Primer Pairs for Identification of Bacillus licheniformis K12 and Usage Thereof

Abstract

The present invention relates to a primer pair that specifically binds to an insertion sequence marker specific to Bacillus licheniformis strains, a composition for identifying Bacillus licheniformis strains comprising said primer pair, and a method for diagnosing Bacillus licheniformis strains by performing PCR using said primer pair and then confirming the PCR product. The composition for the identification and subspecies distinction of Bacillus licheniformis K12 strains and the method for providing strain information using the same according to the present invention not only enables rapid and effective identification of Bacillus licheniformis K12 strains but also allows for subspecies classification, thereby having the effect of deriving scientific and objective Bacillus licheniformis K12 strain search results through DNA analysis.

Description

Technology Field

[0001] The present invention relates to the identification of the Bacillus licheniformis K12 strain, and more specifically, to a primer pair that specifically binds to a specific insertion sequence marker of the Bacillus licheniformis genome, a composition for identifying the Bacillus licheniformis strain comprising the primer pair, and a method for diagnosing the Bacillus licheniformis strain by performing PCR using the primer pair and confirming the PCR product. Background Technology

[0003] Bacillus licheniformis Bacillus licheniformis ), Bacillus subtilis( Bacillus subtilis ), Bacillus amyloliquefaciens( Bacillus amyloliquefaciens Gram-positive bacteria, such as those mentioned above, are frequently used as microbial factories for producing industrial proteins due to their excellent fermentation characteristics and high yields. It is well known that they have been used to produce α-amylase and protease, which are required for textiles, laundry, medical device cleaning, and the pharmaceutical industry. Since these non-pathogenic Gram-positive bacteria produce proteins that do not contain any harmful by-products (e.g., lipopolysaccharides; LPS, also known as endotoxins), they have received a "Qualified Presumption of Safety" assessment from the European Food Safety Authority, and most of the products from these strains have also received a "Generally Recognized As Safe (GRAS)" assessment from the US Food and Drug Administration.

[0004] Therefore, the production of proteins (e.g., enzymes, antibodies, receptors, etc.) within microbial host cells is of particular interest in the field of biotechnology. Likewise, the identification and optimization of Bacillus host cells for the production and secretion of one or more proteins of interest are of great significance in the field of industrial biotechnology.

[0005] In particular, Bacillus licheniformis ( Bacillus licheniformis Since the ) strain is a Bacillus genus host cell of very high industrial importance, the desired Bacillus licheniformis ( among other Bacillus genus strains or Bacillus licheniformis subspecies) Bacillus licheniformis It is necessary to identify and distinguish only the strains.

[0006] Meanwhile, the Bacillus licheniformis K12 strain is a strain isolated from the intestines of rock crabs through screening based on amylase, protease, and cellulase activity. Genome analysis of the Bacillus licheniformis K12 strain revealed that it possesses 33 genes related to antimicrobial substances, 2 genes related to adhesion or film formation, 153 genes related to macromolecular enzymes, and 9 genes related to heavy metal removal, with a total of 195 functional genes included in the entire genome. The inventors are currently planning to establish industrial production facilities using this strain.

[0007] However, genomic research on Bacillus licheniformis strains has not yet been actively conducted, and in particular, Bacillus licheniformis ( Bacillus licheniformis To distinguish ) strains from other Bacillus strains, or to find the desired Bacillus licheniformis ( among Bacillus licheniformis subspecies) Bacillus licheniformis No research has been conducted on markers that can isolate and identify only ) strains.

[0008] Accordingly, the inventors have completed the present invention by discovering a new marker capable of analyzing the Bacillus licheniformis K12 strain, which has high potential for industrial application, between species or subspecies using the genomic DNA sequences of Bacillus strains, and by developing a multi-panel that can reduce the time and cost of genotyping. Prior art literature

[0010] (0001) Patent Publication No. 10-2021-0033369 (March 26, 2021), Primer for molecular identification of Bacillus subtilis related species and method for identifying Bacillus subtilis related species using the same (0002) Patent Registration No. 10-0747701 (August 2, 2007), Antagonistic strain Bacillus licheniformis K11 and plant growth promotion and biological control method using the same The problem to be solved

[0011] One objective of the present invention is Bacillus licheniformis K12 ( Bacillus licheniformis K12) It provides primer pairs for the identification of strains and the differentiation of subspecies.

[0012] Another objective of the present invention is Bacillus licheniformis K12 comprising the primer pair ( Bacillus licheniformis K12) The invention provides a composition for the identification of strains and the distinction of subspecies.

[0013] Another objective of the present invention is to perform PCR using the primer pair and then verify the PCR product, thereby Bacillus licheniformis K12 ( Bacillus licheniformis K12) It provides a method for identifying strains. means of solving the problem

[0015] According to one aspect of the present invention, the present invention comprises a Bacillus licheniformis K12 comprising forward primer and reverse primer pairs of SEQ ID NOs. 1 and 2; SEQ ID NOs. 3 and 4; SEQ ID NOs. 5 and 6; SEQ ID NOs. 7 and 8; and SEQ ID NOs. 9 and 10 ( Bacillus licheniformis K12) Provides a composition for the identification of strains and the distinction of subspecies.

[0016] The term "subspecies" above refers to a biological classification unit that further subdivides a species, and is a group of phenotypically similar populations within a single species.

[0017] The above-mentioned Bacillus licheniformis strain is a gram-positive, spore-forming bacterium, an aerobic rod-shaped bacterium capable of growing through fermentation, and secretes enzymes useful in industrial fields such as protease, amylase, and cellulase, and exists in various fermented foods such as kimchi and soil.

[0018] In addition, Bacillus licheniformis K12 ( Bacillus licheniformis The K12 strain is a lactic acid-producing strain isolated from the intestines of a rock crab and is stored in the strain repository of Pharmims Co., Ltd. Referring to [Fig. 1], Bacillus licheniformis K12 ( Bacillus licheniformis The genome of the K12 strain is 4,338,510 bp long and contains 4,520 open reading frames (ORFs). In addition, it possesses a total of 173 genes, including 1 plasmid gene, 7 integrase-related genes, 11 recombinase-related genes, 35 transposase-related genes, and 120 phage-related gene regions. Furthermore, Bacillus licheniformis K12 ( Bacillus licheniformis K12) The strain possesses 31 genes related to antimicrobial substances, 2 genes related to adhesion or film formation, 153 genes related to polymerases, and 9 genes related to heavy metal removal, totaling 195 types of functional genes within its entire genome, and is evaluated as a strain with very promising potential for industrialization.

[0019] The primer pair of SEQ ID NOs 1 and 2 above is Bacillus licheniformis K12 ( Bacillus licheniformis K12) A primer pair derived based on the BLK1 marker (BGLPMFGA_00209 gene) obtained from the results of genome analysis of the strain, wherein the BLK1 marker shows homology with the Bacillus sp. 7586-K recombinase RecT gene and has a length of 867 bps.

[0020] In addition, the primer pair of SEQ ID NOs 3 and 4 is Bacillus licheniformis K12 ( Bacillus licheniformis K12) A primer pair derived based on the BLK2 marker (BGLPMFGA_02924 gene) obtained from the results of genome analysis of the strain, the BLK2 marker is a gene encoding a recombinase family protein and has a length of 1,632 bps.

[0021] In addition, the primer pair of SEQ ID NOs 5 and 6 is Bacillus licheniformis K12 ( Bacillus licheniformis K12) Primers derived based on the BLK3 marker (BGLPMFGA_00195 gene) obtained from the results of genome analysis of the strain, wherein the BLK3 marker is a Tyr recombinase domain-containing protein gene and has a length of 876 bps.

[0022] In addition, the primer pair of SEQ ID NOs 7 and 8 is Bacillus licheniformis K12 ( Bacillus licheniformis K12) Primers derived based on the BLK4 marker (BGLPMFGA_01007 gene) obtained from the results of genome analysis of the strain, wherein the BLK4 marker is a site-specific integrase gene with a length of 1,125 bps.

[0023] In addition, the primer pair of SEQ ID NOs 9 and 10 is Bacillus licheniformis K12 ( Bacillus licheniformis K12) The primer is derived based on the BLK5 marker (BGLPMFGA_00197 gene) obtained from the results of the genome analysis of the strain, and the BLK5 marker is the SPbeta prophage-derived uncharacterized lipoprotein YonS gene and has a length of 600 bps.

[0024] Referring to a preferred embodiment of the present invention, Bacillus licheniformis K12 (with primer pairs of SEQ ID NOs 1 to 10) Bacillus licheniformis As a result of the PCR reaction on the K12) strain, it was confirmed that the PCR product for the primer pair of SEQ ID NOs 1 and 2 has a length of 474 bps; the PCR product for the primer pair of SEQ ID NOs 3 and 4 has a length of 238 bps; the PCR product for the primer pair of SEQ ID NOs 5 and 6 has a length of 413 bps; the PCR product for the primer pair of SEQ ID NOs 7 and 8 has a length of 371 bps; and the PCR product for the primer pair of SEQ ID NOs 9 and 10 has a length of 407 bps (see FIG. 3b, FIG. 4b, FIG. 5b, FIG. 6b, and FIG. 7b).

[0025] In the composition of the present invention, the forward primer and reverse primer pairs of SEQ ID NOs. 1 and 2; SEQ ID NOs. 3 and 4; and SEQ ID NOs. 7 and 8 are Bacillus licheniformis K12 ( Bacillus licheniformis K12) It is characterized by the ability to analyze the specificity between species of strains.

[0026] Referring to a preferred embodiment of the present invention, using the primer pairs of SEQ ID NOs. 1 to 10 B. licheniformis K12 strain, B. velezensis K10 strain, B. subtilis strain, B. cereus As a result of performing PCR analysis on various species belonging to the genus Bacillus, such as strains, it was observed that various non-specific PCR products were formed in different species of the genus Bacillus depending on the primer pairs of SEQ ID NOs. 1 to 10; however, it was confirmed that for the markers other than the BLK5 marker, there were no PCR products having a size matching or similar to the candidate gene marker (see Fig. 8). Accordingly, the forward and reverse primer pairs of SEQ ID NOs. 1 and 2; SEQ ID NOs. 3 and 4; and SEQ ID NOs. 7 and 8 are Bacillus licheniformis K12 ( Bacillus licheniformis It was confirmed that it can be used for the analysis of interspecies specificity of K12) strains.

[0027] In the composition of the present invention, the forward primer and reverse primer pairs of SEQ ID NOs. 1 and 2; SEQ ID NOs. 5 and 6; and SEQ ID NOs. 9 and 10 are Bacillus licheniformis K12 ( Bacillus licheniformis K12) It is characterized by the ability to analyze specificity between subspecies of strains.

[0028] Referring to a preferred embodiment of the present invention, Bacillus licheniformis (using the primer pairs of SEQ ID NOs 1 to 10) Bacillus licheniformis As a result of performing PCR analysis on various subspecies, including the standard strains KCTC1030, 1659, 1831, and 3056 of the strain, it was confirmed that the forward and reverse primer pairs of SEQ ID NOs. 1 and 2; SEQ ID NOs. 5 and 6; and SEQ ID NOs. 9 and 10 did not form identical PCR products even among subspecies (see FIG. 9). Accordingly, the forward and reverse primer pairs of SEQ ID NOs. 1 and 2; SEQ ID NOs. 5 and 6; and SEQ ID NOs. 9 and 10 were Bacillus licheniformis K12 ( Bacillus licheniformis It was confirmed that it can be used for the analysis of specificity between subspecies of the K12 strain.

[0029] According to another aspect of the present invention, the present invention comprises a Bacillus licheniformis K12 (SEQ NOs 1 and 2; SEQ NOs 3 and 4; SEQ NOs 5 and 6; SEQ NOs 7 and 8; and forward primer and reverse primer pairs of SEQ NOs 9 and 10) Bacillus licheniformis K12) Provides a PCR kit for the identification of strains and the differentiation of subspecies.

[0030] According to another aspect of the present invention, the present invention comprises the steps of: (a) performing PCR using the PCR kit; and (b) identifying the PCR product to identify Bacillus licheniformis K12 ( Bacillus licheniformis Bacillus licheniformis K12 (including the step of identifying the K12) strain and distinguishing subspecies; Bacillus licheniformis K12) Provides a method for providing information about strains.

[0031] Bacillus licheniformis K12 of the present invention ( Bacillus licheniformis K12) In a method for providing information regarding a strain, the PCR may be a multiplex PCR.

[0032] The present invention will be described in detail below.

[0033] As used herein, the term "primer" refers to an oligonucleotide capable of specifically amplifying the nucleic acid sequences of the BLK1 to BLK5 markers selected in the present invention, and any primer that can be designed by a person skilled in the art as long as it is capable of amplifying the nucleic acid sequences of the BLK1 to BLK5 markers selected in the present invention falls under this category.

[0034] As used herein, the term "PCR reaction mixture" refers to a molecular biological reagent used to perform a PCR reaction, including DNA polymerase and a buffer solution.

[0035] The inventors identified Bacillus licheniformis K12 (from the intestines of a rock crab) Bacillus licheniformis To find markers for the identification and subspecies differentiation of the K12) strain, Bacillus licheniformis SCDB 14( Bacillus licheniformis SCDB 14) Through comparative genomic analysis with strains, a focused analysis was performed on regions with high mutation frequency in gene sequences, such as recombinase, integrase, transposase, phage-related gene, and bacterion, and a total of 56 related genes were derived. An interspecies homology analysis was performed on the surrounding regions of the derived genes, and a total of 5 genes were secured as candidate selection markers that showed low homology.

[0036] As a result of performing genetic analysis on the five markers obtained above using fluorescently labeled primers, among the BLK1 to BLK5 markers, BLK1, BLK2, and BLK4 markers were Bacillus licheniformis K12 ( Bacillus licheniformis It can be used for the identification or interspecies differentiation of K12 strains, and the BLK1, BLK3, and BLK5 markers are Bacillus licheniformis K12( Bacillus licheniformis It was confirmed that it can be used to distinguish subspecies of K12) strains.

[0037] Therefore, the Bacillus licheniformis K12 of the present invention ( Bacillus licheniformis K12) The marker for the identification of strains and the distinction of subspecies is one or more markers selected from the group consisting of BLK1 to BLK5.

[0038] In addition, the present invention provides a set of one or more primers selected from a group consisting of primer pairs specific to the BLK1 to BLK5 markers. The primers specific to the BLK1 to BLK5 markers may include each consecutive polynucleotide of 10 to 50 bp located at a 50 to 300 bp site adjacent to the 5' or 3' site of the BLK1 to BLK5 marker DNA.

[0039] Preferably, the primer set is one or more primer sets selected from the group consisting of forward primer and reverse primer pairs of SEQ ID NOs 1 and 2; SEQ ID NOs 3 and 4; SEQ ID NOs 5 and 6; SEQ ID NOs 7 and 8; and SEQ ID NOs 9 and 10.

[0040] Nucleotide sequences in which a portion of the base sequence of the above primer is substituted with other bases, deleted, or in which the position and orientation of a portion of the base sequence are changed also fall within the scope of the primer according to the present invention. In addition, by adding a specific base sequence to the 5' end of the above forward primer to eliminate analysis errors of the amplification product caused by a stuttering band, Bacillus licheniformis K12 ( Bacillus licheniformis K12) It is possible to identify strains and distinguish subspecies.

[0041] In addition, in the case of multiplex PCR, since multiple primers are used during the PCR amplification process, interference occurs between each primer when each primer is amplified, making it difficult to obtain an amplified product with the complete stuttering band removed, as each primer cannot completely synthesize adenosine. Consequently, errors may occur during the genotype determination stage for individual identification, and errors at this stage result in the loss of significance for individual identification. To minimize such errors, a specific nucleotide sequence may be added to the 5' end of the forward primer among the primers specific to the BLK1 to BLK5 markers, and the specific nucleotide sequence may be a sequence used in the industry.

[0042] In addition, the primers of the present invention may be labeled with fluorescent materials for biological analysis. For example, they may be labeled with FAM, VIC, NED, PET, etc., but are not limited thereto.

[0043] From another perspective, the present invention is Bacillus licheniformis K12 ( Bacillus licheniformis K12) A PCR kit for the identification of strains and the differentiation of subspecies is provided, wherein the kit includes a primer set specific to BLK1 to BLK5 markers. Preferably, the primer set is one or more primer sets selected from the group consisting of forward primer and reverse primer pairs of SEQ ID NOs 1 and 2; SEQ ID NOs 3 and 4; SEQ ID NOs 5 and 6; SEQ ID NOs 7 and 8; and SEQ ID NOs 9 and 10.

[0044] In another aspect, the present invention comprises (a) a step of performing PCR using the PCR kit; and (b) confirming the PCR product to identify Bacillus licheniformis K12 ( Bacillus licheniformis Bacillus licheniformis K12 (including the step of identifying the K12) strain and distinguishing subspecies; Bacillus licheniformis K12) Provides a method for providing information about strains.

[0045] Preferably, the primer of step (a) is a set of one or more primers selected from the group consisting of forward primer and reverse primer pairs of SEQ ID NOs 1 and 2; SEQ ID NOs 3 and 4; SEQ ID NOs 5 and 6; SEQ ID NOs 7 and 8; and SEQ ID NOs 9 and 10.

[0046] The nucleic acid sample to be analyzed in step (a) above may be isolated from a biological sample. The biological sample includes solid tissue samples or cells and their offspring derived therefrom. Additionally, the samples also include reagent-treated samples, solubilized samples or cultured cells, cell supernatants, and cell lysates.

[0047] The isolation of nucleic acids from the aforementioned biological samples can be performed according to methods known in the art, and it is also possible to perform the isolation using a commercially available genome DNA extraction kit, as in one embodiment of the present invention.

[0048] The multiplex PCR in step (a) above can be performed under conditions suitable for amplifying each gene according to methods known in the art, and can be performed considering the annealing temperature, the size of the amplification product, etc. For example, it can be performed using the touchdown PCR method, where the touchdown PCR parameters are performed by treating at 95°C for 5 minutes in the initial denaturation step, then repeating the first 10 cycles with 30 seconds at 95°C, 30 seconds at an initial 60°C (the annealing temperature decreases by 1°C with each cycle), and 30 seconds at 72°C as one cycle, and then performing the subsequent 25 cycles under conditions of 30 seconds at 95°C, 30 seconds at 55°C, and 30 seconds at 72°C, followed by extension at 65°C for 30 minutes and termination at 4°C.

[0049] Confirmation or size analysis of the amplified PCR product in step (b) above can be performed by separating the amplified product using an automated gene analysis device and analyzing the size of the alleles for each locus. For example, the size of the amplified product can be confirmed by performing electrophoresis using a conventional method, such as using an agarose gel or an acrylamide gel, and then fluorescently staining the separated DNA fragment. For example, it can be confirmed by performing electrophoresis using the allele analysis mode of a DNA sequencing device from Applied Biosystems and classifying the PCR product by the size of the primer fluorescent substance. The method of analyzing the size of the PCR product in the above step can be easily performed by a person skilled in the art to which the present invention belongs, and can be performed by any conventional method other than the method described as an example above. Effects of the invention

[0051] As explained above, Bacillus licheniformis K12 according to the present invention ( Bacillus licheniformis A composition for the identification and subspecies distinction of strain K12 and a method for providing strain information using the same rapidly and effectively [provides] Bacillus licheniformis K12 ( Bacillus licheniformis Not only can the K12 strain be identified, but subspecies classification is also possible, allowing for scientific and objective Bacillus licheniformis K12( Bacillus licheniformis K12) It has the effect of being able to derive strain search results. Brief explanation of the drawing

[0053] Fig. 1 shows Licheniformis K12 ( Bacillus licheniformis This is the result of the genomic analysis of the K12) strain. Figure 2 is a table summarizing information on BLK1 to BLK5 markers and PCR primer sequences for analyzing them. Figure 3a is an explanatory diagram summarizing the results of the homology comparison analysis for the BLK1 (BGLPMFGA_00209) marker. Figure 3b is an explanatory diagram illustrating the primer design process for the BLK1 (BGLPMFGA_00209) marker. Figure 3c is a schematic diagram showing the surrounding gene sequences around the BLK1 (BGLPMFGA_00209) marker. Figure 4a is an explanatory diagram summarizing the results of the homology comparison analysis for the BLK2 (BGLPMFGA_02924) marker. Figure 4b is an explanatory diagram illustrating the primer design process for the BLK2 (BGLPMFGA_02924) marker. Figure 4c is a schematic diagram showing the surrounding gene sequences around the BLK2 (BGLPMFGA_02924) marker. Figure 5a is an explanatory diagram summarizing the results of the homology comparison analysis for the BLK3 (BGLPMFGA_00195) marker. Figure 5b is an explanatory diagram illustrating the primer design process for the BLK3 (BGLPMFGA_00195) marker. Figure 5c is a schematic diagram showing the surrounding gene sequences around the BLK3 (BGLPMFGA_00195) marker. Figure 6a is an explanatory diagram summarizing the results of the homology comparison analysis for the BLK4 (BGLPMFGA_01007) marker. Figure 6b is an explanatory diagram illustrating the primer design process for the BLK4 (BGLPMFGA_01007) marker. Figure 6c is a schematic diagram showing the surrounding gene sequences around the BLK4 (BGLPMFGA_01007) marker. Figure 7a is an explanatory diagram summarizing the results of the homology comparison analysis for the BLK5 (BGLPMFGA_00197) marker. Figure 7b is an explanatory diagram illustrating the primer design process for the BLK5 (BGLPMFGA_00197) marker. Figure 7c is a schematic diagram showing the surrounding gene sequences around the BLK5 (BGLPMFGA_00197) marker. Figure 8 is a photograph of the PCR reaction results performed on four strains of the genus Bacillus using primer pairs of SEQ ID NOs 1 to 10. Figure 9 is a photograph of the PCR reaction results performed on five sub-species strains of Bacillus licheniformis using primer pairs of SEQ ID NOs. 1 to 10. Specific details for implementing the invention

[0054] The present invention will be described in more detail below through examples. These examples are merely illustrative of the present invention, and therefore the scope of the present invention should not be interpreted as being limited by these examples.

[0056] 실시예 1. 실험재료 및 방법

[0057] 1-1. Strains and Culture Methods

[0058] Bacillus licheniformis The K12 strain is a strain isolated from the internal organs of a rock crab. In addition, Bacillus velezensis The strain is a strain isolated by our research team from the coast, and Bacillus subtilis KCTC2217 strain and Bacillus cereus The ATCC11778 strain was obtained from the Korean Collection for Type Cultures (KCTC) and the American Type Culture Collection (ATCC) and used. B. licheniformis Standard strains, including KCTC1030, 1659, 1831, and 3056, were obtained from the Korea Culture Collection Center (KCTC) and used in this study. The distributed strains were cultured in liquid media such as LB (Luria-Berani Broth) or NB (Nutrient Broth) or agar media at 30°C or 37°C for 16–24 hours for use in this experiment. In addition, reference genomes were obtained from NCBI (https: / / www.ncbi.nlm.nih.gov / ). Bacillus velezensis Genomic and proteomic information from SCDB 14 and GCF_002074095.1 was obtained and utilized.

[0060] 1-2. Genomic Analysis

[0061] ( B. licheniformis Isolation of genomic DNA from samples and preparation for sequencing analysis)

[0062] Genomic DNA was isolated from lactic acid bacteria using the Bacterial Genomic DNA Extraction Kit Ver.3.0 (TaKaRa Korea, Seoul, Republic of Korea), and this DNA was used for DNA sequencing.

[0063] (DNA sequencing)

[0064] The isolated DNA underwent a fragmentation process using a Covaris S2 ultra sonicator system, and a library consisting of 350 bp DNA fragments was constructed using the Truseq Nano DNA sample prep kit (Illumina, Inc., San Diego, CA). The final product was verified using a 2100 BioAnalyzer and sequenced using the Illumina HiSeq platform (Illumina, Inc., San Diego, CA).

[0065] (Bioinformatics analysis for obtaining genomic information)

[0066] The secured sequence reads were obtained by filtering only high-quality reads using the sickle (v1.33) and BWA ver. 0.7.12 programs, and this Bacillus licheniformisIt was used for mapping and genome assembly of the strains. After mapping, local realignment was performed using the GATK ver. 3.1-1 program, and duplicate cleanup was performed using the Picard ver. 1.98 (http: / / picard.sourceforge.net). Sequence variants were called using the GATK ver 3.1-1 program, and annotation for the variants was performed using the SnpEff (v.4.1) program.

[0068] 1-3. Search for Selected Sites and Comparison of Homology

[0069] Analyzed B. licheniformis Based on the genome sequence, genes in regions prone to frequent mutations, such as recombinase, integrase, transposase, phage-related genes, and bacteriophages, were identified. Homology studies were performed on the identified genes using the NCBI (National Center for Biotechnology Information; https: / / www.ncbi.nlm.nih.gov / ), and candidate genes with high potential as selection markers were selected.

[0071] 1-4. Genetic Engineering

[0072] General genetic engineering was performed according to the method of Sambrook et al. (1989). For the genes obtained through the analysis of genomic information and candidate selection markers, primers (Bionia, Korea) were designed and synthesized for PCR amplification. Colony PCR analysis was performed to obtain the genome for PCR. If necessary, genomic DNA was isolated using a genomic prep kit (Solg™ Genomic DNA prep kit; SolGent co., Ltd.) and used as a template for PCR analysis. Each candidate DNA fragment amplified using the synthesized primer set and PCR PreMix (AccuPower PCRMix, Bioneer) was loaded onto an agarose gel to determine the fragment length, and if necessary, the nucleotide sequence was confirmed through gene sequencing.

[0074] Example 2. Genetic information analysis and primer design

[0075] B. licheniformis Referring to [Figure 1], which summarizes the results of the genomic information analysis of the K12 strain, it was confirmed that the genome of the strain has a total length of 4,338,510 bp. Analysis subject B. licheniformis The genome of the K12 strain is a reference genome. B. licheniformis It was analyzed that the genome was 3,956 bps shorter compared to the SCDB 14 strain genome of 4,342,466 bps, but it was found to have 46 more open reading frames (ORFs). In addition, one plasmid was present in each of the two strains, and B. licheniformis For the K12 strain, a single ORF of 5,791 bps length was predicted. However, B. licheniformis In the SCDB 14 strain, the plasmid length was found to be 205,480 bps, so it was estimated that the plasmid affects the total genome length.

[0076] In addition, if functional genes are analyzed, B. licheniformis The K12 strain was found to possess 33 genes related to antimicrobial substances, 2 genes related to adhesion or film formation, 153 genes related to macromolecular enzymes, and 9 genes related to heavy metal removal, with a total of 195 types of functional genes included in the entire genome, and the reference genome is B. licheniformis It was found that it possesses more functional genes compared to the SCDB 14 strain genome.

[0077] In this experiment, possessing a large amount of functional genes as described above B. licheniformis By exploring the specialized regions of strains at the species and subspecies levels B. licheniformis Not only does it induce protection during the industrial utilization of the K12 strain, but also in various microbial communities B. licheniformis It was performed to effectively search for K12 strains.

[0078] Therefore, genomic analysis was performed to search for specific gene sequences for this purpose. B. licheniformis Analysis was performed on regions of the K12 strain's gene sequence expected to have a high mutation frequency, such as recombinase, integrase, transposase, and phage-related genes.

[0079] A total of 173 genes were secured as genes in the relevant region, including 7 integrase-related genes, 11 recombinase-related genes, 35 transposase-related genes, and 120 phage-related gene regions. The reference strain is B. licheniformisIn the case of the SCDB 14 strain, a total of 56 genes were secured, including 2 integrase-related genes, 7 recombinase-related genes, 5 transposase-related genes, and 42 phage-related gene regions.

[0080] thus, B. licheniformis The K12 strain is B. licheniformis It was confirmed to possess a 3.1 times higher variability region compared to SCDB 14 strain.

[0081] Since the variable genes in question are generally associated with gene insertions and deletions, it can be assumed that gene mutations are likely to occur in the genes and surrounding regions. Therefore, NCBI homology was compared for each 1kbps upstream and downstream region of the 173 genes obtained above.

[0082] B. licheniformis Referring to [Figure 2], which summarizes the NCBI blast (https: / / blast.ncbi.nlm.nih.gov / ) analysis results for the variability region of the K12 strain, a total of five genes named BLK1 to BLK5 were obtained as candidate selection markers that were found to have low homology.

[0083] These genes were found to be three recombinase-associated genes (BGLPMFGA_00209, 02924, 00195, BLK1~3), one integrase-associated gene (BGLPMFGA_01007, BLK4), and one phage-associated gene (BGLPMFGA_00197, BLK5). The BGLPMFGA_00195 gene was classified as an integrase-associated gene in the genomic analysis results, but was reclassified as a recombinase-associated gene during the process of re-examining homology.

[0084] (BGLPMFGA_00209 Marker)

[0085] Referring to [Figure 3a], the BGLPMFGA_00209 gene showed homology with the Bacillus sp. 7586-K recombinase RecT gene and has a length of 867 bps. Comparison results of homology of the BGLPMFGA_00209 gene B. licheniformis While the P8_B2 gene was 100% identical, the genes of the remaining strains were found to have less than 94.66% homology in 90% query cover. In particular, it was analyzed that a length of 80 bps from 787 bps at the 3'-terminus to the end point was completely deleted (Fig. 3b).

[0086] In addition, genomic analysis revealed that the BGLPMFGA_00209 gene was located in the [contig 1] region of 176,422–177,288 bps (Fig. 3c). Furthermore, homology analysis extended from both terminal regions to a 1 kb point showed no homology at all (data not shown).

[0087] Since the BGLPMFGA_00209 gene showed 80.4% homology with the Bacillus sp. 7586-K recombinase RecT gene, it is presumed to perform the role of a recombinase. Therefore, the BLK1 gene marker is B. licheniformis The design was made to generate a 474 bps PCR product from the middle region to the 3'-end so as to include the homology-deficient region of the BGLPMFGA_00209 gene of the K12 strain.

[0088] (BGLPMFGA_02924 Marker)

[0089] The BGLPMFGA_02924 gene encodes a recombinase family protein and has a length of 1,632 bps. As a result of analyzing homology between the 1 kbps upstream and 1 kbps downstream regions of the corresponding ORF region, B. licheniformis Among subspecies and some Bacillus species, more than 83% query cover and more than 84.44% homology were observed, but very high variability was observed among Bacillus species (Figs. 4a and 4b). The region showing most homology for Bacillus strains was observed in the 3'-region.

[0090] Referring to [Fig. 4c], which summarizes the genetic analysis results for the BGLPMFGA_02924 gene and surrounding regions, there are genes BGLPMFGA_02922 through 02926. The BGLPMFGA_02924 gene is Bacillus mesophilum It showed 59.1% homology with recombinase family proteins, but further research is needed regarding its precise functionality (data not shown). Among the surrounding regions, relatively low homology was found in the N-terminal region of the BGLPMFGA_02924 ORF gene, whereas high homology was found in the C-terminal region. These results showed a pattern similar to gene sequence comparison. Based on the analysis results, primers were designed for the 5'-region of the BGLPMFGA_02924 ORF gene, which showed relatively low homology, and selected as candidate markers for the BLK2 gene (see Fig. 4b).

[0091] (BGLPMFGA_00195 Marker)

[0092] The BGLPMFGA_00195 gene is a Tyr recombinase domain-containing protein gene with a length of 876 bps. In the genomic analysis results, this gene was classified as a site-specific integrase, but it was reclassified as a recombinase during a review.

[0093] Referring to [Fig. 5a], which summarizes the results of the homology analysis for the extension region of the BGLPMFGA_00195 gene, B. licheniformis Only strains P8_B2 and KS28 showed homology of over 96.24% with 100% query cover. Meanwhile, the others B. licheniformis The strain showed less than 99.76% homology with 26% query cover, indicating that it is a strain-dependent gene with high variability (see Figures 5a and 5b).

[0094] Referring to Fig. 5c, the 3'-terminal region (reverse orientation) of the BGLPMFGA_00195 ORF gene is analyzed to have very low variability because the tRNA gene is mostly located there. On the other hand, the 5'-region was observed to be highly variable. The actual amino acid homology analysis results of the BGLPMFGA_00195 ORF gene, Niallia nealsoniiThe strain showed 62.5% homology with the Tyr recombinase domain-containing protein dp eogo gene, but further research appears necessary regarding the related functionality of the GLPMFGA_00195 ORF gene. The BGLPMFGA_00196 and 00197 genes, corresponding to the N-terminal region of the GLPMFGA_00195 ORF gene, consist of phage-associated genes; however, due to the low homology of these genes as well, further research appears necessary. Therefore, primers were designed to include the 3'-terminal region of the BGLPMFGA_00195 ORF gene, which maintains relatively low homology, and BLK3 primers were constructed to generate a PCR product of 413 bps (see Fig. 5b).

[0095] (BGLPMFGA_01007 Marker)

[0096] The BGLPMFGA_01007 gene is a site-specific integrase gene with a length of 1,125 bps. Referring to [Figure 6a], which analyzes the results of homology comparisons for the extended region, the BGLPMFGA_01007 gene showed 100% homology at the 28% query cover. The BGLPMFGA_01007 gene B. licheniformis Since very low homology was observed even among substrains, it is presumed to have been transferred from specific microorganisms or other regions.

[0097] The 3'-terminus of the BGLPMFGA_01007 ORF gene was found to exhibit very high homology because it is continuously expressed by tRNA; however, it was confirmed that there was a complete lack of homology from the BGLPMFGA_01007 ORF gene to the BGLPMFGA_01010 gene (see Figures 6b and 6c). At the ORF level, the BGLPMFGA_01007 gene showed a relatively low homology of 68.7%, while the BGLPMFGA_01008–01010 genes were analyzed to have homology levels ranging from 35.7% to 76.2%, suggesting that the genes were transferred from a biological species highly specific to that location. Therefore, the BLK4 candidate gene marker was designed in the direction of the 5'-region of the BGLPMFGA_01007 ORF gene, which is completely separated from the homologous region, to generate a PCR product of 371 bps (see Figure 6b).

[0098] (BGLPMFGA_00197 Marker)

[0099] The BGLPMFGA_00197 gene is an SPbeta prophage-derived uncharacterized lipoprotein YonS gene with a length of 600 bps. Referring to [Fig. 7a], the BGLPMFGA_00197 gene is B. licheniformis It exhibits homology of over 95.54% with the P8_B2 and KS28 genomes, with 100% query cover. The BGLPMFGA_00197 gene region was analyzed in a manner similar to the homology comparison of the BGLPMFGA_00195 gene, presumed to show a similar pattern due to being neighboring genes. However, excluding the two subspecies, the remaining Bacillus strains showed homology of less than 66.67% with 17% query cover. While these results are similar to the BGLPMFGA_00195 gene, a difference was observed in the presence of scattered low homology overall (see Fig. 7b). At the ORF level, the BGLPMFGA_00197 gene Bacillus subtilis The SPbeta prophage-derived uncharacterized lipoprotein YonS gene of strain (strain 168) showed less than 32.5% homology (data not shown). In addition, the ORF genes in the surrounding region also showed homology levels of 39.6–62.5%, and the C-terminal side of the BGLPMFGA_00197 ORF gene has a phage-associated gene inserted, so it is presumed to be composed of a specific phage gene or a gene carrying a phage inserted.

[0100] The BLK5 primer was designed to produce a PCR product of 407 bps by designing the primer near the 5'-region of the BGLPMFGA_00197 ORF, which corresponds to a region excluded from the relatively homologous region.

[0102] Example 3. Analysis of species characteristics of selection markers

[0103] Using primers for selected candidate gene markers B. licheniformis K12 strain, B. velezensis K10 strain, B. subtilis strain, B. cereus PCR analysis was performed on various species belonging to the genus Bacillus, including strains.

[0104] Referring to [Figure 8], which summarizes the PCR analysis results, B. licheniformis The strain was found to form a single PCR product in all primer sets.

[0105] However, it was observed that various non-specific PCR products were formed in different species of the genus Bacillus depending on the candidate gene marker, and for the remaining markers excluding the BLK5 marker, no PCR products with a size matching or similar to the candidate gene marker were found.

[0106] In the BLK5 marker B. subtilis strains and B. cereus from the strain B. licheniformis A PCR product with a length similar to 407 bps of the strain appeared as the major product. These results suggest that the genome sequence of the strain, which has not yet been confirmed, B. subtilis strains and B. cereus It is believed that this is because a PCR product is produced that possesses genes similar to the strain or reacts similarly to the primer set used.

[0107] On the other hand, although the BLK3 marker did not generate PCR products of the same size, PCR products of similar size were B. velezensis K10 strain and B. subtilis There is a concern of confusion as it is formed in the strain. However, in additional tests using various lysate preparation methods or genome isolation kits, a 413 bps long product was not clearly formed as the major product, so it was determined that there would be no further concern of confusion.

[0108] but, B. licheniformis In previous studies conducted to develop molecular markers for strains, similar bands B. pumilus strain, B. subtilis strain, B. atrophaeus It has been reported that it is formed in strains, etc. [Almedida, 2014]. Considering these circumstances comprehensively, the primer sets of BLK1, BLK2, and BLK4, excluding BLK3 and BLK5 markers, were selected as suitable gene selection markers for the identification of Bacillus species.

[0110] Example 4. Results of sub-species specificity analysis of selection markers

[0111] The five selected genetic markers B. licheniformis Sub-species specificity was evaluated by applying it to a standard strain.

[0112] Referring to [Fig. 9], B. licheniformisIt was confirmed that the size of the PCR product differed in most strains compared to the K12 strain. Notably, regarding the BLK4 marker, which was identified as species-specific, B. licheniformis K12 strain and B. licheniformis The same PCR product was found in the KCTC1659 strain.

[0113] However, for the BLK1, BLK3, and BLK5 markers, it was confirmed that identical PCR products do not exist even among subspecies. B. licheniformis All used, including positive controls, in conventional PCR analysis targeting sub-strain strains B. licheniformis It was observed that the pattern was very specific compared to the formation of major PCR products in sub-strains [Almedida, 2014].

[0114] Based on these results, B. licheniformis It was confirmed that BLK1, BLK3, and BLK5 markers are suitable for selection of the K12 substrain.

[0115] Overall B. licheniformis Among the total of five primer sets selected as genetic markers for the strain, the BLK1, BLK2, and BLK4 markers are suitable as species-specific markers, whereas, B. licheniformis BLK1, BLK3, and BLK5 markers were found to be suitable as sub-strain-specific markers. In all cases, the most suitable genetic marker was found to be BLK1.

[0116] Foregoing, specific parts of the present invention have been described in detail. It is evident to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. Accordingly, the actual scope of the invention is defined by the appended claims and their equivalents.

[0117] delete

Claims

Claim 1 Bacillus licheniformis K12 comprising forward primer and reverse primer pairs of SEQ ID NOs 1 and 2; SEQ ID NOs 3 and 4; SEQ ID NOs 5 and 6; SEQ ID NOs 7 and 8; and SEQ ID NOs 9 and 10 ( Bacillus licheniformis K12) Composition for the identification of strains and the distinction of subspecies. Claim 2 In claim 1, the forward primer and reverse primer pairs of SEQ ID NOs 1 and 2; SEQ ID NOs 3 and 4; and SEQ ID NOs 7 and 8 are Bacillus licheniformis K12 ( Bacillus licheniformis Bacillus licheniformis K12 ( characterized by the ability to analyze interspecies specificity of the K12) strain Bacillus licheniformis K12) Composition for the identification of strains and the distinction of subspecies. Claim 3 In claim 1, the forward primer and reverse primer pairs of SEQ ID NOs 1 and 2; SEQ ID NOs 5 and 6; and SEQ ID NOs 9 and 10 are Bacillus licheniformis K12 ( Bacillus licheniformis Bacillus licheniformis K12 ( characterized by the ability to analyze specificity between sub-species of the K12) strain Bacillus licheniformis K12) Composition for the identification of strains and the distinction of subspecies. Claim 4 Bacillus licheniformis K12 comprising forward primer and reverse primer pairs of SEQ ID NOs 1 and 2; SEQ ID NOs 3 and 4; SEQ ID NOs 5 and 6; SEQ ID NOs 7 and 8; and SEQ ID NOs 9 and 10 ( Bacillus licheniformis K12) PCR kit for strain identification and subspecies differentiation. Claim 5 (a) a step of performing PCR using the PCR kit of claim 4; and (b) confirming the PCR product to identify Bacillus licheniformis K12 ( Bacillus licheniformis Bacillus licheniformis K12 (including the step of identifying the K12) strain and distinguishing subspecies; Bacillus licheniformis K12) A method for providing information about strains. Claim 6 In claim 5, the Bacillus licheniformis K12 ( Bacillus licheniformis K12) A method for providing information about strains.

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