Substance production of strains

Abstract

Provided is a method for producing a microbial strain that activates a gene group and exhibits desired properties such as high yield and inhibition of by-products. In detail, the present application provides, in one aspect, a method for producing a strain having desired properties, the method comprising the steps of: 1) providing a plurality of constructs comprising a gene cluster containing a plurality of genes, said constructs being formed by monocistronic configuration of promoters; (2) carrying out combinatorial library on the promoter in the construct, and introducing the promoter into a target host cell; and 3) a step for screening a strain having desired properties from the host cell.

Description

TECHNICAL FIELD

[0001] The present application relates to a production of a substance of a strain having a desired property. BACKGROUND

[0002] So far, about half of the drugs developed and used by humans are natural products or substances derived from natural products (non-patent literature 1). It is estimated that the number of natural products available to humans so far has reached saturation, but the results of genome sequencing of microorganisms indicate that there are still a large number of natural products remaining in the genomes of microorganisms, most of which cannot be produced under usual laboratory conditions (dormant state) or have extremely low yields, and therefore cannot be detected (non-patent literature 2). If these biosynthetic gene groups of natural products can be forcibly activated to produce substances efficiently, it is expected to obtain unprecedented new substances, or to obtain useful substances efficiently.

[0003] Among natural products, polyketides (PKs) and non-ribosomal peptides (NRPs) are groups of natural products produced by various microbial species such as actinomycetes, filamentous fungi, and Bacillus subtilis, and these natural products contain many physiologically active molecules such as antibacterial and anticancer molecules that are useful to humans. However, these molecules have very complex structures, and therefore molecules that cannot or are difficult to be produced commercially by chemical synthesis account for a large portion. That is, in order to apply these to industry, a biological production method is required. The gene groups of polyketide synthases (PKSs) or non-ribosomal peptide synthetases (NRPSs) contained in the genomes of microorganisms are usually very long chains, and contain a large number of repetitive sequences, high GC content, or high AT content, and therefore are DNA sequences that are difficult to synthesize by existing methods.

[0004] Therefore, a construct in which a control factor or the like is replaced has been constructed by cloning from the genome of the target microorganism and loading onto a target vector, and used for homologous or heterologous production. As an example thereof, an example of increasing the yield by replacing a stronger promoter than the wild type, achieved by homologous recombination of yeast (non-patent literature 3) or CRISPR-Cas9 (non-patent literature 4, patent literature 1) and the like technologies, has been reported.

[0005] However, the engineering manipulation of the sequence is greatly limited due to the complexity of the sequence of the gene cluster. In addition, in many wild-type PKSs and NRPSs, there are many examples in which a single promoter controls the transcriptional activation of multiple constituent genes or the entire gene cluster. It is presumed that, in the activation of genes in a dormant state or in heterologous production, if the genes are realized in units of genes and the transcription is controlled as a monocistron, the proteins derived from each gene are transcribed and translated at an appropriate time during the proliferation period of the microorganism, which is effective for increasing the yield. However, in the prior art, it is actually difficult to realize due to the aforementioned reasons.

[0006] Prior art documents

[0007] Patent documents

[0008] Patent document 1: KR102301301B1

[0009] Non-patent document 1: D. J. Newman, et. al., J. Nat. Prod., 83, 770 (2020)

[0010] Non-patent document 2: C. C. Brett, et. al., Annu. Rev. Biochem., 90, 763 (2021)

[0011] Non-patent document 3: Kang H-.S., et. al., ACS Synth. Biol., 5(9), 1002 (2016)

[0012] Non-patent document 4: Kim H., et. al., ACS Synth. Biol., 9(1), 175 (2020) SUMMARY

[0013] Technical solution to the problem

[0014] From the above viewpoint, by transcribing each gene constituting a gene cluster of a biosynthesis enzyme of a natural product in a monocistron with a proliferation period-dependent promoter of an appropriate transcription strength, the present application, which produces a target substance in a host cell such as Bacillus subtilis, is completed. That is, the gist of the present application is as described below.

[0015] (Item X1)

[0016] A method of producing a strain having desired properties, the method comprising the steps of:

[0017] 1) a step of providing a construct comprising a gene cluster and a plurality of promoters;

[0018] 2) a step of librarying the promoters in the construct;

[0019] 3) a step of screening a strain having a desired property in the host cell.

[0020] (item X1-1)

[0021] The method according to the above item, wherein the gene cluster contains a plurality of genes.

[0022] (item X1-2)

[0023] The method according to any one of the above items, wherein the promoter is operably linked to the gene.

[0024] (item X1-3)

[0025] The method according to any one of the above items, further comprising the step of 1) providing a construct into which a plurality of promoters are introduced, the construct comprising a gene cluster containing a plurality of genes.

[0026] (item X2)

[0027] The method according to any one of the above items, wherein the plurality of promoters are configured in a monocistronic manner for each gene contained in the gene cluster or in a multicistronic manner for all genes in the gene cluster, or a mixture of the monocistronic and multicistronic configurations for the genes in the gene cluster.

[0028] (item X3)

[0029] The strain having a desired property has at least one or more properties selected from the group consisting of a property of being able to produce a target substance; a property of producing a large amount of a target substance; a property of keeping a target substance inside a cell without secretion; a property of secreting a target substance outside a cell; a property of being easy to cultivate along with production of a target substance; a property of being able to produce a plurality of target substances at a desired ratio; a property of producing a target substance without producing a harmful substance; a property of robustness of the yield of a target substance; a property of no deletion occurring in a gene producing a target substance during cultivation; a property of being able to control production of a target substance; a property of being able to suppress production of a substance other than a target substance; a property of being able to suppress production of a by-product and / or an impurity that is difficult to separate in affinity, ion exchange, gel filtration, reverse phase, normal phase, chiral chromatography, distillation, crystallization, and the like; a property of a low yield of empty capsids in virus vector production, and a property of no deletion occurring in a plasmid.

[0030] (item X3-1)

[0031] The method according to any one of the above items, wherein the empty capsid rate is 80% or less, preferably 70% or less, more preferably 60% or less... 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, most preferably 0%.

[0032] (Item X4)

[0033] A method of performing an action based on a desired property using a strain produced in the method of any of the above items.

[0034] (Item X4-1)

[0035] The method according to any of the above items, wherein the action includes performing a subsequent action of:

[0036] • a property of being able to produce a target substance: an action of producing a target substance

[0037] • a property of being able to produce a target substance at a high yield: an action of producing a target substance

[0038] • a property of keeping a target substance inside a cell without secretion: an action of producing a target substance and collecting it from a cell

[0039] • a property of secreting a target substance outside a cell: an action of producing a target substance and collecting it from a culture solution

[0040] • a property of being easy to culture along with production of a target substance: an action of producing a target substance under usual culture conditions

[0041] • a property of being able to produce multiple target substances at desired ratios: an action of producing multiple target substances

[0042] • a property of producing a target substance without producing a harmful substance: an action of producing a target substance without using special protective equipment

[0043] • a property of robustness of the yield of a target substance: an action of producing a target substance at a constant ratio

[0044] • a property of not occurring deletion in a gene producing a target substance during culture: an action of producing a target substance with a strain not accompanied by deletion of a gene

[0045] • a property of being able to control production of a target substance: an action of producing a target substance under desired conditions (yield, kind / ratio of derivatives)

[0046] • a property of being able to suppress production of a substance other than a target substance: an action of producing only a target substance

[0047] • a by-product difficult to separate in affinity, ion exchange, gel filtration, reverse phase, normal phase, chiral chromatography, distillation, crystallization, etc.

[0048] • a property of being able to suppress generation of impurities: an action of producing only a substance having a target structure (not making derivatives)

[0049] • the property of low yield of empty capsids in viral vector manufacturing: the behavior of producing viral capsids carrying a nucleic acid of interest

[0050] • the property of no deletion occurring in plasmids: the behavior of collecting plasmids having a sequence of interest

[0051] (Item X5)

[0052] The method according to any one of the above items, further comprising the steps of: 4) subjecting the construct of the one or more strains selected in the step 3) to combinatorial library, introducing into a host cell of interest, and 5) again performing the step 3).

[0053] (Item X5-1)

[0054] The method according to any one of the above items, wherein the combinatorial library contains a library of combinatorial plasmids in which a part of the promoter sequence in the construct plasmid is randomized.

[0055] (Item X6)

[0056] A method for producing a substance in a host organism, the method comprising the steps of:

[0057] (A) a step of constructing a construct in which a promoter introduction site is added to a gene cluster of a production subject matter in which recognition sequences of an undesirable restriction enzyme are removed, and a sequence for reconstitution is added;

[0058] (B) a step of introducing a plurality of constructs in which a promoter is introduced into the promoter introduction site into a host organism for combinatorial library;

[0059] (C) a step of combinatorially library the promoter site among the plurality of constructs by Combi-OGAB TM ;

[0060] (D) a step of culturing a production host organism having the combinatorially library construct, and selecting a plurality of strains based on an arbitrary standard;

[0061] (E) a step of again combinatorially library the promoter site among the constructs of the plurality of strains by Combi-OGAB TM ;

[0062] (F) a step of producing a substance using a strain having a desired property.

[0063] (Item X7)

[0064] The method according to any one of the above items, wherein the host organism for combinatorial library is the same as the production host organism.

[0065] (Item X8)

[0066] The method according to any one of the above items, wherein the host organism for the combinatorial library is different from the host organism for the production.

[0067] (Item X9)

[0068] The method according to any one of the above items, wherein the host organism is Bacillus subtilis.

[0069] (Item X10)

[0070] A method for producing a substance in a host organism, the method comprising the step of producing the substance with the strain produced by the method according to any one of the above items.

[0071] (Item X11)

[0072] A method for producing a substance in a host organism, the method comprising the step of producing the substance with the strain obtained by the steps (A) to (E) in the method according to any one of the above items.

[0073] (Item X12)

[0074] The method according to any one of the above items, further comprising modifying the strain.

[0075] (Item X13)

[0076] The method according to any one of the above items, comprising the step of repeating the steps (D) and (E) multiple times.

[0077] (Item X14)

[0078] The method according to any one of the above items, wherein the construction of the construct in the step (A) is performed by OGAB TM .

[0079] (Item X15)

[0080] The method according to any one of the above items, wherein the promoter is a plurality of promoters whose transcription initiation and / or transcription strength are different from each other.

[0081] (Item X16)

[0082] The method according to any one of the above items, wherein the host organism for the production is Bacillus subtilis, actinomycetes, filamentous bacteria, yeast, hydrogen bacteria, rod-shaped bacteria, or animal cells.

[0083] (Item X17)

[0084] The method according to any one of the above items, wherein the host organism is Bacillus subtilis.

[0085] (Item X18)

[0086] The method according to any one of the above items, wherein the undesired restriction enzyme recognition sequence is a Type IIS restriction enzyme recognition sequence selected from the group consisting of Aarl, Bsal, Bbsl, BsmBI, BspQl, and a Sfil recognition sequence for reconstitution.

[0087] (Item X19)

[0088] The method according to any one of the above items, wherein the reconstitution sequence is a Sfil recognition sequence.

[0089] (Item X20)

[0090] The method according to any one of the above items, wherein the gene group is a gene group of Polyketide synthase (PKS) and Non-ribosomal peptide synthase (NRPS).

[0091] Further, the gist of the present application is further described as follows.

[0092] (Item 1)

[0093] A method of producing a strain having a desired property, the method comprising the steps of:

[0094] 1) a step of providing a construct into which a plurality of promoters are introduced, the construct comprising a gene cluster containing a plurality of genes;

[0095] 2) a step of combinatorial librarying the promoters in the construct;

[0096] 3) a step of screening a strain having a desired property in a host cell.

[0097] (Item 2)

[0098] The method according to the above item, wherein the plurality of promoters are configured in a monocistronic configuration.

[0099] (Item 3)

[0100] The method according to any one of the above items, wherein the strain having a desired property is selected from at least one or more of the following strains:

[0101] • a strain capable of producing a target substance

[0102] • Strains that produce a large amount of a target substance

[0103] • Strains that retain a target substance in the cell without secretion

[0104] • Strains that secrete a target substance outside the cell

[0105] • Production strains of a target substance that are easy to culture

[0106] • Strains that can produce multiple target substances at desired ratios

[0107] • Strains that produce a target substance without producing harmful substances

[0108] • Strains in which the yield of a target substance is robust

[0109] • Strains in which no deletion occurs in genes that produce a target substance during cultivation

[0110] • Strains that can control the production of a target substance

[0111] • Strains that can suppress the production of substances other than a target substance

[0112] • By-products that are difficult to separate in affinity, ion exchange, gel filtration, reverse phase, normal phase, chiral chromatography, distillation, crystallization, and the like

[0113] • Strains that can suppress the production of impurities

[0114] • Strains that produce a small amount of empty capsids in virus vector production

[0115] • Strains in which no deletion occurs in plasmids.

[0116] (Item 4)

[0117] A method based on the behavior of a desired property is implemented using a strain produced in any of the above items.

[0118] (Item 5)

[0119] The method according to any of the above items, further comprising the steps of: 4) combining the constructs of the one or more strains selected in the step 3) into a combinatorial library, introducing into a desired host cell, and 5) performing the step 3) again.

[0120] (Item 6)

[0121] A method of producing a substance in a host organism, the method comprising the steps of:

[0122] (A) a step of constructing a construct in which a promoter introduction site is added to a group of genes involved in production of a substance for which removal of an undesirable restriction enzyme recognition sequence is desired, and a sequence for reconstitution is added;

[0123] (B) introducing a plurality of constructs into which a promoter is introduced into the promoter introduction site into a host organism for combinatorial library formation;

[0124] (C) a step of combinatorially library-forming the promoter site among the plurality of constructs by the Combi-OGAB TM method;

[0125] (D) a step of culturing a production host organism having the combinatorially library-formed construct, and screening a plurality of strains based on an arbitrary standard;

[0126] (E) a step of combinatorially library-forming the promoter site among the plurality of strains' constructs again by the Combi-OGAB TM method;

[0127] (F) a step of producing a substance using a strain having a desired property.

[0128] (Item 7)

[0129] The method according to any one of the above items, wherein the host organism for combinatorial library formation is the same as the production host organism.

[0130] (Item 8)

[0131] The method according to any one of the above items, wherein the host organism for combinatorial library formation is different from the production host organism.

[0132] (Item 9)

[0133] The method according to any one of the above items, wherein the host cell is Bacillus subtilis.

[0134] (Item 10)

[0135] A method of producing a substance in a host organism, the method comprising: a step of producing a substance using a strain produced by the method according to any one of the above items.

[0136] (Item 11)

[0137] A method of producing a substance in a host organism, the method comprising: a step of producing a substance using a strain obtained by the steps (A) to (E) in the method according to any one of the above items.

[0138] (Item 12)

[0139] The method according to any one of the above items, further comprising modifying the strain.

[0140] (Item 13)

[0141] The method according to any one of the above items, comprising a step of repeating the steps (D) and (E) multiple times.

[0142] (Item 14)

[0143] The method according to any one of the above items, wherein the construction of the construct in the step (A) is performed by OGAB TM method.

[0144] (Item 15)

[0145] The method according to any one of the above items, wherein the promoter is a plurality of promoters that differ from each other in transcription initiation and / or transcription strength.

[0146] (Item 16)

[0147] The method according to any one of the above items, wherein the production host organism is Bacillus subtilis, actinomycetes, filamentous bacteria, yeast, hydrogen bacteria, coryneform bacteria, or animal cells.

[0148] (Item 17)

[0149] The method according to any one of the above items, wherein the host organism is Bacillus subtilis.

[0150] (Item 18)

[0151] The method according to any one of the above items, wherein the undesirable restriction enzyme recognition sequence is a Type IIS restriction enzyme recognition sequence selected from the group consisting of Aarl, Bsal, Bbsl, BsmBI, Bsp QI, and a Sfil recognition sequence for reconstruction.

[0152] (Item 19)

[0153] The method according to any one of the above items, wherein the sequence for reconstruction is a Sfil recognition sequence.

[0154] (Item 20)

[0155] The method according to any one of the above items, wherein the gene group is a gene group of Polyketides synthase (PKS) and Non-ribosomal peptide synthase (NRPS).

[0156] According to one embodiment of the present application, in order to transcribe a desired gene group such as a natural product biosynthesis enzyme gene group in a single cistron unit, and to combine and screen a library of promoters for each gene, various restriction enzymes can be used. In one embodiment of the present application, in a long and complex sequence such as a natural product biosynthesis enzyme gene group, cloning from the genome of the target microorganism has been the only method in the past, but removing the restriction enzyme recognition sequence one by one is a very time-consuming and high-cost work, but this method is easy to implement, so that the screening cycle can be performed. In one embodiment of the present application, in addition, in the case of adding a new gene pathway to the natural biosynthesis system, the case of a sequence optimization of a biopharmaceutical synthesis-related gene group, and the like, long and complex sequences are often included, but in such a case, applications such as a screening cycle that can be easily performed can also be performed.

[0157] In one embodiment of the present application, OGAB TM method, but this method is an innovative technology for enriching a large number of DNA fragments and synthesizing a long chain DNA. In one application of one embodiment of the present application, this technology can be used to construct a long chain gene group designed on a computer after removing the restriction enzyme recognition sequence described above. In one application of one embodiment of the present application, by using the OGAB TM method, the promoter introduction site can be integrated into the design so that the promoter is not included at the time of construction and is subsequently introduced, and by this strategy, for each plasmid (parent plasmid) into which a promoter that becomes a combination library species is introduced, it is not necessary to enrich a large number of DNA fragments, and the promoter can be introduced into a plasmid constructed by the OGAB TM method. Further, even in the case where it is desired to add a specific promoter species depending on the experimental results, a parent plasmid can be easily prepared, and such advantages can also be applied in one embodiment of the present application.

[0158] In the production of a target substance, the target substance and intermediate substances of the metabolic process, proteins produced by the constituent genes, and the like sometimes affect the growth of the host organism (for example, antibiotics, cytotoxicity, and the like). In such a case, it is expected that by adjusting to cause production in the late proliferation period, self-tolerance and substance production are obtained (Non-Patent Literature 5). For each gene of the constituent gene group, it is not possible to predict in which proliferation period, with what transcription intensity, transcription occurs, and whether it is suitable for producing a target substance, but in one embodiment of the present application, in addition to a promoter that always shows transcription activity, a combination library of proliferation period-dependent promoters with different strengths is constructed for each gene, and screening is performed with yield as an index, and thus a strain with the best promoter combination can be obtained.

[0159] In one embodiment of the present application, B. subtilis is used, but this bacterium is a microorganism that naturally produces NRPS such as surfactin and Plipastatin, and has great potential as a host for producing natural products. In one embodiment of the present application, B. subtilis can be used in OGAB TM method. By using this method, if a propagation period-dependent promoter of B. subtilis (Reference Literature 6) is introduced, the B. subtilis into which the promoter is introduced can be directly used for substance production, and the steps of plasmid extraction, transformation into a host, and the like can be omitted, and a strain producing a target substance can be obtained at high speed.

[0160] Effects of the Invention

[0161] According to the present application, a strain showing desired properties can be obtained quickly and efficiently. The present application can also provide a method for producing a microorganism strain that activates a desired gene group including a gene group constituting a natural product biosynthesis enzyme and shows desired properties such as high productivity and inhibition of by-products, and a new substance or the like that can be produced by this method. BRIEF DESCRIPTION OF DRAWINGS

[0162] Figure 1 is a diagram showing the structure of the plasmid vector pGETS151 deltaBsmB I-pBR used for enrichment.

[0163] Figure 2 is a diagram showing the plasmid enriched with Triketide Pyrone BGC used in Example 1.

[0164] Figure 3 is a diagram showing the convergence of the yield of Pyrone-OMe with the Combi-OGAB TM cycle.

[0165] Figure 4 is a diagram showing the convergence with the increase in the methylation efficiency of BpsB and the Combi-OGAB TM cycle.

[0166] Figure 5 is a diagram showing the plasmid enriched with Gramicidin S BGC used in Example 2.

[0167] Figure 6 is a diagram showing the chromatogram of a strain producing a by-product of Gramicidin S in the strain of the Combi-OGAB TM cycle for the first time.

[0168] Figure 7 is a diagram showing the convergence of the yield of Pyrone-OMe with the Combi-OGAB TMA graph of the yield convergence case of cyclic and short bacitracin S.

[0169] Figure 8 A spectrum of a product of a strain producing a bacitracin S byproduct when analyzed by LC-MS. DETAILED DESCRIPTION

[0170] The present application is explained below while showing the best mode. Throughout the present specification, provided that there is no special mention, an expression in the singular form shall be understood to include the concept in the plural form. Therefore, provided that there is no special mention, the singular form of the article (for example, "a", "an", "the" and the like in the case of English) shall be understood to include the concept in the plural form. Furthermore, provided that there is no special mention, the terms used in the present specification shall be understood to be used in the meanings commonly used in the field. Therefore, provided that there is no other definition, all the professional terms and scientific technical terms used in the present specification have the same meanings as commonly understood by those skilled in the art to which the present application pertains. In the case of a contradiction, the present specification (including the definition) takes precedence.

[0171] The definitions of the terms used particularly in the present specification and / or the basic technical contents are explained below as appropriate.

[0172] In the present specification, the "monocistronic configuration" of a promoter refers to a single promoter configured for a single gene. On the other hand, the "multicistronic configuration" refers to a single promoter configured for multiple genes.

[0173] In the present specification, "combinatorial library" refers to making a comprehensive combination of multiple substances (for example, multiple promoters) to make a library. For example, combinatorial library includes constructing a library of combinatorial plasmids in which the promoter sequence portion is random.

[0174] In the present specification, "desired property" means a beneficial property in the production of a substance. As the desired property, there can be mentioned: a property of being able to produce a target substance; a property of producing a target substance at a high yield; a property of keeping a target substance in a cell without secretion; a property of secreting a target substance outside a cell; a property of being easy to culture along with the production of a target substance (e.g., growing under usual culture conditions (culture medium / temperature / culture time, etc.)); a property of being able to produce a plurality of target substances at a desired ratio; a property of producing a target substance without producing a harmful substance (nontoxicity, etc.); a property of the yield of a target substance being robust; a property of no deletion occurring in a gene producing a target substance during culture; a property of being able to control the production of a target substance; a property of being able to suppress the production of a substance other than a target; a property of being able to suppress the production of a by-product and / or an impurity which is difficult to separate in affinity, ion exchange, gel filtration, reverse phase, normal phase, chiral chromatography, distillation, crystallization, etc.; a property of the yield of empty capsids being small in the manufacture of a viral vector (e.g., the rate of empty capsids is 80% or less, preferably 70% or less, more preferably 60% or less... 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, most preferably 0%); a property of no deletion occurring in a plasmid, etc., but is not limited thereto.

[0175] In the present specification, "property of producing a target substance at a high yield" means a property of the yield being higher than that of a natural strain. The natural strain means a strain in which a substance production gene cluster which has not been subjected to a genetic operation on a sequence (exchange of a promoter, etc.) is added to a production host (described in claim 16). The property of producing a target substance at a high yield can be a property of being at least 10% or more, preferably 20% or more... higher than the yield of the above-described natural strain, but is not limited thereto.

[0176] In the present specification, "property of being easy to culture while producing a target substance" means a property of being able to grow under usual culture conditions used in the culture of a strain to produce a target substance. For example, it is possible to use a medium used in usual microbial culture such as LB medium of a non-selective medium, to culture under usual culture conditions such as shaking culture at 37°C for 24 hours, which indicates "not difficult culturability". Further, as for the handling of a strain, from the viewpoint of pathogenicity, etc., it is not necessary to be biosafety level (BSL) 2 or 3 which requires special equipment, but conditions which are able to be cultured in a BSL 1 laboratory of a usual laboratory are also included in the easiness of culture.

[0177] In the present specification, "a property that the yield of the target substance is robust" means a property that shows high reproducibility in which the same yield is always shown when production research is performed. As one of the methods for verifying robustness, for example, the degree of robustness of the experimental results can be evaluated by using the calculation of the Fan of factor described in Acs Synth. Biol., 11, 1686-1691. (2022).

[0178] In the present specification, "a property that no deletion occurs in the gene" means a property that no deletion of all or a part of the nucleic acid encoding the gene occurs, and no deletion of the gene or a part thereof occurs, and no deletion of the function of the gene occurs due to mutation of a part of the nucleic acid encoding the gene.

[0179] In the present specification, "removal of an undesirable restriction enzyme recognition sequence" means that an undesirable restriction enzyme recognition sequence is deleted, replaced with other bases or sequences, added to other bases or sequences, or changed to a sequence that cannot be recognized by a restriction enzyme in the method of the present application. As the undesirable restriction enzyme recognition sequence, for example, a sequence that can be recognized by OGAB TM TM In the present specification, the type of restriction enzyme used when constructing the library by the Combi-OGAB

[0180] In the present specification, "a promoter introduction site" means a site to which a promoter upstream of each gene in the gene group should be introduced.

[0181] In the present specification, "a reconstruction sequence" means a sequence for forming a restriction enzyme recognition sequence of the enriched nucleic acid. The cut enriched nucleic acid formed is reconstructed to generate a circular plasmid.

[0182] In the present specification, "OGAB TM (Ordered Gene Assembly in Bacillus subtilis, OGAB TM Method") means a method for generating a circular plasmid in a transformant by incorporating an enriched nucleic acid into the transformant. The OGAB TM ​This method allows for the easy preparation of plasmids containing large-sized nucleic acids. In this specification, the nucleic acids incorporated into transforming organisms are referred to as "enriched nucleic acids." Typically, enriched nucleic acids have a tandem repeat structure where one unit and one group of unit nucleic acids of the plasmid appear in the same direction. Using enriched nucleic acids with such structures promotes the generation of plasmids in transforming organisms.

[0183] This manual utilizes "Combi-OGAB" TM "Law", "Combination OGAB" TM The "method" or "combinatorial library construction method of Bacillus subtilis" refers to the method using OGAB. TM This method involves reconstructing a mixture of enriched nucleic acids from multiple constructs into a combination of unit nucleic acids to create a combinatorial library. See also patent number 7101431. It should be noted that "OGAB" TM "and Combi-OGAB" TM "is the trademark of the applicant".

[0184] In this specification, "actinomycetes" refers to the overall morphological characteristics of Gram-positive bacteria that proliferate by forming slender hyphae, represented by various genera under the phylum Actinobacteria. Based on the molecular systematics of the 16S rRNA gene base sequence, it is also classified as a subset of bacilli and cocci.

[0185] In this specification, "filamentous fungi" refers to fungi that multiply by spores through the formation of hyphae and are called molds.

[0186] In this specification, "polyketide synthase (PKS)" refers to a multi-domain enzyme or enzyme complex that synthesizes polyketides. It is found in bacteria, fungi, plants, and a few animals. There are three types of PKS: type I, type II, and type III. Type I PKS is a long, large protein with multiple domains linked together on a single polypeptide; type II PKS is a protein complex with different functions; and type III polyketide synthase is a small protein composed solely of ketone synthase domains. All enzymes catalyze the multiple condensation reactions between an extended-chain substrate (such as malonyl-CoA) and a coenzyme A ester (or ACP body) called the starter substrate. Commonly used starter substrates include acetyl-CoA, fatty acid-CoA esters, benzoyl-CoA, and coumaroyl-CoA.

[0187] In the present specification, "Non-ribosomal peptide synthase (NRPS)" means a group of enzymes that synthesizes a peptide using an amino acid as a substrate without depending on a ribosome, without passing through a nucleic acid such as mRNA. The entity of NRPS is a super-massive protein complex involving several thousands of kDa in molecular weight.

[0188] In the present specification, "Bacillus subtilis" means an aerobic Gram-positive peroxidase-positive bacterium that is ubiquitous in soil or plants, is present in the gastrointestinal tract of ruminants or humans, is 0.7 to 0.8 x 2 to 3 μm in size, is mesophilic, has an optimum growth temperature of 25 to 40°C, forms spores, includes the species Bacillus subtilis or a spore-forming Bacillus genus bacterium such as Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus pumilus, and the like, is not pathogenic, and is any bacterium having a natural transformation ability. In a preferred embodiment, the Bacillus subtilis is the Marburg 168 strain or the RM125 strain of a derivative strain thereof, which is a strain of Bacillus subtilis having a high natural transformation ability. The 168 strain is a Gram-positive bacterium that is most studied in genetics, is harmless to humans and animals, is identified as being applicable to the OGAB TM method, and the like, and is thus suitable for use in the present application.

[0189] In the present specification, "a sequence that replicates in Bacillus subtilis" is used in the same meaning as "a sequence that amplifies in Bacillus subtilis", and means that the nucleic acid sequence replicates (or amplifies) when in a state introduced into Bacillus subtilis. Here, in this context, amplification is used in the same meaning as replication. As a replication mechanism of a nucleic acid (plasmid) in Bacillus subtilis, there are a rolling circle type, a theta type, oriC, a replication mechanism using a phage, and the like, and as a sequence that can replicate in Bacillus subtilis, a sequence using any of the mechanisms can be used. The rolling circle type is a mechanism in which replication of one strand of double-stranded DNA is performed first, and then replication of the other strand is performed, and since the nucleic acid exists as a single-stranded DNA for a long time, there is a tendency that the plasmid becomes unstable. The theta type is a mechanism in which replication of double-stranded DNA (plasmid) is started simultaneously in both directions from the replication origin in the same manner as when the bacterial chromosome replicates, and is preferably used when a long DNA (for example, 10 kb or more) replicates (Janniere, L., A. Gruss, and S. D. Ehrlich. 1993. Plasmids, p. 625-64. In A. L. Sonenshein, J. A. Hoch, and R. Losick (ed.), Bacillus subtilis and other Gram-positive bacteria: biochemistry, physiology and molecular genetics. American Society for Microbiology, Washington, D.C.). The replication mechanism of oriC operates in the same manner as when the host bacterium (for example, Bacillus subtilis) chromosome replicates. As a sequence that replicates in Bacillus subtilis, there are known plasmids or a part thereof or a replication origin, or a modification thereof, which are known to enable the rolling circle type, the theta type, oriC, a replication mechanism using a phage, and the like to operate. As a rolling circle type plasmid, there are known pUB110, pC194, pE194, pT181, and the like, and as a theta type plasmid, there are known pAMβl, pTB19, pLS32, pLS20, and the like. The sequence that replicates in Bacillus subtilis can have the same or similar sequence (for example, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, 99.5% or more of sequence identity) to a known replication origin.For example, after a DNA fragment in which a candidate DNA fragment is linked to a selection marker gene effective in B. subtilis, such as a drug resistance gene, is introduced into B. subtilis, and after culture (addition of a drug, etc.), it can be determined that the candidate DNA fragment has a sequence that replicates within B. subtilis when it replicates extrachromosomally.

[0190] As used in the present specification, "plasmid" refers to a circular DNA that exists independently of a chromosome in a cell or exists independently of a chromosome when introduced into a cell.

[0191] As used in the present specification, "nucleic acid sequence that promotes replication of a plasmid" is used in the same meaning as "nucleic acid sequence that promotes amplification of a plasmid", and refers to any nucleic acid sequence that, when introduced into a host cell (for example, B. subtilis), promotes replication (in this context, the same as amplification) of a plasmid present in the host cell. The nucleic acid sequence that promotes replication of a plasmid is preferably operably linked to the nucleic acid sequence of a plasmid that is the object of encoding, but is not limited thereto. Details of the nucleic acid sequence that promotes replication of a plasmid, the plasmid that is the object, their relationship, and the like are described in other parts of the present specification. As the nucleic acid sequence that promotes replication of a plasmid, a nucleic acid sequence including a replication origin that operates in a host cell (for example, B. subtilis) that is the object can be cited. The nucleic acid sequence that promotes amplification of a plasmid refers to a DNA fragment having a replication origin, and refers to a DNA fragment that can replicate independently of chromosomal DNA. Whether or not these DNA fragments can replicate can be confirmed by, for example, the following method: a selection marker gene, such as a drug resistance gene, is linked to the DNA fragments, and after culture under a drug selection condition, plasmid DNA is purified, and the band of the DNA is observed by electrophoresis, and the like. In addition to the replication origin, the plasmid can include a gene of a rep protein that induces DNA replication enzymes of the host at the replication origin, a partition mechanism gene for reliably partitioning the plasmid into daughter cells, a selection marker gene, and the like. The replication origin can be fused within the gene of the rep protein.

[0192] As used in the present specification, "packaging cell" refers to a cell used for producing a vector plasmid.

[0193] As used in the present specification, "vector plasmid" or "viral vector plasmid" refers to a plasmid that includes a gene carried on a viral vector and is used for producing a viral vector in a production cell. In one embodiment, the vector plasmid includes a sequence of a gene (a desired gene) that causes it to express within a target cell of a viral vector between 2 terminal repeat sequences, and in addition, can include a promoter, and further can include other elements (for example, enhancer, terminator, and the like).

[0194] As used in the present specification, "producer cell" refers to a cell capable of producing a desired viral vector, and can be a cell in which genes necessary for production of the viral vector are expressed from a chromosome and an introduced plasmid. By introducing the vector plasmid of the present application into the producer cell, the desired viral vector can be produced. For example, in the case of an AAV viral vector, production thereof requires El A and El B, but HEK293 has these genes, and thus, these can be removed from the helper plasmid. Even if it is another cell, as long as it is modified in such a way as to have such a helper factor, it can function as a producer cell.

[0195] As used in the present specification, "viral vector" refers to a construct having at least partially a structure derived from a virus, and capable of introducing a nucleic acid into a target cell. Typically, the viral vector is in the form of a virion comprising a viral capsid and a nucleic acid containing a heterologous gene. As used in the present specification, "origin virus" refers to a virus naturally having a structure derived from a virus possessed by a viral vector. In the viral vector, the nucleic acid contained in the capsid (nucleic acid cargo) contains a sequence of a gene (desired gene) to be expressed in a target cell of the viral vector between two terminal repeat sequences, and in addition, can contain a promoter, enhancer, terminator, etc., and can also contain a gene derived from the origin virus.

[0196] In the present specification, "nucleic acid sequence necessary for constituting a viral vector" means a nucleic acid sequence (e.g., a combination of nucleic acid sequences) included in a production cell capable of producing a viral vector. In one embodiment, the nucleic acid sequence necessary for constituting a viral vector includes: a nucleic acid sequence encoding a viral capsid protein; a nucleic acid sequence encoding a protein that packages, transcribes, and replicates the genome of a virus; two terminal repeat sequences of a virus; and a nucleic acid sequence of a helper gene. The "nucleic acid sequence of a helper gene" means, for example, a nucleic acid sequence that can include or essentially constitute any nucleic acid sequence necessary for constituting a viral vector other than the nucleic acid sequence encoding a viral capsid protein; the nucleic acid sequence encoding a protein that packages, transcribes, and replicates the genome of a virus; and the two terminal repeat sequences of a virus. Each of these nucleic acid sequences can be different for each viral vector. Details of these are described in other parts of the present specification. In a preferred embodiment, the nucleic acid sequence necessary for constituting a viral vector can utilize an advantageous sequence common to AAV and adenovirus, and more preferably can utilize a sequence advantageous for AAV. Such a sequence advantageous for AAV and adenovirus common to AAV and adenovirus has the following characteristics: it includes a desired gene between the inverted terminal repeat sequences (ITRs), and can also include a helper gene, rep, cap, or L1, L2, L3, L4, L5. The sequence advantageous for AAV includes a desired gene between the ITRs, and can also include a helper gene, rep, cap. The arrangement of the helper gene, rep, cap has the characteristic that it can also be outside the two ITRs. For example, as such examples, the sequences of specific helper genes E1A, E1B, E2A, E2B, E3, E4 can be given, but are not limited thereto.

[0197] In the present specification, "a protein that packages, transcribes, and replicates the genome of a virus" means a protein that includes a protein that packages a virus genome into a capsid, a protein that transcribes, a protein that replicates a virus genome, a protein that has both functions, and examples include E1A, E1B, E2A, E2B, E4, rep, pol, ψ, APP, MAAP, and rev. Although not intended to be limiting, the protein that packages, transcribes, and replicates the genome of a virus can utilize any one of serotypes 1 to 52 derived from adenovirus or serotypes 1 to 12 derived from adeno-associated virus, or a modified body thereof (rh10, DJ, DJ / 8, PHP.eB, PHP.S, AAV2-retro, AAV2-QuadYF, AAV2.7m8, AAV6.2, rh.74, AAV2.5, AAV-TF, Anc80, and the like). The protein can be natural or artificially introduced with a mutation.

[0198] In the present specification, "capsid" refers to a protein produced by a gene possessed by a virus or a virus present on the surface of a viral vector (enclosed in an envelope as necessary), also referred to as "capsid protein". The capsid can assume infectivity to a cell. Among the capsid proteins, L2, L3, cap, VSV-G, and gag can be cited, but are not limited thereto. Although not intended to be limiting, a nucleic acid sequence encoding a viral capsid protein can utilize any one of serotypes 1 to 52 derived from adenovirus or serotypes 1 to 12 derived from adeno-associated virus, or a modified body thereof (rh10, DJ, DJ / 8, PHP.eB, PHP.S, AAV2-retro, AAV2-QuadYF, AAV2.7m8, AAV6.2, rh.74, AAV2.5, AAV-TT, Anc80, etc.). The capsid can be natural or artificially introduced with a mutation.

[0199] In the present specification, "repeat sequence" or "tandem repeat" (of a nucleic acid sequence) refers to a general term for nucleic acid sequences in which the same sequence is repeated (particularly, several times or more) in a nucleic acid sequence of a biological genome. Any repeat sequence used in the field can be used in the present application. Typically, a promoter sequence is repeated.

[0200] In the present specification, "terminal repeat sequence" refers to a general term for nucleic acid sequences present at the terminal end among sequences in which the same sequence is repeated (particularly, several times or more) in a nucleic acid sequence of a biological genome. As the terminal repeat sequence, an inverted terminal repeat sequence (ITR) or a long chain terminal repeat sequence (LTR), etc. can be cited, but are not limited thereto. The terminal repeat sequence can utilize any one of serotypes 1 to 52 derived from adenovirus or serotypes 1 to 12 derived from adeno-associated virus, or a modified body thereof (rh10, DJ, DJ / 8, PHP.eB, PHP.S, AAV2-retro, AAV2-QuadYF, AAV2.7m8, AAV6.2, rh.74, AAV2.5, AAV-TT, Anc80, etc.). As the terminal repeat sequence, it can be natural or artificially introduced with a mutation.

[0201] In the present specification, "helper gene" refers to a gene that assists in the amplification of a virus that cannot proliferate alone. In the present application, among the helper genes, for example, a nucleic acid sequence encoding a capsid protein of the virus; a nucleic acid sequence of a protein that packages the genome of the virus, encodes transcription, and / or replication; and a nucleic acid sequence of any of the nucleic acid sequences other than the two terminal repeat sequences of the virus that are necessary for the constitution, proliferation, activity improvement, toxicity reduction of the viral vector can be cited, but are not limited thereto. As the helper genes that can be used, for example, E1A, E1B, E2A, E2B, E4, RPE, WRPE, PPT, oPRE, enhancer, insulator, silencer sequence, etc. can be cited, but are not limited thereto.

[0202] In the present specification, "unit nucleic acid" means a nucleic acid molecule or a part of a nucleic acid molecule having a sequence constituting a part of a sequence of an enriched nucleic acid, and a plurality of unit nucleic acids are prepared as unit vectors, and then an enriched nucleic acid is constructed. The unit nucleic acid of the present application can be DNA, RNA, a modified body thereof, or a mixture of these. The method for preparing the unit nucleic acid of the present application or a composition containing the same includes the steps of preparing a solution containing a plurality of unit nucleic acids to which an additional sequence is attached, per kind of the unit nucleic acid; after preparing each solution, measuring the concentration of the unit nucleic acid in each solution in a state where the additional sequence is attached to the unit nucleic acid, and based on the result thereof, fractionating each solution so that the number of moles of the unit nucleic acid in each solution is close to the same as each other. In the present specification, the kind of "unit nucleic acid" is distinguished according to each base sequence. In addition, "unit nucleic acid" is intended to include any of nucleic acids to which a restriction enzyme recognition site is or is not attached. The length of each of the unit nucleic acids is not particularly limited, but when the base sequence of the unit nucleic acid is confirmed, it is preferable that the number of times of base sequence determination be small in order to reduce the cost of time and expense. Therefore, the length of each of the unit nucleic acids is preferably short, and specifically, it is preferably 1600 bp or less, and further preferably 1200 bp or less. In particular, when base sequence determination is performed using an automatic fluorescent sequencer using the Sanger method, about 800 bases in length of the base sequence can be confirmed by one time of base sequence determination, and therefore the length of each of the unit nucleic acids is most preferably 800 bp or less (specifically, 600 bp or less, 500 bp or less, 400 bp or less, 200 bp or less, 100 bp or less, etc.). In this way, when the length of each of the unit nucleic acids is short, a large number of unit nucleic acids are required when they are used in the production of the DNA linker described later. However, when the unit nucleic acids prepared using the method of the present application are used, a large number of unit nucleic acids can be ligated as described below. In addition, when the length of each of the unit nucleic acids is too short, the number of unit nucleic acids increases, and the work efficiency decreases. Therefore, the length of each of the unit nucleic acids is preferably 20 bp or more, more preferably 30 bp or more, and further preferably 50 bp or more. The vector DNA and each of the unit nucleic acids have a structure that can be directly ligated in a state where the order is maintained with each other. In the present specification, "directly ligated in a state where the order is maintained with each other" means a unit nucleic acid having an adjacent sequence on the enriched nucleic acid unit or a combination thereof in which the order and direction are maintained.

[0203] In the present specification, "endotoxin" also referred to as endotoxin, refers to a cell wall component of Gram-positive bacteria such as Bacillus subtilis, and a toxin that is not actively secreted. Quantification can also be performed using a kit such as Pierce LAL Endotoxin Quantitation Kit (Thermo Fisher Scientific, USA). "Endotoxin" is present only in Gram-negative bacteria and is not present in Gram-positive bacteria. "Endotoxin" is also referred to as lipopolysaccharide (LPS), which is present in the outer membrane of Gram-negative bacteria, has a structure of O antigen-core polysaccharide-lipid A, and lipid A has physiological activity. Gram-positive bacteria do not have an outer membrane, and therefore lipid A is not present.

[0204] In the present specification, "loaded nucleic acid" refers to a nucleic acid carried in a viral vector. Whether or not it is a loaded nucleic acid can be confirmed by the following method: DNase treatment is performed on a viral vector preparation, and nucleic acids outside the capsid are digested, after which the DNase is inactivated, and then the nucleic acids extracted from the capsid are confirmed. Whether or not it is a loaded nucleic acid can also be confirmed by investigating the sequence (preferably a sequence of full length or a length close to full length) of the obtained nucleic acid product.

[0205] In the present specification, "host cell" refers to a cell (including the progeny of such a cell) into which foreign nucleic acid or protein or virus or viral vector has been introduced.

[0206] In the present specification, "protein", "polypeptide", and "peptide" are used with the same meaning, and refer to a polymer of amino acids of any length. The polymer can be linear, branched, or cyclic. The amino acids can be natural or non-natural, or modified amino acids. The term also includes naturally or artificially modified polymers. As such modifications, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification (e.g., complex formation with a labeling component) are included.

[0207] If not otherwise indicated, a specific nucleic acid sequence is contemplated to comprise its conservatively modified variants (e.g., degenerate codon substitutions) and com¬plementaries thereof as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with a mixture of base and / or deoxyinosine residues. For example, in modifications of specific wild-type sequences based on Adenovirus serotypes 1-52 and Adeno-associated virus serotypes 1-12, among others, in addition to well-known modifications (e.g., rh10, DJ, DJ / 8, PHP.eB, PHP.S, AAV2-retro, AAV2-QuadYF, AAV2.7m8, AAV6.2, rh.74, AAV2.5, AAV-TT, Anc80, etc.), nucleic acids comprising sequences having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to the original sequence are included.

[0208] In the present specification, a "gene" refers to a nucleic acid portion that exerts a certain biological function. As the biological function, it is possible to cite: encoding a polypeptide or a protein, encoding a protein non-coding functional RNA (rRNA, tRNA, microRNA (miRNA), IncRNA, etc.), a polypeptide, controlling production of a protein or a protein non-coding functional RNA, specifically binding to a specific protein, controlling cleavage or replication of a nucleic acid. Thus, in the gene in the present specification, in addition to a nucleic acid portion encoding a protein or a protein non-coding functional RNA, a transcription translation regulatory sequence such as a promoter, a terminator, an enhancer, an insulator, a silencer, an origin of replication, an internal ribosome entry site, and a nucleic acid portion required for packaging a virion are included. In the present specification, a "gene product" can refer to a polypeptide, a protein, or a protein non-coding functional RNA encoded by a gene.

[0209] In the present specification, when the number of genes is mentioned, one gene refers to a gene having a continuous sequence in a form normally (at the highest frequency or with a probability of 50% or more) present on the genome of a certain organism. For example, 2 exons encoding a certain protein can be 2 genes. For example, when a sequence encoding a promoter sequence and a protein form a continuous sequence, a nucleic acid portion containing the sequence encoding the promoter sequence and the protein can be one gene. For example, when a protein that becomes functional by being cleaved is encoded by a continuous sequence on the genome, the protein can be encoded by one gene. When a gene is mentioned from the functional aspect, the nucleic acid sequence does not need to be a continuous sequence, and for example, a plurality of exons encoding a certain protein are collectively mentioned as the gene of the protein.

[0210] As used herein in the specification, a "deletion" of a gene means that the nucleic acid does not contain the gene, or contains the gene modified such that the gene cannot function normally (e.g., to produce a functional protein).

[0211] As used herein in the specification, "operably linked" means that the expression (operation) of the desired sequence is placed under the control of a transcriptional translational regulatory sequence (e.g., a promoter, enhancer, etc.) or translational regulatory sequence. In order for a promoter to be operably linked to a gene, the promoter is typically placed in close proximity to the gene upstream, but need not necessarily be contiguous.

[0212] As used herein in the specification, "transcriptional translational regulatory sequence" is a collective term for a promoter sequence, a polyadenylation signal, a transcription termination sequence, an upstream regulatory domain, an origin of replication, an enhancer, an IRES, etc., which are capable of cooperatively carrying out replication, transcription, and translation of a coding sequence in a recipient cell. It is not necessary for all of these transcriptional translational regulatory sequences to be present as long as replication, transcription, and translation of the selected coding sequence can be carried out in a suitable host cell. Those skilled in the art can easily identify regulatory nucleic acid sequences from public information. Further, those skilled in the art can identify transcriptional translational regulatory sequences suitable for use for the purpose of use, for example, in vivo, ex vivo, or in vitro.

[0213] As used herein in the specification, "promoter" means a fragment of a nucleic acid sequence that controls transcription of a nucleic acid sequence operably linked thereto. The promoter includes a specific sequence sufficient for recognition, binding, and initiation of transcription by RNA polymerase. The promoter can include a sequence that modulates recognition, binding, or initiation of transcription by RNA polymerase.

[0214] As used herein in the specification, "enhancer" means a fragment of a nucleic acid sequence having a function of increasing the efficiency of expression of a gene of interest.

[0215] As used herein in the specification, "silencer" means a fragment of a nucleic acid sequence having a function of decreasing the efficiency of expression of a gene of interest, as opposed to an enhancer.

[0216] As used herein in the specification, "insulator" means a fragment of a nucleic acid sequence having a function of cis-regulation for regulating expression of a gene located at a distant position on the sequence of DNA.

[0217] As used herein in the specification, "terminator" means a fragment of a nucleic acid sequence located downstream of a region encoding a protein, which is involved in termination of transcription when a nucleic acid is transcribed into mRNA.

[0218] As used in the present specification, "origin of replication" refers to a segment of a nucleic acid sequence that is bound by a protein that recognizes its nucleic acid sequence (e.g., the initiator DnaA protein, etc.) or synthesis of RNA, and causes the DNA double helix to be partially unwound, initiating replication.

[0219] As used in the present specification, "internal ribosome entry site" ("IRES") refers to a segment of a nucleic acid that facilitates entry or retention of a ribosome when translating a nucleic acid sequence downstream thereof.

[0220] As used in the present specification, "homology" of nucleic acids refers to the degree of identity of two or more nucleic acid sequences to each other. Generally, "homology" means a high degree of identity or similarity. Thus, the higher the homology of two nucleic acids, the higher the identity or similarity of the sequences. "Similarity" is a value that, in addition to identity, also includes similar bases in the calculation, where similar bases refer to cases where a portion of the mixed bases (e.g., R = A + G, M = A + C, W = A + T, S = C + G, Y = C + T, K = G + T, H = A + T + C, B = G + T + C, D = G + A + T, V = A + C + G, N = A + C + G + T) are identical. Whether two nucleic acids have homology can be investigated by direct comparison of the sequences or by hybridization under stringent conditions. When two nucleic acid sequences are directly compared, typically at least 50% of the nucleic acid sequences are identical, preferably at least 70% identical, more preferably at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical, and the genes have homology.

[0221] Amino acids can be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, can be referred to by their commonly accepted single-letter codes. In the present specification, comparisons of similarity, identity and homology of amino acid sequences and base sequences can be calculated using BLAST as a tool for sequence analysis, using default parameters. The search for identity, for example, can be performed using BLAST 2.10.1+ (Release 2020.6.18) of NCBI. The value of identity in the present specification refers to the value when the sequence alignment is performed using the above-described BLAST under the default conditions. Among them, when a higher value appears by changing the parameters, the highest value is set as the value of identity. When the identity is evaluated in multiple regions, the highest value among them is taken as the value of identity. Similarity is a value that, in addition to identity, also includes similar amino acids in the calculation.

[0222] In this specification, unless otherwise indicated, a reference to a certain biological material (e.g., a protein, a nucleic acid, a gene) is understood to also refer to variants of that biological material (e.g., variants having modifications in the amino acid sequence) that perform the same function (to the extent possible) as the biological material. Such variants can include molecules that are at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identical when compared to the sequence of the original molecule being aligned by sequence alignment with a fragment of the entire original molecule, an amino acid sequence or nucleic acid sequence of the same size of the original biological material, or by a computer homology program known in the art. Variants can include molecules having modified amino acids (e.g., modifications by disulfide bond formation, glycosylation, lipidation, acetylation, or phosphorylation) or modified nucleotides (e.g., methylation-based modifications).

[0223] In the present specification, "stringent conditions" refer to well-known conditions commonly used in the art. Stringent conditions can be exemplified by the following conditions: (1) low ionic strength and high temperature for washing (e.g., 0.015 M sodium chloride / 0.0015 M sodium citrate / 0.1% sodium dodecyl sulfate at 50°C), (2) use of a denaturant such as formamide in hybridization (e.g., 50% (v / v) formamide with 0.1% bovine serum albumin / 0.1% Ficoll / 0.1% polyvinylpyrrolidone / 50 mM sodium phosphate buffer (pH 6.5) and 750 mM sodium chloride, 75 mM sodium citrate at 42°C), or (3) incubation at 37°C for one night in a solution containing 20% formamide, 5xSSC, 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 mg / ml denatured salmon sperm DNA, followed by washing of the filter at about 37-50°C using 1xSSC. Note that the formamide concentration can also be 50% or more. The washing time can also be 5, 15, 30, 60, or 120 minutes or more. As factors that affect the stringency of the hybridization reaction, temperature, salt concentration, and the like can be considered, and details can be found in Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995). Examples of "highly stringent conditions" are 0.0015 M sodium chloride, 0.0015 M sodium citrate, 65-68°C, or 0.015 M sodium chloride, 0.0015 M sodium citrate, and 50% formamide, 42°C. Hybridization can be performed according to the methods described in Molecular Cloning 2nd ed., Current Protocols in Molecular Biology, Appendix 1-38, DNA Cloning 1: Core Techniques, A Practical Approach, Second Edition, Oxford University Press (1995), and the like. Among these, preferably, sequences containing only A sequences or T sequences are excluded from the sequences that hybridize under stringent conditions.Moderately stringent conditions can be readily determined by one of skill in the art, for example, as described in Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd Ed., Vol. 1, 7.42-7.45, Cold Spring Harbor Laboratory Press, 2001, and for nitrocellulose filters, including the use of a prewash solution of 5 x SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0), hybridization conditions of about 50% formamide, 2 x SSC-6 x SSC (or Stark's solution in about 50% formamide at about 42°C), and wash conditions of about 60°C, 0.5 x SSC, 0.1% SDS. Thus, polypeptides used in the present application also include polypeptides encoded by nucleic acid molecules that hybridize under high or moderately stringent conditions to nucleic acid molecules encoding the polypeptides specifically described in the present application.

[0224] In the present specification, "corresponding" amino acid or nucleic acid refers to an amino acid or nucleic acid in a certain polypeptide molecule or polynucleotide molecule that has or is predicted to have the same function as a specified amino acid or nucleic acid in a polypeptide or polynucleotide used as a comparison standard, particularly an amino acid present in the same position in the active site and contributing to catalytic activity in the case of an enzyme molecule. For example, in the case of an antisense strand molecule, it can be the same portion of the ortholog corresponding to a particular portion of the antisense strand molecule. The corresponding amino acid can be, for example, a particular amino acid that is cysteinylated, glutathionylated, forms an S-S bond, oxidized (for example, oxidation of the methionine side chain), formylated, acetylated, phosphorylated, glycosylated, myristylated, etc. Alternatively, the corresponding amino acid can be an amino acid that assumes dimerization. Such "corresponding" amino acid or nucleic acid can be throughout a certain range of regions or domains. Thus, in this case, the region or domain can be referred to as "corresponding" in the present specification.

[0225] In the present specification, a "corresponding" gene (e.g., polynucleotide sequence or molecule) refers to a gene (e.g., polynucleotide sequence or molecule) in a certain species that has or is predicted to have the same function as a specified gene in a species serving as a standard for comparison, or, in the case where there are a plurality of genes having such a function, refers to a gene that is phylogenetically of the same origin. Thus, a gene corresponding to a certain gene can be an ortholog of the gene. For example, a cap of an AAV of serotype 1 can correspond to a cap of an AAV of serotype 2. For example, a corresponding gene in a certain virus can be found by searching a sequence database of the virus using a viral gene sequence serving as a standard for a corresponding gene as a query sequence.

[0226] According to the present application, the term "activity" refers to a function of a molecule in the broadest sense in the present specification. For the activity, no limitation is intended, but roughly includes a biological function, a biochemical function, a physical function, or a chemical function of a molecule. The activity includes, for example, an enzyme activity, an ability to interact with other molecules and an ability to activate, promote, stabilize, hinder, inhibit, or destabilize a function of other molecules, stability, an ability to be restricted to a specific intracellular location. In cases where applicable, the term also relates to a function of a protein complex in the broadest sense.

[0227] In the present specification, "biological function" when referring to a certain gene or a nucleic acid molecule or a polypeptide related thereto refers to a specific function that the gene, nucleic acid molecule, or polypeptide can have in a living organism, and examples thereof include, but are not limited to, a specific cell surface structure recognition ability, an enzyme activity, a binding ability to a specific protein, and the like. In the present application, examples thereof include a function in which a certain promoter is recognized in a specific host cell, but are not limited thereto. In the present specification, a biological function can be exerted by "biological activity". In the present specification, "biological activity" refers to an activity that a certain factor (e.g., polynucleotide, protein, or the like) can have in a living body, and includes an activity to exert various functions (e.g., transcription-promoting activity), and also includes an activity to activate or inactivate other molecules, for example, by interacting with a certain molecule. For example, when a certain factor is an enzyme, its biological activity includes its enzyme activity. In another example, when a certain factor is a ligand, it includes binding of the ligand to a corresponding receptor. Such a biological activity can be measured by techniques known in the art. Thus, "activity" refers to various measurable indicators that show binding or definite binding (either directly or indirectly); or affect a response (i.e., have a measurable effect on some exposure or stimulus), and examples thereof include an amount of an upstream or downstream protein in a host cell or other similar functional criteria.

[0228] As used herein, the "infectivity" of a virus or viral vector refers to the ability to introduce nucleic acid within the virus or viral vector into a cell by adhesion or membrane fusion of the virus or viral vector to the cell. The Sendai virus vector can have the same replication ability as the wild-type vector, and can also be attenuated by genetic mutation. The "replication ability" of a virus or viral vector refers to the ability to produce infectious virions or viral vector particles within an infected cell.

[0229] As used herein, the terms "transformation", "transduction" and "transfection" are used interchangeably, unless otherwise specified, and refer to the introduction of nucleic acid into a host cell, as necessary via a virus or viral vector. As a method of transformation, any method can be used as long as it is a method for introducing nucleic acid into a host cell, and various known techniques can be used, such as the use of competent cells, electroporation, methods using microprojectile bombardment (gene gun), calcium phosphate method, etc.

[0230] In the present specification, a "purified" substance or biological factor (e.g., nucleic acid or protein, etc.) refers to a substance or biological factor from which at least a portion of the factors naturally accompanying the biological factor has been removed. Thus, the purity of the biological factor in a generally purified biological factor is higher than that of the biological factor in a state in which it is generally present (i.e., is concentrated). The term "purified" as used in the present specification means that preferably at least 75% by weight, more preferably at least 85% by weight, further more preferably at least 95% by weight, and most preferably at least 98% by weight of the same biological factor is present. The substance as used in the present application is preferably a "purified" substance.

[0231] In the present specification, "agent", "agent" or "factor" (all of which correspond to agent in English) are used interchangeably in a broad sense, and can be any substance or other element (e.g., light, radiation, heat, electricity, etc.) as long as the intended purpose can be achieved. As such a substance, for example, proteins, polypeptides, oligopeptides, peptides, polynucleotides, oligonucleotides, nucleotides, nucleic acids (e.g., DNA including cDNA, genomic DNA, RNA such as mRNA), polysaccharides, oligosaccharides, lipids, organic low molecules (e.g., hormones, ligands, information transmitting substances, organic low molecules, molecules synthesized by combinatorial chemistry, low molecules that can be used as pharmaceutical products (e.g., low molecular ligands, etc.), etc.), complex molecules of these, and mixtures of these can be mentioned, but are not limited thereto.

[0232] As used in the present specification, "complex" or "complex molecule" refers to any constitution body containing two or more moieties. For example, when one of the moieties is a polypeptide, the other moiety can be a polypeptide or a substance other than a polypeptide (e.g., a substrate, a sugar, a lipid, a nucleic acid, another hydrocarbon, etc.). In the present specification, two or more moieties constituting a complex can be bound by a covalent bond or by another bond (e.g., a hydrogen bond, an ionic bond, a hydrophobic interaction, a van der Waals force, etc.).

[0233] The term "about" refers to the indicated value plus or minus 10%. When "about" is used in reference to a temperature, it refers to the indicated temperature plus or minus 5°C. When "about" is used in reference to a pH, it refers to the indicated pH plus or minus 0.5.

[0234] (PREFERRED EMBODIMENTS)

[0235] The following describes preferred embodiments of the present application. The embodiments provided below are provided for better understanding of the present application and can be understood as the scope of the present application should not be limited by the following description. Therefore, it is obvious to those skilled in the art that appropriate modifications can be made within the scope of the present application with reference to the description in the present specification. In addition, it can be understood that the following embodiments can be used individually or in combination.

[0236] In one aspect, the present application provides a method for producing a strain having a desired property, the method comprising the steps of: 1) a step of providing a construct containing a gene cluster containing a plurality of genes, i.e., a construct into which a plurality of promoters are introduced; 2) a step of combinatorial library of the promoters in the construct; 3) a step of screening a strain having a desired property in a host cell. In this aspect, the plurality of promoters can be configured in a monocistronic configuration.

[0237] In another aspect, the present application provides a method for producing a substance in a host organism, the method comprising the steps of: (A) a step of constructing a construct in which a promoter introduction site is added to a group of genes involved in production of a substance from which an undesirable restriction enzyme recognition sequence is removed, and a reconstitution sequence; (B) a step of introducing a plurality of constructs into which a promoter is introduced into the promoter introduction site into a combinatorial library into a host organism; (C) a step of combinatorial library of the promoter sites among the plurality of constructs by Combi-OGAB TM method; (D) a step of culturing a production host organism having a construct that has been combinatorially libraryed, and screening a plurality of strains based on any criteria; (E) a step of combinatorially librarying the promoter sites in the constructs of the plurality of strains again by Combi-OGAB TM method; (F) a step of producing a substance using a strain having a desired property.

[0238] As an arbitrary criterion, for example, from among a plurality of strains, it can be the top (e.g., top 5, etc.) in terms of high substance yield, or it can be a prescribed yield, which is appropriately determined by one skilled in the art.

[0239] In one aspect, there is provided a method of producing a strain having a desired property, the method comprising the steps of: 1) a step of providing a construct comprising a gene cluster containing a plurality of genes, i.e., a plurality of constructs in which promoters are mono-cistronically configured; 2) a step of combinatorial library formation of the promoters in the construct, introduction into a host cell of interest; 3) a step of screening for a strain having a desired property in the host cell.

[0240] In another aspect, the present application provides a method of producing a substance in a host organism, the method comprising the steps of: (A) a step of constructing a construct in which a promoter introduction site is added to a group of genes involved in production of a substance in which an undesirable restriction enzyme recognition sequence is removed, and a reconstitution sequence is added; (B) a step of introducing a plurality of constructs in which a promoter is introduced into the promoter introduction site into the host organism; (C) a step of combinatorial library formation of the promoter sites among the plurality of constructs in the cells of the host organism by Combi-OGAB TM ; (D) a step of culturing the host organism and screening for a plurality of strains having a desired property; (E) a step of re-combinatorial library formation of the promoter sites in the constructs of the plurality of strains having a desired property by Combi-OGAB TM ; and (F) a step of using the strain having a desired property to produce a substance.

[0241] In another aspect, the present application provides a method of producing a substance in a host organism, the method comprising: a step of using a strain produced by the method according to the present application to produce a substance.

[0242] In another aspect, the present application provides a method of producing a substance in a host organism, the method comprising: a step of using a strain obtained by the steps (A) to (E) in the method according to the present application to produce a substance.

[0243] The method according to the present application can further comprise modification of the strain according to the present application. The modification can include mutation, addition of a transport protein and / or other metabolic enzyme, and the like. By such modification, the properties of the strain can also be optimized.

[0244] In one embodiment, the strain having a desired property is a strain that produces a target substance, a strain that produces a target substance at a high yield, a strain that retains a target substance in a cell without secretion, a strain that secretes a target substance outside of a cell, a strain that is easy to culture (e.g., grows under usual culture conditions (culture medium / temperature / culture time, etc.)), a strain that produces multiple target substances at a desired ratio, a strain that produces a target substance without producing a harmful substance (non-toxicity, etc.), a strain in which the yield of a target substance is robust, a strain in which no deletion occurs in a gene that produces a target substance during culture, a strain that enables control of production of a target substance, a strain that suppresses production of a substance other than a target substance, a strain that suppresses production of a by-product and / or an impurity that is difficult to separate in affinity, ion exchange, gel filtration, reverse phase, normal phase, chiral chromatography, distillation, crystallization, etc., a strain in which the yield of empty capsids is low in virus vector production, a strain in which no deletion occurs in a plasmid, etc., or any combination of these, but is not limited to these.

[0245] In one embodiment, the strain having a desired property is at least a strain that produces a target substance at a high yield, and can have one or more of the above-described desired properties as needed.

[0246] (Construction of construct)

[0247] A nucleic acid including a nucleic acid sequence having a sequence for replication in a cell and used for production of a virus vector can be introduced into a host cell and form a plasmid in the host cell. In the formation of the plasmid, the OGAB TM method can be appropriately used. In one embodiment, since a strain of Bacillus subtilis or the like can have the ability to form a plasmid from a nucleic acid taken from the outside, in this method, the introduced nucleic acid can also not be a plasmid. For example, when Bacillus subtilis is contacted with a nucleic acid (e.g., a non-circular nucleic acid having a nucleic acid sequence of a tandem repeat), the nucleic acid can be taken up and a plasmid can be formed in Bacillus subtilis (e.g., refer to the OGAB TM method described in the present specification). The construction of a construct can be performed, for example, with reference to International Publication No. 2022 / 097646.

[0248] (Combination library)

[0249] The combination library is described. The following is an example, and a method other than this can also be used. A construct in which a promoter introduction site and a sequence for reconstitution are added is constructed. As described above, the OGAB TMThe sequence for reconstruction can be Sfil, Aarl, AlwNI, Bbsl, BbvI, BcoDI, BfuAI, Bgll, Bsal, BsaXI, BslI, BsmAI, BsmBI, BsmFI, BspMI, BspQI, BstAPI, BstXI, BtgZI, Dralll, Earl, Esp3I, Fokl, Hgal, Mwol, PflMI, SfaNI, SapI recognition sequence, and in addition, a sequence in which the resulting overlapping ends are N can also be used. Various promoters are introduced upstream of each gene in the gene group to construct a master plasmid for combinatorial library. A plurality of promoters having different transcription initiation and / or transcription strengths are selected. After combinatorial library construction, each construct in the library is transduced into a host cell, and one or more strains having desired properties are selected, and based on the selected strains, combinatorial library construction can be performed again. By performing the combinatorial library construction, transduction, and strain selection cycle multiple times, strains having more optimal properties and / or multiple properties can be obtained (fine tuning). For example, by fine tuning the enzymes described in D. B. Basnet, et. al., J. Biotechnol., 2008, 135, 92-96 (EryCIII enzyme reaction, EryK-EryG enzyme reaction); Appl. Microbiol. Biotechnol., 2017, 101, 5951, the yield (content in components) of the desired substance can be improved. The cycle can be performed 2 or more times, for example, 2 times, 3 times, 4 times, or 5 times.

[0250] The selection of strains used in the second cycle of combinatorial library construction can be performed based on any criteria. For example, the top 5 strains having desired properties (e.g., high production of the target substance) or strains satisfying a prescribed property (e.g., yield exceeding the yield of the target substance based on the natural strain) can be selected. The number of strains used in the second cycle can be 2 or more, 50 or more, 100 or more, 1000 or more, 10000 or more. It is preferable that the number of strains used in the second cycle be 2 or more. The number of strains used in the second cycle is the maximum number of transformants that can be obtained, and for example, can be 10, 15, 20, 30, 40, 50, 100. The minimum and maximum values of the number of strains selected can be any number between the above values.

[0251] (Production of enrichment nucleic acids)

[0252] The production steps of the enrichment nucleic acids for incorporation into transformants are described below.

[0253] • Preparation of unit nucleic acids

[0254] Unit nucleic acids for incorporation into the nucleic acid-enriched plasmid are prepared. The unit nucleic acids can be prepared by any known method, for example, by polymerase chain reaction (PCR), chemical synthesis. The unit nucleic acids can have any desired sequence such as a sequence encoding a desired protein (metabolic enzyme, biosynthetic enzyme group of a natural product, artificial enzyme, therapeutic protein, protein constituting a viral vector, etc.), or a part thereof, a sequence controlling a gene (promoter, enhancer, etc.), a sequence for manipulation of nucleic acids (restriction enzyme recognition sequence, etc.), etc. When incorporated into the nucleic acid-enriched plasmid, the ends of each unit nucleic acid can be configured in a manner to produce a specific overhang sequence, so that a plurality of unit nucleic acids are arranged in a specific order and / or direction of characteristics.

[0255] Since a plurality of unit nucleic acids can be ultimately enriched on the plasmid, one or more unit nucleic acids can be designed in a manner to encode one or more genes having a long base length. As a gene having a long base length, for example, a group of genes constituting a series of metabolic pathways, a protein complex composed of a plurality of subunits, etc. can be cited.

[0256] • Preparation of unit vectors

[0257] A unit vector can be prepared by linking a unit nucleic acid and an additional nucleic acid different from the same. By using the unit vector, it becomes possible to more easily manipulate the unit nucleic acid.

[0258] The additional nucleic acid can be a linear nucleic acid or a circular plasmid. In the case of using a circular plasmid as the additional nucleic acid, the unit vector also has a circular structure, and thus can be used for transformation of, for example, Escherichia coli. In one embodiment, the additional nucleic acid can include a replication initiation site to allow the unit vector to replicate in the introduced host. In one embodiment, all of the unit nucleic acids used for construction of a certain enriched nucleic acid can be linked to the same kind of additional nucleic acid, and in this way, the size difference between the unit vectors can be reduced, and the handling of the plurality of unit vectors becomes easy. In one embodiment, the unit nucleic acids used for construction of a certain enriched nucleic acid can be linked to different kinds of additional nucleic acids. In one embodiment, with respect to one or more unit nucleic acids used for construction of a certain enriched nucleic acid, the ratio of (the base length of the unit nucleic acid) / (the base length of the unit vector) or the average thereof can be 50% or less, 40% or less, 30% or less, 20% or less, 15% or less, 10% or less, 7% or less, 5% or less, 2% or less, 1.5% or less, 1% or less, or 0.5% or less. The larger the size of the additional nucleic acid compared to the nucleic acid, the more uniform handling of the different kinds of unit vectors becomes possible. The linking of the unit nucleic acid to the additional nucleic acid can be performed by, for example, ligation using a DNA ligase, TA cloning, or the like. In one embodiment, with respect to one or more unit nucleic acids used for construction of a certain enriched nucleic acid, the ratio of (the base length of the unit nucleic acid) / (the base length of the unit vector) or the average thereof can be 1% or more, 0.3% or more, 0.1% or more, 0.03% or more, 0.01% or more, 0.003% or more, or 0.001% or more, and the handling of the unit vector becomes easy.

[0259] In one embodiment, the length of the unit nucleic acid can be 10 bp or more, 20 bp or more, 50 bp or more, 70 bp or more, 100 bp or more, 200 bp or more, 500 bp or more, 700 bp or more, 1000 bp or more, or 1500 bp or more, and 5000 bp or less, 5000 bp or less, 2000 bp or less, 1500 bp or less, 1200 bp or less, 1000 bp or less, 700 bp or less, or 500 bp or less.

[0260] In one embodiment, the enriched nucleic acid can be constructed from 2 or more, 4 or more, 6 or more, 8 or more, 10 or more, 15 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, or 100 or more, and 1000 or less, 700 or less, 500 or less, 200 or less, 120 or less, 100 or less, 80 or less, 70 or less, 60 or less, or 50 or less, of the unit nucleic acids. By adjusting the number of moles of each unit nucleic acid (or unit vector) to be substantially the same, a desired enriched nucleic acid having a tandem repeat-like structure can be efficiently produced.

[0261] In one embodiment, the unit nucleic acid can have a base length that substantially equally divides the number of unit nucleic acids by the number of one set of repeated sequences in the enriched nucleic acid. In this way, the operation of making the number of moles of each unit nucleic acid (or unit vector) uniform becomes easy. In one embodiment, the unit nucleic acid can have a base length that is increased or decreased by 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 7% or less, or 5% or less, from a base length that substantially equally divides the number of unit nucleic acids by the number of one set of repeated sequences in the enriched nucleic acid.

[0262] In one embodiment, the unit nucleic acid can be designed to have a non-palindromic sequence (a sequence of a non-palindromic sequence) at the end of the unit nucleic acid. For a unit nucleic acid thus designed, when its non-palindromic sequence is made a protruding sequence, a structure in which the unit nucleic acids in the enriched nucleic acid are directly connected while maintaining the order of each other can be easily provided.

[0263] • Production of enriched nucleic acid

[0264] The enriched nucleic acid can be constructed by linking the unit nucleic acids to each other. In one embodiment, the unit nucleic acid can be prepared by cutting from the unit vector using a restriction enzyme or the like. The enriched nucleic acid can contain a group of repetitive sequences of 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more. The repetitive sequences in the enriched nucleic acid can contain the sequences of the unit nucleic acid and the sequences of the enriched vector nucleic acid as needed. The enriched nucleic acid can have a sequence capable of replicating the nucleic acid in a transformed organism. In one embodiment, the sequence capable of replicating the nucleic acid in a transformed organism can contain a replication initiation point effective in the transformed organism (e.g., in a Bacillus genus bacterium (B. subtilis)). The sequence of the replication initiation point effective in B. subtilis is not particularly limited, and, for example, as a sequence having a replication mechanism of the θ type, sequences such as the replication initiation point contained in plasmids such as pTB19 (Imanaka, T., et al. J. Gen. Microbiol. 130, 1399-1408. (1984)), pLS32 (Tanaka, T and Ogra, M. FEBS Lett. 422, 243-246. (1998)), pAMβl (Swinfield, T. J., et al. Gene 87, 79-90. (1990)), and the like can be mentioned.

[0265] The enriched nucleic acid can contain an additional base sequence as needed in addition to the unit nucleic acid. In one embodiment, the enriched nucleic acid can contain a base sequence that controls transcription and translation such as a promoter, an operator, an activator, a terminator, and the like. As a promoter when B. subtilis is used as the host, specifically, Pspac (Yansura, D. and Henner, D. J. Proc. Natl. Acad. Sci, USA 81, 439-443. (1984.)) or Pr promoter (Itaya, M. Biosci. Biotechnol. Biochem. 63, 602-604. (1999)) capable of controlling expression using IPTG (isopropyl β-D-thiogalactopyranoside) or the like can be mentioned.

[0266] The unit nucleic acid can form a repeated structure in which the order and direction are maintained in the enriched nucleic acid. In one embodiment, the unit nucleic acid is constructed in such a manner that the base sequence of the overhanging end of the unit nucleic acid cut from the unit vector is complementary to each other, whereby the repeated structure in which the order and direction are maintained in the enriched nucleic acid can be formed. In one embodiment, the structure of the overhanging end is made unique in each different unit nucleic acid, whereby the repeated structure in which the order and direction are maintained can be efficiently formed. In one embodiment, the overhanging end can have a non-palindromic sequence, and can be either 5' overhang or 3' overhang.

[0267] In one embodiment, the unit nucleic acid having an overhanging end can be cut from the unit vector using a restriction enzyme. In this embodiment, the unit vector can have one or more restriction enzyme recognition sequences. In the case where the unit vector has a plurality of restriction enzyme recognition sequences, each of the restriction enzyme recognition sequences can be recognized by the same restriction enzyme or by different restriction enzymes. In one embodiment, the unit vector can include one pair of regions recognized by the same restriction enzyme, so that the entire region of the unit nucleic acid is included between these regions. In the case where a restriction enzyme that recognizes the region is used, the unit vector can include the region recognized by the restriction enzyme at the end of the region of the unit nucleic acid.

[0268] In one embodiment, in the case where the same kind of restriction enzyme is used for cutting the unit nucleic acid from a plurality of unit vectors, the restriction enzyme treatment can be performed in a solution containing the plurality of unit vectors, whereby the work efficiency can be improved. The kind of restriction enzyme used for making a certain enriched nucleic acid can be, for example, 5 or less, 4 or less, 3 or less, 2 or less, or 1, and by using a smaller number of kinds of restriction enzymes, the molar number imbalance between the unit nucleic acids can be reduced. In one embodiment, the unit nucleic acid cut from the unit vector can be easily purified by any publicly known separation method such as agarose gel electrophoresis.

[0269] The unit nucleic acids and the enrichment carrier nucleic acids as needed can be ligated to each other using a DNA ligase or the like. By doing so, an enrichment nucleic acid can be produced. For example, the ligation of the unit nucleic acids and the enrichment carrier nucleic acids as needed can be performed in the presence of ingredients such as polyethylene glycol (e.g., PEG2000, PEG4000, PEG6000, PEG8000, or the like) and a salt (e.g., an alkali metal of valence 1, sodium chloride, or the like). The concentration of each unit nucleic acid in the ligation reaction solution is not particularly limited and can be 1 fmol / μl or more, or the like. The reaction temperature and time of the ligation are not particularly limited and are 37°C, 30 minutes or more, or the like. In one embodiment, before the unit nucleic acids are ligated to the enrichment carrier nucleic acids as needed, the composition containing the unit nucleic acids and the enrichment carrier nucleic acids as needed can be subjected to an arbitrary condition for inactivating a restriction enzyme (e.g., phenol / chloroform treatment).

[0270] The unit nucleic acids can be adjusted to substantially the same number of moles using the method described in WO2015 / 111248 or the like. By adjusting the unit nucleic acids to substantially the same number of moles, a desired enrichment nucleic acid having a tandem repeat-like structure can be efficiently produced. By measuring the concentration of the unit carrier or the unit nucleic acid, the number of moles of the unit nucleic acids can be adjusted.

[0271] • Production of a plasmid from an enrichment nucleic acid

[0272] By contacting the enriched nucleic acid with a transformant, a plasmid can be formed in the transformant. In one embodiment, as the transformant, there can be mentioned Bacillus bacteria, Streptococcus bacteria, Haemophilus bacteria, Neisseria bacteria, and the like. As the Bacillus bacteria, there can be mentioned B. subtilis, B. megaterium, B. stearothermophilus, and the like, but any transformant capable of forming a plasmid from the enriched nucleic acid can be used. In one embodiment, the transformant to be taken up the enriched nucleic acid is in a competent state, and can actively incorporate the nucleic acid. For example, the B. subtilis in the competent state cleaves a double-stranded nucleic acid as a substrate on the cell, digests either single strand in the double strand from the cleavage point, and takes up the other single strand into the bacterial body. The taken-up single strand can be repaired into a circular double-stranded nucleic acid in the bacterial body. In order to make the transformant in the competent state, any publicly known method can be used, for example, the B. subtilis can be made in the competent state using the method described in Anagnostopoulou, C. and Spizizen, J. J. Bacteriol., 81, 741-46 (1961). The method of transformation can use a publicly known method suitable for each transformant.

[0273] In one embodiment, the present application provides a vector plasmid produced by a packaging cell. In one embodiment, the vector plasmid produced by the packaging cell can be purified using any publicly known method, and the present application also provides a vector plasmid purified as described above. In one embodiment, the purified vector plasmid can be confirmed to have a desired nucleic acid sequence by investigating the size pattern of fragments produced by restriction enzyme cleavage, PCR method, base sequence determination method, and the like. In one embodiment, the vector plasmid-containing composition prepared by the vector plasmid production method of the present application can contain a small amount of endotoxin. In one embodiment, there is provided a B. subtilis comprising the vector plasmid of the present application.

[0274] In the present application, as the transformant (host cell) used, Bacillus subtilis, actinomycetes, filamentous bacteria, yeast, hydrogen bacteria, coryneform bacteria, or animal cells can be selected. The preferred transformant is Bacillus subtilis. These transformants are preferably capable of producing plasmids from the enriched nucleic acids. The transformant used for the combinatorial library production from the enriched nucleic acids and the transformant used for the production of the substance can be the same or different. For example, as the transformant used for the combinatorial library production, Bacillus subtilis can be used, and as the transformant used for the production of the substance, the same Bacillus subtilis can be used, or other transformants, such as actinomycetes, filamentous bacteria, yeast, hydrogen bacteria, coryneform bacteria, or animal cells can be used.

[0275] In one embodiment, the undesirable restriction enzyme recognition sequence can be OGAB TM In the method, the restriction enzyme recognition sequence used in the Combi-OGAB TM In the method, the restriction enzyme recognition sequence used in the Combi-OGAB

[0276] In one embodiment, the gene group related to the production of the substance can be a gene group of polyketide synthase (PKS), non-ribosomal peptide synthase (NRPS), ribosomally synthesized and post-translationally modified peptides (RiPPs), or terpene biosynthetic enzymes of terpenes.

[0277] As the promoter combination in the strain construct having the desired property in the present application, a combination of any of PabrB, PvalS, Phag, PspoVG, PyvyD, PhemA, Pffh, PsdhB, PiolS, Pspo0A, PglyA, PyceC, PsrfAA, PryxA, PytxG, PytvI, PspoVB, PphrK, PphrF, PftsH, Pasd, Pspo0F, PmenB, PgltX, PminC, PphrC, PspoVS, PlicH, Pcdd, PflgM, PcheV, PcitZ, PclpQ, PphrG, PlytR, Phbs, PodhA, PsecA, PphrE, PffsZ, XpaC, PcsbA, PywjC, PyknW, PysdB, PrapI, PyteJ, PureA, PtrxA, Pdps, XynA, PrsbW, PspoIVCA, PyqfY, PyqeZ, PyabR, PkinA, PnadE, PsigH, PresE, PspsA, PgspA, PyflG, Pveg, Pgap, PahpC, PyvrE, PytkL, PyfkJ, PdegS, PpyrAB, PyacL, PywkA, PspoVT, PywtG, PpdhC, PyuzA, PyhdF, PalsD, PpurE, PythP, PcomK, PptsI, PgtaB, PkatX, PydbP, PyqgZ, PydjO, PlytE, PgsiB, PywrE, PopuE, PyoxA, PthrS, PbltD, PyfhL, PyraD, PydaD, PywnJ, PrelA, PydfK, PgerBC, PyitG, PybfO, PcotH, PylnF, PribT, PrapH, PmmgA, PyqfD, PsigW, and the like can be cited, but not limited thereto, and a promoter other than these can also be appropriately used as long as it is a promoter dependent on the proliferation phase of Bacillus subtilis.

[0278] In the present specification, "or" is used when "at least one of the items enumerated in the article can be adopted." The same applies to "and / or." In the present specification, when "within a range" is explicitly described as "2 values," the range also includes the 2 values themselves.

[0279] For the scientific literature, patents, patent applications, and the like cited in the present specification, the entire contents thereof are incorporated by reference in the present specification to the same extent as each is specifically described.

[0280] The present application has been described above based on the preferred embodiments for easy understanding of the present application. The present application is described below based on Examples, but the above description and the following Examples are provided for the purpose of illustration only, and are not provided for the purpose of limiting the present application. Therefore, neither the embodiments specifically described in the present specification nor the Examples limit the scope of the present application, which is limited only by the claims.

[0281] Examples

[0282] The present application is specifically described by the following Examples, but the present disclosure is not limited by these Examples. Reagents are specifically used products described in the Examples, but can be used instead with equivalent products of other manufacturers (Sigma-Aldrich, Wako Pure Chemical Industries, Nacalai, R&D Systems, USCN LifeScience INC, etc.).

[0283] <Reagents and test methods>

[0284] Reagents and the like used in the Examples and general test methods are described below.

[0285] As the host of Bacillus subtilis, BUSY9797 strain derived from Marburg 168 strain and RM125 strain (non-patent literature 7) was used. As a plasmid vector that can replicate in Bacillus subtilis, a modified body of pGETS151-pBR (patent literature 2) was used. Figure 1, SEQ ID NO: 1). Antibiotics carbenicillin, tetracycline, kanamycin were used as products manufactured by Nacalaitesque Co. Lysozyme was purchased from Fuji Photo Film and Otsuka Pharmaceutical Co. Each restriction enzyme was used as products manufactured by New England Biolabs Co. T4 DNA Ligase was purchased from Takara Bio Co. Takara Ligation Kit (Mighty Mix) (Takara Bio Co.) was used for general ligation for plasmid construction of E. coli. KOD Plus polymerase v.2 by Toyobo was used for PCR reaction for unit DNA preparation. On the other hand, Ex-Taq HS manufactured by Takara Bio Co. was used for colony PCR for base sequence determination of DNA cloned into plasmid. Plasmid Safe manufactured by EPICENTER Co. was used for circular plasmid purification. 2-Hydroxyethylagarose of low-melting-point agarose gel for DNA electrophoresis was purchased from Sigma-Aldrich Co. UltraPure Agarose (agarose) by Invitrogen Co. was used for other general electrophoresis. TE saturated phenol (containing 8-Quinolinol) was used as a product manufactured by Nacalai tesque Co. LB medium components and agar were used as products manufactured by BD Co. Each reagent used for extraction operation from bacterial bodies or culture solution after cultivation and analysis with HPLC was used as products manufactured by Nacalai tesque Co.

[0286] E. coli JM109 strain was used for plasmid construction. QIAprep Spin Miniprep Kit by Qiagen Co. was used for small-scale purification of constructed plasmid from E. coli. MinElute Reaction Cleanup Kit by Qiagen Co. was used for DNA purification from enzyme reaction solution. As an ultramicro spectrophotometer, NanoDrop One by Thermo Fisher Co. was used. 3500xL Genetic Analyzer by Applied Biosystems Co. was used for automatic sequencer for base sequence determination.

[0287] As for other general DNA operations, standard operation procedures (Non-patent literature 8) were followed. Transformation of B. subtilis and plasmid extraction by OGAB TM method and the like were performed as reported (Non-patent literature 9).

[0288] 1. Construction of plasmid vector for enrichment

[0289] The plasmid vector pGETS151-pBR used for enrichment is an Escherichia coli-Bacillus subtilis intergeneric shuttle plasmid vector having the replication origin of pBR322 of Escherichia coli and the replication origin of pGETS151 of Bacillus subtilis. The following was used in this example: a promoter was introduced in order to increase the number of copies of the plasmid, and a point mutation was introduced by the BsmBI recognition sequence contained in pGETS151 without cleavage. Figure 1 The structure is shown in FIG. 1, and the base sequence is shown in SEQ ID NO: 1. The plasmid vector was cleaved with the restriction enzyme AarI and purified, and a pGETS151 vector in which the sequence derived from pBR322 was removed and ATTA and AAAA protrusions occurred could be obtained. A Bacillus subtilis plasmid was constructed by introducing a gene cluster of interest using the protrusions.

[0290] 2. Selection of natural product biosynthesis enzyme gene cluster

[0291] The biosynthetic gene cluster (BGC) used in the examples of the present application was targeted at the Triketide Pyrone BGC (bpsA-bpsB) of type III PKS contained in the genome of Bacillus subtilis and the Gramicidin S BGC (grsT-grsA-grsB) of NRPS contained in the genome of Aneurinibacillus migulanus (Aneurinibacillus migulanus) strain DSM2895. The production of the two substances used in this case was not detected in the wild-type strain under normal conditions (Non-patent literature 10, Non-patent literature 11). In the examples shown below, the Triketide Pyrone BGC was produced by Bacillus subtilis Marburg 168 strain carrying the BGC in the genome (homologous production), and the Gramicidin S BGC was studied in Bacillus subtilis BUSY9797 not carrying the BGC in the genome (heterologous production). The most important issue in this study was to produce a substance that is not produced or is produced in only a small amount in the wild-type host in a strain that can produce it homologously or heterologously. Therefore, as a method, the use of a combinatorial library of promoters having different transcription strengths depending on the proliferation phase was used as a unit of each gene constituting the gene cluster.

[0292] Example 1

[0293] (1) Production of the parent plasmid BGC-1 (Triketide Pyrone BGC): In BGC-1, the Triketide Pyrone parent nucleus is prepared by BpsA of type III PKS, and the OH group on the parent nucleus is further methylated by BpsB of the methyltransferase. The aim was to obtain strains that produce high levels of this methylated Triketide Pyrone (Pyrone-OMe) and strains that reduce the residual unmethylated Triketide Pyrone (Pyrone-OH) produced by the intermediate BpsA. Based on the bpsA and bpsB sequences of Bacillus subtilis, sequences were designed to silence the recognition sequences of BbsI and BsmBI. It was anticipated that the ribosome binding site (Shine-Dalgarno) (SD) sequence on the wild-type genome would contain 19 bp upstream of bpsA and 15 bp upstream of bpsB, thus attaching to the upstream of each gene. Furthermore, the promoter insertion site and Combi-OGAB... TM The SfiI recognition sequence (SEQ ID NO: 2) was used for importation. After constructing the sequence using the MAP method (Patent Document 3), it was placed in a Bacillus subtilis vector (…). Figure 2 For this construct, various promoter cassettes (Table 1) were introduced downstream of the BBaK_780000 terminator. The promoters used were the proliferation-phase-dependent promoters described in Non-Patent Document 6. The proliferation-phase-dependent promoters used were: PabrB (strong transcription strength), PvalS (medium transcription strength), Phag (weak transcription strength), PspoVG (strong transcription strength), PsrfAA (medium transcription strength), Pcdd (weak transcription strength), PlytR (strong transcription strength), Pveg (weak transcription strength), PmmgA (strong transcription strength), PyqfD (medium transcription strength), and PsigW (weak transcription strength). These promoter cassettes were first prepared to be introduced into the plasmids bpsA and bpsB as the parent plasmids. Next, these 11 parent plasmids were used via Combi-OGAB TM To facilitate the creation of combinatorial libraries, and to introduce strains that produce high levels of Pyrone-OMe and strains that can inhibit Pyrone-OH residues, the optimal promoter combination for transcriptional strength / initiation was explored.

[0294] [Table 1]

[0295]

[0296] (2) Using Combi-OGAB TM Screening of high-yield strain of methylated Triketide Pyrone

[0297] The 11 parent plasmids were mixed in equal amounts, cut with Sfil, inactivated with TE-saturated phenol, and concentrated with 1-butanol. The water phase was then precipitated with ethanol, and the precipitate was dissolved in TE. The Sfil-treated fragment solution was re-ligated with T4 DNA Ligase, and enriched with Marburg 168 strain. The resulting colonies were inoculated on LB-agar containing 10 μg / mL tetracycline, and incubated at 30°C overnight. The resulting colonies were transferred to 300 μL of LB-liquid medium containing 10 μg / mL tetracycline, and incubated at 30°C overnight. The resulting colonies were then stored at -70°C. To evaluate the amount of Triketide Pyrone biosynthesized by each strain, the stored colonies were transferred to 10 mL of LB-liquid medium containing 10 μg / mL tetracycline, and incubated at 37°C with shaking (1stcycle: 17 hours, 2nd-4thcycles: 24 hours). The culture solution was centrifuged at 8,000 g for 5 minutes, and the precipitate was collected. The precipitate was then suspended in 15 mL of pure water, and centrifuged again. The precipitate was suspended in 5 mL of 150 mM NaCl and 20 mM HC1, and incubated at 80°C for 30 minutes. Then, 5 mL of ethyl acetate was added, and the mixture was stirred with a vortex mixer for 30 seconds. The organic layer was then collected by centrifugation at 10,000 g for 10 minutes. The collected organic layer was dried with an evaporator, and dissolved in 100 μL of methanol. The solution was centrifuged at 15,000 g for 5 minutes, and 20 μL of the supernatant was injected into HPLC to analyze the amount of Triketide Pyrone. The conditions for the analysis in HPLC are shown below.

[0298] Column: COSMOSIL (trademark) 5C 18-MS-II Packed Column 4.6 mm I.D. x 150 mm

[0299] Mobile phase A: 0.1% formic acid-pure water, mobile phase B: methanol

[0300] Gradient: (80% B, 3 minutes) - (80%→98% B, 4 minutes) - (98% B, 18 minutes)

[0301] Flow rate: 1 mL / minute

[0302] Detection: 280 nm

[0303] After the analysis, strains producing a large amount of the objective substance Pyrone-OMe were selected from the obtained data, and were separately cultured. The overnight culture was mixed in equal amounts, and plasmids were extracted. Then, after treatment with Sfil and ligation, transformation of Marburg 168 was performed, and the cycle of analysis was repeated until the yield converged. Note that, in the present study, the peak at m / z 350 was attributed to Pyrone-OMe, and the peak at m / z 336 was attributed to Pyrone-OH, and was quantified, among the Triketide Pyrone derivatives.

[0304] (3) Combi-OGAB TM Change in the ratio of Pyrone-OMe high-yield strains per cycle

[0305] In the first cycle, 144 strains were separately cultured, and as a result, 123 strains grew. Using the described method, plasmids were extracted from these strains, and after HPLC analysis, 13 strains with high yield were used to construct a library for the second cycle. After the second cycle, all strains grew (Table 2). The yield of the final product, Pyrone-OMe, reached an upper limit in three cycles Figure 3 When the promoter sequences of the three strains preceding the high-yield strains of the objective substance were measured in the second cycle and the third cycle, as a result, three out of six were the combination of PvalS-bpsA-PsrfAA-bpsB (Table 3). That is, in order to make a Bacillus subtilis strain with high yield of Pyrone-OMe, the promoter combination of PvalS (Class I-mid) - PsrfAA (Class II-mid) is the best, and it is shown that by using this strain, Pyrone-OMe can be efficiently obtained.

[0306] [Table 2]

[0307]

[0308] [Table 3]

[0309]

[0310] (4) Combi-OGAB TM Change in the ratio of methylation high-efficiency strains per cycle

[0311] At the time of monitoring the production of Pyrone-OMe in each cycle, it was also confirmed that the sample in which the ratio of Pyrone-OMe to the total amount of non-methylated body (Pyrone-OH) produced by BpsA and not subjected to methylation by BpsB and Pyrone-OMe was high. Therefore, based on the passage of each cycle of the production, reanalysis was performed based on the methylation rate of BpsB (100 x Pyrone-OMe production / (Pyrone-OH production + Pyrone-OMe production)). The result thereof was also increased through each cycle, converged in the 3rd cycle Figure 4 ). The combination of Pcdd-bpsA-PvalS-bpsB conferred the highest methylation efficiency in the 3rd cycle (Table 4). That is, in order to make a B. subtilis strain that methylates Pyrone-OH efficiently, the introduction of the promoter combination of Pcdd (Class II-weak) - PvalS (Class I-medium) was optimal. Although the production of Pyrone-OMe with this promoter combination was as much as that of the combination of PvalS-PsrfAA as described, it was shown that Pyrone-OH and Pyrone-OMe detected on HPLC with close elution times could be separated efficiently and simply.

[0312] [Table 4]

[0313]

[0314] Example 2

[0315] (1) Preparation of a parent plasmid of BGC-2 (Gramicidin S BGC)

[0316] As with BGC-1, by exploring the promoters that appropriately regulate the transcription strength and transcription initiation of grsT, grsA, grsB constituting the gramicidin S BGC, the promoter combination optimal for the highest production of gramicidin S was explored. Based on the grsT, grsA, grsB sequences of the DSM2895 strain, sequences in which the recognition sequences of AarI, BsaI, BbsI, BspQI were silenced were designed, and the SD sequence was expected to contain 20 bp upstream of grsT, 22 bp upstream of grsA, and 20 bp upstream of grsB of the DSM2895 strain, thereby being attached to the upstream of each gene. Also, as with BGC-1, Combi-OGAB TM was introduced at the promoter introduction site using the Sfil recognition sequence (SEQ ID NO: 3) for OGAB TM . The sequence was divided into 25 fragments of OGAB TM blocks and synthesized by OGAB Figure 5). Then, as with the BGC-1, a cassette in which any one of the promoters of PvalS (Class I - medium), Phag (Class I - weak), PspoVG (Class II - strong), PsrfAA (Class II - medium), P cdd (Class II - weak), P lytR (Class III - strong), Pveg (Class III - weak), Pmmg A (Class IV - strong), PyqfD (Class IV - medium), and PsigW (Class IV - weak) is connected downstream of the BBaK_780000 terminator was prepared, and the cassette was introduced into the grsT, grsA, grsB plasmid as a parent plasmid. Next, using these 10 kinds of parent plasmids, a combinatorial library was created by Combi-OGAB TM

[0317] (2) Screening of a gramicidin S high-producing strain using Combi-OGAB TM

[0318] The 10 kinds of plasmids were mixed in equal amounts, and after cleavage with Sfil, the enzyme was inactivated with TE-saturated phenol, and the water phase was concentrated with 1-butanol, followed by ethanol precipitation. The resulting precipitate was dissolved in TE. The Sfil-treated fragment solution was again ligated by T4 DNA Ligase, and after enrichment with the BUSY9797 strain into which the plasmid pUB8 (non-patent literature 12) encoding phosphopantetheinyl transferase lpa-8 was introduced, plating was performed on an LB-agar containing 10 pg / mL tetracycline-kanamycin, and overnight incubation was performed at 30°C. The resulting colonies were each transferred to 300 pL of an LB-liquid medium containing 10 pg / mL tetracycline-kanamycin, and after overnight culture at 30°C, they were stored frozen at -70°C. When cultured for evaluation of the amount of gramicidin S biosynthesis, the frozen stock was transferred to 2 mL of a YTG (Bacto yeast extract powder) 50 g / L, Bacto tryptone 50 g / L, glucose 5 g / L) liquid medium containing 10 pg / mL tetracycline-kanamycin, and 72-hour shaking culture was performed at 30°C. To the culture solution, 2 mL of ethyl acetate was added, stirred for 30 seconds with a vortex mixer, centrifuged at 10,000 g for 10 minutes, and the organic layer was collected. The organic layer was dried with an evaporator, and dissolved in 200 pL of 70% methanol-0.5% formic acid. The solution was centrifuged at 15,000 g for 5 minutes, and 20 pL of the supernatant was injected into HPLC and analyzed for the yield of gramicidin S. The measurement conditions in HPLC are shown below.

[0319] ​​Column: COSMOSIL (trademark) 5C18-AR-II Packed Column 4.6 mm I.D. x 150 mm

[0320] Mobile phase A: 0.1% formic acid - pure water, mobile phase B: methanol

[0321] Gradient: (60% B, 5 min) - (60%→78% B, 9 min) - (78%→100% B, 0.1 min) - (100% B, 5 min)

[0322] Flow rate: 1 mL / min

[0323] Detection: 210 nm

[0324] After the analysis, a plurality of high-yield strains were selected from the obtained data, and after re-culturing, the culture solutions were mixed in equal amounts, and plasmids were extracted. Then, the library was reconstructed by Sfil treatment, and the yield of each strain was measured again by Combi-OGAB TM The cycle was repeated until the yield converged.

[0325] (3) Change in the yield of short bacitracin S per cycle

[0326] In the first cycle, 192 strains were cultured respectively, and as a result, 134 strains grew. Using the method described above, the strains were extracted, and when analyzed by HPLC, it was found that among the strains, the strains showing a single peak on the chromatogram and the strains showing a three-peak pattern were Figure 6). When analyzing the peak in LC-MS, it was found that from the sample of the three-peak peak, in addition to the m / z 1141 corresponding to the molecular weight of the substance of interest, 1155 and 1169 were detected. There is a report that the L-Orn selective domain of the surfactin S biosynthesis enzyme gene cluster also has L-Lys selectivity (non-patent literature 13), and there is a report that a strain producing a Lysl residue substitute and a 2 residue substitute exists in a wild type production strain (non-patent literature 14). Since only the promoter sequence was subjected to the library in this study, it was suggested that the transcription initiation of the promoter or the transcription strength was effective for suppressing the production of the Lys substitute of surfactin S. In order to transfer to the 2nd cycle, the re-culturing of the strains that produced surfactin S at a high yield in the 1st cycle was attempted, and half of the strains could not be re-cultured. By this step, it was possible to easily exclude the constructs that were compatible with the host and had low reproducibility of culture. From the 7 strains that could be re-cultured, plasmids were extracted and transferred to the 2nd cycle. Of the 60 strains cultured in the 2nd cycle, 59 strains grew. Among them, 45 strains were surfactin S production strains, and only 2 strains were strains that showed a three-peak peak on the HPLC chromatogram. All strains grew after the 3rd cycle, and no non-production strain of surfactin S was found, nor was a strain that showed a three-peak peak on the HPLC chromatogram (Table 5). As a result of the 4th cycle, it was confirmed that the surfactin S production strains converged in the 3rd cycle Figure 7 When the promoter sequences of the top 5 strains of the 3rd cycle that confirmed the production of surfactin S were confirmed, it was found that PsigW-grsT-Pcdd-grsA-PlytR-grsB was common to all strains. That is, it was suggested that the combination of PsigW (Class IV-weak) -Pcdd (Class II-weak) -PlytR (Class III-strong) was most suitable for the heterologous production of surfactin S in Bacillus subtilis.

[0327] [Table 5]

[0328]

[0329] (4) Analysis of the purity of surfactin S using a high yield strain

[0330] The biosynthetic extract of the C2 strain of the 3rd cycle (3rd-C2) of the highest yield strain of the strain that showed a single-peak peak on the HPLC chromatogram and the 2nd-1C11 strain of the 2nd cycle (2nd-1C11) of the strain that showed a three-peak peak on the HPLC chromatogram were analyzed by LC-MS and LC-MS / MS. In the 3rd-C2 strain, the desired surfactin S: 1 Lys substitute: 2 Lys substitute = 95.56: 3.58: 0.86, and in the 2nd-1C11 strain, 75.63: 14.62: 9.75, it was confirmed that the ratio of the by-product of the Lys substitute was higher Figure 8, Table 6). That is, by optimizing the transcription initiation of the promoter of each gene by the Combi-OGAB TM method, not only the production can be increased, but also the promoter combination which suppresses the production of Lys-substitutes can be explored. The objective short bacitracin S and Lys-substitutes were detected on HPLC with substantially the same elution time. Therefore, a large amount of time and cost is required to improve the purity in the purification step, but by this procedure, it was shown that the promoter combination which not only increases the production but also improves the purity of the objective substance from the production time point was found.

[0331] [Table 6]

[0332]

[0333] PKS and NRPS genes exist not only in Bacillus subtilis, but also in the genomes of actinomycetes and filamentous bacteria in large amounts. Therefore, by conducting the research as in this example, it is considered that the same experimental results can be obtained also in the PKS and NRPS of such actinomycetes and filamentous bacteria.

[0334] (Example 3)

[0335] In this example, it was shown that the configuration pattern of the promoter (monocistronic, multicistronic), the single or multiple pattern of the gene cluster, the single or multiple pattern of the construct, and the like are possible.

[0336] For the short bacitracin S biosynthesis gene cluster described in Example 2, the following 3 kinds of transcription configurations were designed, and plasmids into which various Bacillus subtilis proliferation period-dependent promoters were introduced were prepared similarly to Example 2.

[0337] 1) a construct in which a promoter is configured only in the upstream of grsT, and the entire cluster is transcribed as a multicistron;

[0338] 2) a construct in which a promoter is configured in the upstream of grsT / the upstream of grsA, and grsT is transcribed as a monocistron, and grsA / grsB is transcribed as a multicistron;

[0339] 3) a construct in which a promoter is configured in the upstream of grsT / the upstream of grsB, and grsT / grsA is transcribed as a multicistron, and grsB is transcribed as a monocistron.

[0340] In addition to the above 3 kinds, a plasmid library of 4 kinds of transcription configurations of the plasmid including the construct in which grsT / grsA / grsB is all transcribed as a monocistron which was processed in Example 2 was added, and by combining the plasmids respectively or in mixture by the Combi-OGAB method, a combinatorial library was prepared, and thereby in addition to the screening for the transcription initiation of the promoter of each gene for high production of short bacitracin S, the screening of the transcription unit can be performed.

[0341] REFERENCES

[0342] Reference Patent Literature 1: KR102301301B1

[0343] Reference Patent Literature 2: Japanese Patent Application Publication No. 2021-184730

[0344] Reference Patent Literature 3: Japanese Patent Application Publication No. 2019-198236

[0345] Reference Non-Patent Literature 1: D. J. Newman, et. al., J. Nat. Prod., 83, 770 (2020)

[0346] Reference Non-Patent Literature 2: C. C. Brett, et. al., Annu. Rev. Biochem., 90, 763 (2021)

[0347] Reference Non-Patent Literature 3: Kang H-S. et. al., ACS Synth. Biol., 5(9), 1002 (2016)

[0348] Reference Non-Patent Literature 4: Kim H., et. al., ACS Synth. Bio 1., 9(1), 175 (2020)

[0349] Reference Non-Patent Literature 5: Amagai K., et. al., Sci. Rep., 7, 3382 (2017)

[0350] Reference Non-Patent Literature 6: Yang. S., et al., Appl. Microbiol. Biotechnol., 101(10), 4151-4161 (2017)

[0351] Reference Non-Patent Literature 7: Uozumi, T., et al. Molec. Gen. Genet., 152, 65-69 (1977)

[0352] Reference Non-Patent Literature 8: Sambrook, J., et al., Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989)

[0353] Reference Non-Patent Literature 9: Tsuge, K., et al., Nucleic Acids Res. 31, e133 (2003)

[0354] Reference Non-patent Literature 10: Grubbs, K. J., et al., mSystems. 2(6) (2017)

[0355] Reference Non-patent Literature 11: Berditsch M., et al., App. Environ. Microbiol., 73(20), 6620 (2007)

[0356] Reference Non-patent Literature 12: Tsuge, K., et al., Arch Microbiol. 165, 243-251 (1996)

[0357] Reference Non-patent Literature 13: Stanisic. A., et al., Chem. Sci., 10, 10395-10399 (2019)

[0358] Reference Non-patent Literature 14: Alenezi F., Front. Microbiol., 8, 517-527, (2017)

[0359] (Note)

[0360] As shown above, although the present application is exemplified using the preferred embodiments of the present application, it is to be understood that the scope of the present application should be interpreted only according to the claims. For the patents, patent applications, and documents cited in the present specification, it is to be understood that the contents thereof are themselves specifically recited in the present specification, and the contents are cited as references to the present specification. The present application claims priority to Japanese Patent Application No. 2023-95819 filed to Japan Patent Office, and the contents thereof are entirely cited as references in the present application.

[0361] Industrial applicability

[0362] Provided is a method for producing a microorganism strain that activates a gene cluster of a gene cluster constituting a biosynthesis enzyme gene cluster of a natural product, and exhibits high productivity / inhibits a desired property such as a byproduct, and a new substance that can be produced by the method, and the like, and has applicability in biotechnology such as pharmaceuticals, agriculture, aquaculture, perfumes, food, fibers, chemicals, and the environment.

[0363] [Free text of sequence listing]

[0364] SEQ ID NO: 1: pGETS151ABsmBI-pBR

[0365]

[0366]

[0367]

[0368]

[0369]

[0370]

[0371]

[0372] SEQ ID NO: 2: Triketide Pyorne BGC

[0373]

[0374]

[0375]

[0376] SEQ ID NO: 3: Gramicidin S BGC

[0377]

[0378]

[0379]

[0380]

[0381]

[0382]

[0383]

[0384]

[0385]

[0386]

[0387]

[0388]

[0389]

[0390]

[0391]

[0392]

[0393]

[0394]

[0395]

[0396]

[0397]

[0398] SEQ ID NO: 4: BBaK_780000-PabrB

[0399]

[0400] SEQ ID NO: 5: BBaK_780000-PvalS

[0401]

[0402]

[0403] SEQ ID NO: 6: BBaK_780000-Phag

[0404]

[0405] SEQ ID NO: 7: BBaK_780000-PspoVG

[0406]

[0407] SEQ ID NO: 8: BBaK_780000-PsrfAA

[0408]

[0409] SEQ ID NO: 9: BBaK_780000-Pcdd

[0410]

[0411] SEQ ID NO: 10: BBaK_780000-PlytR

[0412]

[0413]

[0414] SEQ ID NO: 11: BBaK_780000-Pveg

[0415]

[0416] SEQ ID NO: 12: BBaK_780000-PmmgA

[0417]

[0418]

[0419] SEQ ID NO: 13: BBaK_780000-PyqfD

[0420]

[0421] SEQ ID NO: 14: BBaK_780000-PsigW

[0422]

[0423]

Claims

1. A method for producing a strain having a desired property, the method comprising the steps of: 1) a step of providing a construct comprising a gene cluster and a plurality of promoters; 2) a step of combinatorial librarying the promoters in the construct; 3) a step of screening a strain having a desired property in a host cell. The plurality of promoters are configured in a single-cistronic manner for each gene contained in the gene cluster or in a multi-cistronic manner for all genes in the gene cluster, or a mixture of the single-cistronic and multi-cistronic configurations for the genes in the gene cluster. The strain having a desired property has at least one or more properties selected from the group consisting of: a property of being able to produce a substance of interest; a property of high production of a substance of interest; a property of keeping a substance of interest inside a cell without secretion; a property of secreting a substance of interest outside a cell; a property of being easy to cultivate along with production of a substance of interest; a property of being able to produce a plurality of substances of interest at a desired ratio; a property of producing a substance of interest without producing a harmful substance; a property of robustness of the yield of a substance of interest; a property of no deletion occurring in a gene producing a substance of interest during cultivation; a property of being able to control production of a substance of interest; a property of being able to suppress production of a substance other than the interest; a property of being able to suppress production of a by-product and / or impurity that is difficult to separate in affinity, ion exchange, gel filtration, reverse phase, normal phase, chiral chromatography, distillation, crystallization, and the like; a property of low yield of empty capsids in a viral vector manufacturing, and a property of no deletion occurring in a plasmid.

4. A method of performing an action based on a desired property using a strain produced in the method of claim 1.

2. The method of claim 1, wherein, 4) a step of combinatorial librarying the construct of one or more strains screened in the step 3), introducing into a host cell of interest, and 5) a step of repeating the step 3).

3. The method of claim 1, wherein, 6. A method of producing a substance in a host organism, the method comprising the steps of: (A) a step of constructing a construct in which a promoter introduction site is added to a gene group of a substance-producing gene that is involved in removal of an undesirable restriction enzyme recognition sequence, and a sequence for reconstitution is constructed; (B) a step of introducing a plurality of constructs in which a promoter is introduced into the promoter introduction site into a combinatorial librarying host organism; (D) a step of culturing a production host organism having a combinatorial libraryed construct, and screening a plurality of strains based on an arbitrary standard; (F) a step of producing a substance using a strain having a desired property. The combinatorial librarying host organism is the same as the production host organism. The combinatorial librarying host organism is different from the production host organism. The host organism is Bacillus subtilis. A step of producing a substance using a strain produced by the method according to claim 1. A step of producing a substance using a strain obtained by the steps (A) to (E) in the method of claim 6.

12. The method of any one of claims 6-11, further comprising modifying the strain.

13. The method of claim 6, comprising a step of repeating the steps (D) and (E) a plurality of times.

5. The method of claim 1, further comprising the step of: ​ ​ ​ ​ (C) by Combi-OGAB TM Step of librarying the combination of promoter sites among various constructs by Combi-OGAB ​ (E) by Combi-OGAB TM a step of recombining the promoter sites in the constructs of the plurality of strains into a library again by Combi-OGAB ​ 7. The method of claim 6, wherein, ​ 8. The method of claim 6, wherein, ​ 9. The method of claim 7, wherein, ​ 10. A method of producing a substance in a host organism, the method comprising: ​ 11. A method of producing a substance in a host organism, the method comprising: ​ ​ ​ 14. The method of claim 6, wherein, The construction of the construct in step (A) is performed by OGAB TM method.

15. The method of claim 6, wherein, The promoters are a plurality of promoters which differ from each other in transcription initiation and / or transcription strength.

16. The method of claim 6, wherein, The production host organism is Bacillus subtilis, actinomycete, filamentous fungus, yeast, hydrogen bacterium, coryneform bacterium or animal cell.

17. The method of claim 6, wherein, The host organism is Bacillus subtilis.

18. The method of claim 6, wherein, The undesirable restriction enzyme recognition sequence is a Type IIS restriction enzyme recognition sequence selected from the group consisting of Aarl, Bsal, Bbsl, BsmBI, BspQl and a Sfil recognition sequence for reconstruction.

19. The method of claim 6, wherein, The sequence for reconstruction is a Sfil recognition sequence.

20. The method of claim 6, wherein, The gene group is a gene group of polyketide synthase and non-ribosomal peptide synthetase.

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