JP7706628B2 - Modified 5' untranslated region and method for producing target substance using same - Google Patents

Description

The present invention relates to a DNA molecule containing a modified 5' untranslated region or a promoter containing the same, and a method for producing a target substance using the same.

In the industrial production of substances using microorganisms, improving productivity is an important issue. Numerous studies have been conducted in the past on modifying expression control regions such as promoters to improve the expression of target genes.

Patent Document 1 describes that a modified promoter obtained by mutating a catabolite responsive element (cre)-like sequence derived from the alkaline cellulase gene of Bacillus sp. KSM-S237 strain (FERM BP-7875) and KSM-64 strain (FERM BP-2886) improves expression of a target gene. Patent Document 2 describes that a modified promoter obtained by inserting bases between positions 326 and 330 of the nucleotide sequence of the alkaline cellulase gene promoter region of the KSM-64 strain improves expression of a target gene. Patent Documents 3 and 4 describe that a 5' untranslated region (5'UTR) sequence obtained from the Bacillus subtilis aprE gene is operably linked to a heterologous gene to improve expression of the gene. Patent Document 5 describes that a modified mRNA processing/stabilizing sequence with a Shine-Dalgarno sequence added is linked downstream of the promoter region of a target gene and upstream of the ribosome binding region to improve the expression of the target gene. Patent Document 6 describes a method for increasing the production of a target substance by introducing a specific DNA sequence that forms a stem loop 7 nucleotides or more downstream from the transcription start site of a gene related to the synthesis of the target substance. Non-Patent Document 1 describes that the expression of sacB in Bacillus subtilis was controlled by introducing a Shine-Dalgarno-like sequence into the 5'UTR of the sacB repressor gene sacR, and that the expression increased or decreased depending on the number and position of introduction. Non-Patent Document 2 describes that the expression of a target gene in the genus Bacillus was improved by introducing multiple sequences containing a ribosome binding site (RBS) and a start codon into the 5'UTR region of the target gene.

JP 2011-103875 A JP 2000-078981 A Special table 2018-505686 publication Special Publication No. 2020-534821 International Publication No. 2008/140615 Special Publication No. 2009-518019

Lett Appl Microbiol, 2005, 41(2):221-226 Nucleic Acids Research, 2022, 50(20):11979-11990

Improved productivity in the production of target substances by microorganisms is desirable. The present invention relates to a DNA molecule containing a modified 5'UTR that promotes gene expression or a promoter containing the same, and a method for producing a target substance using the same.

The present inventors have found that by replacing at least a portion of the 5'UTR contained in the promoter of a target gene in a microorganism with a specific DNA fragment derived from the 5'UTR of an endoglucanase gene of the genus Bacillus, the expression of the target gene is significantly promoted.

Thus, in one embodiment, the present invention provides a DNA molecule comprising a modified promoter,
The modified promoter comprises a modified 5'UTR containing a heterologous polynucleotide;
The heterologous polynucleotide is a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 70 or a nucleotide sequence having at least 80% identity thereto;
A DNA molecule is provided.
In another embodiment, the present invention provides a transformant containing the DNA molecule.
In yet another embodiment, the present invention provides a method for producing a target substance, comprising culturing the transformant.
In yet another embodiment, the present invention provides a method for producing a modified promoter, comprising the steps of:
The method comprises modifying a 5'UTR contained in a parent promoter,
Modifying the 5'UTR includes replacing part or all of the 5'UTR with a heterologous polynucleotide or adding the heterologous polynucleotide to the 5'UTR;
The heterologous polynucleotide is a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 70 or a nucleotide sequence having at least 80% identity thereto;
The parent promoter is a promoter that comprises a 5'UTR that does not contain the heterologous polynucleotide.
A method is provided.

The present invention can significantly improve the productivity of target substances by microorganisms.

_PSP64 promoter containing the modified 5'UTR constructed in Example 2. The shaded area represents the region replaced with the parent sequence. The spoVG promoter containing the modified 5'UTR constructed in Example 3. The shaded area indicates the region replaced with the parent sequence. The _PSP64 promoter containing the modified 5'UTR and heterologous RBS constructed in Example 7. The shaded area represents the region replaced with the parent sequence, and the black bar represents the heterologous RBS. Method for constructing a prsA overexpression strain. Improved KP43 protease productivity by the _PSP64 promoter containing a modified 5′UTR. Improved KP43 protease productivity by the spoVG promoter containing a modified 5′UTR. Improved amylase productivity by the _PSP64 promoter containing a modified 5′UTR. Improved amylase productivity by the spoVG promoter containing a modified 5′UTR. Improved Cry5B productivity by the _PSP64 promoter containing a modified 5′UTR. Improved lipase productivity by the _PSP64 promoter containing a modified 5′UTR. Improved Cry5B productivity by the _PSP64 promoter containing a modified 5′UTR and additional RBS.

All patents, non-patent literature, and other publications cited herein are hereby incorporated by reference in their entirety.

In this specification, the identity of amino acid sequences and nucleotide sequences is calculated by the Lipman-Pearson method (Science, 1985, 227: 1435-1441). Specifically, it is calculated by performing an analysis using the Search homology program of the genetic information processing software GENETYX Ver. 12 (Nihon Server Co., Ltd.) with the unit size to compare (ktup) set to 2.

As used herein, "at least 80% identity" with respect to amino acid sequences and nucleotide sequences means identity of 80% or more, preferably 85% or more, more preferably 90% or more, even more preferably 95% or more, even more preferably 98% or more, and even more preferably 99% or more.

Unless otherwise defined herein, "one or several" when used in reference to the deletion, substitution, addition, or insertion of an amino acid residue or nucleotide in an amino acid sequence or nucleotide sequence may preferably mean 1 to 5, more preferably 1 to 4, even more preferably 1 to 3, and even more preferably 1 to 2. In this specification, "addition" of an amino acid residue or nucleotide includes the addition of an amino acid residue or nucleotide to one or both ends of a sequence.

In this specification, the "corresponding position" or "corresponding region" on an amino acid sequence or a nucleotide sequence can be determined by aligning a target sequence with a reference sequence (e.g., the amino acid sequence of SEQ ID NO: 3) to give maximum homology. Alignment of amino acid sequences or nucleotide sequences can be performed using known algorithms, and the procedures are known to those skilled in the art. For example, alignment can be performed using the Clustal W multiple alignment program (Thompson, J.D. et al., 1994, Nucleic Acids Res. 22: 4673-4680) with default settings. Clustal W can be used, for example, on the website of the DNA Data Bank of Japan (DDBJ [www.ddbj.nig.ac.jp/searches-j.html]) operated by the National Institute of Genetics. A position of the target sequence that is aligned to any position of the reference sequence by the above alignment is considered to be a "position corresponding to" that position. In addition, a region between corresponding positions or a region consisting of a corresponding motif is considered to be a corresponding region.

In this specification, "amino acid residue" refers to the 20 types of 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).

In this specification, a "promoter" is a DNA sequence that has the function of controlling the expression of a gene (a polynucleotide that codes for an expression product). In general, the "promoter" of a gene is located upstream of the open reading frame (ORF) of the gene and controls the expression of the ORF. A promoter may include regions such as a transcription start point and a 5' untranslated region (5'UTR), or it can be defined as a region that further includes enhancers and cis elements. Enhancers and cis elements are located upstream of the transcription start point, and improve the activity of the promoter by binding transcription factors and the like. Activation of a promoter promotes the transcription of a gene into mRNA. The 5'UTR is a region downstream of the transcription start point up to the start codon, and is itself transcribed into mRNA but not translated, and functions as a regulatory region when translating mRNA into protein. The 5'UTR of Bacillus bacteria includes a Shine-Dalgarno (SD) sequence, which is a ribosome binding site.

As used herein, "open reading frame (ORF)" refers to the region from the initiation codon to the termination codon. The ORF includes the coding sequence (CDS) of a gene.

As used herein, "expression control region" (or simply "control region") refers to a DNA sequence that has the function of controlling the transcription or translation of a gene, and includes the promoter, the 5'UTR that constitutes it, the transcription start site, the ribosome binding site and other sites and regions, enhancers and cis elements that improve the transcriptional activity of the promoter, and the 3' untranslated region (3'UTR).

As used herein, "upstream" and "downstream" as used with respect to a gene or a control region such as a promoter or 5'UTR refer to the 5' and 3' sides of the gene or region, respectively. Unless otherwise defined, the upstream and downstream of a gene are not limited to the upstream and downstream regions adjacent to the ORF of the gene, respectively. Furthermore, unless otherwise defined, the upstream and downstream of a promoter or 5'UTR are not limited to the upstream region adjacent to the 5' end of the region and the downstream region adjacent to the 3' end, respectively.

In this specification, "operably linked" between a control region and a gene means that the gene (ORF) and the control region are linked so that the gene can be expressed under the control of the control region. Also, in this specification, "operably linked" between each region contained in the control region (e.g., promoter and 5'UTR) means that each region is linked within the control region so as to enable control of the expression of the downstream gene (ORF). Therefore, basically, in this specification, a "gene" operably linked to a control region refers to an ORF. The procedure for "operably linked" between a gene and a control region, or each region in a control region, is well known to those skilled in the art.

In this specification, the term "expression cassette" refers to a polynucleotide construct for controlling the expression of a target gene contained therein. Typically, an expression cassette contains a target gene to be expressed and a control region for controlling the expression of the gene. The control region preferably contains a promoter, which is located upstream of the ORF of the target gene and operably linked thereto. For example, when the target gene encodes a polynucleotide encoding a preproprotein containing a signal peptide, the promoter is located upstream of the region encoding the signal peptide, operably linked to the polynucleotide encoding the preproprotein, and controls the expression of the preproprotein. The expression cassette may also contain the 3' untranslated region (3'UTR) of the target gene. Preferably, the expression cassette has a restriction enzyme recognition site at its end to enable the insertion of the expression cassette into a vector or genomic DNA. The expression cassette can be used for constructing an expression vector or introducing a foreign gene into genomic DNA. Preferably, the expression cassette of the present invention is a DNA construct.

As used herein, "heterologous" with respect to polynucleotides such as genes and control regions means that the two elements are not derived from the same polynucleotide strand. For example, when two polynucleotides are "heterologous" to one another, they are derived from separate polynucleotide strands, whether they are derived from genes of the same name or from genes of different names. Thus, when the 5'UTR of a promoter of a gene is replaced with a foreign DNA fragment, the 5'UTR and the DNA fragment are "heterologous" to one another, even if they are derived from genes of the same name, and the DNA fragment is heterologous to the promoter. Thus, "heterologous" with respect to polynucleotides encompasses the meaning that the two elements are not derived from the same gene. In this case, when the 5'UTR of a gene is replaced with a DNA fragment derived from a different gene, the 5'UTR and the DNA fragment are "heterologous" to one another.

In this specification, "bacteria of the genus Bacillus" refers to bacteria of the genus Bacillus in the family Bacillaceae. Examples of bacteria of the genus Bacillus include B. subtilis, B. cereus, B. thuringiensis, B. megaterium, B. amyloliquefaciens, B. pumilus, B. liqueniformis, B. licheniformis, and mutant strains thereof.

As used herein, the term "target substance" refers to any substance (e.g., peptide, protein, metabolic product, etc.) that is desired to be produced by a host cell. As used herein, the term "target gene" refers to a polynucleotide that codes for a target substance or a substance that promotes its production process (e.g., biosynthesis or extracellular transport). For example, target genes include genes that code for proteins or peptides that are target substances, genes that code for proteins involved in the biosynthesis of metabolic products that are target substances, and genes that code for proteins involved in the extracellular transport of target substances. Target genes may be heterologous genes that code for heterologous expression products, homologous genes introduced from the outside, genes that code for expression products that are inherent to the host cell, or genes that code for any other expression products (proteins, peptides, etc.).

Examples of substances encoded by the target gene include enzymes, antibodies, insecticidal proteins, hormones, cytokines, other physiologically active peptides, enzymes involved in the biosynthesis of metabolic products, and transporters involved in the excretion of metabolic products outside the cell, with enzymes and insecticidal proteins being preferred. Examples of the enzyme include oxidoreductase, transferase, hydrolase, lyase, isomerase, and synthetase. Preferred examples include cellulosic biomass decomposition enzymes such as cellulase and hemicellulase, exoglucanase, endoglucanase, β-glucosidase, laccase, lactase, esterase, pectate lyase, pectinase, peroxidase, phytase, pullulanase, PETase, protease, lipase, mannanase, arabinase, galactase, and amylase. More preferred are protease, lipase, and amylase. Examples of insecticidal proteins include Cry proteins produced by Bacillus thuringiensis, preferably Cry proteins classified as Cry4, Cry5, and Cry11, more preferably Cry5B, Cry4Aa, Cry4Ba, and Cry11Aa, and even more preferably Cry5B.

In this specification, the "expression" of a gene includes both the process of transcription from a gene to mRNA and the process of translation from mRNA to a protein, and is used differently depending on the context. Basically, when the expression level of a target gene is improved, the production level of a target substance increases. The expression level of a target gene can be measured by gene expression analysis or by quantifying the production level of a protein expressed from the target gene. For example, when comparing the production levels of the same target substance produced in different cells, the concentrations of the target substances produced may be compared, or if the target substance is an enzyme, the enzyme activity values may be compared. The enzyme activity values and substance concentrations are measured by methods well known to those skilled in the art. In this specification, "improvement" and "promotion" can be used interchangeably with respect to gene expression, and "increase" and "improvement" can be used interchangeably with respect to protein production.

Unless otherwise specified, the names of genes and proteins described in this specification follow the registration information in the Protein Data Bank (PDB) ([www.rcsb.org/]).

The present invention provides modified 5'UTRs, modified promoters containing the same, and their use in the production of target substances by microorganisms.

The modified 5'UTR provided by the present invention can be constructed by modifying the 5'UTR to be modified (hereinafter also referred to as the "parent 5'UTR") using a heterologous polynucleotide. More specifically, the modified 5'UTR of the present invention can be constructed by replacing a part or all of the parent 5'UTR with a heterologous polynucleotide, or by adding a heterologous polynucleotide to the parent 5'UTR. Therefore, the parent 5'UTR of the modified 5'UTR is a 5'UTR that does not contain the heterologous polynucleotide.

The heterologous polynucleotide used to modify the 5'UTR is a polynucleotide that is heterologous (i.e., derived from a different polynucleotide strand) to the parent 5'UTR. Preferably, the heterologous polynucleotide is a fragment derived from the 5'UTR of a Bacillus endoglucanase gene. An example of a Bacillus endoglucanase gene is the endoglucanase (e.g., alkaline cellulase) gene of Bacillus sp. strain KSM-S237 (FERM BP-7875).

The heterologous polynucleotide contains the Shine-Dalgarno (SD) sequence GGAGG, which functions as a ribosome binding site in a Bacillus gene. Preferably, the heterologous polynucleotide contains AGGAGG (SEQ ID NO: 1). More preferably, the heterologous polynucleotide contains AGGAGGTAATATG (SEQ ID NO: 2). Preferably, the heterologous polynucleotide has a length of at least 20 nt. The upper limit of the length of the heterologous polynucleotide is not particularly limited, but is preferably 100 nt or less.

Preferably, the heterologous polynucleotide comprises the nucleotide sequence of WAWWTTWAGGAGGTAATATG (SEQ ID NO: 70, where W is A or T), or a nucleotide sequence having at least 80% identity thereto. However, these heterologous polynucleotides comprise AGGAGG (SEQ ID NO: 1) at positions corresponding to positions 8 to 13 of the nucleotide sequence of SEQ ID NO: 70. Preferably, these heterologous polynucleotides comprise AGGAGGTAATATG (SEQ ID NO: 2) at positions corresponding to positions 8 to 20 of the nucleotide sequence of SEQ ID NO: 70. In one embodiment, the heterologous polynucleotide consists of the nucleotide sequence of SEQ ID NO: 70. In one embodiment, the heterologous polynucleotide has a sequence derived from the 5'UTR of a Bacillus endoglucanase gene adjacent to the 5' end of the nucleotide sequence of SEQ ID NO: 70. For example, the heterologous polynucleotide consists of a fragment (the 3' end consists of the nucleotide sequence of SEQ ID NO: 70) consisting of the 3' terminal region of a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 69 or a sequence having at least 80% identity thereto.

In one embodiment, the heterologous polynucleotide comprises the nucleotide sequence of SEQ ID NO: 3. The nucleotide sequence of SEQ ID NO: 3 is derived from the 5'UTR (SEQ ID NO: 69) of the alkaline cellulase gene of Bacillus sp. KSM-S237 (FERM BP-7875). In a preferred embodiment, the heterologous polynucleotide consists of the nucleotide sequence of SEQ ID NO: 3.

In another embodiment, the heterologous polynucleotide comprises a nucleotide sequence having at least 80% identity to the nucleotide sequence of SEQ ID NO:3. In a preferred embodiment, the heterologous polynucleotide consists of a nucleotide sequence having at least 80% identity to the nucleotide sequence of SEQ ID NO:3. In another preferred embodiment, the heterologous polynucleotide consists of a nucleotide sequence in which one or more nucleotides are deleted, substituted, added, or inserted relative to the nucleotide sequence of SEQ ID NO:3. However, these heterologous polynucleotides contain AGGAGG (SEQ ID NO:1) at positions corresponding to positions 8 to 13 of the nucleotide sequence of SEQ ID NO:3. Preferably, these heterologous polynucleotides contain AGGAGGTAATATG (SEQ ID NO:2) at positions corresponding to positions 8 to 20 of the nucleotide sequence of SEQ ID NO:3.

In another embodiment, the heterologous polynucleotide comprises the nucleotide sequence of SEQ ID NO: 4. The nucleotide sequence of SEQ ID NO: 4 is derived from the 5'UTR (SEQ ID NO: 69) of the alkaline cellulase gene of Bacillus sp. KSM-S237 (FERM BP-7875) and contains the nucleotide sequence of SEQ ID NO: 3. In a preferred embodiment, the heterologous polynucleotide consists of the nucleotide sequence of SEQ ID NO: 4. In another preferred embodiment, the heterologous polynucleotide is a fragment (21-93 nt) of a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 4, including the nucleotide sequence of SEQ ID NO: 3.

In another embodiment, the heterologous polynucleotide comprises a nucleotide sequence having at least 80% identity with the nucleotide sequence of SEQ ID NO: 4 or a fragment thereof. In a preferred embodiment, the heterologous polynucleotide consists of a nucleotide sequence having at least 80% identity with the nucleotide sequence of SEQ ID NO: 4. In another preferred embodiment, the heterologous polynucleotide consists of a nucleotide sequence in which one or several nucleotides are deleted, substituted, added, or inserted with respect to the nucleotide sequence of SEQ ID NO: 4 or a fragment thereof. However, these heterologous polynucleotides contain AGGAGG (SEQ ID NO: 1) at positions corresponding to positions 8 to 13 of the nucleotide sequence of SEQ ID NO: 3. Preferably, these heterologous polynucleotides contain AGGAGGTAATATG (SEQ ID NO: 2) at positions corresponding to positions 8 to 20 of the nucleotide sequence of SEQ ID NO: 3.

In another embodiment, the heterologous polynucleotide comprises the nucleotide sequence of SEQ ID NO:5. In a preferred embodiment, the heterologous polynucleotide consists of the nucleotide sequence of SEQ ID NO:5. In another embodiment, the heterologous polynucleotide comprises a nucleotide sequence having at least 80% identity with the nucleotide sequence of SEQ ID NO:5. In a preferred embodiment, the heterologous polynucleotide consists of a nucleotide sequence having at least 80% identity with the nucleotide sequence of SEQ ID NO:5. In another preferred embodiment, the heterologous polynucleotide consists of a nucleotide sequence in which one or several nucleotides are deleted, substituted, added, or inserted relative to the nucleotide sequence of SEQ ID NO:5. However, these heterologous polynucleotides comprise AGGAGG (SEQ ID NO:1) at positions corresponding to positions 8 to 13 of the nucleotide sequence of SEQ ID NO:5. Preferably, these heterologous polynucleotides comprise AGGAGGTAATATG (SEQ ID NO:2) at positions corresponding to positions 8 to 20 of the nucleotide sequence of SEQ ID NO:5.

In another embodiment, the heterologous polynucleotide comprises the nucleotide sequence of SEQ ID NO:6. In a preferred embodiment, the heterologous polynucleotide consists of the nucleotide sequence of SEQ ID NO:6. In another embodiment, the heterologous polynucleotide comprises a nucleotide sequence having at least 80% identity with the nucleotide sequence of SEQ ID NO:6. In a preferred embodiment, the heterologous polynucleotide consists of a nucleotide sequence having at least 80% identity with the nucleotide sequence of SEQ ID NO:6. In another preferred embodiment, the heterologous polynucleotide consists of a nucleotide sequence in which one or several nucleotides are deleted, substituted, added, or inserted relative to the nucleotide sequence of SEQ ID NO:6. However, these heterologous polynucleotides comprise AGGAGG (SEQ ID NO:1) at positions corresponding to positions 8 to 13 of the nucleotide sequence of SEQ ID NO:6. Preferably, these heterologous polynucleotides comprise AGGAGGTAATATG (SEQ ID NO:2) at positions corresponding to positions 8 to 20 of the nucleotide sequence of SEQ ID NO:6.

SEQ ID NO: 3: AATATTTAGGAGGTAATTG
SEQ ID NO: 4: GTTTTTTTAAAACTTTAACGAAAGCACTTTCGGTAATGCTTATGAATTTAGCTATTTGAT
TCAATTACTTTAAAAATATTTAGGAGGTAATATG
SEQ ID NO: 5: TAATTTTAGGAGGTAATATG
SEQ ID NO: 6: TAATTTAAGGAGGTAATATG

The heterologous polynucleotide can be prepared according to a genetic engineering technique known in the art. For example, the heterologous polynucleotide can be isolated from the genome of a Bacillus bacterium (e.g., KSM-S237) according to a known technique. Alternatively, a heterologous polynucleotide having a desired nucleotide sequence can be prepared by mutagenesis of the isolated polynucleotide according to a known technique. Methods for deleting, substituting, inserting or adding nucleotides to a nucleotide sequence are described, for example, in Dieffenbach et al. (Cold Spring Harbor Laboratory Press, New York, 581-621, 1995). Alternatively, the heterologous polynucleotide can be chemically synthesized. A commercially available DNA synthesis service can be used for chemically synthesizing the polynucleotide.

The parent 5'UTR to be modified with the heterologous polynucleotide is preferably a 5'UTR contained in a promoter of a Bacillus gene. The promoter is preferably a highly functional promoter that exerts high transcription-promoting activity in Bacillus bacteria. Examples of such promoters include genes encoding secreted proteins of Bacillus bacteria, such as the endoglucanase gene of Bacillus sp. KSM-64, the endoglucanase gene of Bacillus sp. KSM-S237, aprE, nprE and amyE of Bacillus subtilis, and the 5'UTR of Bacillus sp. KSM-S237. Examples of the promoter include a promoter of a gene selected from the group consisting of amyL and amyQ of Bacillus licheniformis; a promoter of the spoVG gene of Bacillus subtilis; a promoter of a ribosomal RNA gene and a ribosome-related gene, such as a promoter of a gene selected from the group consisting of rrnO, rrnE, rrnI, rrnJ, rrnB, rpsD, rpsJ, and rpoD; a promoter of a cry crystal protein gene, such as a cryIIIA gene, of B. thuringensis; a P43 promoter, SP82 promoter, or scr promoter of Bacillus genus, etc., provided that the parent 5'UTR does not have the sequence of SEQ ID NO: 70.

Preferably, the parent 5'UTR is derived from a promoter of a gene different from the gene from which the heterologous polynucleotide is derived. For example, when the heterologous polynucleotide comprises the nucleotide sequence of SEQ ID NO: 3 to 6, the parent 5'UTR is preferably not the 5'UTR of the alkaline cellulase gene of Bacillus sp. KSM-S237.

The parent 5'UTR may be present in the genome of the cell, in an isolated polynucleotide fragment (e.g., an isolated promoter-containing region fragment), or may be contained in a vector such as an expression vector. For example, a heterologous polynucleotide can be directly substituted or added to the parent 5'UTR in the genome of the cell by homologous recombination or the like. Alternatively, a modified 5'UTR can be constructed by substituting or adding a heterologous polynucleotide to the parent 5'UTR in a promoter fragment isolated from the genome or to the parent 5'UTR on an expression vector.

The substitution or addition of the heterologous polynucleotide to the parent 5'UTR can be performed according to a conventional method in the art. For example, a fragment containing the parent 5'UTR and a fragment containing the heterologous polynucleotide can be amplified by PCR or the like, and the resulting fragments can be linked by SOE (splicing by overlap extension)-PCR (Gene, 1989, 77(1): pp. 61-68) to construct a modified 5'UTR. Alternatively, the modified 5'UTR can be constructed by inverse PCR using a vector containing the heterologous polynucleotide as a template and a primer encoding the polynucleotide of the modified 5'UTR.

In a preferred embodiment, the resulting modified 5'UTR contains the heterologous polynucleotide at its 3' end. That is, the heterologous polynucleotide is added to the 3' end of the parent 5'UTR, or the 3' end of the parent 5'UTR is replaced with the heterologous polynucleotide. Preferably, the modified 5'UTR is constructed by replacing the 3' end of the parent 5'UTR with the heterologous polynucleotide, and one or more nucleotides at the 3' end of the parent 5'UTR are missing. The length of the 3' end of the parent 5'UTR replaced with the heterologous polynucleotide is not particularly limited, and may be, for example, 20 nt or more, and may be up to the entire parent 5'UTR replaced.

In a preferred embodiment, the modified 5'UTR is constructed by replacing at least 20 nucleotides (1-20 nucleotides from the 3' end) of the parent 5'UTR with the heterologous polynucleotide, and does not include at least 20 nucleotides of the 3' end of the parent 5'UTR.
In another preferred embodiment, the modified 5'UTR is constructed by replacing at least 26 nucleotides (1 to 26 nucleotides from the 3' end) of the parent 5'UTR with the heterologous polynucleotide and does not include at least 26 nucleotides of the 3' end of the parent 5'UTR.
In another preferred embodiment, the modified 5'UTR is constructed by replacing the region from the SD sequence (i.e., the sequence consisting of GGAGG) to the 3' end of the parent 5'UTR with the heterologous polynucleotide, and does not include the region from the SD sequence to the 3' end of the parent 5'UTR.
In another preferred embodiment, the modified 5'UTR is constructed by replacing the region from the sequence consisting of AGGAGG (SEQ ID NO: 1) to the 3' end in the parent 5'UTR with the heterologous polynucleotide, and does not include the region from the sequence consisting of SEQ ID NO: 1 to the 3' end in the parent 5'UTR.
In another preferred embodiment, the modified 5'UTR consists of the heterologous polynucleotide, i.e. the entire parental 5'UTR is replaced with the heterologous polynucleotide.

The modified 5'UTR obtained by the present invention does not need to contain the SD sequence of the parent 5'UTR, but may contain it. The length of the modified 5'UTR obtained by the present invention is not particularly limited as long as it can exert the effect of improving gene expression.

In a further embodiment of the present invention, the modified 5'UTR may further comprise at least one additional ribosome binding region (RBS) (i.e., a heterologous RBS). The heterologous RBS is located upstream of the SD sequence GGAGG contained in the modified 5'UTR. Preferably, the heterologous RBS is located upstream of the region consisting of AGGAGG (SEQ ID NO: 1) contained in the modified 5'UTR. More preferably, the heterologous RBS is located 8 nucleotides or more upstream of the region consisting of AGGAGG (SEQ ID NO: 1) contained in the modified 5'UTR. Even more preferably, the heterologous RBS is located upstream of the heterologous polynucleotide contained in the modified 5'UTR. When there are two or more heterologous RBSs, all of them are located upstream of the SD sequence contained in the modified 5'UTR, preferably upstream of the region consisting of SEQ ID NO: 1, more preferably 8 nucleotides or more upstream of the region consisting of SEQ ID NO: 1, and even more preferably upstream of the heterologous polynucleotide. When the modified 5'UTR contains two or more SD sequences, for example, a SD sequence from a parent 5'UTR and the heterologous polynucleotide, the heterologous RBS is positioned upstream of all of them.
As used herein, the term "positioned upstream of a certain region (or sequence)" refers to the RBS being positioned such that the 3'-end of the RBS is located upstream of the 5'-end of the region (or sequence). Additionally, as used herein, the term "positioned N nucleotides upstream of a certain region (or sequence)" refers to the RBS being positioned such that the 3'-end of the RBS is located N nucleotides upstream of the 5'-end of the region (or sequence).

In one embodiment, the modified 5'UTR does not contain an SD sequence derived from the parent 5'UTR, and the heterologous RBS is located 8 nucleotides or more upstream of the AGGAGG (SEQ ID NO: 1) region derived from the heterologous polynucleotide contained in the modified 5'UTR. When there are two or more heterologous RBSs, all of them are located 8 nucleotides or more upstream of the AGGAGG (SEQ ID NO: 1) region derived from the heterologous polynucleotide.

In another embodiment, the modified 5'UTR does not contain an SD sequence from the parent 5'UTR, and the heterologous RBS is located upstream of the heterologous polynucleotide contained in the modified 5'UTR. When there are two or more heterologous RBSs, all of them are located upstream of the heterologous polynucleotide.

The heterologous RBS is a polynucleotide comprising the nucleotide sequence AGGAGG (SEQ ID NO: 1). Examples of the heterologous RBS include a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1, a polynucleotide consisting of any one of the nucleotide sequences of SEQ ID NO: 7 to 11, and a polynucleotide consisting of a nucleotide sequence comprising any one of the nucleotide sequences of SEQ ID NO: 1 and 7 to 11 in which one or several nucleotides have been deleted, substituted, added or inserted, provided that the polynucleotide comprises AGGAGG (SEQ ID NO: 1).
SEQ ID NO: 7: AGGAGGGA
SEQ ID NO: 8: GAAAGGAGG
SEQ ID NO: 9: GAAAGGAGGGA
SEQ ID NO:10: CGAAAGGAGGGAT
SEQ ID NO: 11: CTTGAAAGGAGGGATGCCTAA

At least one of the heterologous RBSs is located 10 to 100 nucleotides downstream, preferably 10 to 60 nucleotides downstream, and more preferably 13 to 55 nucleotides downstream from the transcription start point upstream of the modified 5'UTR (or the transcription start point of a modified promoter containing the modified 5'UTR, which will be described later). When there are two or more heterologous RBSs, the insertion positions of the remaining RBSs are not particularly limited, but are preferably located 10 to 150 nucleotides downstream, more preferably 10 to 100 nucleotides downstream, even more preferably 10 to 60 nucleotides downstream, and even more preferably 13 to 55 nucleotides downstream from the transcription start point. In this specification, "the RBS is located N nucleotides downstream from the transcription start point" means that the 5' end of the RBS is located N nucleotides downstream from the 3' end of the transcription start point.

The modified 5'UTR containing the heterologous RBS can be constructed by substituting, inserting or adding a polynucleotide of the heterologous RBS to the modified 5'UTR containing the heterologous polynucleotide. For example, the polynucleotide of the heterologous RBS may be substituted, inserted or added upstream of the SD sequence or heterologous polynucleotide region contained in the modified 5'UTR. Alternatively, the modified 5'UTR containing the heterologous RBS can be constructed by substituting, inserting or adding a polynucleotide of the heterologous RBS to the parent 5'UTR, and then substituting or adding the heterologous polynucleotide downstream of the polynucleotide of the parent 5'UTR. Alternatively, the modified 5'UTR containing the heterologous RBS can be constructed by substituting a part or all of the parent 5'UTR with the polynucleotide of the heterologous RBS and a fragment containing the heterologous polynucleotide, starting from the upstream. The procedure of substituting, inserting or adding a polynucleotide of the heterologous RBS to the parent 5'UTR or modified 5'UTR can be performed according to a known method such as the above-mentioned SOE-PCR or inverse PCR.

The modified 5'UTR obtained by the present invention acts to improve the expression of a gene controlled by a promoter containing the modified 5'UTR. Specifically, the modified 5'UTR is positioned downstream of the transcription start point in the promoter and upstream of the ORF of the gene, and is operably linked to the ORF of the gene.

In a preferred embodiment, the present invention produces a modified promoter comprising the modified 5'UTR. The modified promoter is produced by modifying the 5'UTR contained in a promoter (parent promoter) comprising a parent 5'UTR not comprising the heterologous polynucleotide. The modification of the 5'UTR is carried out by replacing a part or all of the 5'UTR with a heterologous polynucleotide, or by adding the heterologous polynucleotide to the 5'UTR. The modification of the 5'UTR may further include the substitution, insertion or addition of a heterologous RBS to the 5'UTR. The heterologous polynucleotide and heterologous RBS used in the modification, and the method of substitution, insertion or addition of a heterologous polynucleotide or a heterologous RBS to the 5'UTR are as described above. Thus, the heterologous polynucleotide and heterologous RBS contained in the modified promoter are heterologous (derived from a different polynucleotide chain) to the parent promoter. Preferably, the heterologous polynucleotide contained in the modified promoter is derived from a gene different from that of the parent promoter.

The parent promoter to be modified may be a promoter derived from the control region of any gene, but is preferably a promoter of a Bacillus gene. The parent promoter is preferably a highly functional promoter capable of exerting high transcription-promoting activity in Bacillus bacteria. Preferred examples of the parent promoter include genes encoding secreted proteins of Bacillus bacteria, such as the endoglucanase gene of Bacillus sp. KSM-64, the endoglucanase gene of Bacillus sp. KSM-S237, aprE, nprE, and amyE of Bacillus subtilis, and the B. licheniformis amyL and amyQ gene promoter; Bacillus subtilis spoVG gene promoter; ribosomal RNA genes and ribosome-related genes, such as rrnO, rrnE, rrnI, rrnJ, rrnB, rpsD, rpsJ and rpoD gene promoter; B. thuringensis cry crystal protein genes, such as cryIIIA gene promoter; P43 promoter, SP82 promoter, scr promoter, and modified promoters derived therefrom. Preferred examples include the promoter derived from the alkaline cellulase gene of Bacillus sp. KSM-64 shown in SEQ ID NO: 72, and the promoter of the Bacillus subtilis spoVG gene shown in SEQ ID NO: 76. Other preferred examples of parent promoters include a polynucleotide having a nucleotide sequence with at least 80% identity to the nucleotide sequence shown in SEQ ID NO: 72 and having the function of controlling gene expression, or a polynucleotide having a nucleotide sequence with at least 80% identity to the nucleotide sequence shown in SEQ ID NO: 76 and having the function of controlling gene expression. The function of the promoter polynucleotide to control gene expression can be measured by known methods.

The parent promoter may be present in the genome of a cell, may be an isolated polynucleotide fragment, or may be contained in a vector, such as an expression vector.
For example, a modified promoter fragment can be constructed by preparing a polynucleotide fragment of a parent promoter from a Bacillus bacterium, and replacing a part or all of the 5'UTR contained in the parent promoter fragment with a heterologous polynucleotide or adding a heterologous polynucleotide to the 5'UTR. Alternatively, a modified promoter can be constructed in the genome of a Bacillus bacterium by modifying the 5'UTR contained in a parent promoter in the genome of the Bacillus bacterium so that a part or all of the 5'UTR is replaced with a heterologous polynucleotide or a heterologous polynucleotide is added to the 5'UTR by homologous recombination or the like. Alternatively, a modified promoter can be constructed in the genome of a Bacillus bacterium by replacing a promoter in the genome of the Bacillus bacterium with a modified promoter containing the modified 5'UTR by homologous recombination or the like.

When the modified 5'UTR is placed in a promoter, it acts to improve gene expression. The modified promoter containing the modified 5'UTR can improve gene expression more than the parent promoter containing the parent 5'UTR before modification (i.e., the promoter before being modified to contain the modified 5'UTR). Preferably, the modified promoter containing the modified 5'UTR increases the expression level of the gene by at least 10%, preferably at least 20%, more preferably at least 25%, and even more preferably at least 30% compared to the parent promoter. When the modified 5'UTR contains the heterologous RBS described above, the modified promoter can further improve the expression level of the gene, preferably by at least 40%, more preferably at least 50%, even more preferably at least 100%, even more preferably at least 200%, even more preferably at least 300%, and even more preferably at least 400% compared to the parent promoter.

The modified 5'UTR and the modified promoter containing the same can be used to improve expression of a gene of interest. Thus, the present invention provides a polynucleotide containing the modified 5'UTR or a modified promoter containing the same, as well as the use thereof for expressing a gene of interest. Preferably, the polynucleotide is DNA.

In one embodiment, the polynucleotide comprising the modified 5'UTR or modified promoter can be directly introduced into the genome of a host cell. For example, the polynucleotide comprising the modified 5'UTR or modified promoter can be introduced into the genome of a host cell and operably linked to the gene of interest.

In one embodiment, the polynucleotide containing the modified 5'UTR or modified promoter is an expression cassette. In addition to the modified 5'UTR or modified promoter, the expression cassette may contain a cis element that improves the transcription activity of the promoter, a 3'UTR, or the like. Furthermore, the expression cassette may contain a selection marker gene such as a drug resistance gene or an auxotrophic marker gene.

In one embodiment, the polynucleotide comprising the modified 5'UTR or modified promoter further comprises a gene of interest. The gene of interest is operably linked to the modified 5'UTR or modified promoter.

In one embodiment, the expression cassette is an expression vector. For example, the expression vector can be prepared by inserting a polynucleotide containing the modified 5'UTR or modified promoter into any vector by a conventional method. For example, the polynucleotide is constructed to have restriction enzyme recognition sequences at both ends. The expression vector of the present invention can be constructed by incorporating this into an expression vector that has been cleaved with a restriction enzyme (restriction enzyme method). Preferably, in the expression vector, the modified 5'UTR or modified promoter is operably linked upstream of the polynucleotide of the target gene.

The type of the vector is not particularly limited, and may be any vector, such as a plasmid, a phage, a phagemid, a cosmid, a virus, a YAC vector, or a shuttle vector. The vector may be a vector for introduction into the genome of a host cell, or a vector that is maintained outside the genome. The vector is preferably a vector that can be amplified in bacteria, more preferably in Bacillus bacteria (e.g., Bacillus subtilis or a mutant thereof). Preferably, the vector is an expression vector that can induce expression of an introduced gene in Bacillus bacteria.

Examples of the vector include pBluescript II SK(-) (Stratagene), pUC18/19, pUC118/119 and other pUC vectors (Takara Bio), pET vectors (Takara Bio), pGEX vectors (GE Healthcare), pCold vectors (Takara Bio), pHY300PLK (Takara Bio), pUB110 (Plasmid, 1986, 15(2):93-103), pBR322 (Takara Bio), and pRS 403 (Stratagene), pMW series vectors such as pMW118/119 and pMW218/219 (Nippon Gene), pRI series vectors such as pRI909/910 (Takara Bio), pBI series vectors (Clontech), IN3 series vectors (Implanta Innovations), pPTR1/2 (Takara Bio), pDJB2 (Gene, 1985, 36: 321-331), pAB4-1 (Mol Gen Genet, 1987, 206: 71-75), pLeu4 (Gene, 1989, 84: 335-343), pPyr225 (Mol Genet Genomics, 2002, 268: 397-406), pFG1 (Curr Genet, 1990, 18: 447-451), yeast expression vector pNAN8142 (Biosci Biotechnol Biochem, 1996, 60: 383-389), pMA91 (Biosci Biotechnol Biochem, 1998, 62: 1615-1618), etc.

The present invention also provides a transformant containing a polynucleotide including the modified 5'UTR or modified promoter. The transformant can be produced by introducing a polynucleotide including the modified 5'UTR or modified promoter into a host.

Examples of hosts for the transformant include microbial cells, preferably bacteria of the genus Bacillus, bacteria of the genus Clostridium, yeast, Escherichia coli, etc., of which bacteria of the genus Bacillus are preferred, and Bacillus subtilis or a mutant thereof is more preferred. Therefore, the transformant of the present invention is preferably a recombinant Bacillus, and more preferably a recombinant of Bacillus subtilis or a mutant thereof.

To introduce a polynucleotide containing the modified 5'UTR or modified promoter into a host cell, well-known transformation techniques such as the calcium phosphate method, electroporation, lipofection, particle gun method, and PEG method can be applied. For example, methods that can be applied to Bacillus subtilis or its mutants include the competent cell transformation method (J Bacteriol, 1967, 93: 1925-1937), the electroporation method (FEMS Microbiol Lett, 1990, 55: 135-138), the protoplast transformation method (Mol Gen Genet, 1979, 168: 111-115), and the Tris-PEG method (J Bacteriol, 1983, 156: 1130-1134).

The transformant can be used to express a gene of interest. In the cells of the transformant, the modified 5'UTR or modified promoter improves the expression of the gene of interest operably linked thereto, thereby improving the productivity of the target substance by the transformant. For example, a transformant cell containing a modified promoter including the modified 5'UTR and an expression cassette including a gene of interest increases the expression level of the gene of interest by preferably at least 10%, more preferably at least 20%, and even more preferably at least 30%, when the expression level of the gene of interest in a cell containing an expression cassette including a parent promoter and a gene of interest is taken as 100%. Furthermore, when the modified 5'UTR contains the heterologous RBS described above, the expression level of the gene of interest can be further improved. For example, a transformed cell containing an expression cassette including a modified promoter containing a modified 5'UTR including a heterologous RBS and a gene of interest increases the expression level of the gene of interest by preferably at least 50%, more preferably at least 100%, even more preferably at least 200%, even more preferably at least 300%, and even more preferably at least 400%, when the expression level of the gene of interest in a cell containing an expression cassette including a parent promoter and a gene of interest is taken as 100%.

Therefore, the present invention also provides a method for producing a target substance, which comprises culturing the transformant. The transformant can be cultured according to a general method in the field. For example, when the transformant is Bacillus subtilis or a mutant thereof, the medium for culturing the transformant may contain a carbon source and an inorganic or organic nitrogen source necessary for the growth of Bacillus subtilis. If necessary, the medium may contain other nutrients, such as inorganic salts, vitamins, antibiotics, etc. The culture conditions, such as temperature, aeration and stirring conditions, pH of the medium, and culture time, may be appropriately selected depending on the species and characteristics of the microorganism, the culture scale, etc.

After the culture, the target substance can be recovered 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 performed according to a known recovery or purification method. For example, the culture may be recovered, and if necessary, cell disruption treatment may be performed using ultrasound or pressure, and then cell components may be removed by decantation, filtration, centrifugation, or the like, and the remaining fraction containing the target substance may be recovered. Alternatively, the target substance can be secreted outside the cells by operably linking a polynucleotide encoding a secretory signal peptide that functions in the transformant to a gene encoding the target substance. In this case, the fraction containing the target substance can be recovered without disrupting the cells.

If necessary, the target substance can be purified by subjecting the recovered fraction containing the target substance to dialysis, salting out, ion exchange, distillation, solvent extraction, or a combination of these methods. In the method for producing a target substance according to the present invention, the culture of the transformant and the recovery of the target substance may be carried out by any of a batch method, a semi-batch method, and a continuous method.

As exemplary embodiments of the present invention, the following substances, manufacturing methods, uses, methods, etc. are further disclosed in this specification. However, the present invention is not limited to these embodiments.

[1] A DNA molecule comprising a modified promoter,
The modified promoter comprises a modified 5'UTR containing a heterologous polynucleotide;
The heterologous polynucleotide is a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 70 or a nucleotide sequence having at least 80% identity thereto;
DNA molecule.
[2] Preferably, the heterologous polynucleotide is any one of the following polynucleotides a), c), and d):
a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:3, or a nucleotide sequence having at least 80% identity thereto;
c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:5, or a nucleotide sequence having at least 80% identity thereto;
d) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:6 or a nucleotide sequence having at least 80% identity thereto;
That is,
The DNA molecule described in [1].
[3] Preferably, the polynucleotide of a) is any one of the following:
b) a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 4 or a nucleotide sequence having at least 80% identity thereto, or a fragment thereof;
The DNA molecule according to [2],
[4] The heterologous polynucleotide is
Preferably, the nucleotide sequence of SEQ ID NO: 1 is contained at positions corresponding to positions 8 to 13 of the nucleotide sequence of SEQ ID NO: 70.
More preferably, the nucleotide sequence of SEQ ID NO: 1 is contained at positions corresponding to positions 8 to 13 of the nucleotide sequence of SEQ ID NO: 3 or positions corresponding to positions 8 to 13 of the nucleotide sequence of SEQ ID NO: 5 or 6.
or,
Preferably, the nucleotide sequence of SEQ ID NO: 2 is contained at positions corresponding to positions 8 to 20 of the nucleotide sequence of SEQ ID NO: 70.
More preferably, the nucleotide sequence of SEQ ID NO: 2 is contained at positions corresponding to positions 8 to 20 of the nucleotide sequence of SEQ ID NO: 3 or at positions corresponding to positions 8 to 20 of the nucleotide sequence of SEQ ID NO: 5 or 6.
The DNA molecule according to any one of [1] to [3].
[5] The DNA molecule according to any one of [1] to [4], wherein the heterologous polynucleotide is preferably 100 nt or less in length.
[6] Preferably, the modified 5'UTR is a DNA sequence in which a part or all of the 5'UTR before modification is replaced with the heterologous polynucleotide, or a DNA sequence in which the heterologous polynucleotide is added to the 5'UTR before modification.
The DNA molecule according to any one of [1] to [5].
[7] The DNA molecule according to any one of [1] to [6], wherein the heterologous polynucleotide is preferably located at the 3' end of the modified 5'UTR.
[8] Preferably, the modified 5'UTR is:
does not contain at least 20 nucleotides at the 3' end of the 5'UTR before modification,
does not contain at least the 3'-terminal 26 nucleotides of the 3'-end of the 5'UTR before modification;
Or,
does not include the region from the Shine-Dalgarno (SD) sequence of the 5'UTR before modification to the 3'end;
[7] The DNA molecule described in [7].
[9] The DNA molecule according to any one of [1] to [8], wherein the modified 5'UTR preferably contains, in addition to the heterologous polynucleotide, a heterologous ribosome binding region, the ribosome binding region being positioned upstream of a region consisting of the nucleotide sequence of SEQ ID NO: 1 contained in the modified 5'UTR.
[10] The DNA molecule according to [9], wherein the heterologous ribosome binding region is preferably located upstream of the heterologous polynucleotide.
[11] Preferably,
the modified 5'UTR contains one heterologous ribosome binding region, the heterologous ribosome binding region being located 10-100 nucleotides downstream from the transcription start point of the modified promoter; or the modified 5'UTR contains two or more heterologous ribosome binding regions, at least one of which is located 10-100 nucleotides downstream from the transcription start point of the modified promoter and the rest are located 10-150 nucleotides downstream from the transcription start point.
The DNA molecule according to [9] or [10].
[12] The DNA molecule according to any one of [1] to [11], wherein the parent promoter of the modified promoter is preferably a promoter derived from the genus Bacillus.
[13] The DNA molecule described in [12], wherein the parent promoter is preferably a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 72 or a nucleotide sequence having at least 80% identity to said sequence, or a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 76 or a nucleotide sequence having at least 80% identity to said sequence.
[14] The DNA molecule according to any one of [1] to [13], wherein the modified promoter preferably consists of the nucleotide sequence of SEQ ID NO: 73, 74, 75, 77, 78, 79, and 80.
[15] Preferably, the vector further comprises a gene of interest,
The gene of interest is a polynucleotide encoding a substance of interest or a substance involved in its biosynthesis or extracellular transport.
The DNA molecule according to any one of [1] to [14].
[16] The target substance,
Preferably, it is an enzyme or an insecticidal protein, more preferably, it is a protease, a lipase, an amylase or Cry5B.
[15] The DNA molecule described in.
[17] The DNA molecule according to [15] or [16], which is preferably an expression cassette or an expression vector.
[18] A transformant comprising the DNA molecule according to any one of [1] to [17].
[19] The transformant contains the DNA molecule according to [17], which is an expression cassette containing the modified promoter and further containing a gene of interest, and
When the expression level of the target gene in a cell containing an expression cassette including the parent promoter of the modified promoter and further including the target gene is taken as 100%,
The transformant increases the expression level of the target gene by preferably at least 10%, more preferably at least 20%, even more preferably at least 30%, even more preferably at least 50%, even more preferably at least 100%, even more preferably at least 200%, even more preferably at least 300%, even more preferably at least 400%.
[18] The transformant described in [18].
[20] The transformant according to [18] or [19], which is preferably a bacterium of the genus Bacillus.
[21] A method for producing a target substance, comprising culturing the transformant according to any one of [18] to [20].

[22] A method for producing a modified promoter, comprising the steps of:
The method comprises modifying a 5'UTR contained in a parent promoter,
Modifying the 5'UTR includes replacing part or all of the 5'UTR with a heterologous polynucleotide or adding the heterologous polynucleotide to the 5'UTR;
The heterologous polynucleotide is a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 70 or a nucleotide sequence having at least 80% identity thereto;
The parent promoter is a promoter that comprises a 5'UTR that does not contain the heterologous polynucleotide.
method.
[23] Preferably, the heterologous polynucleotide is any one of the following polynucleotides a), c), and d):
a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:3, or a nucleotide sequence having at least 80% identity thereto;
c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:5 or a nucleotide sequence having at least 80% identity thereto;
d) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:6 or a nucleotide sequence having at least 80% identity thereto;
That is,
[22] The method described in.
[24] Preferably, the polynucleotide of a) is any of the following:
b) a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 4 or a nucleotide sequence having at least 80% identity thereto, or a fragment thereof;
The method according to [23], comprising:
[25] The heterologous polynucleotide is
Preferably, the nucleotide sequence of SEQ ID NO: 1 is contained at positions corresponding to positions 8 to 13 of the nucleotide sequence of SEQ ID NO: 70.
More preferably, the nucleotide sequence of SEQ ID NO: 1 is contained at positions corresponding to positions 8 to 13 of the nucleotide sequence of SEQ ID NO: 3 or positions corresponding to positions 8 to 13 of the nucleotide sequence of SEQ ID NO: 5 or 6.
or,
Preferably, the nucleotide sequence of SEQ ID NO: 2 is contained at positions corresponding to positions 8 to 20 of the nucleotide sequence of SEQ ID NO: 70.
More preferably, the nucleotide sequence of SEQ ID NO: 2 is contained at positions corresponding to positions 8 to 20 of the nucleotide sequence of SEQ ID NO: 3 or at positions corresponding to positions 8 to 20 of the nucleotide sequence of SEQ ID NO: 5 or 6.
The method according to any one of [22] to [24].
[26] The method according to any one of [22] to [25], wherein the heterologous polynucleotide is preferably 100 nt or less in length.
[27] Preferably, the modification of the 5'UTR comprises substituting or adding the heterologous polynucleotide to the 5'UTR so that it is located at the 3' end of the modified 5'UTR. The method according to any one of [22] to [26].
[28] Preferably, the modification of the 5'UTR is
replacing at least 20 nucleotides at the 3' end of the 5'UTR with the heterologous polynucleotide;
or replacing at least 26 nucleotides at the 3' end of the 5'UTR with the heterologous polynucleotide;
The region from the Shine-Dalgarno (SD) sequence to the 3' end in the 5'UTR is replaced with the heterologous polynucleotide.
The method described in [27].
[29] Preferably, the modification of the 5'UTR further comprises substituting, inserting or adding a ribosome binding region into the 5'UTR, and the ribosome binding region is positioned upstream of a region consisting of the nucleotide sequence of SEQ ID NO: 1 contained in the 5'UTR. The method according to any one of [22] to [28].
[30] The method according to [29], wherein the heterologous ribosome binding region is preferably positioned upstream of the heterologous polynucleotide.
[31] Preferably,
the modification of the 5'UTR comprises replacing, inserting or adding one ribosome binding region into the 5'UTR, the ribosome binding region being positioned 10-100 nucleotides downstream from the transcription start point of the modified promoter; or the modification of the 5'UTR comprises replacing, inserting or adding two or more ribosome binding regions into the 5'UTR, at least one of which is positioned 10-100 nucleotides downstream from the transcription start point of the modified promoter and the rest are positioned 10-150 nucleotides downstream from the transcription start point.
The method described in [29] or [30].
[32] The method according to any one of [22] to [31], wherein the parent promoter is preferably a promoter derived from the genus Bacillus.
[33] The method according to [32], wherein the parent promoter is preferably a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 72 or a nucleotide sequence having at least 80% identity to said sequence, or a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 76 or a nucleotide sequence having at least 80% identity to said sequence.

The present invention will be explained in more detail below using examples. In the following examples and figures, a.u. stands for arbitrary unit.

Example 1 Construction of a Plasmid for Introducing a KP43 Protease Expression Cassette A plasmid for introducing a protease expression cassette into the amyE locus in the B. subtilis genome by homologous recombination was constructed as follows.
A vector fragment was amplified by PCR using the pMW119 plasmid as a template and a forward primer (SEQ ID NO: 12) and a reverse primer (SEQ ID NO: 13). Next, an insert fragment was amplified by PCR using the B. subtisli 168 strain genome as a template and a forward primer (SEQ ID NO: 14) and a reverse primer (SEQ ID NO: 15) to obtain the ORF sequence of the amyE gene. The vector fragment and the insert fragment were ligated by an In-Fusion reaction, and transformed into E. coli to obtain a pMW-amy plasmid.
A vector fragment was amplified by PCR using the pMW-amy plasmid as a template and a forward primer (SEQ ID NO: 16) and a reverse primer (SEQ ID NO: 17). Next, an insert fragment containing the ORF sequence of the spectinomycin resistance gene was amplified by PCR using the kao119 strain genome (Patent No. 6088282) as a template and a forward primer (SEQ ID NO: 18) and a reverse primer (SEQ ID NO: 19). The vector fragment and the insert fragment were ligated by an In-Fusion reaction, and transformed into E. coli to obtain the pMW-amy-sp plasmid.

A vector fragment was amplified by PCR using the pMW-amy-sp plasmid as a template and a forward primer (SEQ ID NO: 16) and a reverse primer (SEQ ID NO: 20). Next, an insert fragment was amplified by PCR using the forward primer (SEQ ID NO: 21) and a reverse primer (SEQ ID NO: 22) using P _SP64 -TS43 (SEQ ID NO: 71) as a template. This insert fragment contained an expression cassette containing a synthetic promoter (P _SP64 , SEQ ID NO: 72) derived from the promoter of the endoglucanase gene of Bacillus sp. KSM-64 and an ORF of a gene encoding alkaline protease (KP43 protease) derived from Bacillus sp. KSM-KP43 (FERM BP-6532). The vector fragment and the insert fragment were ligated by an In-Fusion reaction, and transformed into E. coli to obtain the pMW-TS43 plasmid.

Example 2 Construction of a Plasmid for Introducing a KP43 Protease Expression Cassette Containing a Modified 5'UTR The 5'UTR (UTR ^SP64 ) of the promoter (P _SP64 ) contained in the pMW-TS43 plasmid constructed in Example 1 was modified.
1) Using the pMW-TS43 plasmid as a template, a vector fragment was amplified by PCR using a forward primer (SEQ ID NO: 23) and a reverse primer (SEQ ID NO: 24). Next, an insert fragment containing the sequence of SEQ ID NO: 4 was amplified by PCR using a forward primer (SEQ ID NO: 25) and a reverse primer (SEQ ID NO: 26) using pHY-YR288 (JP Patent Publication 2022-019601) as a template. The sequence of SEQ ID NO: 4 was a partial fragment (94 nt) of the 5'UTR contained in the promoter of the endoglucanase gene derived from KSM-S237. The vector fragment and the insert fragment were combined by an In-Fusion reaction, and transformed into Escherichia coli to obtain the pMW-UTR ^S237 -a-TS43 plasmid. The promoter contained in the pMW-UTR ^S237 -a-TS43 plasmid was designated as P _SP64 -UTR ^S237 -a.

2) Furthermore, a fragment was constructed by inverse PCR using a forward primer (SEQ ID NO: 27) and a reverse primer (SEQ ID NO: 28) with pMW-TS43 as a template, and transformed into E. coli to obtain the pMW-UTR ^S237 -b-TS43 plasmid. Further, a fragment was constructed by inverse PCR using a forward primer (SEQ ID NO: 29) and a reverse primer (SEQ ID NO: 30) with pMW-TS43 as a template, and transformed into E. coli to obtain the pMW-UTR ^S237 -c-TS43 plasmid.

The promoters P _SP64 -UTR ^S237 -a (SEQ ID NO: 73), P SP64 -UTR ^S237 -b (SEQ ID NO: 74), and P SP64 -UTR ^S237 -c (SEQ ID NO: 75) contained in pMW-UTR ^S237 -a-TS43, _pMW- UTR ^S237 -b-TS43, and _pMW -UTR ^S237 -c-TS43, respectively, each had the modified 5'UTR shown in Figure 1. As shown in Figure 1, P _SP64 -UTR ^S237 -a has the sequence of SEQ ID NO: 4 (shaded area) at the 3' end of the 5'UTR region, and P _SP64 -UTR ^S237 -b and P _SP64 -UTR ^S237 -c each have the sequence of SEQ ID NO: 3 (shaded area) at the 3' end of the 5'UTR region.

Example 3 Construction of a plasmid for introducing a spoVG expression cassette containing a modified 5'UTR The promoter of the pMW-TS43 plasmid constructed in Example 1 was replaced with the promoter (P _spoVG , SEQ ID NO: 76) of the spoVG gene of B. subtisli 168 strain. A vector fragment was amplified by PCR using the pMW-TS43 plasmid as a template and a forward primer (SEQ ID NO: 31) and a reverse primer (SEQ ID NO: 32). Next, an insert fragment containing the promoter (P _spoVG ) of the spoVG gene of B. subtisli 168 strain was amplified by PCR using the B. subtisli 168 strain genome as a template and a forward primer (SEQ ID NO: 33) and a reverse primer (SEQ ID NO: 34). The vector fragment and the insert fragment were ligated by an in-fusion reaction, and transformed into E. coli to obtain the pMW-P _spoVG -TS43 plasmid.

The 5'UTR region (UTR ^spoVG ) of P _spoVG in the pMW-P _spoVG -TS43 plasmid was modified. A fragment was constructed by inverse PCR using pMW-P _spoVG -TS43 as a template and a forward primer (SEQ ID NO: 35) and a reverse primer (SEQ ID NO: 36), and the fragment was transformed into E. coli to obtain pMW-P _spoVG -UTR ^S237 -TS43 plasmid. As shown in FIG. 2, the control region P _spoVG -UTR ^S237 (SEQ ID NO: 77) of this plasmid had the entire 5'UTR of the parent promoter replaced with the sequence of SEQ ID NO: 3 (hatched portion).

Example 4 Construction of a Plasmid for Introducing an Amylase Expression Cassette Containing a Modified 5'UTR The YR288 amylase expression cassette was constructed as follows.
A vector fragment was amplified by PCR using the pMW-TS43 plasmid constructed in Example 1 as a template and a forward primer (SEQ ID NO: 23) and a reverse primer (SEQ ID NO: 37). Next, an insert fragment containing the ORF sequence of the YR288 amylase gene was amplified by PCR using pHY-YR288 (JP Patent Publication 2022-019601) as a template and a forward primer (SEQ ID NO: 38) and a reverse primer (SEQ ID NO: 39). The vector fragment and the insert fragment were ligated by an In-Fusion reaction, and transformed into E. coli to obtain the pMW-P _SP64 -YR288 plasmid.

A vector fragment was amplified by PCR using the forward primer (SEQ ID NO: 23) and reverse primer (SEQ ID NO: 40) with the pMW-UTR ^S237 -a-TS43 plasmid constructed in Example 2 as a template. Next, an insert fragment containing the ORF sequence of the YR288 amylase gene was amplified by PCR using the forward primer (SEQ ID NO: 41) and reverse primer (SEQ ID NO: 39) with pHY-YR288 as a template. The vector fragment and the insert fragment were ligated by an In-Fusion reaction, and transformed into E. coli to obtain the pMW-P _SP64 -UTR ^S237 -a-YR288 plasmid.

A vector fragment was amplified by PCR using the forward primer (SEQ ID NO: 23) and reverse primer (SEQ ID NO: 42) and pMW-P _spoVG -TS43 constructed in Example 3 as a template. Next, an insert fragment containing the ORF sequence of the YR288 amylase gene was amplified by PCR using the forward primer (SEQ ID NO: 43) and reverse primer (SEQ ID NO: 39) and pHY-YR288 as a template. The vector fragment and the insert fragment were ligated by an In-Fusion reaction, and transformed into E. coli to obtain the pMW-P _spoVG -YR288 plasmid.

A vector fragment was amplified by PCR using the forward primer (SEQ ID NO: 23) and reverse primer (SEQ ID NO: 44) and pMW-P _spoVG -UTR ^S237 -TS43 constructed in Example 3 as a template. Next, an insert fragment containing the ORF sequence of the YR288 amylase gene was amplified by PCR using the forward primer (SEQ ID NO: 41) and reverse primer (SEQ ID NO: 39) and pHY-YR288 as a template. The vector fragment and the insert fragment were ligated by an In-Fusion reaction, and transformed into E. coli to obtain the pMW-P _spoVG -UTR ^S237 -YR288 plasmid.

A fragment was constructed by inverse PCR using the pMW-P _spoVG -UTR ^S237 -YR288 plasmid as a template and a forward primer (SEQ ID NO: 45) and a reverse primer (SEQ ID NO: 46), and transformed into E. coli to obtain the pMW-P _spoVG -UTR ^S237 -mut1-YR288 plasmid. A fragment was constructed by inverse PCR using the pMW-P _spoVG -UTR ^S237 -YR288 plasmid as a template and a forward primer (SEQ ID NO: 47) and a reverse primer (SEQ ID NO: 48), and transformed into E. coli to obtain the pMW-P _spoVG -UTR ^S237 -mut2-YR288 plasmid. The control regions of these plasmids, P _spoVG -UTR ^S237 -mut1 (SEQ ID NO:78) and P _spoVG -UTR ^S237 -mut2 (SEQ ID NO:79), had the sequences of SEQ ID NO:5 and SEQ ID NO:6, respectively, at the 3' end of the 5'UTR.

Example 5 Construction of a plasmid for introducing a Cry5B expression cassette containing a modified 5'UTR A Cry5B expression cassette was constructed. A vector fragment was amplified by PCR using a forward primer (SEQ ID NO: 23) and a reverse primer (SEQ ID NO: 37) with the pMW-TS43 plasmid constructed in Example 1 as a template. Next, an insert fragment containing the ORF sequence of the Cry5B gene was amplified by PCR using a forward primer (SEQ ID NO: 49) and a reverse primer (SEQ ID NO: 50) with pHY-Pscry5B (Patent No. 7218090) as a template. The vector fragment and the insert fragment were combined by an In-Fusion reaction, and transformed into E. coli to obtain a pMW-P _SP64 -cry5B plasmid.

A vector fragment was amplified by PCR using a forward primer (SEQ ID NO: 23) and a reverse primer (SEQ ID NO: 40) with the pMW-UTR ^S237 -a-TS43 plasmid constructed in Example 2 as a template. Next, an insert fragment containing the ORF sequence of the Cry5B gene was amplified by PCR using a forward primer (SEQ ID NO: 51) and a reverse primer (SEQ ID NO: 50) with pHY-Pscry5B as a template. The vector fragment and the insert fragment were ligated by an In-Fusion reaction, and transformed into E. coli to obtain the pMW-P _SP64 -UTR ^S237 -a-cry5B plasmid.

Example 6 Construction of a Plasmid for Introducing a Lipase Expression Cassette Containing a Modified 5'UTR A lipase expression cassette was constructed. A vector fragment was amplified by PCR using the pMW-TS43 plasmid constructed in Example 1 as a template and a forward primer (SEQ ID NO: 23) and a reverse primer (SEQ ID NO: 37). Next, an insert fragment containing the ORF sequence of the lipase gene was amplified by PCR using the forward primer (SEQ ID NO: 53) and a reverse primer (SEQ ID NO: 54) and pHY-CnLip (SEQ ID NO: 52) as a template. The vector fragment and the insert fragment were ligated by an In-Fusion reaction, and transformed into E. coli to obtain the pMW-P _SP64 -Lip plasmid.

A vector fragment was amplified by PCR using the pMW-UTR ^S237 -a-TS43 plasmid constructed in Example 2 as a template and a forward primer (SEQ ID NO: 23) and a reverse primer (SEQ ID NO: 40). Next, an insert fragment containing the ORF sequence of the lipase gene was amplified by PCR using the forward primer (SEQ ID NO: 55) and a reverse primer (SEQ ID NO: 54) and pHY-CnLip as a template. The vector fragment and the insert fragment were ligated by an In-Fusion reaction, and transformed into E. coli to obtain the pMW-P _SP64 -UTR ^S237 -a-Lip plasmid.

Example 7 Addition of an RBS to a KP43 protease control region containing a modified 5'UTR An investigation was carried out into adding a ribosome binding site (RBS) (CTTGAAAGGAGGGATGCCTAA; SEQ ID NO: 11) to a KP43 protease control region containing a modified 5'UTR. A fragment was constructed by inverse PCR using the pMW-P _SP64 -UTR ^S237 -a-cry5B plasmid constructed in Example 5 as a template and a forward primer (SEQ ID NO: 56) and a reverse primer (SEQ ID NO: 57), and transformed into E. coli to obtain pMW-P _SP64 -UTR ^S237 -a-RBS-cry5B. As shown in Figure 3, the control region P _SP64 -UTR ^S237 -a-RBS (sequence number 80) of this plasmid had an RBS (black bar) added downstream of the transcription start site and had the sequence of SEQ ID NO: 4 (hatched area) at the 3' end of the 5'UTR.

Example 8 Preparation of recombinant Bacillus containing modified 5'UTR 1) Construction of prsA overexpression strain A prsA overexpression strain was prepared by introducing a prsA gene overexpression cassette (SEQ ID NO: 58) into a sigF gene deleted strain of B. subtilis 168 (ΔsigF strain: Japanese Patent No. 4336082).
A DNA fragment for introducing the prsA overexpression cassette was constructed in the following manner. First, the following PCR fragments 1 to 3 were constructed:
PCR fragment 1: forward primer; SEQ ID NO:59, reverse primer; SEQ ID NO:60, template DNA; prsA-Ka strain genomic DNA (Patent No. 4839144);
PCR fragment 2: forward primer; SEQ ID NO: 61, reverse primer; SEQ ID NO: 62), template DNA: MazF cassette (Genet. Syst., 84(4):315-318,2009);
PCR fragment 3: forward primer; sequence number 63, reverse primer; sequence number 64), template DNA: 168 strain genomic DNA.
The PCR fragments 1 to 3 were ligated by SOE-PCR (forward primer: SEQ ID NO: 65, reverse primer: SEQ ID NO: 66) to construct a DNA fragment for introducing a prsA overexpression cassette.
The constructed DNA fragment for introducing the prsA overexpression cassette was introduced into the ΔsigF strain, and the prsA overexpression cassette was introduced into the nprE locus of the ΔsigF strain. Gene introduction was performed by homologous recombination according to the marker-free deletion method developed by Morimoto et al. (Genet. Syst., 84(4):315-318, 2009) (FIG. 4). The obtained prsA overexpression ΔsigF strain (ΔsigF-prsA strain) was used as a parent strain to prepare recombinant Bacillus bacteria into which the following target gene expression cassettes were introduced.

2) Introduction of target gene expression cassette into parent strain genome Protease and amylase expression strains were constructed by the following method. A PCR fragment for genome introduction was obtained by PCR using a forward primer (SEQ ID NO: 67) and a reverse primer (SEQ ID NO: 68) with the target gene expression cassette introduction plasmid constructed in Examples 1 to 4 and 7 as a template. This PCR fragment was introduced into the ΔsigF-prsA strain prepared in 1) above by the competent cell method (see Japanese Patent No. 6088282), and transformed by homologous recombination into the amyE gene locus on the genome.

The Cry5B and lipase expression strains were constructed by the following method. A PCR fragment for genome introduction was obtained by PCR using a forward primer (sequence number 67) and a reverse primer (sequence number 68) with the plasmid for introducing the target gene expression cassette constructed in Examples 5 and 6 as a template. This PCR fragment was introduced into the ΔsigF strain (Patent No. 4336082) by the competent cell method (see Patent No. 6088282) and introduced by homologous recombination into the amyE gene locus on the genome to produce a recombinant Bacillus bacterium with the expression cassette introduced.

Example 9 Production of a target protein by culturing recombinant Bacillus bacteria The recombinant Bacillus bacteria into which the target gene expression cassette obtained in Example 8 had been introduced was cultured overnight at 30°C and 180 rpm in 2 mL of LB medium in a 10 mL round-bottom Spitz tube with shaking. 500 μL of the resulting culture solution was sampled and inoculated into 20 mL of 2×L-maltose medium (2% peptone, 1% yeast extract, 1% NaCl, 7.5% maltose, 7.5 ppm manganese 4-5 hydrate, antifoaming agent; % is v/w%). This was cultured at 32°C (30°C for the Cry5B expression strain and lipase expression strain) and 210 rpm for 72 hours.

Example 10 Measurement of the amount of target protein produced 1) Method The amount of target protein produced by the recombinant Bacillus bacteria prepared in Example 8 was measured.
The amount of protease produced was determined as a protease activity value. After the completion of the culture in Example 9, the protease activity of the culture supernatant from which the cells had been removed was measured by the following procedure. 0.9 mL of 1/15 M phosphate buffer (pH 7.4) and 0.05 mL of 40 mM Glt-Ala-Ala-Pro-Leu-p-nitroanilide/dimethyl sulfoxide solution were placed in a test tube and incubated at 30°C for 5 minutes. 0.05 mL of the culture supernatant was added thereto and reacted at 30°C for 10 minutes, after which 2.0 mL of 5% (w/v) citric acid aqueous solution was added to stop the reaction, and the absorbance at 420 nm was measured using a spectrophotometer to obtain the protease activity value. The protease activity value of the recombinant Bacillus bacteria into which an expression cassette containing a modified 5'UTR had been introduced was expressed as a relative value (au) with the activity of the recombinant Bacillus bacteria into which an expression cassette containing an unmodified 5'UTR had been introduced taken as 1.

The amount of amylase produced was determined as the amylase activity value. Amylase activity measurement was performed as follows. After the end of the culture in Example 9, the amylase activity of the culture supernatant from which the cells had been removed was measured as follows. 0.9 mL of an aqueous solution in which one tablet of Fadebas Amylase Test was suspended in 5 mL of 1/15 M phosphate buffer (pH 7.4) was added to a test tube and incubated at 30°C for 15 minutes. 0.05 mL of an enzyme solution (culture supernatant) appropriately diluted with a 2 mM calcium chloride aqueous solution was added to this, and the reaction was carried out at 30°C for exactly 10 minutes, after which 2 mL of a 5% (w/v) citric acid aqueous solution was added to stop the reaction. The enzyme reaction solution was centrifuged at 3000 rpm for 5 minutes, and the absorbance at 750 nm of the supernatant was measured using a spectrophotometer to determine the amylase activity value. The amylase activity of recombinant Bacillus bacteria into which an expression cassette containing a modified 5'UTR was introduced was expressed as a relative value (a.u.), with the activity of recombinant Bacillus bacteria into which an expression cassette containing an unmodified 5'UTR was introduced being taken as 1.

The amount of Cry5B produced was determined as a protein concentration. The Cry5B protein concentration was measured by the method described in Japanese Patent No. 7218090. Specifically, after the completion of the culture in Example 9, the Cry5B concentration in the culture supernatant from which the bacterial cells had been removed was measured by the following procedure. The culture supernatant and bovine serum albumin (BSA) (manufactured by Wako Pure Chemical Industries, Ltd.) as a standard protein were subjected to SDS-PAGE. The gel was stained with Bio-Safe ^TM Coomassie (BIO-RAD) while shaking for 1 hour, and decolorized with ion-exchanged water. The brightness of each band in the gel image was analyzed using the image software ImageJ (rsb.info.nih.gov/ij/download.html) developed by the National Institutes of Health (NIH). A calibration curve was created from the BSA data. The Cry5B protein concentration was calculated from this calibration curve. The Cry5B protein concentration of recombinant Bacillus bacteria into which an expression cassette containing a modified 5'UTR was introduced is shown as a relative value (au) with the Cry5B protein concentration of recombinant Bacillus bacteria into which an expression cassette containing an unmodified 5'UTR was introduced set at 1.

The amount of lipase produced was determined as the lipase activity value. After the completion of the culture in Example 9, the lipase activity of the culture supernatant from which the cells had been removed was measured by the following method. The lipase activity was determined by measuring the rate of increase in absorbance accompanying the release of p-nitrophenol by the action of lipase. A substrate solution was prepared by adding p-nitrophenylbutyric acid (pNPB) (SIGMA) to a final concentration of 2 mM to 20 mM Tris-HCl (pH 7.0) and mixing the mixture. 4 μL of the culture supernatant appropriately diluted with 20 mM Tris-HCl (pH 7.0) and 100 μL of the substrate solution were mixed in each well of a 96-well assay plate, and the change in absorbance (OD/min) at 405 nm at 30°C was measured. The difference ΔOD/min from the blank (sample without culture supernatant added) was determined. The lipase activity of recombinant Bacillus bacteria into which an expression cassette containing a modified 5'UTR was introduced is shown as a relative value (a.u.) with the lipase activity of recombinant Bacillus bacteria into which an expression cassette containing an unmodified 5'UTR was introduced set at 1.

2) Results Figure 5 shows the improvement in the production of KP43 protease by recombinant Bacillus bacteria into which a KP43 protease expression cassette having the modified P _SP64 promoter (P _SP64 -UTR ^S237 -a, b, and c) containing the modified 5'UTR constructed in Example 2 has been introduced. When the production of KP43 protease when the parent promoter (P _SP64 -UTR ^SP64 ) containing an unmodified 5'UTR is taken as 100%, P _SP64 -UTR ^S237 -a, b, and c containing the modified 5'UTR improved the protease production in the recombinant Bacillus bacteria by 45%, 38%, and 42%, respectively, compared to the parent promoter, when the production of KP43 protease when the parent promoter (P SP64 -UTR SP64 ) containing an unmodified 5'UTR is used. 6 shows the improvement in the production of KP43 protease by recombinant Bacillus bacteria into which a KP43 protease expression cassette having a spoVG promoter (P _spoVG -UTR ^S237 ) containing a modified 5'UTR constructed in Example 3 has been introduced. P _spoVG -UTR ^S237 containing a modified 5'UTR improved the protease production in the recombinant Bacillus bacteria by 4.0 times compared to the parent promoter (P _spoVG -UTR ^spoVG ) containing an unmodified 5'UTR.

7 shows the improvement in amylase production by recombinant Bacillus bacteria into which an amylase expression cassette having the modified P _SP64 promoter (P _SP64 -UTR ^S237 -a) containing the modified 5'UTR constructed in Example 4 has been introduced. P _SP64 -UTR ^S237 -a containing the modified 5'UTR improved the amylase production in the recombinant Bacillus bacteria by 2.7-fold compared to the parent promoter (P _SP64 -UTR ^SP64 ) containing an unmodified 5'UTR. 8 shows the improvement in amylase production by recombinant Bacillus bacteria introduced with an amylase expression cassette having the spoVG promoter ( _PspoVG - ^UTRs237 , _PspoVG - ^UTRs237 -mut1 and _PspoVG - ^UTRs237 -mut2) containing the modified 5'UTR constructed in Example 4. _PspoVG - ^UTRs237 , _PspoVG - ^UTRs237 -mut1 and _PspoVG - ^UTRs237 -mut2 containing the modified 5'UTR improved the amylase production in the recombinant Bacillus bacteria by 7.5-fold, 2.6-fold and 7.8-fold, respectively, compared to the parent promoter ( _PspoVG - ^UTRspoVG ) containing an unmodified 5'UTR.

9 shows the improvement in the production of Cry5B by recombinant Bacillus bacteria into which a Cry5B expression cassette having a modified P _SP64 promoter (P _SP64 -UTR ^S237 -a) containing a modified 5'UTR constructed in Example 5 has been introduced. P _SP64 -UTR ^S237 -a containing a modified 5'UTR improved the production of Cry5B in recombinant Bacillus bacteria by 3.3-fold compared to the parent promoter (P _SP64 -UTR ^SP64 ) containing an unmodified 5'UTR.

10 shows the improvement in the production of lipase by recombinant Bacillus bacteria into which a lipase expression cassette having the modified P _SP64 promoter (P _SP64 -UTR ^S237 -a) containing the modified 5'UTR constructed in Example 6 has been introduced. P _SP64 -UTR ^S237 -a containing the modified 5'UTR improved the production of lipase in the recombinant Bacillus bacteria by 34% compared to the parent promoter (P _SP64 -UTR ^SP64 ) containing an unmodified 5'UTR.

11 shows the improvement in the production of Cry5B by recombinant Bacillus bacteria into which a Cry5B expression cassette having a modified P _SP64 promoter (P _SP64 -UTR ^S237 -a-RBS) including a modified 5'UTR with an additional RBS constructed in Example 7 has been introduced. The modified promoter having an additional RBS (P _SP64 -UTR ^S237 -a-RBS) improved the production of Cry5B by 56% compared to P _SP64 -UTR ^S237 -a, assuming the production of Cry5B when a modified promoter without an additional RBS (P _SP64 -UTR ^S237 -a) is used as 100%. Furthermore, P _SP64 -UTR ^S237 -a-RBS increased the production of Cry5B in the recombinant Bacillus by 5.1-fold compared to the parent promoter (P _SP64 -UTR ^SP64 ) containing the unmodified 5'UTR.

Claims (17)

A DNA molecule comprising a modified promoter,
The modified promoter comprises a modified 5'UTR containing a heterologous polynucleotide;
The heterologous polynucleotide is any one of the following polynucleotides a) to d):
a) a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 3, or a nucleotide sequence having at least 90% identity thereto and comprising the nucleotide sequence AGGAGG at positions corresponding to positions 8 to 13 of the nucleotide sequence of SEQ ID NO: 3;
b) a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 4 or a fragment thereof comprising the nucleotide sequence of SEQ ID NO: 3, or
a polynucleotide having at least 90% identity with said polynucleotide or a fragment thereof and comprising a nucleotide sequence containing the nucleotide sequence AGGAGG at positions corresponding to positions 8 to 13 of the nucleotide sequence of SEQ ID NO:3;
c) a polynucleotide consisting of the nucleotide sequence of SEQ ID NO:5, or a nucleotide sequence having at least 90% identity thereto and comprising the nucleotide sequence AGGAGG at positions corresponding to positions 8 to 13 of the nucleotide sequence of SEQ ID NO:5;
d) a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 6, or a nucleotide sequence having at least 90% identity thereto and comprising the nucleotide sequence AGGAGG at positions corresponding to positions 8 to 13 of the nucleotide sequence of SEQ ID NO: 6;
and
The modified promoter improves the expression level of a gene compared to the parent promoter before it was modified to include the modified 5'UTR.
DNA molecule. The DNA molecule according to claim 1, wherein the polynucleotides a) to d) contain the nucleotide sequence of SEQ ID NO:2 at positions corresponding to positions 8 to 20 of the nucleotide sequence of SEQ ID NO:3. The DNA molecule of claim 1, wherein the 3' end of the heterologous polynucleotide consists of the nucleotide sequence of SEQ ID NO:70. The DNA molecule of claim 1, wherein the heterologous polynucleotide is 100 nt or less in length. The DNA molecule of claim 1, wherein the heterologous polynucleotide is located at the 3' end of the modified 5'UTR. The DNA molecule of claim 1, wherein the modified 5'UTR contains, in addition to the heterologous polynucleotide, a heterologous ribosome binding region, which is positioned upstream of a region consisting of the nucleotide sequence AGGAGG contained in the modified 5'UTR. The DNA molecule of claim 6, wherein the heterologous ribosome binding region is located upstream of the heterologous polynucleotide. The DNA molecule of claim 6, wherein the heterologous ribosome binding region is located 10 to 100 nucleotides downstream from the transcription start site of the modified promoter. The DNA molecule of claim 1, wherein the parent promoter of the modified promoter is a promoter derived from the genus Bacillus. The DNA molecule of claim 9, wherein the parent promoter is a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 72 or a nucleotide sequence having at least 85 % identity thereto, or a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 76 or a nucleotide sequence having at least 85 % identity thereto. The DNA molecule of claim 1, further comprising a gene of interest. The DNA molecule of claim 11, which is an expression cassette or an expression vector. A transformant containing the DNA molecule according to claim 12. The transformant contains the DNA molecule according to claim 1 , which is an expression cassette containing the modified promoter and further containing a gene of interest, and
When the expression level of the target gene in a cell containing an expression cassette including the parent promoter of the modified promoter and further including the target gene is taken as 100%,
The transformant increases the expression level of the target gene by at least 10%.
The transformant according to claim 13. The transformant according to claim 13, which is a bacterium of the genus Bacillus. A method for producing a target substance, comprising culturing the transformant according to claim 13. A method for producing a modified promoter, comprising the steps of:
The method comprises modifying a 5'UTR contained in a parent promoter,
Modifying the 5'UTR includes replacing part or all of the 5'UTR with a heterologous polynucleotide or adding the heterologous polynucleotide to the 5'UTR;
The heterologous polynucleotide is any one of the following polynucleotides a) to d):
a) a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 3, or a nucleotide sequence having at least 90% identity thereto and comprising the nucleotide sequence AGGAGG at positions corresponding to positions 8 to 13 of the nucleotide sequence of SEQ ID NO: 3;
b) a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 4 or a fragment thereof comprising the nucleotide sequence of SEQ ID NO: 3, or
a polynucleotide having at least 90% identity with said polynucleotide or a fragment thereof and comprising a nucleotide sequence containing the nucleotide sequence AGGAGG at positions corresponding to positions 8 to 13 of the nucleotide sequence of SEQ ID NO:3;
c) a polynucleotide consisting of the nucleotide sequence of SEQ ID NO:5, or a nucleotide sequence having at least 90% identity thereto and comprising the nucleotide sequence AGGAGG at positions corresponding to positions 8 to 13 of the nucleotide sequence of SEQ ID NO:5;
d) a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 6, or a nucleotide sequence having at least 90% identity thereto and comprising the nucleotide sequence AGGAGG at positions corresponding to positions 8 to 13 of the nucleotide sequence of SEQ ID NO: 6;
and
the parent promoter is a promoter that comprises a 5'UTR that does not contain the heterologous polynucleotide;
The modified promoter improves the expression level of a gene compared to the parent promoter.
method.