New promoter

Abstract

To provide a novel promoter, and a method for producing a target material using the promoter.SOLUTION: The present invention provides a novel promoter derived from Corynebacterium bacteria, the promoter having high transfer activity.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This invention relates to a novel promoter. [Background technology]

[0002] In the industrial production of substances by microorganisms, improving the productivity of the target substance is one of the important challenges. Biotechnology research aimed at improving the productivity of substances by microorganisms has been conducted. One such area is research on highly active promoters. Patent document 1 discloses a promoter for the elongation factor tu as a highly active promoter that functions in Corynebacterium bacteria.

[0003] Patent Document 2 discloses the analysis of various promoter sequences derived from the genera Escherichia and Corynebacterium, and the synthesis of promoters SPL1, SPL7, and SPL13, which function as highly active promoters in Corynebacterium, from these sequences. [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] Japanese Patent Publication No. 2008-212155 [Patent Document 2] Special Publication No. 2019-528075 [Overview of the Initiative] [Problems that the invention aims to solve]

[0005] The present invention provides a novel promoter and a method for producing a target substance using the promoter. [Means for solving the problem]

[0006] The inventors investigated promoters derived from Corynebacterium species and discovered a novel promoter with high transcriptional activity.

[0007] In one aspect, the present invention provides a DNA having promoter activity, which consists of a nucleotide sequence selected from the group consisting of the following (a) to (c): (a) The nucleotide sequence upstream of the gene encoding a polypeptide consisting of the following amino acid sequence; (b) A nucleotide sequence having at least 90% identity with the nucleotide sequence upstream of the gene encoding a polypeptide consisting of the following amino acid sequence; and (c) A nucleotide sequence in which one or several nucleotides are deleted, substituted, added, or inserted with respect to the nucleotide sequence upstream of the gene encoding a polypeptide consisting of the following amino acid sequence, MX1X2X3FX1IX4QX5IFX1GX6X1X1LX7X1SX8X1X1GAQX9VFX 10 X1X 11 X1X1X 12 SS (where X1 is an arbitrary amino acid residue, X2 is S or A, X3 is V or I, X4 is F or I, X5 is S or A, X6 is V or I, X7 is V or I, X8 is V or I, X9 is G or N, X 10 is D or T, X 11 is I or V, X 12 is A or F). In another aspect, the present invention provides a promoter consisting of a DNA selected from the group consisting of the following (g) to (i): (g) A DNA consisting of the nucleotide sequence upstream of the gene encoding a polypeptide consisting of the following amino acid sequence; (h) A DNA consisting of a nucleotide sequence having at least 90% identity with the nucleotide sequence upstream of the gene encoding a polypeptide consisting of the following amino acid sequence and having promoter activity; and (i) A DNA consisting of a nucleotide sequence in which one or several nucleotides are deleted, substituted, added, or inserted with respect to the nucleotide sequence upstream of the gene encoding a polypeptide consisting of the following amino acid sequence and having promoter activity, MX1X2X3FX1IX4QX5IFX1GX6X1X1LX7X1SX8X1X1GAQX9VFX 10 X1X 11 X1X1X 12 SS (Here, X1 is an arbitrary amino acid residue, X2 is S or A, X3 is V or I, X4 is F or I, X5 is S or A, X6 is V or I, X7 is V or I, X8 is V or I, X9 is G or N, X 10 is D or T, X 11 is I or V, X 12 is A or F) is provided. In another aspect, the present invention provides an expression vector containing the DNA or the promoter. In another aspect, the present invention provides a DNA expression cassette containing the DNA or the promoter. In another aspect, the present invention provides Corynebacterium containing the expression vector or the DNA expression cassette. In another aspect, the present invention culturing the Corynebacterium; and recovering the target substance from the culture obtained by the culturing, provides a method for producing a target substance, including.

Effect of the Invention

[0008] The promoter of the present invention has high transcriptional activity and can significantly improve the transcription level of the gene to be controlled. According to the promoter of the present invention, the efficiency of microbiological production of the target substance can be improved.

Brief Description of the Drawings

[0009] [Figure 1] Conversion activity of HFM122 from ABA to AHBA under the control of the tu promoter, SPL13 promoter, or cg2875 promoter. [Figure 2]AHBA conversion rate by HFM122 under the control of the tu promoter, SPL13 promoter, or cg2875 promoter. [Figure 3] Expression levels of reporter proteins under the control of the tu promoter, SPL13 promoter, or cg2875 promoter (indicated by relative fluorescence intensity relative to the tu promoter). [Figure 4] AHBA conversion rate by HFM122 under the control of the tu promoter, SPL13 promoter, cg2875 promoter, or reduced cg2875 promoter. [Figure 5] AHBA conversion rate by HFM122 under the control of the tu promoter, SPL13 promoter, or a promoter of a homolog of the cg2875 gene. [Modes for carrying out the invention]

[0010] In this specification, the identity of amino acid sequences or nucleotide sequences is calculated using the Lipman-Pearson method (Science, 1985, 227:1435-1441). Specifically, it is calculated by performing the analysis using the homology analysis program of the genetic information processing software GENETYX Ver.12 with a unit size to compare (ktup) of 2.

[0011] In this specification, "at least 90% identity" with respect to the nucleotide sequence means identity of 90% or more, preferably 95% or more, more preferably 96% or more, even more preferably 97% or more, even more preferably 98% or more, and still more preferably 99% or more.

[0012] In this specification, unless otherwise defined, “one or several” as used with respect to the deletion, substitution, addition or insertion of nucleotides in a nucleotide sequence may preferably mean 1 to 20, more preferably 1 to 10, even more preferably 1 to 5, even more preferably 1 to 3, and still more preferably 1 or 2. In this specification, “addition” of nucleotides includes the addition of one or several nucleotides to one end and both ends of a sequence.

[0013] In this specification, "amino acid residue" means the 20 amino acid residues that make up proteins: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine ​​(Cys or C), glutamine (Gln or Q), glutamic acid (Glu or E), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Val or V).

[0014] In this specification, "upstream" and "downstream" with respect to a gene refer to the upstream and downstream directions of the gene's transcription. For example, the "upstream sequence" and "downstream sequence" of a gene refer to the sequences located on the 5' and 3' ends of the gene in the DNA sense strand, respectively. For example, "promoter linked upstream of a gene" means that the promoter is located on the 5' end of the gene in the DNA sense strand. Also, for example, "nucleotide sequence upstream of a gene" means the nucleotide sequence located on the 5' end of the gene in the DNA sense strand, that is, adjacent to the start codon of the gene.

[0015] In this specification, "operable linkage" between a gene and a regulatory region such as a promoter means that the gene and the regulatory region are linked in such a way that the gene can be expressed under the control of the regulatory region. Procedures for "operable linkage" between a gene and a regulatory region are well known to those skilled in the art.

[0016] In this specification, "promoter activity" refers to the activity that promotes the transcription of DNA (genes) into mRNA. Promoter activity can be confirmed by using a suitable reporter gene. For example, promoter activity can be confirmed by ligating a DNA encoding a detectable protein, i.e., a reporter gene, downstream of the promoter and measuring the production amount of the gene product of that reporter gene. Examples of reporter genes include genes for enzymes that act on chromogenic substrates, such as the β-galactosidase (LacZ) gene, β-glucuronidase (GUS) gene, luciferase gene, β-lactamase gene, and the gene encoding EtbC (2,3-dihydroxy-ethylbenzene 1,2-dioxygenase), as well as genes for fluorescent proteins, such as the gene encoding GFP (Green Fluorescent Protein). Alternatively, promoter activity can also be confirmed by measuring the expression level of mRNA transcribed from the reporter gene using quantitative RT-PCR or the like. Alternatively, promoter activity can also be confirmed by measuring the reaction involving the protein encoded by the reporter gene. For example, HFM122 (NCBI Reference Sequence: WP_010920262.1), known as 4-hydroxybenzoic acid-3-monooxygenase (EC1.14.13.2), and its variants (referred to in Japanese Patent Publication No. 2021-73914) possess 4-aminobenzoic acid hydroxylation activity and can convert 4-aminobenzoic acid (4-ABA) to 4-amino-3-hydroxybenzoic acid (4,3-AHBA). When the gene encoding HFM122 is used as a reporter gene, promoter activity can be measured by measuring the concentration of 4-ABA, 4,3-AHBA, or both, or by calculating the conversion rate from 4-ABA to 4,3-AHBA based on these concentrations.

[0017] In this specification, "target substance" means a substance that a cell can produce and whose production or increase in production is desired. Target substances include substances encoded by genes (target genes), substances produced by the action of substances encoded by genes (target genes), and substances whose production is increased by the action of substances encoded by genes (target genes).

[0018] The inventors have found that DNA consisting of the nucleotide sequence (SEQ ID NO: 1) 613 bp upstream of the start codon of the gene indicated by Gene ID: cg2875, derived from Corynebacterium glutamicum, exhibits higher promoter activity compared to known highly active promoters such as the tu promoter and the SPL13 promoter (Figures 1-3). The cg2875 gene in Corynebacterium glutamicum consists of the nucleotide sequence of SEQ ID NO: 2, which encodes the amino acid sequence of SEQ ID NO: 3, and is a gene predicted as an ORF by the ORF prediction software Glimmer. The protein encoded by the cg2875 gene is known to be a membrane protein and to be mycolated, but its function and expression level were previously unknown (Issa, H. et al., PLoS One, 2017, 12(2):e0171955).

[0019] Furthermore, the inventors have found that DNA consisting of the 208 bp upstream nucleotide sequence of the cg2875 gene (SEQ ID NO: 23) exhibits higher promoter activity compared to the tu promoter and the SPL13 promoter (Figure 4). This DNA, if it contains at least 86 bp of nucleotide sequence at its 3' end (SEQ ID NO: 21), exhibits promoter activity equivalent to that of the tu promoter and the SPL13 promoter.

[0020] Furthermore, the inventors found that DNA consisting of upstream nucleotide sequences (SEQ ID NOs. 26-41) of the homolog of the cg2875 gene has higher promoter activity compared to the tu promoter and the SPL13 promoter (Figure 5). Here, "homolog of the cg2875 gene" refers to a gene that is presumed to encode a polypeptide having equivalent function to the cg2875 protein. Examples include a gene downstream of a nucleotide sequence that achieves a query cover of 98% or more in a BLAST search using the nucleotide sequence of the cg2875 gene promoter (SEQ ID NOs. 23) as the query, and a gene encoding an amino acid sequence that achieves a query cover of 100% in a BLAST search using the amino acid sequence of the cg2875 protein (SEQ ID NOs. 3) as the query. The amino acid sequence encoded by the homolog, including the amino acid sequence encoded by the cg2875 gene, can be expressed as follows. MX1X2X3FX1IX4QX5IFX1GX6X1X1LX7X1SX8X1X1GAQX9VFX 10 X1X 11 X1X1X 12 SS (Sequence ID 169) (Here, X1 is any amino acid residue, X2 is S or A, X3 is V or I, X4 is F or I, X5 is S or A, X6 is V or I, X7 is V or I, X8 is V or I, X9 is G or N, X 10 is D or T, X 11 is I or V, X 12 (is A or F).

[0021] Examples of "promoters for homologs of the cg2875 gene" include promoters consisting of nucleotide sequences that achieve a query cover of 98% or more in a BLAST search using the nucleotide sequence of the cg2875 gene promoter (SEQ ID NO: 23) as the query, and promoters consisting of upstream nucleotide sequences of genes encoding amino acid sequences that achieve a query cover of 100% in a BLAST search using the amino acid sequence of the cg2875 protein (SEQ ID NO: 3) as the query.

[0022] The present invention relates to DNA having novel promoter activity, a novel promoter, an expression vector containing the DNA or the promoter, a DNA expression cassette and a transformant, and a method for producing a target substance using the transformant.

[0023] The DNA having promoter activity according to the present invention includes DNA having promoter activity consisting of a nucleotide sequence selected from the group consisting of (a) to (c) below. (a) The upstream nucleotide sequence of the gene encoding the polypeptide consisting of the following amino acid sequence; (b) A nucleotide sequence having at least 90% identity with the upstream nucleotide sequence of a gene encoding a polypeptide consisting of the following amino acid sequence; and (c) A nucleotide sequence in which one or more nucleotides are deleted, substituted, added, or inserted into the upstream nucleotide sequence of a gene encoding a polypeptide consisting of the following amino acid sequence; MX1X2X3FX1IX4QX5IFX1GX6X1X1LX7X1SX8X1X1GAQX9VFX 10 X1X 11 X1X1X 12 SS (Here, X1 is any amino acid residue, X2 is S or A, X3 is V or I, X4 is F or I, X5 is S or A, X6 is V or I, X7 is V or I, X8 is V or I, X9 is G or N, X 10 is D or T, X 11 is I or V, X 12 (is A or F).

[0024] The length of the upstream nucleotide sequence of the gene encoding the polypeptide consisting of the above amino acid sequence is not particularly limited as long as it has promoter activity, but is preferably 85 bp or more, more preferably 100 bp or more, even more preferably 200 bp or more, preferably 800 bp or less, more preferably 650 bp or less, even more preferably 250 bp or less, and even more preferably 208 bp. The range of the nucleotide sequence length is preferably 85 to 800 bp, more preferably 85 to 650 bp, even more preferably 100 to 650 bp, even more preferably 100 to 250 bp, even more preferably 200 to 250 bp, and even more preferably 208 bp.

[0025] Preferably, the DNA having promoter activity of the present invention consists of a nucleotide sequence selected from the group consisting of (d) to (f) below: (d) any of the nucleotide sequences of SEQ ID NOs: 1, 21-23, and 26-41; (e) a nucleotide sequence having at least 90% identity with any of the nucleotide sequences of SEQ ID NOs: 1, 21-23, and 26-41; and (f) a nucleotide sequence in which one or more nucleotides are deleted, substituted, added, or inserted into any of the nucleotide sequences of SEQ ID NOs: 1, 21-23, and 26-41. Here, DNA consisting of any of the nucleotide sequences of SEQ ID NOs: 1, 21-23, and 26-41 has promoter activity as shown in the examples below. The microbial species from which DNA consisting of the nucleotide sequences of SEQ ID NOs: 26-41 originates are shown in Table 7 below.

[0026] More preferably, the DNA having promoter activity of the present invention consists of a nucleotide sequence selected from the group consisting of (d') to (f') below. (d') Nucleotide sequence of sequence number 23; (e') A nucleotide sequence having at least 90% identity with the nucleotide sequence of SEQ ID NO: 23; and (f') A nucleotide sequence in which one or more nucleotides are deleted, substituted, added, or inserted into the nucleotide sequence of SEQ ID NO: 23. More preferably, the DNA having promoter activity of the present invention includes a nucleotide sequence selected from the group consisting of (d') to (f') above, in which the nucleotide sequence selected from the group consisting of (d") to (f") below is included. (d) Nucleotide sequence of Sequence ID No. 21; (e) A nucleotide sequence having at least 90% identity with the nucleotide sequence of Sequence ID No. 21; and (f) A nucleotide sequence in which one or more nucleotides are deleted, substituted, added, or inserted from the nucleotide sequence of SEQ ID NO: 21.

[0027] The DNA having promoter activity of the present invention exhibits promoter activity in at least Corynebacterium. Preferably, the DNA having promoter activity of the present invention exhibits improved promoter activity compared to the tu promoter. More preferably, the DNA having promoter activity of the present invention exhibits improved promoter activity compared to the SPL13 promoter.

[0028] Examples of promoters of the present invention include promoters comprising DNA selected from the group consisting of (g) to (i) below. (g) DNA consisting of the nucleotide sequence upstream of the gene encoding a polypeptide consisting of the following amino acid sequence; (h) DNA having promoter activity, consisting of a nucleotide sequence having at least 90% identity with the upstream nucleotide sequence of a gene encoding a polypeptide consisting of the following amino acid sequence; and (i) DNA having promoter activity, consisting of a nucleotide sequence in which one or more nucleotides are deleted, substituted, added, or inserted into the nucleotide sequence upstream of a gene encoding a polypeptide consisting of the following amino acid sequence; MX1X2X3FX1IX4QX5IFX1GX6X1X1LX7X1SX8X1X1GAQX9VFX 10 X1X 11 X1X1X 12 SS (Here, X1 is any amino acid residue, X2 is S or A, X3 is V or I, X4 is F or I, X5 is S or A, X6 is V or I, X7 is V or I, X8 is V or I, X9 is G or N, X 10 is D or T, X 11 is I or V, X 12 (is A or F).

[0029] The length of the upstream nucleotide sequence of the gene encoding the polypeptide consisting of the above amino acid sequence is not particularly limited as long as it has promoter activity, but is preferably 85 bp or more, more preferably 100 bp or more, even more preferably 200 bp or more, preferably 800 bp or less, more preferably 650 bp or less, even more preferably 250 bp or less, and even more preferably 208 bp. The range of the nucleotide sequence length is preferably 85 to 800 bp, more preferably 85 to 650 bp, even more preferably 100 to 650 bp, even more preferably 100 to 250 bp, even more preferably 200 to 250 bp, and even more preferably 208 bp.

[0030] Preferably, the promoter of the present invention consists of DNA selected from the group consisting of (j) to (l) below. (j) DNA consisting of any of the nucleotide sequences of sequence numbers 1, 21-23, and 26-41; (k) DNA having promoter activity, consisting of a nucleotide sequence having at least 90% identity with any of the nucleotide sequences of sequence numbers 1, 21-23, and 26-41; and (l) DNA having promoter activity, consisting of nucleotide sequences in which one or more nucleotides are deleted, substituted, added, or inserted into any of the nucleotide sequences of SEQ ID NOs: 1, 21-23, and 26-41. Here, DNA consisting of any of the nucleotide sequences of SEQ ID NOs: 1, 21-23, and 26-41 has promoter activity as shown in the examples below. The microbial species from which DNA consisting of the nucleotide sequences of SEQ ID NOs: 26-41 originates are shown in Table 7 below.

[0031] More preferably, the promoter of the present invention consists of DNA selected from the group consisting of (j') to (l') below. (j') DNA consisting of the nucleotide sequence of sequence number 23; (k') DNA consisting of a nucleotide sequence having at least 90% identity with the nucleotide sequence of Sequence ID No. 23, and possessing promoter activity; and (l') DNA having promoter activity, consisting of a nucleotide sequence in which one or more nucleotides are deleted, substituted, added, or inserted into the nucleotide sequence of SEQ ID NO: 23. More preferably, the promoter of the present invention comprises DNA selected from the group consisting of (j') to (l') above, and further comprising DNA selected from the group consisting of (j") to (l") below. (j) DNA consisting of the nucleotide sequence of sequence number 21; DNA consisting of a nucleotide sequence having at least 90% identity with the nucleotide sequence of (k) Sequence ID No. 21, and possessing promoter activity; and (l) DNA having promoter activity, consisting of a nucleotide sequence in which one or more nucleotides are deleted, substituted, added, or inserted into the nucleotide sequence of SEQ ID NO: 21.

[0032] The promoter of the present invention has promoter activity in at least Corynebacterium. Preferably, the promoter of the present invention has improved promoter activity compared to the tu promoter. More preferably, the promoter of the present invention has improved promoter activity compared to the SPL13 promoter. Hereinafter, DNA having the promoter activity of the present invention and the promoter of the present invention will be collectively referred to as "the promoter of the present invention, etc."

[0033] The method for obtaining the promoter, etc. of the present invention is not particularly limited and can be obtained by conventional chemical synthesis methods or genetic engineering techniques. For example, the promoter, etc. of the present invention can be artificially synthesized based on the nucleotide sequence of the promoter, etc. of the present invention (e.g., SEQ ID NOs: 1, 21-23, and 26-41). For artificial synthesis, commercially available DNA synthesis services provided by companies such as GenScript can be used. Alternatively, the nucleotide sequence of the promoter, etc. of the present invention (e.g., SEQ ID NOs: 1, 21-23, and 26-41) can be cloned from microorganisms such as Corynebacterium glutamicum, for example, according to the method described in Molecular Cloning - A LABORATORY MANUAL THIRD EDITION (Joseph Sambrook, David W. Russell, Cold Spring Harbor Laboratory Press, 2001).

[0034] The promoters of the present invention can also be produced by introducing mutations into the DNA of the nucleotide sequences of the promoters of the present invention (e.g., SEQ ID NOs. 1, 21-23, and 26-41). Methods for introducing mutations include, for example, ultraviolet irradiation and site-directed mutagenesis. Site-directed mutagenesis methods include methods utilizing Splicing overlap extension (SOE) PCR (Horton et al., Gene 77, 61-68, 1989), ODA method (Hashimoto-Gotoh et al., Gene 152, 271-276, 1995), and Kunkel method (Kunkel, TA, Proc. Natl. Acad. Sci. USA, 1985, 82, 488). Alternatively, Site-Directed Mutagenesis System Mutan-SuperExpress Km Kit (Takara Bio), Transformer TMCommercially available site-directed mutagenesis kits, such as the Site-Directed Mutagenesis Kit (Clonetech) and the KOD-Plus-Mutagenesis Kit (Toyobo), can also be used. By selecting DNA with the desired promoter activity from the mutated DNA, the promoter of the present invention can be obtained. For example, by activating the linkage of a target gene downstream of the mutated DNA and analyzing the expression level of the target gene, DNA with promoter activity can be selected.

[0035] Alternatively, methods for deleting, substituting, adding, or inserting nucleotides into a nucleotide sequence are described, for example, by Dieffenbach et al. (Cold Spring Harbor Laboratory Press, New York, 581-621, 1995).

[0036] By using the method described above, it is possible to obtain a promoter of the present invention consisting of a nucleotide sequence having at least 90% identity with the nucleotide sequence of the promoter of the present invention (for example, SEQ ID NOs: 1, 21-23, and 26-41), or a promoter of the present invention consisting of a nucleotide sequence in which one or more nucleotides are deleted, substituted, added, or inserted from the nucleotide sequence of the promoter of the present invention (for example, SEQ ID NOs: 1, 21-23, and 26-41).

[0037] The promoter of the present invention has the function of controlling the expression of a gene located downstream thereof. By using the promoter of the present invention, a DNA fragment having an expression regulatory region with excellent transcriptional activity can be obtained. For example, a DNA fragment can be constructed that includes a target gene and the promoter of the present invention operably linked upstream thereof. In addition to the promoter of the present invention and the target gene, the DNA fragment may also contain a cis-element or terminator that enhances the transcriptional activity of the promoter of the present invention. Furthermore, the DNA fragment may also contain a select marker gene such as a drug resistance gene or a nutritional requirement marker gene. Preferably, the DNA fragment containing the target gene and the promoter of the present invention is a DNA expression cassette for expressing the target gene.

[0038] The DNA fragment containing the promoter of the present invention described above can be constructed to have restriction enzyme recognition sequences at both ends. The promoter of the present invention can be introduced into a vector using these restriction enzyme recognition sequences. For example, the promoter of the present invention can be introduced into a vector by cutting a vector with a restriction enzyme and adding a DNA fragment containing the promoter of the present invention and having restriction enzyme cutting sequences at its ends (restriction enzyme method).

[0039] A DNA fragment containing the promoter of the present invention may be directly introduced into the genome of a host cell. For example, a DNA fragment containing the promoter of the present invention may be introduced upstream of a target gene in the genome of a host cell. Alternatively, for example, a DNA expression cassette containing the target gene and the promoter of the present invention may be introduced into the genome of a host cell.

[0040] Alternatively, by incorporating the promoter of the present invention into an expression vector that enables the expression of a target gene, an expression vector can be obtained that can improve the expression of the target gene at the transcriptional level. In such an expression vector, the promoter of the present invention can be operably linked upstream of the DNA encoding the target gene. The expression vector having the promoter of the present invention may be a vector for introduction into the chromosome of a host cell, or a vector that is retained outside the chromosome. It is preferable that it be replicable within the host cell.

[0041] Examples of expression vectors include vectors for E. coli and Corynebacterium, and plasmids, cosmids, phasmids, phages, transposons, and BAC vectors can be used. Preferred vector examples include pET21-a(+), pUC18 / 19, pUC118 / 119, pBR322, pMW218 / 219, pZ1 (Applied and Environmental Microbiology, 1989, 55:684-688), pEKEx1 (Gene, 1991, 102:93-98), pHS2-1 (Gene, 1991, 107:69-74), pCLiK5MCS (JP 2005-522218), pCG2 (JP 58-35197), and pNG2 (FEMS Microbiology). Examples include Letters, 1990, 66:119-124), pAG1 (Japanese Patent Publication No. 61-52290), and pHKPsacB1 (International Publication No. 2014 / 007273).

[0042] The target gene located downstream of the promoter of the present invention in the expression vector or DNA fragment described above is not particularly limited. For example, the target gene is a gene that codes for a target substance or an enzyme involved in its synthesis. The target gene may be a heterologous gene that codes for a heterologous expression product, a homologous gene introduced from an external source, a gene that codes for an expression product naturally present in the host cell, or any other gene that codes for any protein, peptide, nucleic acid, etc. Examples of substances coded by the target gene include enzymes, hormones, cytokines, other physiologically active peptides, transporters, and non-coding RNAs. Examples of enzymes include oxidoreductases, transferases, hydrolases, lyases, isomerases, ligases or synthetases, glycolytic enzymes, pentose phosphate pathway enzymes, TCA pathway enzymes, and enzymes involved in the synthesis of aromatic compounds. Preferred examples of target genes include enzymes involved in the synthesis of aromatic compounds in host cells, such as gallic acid synthase (described in Japanese Patent Publication No. 2009-065839 and Japanese Patent Publication No. 2009-213392), and genes encoding AroF, AroG, PabAB, PabC, etc. Examples of aromatic compounds include gallic acid, protocatechuic acid (PCA), aminohydroxybenzoic acid (AHBA), pyridinedicarboxylic acid (PDCA), catechol, and 4-hydroxybenzoic acid.

[0043] A transformant of the present invention can be obtained by introducing an expression vector or DNA fragment containing the promoter of the present invention into host cells using a general transformation method, such as electroporation, transformation, transfection, conjugation, protoplast, particle gun, or Agrobacterium.

[0044] The host cell into which the above vector or DNA fragment is introduced is not particularly limited as long as the promoter of the present invention can function as a promoter within the cell, but bacteria are preferred, and Corynebacterium is more preferred. Examples of Corynebacterium include Corynebacterium glutamicum, Corynebacterium efficiens, Corynebacterium ammoniagenes, Corynebacterium halotolerans, Corynebacterium alkanolyticum, and Corynebacterium callunae, with Corynebacterium glutamicum being more preferred. According to molecular biological classification, coryneform bacteria such as Brevibacterium flavum, Brevibacterium lactofermentum, Brevibacterium divaricatum, and Corynebacterium lilium have been unified under the name Corynebacterium glutamicum (Liebl W et al, Int J Syst Bacteriol, 1991, 41:255-260; Komagata Kazuo et al, Fermentation and Industry, 1987, 45:944-963).

[0045] The above-mentioned transformants can be used to produce the target substance. For example, a transformant containing an expression vector or DNA fragment having a gene encoding the target substance or an enzyme involved in its synthesis, and the promoter of the present invention operably linked upstream of the gene, can be cultured to express the gene under the control of the promoter of the present invention and produce the target substance.

[0046] Examples of target substances are as described above, but preferred examples include substances encoded by the target gene described above, and products synthesized by the action of said substances, such as aromatic compounds produced by enzymes involved in the production of the aromatic compounds described above. Examples of aromatic compounds include gallic acid, protocatechuic acid (PCA), aminohydroxybenzoic acid (AHBA), pyridinedicarboxylic acid (PDCA), catechol, and 4-hydroxybenzoic acid.

[0047] The culture conditions for the transformant are not particularly limited, as long as they allow for cell proliferation and production of the target substance. For example, if the transformant is Corynebacterium, preferred culture media include LB medium and CGXII medium (Journal of Bacteriology, 1993, 175:5595-5603), and culture conditions include a culture temperature of 15°C to 45°C and a culture time of 1 to 7 days. If the transformant is Escherichia coli, preferred culture media include LB medium and M9 medium, and culture conditions include a culture temperature of 15°C to 45°C and a culture time of 1 to 7 days.

[0048] After culturing, the target substance can be obtained by recovering it from the culture. If necessary, the recovered target substance may be further purified. The method for recovering or purifying the target substance from the culture is not particularly limited and may be carried out according to known recovery or purification methods. For example, the culture can be recovered, and if necessary, cell disruption treatment by ultrasound or pressurization can be performed, then cellular components can be removed by tilting, filtration, centrifugation, etc., and the remaining fraction containing the target substance can be recovered. If necessary, the recovered fraction can be purified by dialysis, salting out, ion exchange, distillation, solvent extraction, or a combination thereof to obtain the target substance. In the method for producing the target substance according to the present invention, the culturing of the transformant and the recovery of the target substance may be carried out by batch, semi-batch, or continuous methods.

[0049] Exemplary embodiments of the present invention are further disclosed herein, including the following substances, manufacturing methods, uses, and methods. However, the present invention is not limited to these embodiments.

[0050] [1] DNA having promoter activity, consisting of a nucleotide sequence selected from the group (a) to (c) below: (a) The upstream nucleotide sequence of the gene encoding the polypeptide consisting of the following amino acid sequence; (b) A nucleotide sequence having at least 90% identity with the upstream nucleotide sequence of a gene encoding a polypeptide consisting of the following amino acid sequence; and (c) A nucleotide sequence in which one or more nucleotides are deleted, substituted, added, or inserted into the upstream nucleotide sequence of a gene encoding a polypeptide consisting of the following amino acid sequence; MX1X2X3FX1IX4QX5IFX1GX6X1X1LX7X1SX8X1X1GAQX9VFX 10 X1X 11 X1X1X 12 SS (Here, X1 is any amino acid residue, X2 is S or A, X3 is V or I, X4 is F or I, X5 is S or A, X6 is V or I, X7 is V or I, X8 is V or I, X9 is G or N, X 10 is D or T, X 11 is I or V, X 12 (is A or F). [2] The DNA according to [1], wherein the length of the upstream nucleotide sequence of the gene encoding the polypeptide consisting of the above amino acid sequence is preferably 85 bp or more, more preferably 100 bp or more, even more preferably 200 bp or more, preferably 800 bp or less, more preferably 650 bp or less, even more preferably 250 bp or less, and even more preferably 208 bp, and the range of the nucleotide sequence length is preferably 85 to 800 bp, more preferably 85 to 650 bp, even more preferably 100 to 650 bp, even more preferably 100 to 250 bp, even more preferably 200 to 250 bp, and even more preferably 208 bp. [3] DNA as described in [1] or [2], consisting of a nucleotide sequence selected from the group consisting of (d) to (f) below: (d) Any nucleotide sequence of sequence numbers 1, 21-23, and 26-41; (e) Nucleotide sequences having at least 90% identity with any of the nucleotide sequences of sequence numbers 1, 21-23, and 26-41; and (f) A nucleotide sequence in which one or more nucleotides are deleted, substituted, added, or inserted into any of the nucleotide sequences of SEQ ID NOs: 1, 21-23, and 26-41. [4] DNA consisting of a nucleotide sequence selected from the group consisting of (d') to (f') below, as described in any one of [1] to [3]: (d') Nucleotide sequence of sequence number 23; (e') A nucleotide sequence having at least 90% identity with the nucleotide sequence of SEQ ID NO: 23; and (f') A nucleotide sequence in which one or more nucleotides are deleted, substituted, added, or inserted into the nucleotide sequence of SEQ ID NO: 23. [5] DNA as described in [4], comprising a nucleotide sequence selected from the group consisting of (d) to (f) below: (d) Nucleotide sequence of Sequence ID No. 21; (e) A nucleotide sequence having at least 90% identity with the nucleotide sequence of Sequence ID No. 21; and (f) A nucleotide sequence in which one or more nucleotides are deleted, substituted, added, or inserted from the nucleotide sequence of SEQ ID NO: 21. [6] A promoter consisting of DNA selected from the group consisting of (g) to (i) below: (g) DNA consisting of the nucleotide sequence upstream of the gene encoding a polypeptide consisting of the following amino acid sequence; (h) DNA having promoter activity, consisting of a nucleotide sequence having at least 90% identity with the upstream nucleotide sequence of a gene encoding a polypeptide consisting of the following amino acid sequence; and (i) DNA having promoter activity, consisting of a nucleotide sequence in which one or more nucleotides are deleted, substituted, added, or inserted into the nucleotide sequence upstream of a gene encoding a polypeptide consisting of the following amino acid sequence; MX1X2X3FX1IX4QX5IFX1GX6X1X1LX7X1SX8X1X1GAQX9VFX 10 X1X 11 X1X1X 12 SS (Here, X1 is any amino acid residue, X2 is S or A, X3 is V or I, X4 is F or I, X5 is S or A, X6 is V or I, X7 is V or I, X8 is V or I, X9 is G or N, X 10 is D or T, X 11 is I or V, X 12 (is A or F). [7] The promoter according to [6], wherein the length of the upstream nucleotide sequence of the gene encoding the polypeptide consisting of the above amino acid sequence is preferably 85 bp or more, more preferably 100 bp or more, even more preferably 200 bp or more, preferably 800 bp or less, more preferably 650 bp or less, even more preferably 250 bp or less, and even more preferably 208 bp, and the range of the nucleotide sequence length is preferably 85 to 800 bp, more preferably 85 to 650 bp, even more preferably 100 to 650 bp, even more preferably 100 to 250 bp, even more preferably 200 to 250 bp, and even more preferably 208 bp. [8] A promoter as described in [6] or [7], consisting of DNA selected from the group consisting of (j) to (l) below: (j) DNA consisting of any of the nucleotide sequences of sequence numbers 1, 21-23, and 26-41; (k) DNA having promoter activity, consisting of a nucleotide sequence having at least 90% identity with any of the nucleotide sequences of sequence numbers 1, 21-23, and 26-41; and (l) DNA having promoter activity, consisting of nucleotide sequences in which one or more nucleotides are deleted, substituted, added, or inserted into any of the nucleotide sequences of SEQ ID NOs: 1, 21-23, and 26-41. [9] A promoter described in any one of the following items [6] to [8], consisting of DNA selected from the group (j') to (l'): (j') DNA consisting of the nucleotide sequence of sequence number 23; (k') DNA consisting of a nucleotide sequence having at least 90% identity with the nucleotide sequence of Sequence ID No. 23, and possessing promoter activity; and (l') DNA having promoter activity, consisting of a nucleotide sequence in which one or more nucleotides are deleted, substituted, added, or inserted into the nucleotide sequence of SEQ ID NO: 23.

[10] The promoter described in [9], comprising DNA selected from the group consisting of (j) to (l) below: (j) DNA consisting of the nucleotide sequence of sequence number 21; DNA consisting of a nucleotide sequence having at least 90% identity with the nucleotide sequence of (k) Sequence ID No. 21, and possessing promoter activity; and (l) DNA having promoter activity, consisting of a nucleotide sequence in which one or more nucleotides are deleted, substituted, added, or inserted into the nucleotide sequence of SEQ ID NO: 21.

[11] Preferably, the DNA described in any one of [1] to [5] or the promoter described in any one of [6] to

[10] , which has promoter activity in Corynebacterium.

[0051] An expression vector comprising the DNA described in any one of items

[12] , [1] to [5], and

[11] , or the promoter described in any one of items [6] to

[11] .

[13] Preferably, the expression vector according to

[12] , comprising a gene encoding a target substance or an enzyme involved in its synthesis, and the DNA or promoter ligated upstream of the gene.

[14] The expression vector according to

[13] , wherein the target substance is preferably an aromatic compound.

[0052]

[15] A DNA fragment containing any one of items [1] to [5] and

[11] or any one of items [6] to

[11] .

[16] Preferably, the DNA fragment according to

[15] , comprising a gene encoding a target substance or an enzyme involved in its synthesis, and the DNA or promoter ligated upstream of the gene.

[17] The DNA fragment according to

[16] , wherein the target substance is preferably an aromatic compound.

[18] Preferably a DNA expression cassette, the DNA fragment according to any one of

[15] to

[17] .

[0053] A transformant comprising an expression vector described in any one of items

[19] ,

[12] , to

[14] , or a DNA fragment described in any one of items

[15] , to

[17] .

[20] The transformant according to

[19] , preferably Corynebacterium, and more preferably Corynebacterium glutamicum.

[0054] Culturing the transformants described in

[21] ,

[19] , or

[20] ; and To recover the target substance from the culture obtained in the said culture, A method for producing the target substance, including the method described above.

[22] The method according to

[21] , wherein the target substance is preferably an aromatic compound, and more preferably at least one selected from the group consisting of gallic acid, protocatechuic acid, aminohydroxybenzoic acid, pyridinedicarboxylic acid, catechol, and 4-hydroxybenzoic acid.

[0055]

[23] DNA having promoter activity, consisting of a nucleotide sequence selected from the group consisting of (d) to (f) below: (d) Any nucleotide sequence of sequence numbers 1, 21-23, and 26-41; (e) Nucleotide sequences having at least 90% identity with any of the nucleotide sequences of sequence numbers 1, 21-23, and 26-41; and (f) A nucleotide sequence in which one or more nucleotides are deleted, substituted, added, or inserted into any of the nucleotide sequences of SEQ ID NOs: 1, 21-23, and 26-41.

[24] DNA as described in

[23] , consisting of a nucleotide sequence selected from the group consisting of (d') to (f') below: (d') Nucleotide sequence of sequence number 23; (e') A nucleotide sequence having at least 90% identity with the nucleotide sequence of SEQ ID NO: 23; and (f') A nucleotide sequence in which one or more nucleotides are deleted, substituted, added, or inserted into the nucleotide sequence of SEQ ID NO: 23.

[25] DNA as described in

[24] , comprising a nucleotide sequence selected from the group consisting of (d) to (f) below: (d) Nucleotide sequence of Sequence ID No. 21; (e) A nucleotide sequence having at least 90% identity with the nucleotide sequence of Sequence ID No. 21; and (f) A nucleotide sequence in which one or more nucleotides are deleted, substituted, added, or inserted from the nucleotide sequence of SEQ ID NO: 21.

[26] A promoter consisting of DNA selected from the group consisting of (j) to (l) below: (j) DNA consisting of any of the nucleotide sequences of sequence numbers 1, 21-23, and 26-41; (k) DNA having promoter activity, consisting of a nucleotide sequence having at least 90% identity with any of the nucleotide sequences of sequence numbers 1, 21-23, and 26-41; and (l) DNA having promoter activity, consisting of nucleotide sequences in which one or more nucleotides are deleted, substituted, added, or inserted into any of the nucleotide sequences of SEQ ID NOs: 1, 21-23, and 26-41.

[27] The promoter described in

[26] , consisting of DNA selected from the group consisting of (j') to (l') below: (j') DNA consisting of the nucleotide sequence of sequence number 23; (k') DNA consisting of a nucleotide sequence having at least 90% identity with the nucleotide sequence of Sequence ID No. 23, and possessing promoter activity; and (l') DNA having promoter activity, consisting of a nucleotide sequence in which one or more nucleotides are deleted, substituted, added, or inserted into the nucleotide sequence of SEQ ID NO: 23.

[28] The promoter described in

[27] , comprising DNA selected from the group consisting of (j) to (l) below: (j) DNA consisting of the nucleotide sequence of sequence number 21; DNA consisting of a nucleotide sequence having at least 90% identity with the nucleotide sequence of (k) Sequence ID No. 21, and possessing promoter activity; and (l) DNA having promoter activity, consisting of a nucleotide sequence in which one or more nucleotides are deleted, substituted, added, or inserted into the nucleotide sequence of SEQ ID NO: 21.

[29] Preferably, a DNA according to any one of

[23] to

[25] or a promoter according to any one of

[26] to

[28] that has promoter activity in Corynebacterium. [Examples]

[0056] The present invention will be described in more detail below based on examples, but the present invention is not limited thereto.

[0057] Example 1: Preparation of plasmids for promoter activity evaluation 1) Fabrication of pECsf_gapS_pabABC_Pcg2875_HFM122_V47L The vector fragment was amplified using pECsf_gapS_pabABC_tuD_HFM122_V47L (see Japanese Patent Publication No. 2021-073914) as a template and primers (SEQ ID NOs: 4 and 5). In pECsf_gapS_pabABC_tuD_HFM122_V47L, under the control of the tu promoter of Corynebacterium glutamicum ATCC13032 strain, the valine at position 47 of HFM122 is replaced with leucine, and a gene encoding HFM122_V47L, which has higher activity than wild-type HFM122, is linked. Furthermore, using the genomic DNA of ATCC13032 strain as a template, the sequence in which the linker sequence of the vector fragment was attached to the promoter sequence (Pcg2875) of Gene ID: cg2875 shown in SEQ ID NO: 1 was amplified using primers (SEQ ID NOs: 6 and 7). The vector fragment and the Pcg2875 fragment were ligated using the In-Fusion HD cloning kit (Clontech) to produce pECsf_gapS_pabABC_Pcg2875_HFM122_V47L. The resulting plasmid solution was used to transform ECOS Competent E.Coli DH5α strain (Nippon Gene), and the resulting cell suspension was spread onto LB agar medium containing kanamycin and incubated overnight at 37°C. Colony PCR was performed using the resulting colonies as templates with KOD One PCR Master Mix (TOYOBO) and primers (SEQ ID NOs. 8 and 9). Transformants in which the plasmid containing the target gene was confirmed were inoculated into 2 mL of LB liquid medium containing kanamycin and incubated overnight at 37°C. Plasmid EasyPure (Takara Bio) was used to purify the culture medium obtained from the resulting culture medium, yielding pECsf_gapS_pabABC_Pcg2875_HFM122_V47L.

[0058] 2) Fabrication of pECsf_gapS_pabABC_SPL13_HFM122_V47L The vector fragment was amplified using pECsf_gapS_pabABC_tuD_HFM122_V47L as a template with primers (SEQ ID NOs: 10 and 11). Meanwhile, the SPL13 promoter sequence (SEQ ID NO: 12) disclosed in Patent Document 2 was chemically synthesized using Eurofins Genomics' artificial gene synthesis service. Using the obtained DNA containing the SPL13 promoter as a template, the sequence in which the linker sequence of the vector fragment was attached to the SPL13 promoter sequence was amplified using primers (SEQ ID NOs: 13 and 14). The vector fragment and the SPL13 promoter fragment were ligated using the In-Fusion HD cloning kit (Clontech) to produce pECsf_gapS_pabABC_SPL13_HFM122_V47L. The obtained plasmid solution was used to transform ECOS Competent E.Coli DH5α strain (Nippon Gene), and the resulting cell suspension was spread onto LB agar medium containing kanamycin and left to stand overnight at 37°C. Colony PCR was performed using the generated colonies as templates with KOD One PCR Master Mix (TOYOBO) and primers (SEQ ID NOs. 9 and 13). Transformants in which the plasmid containing the target gene was confirmed were inoculated into 2 mL of LB liquid medium containing kanamycin and cultured overnight at 37°C. Plasmid purification was performed from the resulting culture medium using NucleoSpin Plasmid EasyPure (Takara Bio) to obtain pECsf_gapS_pabABC_SPL13_HFM122_V47L. Table 1 shows the primers used in 1) and 2) above.

[0059] [Table 1]

[0060] Example 2 Preparation of Transformed Body The plasmids pECsf_gapS_pabABC_Pcg2875_HFM122_V47L and pECsf_gapS_pabABC_SPL13_HFM122_V47L obtained in Example 1, along with pECsf_gapS_pabABC_tuD_HFM122_V47L, were used to transform Corynebacterium glutamicum KC341 strain obtained in Reference Example 1 (described later) by electroporation (Bio-rad). The resulting transformed cell solution was spread onto kanamycin-containing LB agar medium and left to stand at 30°C for 2 days. The resulting colonies were used as transformants.

[0061] Example 3 Comparison of promoter activity 1) Culture of transformed organisms The transformants obtained in Example 2 were inoculated into test tubes containing 10 mL of CGXII medium (containing 50 mg / L kanamycin sulfate) as shown in Table 2, and cultured with shaking at 30°C and 200 rpm for 2 days.

[0062] [Table 2]

[0063] 2) Measurement of promoter activity The quantification of 4-amino-3-hydroxybenzoic acid (4,3-AHBA, hereinafter simply referred to as AHBA) and 4-aminobenzoic acid (4-ABA, hereinafter simply referred to as ABA) was performed by HPLC. The culture medium to be used for HPLC analysis was appropriately diluted with 37 mM sulfuric acid, and then unwanted substances were removed using an AcroPrep 96 filter plate (0.2 μm GHP membrane, Nippon Pall). The HPLC measurement conditions are shown in Table 3. The experiment was performed with N=3.

[0064] [Table 3]

[0065] The AHBA conversion rate was calculated from the AHBA concentration and ABA concentration according to the following formula.

number

[0066] As shown in Figures 1 and 2, strains expressing HFM122_V47L with the cg2875 promoter showed lower concentrations of the substrate ABA, higher concentrations of the product AHBA, and higher AHBA conversion rates compared to strains expressing HFM122_V47L with the tu promoter or the SPL13 promoter. Therefore, it was shown that the activity of the cg2875 promoter is higher than that of the tu promoter and the SPL13 promoter.

[0067] Example 4: Preparation of plasmids for promoter activity evaluation using a reporter protein 1) Creation of pECsf_Pcg2875_mCherry Using the pmCherry Vector purchased from Takara Bio Inc. as a template, the reporter protein mCherry sequence (sequence number 168) was amplified using primers (sequence numbers 156 and 157). Using pECsf_gapS_pabABC_tuD_HFM122_V47L (see Japanese Patent Publication No. 2021-073914) as a template, the vector fragment to which the linker sequence of the mCherry fragment was attached was amplified using primers (sequence numbers 158 and 159). Using the genomic DNA of strain ATCC13032 as a template, the sequence to which the linker sequence of the mCherry fragment and the linker sequence of the vector fragment were attached to the promoter sequence of Gene ID: cg2875 (Pcg2875) shown in sequence number 1 was amplified using primers (sequence numbers 160 and 161). The vector fragment, mCherry fragment, and Pcg2875 fragment were ligated using the In-Fusion HD cloning kit (Clontech) to produce pECsf_Pcg2875_mCherry. The obtained plasmid solution was used to transform ECOS Competent E.Coli DH5α strain (Nippon Gene), and the resulting cell saturation was spread onto LB agar medium containing kanamycin and left to stand overnight at 37°C. Colony PCR was performed using the resulting colonies as templates with KOD One PCR Master Mix (TOYOBO) and primers (SEQ ID NOs. 162 and 163). Transformants in which the plasmid containing the target gene was confirmed were inoculated into 2 mL of LB liquid medium containing kanamycin and cultured overnight at 37°C. Plasmid purification was performed from the resulting culture medium using NucleoSpin Plasmid EasyPure (Takara Bio) to obtain pECsf_Pcg2875_mCherry.

[0068] 2) Creation of pECsf_tuD_mCherry Using the pmCherry Vector purchased from Takara Bio Inc. as a template, the reporter protein mCherry sequence was amplified using primers (SEQ ID NOs: 156 and 157). Using pECsf_gapS_pabABC_tuD_HFM122_V47L (see Japanese Patent Publication No. 2021-073914) as a template, the vector fragment to which the linker sequence of the mCherry fragment was attached was amplified using primers (SEQ ID NOs: 158 and 159). Using pECsf_gapS_pabABC_tuD_HFM122_V47L (see Japanese Patent Publication No. 2021-073914) as a template, the sequence to which the linker sequence of the mCherry fragment and the linker sequence of the vector fragment were attached to the tu promoter sequence of Corynebacterium glutamicum strain ATCC13032 was amplified using primers (SEQ ID NOs: 164 and 165). The vector fragment, mCherry fragment, and tuD fragment were ligated using the In-Fusion HD cloning kit (Clontech) to produce pECsf_tuD_mCherry. The obtained plasmid solution was used to transform ECOS Competent E.Coli DH5α strain (Nippon Gene), and the resulting cell saturation was spread onto LB agar medium containing kanamycin and left to stand overnight at 37°C. Colony PCR was performed using the resulting colonies as templates with KOD One PCR Master Mix (TOYOBO) and primers (SEQ ID NOs. 162 and 163). Transformants in which the plasmid containing the target gene was confirmed were inoculated into 2 mL of LB liquid medium containing kanamycin and cultured overnight at 37°C. Plasmid purification was performed from the resulting culture medium using NucleoSpin Plasmid EasyPure (Takara Bio) to obtain pECsf_tuD_mCherry.

[0069] 3) Creation of pECsf_SPL13_mCherry Using the pmCherry Vector purchased from Takara Bio Inc. as a template, the reporter protein mCherry sequence was amplified using primers (SEQ ID NOs: 156 and 157). Using pECsf_gapS_pabABC_tuD_HFM122_V47L (see Japanese Patent Publication No. 2021-073914) as a template, a vector fragment to which the linker sequence of the mCherry fragment was attached was amplified using primers (SEQ ID NOs: 158 and 159). The SPL13 promoter sequence disclosed in Patent Document 2 was chemically synthesized using Eurofins Genomics' artificial gene synthesis service. Using the obtained DNA containing the SPL13 promoter as a template, a sequence to which the linker sequence of the mCherry fragment and the linker sequence of the vector fragment were attached to the SPL13 promoter sequence was amplified using primers (SEQ ID NOs: 166 and 167). The vector fragment, mCherry fragment, and SPL13 promoter fragment were ligated using the In-Fusion HD cloning kit (Clontech) to produce pECsf_SPL13_mCherry. The obtained plasmid solution was used to transform ECOS Competent E.Coli DH5α strain (Nippon Gene), and the resulting cell saturation was spread onto LB agar medium containing kanamycin and left to stand overnight at 37°C. Colony PCR was performed using the resulting colonies as templates with KOD One PCR Master Mix (TOYOBO) and primers (SEQ ID NOs. 162 and 163). Transformants in which the plasmid containing the target gene was confirmed were inoculated into 2 mL of LB liquid medium containing kanamycin and cultured overnight at 37°C. Plasmid purification was performed from the resulting culture medium using NucleoSpin Plasmid EasyPure (Takara Bio) to obtain pECsf_SPL13_mCherry. Table 4 shows the primers used in steps 1) to 3) above.

[0070] [Table 4]

[0071] Example 5 Preparation of Transformed Body Using the plasmids obtained in Example 4, Corynebacterium glutamicum DRHG145 strain (see Japanese Patent Publication No. 6322576) was transformed by electroporation (Bio-rad). The resulting transformed cell solution was spread onto kanamycin-containing LB agar medium and left to stand at 30°C for 2 days. The resulting colonies were used as transformants.

[0072] Example 6 Comparison of promoter activity 1) Culture of transformed organisms The transformants obtained in Example 5 were inoculated into test tubes containing 10 mL of CGXII medium (containing 50 mg / L kanamycin sulfate) as shown in Table 2, and cultured with shaking at 30°C and 200 rpm for 2 days.

[0073] 2) Measurement of promoter activity The fluorescence intensity of the reporter protein mCherr was measured using a plate reader, Infinite M Plex (TECAN), at an excitation wavelength of 587 nm and an fluorescence wavelength of 610 nm. The experiment was performed with N=3.

[0074] Figure 3 shows the relative fluorescence intensity relative to the tu promoter. The fluorescence intensity was higher in the strain expressing mCherry with the cg2875 promoter than in the strains expressing mCherry with the tu promoter or the SPL13 promoter. Therefore, it was shown that the activity of the cg2875 promoter is higher than that of both the tu promoter and the SPL13 promoter.

[0075] Example 7: Reduction of the promoter region 1) Creation of a plasmid with the promoter region reduced to 80 bp. Inverse PCR was performed using pECsf_gapS_pabABC_Pcg2875_HFM122_V47L as a template and primers (SEQ ID NOs. 15 and 16) to produce a plasmid with the cg2875 promoter (Pcg2875) reduced to 80 bp (SEQ ID NOs. 20). The obtained plasmid solution was used to transform ECOS Competent E.Coli DH5α strain (Nippon Gene Co., Ltd.), and the resulting cell suspension was spread onto LB agar medium containing kanamycin and left to stand overnight at 37°C. Colony PCR was performed using the resulting colonies as templates with KOD One PCR Master Mix (TOYOBO Co., Ltd.) and primers (SEQ ID NOs. 24 and 25). Transformants in which the introduction of the plasmid with the reduced promoter region to 80 bp was confirmed were inoculated into 2 mL of LB liquid medium containing kanamycin and cultured overnight at 37°C. Plasmid EasyPure (Takara Bio) was used to purify the plasmid from the obtained culture medium, yielding pECsf_gapS_pabABC_Pcg2875_S1_HFM122_V47L.

[0076] 2) Creation of a plasmid with the promoter region reduced to 86 bp Plasmid pECsf_gapS_pabABC_Pcg2875_S2_HFM122_V47L was obtained in the same manner as in 1) above, except that primers SEQ ID NOs. 15 and 17 were used as primers for inverse PCR, by reducing the cg2875 promoter to 86 bp (SEQ ID NO. 21).

[0077] 3) Creation of a plasmid with the promoter region reduced to 96 bp Plasmid pECsf_gapS_pabABC_Pcg2875_S3_HFM122_V47L was obtained in the same manner as in 1) above, except that primers SEQ ID NOs. 15 and 18 were used as primers for inverse PCR, by reducing the cg2875 promoter to 96 bp (SEQ ID NO. 22).

[0078] 4) Creation of a plasmid with a reduced promoter region of 208 bp Plasmid pECsf_gapS_pabABC_Pcg2875_S4_HFM122_V47L was obtained in the same manner as in 1) above, except that primers SEQ ID NOs. 15 and 19 were used as primers for inverse PCR, by reducing the cg2875 promoter to 208 bp (SEQ ID NO. 23). Table 5 shows the primers used in steps 1) to 4) above, and Table 6 shows the promoter regions prepared in steps 1) to 4) above.

[0079] [Table 5]

[0080] [Table 6]

[0081] Example 8 Preparation of transformants Transformants were obtained in the same manner as in Example 2, except that the plasmids pECsf_gapS_pabABC_Pcg2875_S1_HFM122_V47L, pECsf_gapS_pabABC_Pcg2875_S2_HFM122_V47L, pECsf_gapS_pabABC_Pcg2875_S3_HFM122_V47L, and pECsf_gapS_pabABC_Pcg2875_S4_HFM122_V47L obtained in Example 7 were used.

[0082] Example 9 Comparison of promoter activity 1) Culture of transformed organisms The transformants were cultured in the same manner as in Example 3, 1), except that the transformants obtained in Examples 2 and 8 were used.

[0083] 2) Measurement of promoter activity The promoter activity of each obtained culture medium was measured in the same manner as in Example 3, part 2). The experiment was conducted with N=3. As shown in Figure 4, strains expressing HFM122_V47L with the cg2875_S4 promoter (a reduced cg2875 promoter to 208 bp) showed a higher AHBA conversion rate than strains expressing HFM122_V47L with the tu or SPL13 promoter. Therefore, it was shown that the activity of the cg2875 promoter is higher than that of the tu and SPL13 promoters when the promoter region is 208 bp long. Furthermore, strains expressing HFM122_V47L with the cg2875_S2 promoter (a reduced cg2875 promoter to 86 bp) showed an AHBA conversion rate equal to or better than that of strains expressing HFM122_V47L with the tu or SPL13 promoter. On the other hand, strains expressing HFM122_V47L with the cg2875_S1 promoter (a reduced cg2875 promoter to 80 bp) showed a lower AHBA conversion rate than strains expressing HFM122_V47L with the tu or SPL13 promoter. Therefore, it was shown that the activity of the cg2875 promoter is equivalent to or greater than that of the tu and SPL13 promoters if the promoter region is 86 bp long.

[0084] Example 10: Search for homologous promoters 1) BLAST search of the nucleotide sequence of the cg2875 promoter The nucleotide sequence of the 208bp cg2875_S4 promoter (SEQ ID NO: 23) was subjected to a BLAST search (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome). From the search results, alignment sequences with Query cover of 98% or more were extracted, yielding 42 nucleotide sequences (promoter sequences). In addition, 42 nucleotide sequences of genes downstream of alignment sequences with Query cover of 98% or more were extracted from each genome sequence.

[0085] 2) BLAST search of the amino acid sequence of cg2875 The amino acid sequence of cg2875 (SEQ ID NO: 3) was subjected to a BLAST search. From the search results, alignment sequences with 100% query cover were extracted, and the amino acid sequences of eight homologs were obtained. In addition, the upstream 208 bp nucleotide sequence (promoter sequence) of the gene encoding the amino acid sequences of the eight homologs was extracted from each genome sequence as the homolog promoter sequence.

[0086] 3) Extraction of homolog promoter sequences From the nucleotide sequence of the 208bp cg2875_S4 promoter, the nucleotide sequences of the 42 promoters in 1) above, and the nucleotide sequences of the 8 promoters in 2) above, a total of 51 nucleotide sequences, duplicate sequences were removed to obtain the nucleotide sequences of 16 homologous promoters (SEQ ID NOs. 23 and 26-41). The origin of each homologous promoter is shown in Table 7, and the amino acid sequences encoded by the genes downstream of each homologous promoter (SEQ ID NOs. 3 and 42-57) are shown in Table 8.

[0087] [Table 7]

[0088] [Table 8]

[0089] The amino acid sequence of the protein encoded by the gene downstream of each homologous promoter can be expressed by the following formula. MX1X2X3FX1IX4QX5IFX1GX6X1X1LX7X1SX8X1X1GAQX9VFX 10 X1X 11 X1X1X 12 SS (Here, X1 is any amino acid residue, X2 is S or A, X3 is V or I, X4 is F or I, X5 is S or A, X6 is V or I, X7 is V or I, X8 is V or I, X9 is G or N, X 10 is D or T, X11 is I or V, X 12 (is A or F).

[0090] 4) Preparation of plasmids for evaluating homologous promoter activity 1 The vector fragment was amplified using pECsf_gapS_pabABC_tuD_HFM122_V47L as a template with primers (SEQ ID NOs: 4 and 5). Meanwhile, the cgUSDA promoter sequence (PcgUSDA, SEQ ID NO: 28) was chemically synthesized using Eurofins Genomics' artificial gene synthesis service. Using the obtained DNA containing the cgUSDA promoter as a template, the sequence in which the linker sequence of the vector fragment was attached to the cgUSDA promoter sequence was amplified using primers (SEQ ID NOs: 58 and 59). The vector fragment and the cgUSDA promoter fragment were ligated using the In-Fusion HD cloning kit (Clontech). The resulting plasmid solution was used to transform ECOS Competent E.Coli DH5α strain (Nippon Gene), and the resulting cell suspension was spread on LB agar medium containing kanamycin and left to stand overnight at 37°C. Colony PCR was performed using the resulting colonies as a template with KOD One PCR Master Mix (TOYOBO) and primers (SEQ ID NOs: 60 and 58). Transformants in which the plasmid containing the target gene was confirmed were inoculated into 2 mL of LB liquid medium containing kanamycin and cultured overnight at 37°C. Plasmid purification was performed from the resulting culture medium using NucleoSpin Plasmid EasyPure (Takara Bio) to obtain pECsf_gapS_pabABC_PcgUSDA_HFM122_V47L.

[0091] cgR promoter (PcgR, SEQ ID NO: 29), cgB414 promoter (PcgB414, SEQ ID NO: 30), bfZL1 promoter (PbfZL1, SEQ ID NO: 31), cgScgG1 promoter (PcgScgG1, SEQ ID NO: 33), cgYI promoter (PcgYI, SEQ ID NO: 34), csN24 promoter (PcsN24, SEQ ID NO: 35), ccjz16 promoter (Pccjz16, SEQ ID NO: 37), cdgimn1 promoter (Pcdgimn1, SEQ ID NO: 37) For the promoters No. 38), cgCS176 promoter (PcgCS176, SEQ ID NO: 39), cgBCo promoter (PcgBCo, SEQ ID NO: 40), and cgKbcgl promoter (PcgKbcgl, SEQ ID NO: 41), the same procedure as for the cgUSDA promoter was used, except that the primers shown in Table 9 below were used for amplifying the sequence in which the linker sequence of the vector fragment was attached to the promoter sequence and for colony PCR. Plasmids with motors: pECsf_gapS_pabABC_PcgR_HFM122_V47L, pECsf_gapS_pabABC_PcgB414_HFM122_V47L, pECsf_gapS_pabABC_PbfZL1_HFM122_V47L, pECsf_gapS_pabABC_PcgScgG1_HFM122_V47L, pECsf_gapS_pabABC_PcgYI_HFM122_V47L, pECsf_gapS_pabABC _PcsN24_HFM122_V47L, pECsf_gapS_pabABC_Pccjz16_HFM122_V47L, pECsf_gapS_pabABC_Pcdgimn1_HFM122_V47L, pECsf_gapS_pabABC_PcgCS176_HFM122_V47L, pECsf_gapS_pabABC_PcgBCo_HFM122_V47L, and pECsf_gapS_pabABC_PcgKbcgl_HFM122_V47L were obtained.

[0092] [Table 9]

[0093] 5) Preparation of plasmids for evaluating homologous promoter activity 2 Inverse PCR was performed using pECsf_gapS_pabABC_Pcg2875_HFM122_V47L as a template with primers (SEQ ID NOs. 83 and 84, or 85 and 86) to construct plasmids in which the cgTQ2223 promoter sequence (PcgTQ2223, SEQ ID NO. 26) or the cgATCC21831 promoter sequence (PcgATCC21831, SEQ ID NO. 27) was ligated with HFM122_V47L. The obtained plasmid solution was used to transform ECOS Competent E.Coli DH5α strain (Nippon Gene Co., Ltd.), and the resulting cell suspension was spread onto LB agar medium containing kanamycin and left to stand overnight at 37°C. The resulting colonies were inoculated into 2 mL of LB liquid medium containing kanamycin and cultured overnight at 37°C. Plasmids were purified from the obtained culture medium using NucleoSpin Plasmid EasyPure (Takara Bio) to obtain pECsf_gapS_pabABC_PcgTQ2223_HFM122_V47L and pECsf_gapS_pabABC_PcgATCC21831_HFM122_V47L.

[0094] 6) Preparation of plasmids for evaluating homologous promoter activity 3 Inverse PCR was performed using pECsf_gapS_pabABC_PbfZL1_HFM122_V47L as a template with primers (SEQ ID NOs. 87 and 88) to produce a plasmid in which the cgmcc1 promoter sequence (Pcgmcc1, SEQ ID NO. 32) was ligated with HFM122_V47L. The obtained plasmid solution was used to transform ECOS Competent E.Coli DH5α strain (Nippon Gene Co., Ltd.), and the resulting cell suspension was spread on LB agar medium containing kanamycin and left to stand overnight at 37°C. The resulting colonies were inoculated into 2 mL of LB liquid medium containing kanamycin and cultured overnight at 37°C. Plasmid purification was performed from the obtained culture medium using NucleoSpin Plasmid EasyPure (Takara Bio) to obtain pECsf_gapS_pabABC_Pcgmcc1_HFM122_V47L.

[0095] 7) Preparation of plasmids for evaluating homologous promoter activity 4 Inverse PCR was performed using pECsf_gapS_pabABC_PcgYI_HFM122_V47L as a template with primers (SEQ ID NOs. 89 and 90) to construct a plasmid in which the cgYImut promoter sequence (PcgYImut, SEQ ID NOs. 91), in which the 138th A of the cgYI promoter sequence (PcgYI, SEQ ID NOs. 34) was substituted with G and the 139th T with C, was ligated with HFM122_V47L. The obtained plasmid solution was used to transform ECOS Competent E.Coli DH5α strain (Nippon Gene Co., Ltd.), and the resulting cell suspension was spread on LB agar medium containing kanamycin and left to stand overnight at 37°C. The resulting colonies were inoculated into 2 mL of LB liquid medium containing kanamycin and cultured overnight at 37°C. Plasmid was purified from the obtained culture medium using NucleoSpin Plasmid EasyPure (Takara Bio) to obtain pECsf_gapS_pabABC_PcgYImut_HFM122_V47L. Next, inverse PCR was performed using pECsf_gapS_pabABC_PcgYImut_HFM122_V47L as a template with primers (SEQ ID NOs. 92 and 93) to produce a plasmid in which the cgB253 promoter sequence (PcgB253, SEQ ID NO. 36) and HFM122_V47L were ligated. The obtained plasmid solution was used to transform ECOS Competent E.Coli DH5α strain, and the resulting cell suspension was spread on LB agar medium containing kanamycin and left to stand overnight at 37°C. The resulting colonies were inoculated into 2 mL of LB liquid medium containing kanamycin and cultured overnight at 37°C. The obtained culture medium was purified using NucleoSpin Plasmid EasyPure to obtain pECsf_gapS_pabABC_PcgB253_HFM122_V47L. Table 10 shows the primers used in steps 5) to 7) above.

[0096] [Table 10]

[0097] Example 11 Preparation of transformants Transformants were obtained in the same manner as in Example 2, except that a total of 16 plasmids obtained in Example 10 4) to 7) were used.

[0098] Example 12 Comparison of promoter activity 1) Culture of transformed organisms The transformants were cultured in the same manner as in Example 3, 1), except that the transformants obtained in Examples 2 and 11 were used.

[0099] 2) Measurement of promoter activity The promoter activity of each obtained culture medium was measured in the same manner as in Example 3, part 2). The experiment was conducted with N=3. As shown in Figure 5, all strains expressing HFM122_V47L with the homolog promoter had higher AHBA conversion rates than strains expressing HFM122_V47L with the tu or SPL13 promoter. Therefore, the activity of the homolog promoter was higher than that of the tu promoter and the SPL13 promoter.

[0100] Reference Example 1: Preparation of a transformant (KC341 strain) 1) Preparation of transformants in which the tu promoter is introduced into the cg2391(aroG) promoter region. i) Construction of a plasmid for introducing the TU promoter into the cg2391 promoter region The 5' upstream region of the cg2391 gene (SEQ ID NO: 94) was amplified using two types of DNA primers (SEQ ID NOs: 95 and 96), and the 5' region of the cg2391 gene ORF (SEQ ID NO: 97) was amplified using two types of DNA primers (SEQ ID NOs: 98 and 99) to obtain DNA fragments. In addition, a DNA fragment (SEQ ID NO: 100) containing the promoter of the tuf gene (cg0587) of Corynebacterium glutamicum ATCC13032 strain (hereinafter referred to as the tu promoter) was amplified using two types of DNA primers (SEQ ID NOs: 101 and 102) with the genome of the ATCC13032 strain as a template to obtain DNA fragments. Furthermore, pHKPsacB1 (see International Publication No. 2014 / 007273) was amplified using two types of DNA primers (SEQ ID NOs: 103 and 104), and the resulting PCR product was treated with DpnI (Takara Bio). The DNA fragments of the four obtained PCR products were purified using NucleoSpin Gel and PCR Clean-up (Takara Bio), and the plasmid pHKPsacB_Ptu-aroG was constructed by ligating them using the In-Fusion HD Cloning Kit (Clontech).

[0101] ii) Creation of a strain in which the TU promoter is introduced into the cg2391 promoter region. Using electroporation-based transformation, the plasmid pHKPsacB_Ptu-aroG described above was introduced into Corynebacterium glutamicum HT23 strain (see International Publication No. 2014 / 007273), and the KC265sr strain was obtained by selecting for kanamycin resistance. The KC265sr strain was analyzed by PCR using primers SEQ ID NOs. 95 and 105 (Sapphire Amp (Takara Bio)), and it was confirmed that the KC265sr strain is a once-crossover homologous recombinant in which plasmid pHKPsacB_Ptu-aroG was introduced into the promoter region of cg2391. The KC265sr strain was cultured for 24 hours in 1 mL of LB liquid medium (10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride), and a portion of the culture medium was streaked onto LB agar containing 20% ​​sucrose to obtain the KC265 strain. PCR using primers SEQ ID NOs. 105 and 106 (Sapphire Amp (Takara Bio)) confirmed that the KC265 strain is a double-crossover homologous recombinant with the tu promoter introduced into the cg2391 (aroG) promoter region, as expected.

[0102] 2) Preparation of transformants in which the tu promoter is introduced into the cg1835(aroE3) promoter region. i) Construction of a plasmid for introducing the TU promoter into the cg1835 promoter region The 5' upstream region of the cg1835 gene (SEQ ID NO: 107) was amplified using two types of DNA primers (SEQ ID NOs: 108 and 109), and the 5' region of the cg1835 gene ORF (SEQ ID NO: 110) was amplified using two types of DNA primers (SEQ ID NOs: 111 and 112) to obtain DNA fragments. In addition, a DNA fragment containing the tu promoter (SEQ ID NO: 100) was amplified using the genome of strain ATCC13032 as a template with two types of DNA primers (SEQ ID NOs: 101 and 102) to obtain DNA fragments. Furthermore, pHKPsacB1 was used as a template and amplified using two types of DNA primers (SEQ ID NOs: 103 and 104), and the resulting PCR product was treated with DpnI (Takara Bio). The DNA fragments of the four obtained PCR products were purified using NucleoSpin Gel and PCR Clean-up (Takara Bio), and the plasmid pHKPsacB_Ptu-aroE3 was constructed by ligating them using the In-Fusion HD Cloning Kit (Clontech).

[0103] ii) Creation of a strain in which the TU promoter is introduced into the CG1835 promoter region. Using electroporation for transformation, the plasmid pHKPsacB_Ptu-aroE3 described above was introduced into the KC265 strain obtained in 1), and the KC282sr strain was obtained by selecting for kanamycin resistance. The KC282sr strain was analyzed by PCR using primers SEQ ID NOs. 108 and 113 (Sapphire Amp (Takara Bio)), and it was confirmed that the KC282sr strain is a once-crossover homologous recombinant in which the plasmid pHKPsacB_Ptu-aroE3 was introduced into the promoter region of cg1835. The KC282sr strain was cultured for 24 hours in 1 mL of LB liquid medium (10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride), and a portion of the culture medium was streaked onto LB agar containing 20% ​​sucrose to obtain the KC282 strain. PCR using primers SEQ ID NOs. 113 and 114 (Sapphire Amp (Takara Bio)) confirmed that the KC282 strain is a double-crossover homologous recombinant with the tu promoter introduced into the cg1835 (aroE3) promoter region, as expected.

[0104] 3) Preparation of transformants in which the tu promoter is introduced into the cg1827(aroB) promoter region. i) Construction of a plasmid for introducing the TU promoter into the cg1827 promoter region The 5' upstream region of the cg1827 gene (SEQ ID NO: 115) was amplified using two types of DNA primers (SEQ ID NOs: 116 and 117), and the 5' region of the cg1827 gene ORF (SEQ ID NO: 118) was amplified using two types of DNA primers (SEQ ID NOs: 119 and 120) to obtain DNA fragments. In addition, a DNA fragment containing the tu promoter (SEQ ID NO: 100) was amplified using the genome of strain ATCC13032 as a template with two types of DNA primers (SEQ ID NOs: 101 and 102) to obtain DNA fragments. Furthermore, pHKPsacB1 was used as a template and amplified using two types of DNA primers (SEQ ID NOs: 103 and 104), and the resulting PCR product was treated with DpnI (Takara Bio). The DNA fragments of the four obtained PCR products were purified using NucleoSpin Gel and PCR Clean-up (Takara Bio), and the plasmid pHKPsacB_Ptu-aroB was constructed by ligating them using the In-Fusion HD Cloning Kit (Clontech).

[0105] ii) Creation of a strain in which the TU promoter is introduced into the CG1827 promoter region. Using electroporation for transformation, the plasmid pHKPsacB_Ptu-aroB described above was introduced into the KC282 strain obtained in 2), and the KC300sr strain was obtained by selecting for kanamycin resistance. The KC300sr strain was analyzed by PCR using primers SEQ ID NOs. 116 and 121 (Sapphire Amp (Takara Bio)), and it was confirmed that the KC300sr strain is a one-time crossover homologous recombinant in which the plasmid pHKPsacB_Ptu-aroB was introduced into the promoter region of cg1827. The KC300sr strain was cultured for 24 hours in 1 mL of LB liquid medium (10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride), and a portion of the culture medium was streaked onto LB agar medium containing 20% ​​sucrose to obtain the KC300 strain. PCR using primers SEQ ID NOs. 121 and 122 (Sapphire Amp (Takara Bio)) confirmed that the KC300 strain is a double-crossover homologous recombinant with the tu promoter introduced into the cg1827 (aroB) promoter region, as expected.

[0106] 4) Preparation of transformants in which the tu promoter is introduced into the cg0873(aroA) promoter region. i) Construction of a plasmid for introducing the TU promoter into the cg0873 promoter region The 5' upstream region of the cg0873 gene (SEQ ID NO: 123) was amplified using two types of DNA primers (SEQ ID NOs: 124 and 125), and the 5' region of the cg0873 gene ORF (SEQ ID NO: 126) was amplified using two types of DNA primers (SEQ ID NOs: 127 and 128) to obtain DNA fragments. In addition, a DNA fragment containing the tu promoter (SEQ ID NO: 100) was amplified using the genome of strain ATCC13032 as a template with two types of DNA primers (SEQ ID NOs: 101 and 102) to obtain DNA fragments. Furthermore, pHKPsacB1 was used as a template and amplified using two types of DNA primers (SEQ ID NOs: 103 and 104), and the resulting PCR product was treated with DpnI (Takara Bio). The DNA fragments of the four obtained PCR products were purified using NucleoSpin Gel and PCR Clean-up (Takara Bio), and the plasmid pHKPsacB_Ptu-aroA was constructed by ligating them using the In-Fusion HD Cloning Kit (Clontech).

[0107] ii) Creation of a strain in which the tu promoter is introduced into the cg0873 promoter region. Using electroporation for transformation, the plasmid pHKPsacB_Ptu-aroA described above was introduced into the KC300 strain obtained in 3), and the KC314sr strain was obtained by selecting for kanamycin resistance. The KC314sr strain was analyzed by PCR using primers SEQ ID NOs. 124 and 129 (Sapphire Amp (Takara Bio)), and it was confirmed that the KC314sr strain is a one-time crossover homologous recombinant in which the plasmid pHKPsacB_Ptu-aroA was introduced into the promoter region of cg0873. The KC314sr strain was cultured for 24 hours in 1 mL of LB liquid medium (10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride), and a portion of the culture medium was streaked onto LB agar containing 20% ​​sucrose to obtain the KC314 strain. PCR using primers SEQ ID NOs. 129 and 130 (Sapphire Amp (Takara Bio)) confirmed that the KC314 strain is a double-crossover homologous recombinant with the tu promoter introduced into the cg0873 (aroA) promoter region, as expected.

[0108] 5) Creation of a transformant in which the cg0503(qsuC) gene, conjugated with the tu promoter, is introduced into the cg1226(pobA) promoter region. i) Construction of a plasmid for introducing the cg0503 gene, linked to the tu promoter, into the cg1226 gene region. The 5' upstream region of the cg1226 gene (SEQ ID NO: 131) was amplified using two types of DNA primers (SEQ ID NOs: 132 and 133), and the 3' region of the cg1226 gene (SEQ ID NO: 134) was amplified using two types of DNA primers (SEQ ID NOs: 135 and 136) to obtain DNA fragments. Furthermore, a DNA fragment containing the tu promoter (SEQ ID NO: 100) was amplified using the genome of the ATCC13032 strain as a template, using two types of DNA primers (SEQ ID NOs: 101 and 102) to obtain DNA fragments. Additionally, a DNA fragment of the cg0503 gene (SEQ ID NO: 137) was amplified using the genome of the ATCC13032 strain as a template, using two types of DNA primers (SEQ ID NOs: 138 and 139) to obtain DNA fragments. Finally, pHKPsacB1 was used as a template for amplification using two types of DNA primers (SEQ ID NOs: 103 and 104), and the resulting PCR products were treated with DpnI (Takara Bio). For the five types of PCR products obtained, each DNA fragment was purified using NucleoSpin Gel and PCR Clean-up (Takara Bio), and the plasmid pHKPsacB_ΔpobA::Ptu-qsuC was constructed by ligating them using the In-Fusion HD Cloning Kit (Clontech).

[0109] ii) Creation of a strain in which the cg0503 gene, linked to the tu promoter, is introduced into the cg1226 gene region. Using electroporation for transformation, the plasmid pHKPsacB_ΔpobA::Ptu-qsuC described above was introduced into the KC314 strain obtained in 4), and the KC315sr strain was obtained by selecting for kanamycin resistance. The KC315sr strain was analyzed by PCR using primers SEQ ID NOs. 132 and 140 (Sapphire Amp (Takara Bio)), and it was confirmed that the KC315sr strain is a one-time crossover homologous recombinant in which the plasmid pHKPsacB_ΔpobA::Ptu-qsuC was introduced into the promoter region of cg1226. The KC315 strain was cultured for 24 hours in 1 mL of LB liquid medium (10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride), and a portion of the culture medium was streaked onto LB agar containing 20% ​​sucrose to obtain the KC315 strain. PCR using primers SEQ ID NOs. 140 and 141 (Sapphire Amp (Takara Bio)) confirmed that the KC315 strain is a double-crossover homologous recombinant in which the cg0503 (qsuC) gene, linked to the tu promoter, is introduced into the cg1226 (pobA) gene region, as expected.

[0110] 6) Creation of transformants into which an E. coli-derived aroG mutant gene, conjugated with a tu promoter, is introduced into the 3' downstream region of the cg0620 gene. i) Construction of a plasmid for introducing an E. coli-derived aroG mutant gene, conjugated with the tu promoter, into the 3' downstream region of the cg0620 gene. The ORF region and the 5' upstream region (SEQ ID NO: 142) of the cg0620 gene were amplified using two types of DNA primers (SEQ ID NO: 143 and 144), and the 3' downstream region (SEQ ID NO: 145) of the cg0620 gene was amplified using two types of DNA primers (SEQ ID NO: 146 and 147) to obtain DNA fragments. In addition, a DNA fragment containing the tu promoter (SEQ ID NO: 148) was synthesized by artificial gene synthesis and amplified using two types of DNA primers (SEQ ID NO: 149 and 150) to obtain DNA fragments. A DNA fragment containing the aroG mutant gene of E. coli (a mutation in which aspartic acid at position 146 is replaced with asparagine) was synthesized by artificial gene synthesis, and this was used as a template to amplify using two types of DNA primers (SEQ ID NO: 151 and 152) to obtain DNA fragments (SEQ ID NO: 153). Furthermore, pHKPsacB1 was used as a template and amplified using two types of DNA primers (SEQ ID NO: 103 and 104), and the resulting PCR product was treated with DpnI (Takara Bio). For the five types of PCR products obtained, each DNA fragment was purified using NucleoSpin Gel and PCR Clean-up (Takara Bio), and the plasmid pHKPsacB_cg0620_Ptu-aroG_D146N was constructed by ligating them using the In-Fusion HD Cloning Kit (Clontech).

[0111] ii) Creation of a strain by introducing an E. coli-derived aroG mutant gene, with the tu promoter linked to the 3' downstream region of the cg0620 gene. Using electroporation for transformation, the plasmid pHKPsacB_cg0620_Ptu-aroG_D146N described above was introduced into the KC315 strain obtained in 5), and the KC341sr strain was obtained by selecting for kanamycin resistance. The KC341sr strain was analyzed by PCR using primers SEQ ID NOs. 143 and 154 (Sapphire Amp (Takara Bio)), and it was confirmed that the KC341sr strain is a once-crossover homologous recombinant in which the plasmid pHKPsacB_cg0620_Ptu-aroG_D146N was introduced into the 3' downstream region of the cg0620 gene. The KC341sr strain was cultured for 24 hours in 1 mL of LB liquid medium (10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride), and a portion of the culture solution was streaked onto LB agar containing 20% ​​sucrose to obtain the KC341 strain. PCR using primers SEQ ID NOs. 154 and 155 (Sapphire Amp (Takara Bio)) confirmed that the KC341 strain is a double-crossover homologous recombinant in which, as expected, an E. coli-derived aroG mutant gene linked to the tu promoter is introduced into the 3' downstream region of the cg0620 gene. By following the above procedure, we obtained the Corynebacterium glutamicum KC341 strain. The primers used above are shown in Table 11.

[0112] [Table 11]

Claims

1. DNA having promoter activity, comprising a nucleotide sequence selected from the group consisting of (a) to (c) below and a nucleotide sequence selected from the group consisting of (d) to (f) below: (a) The upstream nucleotide sequence of the gene encoding a polypeptide consisting of the following amino acid sequence; (b) A nucleotide sequence having at least 90% identity with the upstream nucleotide sequence of a gene encoding a polypeptide consisting of the following amino acid sequence; and (c) A nucleotide sequence in which one or more nucleotides are deleted, substituted, added, or inserted into the upstream nucleotide sequence of a gene encoding a polypeptide consisting of the following amino acid sequence; MX 1 X 2 X 3 FX 1 IX 4 QX 5 IFX 1 GX 6 X 1 X 1 LX 7 X 1 SX 8 X 1 X 1 GAQX 9 VFX 10 X 1 X 11 X 1 X 1 X 12 SS (Here, X 1 is any amino acid residue, X 2 is S or A, X 3 is V or I, X 4 is F or I, X 5 is S or A, X 6 is V or I, X 7 is V or I, X 8 is V or I, X 9 is G or N, X 10 is D or T, X 11 is I or V, X 12 (is A or F) (d) Any nucleotide sequence of sequence numbers 1, 21-23, and 26-41; (e) a nucleotide sequence having at least 90% identity with any of the nucleotide sequences of SEQ ID NOs: 1, 23, and 26-41, or a nucleotide sequence having at least 95% identity with any of the nucleotide sequences of SEQ ID NOs: 21 and 22; and (f) A nucleotide sequence in which 1 to 20 nucleotides are deleted, substituted, added, or inserted into any of the nucleotide sequences of SEQ ID NOs: 1, 23, and 26-41, or a nucleotide sequence in which 1 to 5 nucleotides are deleted, substituted, added, or inserted into any of the nucleotide sequences of SEQ ID NOs: 21 and 22.

2. The DNA according to claim 1, wherein (e) is a nucleotide sequence having at least 95% identity with any of the nucleotide sequences of SEQ ID NOs: 1, 21-23, and 26-41, and (f) is a nucleotide sequence in which 1 to 10 nucleotides are deleted, substituted, added, or inserted from any of the nucleotide sequences of SEQ ID NOs: 1, 23, and 26-41, or a nucleotide sequence in which 1 to 5 nucleotides are deleted, substituted, added, or inserted from any of the nucleotide sequences of SEQ ID NOs: 21 and 22.

3. The DNA according to claim 1, comprising a nucleotide sequence selected from the group consisting of (d') to (f') below: (d') Nucleotide sequence of Sequence ID No. 23; (e') A nucleotide sequence having at least 90% identity with the nucleotide sequence of Sequence ID No. 23; and (f') A nucleotide sequence in which 1 to 20 nucleotides are deleted, substituted, added, or inserted into the nucleotide sequence of SEQ ID NO:

23.

4. The DNA according to claim 3, comprising a nucleotide sequence selected from the group consisting of (d) to (f) below: (d") Nucleotide sequence of SEQ ID NO: 21; (e") A nucleotide sequence having at least 95% identity with the nucleotide sequence of Sequence ID No. 21; and (f'') A nucleotide sequence in which 1 to 5 nucleotides are deleted, substituted, added, or inserted into the nucleotide sequence of SEQ ID NO:

21.

5. The DNA according to claim 1, having promoter activity equivalent to or greater than that of the SPL13 promoter of Sequence ID No.

12.

6. A promoter comprising DNA selected from the group consisting of (g) to (i) below, and DNA selected from the group consisting of (j) to (l) below: (g) DNA consisting of the upstream nucleotide sequence of a gene encoding a polypeptide consisting of the following amino acid sequence; (h) DNA having promoter activity, comprising a nucleotide sequence having at least 90% identity with the upstream nucleotide sequence of a gene encoding a polypeptide consisting of the following amino acid sequence; and (i) DNA having promoter activity, consisting of a nucleotide sequence in which one or more nucleotides are deleted, substituted, added, or inserted into the nucleotide sequence upstream of a gene encoding a polypeptide consisting of the following amino acid sequence; MX 1 X 2 X 3 FX 1 IX 4 QX 5 IFX 1 GX 6 X 1 X 1 LX 7 X 1 SX 8 X 1 X 1 GAQX 9 VFX 10 X 1 X 11 X 1 X 1 X 12 SS (Here, X 1 is any amino acid residue, X 2 is S or A, X 3 is V or I, X 4 is F or I, X 5 is S or A, X 6 is V or I, X 7 is V or I, X 8 is V or I, X 9 is G or N, X 10 is D or T, X 11 is I or V, X 12 (is A or F) (j) DNA consisting of any of the nucleotide sequences of SEQ ID NOs: 1, 21-23, and 26-41; (k) DNA comprising a nucleotide sequence having at least 90% identity with any of the nucleotide sequences of SEQ ID NOs: 1, 23, and 26-41, and possessing promoter activity, or DNA comprising a nucleotide sequence having at least 95% identity with any of the nucleotide sequences of SEQ ID NOs: 21 and 22, and possessing promoter activity; and (l) DNA having promoter activity, comprising a nucleotide sequence in which 1 to 20 nucleotides are deleted, substituted, added, or inserted into any of the nucleotide sequences of SEQ ID NOs: 1, 23, and 26-41, or DNA having promoter activity, comprising a nucleotide sequence in which 1 to 5 nucleotides are deleted, substituted, added, or inserted into any of the nucleotide sequences of SEQ ID NOs: 21 and 22.

7. The promoter according to claim 6, wherein (k) is a DNA having promoter activity and consisting of a nucleotide sequence having at least 95% identity with any of the nucleotide sequences of SEQ ID NOs: 1, 21-23, and 26-41, and (l) is a DNA having promoter activity and consisting of a nucleotide sequence having 1 to 10 nucleotides deleted, substituted, added, or inserted from any of the nucleotide sequences of SEQ ID NOs: 1, 23, and 26-41, or a nucleotide sequence having promoter activity, or a DNA having promoter activity and consisting of a nucleotide sequence having 1 to 5 nucleotides deleted, substituted, added, or inserted from any of the nucleotide sequences of SEQ ID NOs: 21 and 22.

8. The promoter according to claim 6, comprising DNA selected from the group consisting of (j') to (l') below: (j') DNA consisting of the nucleotide sequence of sequence number 23; (k') DNA having promoter activity, comprising a nucleotide sequence having at least 90% identity with the nucleotide sequence of Sequence ID No. 23; and (l') DNA having promoter activity, consisting of a nucleotide sequence in which 1 to 20 nucleotides are deleted, substituted, added, or inserted to the nucleotide sequence of SEQ ID NO:

23.

9. The promoter according to claim 8, comprising DNA selected from the group consisting of (j) to (l) below: (j") DNA consisting of the nucleotide sequence of Sequence ID No. 21; (k'') DNA having promoter activity, comprising a nucleotide sequence having at least 95% identity with the nucleotide sequence of Sequence ID No. 21; and (l") DNA having promoter activity, consisting of a nucleotide sequence in which 1 to 5 nucleotides are deleted, substituted, added, or inserted to the nucleotide sequence of SEQ ID NO:

21.

10. The promoter according to claim 6, wherein the DNA has promoter activity equivalent to or greater than that of the SPL13 promoter of Sequence ID No.

12.

11. An expression vector comprising DNA according to any one of claims 1 to 5 or a promoter according to any one of claims 6 to 10.

12. The expression vector according to claim 11, comprising a gene encoding a target substance or an enzyme involved in its synthesis, and the DNA or promoter ligated upstream of the gene.

13. A DNA expression cassette comprising the DNA according to any one of claims 1 to 5 or the promoter according to any one of claims 6 to 10.

14. The DNA expression cassette according to claim 13, comprising a gene encoding a target substance or an enzyme involved in its synthesis, and the DNA or promoter ligated upstream of the gene.

15. Corynebacterium comprising the expression vector according to claim 11.

16. Corynebacterium comprising the expression vector according to claim 12.

17. Corynebacterium comprising the DNA expression cassette according to claim 13.

18. Corynebacterium comprising the DNA expression cassette according to claim 14.

19. Corynebacterium into which the DNA according to any one of claims 1 to 5 or the promoter according to any one of claims 6 to 10 has been introduced.

20. Corynebacterium according to claim 19, wherein the DNA or promoter is introduced upstream of a gene encoding a target substance or an enzyme involved in its synthesis.

21. Corynebacterium glutamicum as described in claim 15.

22. Corynebacterium glutamicum as described in claim 16.

23. Corynebacterium glutamicum as described in claim 17.

24. Corynebacterium glutamicum as described in claim 18.

25. Corynebacterium glutamicum as described in claim 19.

26. Corynebacterium glutamicum as described in claim 20.

27. Culturing Corynebacterium according to claim 16; and A method for producing a target substance, comprising recovering the target substance from a culture obtained in the culture.

28. Culturing Corynebacterium according to claim 18; and A method for producing a target substance, comprising recovering the target substance from a culture obtained in the culture.

29. Culturing Corynebacterium according to claim 20; and A method for producing a target substance, comprising recovering the target substance from a culture obtained in the culture.

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