CN114644688A

CN114644688A - Bacillus licheniformis for producing lantibiotic peptide and inhaul cable peptide

Info

Publication number: CN114644688A
Application number: CN202011498305.1A
Authority: CN
Inventors: 钟瑾; 刘亚勇; 滕坤玲; 王天威; 夏天奇
Original assignee: Institute of Microbiology of CAS
Current assignee: Institute of Microbiology of CAS
Priority date: 2020-12-17
Filing date: 2020-12-17
Publication date: 2022-06-21

Abstract

The invention discloses a bacillus licheniformis for producing lanthionine lichenicidin II-8 and lagotide lichenin. The bacillus licheniformis is bacillus licheniformis BaC1-8, and the preservation number of the bacillus licheniformis in the common microorganism center of China Committee for culture Collection of microorganisms is CGMCC No. 20653. Experiments prove that the lantibiotic II-8 can inhibit gram-positive bacteria, the inhaul peptide Lichelasin can inhibit bacteria, and the bacillus licheniformis BaC1-8 can inhibit gram-positive bacteria and/or fungi. The lantibiotic II-8, the lichenin Lichelisin and the Bacillus licheniformis BaC1-8 are all bacteriostats obtained by first separation, and have important application value.

Description

Bacillus licheniformis for producing lantibiotic peptide and inhaul cable peptide

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a bacillus licheniformis for producing lantibiotic peptides and inhaul cable peptides.

Background

Bacillus licheniformis (Bacillus licheniformis) is a kind of Bacillus which can produce rich enzymes and active substances and has probiotic function, and can produce various antibacterial substances, including non-ribosomal peptides such as surfactin, iturin, feng jun, bacitracin and the like and lanthionine synthesized by ribosomes such as kukolin, lichen jun. Wherein, lipopeptide secondary metabolites such as surfactin, iturin, and mustargin, etc. can inhibit or kill a plurality of plant pathogenic bacteria, including a plurality of fusarium, aspergillus, etc., and have better application potential in the biological control of plant diseases. Bacitracin is a non-ribosomal peptide consisting of 12 amino acids and having a thiazole ring, and has an inhibitory effect on various gram-positive and gram-negative pathogenic bacteria. The bacillus licheniformis has the characteristics of broad spectrum, safety, high efficiency, stability and the like, and has better application in the aspects of animal feed additives, gastrointestinal tract infection, skin infection and the like. Lantibiotides are ribosome-synthesized post-translationally modified peptides, important members of the natural product family, and most of the lantibiotides have certain bacteriostatic activity. The lantibiotic peptide has strong inhibitory activity to a plurality of clinically relevant gram-positive pathogenic bacteria, including staphylococcus, streptococcus, enterococcus, clostridium and the like, has the characteristics of high thermal stability, small molecular weight, no toxicity, no drug resistance, low immunogenicity and the like, and has great application potential in the aspects of food preservation and fresh-keeping medicine development and the like.

With the development of sequencing technology, a great deal of bacillus licheniformis genome data shows that a plurality of new bacteriocins which are not characterized and explored still exist, including new lanthionine, lagotide, thiopeptide and the like; the bacteriocin which is not easy to generate drug resistance can be used as a novel antibacterial substance without toxicity and side effects, and has important practical significance in the aspects of developing novel antibacterial drugs, solving clinical infection of drug-resistant strains and the like. The bacillus licheniformis has the characteristics of rapid growth, spore generation, strong stress resistance and the like, is widely existed in natural environments such as water, plants, soil and the like, and has remarkable achievements in the industries such as medicine, feed, cultivation and the like as safe probiotics. Therefore, in recent years, researchers at home and abroad have increasingly paid attention to bacillus licheniformis.

Disclosure of Invention

The invention aims to develop a novel bacteriostatic agent.

The invention firstly protects a new bi-component lanthionine, named as lanthionine licenicidin II-8, which consists of polypeptide A1 and polypeptide A2;

the amino acid sequence of the polypeptide A1 is shown as SEQ ID NO. 2;

the amino acid sequence of the polypeptide A2 is shown in SEQ ID NO. 3.

The polypeptide A1 consists of 68 amino acids.

The polypeptide A2 consists of 71 amino acids.

The invention also protects the application of the lantibiotide licenicidin II-8, which can be at least one of b1) and b 2):

b1) inhibiting gram-positive bacteria;

b2) preparing a product for inhibiting gram-positive bacteria.

In the above application, the gram-positive bacterium may be b.cereus 1.260.

The invention also discloses a novel gumline named as gumline Lichelassin, and the amino acid sequence of the gumline Lichelassin is shown in SEQ ID NO. 4.

The dragline peptide Lichelassin consists of 38 amino acids.

The invention also protects the application of the dragline Lichelassin, which can be at least one of c1) and c 2):

c1) inhibiting bacteria;

c2) preparing a product for inhibiting bacteria.

In the above application, the bacteria may be gram-positive bacteria and/or gram-negative bacteria.

The gram-positive bacterium can be bacillus cereus or listeria. The bacillus cereus can be specifically B.cereus1.260 deposited in the China general microbiological culture Collection center. The Listeria can be Listeria monocytogens CGMCC1.10753 deposited in the China general microbiological culture Collection center.

The gram-negative bacteria can be toxigenic escherichia coli or salmonella typhimurium. The Salmonella typhimurium can be Salmonella typhimurium. The toxigenic Escherichia coli can be Escherichia coli K88.

The invention also protects a strain of Bacillus licheniformis, namely Bacillus licheniformis (Bacill licheniformis) BaC1-8, which is preserved in China general microbiological culture Collection center (CGMCC for short, with the address of No. 3 of Beijing city Shangyang Wen Xilu No. 1 of Chaozhong district), and the preservation number is CGMCC No.20653, 9 and 14 days in 2020. Bacillus licheniformis (Bacillus licheniformis) BaC1-8 CGMCC No.20653 is called Bacillus licheniformis BaC1-8 for short.

The invention also protects a microbial inoculum which can contain Bacillus licheniformis (Bacillus licheniformis) BaC1-8 CGMCC No. 20653. The microbial inoculum can be used for at least one of a1) and a 2):

a1) inhibiting gram positive bacteria and/or fungi;

a2) preparing a product for inhibiting gram-positive bacteria and/or fungi.

The active ingredient of the microbial inoculum can be Bacillus licheniformis (Bacillus licheniformis) BaC1-8 CGMCC No. 20653.

The microbial inoculum can specifically consist of Bacillus licheniformis (Bacillus licheniformis) BaC1-8 CGMCC No. 20653.

The invention also discloses a preparation method of the microbial inoculum, which comprises the following steps: inoculating Bacillus licheniformis (Bacillus licheniformis) BaC1-8 CGMCC No.20653 to a culture medium and culturing to obtain a bacterial liquid, namely the microbial inoculum.

The culture medium may be a bacterial culture medium. The bacterial culture medium can be LB liquid culture medium.

The gram-positive bacterium may specifically be Listeria monocytogenes CGMCC1.10753, B.cereus1.260, B.cereus1.1846, Staphylococcus argenteus 1-1, S.argenteus MRSA, Micrococcus luteus 8166, Enterococcus faecalis V583 or Streptococcus agalactia 10465.

The fungus may be a filamentous fungus. The filamentous fungus may in particular be Aspergillus flavus (e.g. Aspergillus flavus LAB08), Aspergillus parasiticus (e.g. A.paraticiticus 3.0124), Aspergillus ochraceus (e.g. A.ochracea 3.6486), Aspergillus sulphureus (e.g. A.sulphlureus 3.5292), Fusarium graminearum (e.g. Fusarium graminearum HWS07) or Fusarium oxysporum (e.g. F.oxysporum HWS 04).

The microbial inoculum may include a carrier in addition to the active ingredient. The carrier may be a solid carrier or a liquid carrier. The solid carrier may be a mineral material, a plant material or a polymeric compound. The mineral material may be at least one of clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica, and diatomaceous earth. The plant material may be at least one of wheat bran, soybean meal, corncob meal, soybean meal and starch. The high molecular compound may be polyvinyl alcohol and/or polyglycol. The liquid carrier can be an organic solvent, vegetable oil, mineral oil, or water. The organic solvent may be decane and/or dodecane. In the microbial inoculum, the active ingredient may be present in the form of cultured living cells, a fermentation broth of living cells, a filtrate of a cell culture, or a mixture of cells and a filtrate. The composition can be prepared into various dosage forms, such as liquid, emulsion, suspending agent, powder, granules, wettable powder or water dispersible granules.

According to the requirement, the microbial inoculum can also be added with a surfactant (such as Tween 20, Tween 80 and the like), a binder, a stabilizer (such as an antioxidant), a pH regulator and the like.

The invention also protects the application of the Bacillus licheniformis (Bacillus licheniformis) BaC1-8 CGMCC No.20653, which can be a1) or a2) at least one of:

a1) inhibiting gram positive bacteria and/or fungi;

a2) preparing a product for inhibiting gram-positive bacteria and/or fungi.

In the above application, the gram-positive bacterium may be Listeria monocytogenes CGMCC1.10753, B.cereus1.260, B.cereus1.1846, Staphylococcus argenteus 1-1, S.argenteus MRSA, Micrococcus luteous 8166, Enterococcus faecalis V583 or Streptococcus agalactiae 10465.

In the above application, the fungus may be a filamentous fungus. The filamentous fungus may in particular be Aspergillus flavus (e.g. Aspergillus flavus LAB08), Aspergillus parasiticus (e.g. A.paraticiticus 3.0124), Aspergillus ochraceus (e.g. A.ochracea 3.6486), Aspergillus sulphureus (e.g. A.sulphlureus 3.5292), Fusarium graminearum (e.g. Fusarium graminearum HWS07) or Fusarium oxysporum (e.g. F.oxysporum HWS 04).

The invention also protects a bacteriostatic agent which contains the lanthionine licenidin II-8, the lichenin and/or Bacillus licheniformis (Bacillus liceniformis) BaC1-8 CGMCC No. 20653; the bacteriostatic agent is used for inhibiting gram-positive bacteria, gram-negative bacteria and/or fungi.

The invention also provides a method for inhibiting gram-positive bacteria, which adopts the lantibiotic II-8 and/or Bacillus licheniformis (Bacillus licheniformis) BaC1-8 CGMCC No.20653 for treatment.

The invention also provides a method for inhibiting bacteria, which adopts the processing of the inhaul peptide Lichelassin.

The invention also provides a method for inhibiting fungi, which adopts Bacillus licheniformis (Bacillus licheniformis) BaC1-8 CGMCC No.20653 for treatment.

Any of the gram-positive bacteria may specifically be Listeria monocytogenes CGMCC1.10753, B.cereus1.260, B.cereus1.1846, Staphylococcus argetenus 1-1, S.argetenus MRSA, Micrococcus luteus 8166, Enterococcus faecalis V583, Streptococcus agalactiae 10465, B.cereus1.260, Bacillus cereus or Listeria. The bacillus cereus can be specifically B.cereus1.260 deposited in the China general microbiological culture Collection center. The Listeria can be Listeria monocytogens CGMCC1.10753 deposited in the China general microbiological culture Collection center.

Any of the above fungi may be filamentous fungi. The filamentous fungus may in particular be Aspergillus flavus (e.g. Aspergillus flavus LAB08), Aspergillus parasiticus (e.g. A.paraticiticus 3.0124), Aspergillus ochraceus (e.g. A.ochracea 3.6486), Aspergillus sulphureus (e.g. A.sulphlureus 3.5292), Fusarium graminearum (e.g. Fusarium graminearum HWS07) or Fusarium oxysporum (e.g. F.oxysporum HWS 04).

Any of the gram-negative bacteria described above may be toxigenic escherichia coli or salmonella typhimurium. The Salmonella typhimurium can be Salmonella typhimurium. The toxigenic Escherichia coli can be Escherichia coli K88.

As described above, the lantibiotic II-8 and the lichenin are both isolated from the fermentation product of Bacillus licheniformis (Bacillus licheniformis) BaC1-8 CGMCC No.20653 for the first time.

Experiments prove that the lantibiotic II-8 can inhibit gram-positive bacteria, the lichenin can inhibit bacteria (such as gram-positive bacteria and gram-negative bacteria), and the Bacillus licheniformis (Bacillus licheniformis) BaC1-8 CGMCC No.20653 can inhibit gram-positive bacteria and/or fungi. The lantibide lichenicidin II-8, the lichenin and the Bacillus licheniformis (Bacillus licheniformis) BaC1-8 CGMCC No.20653 are bacteriostats obtained by first separation, and have important application value.

Drawings

FIG. 1 shows the 16S rDNA clade of Bacillus licheniformis BaC 1-8.

FIG. 2 shows the detection of the bacteriostatic activity of the fermentation supernatant and ammonium sulfate crude extract of Bacillus licheniformis BaC 1-8.

FIG. 3 shows the detection of the bacteriostatic ability of Bacillus licheniformis BaC1-8 on filamentous fungi.

FIG. 4 is an anti SMASH analysis of the genome of Bacillus licheniformis BaC 1-8.

FIG. 5 shows the result of the separation and identification of bacteriostatic substance of Bacillus licheniformis BaC 1-8.

FIG. 6 shows secondary mass spectrometry and bacteriostasis verification of components A1 and A2 of lichenicidin II-8.

FIG. 7 is a secondary mass spectrometric analysis of bacitracin.

FIG. 8 is a first mass spectrometric analysis of the Finerucin.

FIG. 9 shows the structure of the gene cluster of the dragline lichelassin in Bacillus licheniformis BaC 1-8.

FIG. 10 shows the measurement of bacteriostatic ability of lichelassin.

FIG. 11 shows the first-order mass spectrum and structure prediction of lichisin.

FIG. 12 shows the identification of lichisin by secondary mass spectrometry.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 isolation, identification and preservation of Bacillus licheniformis BaC1-8

Commercially available pickle juice was diluted with water, spread on an LB solid medium, and cultured at 37 ℃ for 24 hours to obtain several strains and named. The strain BaC1-8 is subjected to 16SrDNA sequencing, and the sequencing result is shown in SEQ ID NO: 1 is shown.

Converting SEQ ID NO: 1 are aligned at the NCBI.

The alignment results are shown in FIG. 1. The results show that the strain BaC1-8 has the highest homology with the known Bacillus licheniformis DSM13(NR _118996.1) and reaches 99.93 percent. Thus, strain BaC1-8 was identified as Bacillus licheniformis (Bacillus licheniformis).

The strain BaC1-8 has been preserved in China general microbiological culture Collection center (CGMCC, address No. 3 Xilu No. 1 Beijing, Chaoyang, China) in 9-14 months in 2020 with the preservation number of CGMCC No. 20653. The strain BaC1-8 is called Bacillus licheniformis (Bacillus licheniformis) BaC1-8 CGMCC No.20653, and is called Bacillus licheniformis BaC1-8 for short.

Example 2 detection of bacteriostatic ability of Bacillus licheniformis BaC1

Firstly, obtaining fermentation supernatant of bacillus licheniformis BaC1-8 and crude ammonium sulfate extract

1. Inoculating the Bacillus licheniformis BaC1-8 monoclonal to 5mL LB liquid medium, culturing overnight at 37 ℃ and 200rpm to obtain a culture solution 1; the culture bacterial liquid 1 is inoculated (the inoculum size is 1 percent) to 500mL of LB liquid culture medium, and cultured for 20h at 37 ℃ and 200rpm to obtain culture bacterial liquid 2.

2. After the step 1 is finished, taking a culture bacterium liquid 2, centrifuging at 10000rpm for 20min, and collecting a supernatant; the supernatant is the fermentation supernatant.

3. After the step 2 is completed, slowly adding ammonium sulfate into 400mL of fermentation supernatant to obtain a system; the saturation level of ammonium sulfate in the system was 60%. Then, the mixture was stirred thoroughly with a magnetic stirrer at 4 ℃ for 4 hours (for the purpose of ammonium sulfate precipitation).

4. After completion of step 3, the mixture was centrifuged at 10000rpm for 30min at 4 ℃ to collect the precipitate.

5. After the step 4 is completed, taking the precipitate, and carrying out heavy suspension by using 5mL of PBS buffer solution to obtain a heavy suspension; then, the resuspension was dialyzed with a dialysis membrane (MWCO 1kD, Flat Width 31mm, Spectral Por membrane, USA), the dialysis membrane was placed in 0.1 XPBS buffer, stirred with a magnetic stirrer at 4 ℃ and replaced with buffer every 8 hours for 3 times, and ammonium sulfate was sufficiently removed to obtain a dialysate.

6. After completion of step 5, the dialysate was freeze-dried and then resuspended in 10ml of 50% acetonitrile in water containing 0.1% trifluoroacetic acid to obtain a crude ammonium sulfate extract.

Secondly, the bacteriostatic activity of the fermentation supernatant and the ammonium sulfate crude extract of the bacillus licheniformis BaC1-8

The strains to be tested 1 to 8 are gram-positive bacteria. The strains to be tested 9 and 11 are gram-negative bacteria.

The strain 1 to be tested is Listeria monocytogenens CGMCC1.10753 preserved in the common microorganism center of China Committee for culture Collection of microorganisms. Hereinafter, Listeria monocytogens CGMCC1.10753 is abbreviated as l.monocytogens 1.10753.

The strain 2 to be tested is B.cereus1.260 deposited in the China general microbiological culture Collection center.

The strain 3 to be tested is B.cereus1.1846 preserved in the China general microbiological culture Collection center.

The strain 4 to be tested is Staphylococcus argenteus 1-1, which is described in the following documents: wang J, Zhang L, Teng K, Sun S, Sun Z, Zhong J.Cereidins, novel lantibiotics from Bacillus cereus with potential antimicrobial activity. apple Environ Microbiol.2014Apr; 80(8) 2633-43.doi 10.1128/AEM.03751-13. Hereinafter, Staphylococcus argenteus 1-1 is abbreviated as S.argenteus 1-1.

The strain 5 to be tested is s.argenteus MRSA, which is described in the following documents: bogettam K, Vondracek M, Karlsson M, Fang H, GiskeCg. introduction of a hydrolysis probe PCR assay for high-throughput screening of Methucin-resistant Staphylococcus aureus with the ability to include or the expression detection of Staphylococcus argenteus PLoS one.2018Feb 9; 13(2) e0192782.doi: 10.1371/journal.bone.0192782.

The strain 6 to be tested is Micrococcus luteus 8166, which is described in the following documents: wang J, Zhang L, Teng K, Sun S, Sun Z, Zhong J.Cereidins, novel lantibiotics from Bacillus cereus with potential antimicrobial activity. apple Environ Microbiol.2014Apr; 80(8) 2633-43.doi 10.1128/AEM.03751-13. hereinafter, Micrococcus luteus 8166 is abbreviated as M.luteus 8166.

The strain 7 to be tested is Enterococcus faecalis V583, which is described in the following documents: wang J, Zhang L, Teng K, Sun S, Sun Z, Zhong J.Cereidins, novel lantibiotics from Bacillus cereus with potential antimicrobial activity. apple Environ Microbiol.2014Apr; 80(8), 2633-43.doi:10.1128/AEM.03751-13. hereinafter, Enterococcus faecalis V583 is abbreviated as E.faecalis V583.

The strain 8 to be tested is Streptococcus agalactiae 10465 deposited in China center for Industrial culture Collection. Hereinafter, Streptococcus agalactiae 10465 is abbreviated as s.

The strain 9 to be tested is Salmonella typhimurium, which is described in the following documents: Shuai-Cheng W, Ben-Dong F, Xiu-Link C, Jian-Qing S, Yun-Xing F, Zhen-Qiang C, Dao-Xiu X, Zong-Mei W. Subinhibitory con-centrations of alloying repain the vitamin of Salmonella typhimurium and protect against Salmonella typhimurium infection. 109(11): 1503-1512, doi:10.1007/s10482-016-0752-z.

The strain 10 to be tested is Escherichia coli K88, which is described in the following documents: wang Z, Li J, Li J, Li Y, Wang L, Wang Q, Fang L, Ding X, Huang P, Yin J, Yin Y, Yang H.protective effect of chicken egg yolk immunoglobulins (IgY) against original organic Escherichia coli K88 addition in weighed pig.BMC Vet Res.2019Jul 8; 15(1): 234, doi:10.1186/s 12917-019-.

The strain 11 to be tested was Stenotrophoromonas maltophilia 1.1788 deposited in the general microbiological center of China Committee for culture Collection of microorganisms.

The bacteriostatic activity (namely the diameter of a bacteriostatic ring) of each strain to be detected is detected, the experiment is repeated three times, and the steps of each time are as follows:

1. and (3) inoculating the monoclonal of the strain to be detected to an LB liquid culture medium, and culturing at 37 ℃ and 210rpm to obtain a bacterial suspension of the strain to be detected. The concentration of the bacterial suspension of the bacteria to be tested is 10⁸cfu/mL。

2. Pouring 20 μ L of bacterial suspension of bacteria to be tested into 15-20mLLB solid culture medium (LB solid culture medium temperature is about 50-55 deg.C), and mixing well to obtain mixed solution.

3. Placing 6 Oxford cups in a culture dish, wherein the distance between the centers of the Oxford cups is more than 25mm, the distance between the centers of the Oxford cups and the periphery of the culture dish is more than 15mm, and the distances are uniform; then the mixed solution is poured into the reactor and cooled.

4. After completing step 3, 1mL of the fermentation supernatant obtained in step one 2 or the crude ammonium sulfate extract obtained in step one 6 (corresponding to the height of the liquid level of the mixed solution in the culture dish) was added to the Oxford cup, the culture dish was incubated at 37 ℃ for 20 hours, and then the diameter of the antibacterial ring was measured with a vernier caliper.

5. After completing step 3, 1mL of sterile water (corresponding to the height of the liquid level of the mixture in the culture dish) was added to the oxford cup, the culture dish was placed at 37 ℃ for culturing for 20h, and then the diameter of the antibacterial ring was measured with a vernier caliper as a negative control.

6. After completing step 3, 1mL of Nisin solution (with a concentration of 50. mu.g/mL) was added to the Oxford cup (consistent with the height of the liquid surface of the mixed solution in the culture dish), the culture dish was incubated at 37 ℃ for 20h, and then the diameter of the antibacterial ring was measured with a vernier caliper as a positive control. Nisin, Nisin, is a natural bioactive antibacterial peptide, is a purely natural, efficient and safe polypeptide active substance extracted by biotechnology, and can inhibit a plurality of gram-positive bacteria.

Then, the following judgment is made:

(1) if the diameter of the bacteriostatic ring is more than 0mm, judging that the bacteriostatic ring has bacteriostatic effect; otherwise, judging that no bacteriostatic effect exists;

(2) if the three repeated tests have the bacteriostatic action result, the strain to be detected is judged to have the bacteriostatic function;

(3) the average diameter of the bacteriostatic rings of the three repeated tests is more than 6mm, and the bacteriostatic effect is judged to be achieved and is represented by +++; the average diameters of the bacteriostatic rings of the three repeated tests are more than 3mm and less than 6mm, and the bacteriostatic effect is judged to be achieved and is expressed by + + in the specification; the average diameters of the bacteriostatic rings of the three repeated tests are all larger than 0mm and smaller than 3mm, and the bacteriostatic effect is judged to be present and is represented by + in the specification.

(4) Negative control should not have the bacteriostatic ring to produce, positive control should have the bacteriostatic ring to produce, otherwise the test is invalid.

The results of the partial tests are shown in FIG. 2 (the supernatant is fermentation supernatant, the crude extract is ammonium sulfate crude extract, and Nisin is positive control) and Table 1. The results show that the fermentation supernatant and the ammonium sulfate crude extract of the bacillus licheniformis BaC1-8 can obviously inhibit 8 gram-positive bacteria, but cannot inhibit 3 gram-negative bacteria.

TABLE 1

	Fermented supernatant	Crude ammonium sulfate extract
			L.monocytogenes 1.10753	+	+
B.cereus1.260	+	++
			B.cereus1.1846	++	+++
S.argenteus 1-1	+	++
			M.luteus 8166	++	++
E.faecalis V583	++	++
			S.agalactiae 10465	+	++

Thirdly, detecting the bacteriostatic ability of the bacillus licheniformis BaC1-8 on filamentous fungi

Aspergillus flavus LAB08 and f.oxysporum HWS04 are both described in the following documents: liu Y, Teng K, Wang T, Dong E, Zhang M, Tao Y, Zhong J. antibacterial Bacillus velezensis HC 6: production of proteins of peptides and biocontrol potential in mail.J. Appl Microbiol.2020 Jan; 128(1): 242-254. doi: 10.1111/jam.14459.

Paraticicus 3.0124, achraceus 3.6486, sulphleureus 3.5292 and Fusarium graminearum HWS07 are all deposited at the china general microbiological culture collection center.

The bacteriostatic ability of Bacillus licheniformis BaC1-8 against filamentous fungi (Aspergillus flavus LAB08, A. paraticus 3.0124, A. ochracea 3.6486, A. subplurus 3.5292, Fusarium grandium HWS07 or F. oxysporum HWS04) was tested by plate co-culture (described in Liu Y, Teng K, Wang T, Dong E, Zhang M, Tao Y, Zhong J. antibacterial Bacillus velezensis HC 6: production of peptides and biocontrol pore in mail. J. apple. Microbiol. 0. 202Jan; 128 (1): 242-254. doi: 10.1111/jam.14459.). Bacillus licheniformis BaC1-8 was replaced with water as a blank control.

The results are shown in fig. 3(Control is a blank Control, a is Aspergillus flavus LAB08, B is Aspergillus parasiticus 3.0124, C is Aspergillus ochraceus 3.6486, D is Aspergillus thiopicus 3.5292, E is Fusarium graminearum HWS07, and F is Fusarium oxysporum HWS 04). The results indicate that bacillus licheniformis BaC1-8 can inhibit filamentous fungi such as Aspergillus flavus LAB08, a. parasiticus 3.0124, a. ochracea 3.6486, a. sulpholeurus 3.5292, Fusarium graminearum HWS07 or f.oxysporum HWS 04.

Example 3 isolation and characterization of the metabolite of Bacillus licheniformis BaC1-8 in its Natural form

First, anti SMASH analysis of Bacillus licheniformis BaC1-8 genome

Bacillus licheniformis BaC1-8 was subjected to genomic sequencing and anti SMASH analysis.

The results of the analysis are shown in FIG. 4. The result shows that bacillus licheniformis BaC1-8 has 16 metabolite gene clusters, mainly comprising 9 NRPS gene clusters, 1 novel dragline peptide (lasso peptide), 1 bacteriocin gene cluster and 1 novel bi-component lanthionine gene cluster.

Second, separation and identification of metabolite of Bacillus licheniformis BaC1-8 in natural state

In order to determine whether the bacillus licheniformis BaC1-8 produces metabolites analyzed in the genome in the natural state, the bacteriostatic substances produced by the bacillus licheniformis BaC1-8 are separated and purified. The method comprises the following specific steps:

1. obtaining extract (i.e. ammonium sulfate crude extract of Bacillus licheniformis BaC 1-8)

The same procedure as in the first step of example 2.

2. The extracts were separated by HPLC and identified by mass spectrometry.

The results of the isolation and identification are shown in FIG. 5(bacitracin is bacitracin and fengycin is fengesin). The results showed that the extract contained bacitracin, carmustine, a novel two-component lanthionine (consisting of A1 and A2) which was named lichenicidin II-8.

3. Secondary mass spectrometric analysis of A1 and A2 fractions of lichenicidin II-8

(1) The results of secondary mass spectrometry analysis of A1 and A2 fractions of lichenicidin II-8 are shown in A of FIG. 6.

The result shows that the licenicidin II-8 is similar to the licenicidin by 50 percent, the molecular weights of A1 and A2 are 3254Da and 2510Da respectively, and the secondary mass spectrum preliminary judgment of the two components is consistent with the prediction.

The amino acid sequence of a1 is:

MSKIEAWKNPVARMNSQIVSPAGDLMDELSDSEMEMLAGGCEWYNISCQLGNKGQWCTLTKECQRSCK(SEQ ID NO:2)。

the amino acid sequence of a2 is:

MSHREMAAIYRDANKRANLEFSNPVGEVNEEELKNLAGAADVTPHTTPSSLPCGTLVTAVWCPSNACTSDC(SEQ ID NO:3)。

(2) the bacteriostatic activity of lichenicidin II-8 on B.cereus1.260 was tested according to the method of step two in example 2.

The detection result is shown in B in FIG. 6. The result shows that the two components play a synergistic role and can obviously inhibit B.cereus 1.260.

4. Secondary mass spectrometric analysis of bacitracin

The bacillus licheniformis BaC1-8 has a complete gene cluster of bacitracin, and mass spectrometry identifies a substance with the molecular weight of 1422Da, which is consistent with the molecular weight of bacitracin.

The substance was found to have a fragment peak of bacitracin intact by secondary mass spectrometry (see FIG. 7), thus confirming that bacitracin is produced by Bacillus licheniformis BaC1-8 in its native state. Bacitracin has strong bacteriostatic activity against gram-positive bacteria and some gram-negative bacteria, and has no pathogenicity to human and animals, thus being widely applied to clinical medicine and used as a commonly used animal feed additive.

5. First-order mass spectrometry analysis of Finesin

Bacillus licheniformis BaC1-8 has the gene cluster of fengycin and can produce abundant fengycin in a natural state. fengycin is a kind of lipopeptide synthesized by non-ribosome, is composed of 10 amino acids and fatty chain, and has strong bacteriostasis capacity to filamentous fungi.

The results of the primary mass spectrometry of Fengycin are shown in FIG. 8. The result shows that the metabolite of the Bacillus licheniformis BaC1-8 contains C16Fengycin A [ M + Na ]]⁺、C17Fengycin A[M+H]⁺、C17 Fengycin A[M+Na]⁺、C16FengycinB[M+H]⁺、C17FengycinB[M+H]⁺And 5 kinds of carmustine.

Gene cluster of dragline lichelsin and its bacteriostasis capacity

1. According to the result of the first step, the Bacillus licheniformis BaC1-8 is found to have a new inhaul cable peptide gene cluster (see figure 9). The most of the inhaul peptide is from actinomycetes and proteobacteria, and only one kind of inhaul peptide paeninodinodinodinodinodinidin is reported in firmicutes, which indicates that the inhaul peptide licelisin is very new in source.

The amino acid sequence of the gumlossin helichassin is MRKLESAQVKNYGTFLTVTLSKKSKPGDGKFGRGVKRG (SEQ ID NO: 4).

2. Obtaining supernatant and crude extract of lichelassin

(1) Constructing recombinant Escherichia coli containing the whole gene cluster of the gumlosin lichelsin. Heterologous expression of the dragline peptide lichelapsin in recombinant Escherichia coli.

(2) Inoculating the recombinant escherichia coli to 5mL of LB liquid culture medium, and culturing at 37 ℃ and 180rpm overnight to obtain a culture bacterial liquid; then, the culture broth was inoculated into 500mL of LB liquid medium and induced at 37 ℃ and 180rpm for 24 hours.

(3) After completion of step (2), centrifugation was carried out at 5000rpm for 30min, and the supernatant was collected.

(4) After the step (3) is completed, adding 200g of Amberlite XAD-7HP into the supernatant, and oscillating for 12 h; then washing with 2L of distilled water and 1L of 30% (v/v) ethanol aqueous solution in sequence; eluting with 500ml of ethanol (80% (v/v) and pH2.0) to obtain active substance; finally, condensing in a rotary evaporator at 42 ℃ under a vacuum condition to obtain a concentrated solution; the concentrated solution is the crude lichelassin extract.

3. Detection of the crude extract and supernatant of the lichenin obtained in step 2 with reference to the method of step two in example 2 against Bacillus cereus (specific strain B.cereus1.260 deposited in the center of general microorganisms of the China Committee for culture Collection of microorganisms), Listeria (specific strain Listeria monocytogenes CGMCC1.10753 deposited in the center of general microorganisms of the China Committee for culture Collection of microorganisms, hereinafter Listeria monocytogenes CGMCC1.10753 is abbreviated as L.monoclonal genes 1.10753), Salmonella typhimurium (specific strain Salmonella typhimurium described in Shuai-Cheng W, Ben-Dong F, Xiu-Ling C, Jian-Qirating S, Yun-Xiing F, Zhen-Qiang C, Zoo-Xiu X, Zong-Mei W.Subingy restriction of Bacillus cereus 32. 12. and Salmonella typhimurium 32. 201632 z.) and toxigenic Escherichia coli (a specific strain is Escherichia coli K88, which is described in: wang Z, Li J, Li J, Li Y, Wang L, Wang Q, Fan L, Ding X, Huang P, Yin J, Yin Y, Yang H.protective effect of chicken egg yolk immunoglobulins (IgY) against an antibiotic organic Escherichia coli K88 addition in a weighted piglets.BMC Vet Res.201Jul 8; 15(1): 234, doi:10.1186/s 12917-019-1958-x).

The results of some of the tests are shown in FIG. 10. The result shows that the crude lichelassin extract and the supernatant obtained in the step 2 not only have obvious bacteriostatic ability on gram-positive bacteria such as bacillus cereus, listeria and the like, but also have good inhibitory effect on gram-negative bacteria such as toxigenic escherichia coli, salmonella typhimurium and the like. Therefore, the dragline lichelassin has a special bacteriostatic mechanism and has good application potential.

The results of the first-order mass spectrometric detection of Lichelassin are shown in the left panel of FIG. 11. The results showed that the molecular weight of Lichelassin was 1871Da, in agreement with the calculations. The structure prediction lichelsin presents a typical dragline structure with a Ring structure, a loop structure, and a Tail structure (see the right diagram in fig. 11).

The results of the second-order mass spectrometry of Lichelassin are shown in FIG. 12. The results showed that the amino acid sequence of Lichelasin is shown in SEQ ID NO:4, and it was confirmed that it is possible that the first eight amino acids of Lichelasin form a cyclic structure.

The above results show that Bacillus licheniformis BaC1-8 can produce abundant bacteriostatic substances, including lantibiotic II-8 and inhaul-peptide lichenin and two NRPSs (fengycin and bacitracin). The bacillus licheniformis BaC1-8, the lantibiotic II-8 and the inhaul peptide lichenin have important application potential in the aspects of antibiosis, mildew prevention, drug development and the like.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

<110> institute of microbiology of Chinese academy of sciences

<120> a strain of Bacillus licheniformis for producing lantibiotic peptide and inhaul cable peptide

<160> 4

<170> PatentIn version 3.5

<210> 1

<211> 1418

<212> DNA

<213> Artificial sequence

<400> 1

agtcgagcgg accgacggga gcttgctccc ttaggtcagc ggcggacggg tgagtaacac 60

gtgggtaacc tgcctgtaag actgggataa ctccgggaaa ccggggctaa taccggatgc 120

ttgattgaac cgcatggttc aatnataaaa ggtggctttt agctaccact tacagatgga 180

cccgcggcgc attagctagt tggtgaggta acggctcacc aaggcgacga tgcgtagccg 240

acctgagagg gtgatcggcc acactgggac tgagacacgg cccagactcc tacgggaggc 300

agcagtaggg aatcttccgc aatggacgaa agtctgacgg agcaacgccg cgtgagtgat 360

gaaggttttc ggatcgtaaa actctgttgt tagggaagaa caagtaccgt tcgaataggg 420

cggtaccttg acggtaccta accagaaagc cacggctaac tacgtgccag cagccgcggt 480

aatacgtagg tggcaagcgt tgtccggaat tattgggcgt aaagcgcgcg caggcggttt 540

cttaagtctg atgtgaaagc ccccggctca accggggagg gtcattggaa actggggaac 600

ttgagtgcag aagaggagag tggaattcca cgtgtagcgg tgaaatgcgt agagatgtgg 660

aggaacacca gtggcgaagg cgactctctg gtctgtaact gacgctgagg cgcgaaagcg 720

tggggagcga acaggattag ataccctggt agtccacgcc gtaaacgatg agtgctaagt 780

gttagagggt ttccgccctt tagtgctgca gcaaacgcat taagcactcc gcctggggag 840

tacggtcgca agactgaaac tcaaaggaat tgacgggggc ccgcacaagc ggtggagcat 900

gtggtttaat tcgaagcaac gcgaagaacc ttaccaggtc ttgacatcct ctgacaaccc 960

tagagatagg gcttcccctt cgggggcaga gtgacaggtg gtgcatggtt gtcgtcagct 1020

cgtgtcgtga gatgttgggt taagtcccgc aacgagcgca acccttgatc ttagttgcca 1080

gcattcagtt gggcactcta aggtgactgc cggtgacaaa ccggaggaag gtggggatga 1140

cgtcaaatca tcatgcccct tatgacctgg gctacacacg tgctacaatg ggcagaacaa 1200

agggcagcga agccgcgagg ctaagccaat cccacaaatc tgttctcagt tcggatcgca 1260

gtctgcaact cgactgcgtg aagctggaat cgctagtaat cgcggatcag catgccgcgg 1320

tgaatacgtt cccgggcctt gtacacaccg cccgtcacac cacgagagtt tgtaacaccc 1380

gaagtcggtg aggtaacctt ttggagccag ccgccgaa 1418

<210> 2

<211> 68

<212> PRT

<213> Artificial sequence

<400> 2

Met Ser Lys Ile Glu Ala Trp Lys Asn Pro Val Ala Arg Met Asn Ser

1 5 10 15

Gln Ile Val Ser Pro Ala Gly Asp Leu Met Asp Glu Leu Ser Asp Ser

20 25 30

Glu Met Glu Met Leu Ala Gly Gly Cys Glu Trp Tyr Asn Ile Ser Cys

35 40 45

Gln Leu Gly Asn Lys Gly Gln Trp Cys Thr Leu Thr Lys Glu Cys Gln

50 55 60

Arg Ser Cys Lys

65

<210> 3

<211> 71

<212> PRT

<213> Artificial sequence

<400> 3

Met Ser His Arg Glu Met Ala Ala Ile Tyr Arg Asp Ala Asn Lys Arg

1 5 10 15

Ala Asn Leu Glu Phe Ser Asn Pro Val Gly Glu Val Asn Glu Glu Glu

20 25 30

Leu Lys Asn Leu Ala Gly Ala Ala Asp Val Thr Pro His Thr Thr Pro

35 40 45

Ser Ser Leu Pro Cys Gly Thr Leu Val Thr Ala Val Trp Cys Pro Ser

50 55 60

Asn Ala Cys Thr Ser Asp Cys

65 70

<210> 4

<211> 38

<212> PRT

<213> Artificial sequence

<400> 4

Met Arg Lys Leu Glu Ser Ala Gln Val Lys Asn Tyr Gly Thr Phe Leu

1 5 10 15

Thr Val Thr Leu Ser Lys Lys Ser Lys Pro Gly Asp Gly Lys Phe Gly

20 25 30

Arg Gly Val Lys Arg Gly

35

Claims

1. The lanthionine lichenicidin II-8 consists of polypeptide A1 and polypeptide A2;

the amino acid sequence of the polypeptide A1 is shown as SEQ ID NO. 2;

the amino acid sequence of the polypeptide A2 is shown in SEQ ID NO. 3.

2. Use of the lantibiotic idin lichenicidin ii-8 as claimed in claim 1, being at least one of b1) and b 2):

b1) inhibiting gram-positive bacteria;

b2) preparing a product for inhibiting gram-positive bacteria.

3. The amino acid sequence of the stay peptide Lichelassin is shown in SEQ ID NO. 4.

4. The use of the inhaul cable peptide Lichelisin according to claim 3, which is at least one of c1) and c 2):

c1) inhibiting bacteria;

c2) preparing a product for inhibiting bacteria.

5. Bacillus licheniformis (Bacillus licheniformis) BaC1-8, the preservation number of which in China general microbiological culture Collection center is CGMCC No. 20653.

6. A microbial preparation comprising the Bacillus licheniformis (Bacillus licheniformis) BaC1-8 CGMCC No.20653 according to claim 5.

7. Application of Bacillus licheniformis (Bacillus licheniformis) BaC1-8 CGMCC No.20653, a1) and a2) is as follows:

a1) inhibiting gram positive bacteria and/or fungi;

a2) preparing a product for inhibiting gram-positive bacteria and/or fungi.

8. A bacteriostatic agent comprising the lanthionine lichenicidin ii-8 of claim 1, the lecithsin of claim 3 and/or the Bacillus licheniformis (Bacillus licheniformis) BaC1-8 CGMCC No.20653 of claim 5; the bacteriostatic agent is used for inhibiting gram-positive bacteria, gram-negative bacteria and/or fungi.

9. A method for inhibiting gram positive bacteria by treatment with the lantibiotic peptide licenicidin II-8 of claim 1 and/or the Bacillus licheniformis (Bacillus liceniformis) BaC1-8 CGMCC No.20653 of claim 5.

10. A method for inhibiting bacteria, which is to treat the inhaul peptide Lichelasin according to claim 3.

11. A method for inhibiting fungi by treating with Bacillus licheniformis (Bacillus licheniformis) BaC1-8 CGMCC No.20653 according to claim 5.