Plasmid for genetic manipulation of bacillus licheniformis and application thereof

Abstract

The invention belongs to the technical field of biology, and discloses a plasmid for genetic manipulation of bacillus licheniformis and application of the plasmid. The temperature-sensitive plasmid pTSMK created by the invention can be transferred into bacillus licheniformis in a conjugational transfer manner, and has both high efficiency and universality. The plasmid is used for carrying target DNA (deoxyribonucleic acid), so that the genetic manipulation of the bacillus licheniformis can be realized, such as DNA knockout, DNA knockin and transposon random insertion mutation.

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 B. licheniformis. Such plasmids can replicate in B. licheniformis below a certain temperature threshold, but cannot replicate above the threshold [Horinouchi et al. 1982. J Bacteriol. 150(2):804-14. doi: 10.1128 / jb.150.2.804-814.]. With this property, after the target DNA fragment is carried into the host bacteria by such plasmids, the strains with the target mutation can be screened by increasing the culture temperature and adding antibiotics. Therefore, the key to establishing a general genetic manipulation method is to introduce temperature-sensitive plasmids into different B. licheniformis strains.

[0004] Currently, two methods have been reported for introducing temperature-sensitive plasmids into B. licheniformis, namely electroporation [Nijland et al. 2010. PLoS One. 16;5(3):e9724. doi: 10.1371 / journal.pone.0009724.] and conjugation [Rachinger et al. 2013. J Biotechnol. 167(4):365-9. doi: 10.1016 / j.jbiotec.2013.07.026.]. However, both methods have the problem of low applicability. Specifically, the efficiency of electroporation is generally low and many strains are difficult to successfully transform [Jeong et al. 2022. Front Microbiol. 26:13:802040. doi: 10.3389 / fmicb.2022.802040.]. Conjugation is to transfer temperature-sensitive plasmids from E. coli to B. licheniformis by the conjugation system of E. coli. This method has the problems of low transfer efficiency (10 -7 -10 -5The conjugation system from Bacillus is more efficient than the conjugation system from E. coli in transferring plasmids from B. subtilis to B. licheniformis [Koehler et al. 1987. J Bacteriol. 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, there is no report on the use of the Bacillus conjugation system to transfer temperature-sensitive plasmids. Therefore, there is an urgent need to provide a universal temperature-sensitive plasmid that can meet the needs of various common genetic manipulation operations. SUMMARY

[0005] To solve the problem of the lack of universal and efficient genetic manipulation methods in B. licheniformis, the present application creates a temperature-sensitive and conjugation-transferrable plasmid pTSMK and its application, which can be efficiently and universally transferred to different B. licheniformis strains by the Bacillus conjugation system, thereby meeting the needs of various genetic manipulations for B. licheniformis, including DNA knockout, DNA knock-in, and transposon random insertion mutation.

[0006] The object of the present application can be achieved by the following technical solutions: A plasmid for genetic manipulation of B. licheniformis, the sequence of which is shown in SEQ ID NO. 1; the plasmid for genetic manipulation of B. 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.

[0007] The application also provides a construction method of the above-mentioned plasmid for genetic manipulation of Bacillus licheniformis, in which three fragments of a replicon fragment with a sequence as shown in SEQ ID NO. 2, a transfer initiation site fragment with a sequence as shown in SEQ ID NO. 3 and a kanamycin resistance marker fragment as shown in 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.

[0008] The application also provides application of the above-mentioned plasmid in DNA knockout, DNA knock-in and transposon random insertion mutation of Bacillus licheniformis.

[0009] The application also provides application of the above-mentioned plasmid in DNA knockout of Bacillus licheniformis.

[0010] Further, the DNA knockout is to construct a Bacillus licheniformis strain with knockout of poly-gamma-glutamate synthesis genes pgsC and three extracellular protease genes aprE, bprA and vpr .

[0011] The application also provides application of the above-mentioned plasmid in DNA knock-in of Bacillus licheniformis.

[0012] Further, the DNA knock-in is to knock in a gfp expression cassette fragment to construct a green fluorescent labeled Bacillus licheniformis strain, and the sequence of the gfp expression cassette fragment is as shown in SEQ ID NO. 5.

[0013] The application also provides application of the above-mentioned plasmid in transposon random insertion mutation.

[0014] The application also provides a recombinant Bacillus licheniformis constructed by using the above-mentioned plasmid.

[0015] Further, genetic manipulation is performed by using the above-mentioned plasmid, and the genetic manipulation includes DNA knockout, DNA knock-in and transposon random insertion mutation.

[0016] Beneficial effects

[0017] The temperature-sensitive plasmid pTSMK created by the application can be transferred into Bacillus licheniformis by conjugation, and has high efficiency and universality. The plasmid can be conjugated and cannot be replicated at high temperature. By virtue of this characteristic, after the plasmid carrying a target DNA fragment enters a host bacterium, a strain with a target mutation can be screened by increasing the culture temperature and adding antibiotics. The genetic manipulation of Bacillus licheniformis, such as DNA knockout, DNA knock-in and transposon random insertion mutation, can be realized by using the plasmid carrying the target DNA, and false positive genetic manipulation interference caused by plasmid replication can be excluded. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 Genetic map and conjugation of plasmid pTSMK Figure 1 a is the genetic map of pTSMK; oriTLs20 is the transfer origin site from pLS20; rep is the temperature-sensitive replicon from pE194; kmR is the kanamycin resistance marker Figure 1 b is the schematic diagram of conjugation of plasmid pTSMK; SCK6D is a D-alanine auxotroph; DPF and MPF represent DNA processing factor and mating pair formation factor, respectively Figure 1 c is the transfer efficiency of pTSMK; the transfer efficiency is the ratio of conjugants to recipient bacteria Figure 2 Identification results of wild-type Bacillus licheniformis isolated in the application Figure 2 a is a phylogenetic tree constructed based on 16s rRNA gene sequences of Bacillus licheniformis strains and related species; GenBank accession numbers of each model strain used in the analysis are marked after the species name Figure 2 b is a phylogenetic tree constructed based on 16s rRNA gene sequences of Bacillus licheniformis strains and related species gyrA GenBank accession numbers of each model strain used in the analysis are marked after the species name Figure 3 Gene knockout and knock-in effects by means of pTSMK Figure 3 a is a multiple sequence alignment of partial sequences of PheS of different Bacillus species; asterisks mark conserved amino acids; red font 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 b is a map of the mobile vector pTSMKP Figure 3 c is the survival of B. licheniformis overexpressing pheS* on LB plates containing 5 mM p-Cl-phe Figure 3 d is the operation procedure for knockout in B. licheniformis Figure 3 e is verification of knockout effect by PCR amplification Figure 3 f is verification of knock-in effect by PCR and GFP fluorescence

[0019] Figure 4 Results of transposon random insertion mutation by means of pTSMK Figure 4 a is a map of the mobile transposon vector pTSMT Figure 4 b is the operation procedure for transposon mutagenesis; colonies cultured at 50℃ are randomly picked onto LB plates containing kanamycin and zeocin, respectively Figure 4C is the screening of transposon in the chromosomal localization of kanamycin resistance marker. oriC, origin of replication; ter, termination of replication. DETAILED DESCRIPTION

[0020] Example 1

[0021] This example provides a plasmid pTSMK (SEQ ID NO. 1) which is temperature sensitive and can be transferred by Bacillus conjugation system. Figure 1 In a), it is composed 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.] (SEQ ID NO. 2); ② a transfer initiation site fragment from conjugative plasmid pLS20 [Ramachandran et al. 2017. PLoS Genet. 13(2):e1006586. doi: 10.1371 / journal.pgen.1006586. eCollection 2017 Feb.] (SEQ ID NO. 3); ③ a kanamycin resistance marker fragment from plasmid pUB110 [McKenzie et al. 1986. Plasmid. 15(2):93-103. doi: 10.1016 / 0147-619x(86)90046-6.] (SEQ ID NO. 4). The three DNA fragments were synthesized by the Commissioned Biotechnology Company, and then the three fragments were ligated by overlap extension PCR method [You et al. 2012. Appl Environ Microbiol. 78(5):1593-5. doi: 10.1128 / AEM.07105-11.], and the ligated fragments were introduced into Bacillus subtilis SCK6 [Zhang et al. 2011. Microb Biotechnol. 4(1):98-105. doi: 10.1111 / j.1751-7915.2010.00230.x] to obtain plasmid pTSMK (SEQ ID NO. 1). rep oriT LS20 Km R

[0022] The principle of transferring plasmid pTSMK into B. licheniformis by Bacillus conjugation system is as follows: Figure 1 ​​​As 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.

[0023] 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: taking plasmid pLS20 (GenBank Number NC_015148.1) as a template, the upstream homologous arm and the downstream homologous arm are amplified by primers LS20LF-F / LS20LF-R and LS20RF-F / LS20RF-R, respectively. Taking 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 is amplified by primers SPC-F / SPC-R. Then the three fragments are fused into fragment LF-SPC-RF by overlap extension PCR using primers LS20LF-F / LS20RF-R. Then the fragment is transformed into SCK6D (pLS20) to obtain strain SCK6D (pLS20 spc ).

[0024] To verify the transfer efficiency and universality of pTSMK, it is introduced into strain SCK6D (pLS20 spc ) to obtain strain SCK6D (pLS20 spc , pTSMK). According to the method reported in the literature [Mitsuhiro et al. 2006. Biosci Biotechnol Biochem. 70(3):740-2. doi: 10.1271 / bbb.70.740.], the donor strain SCK6D (pLS20 spc , pTSMK) is paired with 10 strains of different sources of Bacillus licheniformis (see Table 1 and Figure 2 ), and the Bacillus licheniformis that successfully received pTSMK is screened on LB medium containing kanamycin. As shown in c of Figure 1 , pTSMK can be efficiently transferred from the donor strain to all 10 strains of Bacillus licheniformis, with a transfer efficiency of 10 -5 ~10 -3 conjugants / receptor bacteria. This result shows that pTSMK has ideal transfer efficiency and universality, and far exceeds the method based on the E. coli conjugation system [Rachinger et al. 2013. J Biotechnol. 167(4):365-9. doi:10.1016 / j.jbiotec.2013.07.026.] in both aspects. In addition, about 80% of the conjugants have only kanamycin resistance and no spectinomycin resistance, indicating that most of the conjugants only obtain pTSMK and do not obtain pLS20 spc , which has little interference with subsequent genetic manipulation.

[0025] Example 2

[0026] Genetic knockout of B. licheniformis by means of plasmid pTSMK To achieve a genetic knockout without resistance marker residues, an expression cassette containing the counter-selection marker pheS* was introduced into the B. licheniformis genome by means of the plasmid pTSMK (Figure 1). Figure 3 a) Construction of plasmid pTSMKP on plasmid pTSMK by PCR method [You et al. 2012. Appl Environ Microbiol. 78(5): 1593-5. doi: 10.1128 / AEM.07105-11.] using primers pTSMK-5' (SEQ ID NO: 1) and pTSMK-3' (SEQ ID NO: 2) (Figure 2). Figure 3b). In which, the codons of 255 and 309 of pheS* were changed from ACA and GCT to TCA and GGT, respectively, in wild-type pheS [the gene is located at BLi03016 in the genome (GenBank accession number AE017333.1)]. The construction process is as follows: first, take B. licheniformis 14580 [Rey et al. 2004. Genome Biol. 5(r77). doi.org / 10.1186 / gb-2004-5-10-r77] as the template, and use primers pheS*1-F / pheS*1-R, pheS*2-F / pheS*2-R and pheS*3-F / pheS*3-R to amplify three parts of the gene pheS [the gene is located at BLi03016 in the genome (GenBank accession number AE017333.1)], respectively, in which the base mutations are introduced by pheS*2-F and pheS*3-F, and fuse the three parts into a PheS* that can recognize p-chlorophenylalanine; at the same time, take plasmid pTPC as the template, and use primers pTPC-F / pTPC-R to amplify the vector pTPC; then, transform PheS* and the vector pTPC [Zhou et al. 2017. Appl Microbiol Biotechnol. 101(1):217-227. doi: 10.1007 / s00253-016-7906-9.] into E. coli DH5a by homologous recombination to obtain plasmid pTPC-pheS*; take plasmids pTPC-pheS* and pTSMK as templates, respectively, and use primers P-F / P-R and TSMK-F / TSMK-R to amplify the pheS* expression cassette and the vector TSMK; finally, obtain the multimer of plasmid pTSMKP by PCR method [You et al. 2012. Appl Environ Microbiol. 78(5):1593-5. doi: 10.1128 / AEM.07105-11.], and transform it into B. subtilis SCK6D (pLS20 spc ) to obtain plasmid pTSMKP. Use the above conjugation method to transfer plasmid pTSMKP from donor bacteria SCK6D (pLS20 spc , pTSMKP) into ten strains of B. licheniformis, respectively. The results show that all B. licheniformis strains carrying plasmid pTSMKP are sensitive to p-chlorophenylalanine, and the survival rate is less than 10 -4 ​Figure 3 (c) indicates that the reverse filtering marker pheS* has excellent working efficiency.

[0027] 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 location of this gene in the genome (GenBank Accession No. AE017333.1) is BLi03838] and three extracellular protease genes aprE [The location of this gene in the genome (GenBank Accession No. AE017333.1) is BLi01109] , bprA [The location of this gene in the genome (GenBank Accession No. AE017333.1) is BLi01747] and vpr [The location of this gene in the genome (GenBank Accession No. AE017333.1) is BLi04019] ， Finally, strain B9D5 was obtained, which cannot synthesize poly-γ-glutamic acid and amylase, and has reduced extracellular protease activity, and can be used as a chassis cell for synthesis of γ-glutamic acid, starch or partial enzyme preparation.

[0028] Example 3

[0029] Gene knock-in of Bacillus licheniformis by means of plasmid pTSMK In order to realize insertion of the target gene expression cassette into the Bacillus licheniformis chromosome, the vector pTSMKP-amyS was amplified using primers (amyS)MKP-F / (amyS)MKP-R with plasmid pTSMKP-ΔamyS as the template. The gfp expression cassette fragment was obtained by DNA synthesis (SEQ ID NO. 5). Then the pTSMKP-gfp was obtained by the method of multimeric PCR [You et al. 2012. Appl Environ Microbiol. 78(5): 1593-5. doi: 10.1128 / AEM.07105-11.]. amyS- gfp Plasmid multimer. Then the same method as in Example 2 was followed to obtain the knock-in strain with the gfp expression cassette inserted into the amylase site of Bacillus licheniformis by two single exchanges (B9D5Gf), and finally the strain B9D5G was obtained, which produces green fluorescence. Figure 3

[0030] Example 4

[0031] Random transposon insertion mutation of Bacillus licheniformis by means of plasmid pTSMK The transposon element and the blasticidin resistance marker were constructed on plasmid pTSMK by multimeric plasmid PCR [You et al. 2012. Appl Environ Microbiol. 78(5): 1593-5. doi: 10.1128 / AEM.07105-11.] to obtain plasmid pTSMT (pTSMK-Tn7-Blas). 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, respectively, and using primers trans-F / trans-R, zeo-F / zeo-R, TSMT-F / TSMT-R, the fragments trans, zeo and TSMT are amplified. Then the above three fragments are used to obtain the pTMT plasmid multimer by multimer PCR, which is transformed into Bacillus subtilis SCK6D (pLS20 spc ) to obtain plasmid pTMT Figure 4 a).

[0032] To evaluate the effect of transposon random insertion, pTSMT is transferred from the donor strain SCK6D (pLS20 spc , pTMT) to B. licheniformis B9 by conjugation, and strain B9 (pTSMT) is obtained after resistance screening (25 mg / L kanamycin and 25 mg / L bleomycin). Figure 4 b): strain B9 (pTSMT) is inoculated into LB liquid medium and cultured at 30°C for 10 h. Then the cultured cells are spread on LB plates containing kanamycin and incubated at 50°C until colonies grow, and the grown colonies are streaked onto LB plates containing kanamycin or bleomycin, respectively.

[0033] Ten colonies that can grow on LB plates containing kanamycin but cannot grow on LB plates containing bleomycin are selected. Identification is performed by Arbitrary PCR (Black et al.. 1993. Insect Mol Biol. 2:1-6. doi: 10.1111 / j.1365-2583.1993.tb00118.x.), and the insertion positions of the kanamycin resistance marker in the 10 mutant strains are ten different positions Figure 4 c), which proves that the transposon random insertion is successful and can be used for the construction of a transposon random mutation library of B. licheniformis.

[0034] Table 1 Strains used in the present application

[0035] Table 2 Primers used in the present application

[0036]

[0037]

[0038] SEQ ID NO. 1

[0039] SEQ ID NO. 2

[0040] SEQ ID NO. 3 Aaagagcaatctcgtcatcgaagactaaatttctgtatggaaaacagttatttttcgtgtgcataaaataaagatttatgtgcatttagttctaaatcacctaaataatggttgaacataaatgttttttttatgtccattcagcaataaatctggtaccacgaaaaaacaaaccgcactgcggtttcaccatgcaaatggtgccagtttccccttatgctctttccttttccaccccctccttccttcggaatcgggggccggctttttgctgccgcaaaaagccttggaaaaagaaacacttatttgaacagatcgttgcaaagtaatgtgcagaaatcgatgaaaaggagcgttaacaa.

[0041] SEQ ID NO. 4

[0042] SEQ ID NO.5

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 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 mutation.

8. A recombinant Bacillus licheniformis strain, characterized in that, Constructed by 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, DNA knock-in and transposon random insertion mutation.

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