A plasmid for genetic manipulation of Bacillus licheniformis and its application

Abstract

This invention belongs to the field of biotechnology and discloses a plasmid for genetic manipulation of Bacillus licheniformis and its applications. The temperature-sensitive plasmid pTSMK created in this invention can be transferred into Bacillus licheniformis via conjugation transfer, exhibiting both high efficiency and versatility. Using this plasmid to carry target DNA, genetic manipulation of Bacillus licheniformis can be achieved, such as DNA knockout, DNA knock-in, and random transposon insertion mutations.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and discloses a plasmid for genetic manipulation of Bacillus licheniformis and its application. Background Technology

[0002] Bacillus licheniformis ( Bacillus licheniformis Bacillus licheniformis is a recognized safe bacterium widely used in agriculture, industry, environmental science, and medicine. In its application, genetic manipulation of Bacillus licheniformis is often necessary to enhance certain properties. Although some genetic manipulation methods for Bacillus licheniformis have been reported [Nijland et al. 2010. PLoS One. 16;5(3):e9724. doi: 10.1371 / journal.pone.0009724.;Xue et al. 1999. J. Microbiol. Methods. 34(3), 183-191. doi.org / 10.1016 / S0167-7012(98)00087-6.;Hoffmann et al. 2010. Appl Environ Microbiol. 76(15):5046-57. doi: 10.1128 / AEM.00660-10.; Brophy et al. 2018. Nat Microbiol. 3(9):1043-1053. doi: [10.1038 / s41564-018-0216-5.], but these methods are often only applicable to model strains or individual wild strains, and lack versatility. In recent years, a large number of wild Bacillus licheniformis with important functions have been isolated and reported [Muraset al. 2021. Crit Rev Biotechnol. 41(4):609-627. doi: 10.1080 / 07388551.2021.1873239.], and there is an urgent need for genetic manipulation methods with strong versatility to enable the modification of different strains.

[0003] Temperature-sensitive plasmids are commonly used tools in genetic manipulation of Bacillus licheniformis. These plasmids can replicate in Bacillus licheniformis at temperatures below a certain threshold, but cannot replicate above that threshold [Horinouchi et al. 1982. JBacteriol. 150(2):804-14. doi: 10.1128 / jb.150.2.804-814.]. Utilizing this characteristic, after introducing the target DNA fragment into the host bacterium using this type of plasmid, strains exhibiting the target mutation can be screened by increasing the culture temperature and adding antibiotics. Therefore, the key to establishing a universal genetic manipulation method is the ability to introduce temperature-sensitive plasmids into different Bacillus licheniformis strains.

[0004] Two methods for introducing temperature-sensitive plasmids into Bacillus licheniformis have been reported: electroporation transformation [Nijland et al. 2010. PLoS One. 16;5(3):e9724. doi: 10.1371 / journal.pone.0009724.] and conjugation transfer [Rachinger et al. 2013. J Biotechnol. 167(4):365-9. doi: 10.1016 / j.jbiotec.2013.07.026.]. However, both methods suffer from limited applicability. Specifically, electroporation transformation generally has low efficiency and is difficult to achieve with many strains [Jeong et al. 2022. FrontMicrobiol. 26:13:802040. doi: 10.3389 / fmicb.2022.802040.]. Conjugation transfer involves transferring temperature-sensitive plasmids from *E. coli* to *Bacillus licheniformis* using the *E. coli* conjugation system. This method suffers from low transfer efficiency (10...). -7 -10 -5The problems of single conjugate / recipient bacteria and poor universality (achieved only in 2 strains of Bacillus licheniformis) [Rachinger et al. 2013. J Biotechnol. 167(4):365-9. doi: 10.1016 / j.jbiotec.2013.07.026.]. Compared to Escherichia coli conjugation systems, Bacillus conjugation systems can more efficiently transfer plasmids from Bacillus subtilis to Bacillus licheniformis [Koehler et al. 1987. JBacteriol. 169(11):5271-8. doi: 10.1128 / jb.169.11.5271-5278.][Brophy et al. 2018. Nat Microbiol. 3(9):1043-1053. doi: 10.1038 / s41564-018-0216-5.]. However, to date, there are no reports on the transfer of temperature-sensitive plasmids using Bacillus conjugation systems. Therefore, there is an urgent need to provide a universal temperature-sensitive plasmid that can meet the needs of various common genetic operations. Summary of the Invention

[0005] To address the lack of universal and efficient genetic manipulation methods for Bacillus licheniformis, this invention creates a temperature-sensitive plasmid pTSMK that can be conjugated and transferred, and its applications. This plasmid can be efficiently and universally transferred to different Bacillus licheniformis strains using a Bacillus conjugation system, thereby satisfying various genetic manipulations for Bacillus licheniformis, including DNA knockout, DNA knock-in, and transposon random insertion mutations.

[0006] The objective of this invention can be achieved through the following technical solutions:

[0007] A plasmid for genetic manipulation of Bacillus licheniformis, the sequence of which is shown in SEQ ID NO.1; the plasmid for genetic manipulation of Bacillus licheniformis comprises a replicon fragment, a transfer initiation site fragment, and a kanamycin resistance marker fragment; the sequence of the replicon fragment is shown in SEQ ID NO.2; the sequence of the transfer initiation site fragment is shown in SEQ ID NO.3; and the sequence of the kanamycin resistance marker fragment is shown in SEQ ID NO.4.

[0008] The present invention also provides a method for constructing the plasmid for genetic manipulation of Bacillus licheniformis, wherein three fragments are ligated by PCR: a replicon fragment with a sequence as shown in SEQ ID NO.2, a transfer origin fragment with a sequence as shown in SEQ ID NO.3, and a kanamycin resistance marker fragment with a sequence as shown in SEQ ID NO.4. The ligated fragment is then introduced into Bacillus subtilis to obtain the plasmid for genetic manipulation of Bacillus licheniformis.

[0009] The present invention also provides the application of the above-mentioned plasmids in DNA knockout, DNA knock-in and transposon random insertion mutations in Bacillus licheniformis.

[0010] The present invention also provides the application of the above-described plasmid in DNA knockout of Bacillus licheniformis.

[0011] Furthermore, the DNA knockout refers to constructing a gene that knocks out polyγ-glutamic acid synthesis. pgsC and three extracellular protease genes aprE, bprA and vpr Bacillus licheniformis strain.

[0012] The present invention also provides the application of the above-described plasmid in DNA knock-in of Bacillus licheniformis.

[0013] Furthermore, the DNA knock-in involves knocking in a gfp expression cassette fragment to construct a green fluorescently labeled Bacillus licheniformis strain, the sequence of which is shown in SEQ ID NO.5.

[0014] The present invention also provides the application of the above-described plasmids in transposon random insertion mutations.

[0015] The present invention also provides a recombinant Bacillus licheniformis strain constructed using the aforementioned plasmid.

[0016] Furthermore, genetic operations are performed using the plasmids described above, including DNA knockout, DNA knock-in, and transposon random insertion mutations.

[0017] Beneficial effects

[0018] The temperature-sensitive plasmid pTSMK created in this invention can be transferred to Bacillus licheniformis via conjugation transfer, exhibiting both high efficiency and universality. This plasmid can be transferred via conjugation and cannot replicate at high temperatures. Utilizing this characteristic, after carrying the target DNA fragment into the host bacterium with this type of plasmid, strains exhibiting the target mutation can be screened by increasing the culture temperature and adding antibiotics. Using this plasmid to carry the target DNA allows for genetic manipulation of Bacillus licheniformis, such as DNA knockout, DNA knock-in, and random transposon insertion mutations, while eliminating false-positive interference from plasmid replication. Attached Figure Description

[0019] Figure 1 Genetic map and conjugation transfer of plasmid pTSMK; Figure 1 In the diagram, 'a' represents the pTSMK genetic map; 'oriTLs20' is the transfer origin site derived from pLS20; 'rep' is the thermosensitive replicon of pE194; and 'kmR' is the kanamycin resistance marker. Figure 1 b is a schematic diagram of conjugation and transfer of plasmid pTSMK; strain SCK6D is a D-alanine auxotroph. DPF and MPF represent DNA processing factor and mating formation factor, respectively; Figure 1 In the figure, c represents the transfer efficiency of pTSMK; the transfer efficiency is the ratio of conjugate to recipient bacteria.

[0020] Figure 2 The identification results of the wild-type Bacillus licheniformis isolated in this invention; Figure 2 In the middle section, 'a' represents a phylogenetic tree constructed based on the 16S rRNA gene sequences of Bacillus licheniformis strains and related species; the GenBank accession numbers of each type strain used in the analysis are indicated after the species name. Figure 2 b is based on Bacillus licheniformis strains and related species. gyrA Phylogenetic tree constructed from gene sequences; the phylogenetic tree was constructed using the neighbor-joining method with MAGEX software and generated with 1000 repetitions;

[0021] Figure 3 To achieve gene knockout and knock-in effects using pTSMK; Figure 3 In the middle, 'a' represents multiple sequence alignment of partial PheS sequences from different Bacillus species; asterisks mark conserved amino acids; red text indicates conserved threonine and alanine residues, which can be mutated to serine and glycine residues, respectively, to produce sensitivity to p-Cl-phe. Figure 3 In the image, b represents the spectrum of the mobile carrier pTSMKP. Figure 3 c represents the survival of Bacillus licheniformis overexpressing pheS* on LB plates containing 5 mM p-Cl-phe; Figure 3 In the diagram, 'd' represents the procedure for knocking out Bacillus licheniformis. Figure 3 In the middle, 'e' represents the validation of the knockout effect via PCR amplification; Figure 3 f represents the knock-in effect verified by PCR and GFP fluorescence.

[0022] Figure 4 To obtain the results of random insertion mutations of transposons using pTSMK; Figure 4 In the image, 'a' represents the pTSMT (plasma transposable carrier). Figure 4 In section b, the transposon mutagenesis procedure is described; colonies cultured at 50°C are randomly picked and placed onto LB agar plates containing kanamycin and zomycin, respectively. Figure 4In the text, 'c' indicates the chromosomal location for selecting kanamycin resistance markers in transposons. 'oriC' stands for origin of replication; 'ter' for endpoint of replication. Detailed Implementation

[0023] Example 1

[0024] This embodiment provides a temperature-sensitive plasmid pTSMK that can be transferred by a Bacillus conjugation system. Figure 1 (a) consists of three fragments: ① a temperature-sensitive replicon fragment from plasmid pE194 [Horinouchi et al. 1982. J Bacteriol. 150(2):804-14. doi: 10.1128 / jb.150.2.804-814.]. rep (SEQ ID NO.2); ② A fragment from the transfer origin site of the conjugation plasmid pLS20 [Ramachandran et al. 2017. PLoS Genet. 13(2):e1006586.doi: 10.1371 / journal.pgen.1006586.eCollection 2017 Feb.]. oriT LS20 (SEQ ID NO.3); ③ Kanamycin resistance marker fragment from plasmid pUB110 [McKenzie et al. 1986. Plasmid.15(2):93-103. doi: 10.1016 / 0147-619x(86)90046-6.] Km R (SEQ ID NO.4). The three DNA fragments were synthesized by a biotechnology company and then ligated by overlap extension PCR [You et al. 2012. Appl Environ Microbiol. 78(5):1593-5. doi: 10.1128 / AEM.07105-11.]. The ligated fragments were then introduced into Bacillus subtilis SCK6 [Zhang et al. 2011. MicrobBiotechnol. 4(1):98-105. doi: 10.1111 / j.1751-7915.2010.00230.x] to obtain plasmid pTSMK (SEQ ID NO.1).

[0025] The principle of plasmid pTSMK being transferred into Bacillus licheniformis via the Bacillus conjugation system is as follows: Figure 1As shown in b. The helper plasmid pLS20 encodes a conjugation transfer system [Ramachandran et al. 2017. PLoS Genet. 13(2):e1006586.doi: 10.1371 / journal.pgen.1006586.eCollection 2017 Feb.], which includes three functional modules: an intercellular channel formation module, a DNA transfer module, and a transfer initiation site module. After the intercellular channel is formed, the proteins in the DNA transfer module recognize the transfer initiation site and begin transferring DNA to the recipient bacteria from there. Because the plasmid pTSMK carries the transfer initiation site module, pTSMK can be conjugated and transferred to the recipient strain when it coexists with pLS20 in a cell.

[0026] To verify whether pTSMK could be transferred, we first constructed a D-alanine auxotrophic donor bacterium, SCK6D, which could not grow on a D-alanine-deficient medium. The specific procedures were as follows: Using Bacillus subtilis SCK6 as the starting strain, the D-alanine racemase gene was knocked out using the method of Zhou et al. (Zhou et al. 2017. Appl Microbiol Biotechnol. 101(1), 217-227. DOI: 10.1007 / s00253-016-7906-9). alrA The D-alanine auxotrophic strain SCK6D was obtained. Using strain SCK6 as a template, the upstream homologous arm, the forward repeat sequence, and the downstream homologous arm were amplified using primers LF-F / LF-R, DR-F / DR-R, and RF-F / RF-R, respectively. The PC cassette was amplified using primer PC-F / PC-R with pTPC [Zhou et al.2017. Appl Microbiol Biotechnol. 101(1), 217-227. DOI: 10.1007 / s00253-016-7906-9] as a template. Then, these four fragments were fused into a fragment LF-DR-PC cassette-RF using overlap extension PCR with primer LF-F / RF-R. This fragment was then transformed into SCK6, and strain SCK6D was obtained after one chloramphenicol selection and one p-chlorophenylalanine selection. Secondly, we inserted a spectinomycin resistance marker into plasmid pLS20 to obtain plasmid pLS20. spcThe specific process is as follows: Using plasmid pLS20 (GenBank Number NC_015148.1) as a template, the upstream and downstream homologous arms were amplified using primers LS20LF-F / LS20LF-R and LS20RF-F / LS20RF-R, respectively. Using plasmid pUS20 [Ye et al.. 2018. J Biotechnol. 20:284:57-62. doi: 10.1016 / j.jbiotec.2018.08.001.] as a template, the spectinomycin resistance marker was amplified using primers SPC-F / SPC-R. Then, the three fragments were fused into the fragment LF-SPC-RF using overlap extension PCR with primers LS20LF-F / LS20RF-R. This fragment was then transformed into SCK6D (pLS20), and strain SCK6D (pLS20) was obtained after spectinomycin selection. spc ).

[0027] To verify the transfer efficiency and universality of pTSMK, it was introduced into strain SCK6D (pLS20). spc The strain SCK6D (pLS20) was obtained from ) spc According to the method reported in the literature [Mitsuhiro et al. 2006. Biosci BiotechnolBiochem. 70(3):740-2. doi: 10.1271 / bbb.70.740.], the donor bacteria SCK6D (pLS20) were used. spc (pTSMK) was paired with 10 strains of Bacillus licheniformis from different sources (strain information is shown in Table 1 and 2). Figure 2 Bacillus licheniformis that successfully received pTSMK were screened on LB medium containing kanamycin. Figure 1 The results showed that pTSMK could be efficiently transferred from the donor bacteria to all 10 strains of Bacillus licheniformis, with a transfer efficiency of 10%. -5 ~10 -3 The results showed that pTSMK had ideal transfer efficiency and universality, and far surpassed methods based on E. coli conjugation systems in both aspects [Rachinger et al. 2013. J Biotechnol. 167(4):365-9. doi:10.1016 / j.jbiotec.2013.07.026.]. Furthermore, approximately 80% of the conjugates showed only kanamycin resistance and no spectinomycin resistance, indicating that most conjugates yielded pTSMK but not pLS20. spc This has minimal impact on subsequent genetic manipulation.

[0028] Example 2

[0029] Gene knockout of Bacillus licheniformis using plasmid pTSMK

[0030] To achieve gene knockout without residual resistance markers, expression cassettes containing the reverse selection marker pheS* ( Figure 3 a) Plasmid pTSMKP was constructed on plasmid pTSMK by PCR [You et al. 2012. Appl Environ Microbiol. 78(5):1593-5. doi:10.1128 / AEM.07105-11.]. Figure 3(b). Among them, codons 255 and 309 of pheS* are changed from ACA and GCT of wild-type pheS [the gene is located at BLi03016 in the genome (GenBank accession number AE017333.1)] to TCA and GGT [Zhou et al. 2017. Appl Microbiol Biotechnol. 101(1), 217-227. DOI: 10.1007 / s00253-016-7906-9]. The construction process is as follows: First, using *Bacillus licheniformis* 14580 [Rey et al. 2004. Genome Biol. 5(r77). doi.org / 10.1186 / gb-2004-5-10-r77] as a template, the three parts of the gene pheS [located at BLi03016 in the genome (GenBank accession number AE017333.1)] were amplified using primers pheS*1-F / pheS*1-R, pheS*2-F / pheS*2-R, and pheS*3-F / pheS*3-R, respectively. Base mutations were introduced by pheS*2-F and pheS*3-F. These three parts were then fused into PheS*, which recognizes p-chlorophenylalanine. Simultaneously, using plasmid pTPC as a template, the vector pTPC was amplified using primers pTPC-F / pTPC-R. Then, PheS* and the vector pTPC [Zhou et al.] were used to amplify the gene. [You et al. 2017. Appl Microbiol Biotechnol. 101(1):217-227. doi: 10.1007 / s00253-016-7906-9.] Plasmid pTPC-pheS* was obtained by homologous recombination in Escherichia coli DH5α; using plasmid pTPC-pheS* and pTSMK as templates, the pheS* expression cassette and vector TSMK were amplified using primers PF / PR and TSMK-F / TSMK-R, respectively; finally, the multimer of plasmid pTSMKP was obtained by PCR [You et al. 2012. Appl Environ Microbiol. 78(5):1593-5. doi: 10.1128 / AEM.07105-11.] and transformed into Bacillus subtilis SCK6D (pLS20). spc The plasmid pTSMKP was obtained from the donor bacterium SCK6D (pLS20). Using the conjugation method described above, the plasmid pTSMKP was separated from the donor bacterium SCK6D (pLS20). spc The plasmid pTSMKP was conjugated into ten strains of Bacillus licheniformis. The results showed that all Bacillus licheniformis carrying pTSMKP were sensitive to p-chlorophenylalanine, with a survival rate of less than 10%. -4 ( Figure 3 (c) indicates that the reverse filtering marker pheS* has excellent working efficiency.

[0031] To achieve the knockout of the target gene, the gene to be knocked out (such as the amylase gene) is... amyS For example, the upstream and downstream homologous arms of the plasmid pTSMK were constructed on the plasmid pTSMK using the same method [You et al. 2012. Appl Environ Microbiol. 78(5):1593-5. doi:10.1128 / AEM.07105-11.] to obtain the knockout plasmid pTSMKP-Δ. amyS After the knockout plasmid was transferred into Bacillus licheniformis B9 via conjugation, the target gene was knocked out through two single crossovers. Figure 3 (d). The specific procedure is as follows: First, using the Bacillus licheniformis B9 genome and plasmid pTSMKP as templates, primers amySLF-F / amySLF-R, amySRF-F / amySRF-R, and (amyS)MKP-F / (amyS)MKP-R were used to amplify the... amyS The upstream and downstream homologous arms of the gene and the vector TSMKP were then obtained by multimeric PCR [You et al. 2012. ApplEnviron Microbiol. 78(5):1593-5. doi: 10.1128 / AEM.07105-11.]. amyS Plasmid multimers were then transformed into SCK6D (pLS20). spc In the process of conjugation transformation, the knockout plasmid pTSMKP-Δ was converted into a plasmid. amyS Transferred to Bacillus licheniformis, then selected pLS20 carrying the knockout plasmid but without the helper plasmid. spc Bacillus licheniformis B9 was inoculated into LB medium and cultured at 50 °C for 6 h. The cultured cells were then diluted and plated onto LB agar plates containing 25 mg / L kanamycin. The next day, colonies containing the single crossover genotype were selected and cultured at 37 °C for 6 h. The single crossover genotype was verified using primers MKPYZ-F / (ΔamyS)YZ-R. The cultured cells were then diluted and plated onto MGY plates containing 5 mM p-chlorophenylalanine. The knockout mutant strain was finally verified by PCR. Verification of the ΔamyS genotype in the knockout strain was performed using primers (ΔamyS)YZ-F / (ΔamyS)YZ-R. Figure 3 As shown in Figure e, this procedure was used to further knock out four genes, namely the polygamma-glutamic acid synthesis gene. pgsC[The gene's location in the genome (GenBank accession number AE017333.1) is BLi03838] and three extracellular protease genes. aprE [The location of this gene in the genome (GenBank accession number AE017333.1) is BLi01109] bprA [The gene's location in the genome (GenBank accession number AE017333.1) is BLi01747] and vpr [The location of this gene in the genome (GenBank accession number AE017333.1) is BLi04019] ， The final strain obtained was B9D5, which cannot synthesize poly-γ-glutamic acid and amylase, and has reduced extracellular protease activity. It can be used as a chassis cell for the synthesis of γ-glutamic acid, starch, or some enzyme preparations.

[0032] Example 3

[0033] Gene knock-in of Bacillus licheniformis using plasmid pTSMK

[0034] To insert the target gene expression cassette into the Bacillus licheniformis chromosome, the vector pTSMKP-amyS was amplified using plasmid pTSMKP-ΔamyS as a template and primers (amyS)MKP-F / (amyS)MKP-R. The gfp expression cassette fragment was obtained through DNA synthesis (SEQ ID NO. 5). Then, pTSMKP-ΔamyS was obtained by multimeric PCR [You et al. 2012. ApplEnviron Microbiol. 78(5):1593-5. doi: 10.1128 / AEM.07105-11.]. amyS- gfp Plasmid multimers. Then, following the same method as in Example 2, knock-in strains with gfp expression cassettes inserted into the Bacillus licheniformis amylase site were obtained through two single exchanges. Figure 3 (f) Finally, strain B9D5G was obtained, which produces green fluorescence.

[0035] Example 4

[0036] Random transposon insertion mutations of Bacillus licheniformis using plasmid pTSMK

[0037] Transposon elements and bleomycin resistance markers were constructed on plasmid pTSMK via multimeric plasmid PCR [You et al. 2012. Appl Environ Microbiol. 78(5):1593-5. doi: 10.1128 / AEM.07105-11.] to obtain plasmid pTSMT. Figure 4 (a). The construction process is as follows: First, using plasmids pMarA (Breton et al. 2006. Appl Environ Microbiol. 72(1):327-33. doi: 10.1128 / AEM.72.1.327-333.2006.), pUB110, and pTSMK as templates, the fragments trans, zeo, and TSMT were amplified using primers trans-F / trans-R, zeo-F / zeo-R, and TSMT-F / TSMT-R. Then, the above three fragments were used to obtain pTMT plasmid multimers by multimer PCR, and transformed into Bacillus subtilis SCK6D (pLS20). spc plasmid pTMT () was obtained Figure 4 (a)

[0038] To evaluate the effect of transposon random insertion, pTSMT was extracted from the donor strain SCK6D (pLS20) via conjugation. spc pTSMT was transferred into Bacillus licheniformis B9, and strain B9 (pTSMT) was obtained after resistance selection (25 mg / L kanamycin and 25 mg / L bleomycin). The strain was then processed according to the following procedure ( Figure 4 (b) Inoculate strain B9 (pTSMT) into LB liquid medium and incubate at 30°C for 10 h. Then spread the cultured cells onto LB plates containing kanamycin and incubate at 50°C until colonies grow. Streak the grown colonies onto LB plates containing kanamycin or bleomycin.

[0039] Ten colonies that could grow on LB agar plates containing kanamycin but not on LB agar plates containing bleomycin were selected. Arbitrary PCR (Black et al. 1993. Insect Mol Biol. 2:1-6. Doi: 10.1111 / j.1365-2583.1993.tb00118.x.) identified ten different insertion sites for the kanamycin resistance marker in the ten mutant strains. Figure 4 (c) This demonstrates that the transposon random insertion was successful and can be used to construct a Bacillus licheniformis transposon random mutation library.

[0040] Table 1. Strains used in this invention

[0041]

[0042] Table 2 Primers used in this invention

[0043]

[0044]

[0045]

[0046] SEQ ID NO.1

[0047]

[0048] SEQ ID NO.2

[0049]

[0050] SEQ ID NO.3

[0051] Aaagagcaatctcgtcatcgaagactaaatttctgtatggaaaacagttatttttcgtgtgcataaaataaagatttatgtgcatttagttctaaatcacctaaataatggttgaacataaatgttttttttatgtccattcagcaataaatctggtaccacgaaaaaacaaaccgcactgcggtttcaccatgcaaatggtgccagtttccccttatgctctttccttttccaccccctccttccttcggaatcgggggccggctttttgctgccgcaaaaagccttggaaaaagaaacacttatttgaacagatcgttgcaaagtaatgtgcagaaatcgatgaaaaggagcgttaacaa。

[0052] SEQ ID NO.4

[0053]

[0054] SEQ ID NO.5

[0055]

Claims

1. A plasmid for genetic manipulation of Bacillus licheniformis, characterized in that, The sequence of the plasmid for genetic manipulation of Bacillus licheniformis is shown as SEQ ID NO. 1; the plasmid for genetic manipulation of Bacillus licheniformis comprises a replicon fragment, a transfer initiation site fragment and a kanamycin resistance marker fragment; the sequence of the replicon fragment is shown as SEQ ID NO. 2; the sequence of the transfer initiation site fragment is shown as SEQ ID NO. 3; the sequence of the kanamycin resistance marker fragment is shown as SEQ ID NO.

4.

2. The method for constructing a plasmid for genetic manipulation of Bacillus licheniformis according to claim 1, characterized in that, The three fragments of the replicon fragment with the sequence shown as SEQ ID NO. 2, the transfer initiation site fragment with the sequence shown as SEQ ID NO. 3 and the kanamycin resistance marker fragment shown as SEQ ID NO. 4 are connected by a PCR method, and the connected fragments are introduced into Bacillus subtilis to obtain the plasmid for genetic manipulation of Bacillus licheniformis.

3. Use of the plasmid of claim 1 in DNA knockout of Bacillus licheniformis.

4. Use according to claim 3, characterized in that, The DNA knockout is constructing a knockout poly-gamma glutamate synthesis gene pgsC and three extracellular protease genes aprE, bprA and vpr of a bacillus licheniformis strain.

5. Use of the plasmid of claim 1 in DNA knock-in of Bacillus licheniformis.

6. Use according to claim 5, characterized in that, The DNA knock-in is knock-in of a gfp expression cassette fragment to construct a green fluorescent labeled Bacillus licheniformis strain, and the sequence of the gfp expression cassette fragment is shown as SEQ ID NO.

5.

7. Use of the plasmid of claim 1 in transposon random insertion mutagenesis, characterized in that, The plasmid of claim 1 is transferred into Bacillus licheniformis by means of a Bacillus conjugation system, thereby performing transposon random insertion mutation on Bacillus licheniformis.

8. A recombinant Bacillus licheniformis strain, characterized in that, Constructed by using the plasmid of claim 1.

9. The recombinant B. licheniformis of claim 8, wherein, Genetic manipulation is performed by using the plasmid of claim 1, and the genetic manipulation comprises DNA knockout and DNA knock-in.

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