Modified promoter, expression vector, microorganism, method for producing substance, modified cyanobacteria, and method for producing modified promoter

Abstract

The present disclosure provides a modified promoter capable of flexibly adjusting the expression level of a gene encoding a protein. The disclosed modified promoter is a base sequence (SEQ ID NO: 1) of a 348 base upstream of an initiation codon of an slr1841 gene, or a base sequence having 80% or more homology with the base sequence represented by SEQ ID NO: 1. And a promoter modified by at least one method selected from the group consisting of (i) substitution of one or more bases contained in the base sequence with another base, (ii) deletion of one or more bases from the base sequence, and (iii) insertion of one or more bases into the base sequence.

Description

Technical Field

[0001] The present disclosure relates to a modified promoter, an expression vector comprising the modified promoter, a microorganism comprising the expression vector, a method for producing a substance using the microorganism, a modified cyanobacterium into which the modified promoter is introduced into its genome, and a method for producing the modified promoter. Background Art

[0002] In order to improve the productivity of substances using microorganisms, development of promoters capable of increasing the expression level of genes encoding any substances (eg, proteins, etc.) produced by microorganisms has been conducted.

[0003] For example, Patent Document 1 discloses a modified promoter having enhanced promoter activity and capable of strong expression induction obtained by modifying the base sequence of the promoter of the alkaline cellulase gene of Bacillus subtilis, and a method for efficiently secreting and producing alkaline cellulase using a microorganism using the modified promoter.

[0004] In addition, for example, Patent Document 2 discloses a method for efficiently producing polyhydroxyalkanoate copolymers with a controlled monomer composition ratio using a microorganism using the modified promoter by modifying the base sequence of the promoter of the R structure-specific enoyl-CoA hydratase gene of Aeromonas bacteria to produce a modified promoter capable of regulating the expression of the gene.

[0005] Prior art literature

[0006] Patent Literature

[0007] Patent Document 1: Japanese Patent Application Publication No. 2005-278644

[0008] Patent Document 2: International Publication No. 2015 / 115619 Summary of the invention

[0009] Problems to be solved by the invention

[0010] However, depending on the substance, even if a modified promoter with high promoter activity is used, the expression level of the gene encoding the substance may decrease. Therefore, it is necessary to appropriately regulate the promoter activity according to the substance to be produced by the microorganism, but regulation is difficult.

[0011] Therefore, the present disclosure provides a modified promoter, an expression vector, a microorganism, a method for producing a substance, a modified cyanobacterium, and a method for producing a modified promoter that can flexibly regulate the expression amount of a gene encoding an arbitrary substance.

[0012] Means for solving problems

[0013] The modified promoter involved in one scheme of the present invention is a promoter obtained by modifying a base sequence of 348 bases upstream of the start codon of the slr1841 gene (sequence number 1), or a base sequence having more than 80% homology with the base sequence shown in sequence number 1, by (i) replacing one or more bases contained in the base sequence with other bases, (ii) deleting one or more bases from the base sequence, or (iii) inserting one or more bases into the base sequence.

[0014] Effects of the Invention

[0015] According to the disclosed transformation promoter and the manufacture method of the transformation promoter, the transformation promoter that can flexibly regulate the expression of the gene of any material of coding can be provided. In addition, according to the disclosed expression vector, the expression vector that can flexibly regulate the expression of the gene of any material of coding can be provided in the transformed cell. In addition, according to the disclosed microorganism, the production method of material and the transformation cyanobacteria, by the expression of the gene of any material of coding, the microorganism, the production method of material and the transformation cyanobacteria that can produce the material by this gene encoding with desired efficiency can be provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 is a schematic diagram showing the promoter region of slr1841.

[0017] Figure 2 The figure shows the position of the transcription start site (TSS) of the slr1841 gene.

[0018] Figure 3 This is a flowchart showing an example of a method for preparing a modified promoter.

[0019] Figure 4 It is a schematic diagram showing an example of an expression vector.

[0020] Figure 5 Electrophoresis images of fragments obtained by 5'-RACE (5'-rapid amplification of cDNA ends).

[0021] Figure 6 It is a graph showing the results of measuring the fluorescence intensity of the culture solutions of cyanobacteria and Escherichia coli after transformation.

[0022] Fig. 7A The graph shows the structure of a modified promoter in which a mutation is introduced into the upstream region of the transcription start site (TSS) of the slr1841 promoter and the results of measuring the fluorescence intensity of the culture fluid of the transformed cells into which the modified promoter is introduced.

[0023] Figure 7BThe graph shows the base sequence of the -10 region into which a mutation has been introduced and the results of measuring the fluorescence intensity of the culture solution of the transformed cells into which the modified promoter into which the mutation has been introduced has been introduced.

[0024] Figure 8 It is a figure which shows the analysis result of the culture supernatant by LC-MS (Liquid Chromatograph-Mass Spectrometry).

[0025] Fig. 9 The graph shows the results of antibiotic resistance tests of transformed strains and wild-type strains.

[0026] Fig.10 Displays serial number 1 to serial number 2. DETAILED DESCRIPTION

[0027] (Insights that form the basis of this disclosure)

[0028] The production of substances using microorganisms (particularly fermentation) has been carried out since ancient times. For example, in the production of useful substances using fermentation, microorganisms produce fermented foods and alcoholic beverages containing substances useful to humans (specifically organic substances) in the process of obtaining energy by anaerobically metabolizing organic substances taken in as nutrients. At present, the production of substances using microorganisms is not limited to such traditional fermentation production, but is flexibly used in the production of a variety of substances such as amino acids, nucleic acids, antibiotics, and proteins.

[0029] In addition, there are a large number of unknown microorganisms in nature, and the microorganisms discovered now can be said to be about a few percent of the total. Therefore, microorganisms are considered to be rich genetic resources. Therefore, by exploring microorganisms with useful functions from nature, such as microorganisms with functions that contribute to improving the production efficiency of target substances (hereinafter referred to as useful microorganisms), and isolating and breeding them, it is expected to build a system that can produce useful substances more efficiently than before.

[0030] Furthermore, in recent years, due to the development of biotechnology, microorganisms into which specific genes of useful microorganisms have been integrated (hereinafter referred to as genetically modified microorganisms) have been produced using genetically modified techniques and the development of production technologies for useful substances using genetically modified microorganisms has been actively carried out.

[0031] In order to make genetically modified microorganisms produce useful substances (for example, useful proteins), it is necessary not only to introduce the gene encoding the useful substance (useful protein) into the genome of the microorganism, but also to introduce an appropriate promoter upstream of the gene to induce the expression of the gene.

[0032] The so-called promoter is a region present along with all genes on the genome (hereinafter also referred to as genomic DNA (deoxyribonucleic acid)) present in the cell, and plays an important role in appropriately inducing the expression of each gene in the upstream region of each gene and regulating the expression amount (so-called transcription amount) of the gene. Therefore, in order to make microorganisms produce useful substances (useful proteins), it is essential to select or develop a promoter suitable for efficient transcription of the gene encoding the useful substance (useful protein).

[0033] It is not difficult to imagine that the stronger the activity of the promoter, the more the transcription amount of the gene located downstream thereof (more specifically, the gene encoding the target useful protein), and the more efficient the production of the target useful protein. Of course, there are a large number of such examples, but on the other hand, a strong promoter may not be suitable for the production of all microorganisms (also referred to as cells) or all proteins. If the activity of the promoter is too strong, sometimes the amount of protein produced exceeds the processing capacity of the cell, and it becomes difficult to fold the protein into a normal three-dimensional structure. In this way, sometimes not only can the production of effective useful proteins not be achieved, but it also brings obstacles to the growth and reproduction of the cells themselves.

[0034] Therefore, when using genetically recombinant microorganisms to produce useful proteins, considering the balance between the processing capacity and production efficiency of the microorganism, it is required to develop a promoter that can freely regulate the activity of inducing the expression of the protein. If such a promoter is developed, it becomes easy to select a promoter suitable for the production of the protein of interest, and it is expected that the effective production of the protein can be achieved.

[0035] However, there is almost no knowledge about promoters for microorganisms such as cyanobacteria, and promoters that can be flexibly used in the production of proteins using these microorganisms have not yet been developed (Non-patent Document 1: Marnix H. Medema et al., "Synthetic biology in streptomyces bacteria", Methods in Enzymology, Volume 497, 2011, Pages 485-502, ISSN: 0076-6879).

[0036] The inventors of the present application have conducted in-depth research and have focused on the promoter of the slr1841 gene, which is one of the proteins with the highest expression in cyanobacteria. Then, the inventors of the present application have identified a base sequence that is particularly important for the function of the promoter, and by transforming the base sequence, have found a modified promoter that can adjust the activity of the promoter to a few % to about 280%. Thus, it is possible to easily select a promoter suitable for regulating the expression of any gene in various microorganisms represented by, for example, cyanobacteria. For example, by applying the modified promoter disclosed in the present invention to cyanobacteria, the expression of a gene (for example, the slr1841 gene) originally possessed by cyanobacteria can be reduced to a desired expression. As a result, the outer membrane can be peeled off to the extent that cyanobacteria can produce substances while maintaining life activities.

[0037] Therefore, the present disclosure provides the active transformation promoter that can flexibly regulate the expression of the induction target protein by transforming the gene sequence (so-called base sequence) of the promoter in cyanobacteria source. In addition, the present disclosure provides the expression vector that can regulate the expression amount of the target substance (protein) in the various microorganisms represented by cyanobacteria. In addition, the present disclosure provides the production method of the effective material (in other words, useful substance) produced by this microorganism.

[0038] (Overview of the present disclosure)

[0039] An outline of one aspect of the present disclosure is as follows.

[0040] The modified promoter involved in one scheme of the present invention is a promoter obtained by modifying a base sequence of 348 bases upstream of the start codon of the slr1841 gene (sequence number 1), or a base sequence having more than 80% homology with the base sequence shown in sequence number 1, by at least one of the following methods: (i) replacing one or more bases contained in the base sequence with other bases, (ii) deleting one or more bases from the base sequence, and (iii) inserting one or more bases into the base sequence.

[0041] Thus, the modified promoter can flexibly regulate the expression level of any gene downstream of the modified promoter.

[0042] For example, a modified promoter involved in one embodiment of the present invention is a promoter obtained by modifying a region of a base sequence from 54 bases upstream to 94 bases upstream of the start codon in the base sequence shown in sequence number 1 (sequence number 2), or a region equivalent to the region of the base sequence shown in sequence number 2, by at least one of (i) replacing one or more bases contained in the region with other bases, (ii) deleting one or more bases contained in the region, and (iii) inserting one or more bases into the region.

[0043] Thus, the modified promoter is more susceptible to promoter activity because the region of the base sequence shown in SEQ ID NO: 2, which is the core region of the slr1841 promoter, or a region equivalent to the region, is modified. Therefore, the modified promoter can more flexibly regulate the expression level of any gene downstream of the modified promoter.

[0044] The modified promoter involved in one embodiment of the present invention is a promoter obtained by modifying the region of the base sequence TACAAT from 62 bases upstream to 67 bases upstream of the start codon in the base sequence shown in sequence number 1, or a region equivalent to the region of the base sequence TACAAT, by at least one of (i) replacing one or more bases contained in the region with other bases, (ii) deleting one or more bases contained in the region, and (iii) inserting one or more bases into the region.

[0045] Thus, the modified promoter is modified by modifying the TACAAT region of the base sequence of the -10 region of the slr1841 gene promoter, or a region equivalent to the region, so that the binding specificity of the basic transcription factor and the promoter can be easily changed. Therefore, since the promoter activity of the modified promoter is easily regulated, the expression level of any gene downstream of the modified promoter can be more flexibly regulated.

[0046] The modified promoter involved in one embodiment of the present invention is a promoter obtained by modifying the base sequence TTCTCC from 89 bases upstream to 94 bases upstream of the start codon in the base sequence shown in sequence number 1, or a region equivalent to the base sequence TTCTCC, by at least one of (i) replacing one or more bases contained in the region with other bases, (ii) deleting one or more bases contained in the region, and (iii) inserting one or more bases into the region.

[0047] Thus, the modified promoter is modified by modifying the region of the base sequence TTCTCC, which is the -35 region of the slr1841 gene promoter, or a region equivalent to this region, so that the binding specificity of the basic transcription factor (transcription initiation factor) and the promoter can be easily changed. Therefore, since the promoter activity of the modified promoter is easily regulated, the expression level of any gene downstream of the modified promoter can be more flexibly regulated.

[0048] An expression vector according to one embodiment of the present disclosure comprises any of the modified promoters described above.

[0049] Thus, the expression vector can express any gene downstream of the modified promoter at a desired expression efficiency in transformed cells.

[0050] A microorganism according to one embodiment of the present disclosure comprises the above-mentioned expression vector.

[0051] Thereby, the microorganism can produce the substance encoded by the gene expressed by the above-mentioned expression vector with desired efficiency.

[0052] A microorganism according to one embodiment of the present disclosure has any of the above-mentioned modified promoters introduced into its genome.

[0053] Thus, the microorganism can produce a substance encoded by an arbitrary gene at a desired efficiency by modifying the expression of the gene downstream of the promoter.

[0054] The modified cyanobacterium according to one embodiment of the present disclosure has any of the above-mentioned modified promoters introduced into its genome.

[0055] Thus, the modified cyanobacteria can express Slr1841 at a desired expression level because the modified promoter whose expression level of the slr1841 gene is flexibly regulated is included in the genome. For example, if the modified cyanobacteria is introduced into the genome with the promoter activity lowered to a degree that can maintain life activities, the outer membrane can be peeled off while maintaining life activities, and the metabolites produced in the cell become easy to leak out of the cell.

[0056] A method for producing a substance according to one embodiment of the present disclosure comprises culturing a microorganism comprising the above-mentioned expression vector or a microorganism into which any of the above-mentioned modified promoters has been introduced into its genome, thereby causing the microorganism to produce a substance.

[0057] Therefore, the method for producing a substance can produce a substance by culturing a microorganism, and thus any substance can be produced efficiently.

[0058] A method for producing a substance according to one embodiment of the present disclosure involves culturing the modified cyanobacteria described above, thereby causing the modified cyanobacteria to produce a substance.

[0059] Therefore, the method for producing a substance can produce a substance by culturing and modifying cyanobacteria, and thus any substance can be produced efficiently.

[0060] A method for producing a modified promoter involved in one embodiment of the present invention is to produce a modified promoter by at least one of (i) replacing one or more bases contained in the base sequence with other bases, (ii) deleting one or more bases from the base sequence, and (iii) inserting one or more bases into the base sequence, for a base sequence of 348 bases upstream of the start codon of the slr1841 gene (sequence number 1), or a base sequence having more than 80% homology with the base sequence shown in sequence number 1.

[0061] Thus, the method for producing a modified promoter can produce a modified promoter that can flexibly regulate the expression level of any gene downstream of the modified promoter.

[0062] Hereinafter, embodiments will be described in detail with reference to the drawings.

[0063] In addition, the embodiments described below all show general or specific examples. The numerical values, materials, steps, order of steps, etc. shown in the following embodiments are examples and do not limit the subject matter of the present disclosure. In addition, the constituent elements not recorded in the independent claims showing the highest concept among the constituent elements in the following embodiments are described as arbitrary constituent elements.

[0064] In addition, each figure does not necessarily illustrate the exact details. In each figure, the same reference numerals are given to substantially the same components, and the overlapping description may be omitted or simplified.

[0065] In addition, the numerical ranges below do not only represent strict meanings but also include substantially equivalent ranges, for example, measuring the expression amount (eg, number or concentration, etc.) of a protein or a range thereof.

[0066] In this specification, the term "cell" refers to an individual microorganism (for example, cyanobacteria, etc.).

[0067] (Implementation Method)

[0068] [1. Definition]

[0069] In this specification, the homology of the base sequence and the amino acid sequence is calculated by the BLAST (Basic Local Alignment Search Tool) algorithm. Specifically, it is calculated by pairwise analysis using the BLAST algorithm that can be used on the website of NCBI (National Center for Biotechnology Information) (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi). Information on the base sequence of a gene and the amino acid sequence of the protein encoded by the gene is disclosed on the NCBI website, for example, and can be referenced at any time.

[0070] In addition, the so-called promoter is a region that functions as a transcription initiation portion when a gene is transcribed from a genome. In other words, a promoter is a regulatory sequence (regulatory region) related to the efficiency of the transcription initiation reaction of a gene, and has a broad meaning that includes both regulatory sequences (regulatory regions) that exist relatively near the transcription initiation site and function, and regulatory sequences (regulatory regions) that exist away from the transcription initiation site and function. For example, in true bacteria, a promoter generally contains sequences from the -35 region 35 bases upstream of the transcription initiation site of a gene to the -10 region 10 bases upstream, and the sequences of these regions are varied. For example, it is known that the common sequences of the -35 region and the -10 region in Escherichia coli are, for example, TTGACA and TATAAT (Non-patent literature 2: Calvin B. Harley et al., "Analysis of E. colipromoter sequences", Nucleic Acid Research, Volume 15, Issue 5, 11 March 1987, Pages 2343-2361, https: / / doi.org / 10.1093 / nar / 15.5.2343).

[0071] [2. Modification of promoter]

[0072] Next, the modified promoter involved in the present embodiment is described. The modified promoter involved in the present embodiment is a promoter modified by replacing, deleting or inserting one or more bases in the base sequence of the promoter of the slr1841 gene present in the genomic DNA of cyanobacteria. The modified promoter can flexibly regulate the promoter activity.

[0073] Here, the slr1841 promoter will be described. Figure 1 is a schematic diagram showing the promoter region of slr1841. Figure 2 The figure shows the position of the transcription start site (TSS) of the slr1841 gene.

[0074] like Figure 1 As shown, the slr1841 promoter region is a region of a 348-base base sequence (sequence number 1) existing upstream of the start codon of the slr1841 gene, and is a region that plays an important role in controlling the timing and expression amount of the slr1841 gene expression. There has been no knowledge about the base sequence of the slr1841 promoter of cyanobacteria and the activity of the promoter, but the inventors of the present application analyzed the base sequence of the slr1841 promoter by 5'-RACE (5'-rapid amplification of cDNA ends).

[0075] like Figure 2 As shown, the transcription start site (TSS) of the slr1841 gene is located 54 bases upstream of the start codon. The region from the transcription start site to -35 is called the core region (core promoter), specifically the region of the base sequence (sequence number 2) from 54 bases upstream to 94 bases upstream of the start codon. The -35 region of the slr1841 promoter is the region of the base sequence TTCTCC from 89 bases upstream to 94 bases upstream of the start codon. In addition, the -10 region of the slr1841 promoter is the region of the base sequence TACAAT from 62 bases upstream to 67 bases upstream of the start codon.

[0076] In addition, the base sequence of the modified promoter can be a base sequence having more than 80% homology with the base sequence of the slr1841 promoter (sequence number 1), or a base sequence having preferably more than 90%, more preferably more than 95%, and particularly preferably more than 98% homology.

[0077] The slr1841 gene is a gene encoding Slr1841, which is one of the proteins with the highest expression in the cells of cyanobacteria. Slr1841 is an SLH domain-retaining outer membrane protein, including a C-terminal side region buried in the lipid membrane (also called outer membrane) and an SLH domain on the N-terminal side protruding from the lipid membrane (so-called outer membrane). It is widely distributed in cyanobacteria and bacteria of the Negativicutes class, which is a group of Gram-negative bacteria. The region of the SLH domain-retaining protein buried in the lipid membrane (i.e., outer membrane) forms a channel that enables the outer membrane permeation of hydrophilic substances, and the SLH domain has the function of binding to the cell wall. Examples of genes encoding SLH domain-retaining outer membrane proteins include slr1841 or slr1908 retained by Synechocystis sp. PCC 6803, or oprB retained by Anabaena sp. 90.

[0078] In addition, the promoter is not particularly limited as long as it is a promoter of a gene that is constant and strongly induced to be expressed among genes possessed by true bacteria. For example, the promoter may be a base sequence of a promoter that induces a series of proteins that are expressed in the cell in the largest amount, such as slr1841, slr0688, and slr1908, which are membrane proteins, maintained by the cyanobacterium Synechocystis sp. PCC 6803, or a region equivalent to the base sequence of the above promoter maintained by the cyanobacteria genus Synechococcus, genus Anabaena, and genus Nostoc.

[0079] [3. Production of modified promoter (also called construction)]

[0080] The modified promoter can be produced as follows. Figure 3 This is a flowchart showing an example of a method for preparing a modified promoter.

[0081] like Figure 3 As shown, the modification position and modification content in the base sequence of slr1841 promoter (SEQ ID NO: 1) or a base sequence having 80% or more homology with the base sequence of slr1841 promoter (SEQ ID NO: 1) are selected (step S1).

[0082] For example, the modification position may be within the promoter region, and the modification method is not particularly limited. For example, the modification position is within the region of the base sequence shown in sequence number 1 (i.e., the slr1841 promoter region), preferably within the region of the base sequence shown in sequence number 2 corresponding to the main body of the promoter (i.e., the core region), more preferably within the region of the base sequence TACAAT corresponding to the -10 region or the region of the base sequence TTCTCC corresponding to the -35 region. Thus, the effect on the promoter activity becomes greater.

[0083] Next, the base sequence of the slr1841 promoter (SEQ ID NO. 1) or a base sequence having 80% or more homology to the base sequence of the slr1841 promoter (SEQ ID NO. 1) is modified by (i) replacing, (ii) deleting, or (iii) inserting one or more bases (step S2). Thus, a modified promoter having a modified base sequence in the promoter region is prepared.

[0084] In addition, the modification position may be within a region of a base sequence having 80% or more homology with any of these base sequences. For example, the modification method may be a method of modifying by (i) replacing one or more (e.g., about 1 to 3 bases) bases contained in the base sequence of the promoter with other bases, (ii) deleting one or more (e.g., about 1 to 3 bases) bases from the base sequence of the promoter, or (iii) inserting one or more (e.g., about 1 to 3 bases) bases into the base sequence of the promoter.

[0085] As above, the transformation promoter can be constructed by artificial gene synthesis, and the means of construction is not particularly limited. For example, the transformation promoter can be by extracting genomic DNA from cells (for example, cyanobacteria), by the genomic DNA by PCR (Polymerase Chain Reaction) method amplification of the base sequence of the target promoter region (for example, slr1841 promoter region), the resulting gene fragment is imported into the plasmid, then transformed, so as to construct. Site-directed mutagenesis is a useful method for introducing mutations at specific sites in the plasmid, and gene replacement, deletion or insertion can be performed. Such mutation introduction generally uses the inverse PCR method, and it is easy to use the site-directed mutagenesis kits such as PrimeSTAR (registered trademark) Mutagenesis Basal Kit of タカラバイオ or KOD-Plus-Mutagenesis Kit of Toyobo.

[0086] [4. Evaluation of modified promoter activity]

[0087] As a method for evaluating the activity of the modified promoter, the expression vector to which the base sequence encoding any protein is added downstream of the modified promoter can be introduced into the cell, and the expression of the protein can be analyzed to evaluate. The type of the cell into which the expression vector is introduced and the method for introducing the expression vector into the cell are not particularly limited, but it is simple to introduce the cells of commercially available Escherichia coli with heat shock methods. In addition, the type of protein that makes the modified promoter express the inducible gene is not particularly limited, for example, it can be a protein with a relatively stable three-dimensional structure such as green fluorescent protein (GFP). In addition, the analysis method of the expression of the protein is not particularly limited. For example, when the protein is GFP, by carrying out the determination of the fluorescence intensity emitted from the cell or using GFPtag antibody to carry out protein imprinting to the total protein extracted from the cell, the expression of GFP can be analyzed.

[0088] [5. Application examples of modified promoters]

[0089] Examples of applications of modified promoters include, but are not limited to, efficient production of useful substances using microorganisms, promotion of secretion of substances produced in cells, improvement of drug resistance, and improvement of environmental resistance. Microorganisms will be described later.

[0090] The above-mentioned useful substances and substances produced in cells can be exemplified by proteins, amino acids, nucleic acid-related substances, and antibiotics. In addition, the production method of the substance can be a method of producing by culturing microorganisms so that the microorganisms secrete the target substance (useful substances and substances produced in cells) outside the cells, and appropriately separating the target substance from the culture solution, but it is not particularly limited. For example, the method of appropriately separating the target substance from the culture solution can be recovered by using a permeable membrane that passes through the target substance. In this way, since the target substance secreted into the culture solution can be recovered while culturing the microorganism, there is no need to remove the microorganism from the culture solution. Therefore, the target substance can be produced more simply and efficiently.

[0091] [6. Expression vector]

[0092] The expression vector involved in this embodiment includes a modified promoter. More specifically, the expression vector is a plasmid or virus designed for gene expression in cells. For example, the expression vector is introduced into the plasmid with a gene fragment having a base sequence encoding any protein added downstream of the modified promoter.

[0093] Figure 4 is a schematic diagram of an example of an expression vector. Figure 4 In the invention, the arbitrary protein whose expression is regulated by the modified promoter is GFP. Figure 4The expression vector shown is constructed by inserting a gene encoding GFP into a plasmid downstream of the slr1841 promoter. The activity of the modified promoter can be evaluated by introducing the expression vector into Escherichia coli and measuring the fluorescence of GFP expressed in the transformed cells.

[0094] In addition, the location for designing and constructing the modified promoter is not limited to the outside of the microorganism cell, such as the construction of an expression vector, but the modified promoter can be constructed directly from the target promoter on the genomic DNA in the cell, so that the modified promoter functions in the cell. The method for modifying the genomic DNA is not particularly limited, and can be any of gene recombination, genome editing, or mutation introduction. For example, as a method for modifying the genomic DNA of cyanobacteria, a gene recombination method using natural transformation can be used (Non-patent document 3: Hirose Yura, Photosynthesis Research Methods, Low Temperature Science, Vol. 67, Institute of Low Temperature Science, Hokkaido University, 2008, pp. 9-15, ISSN: 1880-7593, http: / / hdl.handle.net / 2115 / 39084), or a genome editing method using the CRISPR-Cpf1 system (Non-patent document 4: Justin Ungerer et al., "Cpf1 Is A Versatile Tool for CRISPR Genome Editing Across Diverse Species of Cyanobacteria", Scientific Reports, Volume 6, 2016, Article number: 39681).

[0095] [7. Microorganisms]

[0096] The microorganism involved in the present embodiment can contain the expression vector that comprises the transformation promoter in cell, and can also be imported into the transformation promoter on the genome.Microorganism can be for example, the true bacteria that operations such as escherichia coli, subtilis or cyanobacteria are easier, but is not limited to these examples. In addition, when microorganism is cyanobacteria, the cyanobacteria that has imported the transformation promoter on the genome is called transformation cyanobacteria. These microorganisms can bring into play various functions by the different types of genes that are inserted into the downstream of the transformation promoter. For example, the productivity of this microorganism can be improved, and the defense function of the cell like drug resistance or environmental tolerance can also be improved.

[0097] [8. Effects, etc.]

[0098] As described above, the modified promoter involved in the present embodiment is a promoter obtained by modifying a base sequence of 348 bases upstream of the start codon of the slr1841 gene (sequence number 1), or a base sequence having more than 80% homology with the base sequence shown in sequence number 1, by at least one of (i) replacing one or more bases contained in the base sequence with other bases, (ii) deleting one or more bases from the base sequence, and (iii) inserting one or more bases into the base sequence.

[0099] Thus, the modified promoter can flexibly regulate the expression level of any gene downstream of the modified promoter.

[0100] For example, the modified promoter involved in the present embodiment can be a promoter obtained by modifying the region of the base sequence from 54 bases upstream to 94 bases upstream of the start codon in the base sequence shown in sequence number 1 (sequence number 2), or a region equivalent to the region of the base sequence shown in sequence number 2, by at least one of (i) replacing one or more bases contained in the region with other bases, (ii) deleting one or more bases contained in the region, and (iii) inserting one or more bases into the region.

[0101] Thus, the modified promoter is likely to be greatly affected by promoter activity because the region of the base sequence shown in SEQ ID NO: 2, which is the core region of the slr1841 promoter, or a region equivalent to the region, is modified. Therefore, the modified promoter can more flexibly regulate the expression level of any gene downstream of the modified promoter.

[0102] The modified promoter involved in the present embodiment can be a promoter obtained by modifying the region of the base sequence TACAAT from 62 bases upstream to 67 bases upstream of the start codon in the base sequence shown in sequence number 1, or a region equivalent to the region of the base sequence TACAAT, by at least one of (i) replacing one or more bases contained in the region with other bases, (ii) deleting one or more bases contained in the region, and (iii) inserting one or more bases into the region.

[0103] Thus, the modified promoter is modified by modifying the region of the base sequence TACAAT, which is the -10 region of the slr1841 gene promoter, or a region equivalent to the region, so that the binding specificity of the basic transcription factor and the promoter can be easily changed. Therefore, since the promoter activity of the modified promoter is easily regulated, the expression level of any gene downstream of the modified promoter can be more flexibly regulated.

[0104] The modified promoter involved in the present embodiment can be a promoter obtained by modifying the base sequence TTCTCC from 89 bases upstream to 94 bases upstream of the start codon in the base sequence shown in sequence number 1, or a region equivalent to the base sequence TTCTCC, by at least one of (i) replacing one or more bases contained in the region with other bases, (ii) deleting one or more bases contained in the region, and (iii) inserting one or more bases into the region.

[0105] Thus, the modified promoter is modified by modifying the region of the base sequence TTCTCC, which is the -35 region of the slr1841 gene promoter, or a region equivalent to this region, so that the binding specificity of the basic transcription factor (transcription initiation factor) to the promoter can be easily changed. Therefore, since the promoter activity of the modified promoter is easily regulated, the expression level of any gene downstream of the modified promoter can be more flexibly regulated.

[0106] The expression vector involved in this embodiment contains any of the modified promoters described above.

[0107] Thus, the expression vector can express any gene downstream of the modified promoter at a desired expression efficiency in transformed cells.

[0108] The microorganism according to this embodiment includes the above-mentioned expression vector.

[0109] Thus, the microorganism can produce the substance encoded by the gene expressed by the above-mentioned expression vector with desired efficiency.

[0110] The microorganism according to the present embodiment has any of the above-mentioned modified promoters introduced into its genome.

[0111] Thus, by modifying the expression of any gene downstream of the promoter, the microorganism can produce a substance encoded by the gene at a desired efficiency.

[0112] The modified cyanobacterium according to the present embodiment has any of the above-mentioned modified promoters introduced into its genome.

[0113] Thus, the modified cyanobacteria can express Slr1841 at a desired expression level because the modified promoter that can flexibly regulate the expression level of the slr1841 gene is included in the genome. For example, if the modified cyanobacteria is introduced into the genome and the promoter activity is regulated to a level as low as that capable of maintaining life activities, the outer membrane is peeled off while maintaining life activities, and the metabolites produced in the cell are easily leaked out of the cell.

[0114] The method for producing a substance according to the present embodiment is to culture a microorganism containing the above-mentioned expression vector or a microorganism into which any of the above-mentioned modified promoters has been introduced into its genome, thereby causing the microorganism to produce a substance.

[0115] Therefore, the method for producing a substance can produce a substance by culturing a microorganism, and thus any substance can be produced efficiently.

[0116] The method for producing a substance according to the present embodiment cultivates the above-mentioned modified cyanobacteria to cause the modified cyanobacteria to produce a substance.

[0117] Therefore, the method for producing a substance can produce a substance by culturing and modifying cyanobacteria, and thus any substance can be produced efficiently.

[0118] The method for producing a modified promoter involved in the present embodiment is to produce a modified promoter by selecting at least one of (i) replacing one or more bases contained in the base sequence with other bases, (ii) deleting one or more bases from the base sequence, and (iii) inserting one or more bases into the base sequence for a base sequence of 348 bases upstream of the start codon of the slr1841 gene (sequence number 1), or a base sequence having more than 80% homology with the base sequence shown in sequence number 1, to produce the modified promoter.

[0119] Thus, the method for producing a modified promoter can produce a modified promoter that can flexibly regulate the expression level of any gene downstream of the modified promoter.

[0120] Example

[0121] Hereinafter, the modified promoter, expression vector, microorganism, production method of a substance, modified cyanobacteria, and preparation method of the modified promoter of the present disclosure will be specifically described through examples, but the present disclosure is not limited to the following examples and is not subject to any limitation of the following examples.

[0122] (Example 1) Sequence determination of slr1841 promoter

[0123] In Example 1, the sequence of the promoter of the slr1841 gene encoding the SLH domain-retaining outer membrane protein was determined.

[0124] Specifically, first, total RNA (ribonucleic acid) was extracted from cells of the cyanobacterium Synechocystis sp. PCC 6803. Then, cDNA was synthesized from the obtained total RNA, and after the ends were blunted, the adapter was combined to complete the PCR library. The cDNA was diluted and used as a template, and the obtained fragment was subcloned into a T-vector by 5'-RACE, and the sequence was analyzed. Figure 5This is an electrophoresis image of the fragments obtained by 5'-RACE. Figure 5 The left end of the screen shows the electrophoresis image of the molecular weight marker (Gene Ladder Wide2, Nippondin), and the second image from the left shows the electrophoresis image of the control (SMARTer (registered trademark) RACE 5' / 3' Kit, Clontech). Figure 5 As shown, it was confirmed that the amplified fragment of the slr1841 gene was obtained by 5'-RACE.

[0125] In addition, the results of sequence analysis determined the base sequence of the slr1841 promoter (SEQ ID NO: 1). Furthermore, the transcription start site (TSS) of the slr1841 gene was determined at a position 54 bases upstream of the start codon (refer to Figure 1 , Figure 2 ), the region of the base sequence TTCTCC from 89 bases upstream to 94 bases upstream of the start codon was inferred as the -35 region, and the region of the base sequence TACAAT from 62 bases upstream to 67 bases upstream of the start codon was inferred as the -10 region.

[0126] Next, a plasmid DNA containing a sequence from the start codon to 348 bases upstream (SEQ ID NO: 1) including the main part of the promoter located upstream of the transcription start site (TSS) was prepared, and a DNA sequence encoding a gene for green fluorescent protein (GFP) was introduced immediately downstream thereof (see Figure 3 The constructed GFP expression vector using the slr1841 promoter was transformed into Escherichia coli HST08 cells and cyanobacterium Synechocystis sp. PCC6803 cells, and the obtained cells were cultured to confirm whether they emitted fluorescence. The results are shown in Figure 6 .

[0127] Figure 6 It is a graph showing the results of measuring the fluorescence intensity of the culture solutions of cyanobacteria and Escherichia coli after transformation. Figure 6 (a) shows the results of measuring the fluorescence intensity of the culture solution of untransformed Escherichia coli (WT in the figure) and the culture solution of transformed Escherichia coli (GFP in the figure). Figure 6 (b) shows the results of measuring the fluorescence intensity of the culture solution of untransformed cyanobacteria (WT in the figure) and the culture solution of transformed cyanobacteria (GFP). The fluorescence intensity is the intensity value of the fluorescence of GFP generated by the excitation light of wavelength 488nm using a fluorescence spectrophotometer. Figure 6 (a) and Figure 6 As shown in (b), it was confirmed that all transformed cells of Escherichia coli and cyanobacteria emitted fluorescence.

[0128] (Example 2) Production of modified promoter

[0129] In Example 2, the slr1841 promoter obtained in Example 1 was used to prepare a modified promoter.

[0130] Specifically, the plasmid containing the slr1841 promoter obtained in Example 1 was used to introduce mutations into the promoter by reverse PCR. The position of introduction of the mutation focused on the main part of the promoter located upstream of the transcription start site (TSS), especially the part presumed to be the -10 region and the -35 region, and mutations caused by substitutions or defects with a width of 1 to several hundred bases were introduced at various positions represented by the surrounding parts. The GFP expression vector containing the modified promoter prepared in this way was transformed into the cells of Escherichia coli HST08, and the activity of the promoter was evaluated by comparing the cells of the transformed Escherichia coli of Example 1 with the fluorescence intensity. The fluorescence intensity of each transformed cell is the fluorescence intensity of the culture solution of each transformed cell, which is the average value obtained by measuring the fluorescence intensity value of GFP generated by excitation light of a wavelength of 488nm at 525nm three times using a fluorescence spectrophotometer. In Example 2, the promoter activity was evaluated by comparing the fluorescence intensity of the culture solution of each transformed cell with the fluorescence intensity value (525 nm) of the culture solution of the transformed Escherichia coli cells in Example 1. The results are shown in Fig. 7A and Figure 7B . Fig. 7A The graph shows the structure of a modified promoter in which a mutation is introduced into the upstream region of the transcription start site (TSS) of the slr1841 promoter and the results of measuring the fluorescence intensity of the culture fluid of the transformed cells into which the modified promoter is introduced. Figure 7B This is a graph showing the base sequence of the -10 region into which a mutation has been introduced, and the results of measuring the fluorescence intensity of the culture fluid of the transformed cell into which the modified promoter into which the mutation has been introduced has been introduced. Fig. 7A No.1 and Figure 7B No. 21 shows the result of introducing the original promoter (ie, slr1841 promoter) without modification into E. coli.

[0131] like Fig. 7A As shown in , if a mutation is introduced into the main part of the promoter located upstream of the transcription start site (TSS), various modified promoters with promoter activities ranging from several % to about 280% are obtained. Figure 7B As shown, even when a mutation was introduced into the -10 region, various modified promoters with promoter activities ranging from several % to 60% were obtained.

[0132] (Example 3) Genome modification of cyanobacteria

[0133] In Example 3, the base sequence of the slr1841 promoter on the genome of cyanobacteria was modified to form a modified promoter structure into which a mutation that reduces the activity of the promoter was introduced in Example 2. Thus, a modified cyanobacterium in which the expression level of the SLH domain-retaining outer membrane protein of the cyanobacterium was reduced and outer membrane stripping was induced was prepared.

[0134] Specifically, in the genomic DNA of the cyanobacterium Synechocystis sp. PCC 6803, a mutation that reduced the promoter activity in the modified promoter obtained in Example 2 was introduced into the region of the slr1841 promoter that originally existed by genome editing using CRISPR-Cpf1. The mutation introduced into the genomic DNA of the cyanobacterium was Fig. 7A The mutation introduced into the slr1841 promoter in No.2 Figure 7B The mutation introduced into the -10 region in No.29 of Figure 7B A mutation was introduced into the -10 region in No.34.

[0135] The modified cyanobacteria (referred to as No. 2 strain, No. 29 strain, and No. 34 strain) and the cyanobacterium Synechocystis sp. PCC 6803 (referred to as WT strain) obtained by introducing the above mutations were cultured separately. Then, the concentration of amino acids contained in the culture supernatant of these cells was analyzed by LC-MS (Liquid Chromatograph-Mass Spectrometry). The analysis results are shown in Figure 8 . Figure 8 It is a figure which shows the analysis result of the culture supernatant by LC-MS.

[0136] like Figure 8 As shown, the concentrations of seven amino acids, namely, isoleucine (Ile), leucine (Leu), phenylalanine (Phe), arginine (Arg), valine (Val), tyrosine (Tyr), and serine (Ser), were analyzed. The culture supernatants of strains No. 2, No. 29, and No. 34 all contained more types of amino acids than the WT strain.

[0137] The promoter activity of strain No. 34 was 27% (ref. Figure 7B ), the types of amino acids leaked out of the cells were the largest in the three modified cyanobacteria prepared in Example 3, and the concentrations of any amino acid were higher than those of the WT strain.

[0138] The promoter activity of strain No. 29 was 24% (ref. Figure 7B), the types of amino acids leaked out of the cells were the largest as in No.34, but arginine (Arg) was equivalent to that of the WT strain.

[0139] In addition, the promoter activity of strain No. 2 was 41% (ref. Fig. 7A ), the three modified cyanobacteria prepared in Example 3 had the least number of amino acids leaked out of the cell, but it was also one more than the WT strain.

[0140] Next, the antibiotic ampicillin (concentrations: 0 μg / mL, 0.01 μg / mL, 0.1 μg / mL, 1 μg / mL) was added to the culture medium of the modified cyanobacteria (referred to as the modified strain) and the cyanobacterium Synechocystis sp. PCC 6803 (referred to as the wild strain) with outer membrane peeling, and the antibiotic resistance of the modified strain and the wild strain was confirmed. Figure 7B The results are shown in Fig. 9 . Fig. 9 The graph shows the results of antibiotic resistance tests of transformed strains and wild-type strains.

[0141] like Fig. 9 As shown, when 0.1 μg / mL of the antibiotic ampicillin was added to the culture medium, the wild-type strain grew and multiplied without problems, whereas the modified strain was killed. These results suggest that if the expression level of slr1841 is reduced by modifying the slr1841 promoter on the genomic DNA, the outer membrane is separated and the secretion and production of useful substances are promoted, while the resistance to antibiotics from outside the cell is reduced.

[0142] Industrial Availability

[0143] According to the present disclosure, the activity of the modified promoter can be flexibly adjusted, so a promoter having an activity suitable for the type of microorganism and the target substance can be easily selected. Therefore, the present disclosure can be applied not only in the fields of food, chemistry, and medicine, but also in a wide range of fields such as the environmental field.

Claims

1. A modified promoter, which is a base sequence of 348 bases upstream of the start codon of the slr1841 gene, i.e., sequence number 1, or a base sequence having more than 80% homology with the base sequence shown in sequence number 1, by modifying the base sequence by at least one of (i) replacing one or more bases contained in the base sequence with other bases, (ii) deleting one or more bases from the base sequence, and (iii) inserting one or more bases into the base sequence.

2. The modified promoter according to claim 1 is a promoter obtained by modifying the base sequence 54 to 94 bases upstream of the start codon in the base sequence shown in sequence number 1, i.e., the region of sequence number 2, or a region equivalent to the region of the base sequence shown in sequence number 2, by at least one of (i) replacing one or more bases contained in the region with other bases, (ii) deleting one or more bases contained in the region, and (iii) inserting one or more bases into the region.

3. The modified promoter according to claim 1 is a promoter obtained by modifying a region of the base sequence TACAAT located 62 to 67 bases upstream of the start codon in the base sequence shown in sequence number 1, or a region equivalent to the region of the base sequence TACAAT, by at least one of the following methods: (i) replacing one or more bases contained in the region with other bases, (ii) deleting one or more bases contained in the region, and (iii) inserting one or more bases into the region.

4. The modified promoter according to claim 1 is a promoter obtained by modifying the base sequence TTCTCC from 89 to 94 bases upstream of the start codon in the base sequence shown in sequence number 1, or a region equivalent to the base sequence TTCTCC, by at least one of (i) replacing one or more bases contained in the region with other bases, (ii) deleting one or more bases contained in the region, and (iii) inserting one or more bases into the region.

5. An expression vector comprising the modified promoter according to any one of claims 1 to 4. A microorganism comprising the expression vector according to claim 5.

7. A microorganism, wherein the modified promoter according to any one of claims 1 to 4 is introduced into its genome.

8. A modified cyanobacterium, wherein the modified promoter according to any one of claims 1 to 4 is introduced into the genome.

9. A method for producing a substance, wherein: The microorganism according to claim 6 is cultured to allow the microorganism to produce a substance.

10. A method for producing a substance, wherein: The microorganism according to claim 7 is cultured so that the microorganism produces a substance.

11. A method for producing a substance, wherein: The modified cyanobacteria according to claim 8 are cultivated so that the modified cyanobacteria produce substances.

12. A method for producing a modified promoter, wherein: For the base sequence of 348 bases upstream of the start codon of the slr1841 gene, i.e., sequence number 1, or a base sequence having more than 80% homology with the base sequence shown in sequence number 1, a modified promoter is produced by at least one of (i) replacing one or more bases contained in the base sequence with other bases, (ii) deleting one or more bases from the base sequence, and (iii) inserting one or more bases into the base sequence.

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