CN114874950A

CN114874950A - Method for improving bacillus licheniformis bacillus production peptide

Info

Publication number: CN114874950A
Application number: CN202210638715.4A
Authority: CN
Inventors: 徐美娟; 饶志明; 吴洁琴; 夏博雅; 杨套伟; 张显
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2022-06-07
Filing date: 2022-06-07
Publication date: 2022-08-09
Anticipated expiration: 2042-06-07
Also published as: CN114874950B

Abstract

The invention discloses a method for improving bacillus licheniformis to produce bacitracin, and belongs to the technical field of microorganisms. The invention screens and obtains bacillus licheniformis ATH with high yield of bacitracin from a feed sample, expresses acetyl coenzyme A synthetase Bsacs gene from bacillus subtilis in the bacillus licheniformis ATH by a genetic engineering method, increases the supply of amino acid consisting of intracellular bacitracin and improves the titer of the bacitracin. After the obtained recombinant bacillus licheniformis is inoculated into a fermentation medium containing soybean meal for fermentation culture for 42 hours, the obtained bacitracin titer is 1928IU/mL, is improved by 46.16 percent compared with the original strain ATH bacitracin titer, and simultaneously, the content of bacitracin A is 1081IU/mL, is improved by 37.2 percent compared with the original strain. The whole fermentation process has high bacitracin production efficiency, low raw material cost, simple method and easy operation.

Description

Method for improving bacillus licheniformis bacillus production peptide

Technical Field

The invention relates to a method for improving bacillus licheniformis bacillus production of bacitracin, and belongs to the technical field of biology.

Background

The bacitracin is a polypeptide spectrum antibiotic obtained by fermenting Bacillus subtilis and Bacillus licheniformis, comprises Orn, D-Phe, His, D-Asp, Asn, Lys, D-Glu, Cys, Leu and Ile, and consists of a plurality of cyclic homologues respectively including bacitracin A, B1, B2, B3, C1, C2, C3 and F, wherein bacitracin A and B account for 95 percent of activity, the yield synthesis is determined by polygene, the metabolic network is complex, the main production strains are Bacillus subtilis and Bacillus licheniformis, the bacteriostatic action of the bacitracin is that the bacitracin inhibits the formation of bacterial cell walls, so gram-positive bacteria and part of gram-negative bacteria, and the bacitracin is widely applied to feed addition in the animal breeding process.

Amino acids required by bacitracin synthesis are derived from an EMP pathway and TCA cycle, carbon and nitrogen metabolism in Bacillus licheniformis is strictly regulated, a large amount of carbon sources are converted into pyruvic acid and acetyl coenzyme A through the EMP pathway and then into overflow metabolites such as lactic acid, acetic acid and acetoin, and the acetyl coenzyme A is used as an important intermediate product of intracellular metabolism and is an important precursor for synthesizing various bio-based chemicals. While at the same time the accumulation of acetic acid on the one hand inhibits the growth and metabolism of E.coli and on the other hand leads to carbon atom losses and a reduced yield of the desired product. Therefore, it is urgently needed to find a method for reducing the decomposition path of acetyl CoA, reducing the accumulation of acetic acid and improving the bacillus production peptide of bacillus licheniformis.

Disclosure of Invention

In order to solve the technical problems, the invention provides a Bacillus licheniformis (ATH) which has been preserved in the China center for type culture collection in 2022, 1 month and 19 days, and the preservation number is CCTCC NO: m2022094, the preservation address is China, Wuhan university.

In one embodiment of the present invention, the ATH for Bacillus licheniformis is derived from feed samples, the screened strain is sequenced through 16S rDNA, the strain is identified as Bacillus licheniformis (Bacillus licheniformis), and the strain is named as Bacillus licheniformis ATH.

The invention also provides a microbial agent which contains the bacillus licheniformis ATH.

The invention also provides a product, which contains the bacillus licheniformis ATH.

In one embodiment of the invention, the product includes, but is not limited to, bacteriostatic agents, pesticides.

The invention also provides application of the bacillus licheniformis ATH in preparation of a product for inhibiting bacteria.

In one embodiment of the invention, the bacteria include, but are not limited to, escherichia coli, corynebacterium glutamicum, staphylococcus aureus, bacillus cereus.

The invention also provides a recombinant bacillus licheniformis, and the recombinant bacillus licheniformis takes the bacillus licheniformis ATH as a host cell and overexpresses acetyl coenzyme A synthetase ACS.

In one embodiment of the invention, the amino acid sequence of ACS is shown in SEQ ID NO. 2.

In one embodiment of the invention, the nucleotide sequence encoding the ACS is shown in SEQ ID NO. 1.

In one embodiment of the invention, pHY300PLK is used as the expression vector.

The invention also provides application of the acetyl coenzyme A synthetase ACS with the amino acid sequence shown as SEQ ID NO.2 in improving the yield of bacillus licheniformis bacitracin.

The invention also provides a method for preparing bacitracin, which is prepared by fermenting the recombinant bacillus licheniformis.

In one embodiment of the invention, the recombinant bacillus licheniformis seed solution is inoculated into a fermentation medium according to the inoculation amount of 10-15% and prepared by fermentation.

In an embodiment of the invention, the method comprises the steps of inoculating the recombinant bacillus licheniformis to a fermentation medium for fermentation to obtain a fermentation liquid containing bacitracin, and then separating the fermentation liquid containing bacitracin to obtain bacitracin.

In one embodiment of the present invention, the fermentation conditions are a temperature of 30 to 37 ℃, a rotation speed of 180 to 250rpm, and a pH of 7.0 to 7.5.

In one embodiment of the present invention, the fermentation conditions are 37 ℃ temperature, 200rpm rotation speed and 7.0 pH.

In one embodiment of the invention, the fermentation medium comprises: 30-50 g/L of soybean meal and 30-40 g/L, CaCO of soluble starch ₃ 20～30g/L、(NH ₄ ) ₂ SO ₄ 0.5～1g/L、ZnSO ₄ 5～10g/L and 10-15 g/L of peptone.

In one embodiment of the invention, the fermentation medium comprises: 50g/L of soybean meal and 40g/L, CaCO of soluble starch ₃ 24g/L、(NH ₄ ) ₂ SO ₄ 1g/L、ZnSO ₄ 5g/L and 10g/L of peptone.

The invention also provides the application of the Bacillus licheniformis ATH, the recombinant Bacillus licheniformis, the application or the method for preparing bacitracin in preparing products containing bacitracin.

In one embodiment of the invention, the product is a drug, health product, feed additive.

The invention also provides a screening method of the high-yield bacitracin bacillus licheniformis strain, which comprises the following steps:

(1) coating the strains to be screened on a solid plate culture medium, and selecting single colonies to inoculate in a seed culture medium for culture after the colonies are formed;

(2) transferring the culture solution obtained in the step (1) to a fermentation medium for fermentation;

(3) coating the staphylococcus aureus indicator on an LB solid plate culture medium, and putting into an oxford cup;

(4) and (3) centrifuging the fermentation liquor obtained in the step (2), taking supernatant liquid on an indicator bacterium plate in the step (3), and taking the strain with the largest transparent circle as a high-yield strain and the strain with the highest bacteriostatic effect as an initial strain as shown in figure 1.

The invention also provides the bacitracin prepared by the method.

The invention also provides a product containing the bacitracin.

In one embodiment of the invention, the product is a pharmaceutical, nutraceutical or feed additive.

Advantageous effects

(1) According to the invention, a high-efficiency antibacterial Bacillus licheniformis (ATH) is successfully screened, and the Bacillus licheniformis (ATH) is used as a host cell to construct a recombinant Bacillus licheniformis strain, so that the recombinant strain effectively utilizes acetic acid and can improve the yield of bacitracin; not only can reduce the toxic action of acetic acid on the growth of the bacillus licheniformis, but also can effectively utilize the acetic acid to generate acetyl coenzyme A, and has higher application value in the production of bacitracin.

(2) The invention provides a recombinant bacillus licheniformis ATH/pHY300PLK-Bsacs, which is inoculated to soybean meal powder and 5% ZnSO ₄ The fermentation culture is carried out for 36h in a fermentation culture medium, fermentation supernatant is taken to detect that the titer of bacitracin is 1928IU/mL, the titer of bacitracin of the original strain is 1039IU/mL and is improved by 46.16%, and meanwhile, the content of bacitracin A is 1081IU/mL and is improved by 37.28% compared with the content of bacitracin of the original strain of 678IU/mL, so that the recombinant bacillus licheniformis ATH/pHY300PLK-Bsacs is used for producing bacitracin, the efficiency is high, the raw material cost is low, and the operation is easy.

(3) The method of the invention not only reduces the toxic action of the byproduct acetic acid on the growth of the bacterial strain, but also can effectively utilize the acetic acid to improve the synthesis of acetyl coenzyme A, greatly reduces the production cost, improves the production of the product and lays a good foundation for the industrial production of bacitracin.

Biological material preservation

A Bacillus licheniformis (ATH) strain is preserved in China center for type culture Collection in 2022, 1 month and 19 days, and the preservation number is CCTCC NO: m2022094, class name: the Bacillus licheniformis ATH has the preservation address as follows: china, wuhan university.

Drawings

FIG. 1: colony diagram of ATH plate of Bacillus licheniformis.

FIG. 2: and (4) an ATH single-staining microscopic result chart of the bacillus licheniformis.

FIG. 3: an experimental graph of an antibacterial plate indicating staphylococcus aureus strains, wherein three antibacterial zones are all the antibacterial zones of the staphylococcus aureus strains.

FIG. 4 is a schematic view of: and (3) a bacteriostatic circle graph of bacitracin in the fermentation liquor, wherein the three bacteriostatic circles are bacteriostatic circles of the original ATH of the bacillus licheniformis.

FIG. 5: amplification result of the acetyl coenzyme A synthetase coding gene Bsacs.

FIG. 6: and (3) carrying out colony PCR verification on the recombinant strain bacillus licheniformis ATH/pHY300 PLK-Bsacs.

FIG. 7: the bacteriostatic circle graphs of the bacitracin standard product are respectively bacteriostatic circles with gradient titer of 1000, 800, 600, 400 and 200 IU/mL.

FIG. 8: and (3) an inhibition zone graph of bacitracin in the fermentation liquor, wherein the three inhibition zones are inhibition zones of bacillus licheniformis ATH/pHY300 PLK-Bsacs.

FIG. 9: standard curve of bacitracin standard.

FIG. 10: HPLC liquid phase diagram of bacitracin standard.

FIG. 11: HPLC liquid phase diagram of bacitracin in fermentation broth.

Detailed Description

The invention is further illustrated below with reference to specific examples.

Bacitracin standards referred to in the following examples were purchased from alatin bioreagent; the pHY300PLK plasmid referred to in the examples below was purchased from Biovector plasmid vector bacterial cell Gene Collection; coli, Bacillus cereus, staphylococcus aureus, Bacillus subtilis subsp 168, referred to in the examples below, were purchased from the BioVector plasmid vector bacterial cell gene collection center.

The media and the required solutions referred to in the following examples are as follows:

bacitracin screening medium: 10g/L of peptone, 5g/L, NaCl 10g/L of yeast extract powder and 10U/mL of bacitracin zinc.

LB liquid medium: 10g/L of peptone and 5g/L, NaCl 10g/L of yeast extract.

LB solid Medium: 10g/L of peptone, 5g/L, NaCl 10g/L of yeast extract powder and 15-20 g/L of agar powder.

Seed culture medium: the seed culture medium comprises 50g/L of peptone, 3g/L of beef extract, 2g/L of yeast powder and 5g/L of NaCl.

Fermentation medium: 50g/L of soybean meal and 40g/L, CaCO g of soluble starch ₃ 24g/L、(NH ₄ ) ₂ SO ₄ 1g/L、ZnSO ₄ 5g/L and 10g/L of peptone.

The detection methods referred to in the following examples are as follows:

the bacitracin detection method comprises the following steps: selecting a C18 chromatographic column; 0.05mol/L acetate buffer (pH 6.4) -50% acetonitrile solution (75:25) as a mobile phase; the detection wavelength is 254 nm; the column temperature was 30 ℃.

Example 1: screening of Bacillus licheniformis ATH Strain

(1) And (3) culturing the feed sample in a 28 ℃ incubator for 24 hours, carrying out water bath treatment in a water bath kettle at 80-85 ℃ for 15min, cooling to room temperature, and carrying out gradient dilution by using normal saline. And coating the sample diluent on a bacitracin screening culture medium plate, culturing at the constant temperature of 37 ℃ for 24 hours, and then picking a colony growing on the culture medium and plump on an LB liquid culture medium for culturing.

(2) The screened single colony is shown in figure 1, the colony is round, irregular in edge, thin, rough in surface, opaque and grey-white, and the single staining microscopy result is shown in figure 2, the length of the strain is about 1.5-3.0 mu m, the width of the strain is about 0.6-0.8 mu m, and the gram staining of the strain is positive. The screened strain is sequenced through 16S rDNA (nucleotide sequence is shown as SEQ ID NO. 3), the strain is identified as Bacillus licheniformis (ATH), and the strain is deposited in the China center for type culture Collection on 1-19/2022.

Example 2: bacteriostatic experiment of bacillus licheniformis ATH

(1) Screening of indicator strains

In order to verify the bacteriostatic effect of bacillus licheniformis ATH, escherichia coli, bacillus cereus, corynebacterium glutamicum and staphylococcus aureus are selected as indicator strains in the experiment;

after the above-mentioned indicator strains were cultured overnight at 37 ℃ for 12 hours, respectively, the OD was diluted with sterile water at a super clean bench to 0.8, and 200 μ L of each was applied to an LB plate as a bacteriostatic indicator plate.

(2) Inoculating bacillus licheniformis ATH into an LB liquid culture medium, culturing overnight at 37 ℃, diluting the bacillus licheniformis ATH to OD600 ═ 1.0, filtering and sterilizing by using a 0.22 mu m microporous filter membrane in a super clean bench, sucking quantitative bacteria liquid by using a sterilizing filter paper sheet with the diameter of 2cm, respectively inoculating the bacteria liquid on a bacteriostasis indicating plate, culturing overnight at 37 ℃ for 12h, and then, obtaining the bacillus peptide with the best bacteriostasis effect on staphylococcus aureus (shown in figure 3), and then, corynebacterium glutamicum, bacillus cereus and escherichia coli. In conclusion, gram-positive bacteria staphylococcus aureus is selected as an indicator bacterium, and the bacteriostatic effect of bacillus licheniformis ATH is further analyzed.

(3) According to the experimental result of the step (2), staphylococcus aureus is used as an indicator bacterium, after overnight culture at 37 ℃, the staphylococcus aureus is diluted by a certain multiple with sterile water, 200 mu L of the sample is sucked to coat the plate, and the concentration of the staphylococcus aureus is as follows: OD600 ═ 0.8.

(4) Inoculating bacillus licheniformis ATH into an LB liquid culture medium, carrying out overnight culture at 37 ℃, separating and streaking, inoculating a single colony into a seed culture medium, and carrying out culture at 37 ℃ and 200rpm for 8-12 h to prepare a seed solution; adding the prepared seed solution into 25mL of fermentation medium according to the inoculation amount of 10% (v/v), culturing at 37 deg.C and 200rpm for 36h, and adding ZnSO with mass fraction of 5% ₄ The culture was continued for 42 hours, and the supernatant was collected by centrifugation.

(5) Taking an oxford cup and placing the oxford cup on an LB plate containing an indicating strain (OD600 ═ 0.8);

filtering and sterilizing the clear fermentation liquid obtained in the step (4) in a super clean bench by using a 0.22 mu m microporous filter membrane, taking 10 mu L of sterilized fermentation liquid (OD600 is 0.8), putting the fermentation liquid into an Oxford cup, and culturing for 12h at 37 ℃;

the result is shown in figure 4, the bacterial strain bacillus licheniformis ATH produces bacitracin with good bacteriostatic effect, and the diameter of the bacteriostatic circle is measured to be 13.8 mm.

(6) The detection method of the diameter and the titer of the bacteriostatic circle of the bacitracin standard product comprises the following steps:

the method comprises the following specific steps:

coating an LB flat plate with staphylococcus aureus as an indicator according to the bacteriostatic experiment result in the step (2); placing an oxford cup on the LB flat plate; wherein the concentration of the indicator bacteria is as follows: OD600 ═ 0.8;

1) diluting the titer of the bacitracin standard product to 1000IU/mL, 800IU/mL, 600IU/mL, 400IU/mL, 200 IU/mL; respectively sucking 10 mu L of bacitracin solutions with different titers, and placing the bacitracin solutions in different oxford cups; culturing at 37 deg.C for 12h, and determining the diameter of inhibition zone, the results are shown in Table 1 and FIG. 7;

2) and (3) filtering and sterilizing the fermentation liquor prepared in the step (4) by using a 0.22-micron microporous filter membrane in a super-clean workbench, and taking 10 mu L of sterilized bacterial liquid (OD value is: 1.0) in an oxford cup; the cells were cultured at 37 ℃ for 12 hours, and the diameter of the zone of inhibition was measured, the results are shown in Table 1.

Standard curves (as shown in FIG. 9) were plotted from the results of diameter size and bacitracin standard titer concentration in Table 1 and fitted to the functional equation.

Table 1: titer and diameter of bacteriostatic circle of bacitracin standard sample

According to the peptide standard curve of bacillus licheniformis and the measured diameter of the ATH inhibition zone of the original strain of bacillus licheniformis, the result shows that the titer of the ATH peptide of the original strain of bacillus licheniformis is 1039IU/mL, and the HPLC detection shows that the content of the bacitracin A in the fermentation liquor is 678 IU/mL.

Example 3: construction of expression vector for acetyl-CoA synthetase

The method comprises the following specific steps:

(1) taking genome DNA of Bacillus subtilis subsp 168 as a template, and taking F and R as primers to carry out PCR amplification to obtain acetyl coenzyme A synthetase gene Bsacs (the nucleotide sequence is shown as SEQ ID NO. 1) from heterologous sources; the primer sequences used were as follows:

F：5-ATGGGTCGCGGATCCGATGAACTTGAAAGCGTTAC-3’；

R：5-CGAGTGCGGCCGCAATTAATCCTCCATTGTTGACAG-3’。

the PCR amplification conditions were: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃, 30s, annealing at 58 ℃, 30s, extension at 72 ℃, 90s, and 30 cycles; final extension at 72 ℃ for 10 min.

The PCR amplification system is as follows: 0.5. mu.L of template, 0.5. mu.L of each of the upstream and downstream primers, 23.5. mu.L of sterilized double distilled water, and 25. mu.L of Primer StarDNA polymerase.

(2) The gene Bsacs (obtained in the step (1) and used for coding the acetyl coenzyme A synthetase are cut by restriction enzymes EcoRI and BamHI and then are connected with pHY300PLK plasmid to obtain a connection product, the connection product is transformed into Escherichia coli (Escherichia coli) JM109 to obtain a transformation product, and the transformation product is coated on an LB solid culture medium (containing 10 mu g/mL) ^-1 Ampicillin), and performing inverted culture in a constant-temperature incubator at 37 ℃ for 8-12 h to obtain a transformant; and (3) colony PCR verification, selecting a transformant which is verified to be correct, inoculating the transformant to an LB liquid culture medium, performing shake-flask culture for 8-12 h under the conditions of 37 ℃ and 120-180 rpm, performing colony PCR verification, selecting a transformant which is verified to be correct, inoculating the transformant to the LB liquid culture medium, performing shake-flask culture for 8-12 h under the conditions of 37 ℃ and 120-180 rpm, extracting plasmids, performing PCR amplification verification (shown in figure 5) and sequencing verification, and obtaining the recombinant plasmid pHY300PLK-Bsacs which is successfully transformed after verification is correct.

Example 4: construction of recombinant Bacillus licheniformis

The method comprises the following specific steps:

respectively electrically transforming the recombinant plasmid pHY300PLK-Bsacs obtained in the example 3 into the ATH of the bacillus licheniformis to obtain a transformation product; the respective transformed products were applied to LB solid medium (containing 10. mu.g. multidot.mL) ^-1 Ampicillin) and is inversely cultured in a constant temperature incubator at 37 ℃ for 12h to respectively obtain transformants; as a result of the colony PCR verification, positive transformants were obtained as shown in FIG. 6, and the transformants verified to be correct were selected and inoculated into LB liquid medium (containing 10. mu.g.mL) ^-1 Ampicillin), shake-flask culturing for 12h in a constant-temperature incubator at 37 ℃ under the condition of 120-180 rpm to obtain fermentation liquor, extracting plasmids respectively for PCR verification and sequencing verification, and obtaining the recombinant bacillus licheniformis after verification is correct: ATH/pHY300 PLK-Bsacs.

Example 5: recombinant bacillus licheniformis bacitracin

The method comprises the following specific steps:

(1) streaking and activating the recombinant bacillus licheniformis ATH/pHY300PLK-Bsacs prepared in the embodiment 4 on a flat plate, inoculating the activated recombinant bacillus licheniformis ATH/pHY300PLK-Bsacs into an LB liquid culture medium, and culturing at 37 ℃ for 8-10 h to prepare a seed solution;

(2) adding the prepared seed solution into 25mL of fermentation medium according to the inoculation amount of 10% (v/v), culturing at 37 deg.C and 200rpm for 36h, and adding ZnSO with mass fraction of 5% ₄ Culturing the solution for 42 hours, and centrifuging to collect supernatant;

(3) coating an LB flat plate by taking staphylococcus aureus as an indicator bacterium; placing an oxford cup on the LB flat plate; wherein the concentration of the indicator bacteria is as follows: OD600 ═ 0.8;

filtering the supernatant in a superclean bench with 0.22 μm microporous filter membrane for sterilization, sucking 10 μ L of the supernatant (OD value: 1.0), placing in Oxford cup, culturing at 7 deg.C for 12 hr, and measuring the antibacterial zone;

the results showed that the recombinant strain had a zone diameter of 19.9mm (as shown in FIG. 8).

According to the titer detection method of the example 2, the bacitracin titer of the recombinant bacillus licheniformis ATH/pHY300PLK-Bsacs is 1928IU/mL through a function equation; HPLC is adopted to detect the bacitracin A content in the fermentation liquor to be 1081IU/mL (as shown in figures 10-11).

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

SEQUENCE LISTING

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<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)..(3)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (5)..(7)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (11)..(11)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (1067)..(1067)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (1088)..(1090)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (1099)..(1099)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (1136)..(1137)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (1145)..(1145)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (1150)..(1150)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (1152)..(1152)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (1163)..(1164)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (1168)..(1168)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (1181)..(1181)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (1183)..(1183)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (1185)..(1188)

<223> n is a, c, g, or t

<400> 3

nnncnnnggg ngctatacat gcagtcgagc ggacagatgg gagcttgctc cctgatgtta 60

gcggcggacg ggtgagtaac acgtgggtaa cctgcctgta agactgggat aactccggga 120

aaccggggct aataccggat gcttgattga accgcatggt tcaattataa aaggtggctt 180

ttagctacca cttacagatg gacccgcggc gcattagcta gttggtgagg taacggctca 240

ccaaggcaac gatgcgtagc cgacctgaga gggtgatcgg ccacactggg actgagacac 300

ggcccagact cctacgggag gcagcagtag ggaatcttcc gcaatggacg aaagtctgac 360

ggagcaacgc cgcgtgagtg atgaaggttt tcggatcgta aaactctgtt gttagggaag 420

aacaagtacc gttcgaatag ggcggtacct tgacggtacc taaccagaaa gccacggcta 480

actacgtgcc agcagccgcg gtaatacgta ggtggcaagc gttgtccgga attattgggc 540

gtaaagcgcg cgcaggcggt ttcttaagtc tgatgtgaaa gcccccggct caaccgggga 600

gggtcattgg aaactgggga acttgagtgc agaagaggag agtggaattc cacgtgtagc 660

ggtgaaatgc gtagagatgt ggaggaacac cagtggcgaa ggcgactctc tggtctgtaa 720

ctgacgctga ggcgcgaaag cgtggggagc gaacaggatt agataccctg gtagtccacg 780

ccgtaaacga tgagtgctaa gtgttagagg gtttccgccc tttagtgctg cagcaaacgc 840

attaagcact ccgcctgggg agtacggtcg caagactgaa actcaaagga attgacgggg 900

ggcccgcaca agcggtggag catgtggttt aattcgaagc aacgcgaaga acccttacca 960

ggtcttgaca tcctcttgac aaccctagag atagggactt cccctttcgg ggcagaggtg 1020

acaggtggtg catggttgtc gtcagctcgg tgtcgtgaga tgttggntta ggtcccgcac 1080

gagcgcannn tgatcttant tgcagcattc agttgggcac tcttaggtga ctgccnntga 1140

caaanccggn gnaaggtggg atnnacgntc aattcaatcc ntngnnnn 1188

Claims

1. A Bacillus licheniformis (ATH) strain is preserved in China center for type culture Collection in 2022, 1 month and 19 days, and the preservation number is CCTCC NO: m2022094, the preservation address is China, Wuhan university.

2. A bacteriostatic agent comprising the bacillus licheniformis ATH of claim 1, or a fermentation broth thereof.

3. Use of the bacillus licheniformis ATH according to claim 1 for the preparation of a bacteria inhibiting product.

4. The use of claim 3, wherein the bacteria include, but are not limited to, Escherichia coli, Corynebacterium glutamicum, Staphylococcus aureus, Bacillus cereus.

5. A recombinant Bacillus licheniformis strain characterized in that, the Bacillus licheniformis ATH of claim 1 is used as host cell, expressing acetyl-CoA synthetase with amino acid sequence as shown in SEQ ID NO. 2.

6. Use of an acetyl-coa synthetase having an amino acid sequence as shown in SEQ ID No.2 for increasing the yield of bacitracin from bacillus licheniformis as claimed in claim 1.

7. A method for preparing bacitracin, which comprises preparing a fermentation broth by fermenting the ATH of Bacillus licheniformis of claim 1 or the recombinant Bacillus licheniformis of claim 5, and extracting and separating the fermentation broth.

8. Bacitracin prepared by the method of claim 7.

9. A pharmaceutical, health product or feed additive comprising the bacitracin of claim 8.

10. Use of the bacillus licheniformis ATH of claim 1 or the recombinant bacillus licheniformis of claim 5 or the process of claim 7 for the preparation of a bacitracin containing product.