burkholderia bacterium, but when used in Burkholderia bacteria in which the function of a gene on a specific chromosome is inhibited, they show significantly higher promoter activity compared to when used in wild-type strains.

[0045] The 14020 promoter is one of the following DNAs (i) to (iv): (i) DNA having promoter activity, consisting of more than approximately 80 consecutive base pairs in the base sequence of Sequence ID No. 7, for example, 90 or more, or 100 or more; (ii) DNA that can hybridize under stringent conditions with DNA consisting of more than approximately 80 consecutive complementary base sequences, for example, 90 or more, or 100 or more, in the base sequence of Sequence ID No. 7, and that has promoter activity; (iii) DNA having promoter activity, comprising a base sequence in which more than 80 consecutive bases, for example, 90 or more, 100 or more bases, preferably one or several bases, are deleted, substituted, or added in the base sequence of Sequence ID No. 7; or (iv) DNA having promoter activity, comprising more than 80 consecutive base sequences, for example, 90 or more, 100 or more base sequences, and having at least 80% sequence homology, preferably 90% or more, and particularly preferably sequence identity. It may include.

[0046] In a preferred embodiment, the 14020 promoter is, for example, one of the following DNAs (i) to (iv): (i) DNA consisting of the base sequence of Sequence ID No. 8; (ii) DNA that can hybridize under stringent conditions with DNA having a nucleotide sequence complementary to the nucleotide sequence of Sequence ID No. 8 and possesses promoter activity; (iii) DNA having promoter activity, consisting of a nucleotide sequence in which one or more nucleotides are deleted, substituted, or added in the nucleotide sequence of Sequence ID No. 8; (iv) A DNA having promoter activity, consisting of a base sequence having at least 80%, preferably 90%, or more sequence homology, preferably sequence identity, with SEQ ID NO: 8. It may include.

[0047] The 48230 promoter is one of the following DNAs (i) to (iv): (i) DNA having promoter activity, consisting of more than approximately 100 consecutive base pairs in the base sequence of Sequence ID No. 9, for example, 110 or more, or 120 or more; (ii) DNA that can hybridize under stringent conditions with DNA consisting of more than approximately 100 consecutive complementary base sequences, for example, 110 or more, or 120 or more, in the base sequence of Sequence ID No. 9, and that has promoter activity; (iii) DNA having promoter activity, which consists of a base sequence in which one or more, preferably one or several, bases in a sequence of more than 100 consecutive bases, for example, 110 or more, 120 or more bases, is deleted, substituted, or added in the base sequence of Sequence ID No. 9; or (iv) DNA having promoter activity, comprising more than 100 consecutive base sequences, for example, 110 or more, 120 or more base sequences, and having at least 80%, preferably 90% or more sequence homology, preferably sequence identity, It may include.

[0048] In a preferred embodiment, the 48230 promoter is, for example, one of the following DNAs (i) to (iv): (i) DNA consisting of the base sequence of Sequence ID No. 10; (ii) DNA that can hybridize under stringent conditions with DNA having a nucleotide sequence complementary to the nucleotide sequence of Sequence ID No. 10 and that possesses promoter activity; (iii) DNA having promoter activity, consisting of a nucleotide sequence in which one or more nucleotides are deleted, substituted, or added in the nucleotide sequence of Sequence ID No. 10; (iv) A DNA having a base sequence having at least 80%, preferably 90%, or more sequence homology, preferably sequence identity, with SEQ ID NO: 10, and possessing promoter activity. It may include.

[0049] In a third embodiment, a mutant strain of the genus Burkholderia is provided, which is modified to inhibit the expression of one or more genes selected from the group consisting of, for example, BSFP_068740, BSFP_068730, and BSFP_068720, on a specific chromosome, or the expression of the protein encoded by such gene.

[0050] The Burkholderia bacteria used may be naturally occurring or artificially created, as long as the function of genes on specific chromosomes that disrupt the stability of expression vectors such as plasmids is inhibited.

[0051] In a preferred embodiment, a gene on a specific chromosome is either disrupted or modified to inhibit the expression of the gene or the protein encoded by the gene.

[0052] Such inhibition can be achieved using known methods that cause a loss of function in the target gene, such as gene disruption by genetic engineering or induction of functional loss by mutation. Desired mutations may also be induced in existing natural or mutant Burkholderia bacteria by treatment with mutagens such as nitrosoguanidine, or by physical treatment such as irradiation with ultraviolet light, X-rays, or gamma rays. Alternatively, the expression of the target gene may be inhibited by deleting the promoter region upstream of the target gene or by causing a loss of promoter function.

[0053] Methods for deleting the chromosome itself may include those used in Burkholderia cenocepacia (Agnoli et al., Mol Microbiol., 83(2), 362-378(2012)) or those used in Burkholderia ubonensis (Price et al., PLoS Negl Trop Dis.. 11(9) :e0005928 (2017)).

[0054] Microorganisms classified under the genus Burkholderia include Burkholderia stabilis mentioned above, as well as Burkholderia multivorans, Burkholderia cepacia, Burkholderia mallei, Burkholderia glumae, Burkholderia ambifaria, Burkholderia dolosa, Burkholderia gladioli, and Burkholderia plantarii (AZEGAMI et al., Int J Syst Evol Microbiol, 37(2): 144-152(1987); Seo et al., BMC Genomics, 16:346(2015)). Burkholderia stabilis or Burkholderia silva trantica are preferred.

[0055] Because bacteria of the genus Burkholderia exist in a variety of natural ecosystems, the culture media and conditions for culturing them vary. For example, to culture Burkholderia stabilis, you can culture it in LB medium (1% Bacto(trademark) Tryptone, 0.5% Bacto(trademark) Yeast extract, 0.5% sodium chloride) at 30°C. Furthermore, for strains available for purchase from DSMZ, DSMZ publishes its recommended culture media and conditions as "Cultivation conditions," which you should refer to.

[0056] In a fourth embodiment, a method is provided for maintaining an expression construct in Burkholderia bacteria, comprising the step of culturing Burkholderia bacteria containing an expression construct, in which Burkholderia bacteria lack one or more genes selected from the group consisting of specific chromosome genes, such as BSFP_068740, BSFP_068730, and BSFP_068720, or in which the expression of such genes or the expression of the protein encoded by such genes is inhibited.

[0057] In a preferred embodiment, the copy number of expression constructs in cultured Burkholderia bacteria may be increased, and / or their stability may be increased, compared to Burkholderia bacteria in which the function of a gene on a specific chromosome is not inhibited.

[0058] Increased copy number or stability of the expression construct enables high expression of the target protein. The stability of the expression construct can be evaluated using the stability of the expression construct in the host, for example, the number of colonies grown in a medium containing antibiotics compared to the number of colonies grown in a medium without antibiotics (expression construct retention rate).

[0059] In a fifth embodiment, a method is provided for increasing the stability of an expression construct in Burkholderia bacteria, comprising the step of culturing Burkholderia bacteria containing an expression construct, in which Burkholderia bacteria lack one or more genes selected from the group consisting of specific chromosome genes, such as BSFP_068740, BSFP_068730, and BSFP_068720, or in which the expression of such genes or the expression of the protein encoded by such genes is inhibited. [Examples]

[0060] The present invention will be described below based on examples, but the scope of the present invention is not limited to the following examples. Furthermore, the measured values ​​shown below may vary depending on the measurement conditions and the accuracy of the equipment used. In the agarose gel electrophoresis in the examples, GeneDireX 1Kb plus DNA ladder marker RTU was used as the molecular weight marker. This marker consists of 13 fragments, with molecular weights of 100bp, 250bp, 500bp, 750bp, 1000bp, 1500bp, 2000bp, 3000bp, 4000bp, 5000bp, 6000bp, 8000bp, and 10000bp, from smallest to largest.

[0061] Example 1: Preparation of gene knockout strains (homologous recombination) 500 μL of a bacterial suspension of Burkholderia stabilis (accession number: NITE BP-02704), cultured to the stationary phase in LB medium (1% Bacto® Tryptone, 0.5% Bacto® Yeast extract, 0.5% sodium chloride) with shaking at 30°C, was added to 10 mL of LB medium and incubated at 30°C for 12 hours.

[0062] After culturing, the bacteria were collected by centrifugation (3,000g, 10 minutes, room temperature), and total DNA was extracted using the DNeasy® Blood & Tissue Kit (QIAGEN).

[0063] Using the obtained total DNA of Burkholderia stabilis as a template, DNA amplification was performed by polymerase chain reaction (PCR) using synthetic oligodeoxyribonucleotide primers (hereinafter abbreviated as primers) of SEQ ID NOs. 17 and 18 (Saiki et al., Science, 239 487-491 (1988)). The PCR enzyme used was KOD FX Neo (Toyobo). As a result, DNA consisting of SEQ ID NO. 19 was obtained. These DNA fragments amplified by the PCR reaction were subjected to agarose gel electrophoresis, excised from the gel, and purified using the QIAquick Gel Extraction Kit (QIAGEN).

[0064] Furthermore, using pK18mobsacB (SCHAFER et al., Gene, 145(1) 69-73(1994)) as a template, DNA amplification was performed by PCR using primers SEQ ID NO: 20 and SEQ ID NO: 21. As a result, DNA consisting of approximately 4.1 kb was obtained. The DNA fragment amplified by the PCR reaction was purified by agarose gel electrophoresis in the same manner.

[0065] Each purified DNA fragment, consisting of Sequence ID No. 19 and approximately 4.1 kb, was ligated using the In-Fusion® HD Cloning Kit (TaKaRa), transformed into E. coli DH5α, and then spread onto LB agar medium containing 50 μg / mL kanamycin. After incubation at 30°C for 24 hours, the resulting colonies were subcultured in LB medium containing 50 μg / mL kanamycin and incubated at 37°C for 12 hours. Plasmid was then extracted from the culture medium using the QIAprep Spin Miniprep Kit (QIAGEN) to obtain pK18mobsacB-P0205 for disruption strain production (Figure 1).

[0066] Using the total DNA of Burkholderia stabilis as a template, DNA amplification was performed by PCR using primers number 22 and 23, 24 and 25, 26 and 27, 28 and 29, 30 and 31, and 32 and 33. The DNA fragments amplified by the PCR reaction were purified by agarose gel electrophoresis to obtain each DNA sequence. These sequences are denoted as BSFP_068720A, BSFP_068720B, BSFP_068730A, BSFP_068730B, BSFP_068740A, and BSFP_068740B, respectively.

[0067] pK18mobsacB-P0205 was cleaved using EcoRI and HindIII (Takara Bio Inc.) and purified by agarose gel electrophoresis. The obtained pK18mobsacB-P0205 fragment and the three fragments BSFP_068720A and BSFP_068720B were ligated using the In-Fusion® HD Cloning Kit (Takara Bio Inc.) and transformed into E. coli DH5α. Similarly, the pK18mobsacB-P0205 fragment and the three fragments BSFP_068730A and BSFP_068730B, and the pK18mobsacB-P0205 fragment and the three fragments BSFP_068740A and BSFP_068740B were also ligated using the In-Fusion® HD Cloning Kit (Takara Bio Inc.) and transformed into E. coli DH5α. E. coli containing the three transformed cells was spread onto LB agar medium containing 50 μg / mL kanamycin. After incubation at 30°C for 24 hours, the resulting colonies were subcultured onto LB medium containing 50 μg / mL kanamycin and incubated at 37°C for 12 hours. Plasmids were then extracted from the culture medium to obtain the disruption vectors pK18mobsacB-P0205-BSFP_068720, pK18mobsacB-P0205-BSFP_068730, and pK18mobsacB-P0205-BSFP_068740.

[0068] Each of the obtained vectors was transformed into E. coli DH5α to obtain DH5α(pK18mobsacB-P0205-BSFP_068720), DH5α(pK18mobsacB-P0205-BSFP_068730), and DH5α(pK18mobsacB-P0205-BSFP_068740). The helper plasmid pRK2013(ATCC37159) was transformed into E. coli HB101 to obtain HB101(pRK2013).

[0069] Burkholderia stabilis was inoculated into 5 mL of LB medium and incubated at 30°C for 12 hours to obtain a Burkholderia stabilis culture. In addition, DH5α (pK18mobsacB-P0205-BSFP_068720), DH5α (pK18mobsacB-P0205-BSFP_068730), DH5α (pK18mobsacB-P0205-BSFP_068740), and HB101 (pRK2013) were inoculated into 10 mL of LB medium containing 20 μg / mL of kanamycin and incubated at 37°C for 12 hours to obtain each E. coli culture.

[0070] 1.5 mL of Burkholderia stabilis culture solution was centrifuged (15,000 g, 1 minute, 4°C) to obtain Burkholderia stabilis cells.

[0071] Furthermore, 2 mL of DH5α (pK18mobsacB-P0205-BSFP_068720), DH5α (pK18mobsacB-P0205-BSFP_068730), DH5α (pK18mobsacB-P0205-BSFP_068740), and HB101 (pRK2013) culture medium was centrifuged (15,000 g, 1 minute, 4°C), the supernatant was removed, and LB medium was prepared. 2 mL of [the substance] was added and the cells were suspended. The cells were then centrifuged again (15,000 g, 1 minute, 4°C) to obtain DH5α (pK18mobsacB-P0205-BSFP_068720), DH5α (pK18mobsacB-P0205-BSFP_068730), DH5α (pK18mobsacB-P0205-BSFP_068740), and HB101 (pRK2013) cells.

[0072] Burkholderia stabilis cells, DH5α (pK18mobsacB-P0205-BSFP_068720) cells, and HB101 (pRK2013) cells were suspended and mixed in 100 μL of LB medium and spread onto LB agar plates. Similarly, combinations of Burkholderia stabilis cells, DH5α (pK18mobsacB-P0205-BSFP_068730) cells, and HB101 (pRK2013) cells, and combinations of Burkholderia stabilis cells, DH5α (pK18mobsacB-P0205-BSFP_068740) cells, and HB101 (pRK2013) cells were suspended and spread onto LB agar plates. Each agar plate was incubated overnight at 30°C.

[0073] After one night, each of the grown bacterial cells was scraped off with a Conlarger rod and suspended in 1 mL of 10 mM magnesium sulfate solution. The suspension was diluted 10-fold, and 100 μL of the diluted solution was inoculated onto Nutrient Broth (Becton, Dickinson & Co.) agar medium containing 200 μg / mL kanamycin and 50 μg / mL ampicillin. The cultures were incubated at 30°C for 2 days to obtain one-time recombinants for disrupting BSFP_068720, BSFP_068730, and BSFP_068740.

[0074] Next, each recombinant was inoculated once into 5 mL of LB medium and incubated at 30°C for 12 hours. The resulting culture solution was then mixed with LB medium for 10 minutes. 3 ~10 5 The solution was diluted twice and inoculated onto an agar medium containing 0.33% Bacto(trademark) Tryptone, 0.17% Bacto(trademark) Yeast extract, 0.5% sodium chloride, 10% sucrose, and 1.5% agar, and incubated at 30°C for 2 days.

[0075] The obtained colonies were inoculated onto Nutrient Broth agar and Nutrient Broth agar containing 200 μg / mL kanamycin. Colonies that lost kanamycin resistance were selected, and PCR was performed on the colonies derived from single recombination of BSFP_068720 using the primers described in SEQ ID NOs. 34 and 35, on the colonies derived from single recombination of BSFP_068730 using the primers described in SEQ ID NOs. 36 and 37, and on the colonies derived from single recombination of BSFP_068740 using the primers described in SEQ ID NOs. 38 and 39. Agarose gel electrophoresis was performed, and it was confirmed that the lengths were shorter compared to the wild-type strain (Figure 2), indicating that each gene had been shortened and rendered non-functional. These strains were designated as BSFP_068720 knockout strain, BSFP_068730 knockout strain, and BSFP_068740 knockout strain, respectively.

[0076] Example 2: Creation of gene knockout strains (mutation induction) Burkholderia stabilis was inoculated into 100 mL of LB medium and incubated at 28°C for 20 hours. 30 mL of the culture was centrifuged, the supernatant was removed, and the cells were obtained. 30 mL of 0.85% sodium chloride aqueous solution was added to the cells and they were suspended. 0.5 mL of Tris-maleate buffer (2M Tris, 2M maleic acid, pH 6.2) was added to 9.5 mL of the suspended cells, followed by 50 mg / mL of 1-methyl-3-nitro-1-nitrosoguanidine and 1 mL of N,N-dimethylformamide. The mixture was shaken at 200 rpm at 28°C for 60 minutes. After shaking, the mixture was centrifuged, the supernatant was removed, and the obtained cells were suspended in 10 mL of 0.85% sodium chloride aqueous solution. The mixture was similarly centrifuged, the supernatant was removed, and the obtained cells were suspended in 10 mL of 0.85% sodium chloride aqueous solution. The mixture was centrifuged again and centrifuged to obtain the cells. 10 mL of LB medium was added to the bacterial cells, and the culture was incubated at 28°C at 200 rpm with shaking for 2 hours. 100 μL of the culture was then inoculated onto LB agar plates at varying dilution concentrations and incubated at 28°C for 48 hours.

[0077] The obtained colonies were suspended in 20 μL of Buffer P1 from the QIAprep Spin Miniprep Kit (QIAGEN), and 20 μL of Buffer P2 was added. Using the resulting solution as a template, DNA amplification was performed by PCR using primers SEQ ID NOs. 40 and 41. The PCR enzyme used was KOD FX Neo (Toyobo). Strains that did not amplify with these primer sets were cultured in LB medium with shaking at 30°C until they reached the stationary phase. 500 μL of this bacterial suspension was added to 10 mL of LB medium and incubated at 30°C for 12 hours. After incubation, the cells were collected by centrifugation, resuspended in 500 μL of Tris-EDTA (TE) buffer, 50 μL of proteinase K (20 mg / mL) and 25 μL of 10% SDS were added, and the mixture was incubated at 55°C for 30 minutes. After the reaction, 600 μL of a phenol, chloroform, and isoamyl alcohol mixture (50:49:1) was added, the mixture was stirred, and then centrifuged. The supernatant was transferred to another tube and re-extracted with the phenol, chloroform, and isoamyl alcohol mixture. 50 μL of 3M sodium acetate and 350 μL of isopropanol were added to the supernatant to precipitate the DNA, and the recovered DNA was washed twice with 500 μL of 70% ethanol. The DNA was air-dried at room temperature for 10 minutes and dissolved in 200 μL of deionized water. The obtained DNA was sequenced according to the standard protocol (350 bp insert) of the TruSeq DNA PCR-Free Library Prep Kit (Illumina). Subsequently, DNA sequencing was performed using a HiSEq next-generation sequencer with paired ends and 100 bases / read. The output read sequences confirmed the absence of chromosome 3. This strain was designated as a chromosome 3-deficient strain.

[0078] Example 3: Preparation of a cholesterol esterase expression vector A cholesterol esterase expression vector described in Japanese Patent Publication No. 2019-205402 (shown above) was prepared by the following method. Using the total DNA of Burkholderia stabilis obtained in Example 1 as a template, DNA amplification was performed by PCR using the primers of SEQ ID NOs. 42 and 43, SEQ ID NOs. 44 and 45, and SEQ ID NOs. 46 and 47. As a result, two types of terminators (SEQ ID NOs. 48 and 49) and a multi-cloning site (BglII, NheI, SnaBI, SacI, KpnI, SpeI, BamHI, XbaI, NdeI) fragment (SEQ ID NO. 50) were obtained. Similarly, using the pBBR122 plasmid (MoBiTec) as a template, DNA amplification was performed using the primers of SEQ ID NOs. 51 and 52. These DNA fragments amplified by the PCR reaction were subjected to agarose gel electrophoresis, excised from the gel, and purified using the QIAquick Gel Extraction Kit (QIAGEN). Purified DNA fragments were ligated using the In-Fusion® HD Cloning Kit (Takara Bio Inc.), transformed into E. coli DH5α, and then spread onto LB agar medium containing 50 μg / mL kanamycin. After incubation at 30°C for 24 hours, the resulting colonies were subcultured into LB medium containing 50 μg / mL kanamycin and incubated at 37°C for 12 hours. Plasmid was then extracted from the culture medium using the QIAprep Spin Miniprep Kit (QIAGEN Inc.) to obtain pBBR122-T-MCS-T.

[0079] Using the total DNA of Burkholderia stabilis obtained in Example 1 as a template, DNA amplification was performed by PCR using primers SEQ ID NOs. 53, 54, 55, and 56 in the sequence listing. As a result, DNA fragments of the genes encoding cholesterol esterase and its chaperone, and a DNA fragment of the promoter region were obtained. The obtained DNA fragments were subjected to agarose gel electrophoresis, excised from the gel, and purified using the QIAquick Gel Extraction Kit (QIAGEN). Using pBBR122-T-MCS-T as a template, DNA amplification was performed using primers SEQ ID NOs. 57 and 58 in the sequence listing to amplify the DNA fragment of the expression vector. The purified DNA, consisting of the obtained expression vector DNA fragment and the previously obtained DNA fragments of the genes encoding cholesterol esterase and its chaperone, and the promoter region DNA fragment, was ligated using the In-Fusion® HD Cloning Kit (Takara Bio). The ligated DNA was transformed into E. coli DH5α and then spread onto LB agar medium containing 50 μg / mL kanamycin. After culturing at 30°C for 24 hours, the obtained colonies were subcultured in LB medium containing 50 μg / mL kanamycin and cultured at 37°C for 12 hours. Plasmids were then extracted from the culture medium using the QIAprep Spin Miniprep Kit (QIAGEN) to obtain pBBR122-182 (Figure 3).

[0080] Example 4 Preparation of Recombinant Plants Burkholderia stabilis wild strain and the BSFP_068720, BSFP_068730, BSFP_068740, and chromosome 3 knockout strains obtained in Examples 1 and 2 were cultured in 100 mL of LB medium at 30°C with shaking until the logarithmic growth phase, and then the cells were collected by centrifugation. 100 mL of ice-cold sterile water was added to the collected cells, and after suspension, the cells were collected again by centrifugation. This procedure was repeated once more, and then 5 mL of chilled 10% glycerin solution was added to the collected cells, the cells were thoroughly suspended, and the cells were collected by centrifugation.

[0081] The recovered bacterial cells were suspended in 5 mL of ice-cold 10% glycerin solution, dispensed into 40 μL portions, and frozen at -80°C to obtain competent cells of the wild-type strain, BSFP_068720 knockout strain, BSFP_068730 knockout strain, BSFP_068740 knockout strain, and chromosome 3 knockout strain. The obtained competent cells were thawed on ice, and approximately 200-400 ng each of pBBR122 and pBBR122-182 obtained in Example 3 were added and mixed. This mixture was transferred to a 0.2 cm wide electroporation cuvette (Bio-Rad), and electrical pulses were applied using a GenePulser II gene transfer device with an electric field strength of 12.5 kV / cm, capacitance of 25 μF, and external resistance of 200 Ω. A mixture of electrically pulsed bacterial cells and DNA was mixed into 1 mL of LB medium and incubated at 30°C for 1 hour. Then, 200 μL of the bacterial suspension was spread onto LB medium containing 50 μg / mL of kanamycin and incubated at 30°C for 1 day. Transformations of each expression vector were obtained: wild-type-pBBR122, wild-type-pBBR122-182, BSFP_068720 disruption-pBBR122, and BS. We obtained FP_068720 disruption strain-pBBR122-182, BSFP_068730 disruption strain-pBBR122, BSFP_068730 disruption strain-pBBR122-182, BSFP_068740 disruption strain-pBBR122, BSFP_068740 disruption strain-pBBR122-182, chromosome 3 deletion strain-pBBR122, and chromosome 3 deletion strain-pBBR122-182.

[0082] Example 5: Confirmation of plasmid copy number The transformants obtained in Example 4—wild-type-pBBR122, BSFP_068720-disruption-pBBR122, BSFP_068730-disruption-pBBR122, BSFP_068740-disruption-pBBR122, and chromosome 3-deficient-pBBR122—were inoculated into 5 mL of LB medium containing 100 μg / mL kanamycin and cultured at 28°C for 12 hours. This bacterial suspension was added to 5 mL of LB medium containing 100 μg / mL kanamycin to achieve a bacterial turbidity OD = 0.2, and cultured at 28°C for 24 hours and 48 hours, respectively, to obtain bacterial cells with different culture times. The bacterial cells cultured for 24 hours and 48 hours were recovered by centrifugation (3,000 g, 10 minutes, room temperature).

[0083] For bacterial cells cultured for 24 hours, total DNA was extracted using the QIAamp DNA Mini Kit (manufactured by QIAGEN).

[0084] To confirm the copy number of the plasmid, real-time PCR was performed on each total DNA obtained using the method described in Choi KH et.al., Appl. Environ. Microbiol., 74(4) 1064-75 (2008). Specifically, the aspartate-semialdehyde dehydrogenase gene was amplified as a chromosomal gene using the primer set of SEQ ID NO: 59 and SEQ ID NO: 60. The obtained DNA fragments were subjected to agarose gel electrophoresis, excised from the gel, and purified using the QIAquick Gel Extraction Kit (QIAGEN) to obtain DNA fragments. These DNA fragments and DNA fragments obtained by cutting pBBR122 with SmaI (Takara Bio) were ligated using the In-Fusion® HD Cloning Kit (Takara Bio). The ligated DNA was transformed into E. coli DH5α and then spread on LB agar medium containing 50 μg / mL chloramphenicol. After culturing at 30°C for 24 hours, the obtained colonies were subcultured in LB medium containing 50 μg / mL chloramphenicol and cultured at 37°C for 12 hours. Plasmid was then extracted from the culture medium using the QIAprep Spin Miniprep Kit (QIAGEN) to obtain pBBR122-asd. Samples were prepared by mixing total DNA, pBBR122-asd, and each primer set (primer set for SEQ ID NOs. 61 and 62 for chromosome copy number calculation, and primer set for SEQ ID NOs. 63 and 64 for plasmid copy number calculation) according to the LightCycler FastStart DNA Master Plus SYBR Green I (Roche) protocol, and real-time PCR was performed using a LightCycler 480 (Roche). The PCR conditions were denaturation at 95°C for 10 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 1 second. The Cp value calculated for each gene using the 2nd Derivative Maximum method was output. A calibration curve was created using the Cp value of pBBR122-asd, and the copy number of plasmids for each strain was calculated (Figure 4).As a result, the pBBR122 count in the wild-type strain was approximately 30 copies, while in the BSFP_068720 knockout strain, BSFP_068730 knockout strain, BSFP_068740 knockout strain, and chromosome 3 deletion strain, the pBBR122 count was approximately 300-400 copies. This indicates that the loss of function of at least one of BSFP_068720, BSFP_068730, or BSFP_068740 increases the plasmid copy number.

[0085] Plasmids were extracted from bacterial cells cultured for 48 hours using the QIAprep Spin Miniprep Kit (QIAGEN). The obtained plasmids were cleaved with XhoI (Takara Bio), and agarose gel electrophoresis was performed (Figure 5). As a result, a single signal originating from the plasmid was observed, and the signal was stronger in the BSFP_068720 knockout strain-pBBR122, BSFP_068730 knockout strain-pBBR122, BSFP_068740 knockout strain-pBBR122, and chromosome 3 knockout strain-pBBR122 compared to the wild-type strain-pBBR122.

[0086] The results above show that strains into which pBBR12 was introduced after disrupting BSFP_068720, BSFP_068730, and BSFP_068740 respectively showed stronger signals by agarose electrophoresis and a higher plasmid copy number compared to strains into which pBBR122 was introduced after introducing pBBR122 into wild-type strains. Furthermore, strains into which pBBR122 was introduced after deleting chromosome 3 containing BSFP_068720, BSFP_068730, and BSFP_068740 also showed a higher plasmid copy number compared to strains into which pBBR122 was introduced after introducing pBBR122 into wild-type strains. This also indicates that the loss of function of at least one of BSFP_068720, BSFP_068730, or BSFP_068740 leads to an increase in plasmid copy number.

[0087] Example 6 Confirmation of plasmid stability The transformants obtained in Example 4—wild-type-pBBR122-182, BSFP_068720-destroyed-pBBR122-182, BSFP_068730-destroyed-pBBR122-182, BSFP_068740-destroyed-pBBR122-182, and chromosome 3-deficient-pBBR122-182—were inoculated into 5 mL of LB medium and cultured at 28°C for 24 hours. The culture solution obtained after 24 hours was designated as the first generation, and 2 μL of the first-generation culture solution was inoculated into a new 5 mL of LB medium and cultured at 28°C for 24 hours. The resulting culture solution was designated as the second generation, and 2 μL of the second-generation culture solution was inoculated into a new 5 mL of LB medium and cultured at 28°C for 24 hours. The resulting culture solution was designated as the third generation. The first-generation culture solution and the third-generation culture solution were each mixed with LB medium for 10 minutes. 7 The solution was diluted 1:1 and inoculated onto LB agar and LB agar containing 100 μg / mL kanamycin. The cultures were incubated at 30°C for 2 days, and the number of colonies grown was counted and defined as Colony Forming Units (CFU). Since colonies growing on LB agar represent the total bacterial count, and colonies growing on LB agar containing kanamycin represent the number of bacteria carrying the plasmid, the plasmid retention rate was calculated using the following formula.

[0088] (Formula) Plasmid retention rate (%) = CFU in LB agar medium containing kanamycin / CFU in LB agar medium × 100

[0089] [Table 1]

[0090] The numbers in the table are 10 7 The table shows the CFU of the culture medium diluted to 1 / 20. In addition, Km(-) in the table indicates the CFU in LB agar medium, and Km(+) indicates the CFU in LB agar medium containing 100 μg / mL of kanamycin.

[0091] As shown in Table 1, strains in which the expression vector was inserted into the wild-type strain lost the plasmid after being cultured once in kanamycin-free LB liquid medium. On the other hand, strains in which any of BSFP_068720, BSFP_068730, or BSFP_068740 was disrupted, or strains in which chromosome 3 containing these three genes was deleted, were less likely to lose the plasmid even after being subculturished three times in kanamycin-free LB medium. These results indicate that the plasmid is stably retained within the bacterial cell when at least one of BSFP_068720, BSFP_068730, or BSFP_068740 loses its function.

[0092] Example 7 Confirmation of cholesterol esterase productivity Of the transformants obtained in Example 4, wild-type-pBBR122-182, BSFP_068720-disrupted-pBBR122-182, BSFP_068730-disrupted-pBBR122-182, BSFP_068740-disrupted-pBBR122-182, and chromosome 3-deficient-pBBR122-182 were each inoculated into 5 mL of YSO medium (3% yeast extract, 3% sorbitol, 3% methyl oleate, pH 7.0) containing 100 μg / mL of kanamycin, and cultured at 28°C for 72 hours. After culturing, the amount of cholesterol esterase produced by each strain was evaluated using the following enzyme activity measurement method.

[0093] <Method for measuring enzyme activity> A reaction reagent was prepared containing the following reagents at the concentrations specified below. [Reaction reagents] 40 mM potassium phosphate buffer (pH 6.8) 0.02% TODB (Dojindo Chemical Research Institute) 10 U / mL peroxidase 0.3% Triton X-100 2.5 U / mL cholesterol oxidase 10% calf serum solution 0.035% 4-aminoantipyrine

[0094] The reaction reagent was preheated at 37°C for 10 minutes. Using a Hitachi 7080 automated analyzer (manufactured by Hitachi, Ltd.), 3 μL of recombinant cholesterol esterase solution was added to 150 μL of the reaction reagent, and the reaction was carried out at 37°C. The absorbance was measured at a primary wavelength of 546 nm and a secondary wavelength of 660 nm from 76.69 seconds to 119.53 seconds after addition, and the change in absorbance per unit time (ΔA / min) was obtained. The value obtained from the following formula was defined as cholesterol esterase activity, and the cholesterol esterase activity was calculated.

[0095] (formula) Cholesterol esterase activity (U / mL) = ΔA / min ÷ 18 x (150 + 3) ÷ 3 ÷ 1000 {In the formula, ΔA / min represents the change in absorbance per unit time obtained by measuring the absorbance at a primary wavelength of 546 nm and a secondary wavelength of 660 nm from 76.69 seconds to 119.53 seconds after the addition of the above reaction reagent. 18 is the millimolar molecular extinction coefficient at 550 nm of the oxidative condensation reaction product of TODB and 4-aminoantipyrine, 150+3 is the total reaction volume (volume of reaction reagent + volume of recombinant lipase solution), and 3 is the volume of the above recombinant cholesterol esterase solution.}

[0096] Each strain was cultured independently three times, and the activity of cholesterol esterase produced by each strain was measured (Figure 6).

[0097] As shown in Figure 6, compared to the wild-type strain into which the cholesterol esterase expression vector was introduced, the strains into which the cholesterol esterase expression vector was introduced after disrupting BSFP_068720, BSFP_068730, and BSFP_068740 genes were respectively disrupted showed higher cholesterol esterase activity and improved cholesterol esterase production. Furthermore, in the strains into which the cholesterol esterase expression vector was introduced after deleting chromosome 3 containing BSFP_068720, BSFP_068730, and BSFP_068740 genes were deleted, cholesterol esterase activity was higher and cholesterol esterase production was improved compared to the wild-type strain into which the cholesterol esterase expression vector was introduced. From this, it was found that if at least one of BSFP_068720, BSFP_068730, or BSFP_068740 is disrupted and loses its function, cholesterol esterase production will improve even if other genes are disrupted and lose their function. In Examples 5 and 6, it is thought that the cholesterol esterase production improved due to an increase in plasmid copy number and increased stability.

[0098] Example 8: Confirmation of the effect with other plasmids To confirm the effects of disrupting BSFP_068720, BSFP_068730, and BSFP_068740 with plasmids other than pBBR122, plasmid stability was confirmed using plasmid pSa (Datta, American Society for Microbiology, Washington, DC, pp9-15 (1975), Gorai et al., Plasmid 2 489-492 (1979), Ward and Grinsted, Plasmid 7 239-250 (1982), Tait et al., Mol. Gen. Genet. 186 10-15 (1982)), which has a different replication origin than pBBR122. A region containing the origin of pSa and the same kanamycin resistance gene and chloramphenicol resistance gene as pBBR122 was synthesized to obtain the pSa plasmid shown in Sequence ID No. 65.

[0099] Similar to Example 4, pSa plasmids were introduced into competent cells of the wild-type strain, BSFP_068720 disruptor strain, BSFP_068730 disruptor strain, BSFP_068740 disruptor strain, and chromosome 3 knockout strain by electroporation to obtain transformants: wild-type-pSa, BSFP_068720 disruptor strain-pSa, BSFP_068730 disruptor strain-pSa, BSFP_068740 disruptor strain-pSa, and chromosome 3 knockout strain-pSa.

[0100] These transformants were inoculated into 5 mL of LB medium containing 100 μg / mL kanamycin and cultured at 28°C for 12 hours. This bacterial suspension was added to 5 mL of LB medium containing 100 μg / mL kanamycin to achieve a cell turbidity OD=0.2, and cultured at 28°C for 24 hours and 48 hours, yielding cells with different culture times. Plasmids were extracted from the cells cultured for different durations using the QIAprep Spin Miniprep Kit (QIAGEN). The obtained plasmids were cut with XhoI (Takara Bio), and agarose gel electrophoresis was performed (Figure 7). As a result, no plasmid-derived signal was observed in the wild-type strain-pSa, but a single plasmid-derived signal was observed in the BSFP_068720 knockout strain-pSa, BSFP_068730 knockout strain-pSa, BSFP_068740 knockout strain-pSa, and chromosome 3 knockout strain-pSa.

[0101] The results above indicate that strains in which pSa was introduced into strains with disrupted BSFP_068720, BSFP_068730, and BSFP_068740 respectively showed stronger signals by agarose electrophoresis compared to strains in which pSa was introduced into wild-type strains. Furthermore, strains in which pSa was introduced into strains lacking chromosome 3, including BSFP_068720, BSFP_068730, and BSFP_068740, also showed a higher plasmid copy number compared to strains in which pSa was introduced into wild-type strains. This suggests that, not only with pBBR122 but also with the pSa plasmid, the loss of function of at least one of BSFP_068720, BSFP_068730, or BSFP_068740 increases the plasmid copy number.

[0102] Furthermore, to confirm the effect with another plasmid, cholesterol esterase activity was examined using a plasmid with RK2ori, a replication origin different from pBBR122 (TJ Schmidhauser et al., Plasmid. 9(3) 325-30(1983)). The necessary regions for the plasmid with RK2ori, including the region containing RK2ori and trpA, the region containing the kanamycin resistance gene of pBBR122, the 182 promoter, cholesterol esterase and its foldase, and the terminator region were totally synthesized to obtain the pRK2-182 plasmid shown in SEQ ID NO: 66. Each knockout strain was transformed in the same manner as in Example 4, and cholesterol esterase activity after 72 hours of incubation at 28°C was examined in the same manner as in Example 7. As shown in Figure 8, compared to the wild-type strain into which a cholesterol esterase expression vector was introduced, strains into which cholesterol esterase expression vectors were introduced after disruption of BSFP_068720, BSFP_068730, and BSFP_068740 respectively showed higher cholesterol esterase activity and improved cholesterol esterase production. Furthermore, strains into which cholesterol esterase expression vectors were introduced after deletion of chromosome 3 containing BSFP_068720, BSFP_068730, and BSFP_068740 also showed higher cholesterol esterase activity and improved cholesterol esterase production compared to the wild-type strain into which a cholesterol esterase expression vector was introduced. From this, it was found that if at least one of BSFP_068720, BSFP_068730, or BSFP_068740 is disrupted and loses its function, cholesterol esterase production will improve even if other genes are disrupted and lose their function. This indicates that, not only in pBBR122 but also in the pRK2 plasmid, the loss of function of at least one of BSFP_068720, BSFP_068730, or BSFP_068740 improves cholesterol esterase productivity.

[0103] Based on these results, similar copy number increases were observed in plasmids other than pBBR122, such as pSa and pRK2, suggesting that this improved cholesterol esterase productivity.

[0104] Example 9: Confirmation of the effects of other protein expression To confirm the effects of disrupting BSFP_068720, BSFP_068730, and BSFP_068740 in the expression of proteins other than cholesterol esterase, the DNA sequence containing the lipase gene and its chaperone gene derived from Burkholderia plantarii (ATCC43733), as shown in SEQ ID NO: 67, was totally synthesized. Using the totally synthesized gene as a template, PCR was performed using the primers shown in SEQ ID NO: 68 and 69, and the amplified fragment was excised by agarose gel electrophoresis. The fragment was purified using the QIAquick Gel Extraction Kit (QIAGEN) to obtain the BpLip (Burkholderia plantarii lipase) fragment. Using pBBR122-182 as a template, PCR was performed using the primers shown in SEQ ID NO: 70 and 71, and similarly, agarose gel electrophoresis and purification using the QIAquick Gel Extraction Kit (QIAGEN) were performed. The obtained sequence and BpLip fragment were ligated using the In-Fusion® HD Cloning Kit (Takara Bio Inc.), transformed into E. coli DH5α, and then spread onto LB agar medium containing 50 μg / mL kanamycin. After incubation at 30°C for 24 hours, the resulting colonies were subcultured into LB medium containing 50 μg / mL kanamycin and incubated at 37°C for 12 hours. Plasmid was then extracted from the culture medium using the QIAprep Spin Miniprep Kit (QIAGEN Inc.) to obtain pBBR122-BpLip.

[0105] Furthermore, the DNA sequence containing the lipase gene derived from Burkholderia cepacia, as shown in SEQ ID NO: 72, was totally synthesized. Using the totally synthesized gene as a template, PCR was performed using the primers shown in SEQ ID NO: 73 and 74. The amplified fragment was excised by agarose gel electrophoresis and purified using the QIAquick Gel Extraction Kit (QIAGEN) to obtain the BcLip (Burkholderia cepacia lipase) fragment. Using pBBR122-182 as a template, PCR was performed using the primers shown in SEQ ID NO: 70 and 75, and similarly, agarose gel electrophoresis and purification using the QIAquick Gel Extraction Kit (QIAGEN) were performed. The obtained sequence and BcLip fragment were ligated using the In-Fusion® HD Cloning Kit (Takara Bio Inc.), transformed into E. coli DH5α, and then plated on LB agar medium containing 50 μg / mL kanamycin. After culturing at 30°C for 24 hours, the obtained colonies were subcultured in LB medium containing 50 μg / mL kanamycin and cultured at 37°C for 12 hours. Plasmid was then extracted from the culture medium using the QIAprep Spin Miniprep Kit (QIAGEN) to obtain pBBR122-BcLip.

[0106] Similar to Example 4, pBBR122-BpLip or pBBR122-BcLip was introduced into competent cells of the wild-type and chromosome 3-deficient strains by electroporation to obtain transformants: wild-type-pBBR122-BpLip, chromosome 3-deficient-pBBR122-BpLip, wild-type-pBBR122-BcLip, and chromosome 3-deficient-pBBR122-BcLip. Each transformant was inoculated into 5 mL of YS medium (3% yeast extract, 3% sorbitol, pH 7.0) containing 100 μg / mL of kanamycin and cultured at 28°C for 72 hours. After culturing, the amount of lipase produced by each strain was evaluated using the enzyme activity measurement method described in T. Yamaguchi et al., Agr. Biol. Chem. 37 (1973), and the activity of the lipase produced by each strain was measured (Figures 9 and 10).

[0107] As shown in Figures 9 and 10, compared to strains into which expression vectors containing Burkholderia plantarii-derived lipase or Burkholderia cepacia-derived lipase were introduced into wild-type strains, strains in which the same expression vector was introduced into strains lacking chromosome 3 containing BSFP_068720, BSFP_068730, and BSFP_068740 showed higher lipase activity and improved lipase production. This indicates that if at least one of BSFP_068720, BSFP_068730, or BSFP_068740 is disrupted and loses its function, lipase production improves even if other genes are disrupted and lose their function. These results demonstrate that the loss of function of at least one of BSFP_068720, BSFP_068730, or BSFP_068740 improves the production of the target protein introduced into the expression vector.

[0108] Example 10: Preparation of an expression vector using a novel promoter Using the total DNA of Burkholderia stabilis obtained in Example 1 as a template, DNA amplification was performed by PCR using primers SEQ ID NOs. 76 and 77, and 78 and 79 in the sequence listing. As a result, the 14020 promoter DNA fragment and the 48230 promoter DNA fragment, which are DNA fragments of the promoter region, were obtained. The obtained DNA fragments were subjected to agarose gel electrophoresis, excised from the gel, and purified using the QIAquick Gel Extraction Kit (QIAGEN). Using pBBR122-182 as a template, DNA amplification was performed using primers SEQ ID NOs. 80 and 81 to amplify the DNA fragment of the expression vector. The obtained expression vector DNA fragment and the 14020 promoter DNA fragment, and the expression vector DNA fragment and the 48230 promoter DNA fragment were ligated using the In-Fusion® HD Cloning Kit (Takara Bio). After transforming Escherichia coli DH5α with each of the ligated DNAs, they were spread on LB agar medium containing 50 μg / mL kanamycin. After culturing at 30°C for 24 hours, the obtained colonies were subcultured in LB medium containing 50 μg / mL kanamycin and cultured at 37°C for 12 hours. Plasmids were then extracted from the culture medium using the QIAprep Spin Miniprep Kit (QIAGEN), yielding pBBR122-14020 and pBBR122-48230, expression vectors in which the promoter upstream of the cholesterol esterase gene was replaced with a novel promoter.

[0109] Example 11 Confirmation of cholesterol esterase activity produced by a novel promoter Competent cells of the chromosome 3-deficient strain prepared in Example 4 were thawed on ice, and approximately 200-400 ng each of pBBR122-14020 or pBBR122-48230 obtained in Example 10 were added and mixed. This mixture was transferred to a 0.2 cm wide electroporation cuvette (Bio-Rad), and electrical pulses were applied using a GenePulser II gene transduction device with an electric field strength of 12.5 kV / cm, capacitance of 25 μF, and external resistance of 200 Ω. The mixture of the electrically pulsed cells and DNA was mixed with 1 mL of LB medium and cultured at 30°C for 1 hour. Then, 200 μL of the bacterial suspension was spread onto LB medium containing 50 μg / mL of kanamycin and cultured at 30°C for 1 day to obtain the transformants chromosome 3-deficient strain-pBBR122-14020 and chromosome 3-deficient strain-pBBR122-48230.

[0110] These transformants and the chromosome 3 knockout strain-pBBR122-182 obtained in Example 4 were each inoculated into 5 mL of culture medium containing 100 μg / mL kanamycin (4% yeast extract, 1.5% sorbitol, 1.5% glycerol, 5% methyl oleate, pH 6.5) and cultured at 28°C for 72 hours. After culturing, the amount of cholesterol esterase produced by each strain was evaluated using the enzyme activity measurement method described in Example 7.

[0111] [Table 2]

[0112] As shown in Table 2, the cholesterol esterase production was higher in transformants using the 14020 promoter and the 48230 promoter than in transformants using pBBR122-182, a cholesterol esterase expression vector described in Japanese Patent Publication No. 2019-205402 (mentioned above). This indicates that the 14020 promoter and the 48230 promoter are very useful for the expression of the target protein.

[0113] Example 12 Confirmation of expression levels based on differences in the length of the novel promoter An expression vector with a shortened novel promoter was prepared by the following method. Using the total DNA of Burkholderia stabilis obtained in Example 1 as a template, DNA amplification by PCR, agarose electrophoresis, and extraction from gel were performed using primer sets SEQ ID NOs. 82 and 77, 83 and 77, 84 and 77, 85 and 77, 86 and 77, 87 and 77, 88 and 77, 89 and 77, 90 and 77, 91 and 77, 92 and 77, 93 and 77, 94 and 77, 95 and 77, 96 and 77, and 97 and 77, as in Example 10, to obtain DNA fragments. Let the respective DNA fragments be 14020-250, 14020-200, 14020-150, 14020-100, 14020-90, 14020-80, 14020-70, 14020-60, 14020-50, 48230-250, 48230-200, 48230-150, 48230-140, 48230-130, 48230-120, and 48230-110. Each represents "promoter name - promoter length," for example, 14020-250 is a DNA fragment used to create a 14020 promoter with a length of 250 bp.

[0114] In the same manner as in Example 10, the DNA fragments 14020-250, 14020-200, 14020-150, 14020-100, 14020-90, 14020-80, 14020-70, 14020-60, 14020-50, 48230-250, 48230-200, 48230-150, 48230-140, 48230-130, 48230-120, and 48230-110, along with pBBR122-182 prepared in Example 10, were used as templates to amplify the DNA fragments of the expression vector obtained by using primers SEQ ID NOs.80 and 81. The plasmids were linked using the kit (manufactured by Takara Bio Inc.), and transformation, culture, and plasmid extraction were performed on E. coli in the same manner as in Example 10, resulting in the expression of the following vectors: pBBR122-14020-250, pBBR122-14020-200, pBBR122-14020-150, pBBR122-14020-100, pBBR122-14020-90, pBBR122-14020-80, pBBR We obtained 122-14020-70, pBBR122-14020-60, pBBR122-14020-50, pBBR122-48230-250, pBBR122-48230-200, pBBR122-48230-150, pBBR122-48230-140, pBBR122-48230-130, pBBR122-48230-120, and pBBR122-48230-110.

[0115] These expression vectors were used to transform competent cells of the chromosome 3 knockout strain in the same manner as in Example 11, resulting in transformants with shortened promoter lengths: chromosome 3 knockout strain-pBBR122-14020-250, chromosome 3 knockout strain-pBBR122-14020-200, chromosome 3 knockout strain-pBBR122-14020-150, chromosome 3 knockout strain-pBBR122-14020-100, chromosome 3 knockout strain-pBBR122-14020-90, chromosome 3 knockout strain-pBBR122-14020-80, and chromosome 3 knockout strain-pBBR122-14020. We obtained the following chromosome 3-deficient strains: -70, chromosome 3-deficient strain -pBBR122-14020-60, chromosome 3-deficient strain -pBBR122-14020-50, chromosome 3-deficient strain -pBBR122-48230-250, chromosome 3-deficient strain -pBBR122-48230-200, chromosome 3-deficient strain -pBBR122-48230-150, chromosome 3-deficient strain -pBBR122-48230-140, chromosome 3-deficient strain -pBBR122-48230-130, chromosome 3-deficient strain -pBBR122-48230-120, and chromosome 3-deficient strain -pBBR122-48230-110. These transformants, along with the chromosome 3 knockout strains pBBR122-14020 and pBBR122-48230 obtained in Example 10, were cultured in the same manner as in Example 11, and the amount of cholesterol esterase produced by each strain was evaluated using the enzyme activity measurement method described in Example 7. For the 14020 promoter, the relative cholesterol esterase activity relative to a 300 bp long promoter was determined using the following formula (Figure 11).

[0116] (Formula) (Cholesterol esterase activity produced by the above transformant) ÷ (Cholesterol esterase activity produced by the chromosome 3 knockout strain -pBBR122-14020) × 100

[0117] Similarly, for the 48230 promoter, the relative cholesterol esterase activity relative to a 300 bp promoter was determined using the following formula.

[0118] (Formula) (Cholesterol esterase activity produced by the above transformant) ÷ (Cholesterol esterase activity produced by the chromosome 3 knockout strain -pBBR122-48230) × 100

[0119] As shown in Figure 11, the 14020 promoter and the 48230 promoter produced cholesterol esterase even when shortened to less than 300 bp.

[0120] Example 13: Confirmation of the effect on other Burkholderia bacteria To confirm that the present invention is applicable to other Burkholderia bacteria, we investigated whether other Burkholderia bacteria besides Burkholderia stabilis possess BSFP_068720, BSFP_068730, and BSFP_068740. In the same manner as in Example 1, total DNA was extracted from Burkholderia silva tranthica (NBRC106337). Using the obtained total DNA of Burkholderia silva tranthica or the total DNA of Burkholderia stabilis obtained in Example 1 as templates, DNA amplification was performed by PCR using primers SEQ ID NOs. 98 and 99, which are capable of amplifying the regions containing BSFP_068720, BSFP_068730, and BSFP_068740. When the obtained DNA fragments were subjected to agarose gel electrophoresis, DNA amplification was observed in Burkholderia silva tranthica, similar to Burkholderia stabilis (Figure 12). These results suggest that Burkholderia silva tranthica possesses homologous genes BSFP_068720, BSFP_068730, and BSFP_068740, and disrupting these genes is expected to lead to increased productivity and an increase in the copy number of the expression construct.

Claims

1. A method for producing a protein encoded by a target gene, A method comprising the step of expressing a target gene in a Burkholderia bacterium that, in the wild strain, has the genes BSFP_068740, BSFP_068730, and BSFP_068720, and which does not have one or more genes selected from the group consisting of BSFP_068740, BSFP_068730, and BSFP_068720, or in which the expression of such genes or the expression of the protein encoded by such genes is inhibited.

2. The gene BSFP_068740 contains one of the following DNAs (i) to (vi): (i) DNA consisting of the base sequence of Sequence ID No. 1; (ii) DNA comprising a base sequence in which 1 to 10 bases are deleted, substituted, or added per 100 bases in the base sequence of Sequence ID No. 1, wherein the protein encoded thereby has the function of reducing the copy number of the expression construct in Burkholderia bacteria or reducing its stability; (iii) DNA comprising a nucleotide sequence having at least 90% sequence identity with Sequence ID No. 1, wherein the protein encoded thereby has the function of reducing the copy number of the expression construct in Burkholderia bacteria or reducing its stability; (iv) DNA consisting of a base sequence that codes for a protein consisting of the amino acid sequence of Sequence ID No. 2; (v) DNA comprising a protein encoding a protein having the function of reducing the copy number of the expression construct in Burkholderia bacteria or reducing its stability, wherein the amino acid sequence of SEQ ID NO: 2 has 1 to 10 amino acids deleted, substituted, or added per 100 amino acid sequences; or (vi) DNA comprising a protein encoding a protein having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 2, and having the function of reducing the copy number of the expression construct in Burkholderia bacteria or reducing its stability. The method according to claim 1, including the method described in claim 1.

3. The gene BSFP_068730 contains one of the following DNAs (i) to (vi): (i) DNA consisting of the base sequence of Sequence ID No. 3; (ii) DNA having a base sequence in which 1 to 10 bases are deleted, substituted, or added per 100 bases in the base sequence of Sequence ID No. 3, wherein the protein encoded thereby has the function of reducing the copy number of the expression construct in Burkholderia bacteria or reducing its stability; (iii) DNA comprising a nucleotide sequence having at least 90% sequence identity with Sequence ID No. 3, wherein the protein encoded thereby has the function of reducing the copy number of the expression construct in Burkholderia bacteria or reducing its stability; (iv) DNA consisting of a base sequence that codes for a protein consisting of the amino acid sequence of Sequence ID No. 4; (v) DNA comprising a protein encoding a protein having the function of reducing the copy number of the expression construct in Burkholderia bacteria or reducing its stability, wherein the amino acid sequence of SEQ ID NO: 4 has 1 to 10 amino acids deleted, substituted, or added per 100 amino acid sequences; or (vi) A protein comprising an amino acid sequence having at least 90% sequence identity with Sequence ID No. 4, and having the function of reducing the copy number of the expression construct in Burkholderia bacteria or reducing its stability, DNA comprising a protein encoding base sequence, The method according to claim 1 or 2, including the method described in claim 1 or 2.

4. The gene BSFP_068720 contains one of the following DNAs (i) to (vi): (i) DNA consisting of the base sequence of Sequence ID No. 5; (ii) DNA comprising a base sequence in which 1 to 10 bases are deleted, substituted, or added per 100 bases in the base sequence of Sequence ID No. 5, wherein the protein encoded thereby has the function of reducing the copy number of the expression construct in Burkholderia bacteria or reducing its stability; (iii) DNA comprising a nucleotide sequence having at least 90% sequence identity with Sequence ID No. 5, wherein the protein encoded thereby has the function of reducing the copy number of the expression construct in Burkholderia bacteria or reducing its stability; (iv) DNA consisting of a base sequence that codes for a protein consisting of the amino acid sequence of SEQ ID NO: 6; (v) DNA comprising a protein encoding a protein having the function of reducing the copy number of the expression construct in Burkholderia bacteria or reducing its stability, wherein the amino acid sequence of SEQ ID NO: 6 has 1 to 10 amino acids deleted, substituted, or added per 100 amino acid sequences; or (vi) A protein comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 6, and having the function of reducing the copy number of the expression construct in Burkholderia bacteria or reducing its stability, comprising a DNA base sequence encoding a protein. The method according to any one of claims 1 to 3, including

5. The method according to any one of claims 1 to 4, wherein the target gene is linked to an expression construct so that it can be expressed.

6. The method according to any one of claims 2 to 5, wherein the expression construct is an expression vector.

7. The method according to claim 6, wherein the expression construct is a plasmid that can be replicated in bacteria of the genus Burkholderia.

8. The method according to claim 7, wherein the plasmid is a broad-spectrum host vector.

9. The method according to claim 8, wherein the broad-area host vector is pBBR122, pSa, or RK2.

10. The method according to any one of claims 2 to 9, wherein the expression construct includes a promoter that controls the expression of a target gene.

11. The method according to claim 10, wherein the promoter is derived from a bacterium of the genus Burkholderia.

12. The promoter is one of the following DNAs (i) to (iii): (i) DNA having promoter activity, consisting of more than 80 consecutive base sequences in the base sequence of Sequence ID No. 7; (ii) DNA having promoter activity, which consists of a sequence in which, in the sequence of Sequence ID No. 7, 1 to 10 bases are deleted, substituted, or added for every 100 bases in a sequence of more than 80 consecutive bases; or (iii) DNA having promoter activity, consisting of more than 80 consecutive base sequences and base sequences having at least 90% sequence identity, in sequence number 7 A method according to any one of claims 1 to 11, including the method described in any one of claims 1 to 11.

13. The aforementioned promoter is one of the following DNAs (i) to (iii): (i) DNA consisting of the base sequence of Sequence ID No. 8; (ii) DNA having promoter activity, wherein the base sequence of Sequence ID No. 8 consists of a base sequence in which 1 to 10 bases are deleted, substituted, or added for every 100 bases; (iii) DNA having a base sequence that is at least 90% identical to sequence number 8 and possesses promoter activity. The method according to claim 12, including the method described in claim 12.

14. The promoter is one of the following DNAs (i) to (iii): (i) DNA having promoter activity, consisting of more than 100 consecutive base sequences in the base sequence of Sequence ID No. 9; (ii) DNA having promoter activity, wherein in the base sequence of Sequence ID No. 9, 1 to 10 bases are deleted, substituted, or added for each 100 bases in a sequence of more than 100 consecutive bases; or (iii) DNA having promoter activity, consisting of more than 100 consecutive base sequences and base sequences having at least 90% sequence identity, The method according to any one of claims 1 to 13, including the method described in any one of claims 1 to 13.

15. The aforementioned promoter is one of the following DNAs (i) to (iii): (i) DNA consisting of the base sequence of Sequence ID No. 10; (ii) DNA having promoter activity, wherein the base sequence of sequence number 10 consists of a base sequence in which 1 to 10 bases are deleted, substituted, or added for every 100 bases; (iii) DNA having a base sequence that is at least 90% identical to sequence number 10 and possessing promoter activity, The method according to claim 14, including the method described in claim 14.

16. The method according to any one of claims 1 to 15, wherein the target gene encodes an esterase.

17. The gene encoding the esterase is one of the following DNAs (i) to (iii): (i) DNA consisting of one of the base sequences from sequence numbers 11 to 13; (ii) DNA having a base sequence in which, in any one of sequence numbers 11 to 13, 1 to 10 bases are deleted, substituted, or added per 100 base sequences, wherein the protein encoded thereby has esterase activity; or (iii) DNA comprising a base sequence having at least 90% sequence identity with any one of sequence numbers 11 to 13, wherein the protein encoded thereby has esterase activity, The method according to claim 16, including the method described in claim 16.

18. The method according to any one of claims 1 to 17, wherein the expression construct comprises DNA encoding a foldase.

19. The DNA encoding foldase is one of the following DNAs (i) to (iii): (i) DNA consisting of one of the base sequences from sequence numbers 14 to 16; (ii) DNA having a base sequence in which, in any one of sequence numbers 14 to 16, 1 to 10 bases are deleted, substituted, or added per 100 bases, wherein the protein encoded thereby has foldase activity; or (iii) DNA comprising a base sequence having at least 90% sequence identity with any one of sequence numbers 14 to 16, wherein the protein encoded thereby has foldase activity, The method according to claim 18, including the method described in claim 18.

20. The method according to any one of claims 1 to 19, wherein the target gene further encodes a signal sequence.

21. The method according to any one of claims 1 to 20, wherein the bacterium of the genus Burkholderia is Burkholderia stabilis.

22. One of the following DNAs (i) through (iii): (i) DNA having promoter activity, consisting of more than 80 consecutive base sequences in the base sequence of Sequence ID No. 7; (ii) DNA having promoter activity, which consists of a sequence in which, in the sequence of Sequence ID No. 7, 1 to 10 bases are deleted, substituted, or added for every 100 bases in a sequence of more than 80 consecutive bases; or (iii) DNA having promoter activity, consisting of more than 80 consecutive base sequences and base sequences having at least 90% sequence identity, in sequence number 7 A promoter consisting of [something].

23. The aforementioned promoter is one of the following DNAs (i) to (iii): (i) DNA consisting of the base sequence of Sequence ID No. 8; (ii) DNA having promoter activity, wherein the base sequence of Sequence ID No. 8 consists of a base sequence in which 1 to 10 bases are deleted, substituted, or added for every 100 bases; (iii) DNA having a base sequence that is at least 90% identical to sequence number 8 and possesses promoter activity. The promoter according to claim 22, including the following:

24. One of the following DNAs (i) through (iii): (i) DNA having promoter activity, consisting of more than 100 consecutive base sequences in the base sequence of Sequence ID No. 9; (ii) DNA having promoter activity, wherein in the base sequence of Sequence ID No. 9, 1 to 10 bases are deleted, substituted, or added for each 100 bases in a sequence of more than 100 consecutive bases; or (iii) DNA having promoter activity, consisting of more than 100 consecutive base sequences and base sequences having at least 90% sequence identity, A promoter consisting of [something].

25. The aforementioned promoter is one of the following DNAs (i) to (iii): (i) DNA consisting of the base sequence of Sequence ID No. 10; (ii) DNA having promoter activity, wherein the base sequence of sequence number 10 consists of a base sequence in which 1 to 10 bases are deleted, substituted, or added for every 100 bases; (iii) DNA having a base sequence that is at least 90% identical to sequence number 10 and possessing promoter activity, The promoter according to claim 24, including the following:

26. A mutant strain of a bacterium of the genus Burkholderia, which is modified to inhibit the expression of one or more genes selected from the group consisting of BSFP_068740, BSFP_068730, and BSFP_068720, or the expression of a protein encoded by said gene, wherein the mutant strain includes an expression construct, and the expression construct includes a promoter that controls the expression of a target gene.

27. The mutant strain according to claim 26, wherein the promoter is the promoter described in any one of claims 22 to 25.

28. A method for maintaining expression constructs in bacteria of the genus Burkholderia, A method comprising the step of culturing a Burkholderia bacterium that does not have one or more genes selected from the group consisting of BSFP_068740, BSFP_068730, and BSFP_068720, or in which the expression of said genes or the expression of the protein encoded by said genes is inhibited, the Burkholderia bacterium containing an expression construct.

29. The method according to claim 28, wherein the copy number of the expression construct is increased or its stability is increased in cultured Burkholderia bacteria.

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