Method for screening DNA sequences

Abstract

To provide a method for screening a DNA sequence by which the optimum combination of the expression intensities of individual genes can readily be found even when the optimum combination of the expression intensities of the individual genes in a cluster containing a plurality of genes encoding a multimodule type biosynthetic enzyme is unknown.SOLUTION: The method for screening a DNA sequence of the present invention comprises at least the steps of: (a) assembling DNA fragments using the OGAB method to prepare a plasmid containing a plurality of DNA fragments encoding multimodular biosynthetic enzymes; (b) transforming the plasmid into a host cell to allow the host cell to produce the multimodular biosynthetic enzymes; and (c) screening a plasmid containing a DNA fragment based on the properties of the produced multimodular biosynthetic enzymes or the properties of the transformed host cell.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for screening DNA sequences. [Background technology]

[0002] Natural compounds produced by microorganisms, such as actinomycetes and filamentous fungi, are known to be useful substances with a wide variety of structures and biological activities. Decoding the genomes of producing microorganisms has now made it easier to identify biosynthetic gene clusters. It has also become clear that there are many gene clusters for useful substances that have not yet been exploited by humans. Research on biosynthetic gene clusters of microbial secondary metabolites has focused on industrially important polyketide and peptide compounds. For example, type I polyketide synthases (PKSs), a type of multimodular biosynthetic enzyme used in the biosynthesis of macrolide compounds such as erythromycin, FK-506 (tacrolimus), rapamycin, and avermectin, which are clinically used as secondary metabolites produced by actinomycetes, are known.

[0003] For example, given the difficulty of producing polyketide compounds by traditional chemical methods and the typically low production of polyketides in wild-type cells, there has been considerable interest in finding improved or alternative means for producing polyketide compounds. For these reasons, attempts have been made to introduce the gene clusters required for biosynthesis from the original bacterial strain into other cells and to produce the compounds heterologously.

[0004] However, heterologous production often results in low productivity. One reason for this is that the optimal combination of expression intensities of individual genes within the target multi-modular biosynthetic enzyme cluster is usually unknown. Therefore, efforts to optimize productivity usually require a comprehensive search of possible combinations of various factors and their strengths. While there are several approaches for heterologous expression of gene clusters, such as cloning existing clusters from genomic DNA samples and de novo assembly from synthetic DNA fragments, the number of combinations to search is enormous, and simplification of this task is needed. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] US Patent Application Publication No. 2010 / 0291633 [Patent Document 2] U.S. Patent No. 7,723,077 [Patent Document 3] Special Publication No. 2011-512140 [Patent Document 4] Japanese Patent Application Laid-Open No. 2004-129654 [Patent Document 5] WO2020 / 203496 [Non-patent literature]

[0006] [Non-Patent Document 1] PLoS One,2009,4(5),e5553 [Non-patent document 2] Chemistry and Biology, 2016, Vol. 54, No. 10, pp. 74 Summary of the Invention [Problem to be solved by the invention]

[0007] An object of the present invention is to provide a method for screening DNA sequences that can easily find the optimal combination of expression intensities of individual genes within a cluster containing multiple genes encoding multi-module biosynthetic enzymes, even if the optimal combination of expression intensities of individual genes within the cluster is unknown. [Means for solving the problem]

[0008] The present inventors have found that an optimal combination of expression intensities of individual genes can be easily found by using a screening method including the steps of: (a) integrating DNA fragments using the OGAB method to prepare a plasmid containing multiple DNA fragments encoding multimodule biosynthetic enzymes; (b) transforming the plasmid into a host cell and causing the host cell to produce the multimodule biosynthetic enzyme; and (c) screening DNA sequences based on the properties of the produced multimodule biosynthetic enzyme or the properties of the transformed host cell. The present invention is based on this novel finding.

[0009] The present invention provides, for example, the following inventions. [1] A method for screening a DNA sequence, comprising at least the following steps (a) to (c): (a) A step of assembling DNA fragments using the OGAB method to prepare a plasmid containing multiple DNA fragments encoding multi-module biosynthetic enzymes. (b) transforming the plasmid into a host cell and causing the host cell to produce the multi-module biosynthetic enzyme; (c) screening the DNA sequences by screening the plasmids containing the DNA fragments based on the properties of the produced multi-module biosynthetic enzymes or the properties of the transformed host cells; [2] The method according to [1], wherein the OGAB method is a combi-OGAB method. [3] The method according to [1] or [2], wherein the plasmid is a plasmid containing a replication origin for Bacillus subtilis, a replication origin for Escherichia coli, and a conjugation initiation sequence for actinomycetes. [4] The method according to any one of [1] to [3], wherein the step (b) comprises conjugative transfer of the plasmid prepared in the step (a) from Escherichia coli to actinomycetes. [5] The method according to any one of [1] to [4], wherein the host cell in the step (b) is an actinomycete. [6] The method according to any one of [1] to [5], wherein the multi-module biosynthetic enzyme is a type I polyketide synthase (PKS). [7] The method according to any one of [1] to [6], wherein the screening in the step (c) uses one or more of the following properties as indicators: (i) Colony size of cultured transformed host cells (ii) the turbidity or density of a culture of transformed host cells (iii) Antibacterial activity (iv) Drug resistance [8] The method according to any one of [1] to [6], wherein the screening in the step (c) uses antibacterial activity as an index. [9] The method according to any one of [1] to [8], wherein step (c) is carried out two or more times.

[10] A plasmid obtained by the screening method according to any one of [1] to [9].

[11] A host cell harboring the plasmid described in

[10] .

[12] A multi-module biosynthetic enzyme obtained using the plasmid described in

[10] or the host cell described in

[11] . [Effects of the Invention]

[0010] The DNA sequence screening method of the present invention has the advantage that it can easily find the optimal combination of expression intensities of individual genes even if the optimal combination of expression intensities of individual genes in a cluster containing multiple genes encoding multi-module biosynthetic enzymes is unknown. [Brief explanation of the drawings]

[0011] [Figure 1] FIG. 1 shows the plasmid map of OGAB vector 2.0. [Figure 2] FIG. 2 shows the flow chart of the plasmid preparation method and gene expression in heterologous expression host cells. [Figure 3] Figure 3 is a schematic diagram of the conventional combi-OGAB method. [Figure 4] Figure 4 is a schematic diagram of the Direct combi-OGAB method. [Figure 5] FIG. 5 shows the results of digesting with NotI the plasmids that were enriched and purified using Direct combi-OGAB in the Examples. [Figure 6] FIG. 6 is a table showing the results of identifying promoters in a combinatorial library and evaluating the diversity of the library in the Examples. DETAILED DESCRIPTION OF THE INVENTION

[0012] Hereinafter, embodiments of the present invention will be described in detail, but the present invention is not limited to the following embodiments.

[0013] [Multi-module biosynthetic enzymes] In this specification, examples of multi-module biosynthetic enzymes include type I polyketide synthases (PKSs) and nonribosomal peptide synthetases.

[0014] The DNA encoding a PKS or nonribosomal peptide synthetase contained in the plasmid of the present invention may be wild-type (a gene encoding such an enzyme or its cDNA), or may be a mutant DNA with modified codon usage, or may result in modification of one or more amino acids. In one preferred embodiment, a PKS derived from a bacterium of the genus Streptomyces contains products of three open reading frames (ORF1, ORF2, and ORF3). The PKS contains three domains: a ketosynthase (KS) domain, an acyltransferase (AT) domain, and an acyl carrier protein (ACP), and these three domains enable polyketide chain elongation. The PKS may further contain domains involved in main chain modification, such as a ketoreductase (KR) domain, a dehydratase (DH) domain, or an enoyl reductase (ER) domain. Compounds prepared by PKS include 6-deoxyerythronolide B (6-dEB), frenolicin, granaticin, tetracenomycin, 6-methylsalicylic acid, oxytetracycline, tetracycline, erythromycin, griseusin, nanaomycin, medelmicin, daunorubicin, tyrosine, carbomycin, spiramycin, avermectin, monensin, nonactin, curamycin, lipomycin, rifamycin, and candicidin.

[0015] The type I polyketide synthase (PKS) is not particularly limited, but examples include a PKS encoded by the DNA sequence shown in SEQ ID NO: 1, and may be a protein encoded by a DNA fragment that has 80% or more, 85% or more, 90% or more, 93% or more, 95% or more, 97% or more, or 99% or more homology to SEQ ID NO: 1.

[0016] Nonribosomal peptides refer to, but are not limited to, a class of peptides that belong to a family of complex natural products composed of simple amino acid monomers. They are synthesized in many bacteria and fungi by large, multifunctional proteins called nonribosomal peptide synthetases (NRPSs). NRPS systems are characterized by their ability to synthesize peptides containing both proteinogenic and nonproteinogenic amino acids.

[0017] Nonribosomal peptide synthetases (NRPSs) refer to, but are not limited to, large multifunctional proteins organized into cooperative groups of active sites called modules, where each module is required to catalyze one cycle of peptide elongation and functional group modification. The number and order of modules, as well as the types of domains present within each NRPS module, determine the structural variation of the resulting peptide product by directing the number, order, and selection of amino acids incorporated and the specific type of modification associated with elongation.

[0018] [Method for screening DNA sequences] The method for screening a DNA sequence of the present invention comprises at least the following steps (a) to (c): (a) a step of integrating DNA fragments using the OGAB method to prepare a plasmid containing multiple DNA fragments encoding multi-module biosynthetic enzymes; (b) transforming the plasmid into a host cell and causing the host cell to produce the multi-module biosynthetic enzyme; and (c) A step of screening the DNA sequences by screening the plasmids containing the DNA fragments based on the properties of the produced multi-module biosynthetic enzymes or the properties of the transformed host cells.

[0019] The above step (a) is a step of accumulating DNA fragments using the OGAB method, the above step (b) is a step of transforming the plasmid into a host cell, and the above step (c) is a step of screening for plasmids containing the DNA fragments. Each step will be explained in detail below.

[0020] (DNA fragment accumulation process using the OGAB method) As used herein, the OGAB method (Ordered Gene Assembly in Bacillus subtilis method) refers to a method for assembling multiple DNA fragments using the Bacillus subtilis plasmid transformation system. Specifically, the DNA fragments to be assembled and the assembly plasmid vector are prepared so that they have specific overhangs of 3 to 4 bases, and the complementary base sequences of these overhangs are used to specify the order and orientation of the DNA fragments to be ligated.

[0021] For example, the methods described in Patent Document 4 include a method for easily obtaining plasmid DNA in which multiple DNA fragments are linked and accumulated in a specific order and orientation by utilizing the DNA uptake ability and homologous recombination ability of microorganisms such as Bacillus bacteria, and which can be amplified in microorganisms, as well as a method for obtaining microorganisms whose genomic DNA contains DNA fragments in which multiple DNA fragments are linked and accumulated in a specific order and orientation.

[0022] Taking advantage of the fact that the three-base protruding ends generated by DNA digestion with the restriction enzyme SfiI can be designated as arbitrary sequences, SfiI cleavage sites can be designed and prepared by generating ends that allow the DNA fragments of the components to be assembled and the linear plasmid vector fragments with an effective replication mechanism in Bacillus subtilis to be ligated in order once within a single DNA assembly unit. These SfiI fragments are mixed at equimolar concentrations and then ligated in the presence of polyethylene glycol and salt to produce linear high-molecular-weight DNA with a structure in which this DNA ligation unit is multiply repeated. This can be transformed into Bacillus subtilis competent cells, allowing DNA to be ligated in the desired order and direction into a Bacillus subtilis plasmid. Furthermore, co-culturing Bacillus subtilis competent cells in which a sequence common to that in the plasmid has been inserted into their genomic DNA with the linear high-molecular-weight DNA obtained above with a structure in which the DNA ligation unit is multiply repeated allows DNA to be ligated in the desired order and direction into Bacillus subtilis genomic DNA.

[0023] The combi-OGAB method is known as an example of the OGAB method. As described in Patent Document 5, the combi-OGAB method is a gene accumulation method (OGAB method) that utilizes the Bacillus subtilis plasmid transformation system, in which the molar concentration ratio of all DNA fragments used to accumulate a combinatorial library is as close to 1 as possible. Specifically, a seed plasmid is constructed by ligating all of the option gene fragments to be combinatorialized. Separate seed plasmids are then constructed for other option gene fragments, resulting in a number of seed plasmids equal to the maximum number of options. Various plasmids are digested with restriction enzymes to obtain a solution in which the gene fragments are mixed equimolarly. This solution maintains equimolarity even when mixed with other seed plasmids. The various gene fragments contained in these solutions are then linearly ligated to obtain high-molecular-weight DNA in a pseudo-tandem repeat state, in which the plasmid vector portion appears periodically. This DNA is then used to transform Bacillus subtilis. A combinatorial library is efficiently constructed by circularizing the plasmid vector portion within Bacillus subtilis using homology within the plasmid vector portion. This combi-OGAB method is referred to herein as the traditional combi-OGAB method (or standard combi-OGAB), and its schematic diagram is shown in Figure 3. Figure 3 shows, as an example, the construction of a promoter library featuring three different promoters at each position. When using traditional combi-OGAB, three individual OGAB reactions are required to generate individual gene clusters with promoter candidates, followed by an additional OGAB reaction to construct the library.

[0024] This method has the advantage that the equimolar concentrations of gene fragments required for constructing a combinatorial library can be prepared extremely simply and reliably, and that the scale of library construction can be made larger than ever before.

[0025] In one embodiment, the OGAB method may be a direct combi-OGAB method, which is an improved version of the conventional combi-OGAB method. A schematic diagram of the direct combi-OGAB method is shown in Figure 4. When using the direct combi-OGAB method, construction of a combinatorial library is performed in a single reaction by combining all necessary library fragments at appropriate concentrations and conditions. The direct combi-OGAB method requires only a single OGAB assembly reaction to construct a combinatorial library of any size, whereas the conventional combi-OGAB method requires multiple OGAB assembly reactions, and the number of required reactions increases with library size. Therefore, constructing a combinatorial library using the direct combi-OGAB method is much faster than using the conventional combi-OGAB method, thereby providing the advantage of simplifying the construction of a combinatorial library. An example of the direct combi-OGAB method is shown in the Examples.

[0026] The plasmid contains a control sequence operably linked to DNA encoding the desired multimodular biosynthetic enzyme, such as a PKS. Suitable expression systems for use in the present invention include systems that function in eukaryotic and prokaryotic host cells. However, as explained above, prokaryotic systems are preferred, and systems compatible with bacteria of the genus Streptomyces are particularly important. Control sequences for use in such systems include promoters, ribosome binding sites, terminators, enhancers, and the like. Useful promoters are those that function in Streptomyces host cells, including, but not limited to, pGapdh, pErmE, and pKasO.

[0027] A selectable marker may also be included in the plasmid. A variety of markers are known that are useful in selecting transformed cell lines and generally contain genes whose expression confers a selectable phenotype on transformed cells when the cells are grown in an appropriate selective medium. Such markers include, for example, genes that confer antibiotic resistance or sensitivity to the plasmid. Alternatively, some polyketides are naturally colored, and this characteristic provides a built-in marker for selecting cells successfully transformed with the constructs of the present invention.

[0028] The plasmid containing multiple genes encoding the multimodule biosynthetic enzyme of the present invention contains DNA encoding the domains contained in the multimodule biosynthetic enzyme, and the type and size of the DNA are not particularly limited. The DNA fragment encoding the domains contained in the multimodule biosynthetic enzyme may be a wild-type gene or its cDNA from a microorganism, or an artificially designed or synthesized DNA fragment, and is not particularly limited. A preferred example is a gene cluster constituting a PKS or NRPS. In a given originating organism, a wild-type DNA fragment primarily uses one codon to express the corresponding amino acid. However, when heterologously expressed, the codon usage must be adjusted to match the host's codon usage. Other factors that may affect the results of heterologous expression include GC content (the content of guanine and cytosine bases in the sequence) and repetitive sequences. Repetitive sequences reduce genetic stability, increase the risk of erroneous hybridization, and inhibit the synthesis of repetitive segments. Therefore, synthetic genes must be optimized in terms of codon usage and GC content. However, these requirements are usually difficult to achieve simultaneously. For example, codon optimization may result in highly repetitive DNA fragments or a high GC content. In the present invention, the GC content is 30 to 70%. It is preferably 70% or less, 68% or less, 65% or less, or 60% or less. By using the present invention, the target plasmid can be synthesized with high efficiency even when the GC content is 50% or more, 52% or more, 55% or more, 58% or more, or 60% or more. Codons are preferably optimized to avoid repeats of nucleotide sequences of 20 bp or more. It is preferable to avoid extreme differences in GC content within a gene. For example, the difference in GC content between the highest and lowest 50 bp stretches is preferably 52% or less. It is preferable to minimize homopolymers. It is preferable to minimize the number and length of small repeats scattered throughout the DNA fragment.

[0029] The plasmid of the present invention contains a replication origin for Bacillus subtilis and may contain a replication origin other than that for Bacillus subtilis, and may contain a prokaryotic F factor partitioning system for single copy maintenance in E. coli other than the replication origin, a conjugation initiation sequence for actinomycetes, a site-specific recombination system that allows the vector to be integrated into the genome of a recipient host at a defined location, and one or more selection markers that function in Bacillus subtilis, E. coli, and actinomycete expression hosts. In one embodiment, the plasmid may be a plasmid that contains a replication origin for Bacillus subtilis, a replication origin for E. coli, and a conjugation initiation sequence for actinomycetes.

[0030] The replication origin of Bacillus subtilis of the present invention is not particularly limited as long as it can exhibit its function. The replication origin of Bacillus subtilis is not particularly limited, but examples thereof include the one shown in SEQ ID NO: 2, and may be one having a homology of 80% or more, 85% or more, 90% or more, 93% or more, 95% or more, 97% or more, or 99% or more to SEQ ID NO: 2.

[0031] Replication origins other than those of Bacillus subtilis are not particularly limited, but include the replication origin of Escherichia coli. The replication origin of Escherichia coli of the present invention may be any that can exhibit its function, and is not particularly limited, but examples thereof include RepA. For example, an example of an Escherichia coli replication origin is that shown in SEQ ID NO: 3, and it may also be one that has a homology of 80% or more, 85% or more, 90% or more, 93% or more, 95% or more, 97% or more, or 99% or more to SEQ ID NO: 3.

[0032] The prokaryotic F factor partitioning system for single copy maintenance in E. coli is not particularly limited, but examples include the system shown in SEQ ID NO: 4, and may have a homology of 80% or more, 85% or more, 90% or more, 93% or more, 95% or more, 97% or more, or 99% or more to SEQ ID NO: 4.

[0033] The conjugation initiation sequence to actinomycetes may be any sequence that can perform its function, and is not particularly limited. For example, the sequence shown in SEQ ID NO: 5 may be used, and the sequence may have a homology of 80% or more, 85% or more, 90% or more, 93% or more, 95% or more, 97% or more, or 99% or more to SEQ ID NO: 5.

[0034] The site-specific recombination system that enables integration of a vector into the genome of a recipient host at a defined location is not particularly limited, but includes, for example, the system shown in SEQ ID NO: 6, and may have 80% or more, 85% or more, 90% or more, 93% or more, 95% or more, 97% or more, or 99% or more homology to SEQ ID NO: 6.

[0035] The one or more selectable markers that function in expression hosts such as Bacillus subtilis, Escherichia coli, and actinomycetes are not particularly limited, but include, for example, the one shown in SEQ ID NO: 7, and may have a homology of 80% or more, 85% or more, 90% or more, 93% or more, 95% or more, 97% or more, or 99% or more to SEQ ID NO: 7.

[0036] In one embodiment, the OGAB vectors include OGAB vector 1.0 (sequence number 8), OGAB vector 2.0 (sequence number 9), OGAB vector 2.1 (sequence number 10), and OGAB vector 2.2 (sequence number 11), and may have a homology of 80% or more, 85% or more, 90% or more, 93% or more, 95% or more, 97% or more, or 99% or more to SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11.

[0037] By using a plasmid containing a conjugation initiation sequence for Bacillus subtilis, Escherichia coli, or actinomycete as the host cell for producing the multi-module biosynthetic enzyme, it is not necessary to modify the plasmid for each host cell to be transformed, and the risk of mutations such as insertions and deletions that occur when amplifying the plasmid can be reduced.

[0038] (Host cell transformation process) Methods for introducing the plasmids of the present invention into suitable hosts are known to those of skill in the art and typically involve the use of CaCl or other divalent cations and other agents such as DMSO. DNA can also be introduced into bacterial cells by electroporation. Once the multimodular biosynthetic enzyme PKS is expressed, polyketide-producing colonies can be identified and isolated using known techniques. Plasmids of the present invention can also be introduced into host cells using conjugative transfer between bacteria. In a preferred embodiment of the present invention, the PKS-encoding nucleotide sequence is transferred from E. coli to actinomycetes by conjugation. The PKS-encoding DNA is then integrated into the genome of the host cell, such as actinomycetes. When the host cell is an actinomycete, the genus Streptomyces is preferred. The greatest advantages of using Streptomyces host cells include higher production titers compared to heterologous expression using E. coli, and the presence of post-translational modification systems essential for active expression of type I PKS. Specific examples include S. albus, S. ambofaciens, S. avermitilis, S. azureus, S. cinnamonensis, S. coelicolor, S. curacoi, S. erythraeus, S. fradiae, S. galilaeus, S. glaucescens, S. hygroscopicus, S. lividans, S. parvulus, S. peucetius, S. rimosus, S. roseofulvus, S. thermotolerans, and S. violaceoruber, with S. albus being preferred.

[0039] DNA fragments are accumulated using the OGAB method, and a plasmid containing multiple DNA fragments encoding a multimodule biosynthetic enzyme is transformed into a host cell, allowing the host cell to produce the multimodule biosynthetic enzyme. The method for producing the multimodule biosynthetic enzyme in the host cell may be a known method. In one embodiment, a transformant into which the plasmid has been introduced is cultured, and the multimodule biosynthetic enzyme can be obtained from the culture. The term "culture" refers to any of the culture supernatant, cultured cells, cultured bacterial cells, or cell or bacterial lysate. The transformant of the present invention can be cultured according to a conventional method used for culturing a host.

[0040] The medium for culturing the transformant of the present invention may be either a natural medium or a synthetic medium, as long as it contains a carbon source, a nitrogen source, inorganic salts, etc. that can be utilized by the host and allows efficient cultivation of the transformant. Examples of carbon sources include carbohydrates such as glucose, galactose, fructose, sucrose, raffinose, and starch; organic acids such as acetic acid and propionic acid; and alcohols such as ethanol and propanol. Examples of nitrogen sources include inorganic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, and ammonium phosphate, or ammonium salts of organic acids, or other nitrogen-containing compounds. Other examples include peptone, meat extract, corn steep liquor, and various amino acids. Examples of inorganic substances include monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate.

[0041] The culture is usually carried out under aerobic conditions such as shaking culture or aeration and agitation culture at 28 to 38° C. The pH is adjusted using an inorganic or organic acid, an alkaline solution, or the like.

[0042] Cultivation under the above culture conditions allows for the production of multi-module biosynthetic enzymes in high yield.

[0043] After cultivation, if the multimodule biosynthetic enzyme is produced intracellularly or intracellularly, the expression product can be collected by disrupting the bacterial cells or cells using a homogenizer or the like. On the other hand, if the polyketide is transported extracellularly or extracellularly, the culture medium is used as is, or the bacterial cells or cells are removed by centrifugation or the like. Thereafter, the expression product is collected from the culture by extraction using ammonium sulfate precipitation or the like, and further isolated and purified using various types of chromatography or the like, as necessary.

[0044] (Screening process) The screening method is based on at least one of the properties of the produced multi-module biosynthetic enzyme and the properties of the transformed host cell. A combination of the properties of the produced multi-module biosynthetic enzyme and the properties of the transformed host cell may also be used as an index.

[0045] The properties of the produced multimodule biosynthetic enzyme refer to general properties of multimodule biosynthetic enzymes and properties of the multimodule biosynthetic enzyme produced in the present invention, and are not particularly limited. Examples include antibacterial activity, and methods for evaluating antibacterial activity may be known methods and are not particularly limited. Examples include the halo method (JIS L1902) and the film adhesion method (JIS Z2801). Antibacterial activity refers to the ability to inhibit protein synthesis against bacteria such as Staphylococcus, and is based on the properties of macrolide antibiotics such as erythromycin.

[0046] The properties of transformed host cells refer to properties inherent to the host cells and properties of the host cells transformed in the present invention, and are not particularly limited. Examples include colony size, bacterial cell turbidity, bacterial cell density, and drug resistance of cultured cells. Methods for evaluating colony size, bacterial cell turbidity, bacterial cell density, and drug resistance may be known methods and are not particularly limited. Examples include visual evaluation and methods using a spectrophotometer. Drug resistance generally refers to resistance to antibiotics such as erythromycin, tetracycline, streptomycin, penicillin, actinomycin, rifampicin, fosfomycin, vancomycin, and chloramphenicol. Drug resistance is based on the original properties of the host cells or on properties acquired by transformation of a plasmid into which drug resistance has been introduced.

[0047] The screening step may be performed only once or multiple times. By performing screening multiple times, a plasmid containing a more optimized combination of DNA fragments can be screened. The number of times is not particularly limited, and may be, for example, 2 or more, 3 or more, 5 or more, 10 or more, 50 or more, or 100 or more. By increasing the number of screenings, a plasmid containing a more optimized combination of DNA fragments can be obtained.

[0048] The combinations of DNA fragments obtained by screening are industrially useful, but the plasmids, host cells, and multimodule biosynthetic enzymes produced during the screening process are also industrially useful. Multimodule biosynthetic enzymes can be produced using the plasmids obtained by the screening method or host cells harboring the plasmids.

[0049] The present invention will be described in more detail below with reference to examples, etc. However, the present invention is not limited to the following examples. [Example]

[0050] Accumulation of DNA fragments in Bacillus subtilis using the Direct combi-OGAB method DNA fragments were collected using the following protocol. The collection method scheme is shown in Figure 2. 1) All DNA fragments of the target PKS cluster construct, fragments 1 to 23 (SEQ ID NOs: 12 to 34) derived from Kitasatospora aureofaciens, were amplified and purified using E. coli. The PKS backbone DNA fragments were fragments 02, 04, 05, 07, 08, 10, 11, and 13, and the PKS promoter DNA fragments were fragments 01, 03, 06, 09, 12, 14, 15, 16, 17, 18, 19, 20, 21, 22, and 23; 2) The concentrations of all DNA fragments (fragments 01–23) were measured using a UV spectrophotometer (Thermofisher Nanodrop) and prepared to produce equimolar fragment mixtures; 3) OGAB Vector 2.0 Plasmid TM It was treated with ATP-Dependent DNase E3101K (Lucigen Corporation), and DNA was purified with Qiagen MinElute; 4) The concentration of each DNA fragment was standardized to 100 ng / μl; 5) 900 ng of each backbone DNA fragment (i.e., 900 ng each of filaments 02, 04, 05, 07, 08, 10, 11, and 13) and 300 ng of each promoter DNA fragment (i.e., 300 ng each of filaments 01, 03, 06, 09, 12, 14, 15, 16, 17, 18, 19, 20, 21, 22, and 23) were mixed in a tube and treated with BsmbI-HFv2(NEB); 6) To purify the digested DNA, phenol-chloroform treatment, butanol treatment, and ethanol precipitation were performed; 7) Gel extraction was performed using a dialysis tube to excise the target fragment from the digested plasmid mixture, and the excised target fragment was purified by ethanol precipitation; 8) The excised fragment was mixed with the cleaved OGAB vector 2.0 (SEQ ID NO: 9), 1 μl of T4 DNA ligase (Takara Bio Inc.), and ligation buffer, and incubated at 37°C for 3 hours to ligate the fragment, thereby obtaining a DNA construct containing a tandem repeat of DNA encoding the target PKS cluster; 9) The reaction solution containing the DNA construct was mixed with Bacillus subtilis competent cells, mixed for a short time at 37°C, incubated for a predetermined time, and then spread onto a tetracycline selection dish; 10) After colony growth, a transformant colony was picked from one culture dish and grown in 2 ml of LB at 37°C overnight. Cells were scraped from the remaining culture dishes, and the plasmid library was purified according to conventional protocols; 11) Plasmids were extracted from overnight cultures according to conventional protocols, and the resulting DNA was examined by restriction enzyme digestion to verify whether it was constructed as expected.

[0051] [Evaluation of Direct combi-OGAB library diversity] 1) All promoter regions were amplified by PCR from the plasmids that were confirmed to be correctly assembled by restriction enzyme digestion; 2) the amplified promoter region was sequenced using Sanger sequencing; 3) The identity of each promoter was determined by Sanger sequencing using GeneeousPrime.

[0052] The efficiency of Direct combi-OGAB integration was confirmed by digesting the purified plasmid with the Direct combi-OGAB using the NotI restriction enzyme. The results are shown in Figure 5. The presence of the expected fragment pattern indicates successful assembly. Lanes 1, 2, 4, and 7-16 in Figure 5 show the expected fragment patterns, indicating successful OGAB integration in these samples. On the other hand, lanes 3, 5, and 6 show fragment patterns that differ from the expected patterns, indicating that OGAB integration was not successful in these samples.

[0053] The sequencing results were used to identify promoters in the combinatorial library and evaluate library diversity. The 12 plasmids purified after Direct combi-OGAB were sequenced using Sanger sequencing to confirm their promoter identity at each promoter position. The promoter ratios at the five promoter positions are also shown (Figure 6A). The results for the combinatorial library enriched using conventional combi-OGAB are also shown (Figure 6B).

Claims

1. A method for screening a DNA sequence, comprising at least the following steps (a) to (c): (a) A step of assembling DNA fragments using the OGAB method to prepare a plasmid containing multiple DNA fragments encoding multi-module biosynthetic enzymes. (b) transforming the plasmid into a host cell and causing the host cell to produce the multimodule biosynthetic enzyme; (c) screening the DNA sequences by screening the plasmids containing the DNA fragments based on the properties of the produced multi-module biosynthetic enzymes or the properties of the transformed host cells;

2. The method according to claim 1, wherein the OGAB method is a combi-OGAB method.

3. 3. The method according to claim 1, wherein the plasmid comprises a replication origin for Bacillus subtilis, a replication origin for Escherichia coli, and a conjugation initiation sequence for actinomycetes.

4. The method according to claim 1 or 1, wherein the step (b) comprises a step of conjugatively transferring the plasmid prepared in the step (a) from Escherichia coli to an actinomycete.

5. The method according to claim 1 or 2, wherein the host cell in step (b) is an actinomycete.

6. The method according to claim 1 or 2, wherein the multi-module biosynthetic enzyme is a type I polyketide synthase (PKS).

7. The method according to claim 1 or 2, wherein the screening in step (c) uses at least one or more of the following properties as an index: (i) Colony size of cultured transformed host cells (ii) Turbidity or density of a culture of transformed host cells (iii) Antibacterial activity (iv) Drug resistance

8. The method according to claim 1 or 2, wherein the screening in step (c) uses antibacterial activity as an index.

9. The method according to claim 1 or 2, wherein step (c) is carried out two or more times.

10. A plasmid obtained by the screening method of claim 1.

11. A host cell comprising the plasmid of claim 10.

12. A multi-module biosynthetic enzyme obtained using the plasmid of claim 10 or the host cell of claim 11.

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