阅读说明：本技术 一种新型抗菌羊毛硫肽AmylocinC及其制备方法和应用 (Novel antibacterial lantibide AmylocinC and preparation method and application thereof ) 是由 刘洪伟 张丽萍 姚彩苗 赵雯雅 王雅娜 张飞燕 于 2021-02-03 设计创作，主要内容包括：本发明公开了一种新型抗菌羊毛硫肽AmylocinC及其制备方法和应用,属于抗菌肽制备领域。本发明通过将抗菌羊毛硫肽AmylocinC前体肽AmyC、合成酶AmyMB和肽酶结构域AmyT150的基因通过热激转化法共转化至大肠杆菌感受态细胞,通过大肠杆菌异源表达获得抗菌羊毛硫肽AmylocinC。本发明公开的抗菌羊毛硫肽AmylocinC具有抗菌活性,能够抑制革兰氏阳性菌生长。(The invention discloses a novel antibacterial lantibide Amylocin C, a preparation method and application thereof, and belongs to the field of antibacterial peptide preparation. The invention co-transforms the genes of the precursor peptide AmyC of the antibacterial lantibiotic Amylocin C, the synthetase AmyMB and the peptidase structural domain AmyT150 into escherichia coli competent cells by a heat shock transformation method, and obtains the antibacterial lantibiotic Amylocin C by escherichia coli heterologous expression. The antibacterial lantibiotic Amylocin C disclosed by the invention has antibacterial activity and can inhibit the growth of gram-positive bacteria.)

1. A lantibide Amylocin C, having a molecular structural formula:

2. a preparation method of the antibacterial lantibiotic AmylocinC as claimed in claim 1, characterized in that the genes of the precursor peptide AmyC of the antibacterial lantibiotic AmylocinC, the synthetase AmyMB and the peptidase domain AmyT150 are co-transformed into Escherichia coli competent cells, and the antibacterial lantibiotic AmylocinC is obtained by heterologous expression of Escherichia coli.

3. The method of claim 2, wherein the lantibiotic precursor peptide, AmyC, gene sequence is at least one of:

1) a gene sequence shown as SEQ ID NO. 1;

2) a DNA molecule which is hybridized with the DNA molecule defined by 1) under strict conditions and codes a polypeptide sequence shown in SEQ ID NO. 2;

3) DNA molecule which has more than 90% of identity with the DNA molecule defined in 1) or 2) and codes the polypeptide sequence shown in SEQ ID NO. 2.

4. The method of claim 2, wherein the gene sequence of the synthetase AmyMB is at least one of the following:

1) a gene sequence shown as SEQ ID NO. 3;

2) a gene sequence which hybridizes with the DNA molecule defined in 1) under strict conditions and codes for a protein with the activity of the synthetase AmyMB;

3) a gene sequence which has 90% or more identity with the DNA molecule defined in 1) or 2) and encodes a protein having the activity of the synthetase AmyMB.

5. The method of claim 2, wherein the peptidase domain AmyT150 has at least one of the following sequences:

1) a gene sequence shown as SEQ ID NO. 4;

2) a gene sequence which hybridizes with the DNA molecule defined in 1) under stringent conditions and codes for a protein with the activity of the peptidase domain AmyT 150;

3) a gene sequence which has 90% or more identity with the DNA molecule defined in 1) or 2) and encodes a protein having the peptidase domain AmyT150 activity.

6. The method of claim 2, wherein the heterologous expression of lantibiotide in the co-expression strain is induced by the addition of IPTG.

7. The method of claim 2, wherein the heterologous expression is followed by a separation and purification step, wherein the separation and purification step is performed by semi-preparative high performance liquid chromatography, and the lantibiotic Amylocin C elutes at 23 min.

8. Use of the antibacterial lantibiotic AmylocinC of claim 1 against gram-positive bacteria.

9. An antibacterial pharmaceutical composition comprising the antibacterial lantibiotic AmylocinC of claim 1 and a pharmaceutically acceptable excipient.

10. A framework amino acid sequence of the lantibiotic amylocin c according to claim 1, wherein the framework amino acid sequence is as set forth in SEQ ID No. 5.

Technical Field

The invention belongs to the field of antibacterial peptide preparation, and particularly relates to novel antibacterial lantibide AmylocinC, and a preparation method and application thereof.

Background

Antibiotics are the most commonly prescribed drugs worldwide, but their use faces increasingly serious challenges in clinical use due to the emergence of drug-resistant bacteria. At present, the problem of indiscriminate use of antibiotics for resisting pathogenic bacteria is becoming serious all the year round, and therefore, the search for some novel and environment-friendly antibacterial substances to replace antibiotics becomes a research hotspot. Antibacterial peptides, which can rapidly and effectively kill bacteria resistant to antibiotics, are considered to be the best alternatives to conventional antibiotics.

Lantibide is a cyclic micromolecular polypeptide generated by gram-positive bacteria, and has a certain inhibiting effect on various food-borne pathogenic bacteria and multi-drug resistant bacteria. The action target of the lantibiotic peptides is usually positioned on a cell wall or a cell membrane, so that the structure of the cell wall or the cell membrane is incomplete, holes are formed, and bacteria are killed, and the sterilization mechanism also causes the drug resistance of the lantibiotic peptides to rarely appear. Due to the special target recognition function, the lantibiotic peptides hardly generate resistance and can be used as traditional antibiotics to replace drugs. About 50 kinds of lantipeptide antimicrobial peptides have been discovered and reported, among which NVB302, Mutacin1140, Duramycin, Gallidermin, Nisin and Microbisporin have been clinically studied. The high conversion rate of clinical research makes the lantipeptide antibacterial peptide have great prospect in drug research and development.

However, the lantibiotide antibacterial peptides generally have the defects of narrow antibacterial spectrum and insufficient stress resistance, so that the development and research of novel efficient lantibide antibacterial peptides with strong stress resistance have important value. With the rapid development of high-throughput sequencing technology, based on a large amount of microbial genome data, the potential of bacillus amyloliquefaciens in the synthesis of various novel lanthionine antibacterial peptides is far beyond the past cognition. A plurality of biosynthetic gene clusters for coding the lantibiotic peptides exist in the bacillus amyloliquefaciens, the gene clusters are usually silent in wild strains, and the production of the lantibiotic peptides by a heterologous expression method is of great significance for developing novel efficient lantibiotic peptides.

Disclosure of Invention

The invention aims to provide a novel efficient lantibide Amylocin C and a preparation method thereof, so as to solve the problems in the prior art, further expand the types of antibacterial lantibide and provide a new raw material for further research and development of antibacterial drugs.

In order to achieve the purpose, the invention provides the following scheme:

in a first aspect, there is provided a lantibide, amylocin c, having a molecular structural formula:

the framework amino acid sequence of the antibacterial lanthionine AmylocinC is shown in SEQ ID No.5, threonine at the 2 nd, 7 th, 9 th and 10 th positions is dehydrated to form dehydro-buthionine, serine at the 16 th position is dehydrated and forms a thioether ring with cysteine at the 20 th position, and serine at the 27 th position is dehydrated and forms a thioether ring with cysteine at the 32 th position.

In a second aspect, the preparation method of the antibacterial lantibiotic Amylocin C is provided, the genes of the precursor peptide AmyC of the antibacterial lantibiotic Amylocin C, the synthetase AmyMB and the peptidase domain AmyT150 are co-transformed into escherichia coli competent cells, and the antibacterial lantibiotic Amylocin C is obtained through escherichia coli heterologous expression.

Preferably, the lantibiotic precursor peptide AmyC gene sequence is at least one of:

1) a gene sequence shown as SEQ ID NO. 1;

2) a DNA molecule which is hybridized with the DNA molecule defined by 1) under strict conditions and codes a polypeptide sequence shown in SEQ ID NO. 2;

3) DNA molecule which has more than 90% of identity with the DNA molecule defined in 1) or 2) and codes the polypeptide sequence shown in SEQ ID NO. 2.

Preferably, the gene sequence of the synthetase AmyMB is at least one of the following:

1) a gene sequence shown as SEQ ID NO. 3;

2) a gene sequence which hybridizes with the DNA molecule defined in 1) under strict conditions and codes for a protein with the activity of the synthetase AmyMB;

3) a gene sequence which has 90% or more identity with the DNA molecule defined in 1) or 2) and encodes a protein having the activity of the synthetase AmyMB.

Preferably, the gene sequence of the peptidase domain AmyT150 is at least one of the following:

1) a gene sequence shown as SEQ ID NO. 4;

2) a gene sequence which hybridizes with the DNA molecule defined in 1) under stringent conditions and codes for a protein with the activity of the peptidase domain AmyT 150;

3) a gene sequence which has 90% or more identity with the DNA molecule defined in 1) or 2) and encodes a protein having the peptidase domain AmyT150 activity.

Preferably, heterologous expression of the lantibiotide in the co-expression strain is induced by the addition of IPTG.

Preferably, the heterologous expression is followed by a separation and purification step, wherein the separation and purification step is performed by using semi-preparative high performance liquid chromatography, and the lantibide Amylocin C elutes at 23 min.

In a third aspect, the application of the antibacterial lantibiotic AmylocinC in resisting gram-positive bacteria is provided.

In a fourth aspect, there is provided an antibacterial pharmaceutical composition comprising the antibacterial lantibiotic amylocin c according to claim 1 and a pharmaceutically acceptable excipient.

In a fifth aspect, a framework amino acid sequence of the lantibiotic AmylocinC is provided, and the framework amino acid sequence is shown as SEQ ID NO. 5.

The technical scheme has the following technical effects:

the novel lantibide AmylocinC identified and synthesized from the bacillus amyloliquefaciens can provide a new alternative medicine for treating multiple drug-resistant bacteria and provide technical support for heterologous biosynthesis of the lantibide. The novel lantibide AmylocinC has a strong inhibition effect on gram-positive bacteria, and provides a new tool for treating and preventing the gram-positive bacteria.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.

FIG. 1 shows the results of PCR amplification products of co-expressed strains;

FIG. 2 is a diagram of high performance liquid purification of lantibiotic Amylocin C;

FIG. 3 is a mass spectrum of lantibiotic AmylocinC;

FIG. 4 is a schematic diagram of the structure of lantibiotic AmylocinC;

FIG. 5 is the chemical structural formula of lantibiotic AmylocinC.

Detailed Description

Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The specification and examples are exemplary only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.

Unless otherwise indicated, the biochemical techniques used in the present invention are conventional in the art.

Unless otherwise specified, the materials, reagents and the like used in the present invention are commercially available.

LB culture medium: culturing Escherichia coli;

10g of tryptone, 5g of NaCl and 5g of yeast extract, adding deionized water to reach the constant volume of 1000mL, and adjusting the pH value to 7.0-7.2.

Strains and vectors used in the following examples:

coli BL21(DE3) was purchased from TransGen Biotech, Inc., Beijing Korea. And (3) indication bacteria: staphylococcus aureus, Klebsiella pneumoniae, Bacillus licheniformis, Bacillus subtilis, Sphingomonas, Escherichia coli, and Pseudomonas fluorescens were deposited by the institute of sciences, Hebei province. pET-28a, pCDFDuet-1 and PACYCDuet-1 vectors were purchased from Tiannze Biotech Inc. (TIANDZ).

Bioinformatics tools used in the following examples:

a database: NCBI (national Center for Biotechnology Information Search database) (http:// www.ncbi.nlm.nih.gov /);

analysis software: anti SMASH 4.0(https:// anti marsh. second microorganisms. org /);

BAGEL 3(http://bagel.molgenrug.nl/)；

Morpheus(https://software.broadinstitute.org/morpheus/)；

local software: EditSeq software, Primer 5.0 software, MegAlign software.

In the related sequences, the coding gene sequence of the lantibiotic AmylocinC precursor peptide AmyC is shown in SEQ ID NO.1, and the polypeptide sequence thereof is shown in SEQ ID NO. 2; the gene of the lantibiotic peptide synthetase is shown as SEQ ID NO. 3; the structure domain of the lantibiotide peptidase is shown in SEQ ID NO. 4; the framework amino acid sequence of the lantibiotic AmylocinC is shown in SEQ ID NO. 5.

Example 1: sources and screening process for lantipeptide AmylocinC

1. Inputting 'Bacillus amyloliquefaciens' under the 'Genome' entry in an NCBI database to search basic information of all strains of the Bacillus amyloliquefaciens and obtain NCBI accession numbers of all strains.

2. Genome data mining was performed by mining 27 strains of bacillus amyloliquefaciens through bioinformatics software anti smash 4.0 and BAGEL 3, and a total of 314 gene clusters were predicted and mainly divided into 16 types. And found that 12 strains of DSM 7, TA208 and the like contain 18 second type lantipeptide synthetic gene clusters, and 21 potential second type lantipeptides are excavated together, wherein precursor peptide genes for synthesizing the novel lantipeptide AmylocinC exist. The synthetase amyMB and peptidase domain amyT150 genes were obtained from the Bacillus amyloliquefaciens WS-8 genomic sequence.

Example 2

Heterologous expression of lantibiotide AmylocinC

1. The nucleotide sequences of the lanthionine precursor peptide AmyC, the synthetase AmyMB, and the peptidase domain AmyT150 gene excavated in example 1 were sent to NovoPro for synthesis. And the precursor peptide gene is constructed on a pET28a carrier, the synthetase gene is constructed on a pCDFDuet-1 carrier, and the peptidase structure domain gene is constructed on a pACYCDuet-1 carrier.

2. The three recombinant vectors containing the lanthionine precursor peptide AmyC, the synthetase AmyMB and the peptidase structural domain AmyT150 gene are co-transferred into a competent cell E.coli BL21(DE3) by a heat shock transformation method to construct a co-expression strain.

3. An LB plate (kanamycin, spectinomycin and chloramphenicol) containing three antibiotics is adopted to screen out positive co-expression strains.

4. And identifying positive colonies of the co-expression strain by adopting a colony PCR method.

The primers used are as follows (SEQ ID NO.6-SEQ ID NO. 11):

AmyC F1：TAATACGACTCACTATAGGG

AmyC R1：TAGTCGTTACATGGACTGAC

AmyMB F1：CCTGGGTGATATCTATGAAAACTAA

AmyMB R1：TTAATCCAGTGCCAGCGGTGTCG

AmyT150 F1：CACCATCATCACCACCTG

AmyT150 R1：TTAGGCGGTTTTATCTTTGCGTTCC

the PCR reaction system is as follows:

PCR verification conditions of the coexpression strain containing the precursor peptide AmyC are as follows: at 95 ℃ for 2 min; 95 ℃, 30s, Tm55 ℃, 40s,72 ℃, 30s, 32 cycles total; 72 ℃ for 10 min; and preserving at 4 ℃.

PCR verification conditions of coexpression strain containing synthetase AmyMB: at 95 ℃ for 2 min; 95 ℃, 30s, Tm53 ℃, 40s,72 ℃, 2min, 32 cycles in total; 72 ℃ for 10 min; and preserving at 4 ℃.

PCR verification conditions of the coexpression strain AmyT150 containing the peptidase domain are as follows: at 95 ℃ for 2 min; 95 ℃, 30s, Tm50 ℃, 40s,72 ℃, 1min, 32 cycles in total; 72 ℃ for 10 min; and preserving at 4 ℃.

The verification result of the co-expression strain is shown in figure 1, and the PCR amplification product band is consistent with the target band, which indicates that the co-expression strain is successfully constructed.

5. Colonies of the co-expression strain were picked and inoculated into 100mL of LB liquid medium containing three antibiotics (kanamycin, spectinomycin, chloramphenicol), cultured at 37 ℃ for 12 hours, and continuously shaken at 180 rpm.

6. The medium was inoculated into 800mL of LB liquid medium in a roller bottle at an inoculum size of 5%, and cultured at 37 ℃ with continuous shaking. After culturing for 90min, OD was measured₆₀₀At a value of 0.6-0.8, the temperature of the shaker was lowered to 25 ℃ and after half an hour IPTG was added to a final concentration of 0.2mM/mL on a clean bench.

7. After culturing at 25 ℃ and 180rpm with continuous shaking for 24 hours, the cells were collected by centrifugation at 8000rpm and 4 ℃ for 20 min. The thalli is deposited and stored at the temperature of 20 ℃ below zero for standby.

Example 3

Purification and identification of lantibiotide AmylocinC

1. The cells obtained in example 2 were extracted with methanol for 4 hours, and the methanol extract was collected, filtered through a 0.22 μm organic filter and then purified by semi-preparative high performance liquid chromatography (HPLC: SHIMADZU LC-20A, Japan).

The C18 column model is Shim-pack VP-ODS, the inner diameter is 20mm, the column length is 250mm, the packing medium is C18, and the detection wavelength range is (190-.

The mobile phase consists of a liquid A and a liquid B;

solution A: from H₂O and trifluoroacetic acid, wherein the volume percentage content of the trifluoroacetic acid in the solution A is 0.1 percent;

and B, liquid B: consists of acetonitrile and trifluoroacetic acid, wherein the volume percentage content of the trifluoroacetic acid in the solution B is 0.1 percent;

the elution conditions were: performing linear gradient elution (0-13 min) on 10% -35% acetonitrile; performing 35-100% acetonitrile linear gradient elution (13-43 min); isocratic elution with 100% acetonitrile (43-53 min); performing linear gradient elution (53-63 min) on 100% -10% acetonitrile; isocratic elution with 10% acetonitrile (63-78 min); the sample loading was 5000. mu.L, the flow rate was 5mL/min, the detection wavelength was 214nm, and the column temperature was 30 ℃.

The high performance liquid purification map of the heterologous expression product of the co-expression strain 3BT is shown in figure 2, wherein the chromatographic peak marked by an asterisk in the map is amylocin C, and the peak-off time is 23 min.

2. Mass spectrometry was performed on the purified AmylocinC, and the results are shown in FIG. 3.

Identifying the AmylocinC structure by QTOF MS/MS: on the basis of the primary mass spectrum, a parent ion with the corresponding molecular weight of 3053.51Da is selected for secondary tandem mass spectrum analysis, and the result of the tandem mass spectrum analysis is shown in FIG. 3.

The structure of lanthionine AmylocinC is schematically shown in FIG. 4, in which threonine at positions 2, 7, 9 and 10 is dehydrated to form dehydrobuthionine, serine at position 16 is dehydrated to form a thioether ring with cysteine at position 20, and serine at position 27 is dehydrated to form a thioether ring with cysteine at position 32.

The chemical structural formula of the lantibiotic AmylocinC is shown in figure 5.

Example 5: determination of biological activity of lantibiotide AmylocinC

The plate confrontation experiment is carried out by taking staphylococcus aureus, klebsiella pneumoniae, bacillus licheniformis, bacillus subtilis, sphingomonas, escherichia coli and pseudomonas fluorescens as indicator bacteria. The prepared bacterial suspension is poured into a preheated and melted solid LB culture medium and evenly mixed, and then the mixture is placed on plates, and each plate is poured with 30mL in a fixed amount. After the plate solidified, a punch with a diameter of 8mm was used to punch the holes. 100uL of separation component is added into each hole, and the separation component is finally placed in a constant temperature incubator at 37 ℃ to be cultured for 1 day, and then the result is observed, the solvent (acetonitrile) with the same volume is used as a contrast, the experimental result is shown in table 1, and the result shows that the AmylocinC has stronger bacteriostatic activity on gram-positive bacteria such as staphylococcus aureus, klebsiella pneumoniae, bacillus licheniformis, bacillus subtilis and the like.

TABLE 1 determination of lantibiotic AmylocinC bioactivity

The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.

Sequence listing

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<210> 6

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

taatacgact cactataggg 20

<210> 7

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

tagtcgttac atggactgac 20

<210> 8

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

cctgggtgat atctatgaaa actaa 25

<210> 9

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

ttaatccagt gccagcggtg tcg 23

<210> 10

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

caccatcatc accacctg 18

<210> 11

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

ttaggcggtt ttatctttgc gttcc 25