CN112851788B - Lanthionine LichencinA3, and preparation method and application thereof - Google Patents
Lanthionine LichencinA3, and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a lantibiotide LichencinA3, a preparation method and application thereof, belonging to the application field of peptide antibiotics; the biosynthetic gene cluster of the lantibiotic LichencinA3 is derived from the excavation of a Bacillus licheniformis public database, namely, the excavated lantibiotic precursor peptide LicA3, synthetase LicMB and peptidase structural domain LicT150 genes are co-transformed into competent cells E.coli BL21 (DE 3), a co-expression strain is constructed, and the lantibiotic LichencinA3 is obtained through a heterologous expression co-expression strain; the lanthionine LichencinA3 has a broad-spectrum bactericidal effect, has a strong inhibiting effect on gram-positive bacteria such as staphylococcus aureus and the like, and can inhibit gram-negative pathogenic bacteria such as sphingomonas and klebsiella pneumoniae.
Description
Technical Field
The invention relates to the field of application of peptide antibiotics, in particular to lantibiotide LichencinA3 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 over the world, 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.
Lanthionine 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 medicines. 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 licheniformis in synthesizing various novel lantibiotic peptides is far beyond the past cognition. The bacillus licheniformis has a plurality of biosynthetic gene clusters for coding the lanthionine antibacterial peptides, the gene clusters are usually silent in wild strains, and the production of the lanthionine antibacterial peptides by a heterologous expression method is of great significance for developing novel efficient lanthionine antibacterial peptides.
Disclosure of Invention
The invention aims to provide a lanthionine LichencinA3, a preparation method and application thereof, so as to solve the problems in the prior art.
In order to achieve the purpose, the invention provides the following scheme:
the invention provides a lanthionine LichencinA3, which has the following molecular structural formula:
the invention provides a preparation method of lantibiotide LichencinA3, which specifically comprises the following steps:
co-transforming the genes of the lantibiotic peptide precursor peptide LicA3, the synthetase LicMB and the peptidase structural domain LicT150 into competent cells, constructing a co-expression strain, and purifying the co-expression strain after heterologous expression to obtain the lantibiotic peptide LichencinA3.
Further, the nucleotide sequence of the gene fragment of the lanthionine precursor peptide LicA3 in the step (1) is shown in SEQ ID NO. 2.
Further, the amino acid sequence of the lantibiotic peptide precursor peptide LicA3 in the step (1) is shown as SEQ ID NO: 1.
Further, the nucleotide sequence of the synthetase LicMB gene segment in the step (1) is shown as SEQ ID NO. 3.
Further, the nucleotide sequence of the peptidase domain LicT150 gene fragment in the step (1) is shown as SEQ ID NO. 4.
Further, the specific method for purification is semi-preparative high performance liquid chromatography, and the lantibiotic LichencinA3 elutes out of the peak at 44 min.
The invention also provides application of the lantibiotic LichencinA3 in preparation of an antibacterial preparation.
The invention also provides a pharmaceutical composition which takes the lanthionine LichencinA3 or pharmaceutically acceptable salts thereof as an active ingredient.
The invention also provides a framework amino acid sequence of the lanthionine LichencinA3, wherein the framework amino acid sequence is shown in SEQ ID NO. 11.
The invention discloses the following technical effects:
the novel lantibiotide LichencinA3 is identified and synthesized from bacillus licheniformis, a novel substitute drug is provided for the treatment of multi-drug resistant bacteria, and technical support is provided for heterologous biosynthesis of the lantibiotide. The novel lantibide LichencinA3 has broad-spectrum bactericidal action, has strong inhibition effect on gram-positive bacteria such as staphylococcus aureus and the like, and can inhibit gram-negative pathogenic bacteria such as sphingomonas and klebsiella pneumoniae.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed to be used 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 inventive exercise.
FIG. 1 shows the results of the verification of PCR amplification products of co-expressing strains;
FIG. 2 is a diagram of high performance liquid purification of lantibiotide LichencinA3;
FIG. 3 is a mass spectrum of lanthionine LichencinA3;
FIG. 4 is a schematic structural diagram of lanthionine LichencinA3;
FIG. 5 is a chemical structural formula of lanthionine LichencinA3;
FIG. 6 shows the bacteriostatic activity of lanthionine LichencinA3.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.
In order to make the aforementioned objects, features and advantages of the present invention more comprehensible, the present invention is described in detail with reference to the accompanying drawings and the detailed description thereof.
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;
the culture medium comprises the following specific components: 10 g 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.
Coli BL21 (DE 3) was purchased from TransGen Biotech, beijing Kogyo gold Biotech. And (3) indication bacteria: staphylococcus aureus, klebsiella pneumoniae and Sphingomonas bacteria were deposited by the institute of sciences, hebei province. pET-28a, pCDFDuet-1 and PACYCDuet-1 vectors were purchased from Tiannze Biotechnology 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 sympatholiness. Org /);
BAGEL 3 (http://bagel.molgenrug.nl/)；
Morpheus (https://software.broadinstitute.org/morpheus/)；
the local software comprises: editSeq software, primer 5.0 software, megAlign software.
Example 1: source and screening process of lanthionine LichencinA3
1. Inputting 'Bacillus licheniformis' under 'Genome' entry in NCBI database to search basic information of all strains of Bacillus licheniformis strain and obtain NCBI accession numbers of all strains.
2. Through mining 18 strains of completely sequenced bacillus licheniformis in the genome by bioinformatics software anti SMASH 4.0 and BAGEL 3, 107 lanthionine precursor peptides coexist, and all belong to the second type of lanthionine. Through the sequence comparison of the gene clusters of the excavated 18 lantibiotide precursor peptides with the gene clusters in the database by local Blast, the existence of a novel lantibiotide LicA3 (the amino acid sequence is shown as SEQ ID NO: 1) in the Bacillus licheniformis SCDB34 (the source of strain information: https:// www.ncbi.nlm.nih.gov/bioproject/PRJNA 290971), namely the existence of a biosynthetic gene cluster for synthesizing the lantibide LichencinA3, is discovered.
Example 2: construction of Gene expression vectors
The nucleotide sequences of the lanthionine precursor peptide LicA3, the synthetase LicMB and the peptidase domain LicT150 gene excavated in example 1 were delivered to NovoPro and synthesized. And a lanthionine precursor peptide LicA3 gene fragment (shown as SEQ ID NO: 2) is constructed on a pET28a vector, a synthetase LicMB gene fragment (shown as SEQ ID NO: 3) is constructed on a pCDFDuet-1 vector, and a peptidase domain LicT150 gene fragment (shown as SEQ ID NO: 4) is constructed on a pACYCDuet-1 vector.
Example 3: construction of Co-expression Strain and heterologous expression
1. The three recombinant vectors containing the lanthionine precursor peptide LicA3, the synthetase LicMB, and the peptidase domain LicT150 gene in example 2 were co-transformed into competent cells E.coli BL21 (DE 3) by heat shock transformation to construct a co-expression strain.
2. An LB plate (kanamycin, spectinomycin and chloramphenicol) containing three antibiotics is adopted to screen out positive co-expression strains.
3. And identifying positive colonies of the co-expression strain by adopting a colony PCR method.
The primers used for PCR (shown as SEQ ID NO:5 to SEQ ID NO:
LicA3 F1：CACATCGTGAAATGGCTG
LicA3 R1：ATGCGTTAGACGGACACC
LicMB F1：CCATCATCACCACACCGT
LicMB R1：CGCCAGCCATAGGTTTTT
LicT150 F1：CACCATCATCACCACCTG
LicT150 R1：GCTTTTATTTGCGGCTAC
the PCR reaction system is as follows:
1.5 μ L of the upstream and downstream primers, 1 μ L of template DNA,5 μ L of 10 XBuffer, 5 μ L of 2 mM dNTPs,2 μ L of 25 mM MgSO4，ddH2O is added to 50 mu L of constant volume;
PCR verification conditions of the LicA3 co-expression strain containing the precursor peptide: 95. 2 min at the temperature of; 95. at the temperature of 30 s; tm is 55 ℃ and 40 s; 72. 30 s at the temperature; 72. 10 min at the temperature; 4. preserving at the temperature of.
PCR verification conditions of coexpression strain containing synthetase LicMB: 95. 2 min at the temperature; 95. at the temperature of 30 s; tm is 53 ℃ and 40 s; 72. 2 min at the temperature; 72. 10 min at the temperature; 4. preserving at the temperature of.
PCR verification conditions of the co-expression strain LicT150 containing the peptidase domain are as follows: 95. 2 min at the temperature of; 95. 30 s at the temperature; tm is 50 ℃ and 40 s; 72. 1 min at the temperature; 72. 10 min at the temperature; 4. preserving at the temperature of.
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.
4. Colonies of the co-expression strains were picked and inoculated into 100 mL of LB liquid medium containing three antibiotics (kanamycin, spectinomycin, chloramphenicol), cultured at 37 ℃ for 12 hours, and continuously shaken at 180 rpm.
5. The medium was inoculated into 800 mL of LB liquid medium in a roller bottle at an inoculum size of 5%, and cultured at 37 ℃ with continuous shaking. After 90 min of incubation, 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 0.2 mM/mL on a clean bench.
6. After culturing at 25 ℃ and 180 rpm with continuous shaking for 24 hours, the cells were centrifuged at 8000 rpm and 4 ℃ for 20 minutes, and the cells were collected. The thalli is deposited and stored at the temperature of 20 ℃ below zero for standby.
Example 4: purification and characterization of lantibiotide LichencinA3
1. Extracting the above thallus with methanol for 4 hr, collecting methanol extract, filtering with 0.22 μm organic filter membrane, and separating and purifying by semi-preparative high performance liquid chromatography (HPLC: SHIMADZU LC-20A, japan).
The model of the C18 column is Shim-pack VP-ODS, the inner diameter is 20 mm, the column length is 250 mm, the packing medium is C18, and the detection wavelength range is (190-800 nm).
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; 35 to 100 percent acetonitrile linear gradient elution (13 to 43 min); isocratic elution with 100% acetonitrile (43-53 min); performing linear gradient elution with 100-10% acetonitrile (53-63 min); isocratic elution with 10% acetonitrile (63-78 min); the sample loading was 5000. Mu.L, the flow rate was 5 mL/min, the detection wavelength was 214 nm, and the column temperature was 30 ℃.
The high performance liquid purification map of the heterologous expression product of the co-expression strain is shown in figure 2, wherein the chromatographic peak marked by asterisk in the map is LichencinA3, and the peak-off time is 44 min.
2. Carrying out mass spectrum identification on the purified LichencinA3
Identifying the structure of LichencinA3 by QTOF MS/MS: on the basis of the primary mass spectrum, a parent ion with the corresponding molecular weight of 3021.31Da is selected for secondary tandem mass spectrometry, and the tandem mass spectrometry result is shown in FIG. 3.
The structural schematic diagram of the lanthionine LichencinA3 is shown in FIG. 4, the framework sequence of the lanthionine AmylocinA3 is shown in SEQ ID NO:11, threonine at positions 6, 7, 15 and 18 is dehydrated to form dehydrobuthionine, serine at position 9 is dehydrated to form a thioether ring with cysteine at position 13, and serine at position 24 is dehydrated to form a thioether ring with cysteine at position 31. The chemical structural formula of the lantibiotide LichencinA3 is shown in figure 5.
Example 5: determination of bacteriostatic activity of lanthionine LichencinA3
The antibacterial activity of the lantibiotide LichencinA3 is determined by adopting a plate confrontation method.
1. Using LB liquid culture medium to culture the indicator strains of staphylococcus aureus, klebsiella pneumoniae and sphingomonas in a suspension manner to obtain OD600A value of 0.3.
2. And (2) respectively taking 100 mu L of the indicator bacterium suspension obtained in the step (1), uniformly mixing the indicator bacterium suspension with 30mL of LB solid medium in a 50 ℃ molten state, paving a flat plate, solidifying the flat plate, and perforating by using a sterilized perforator with the diameter of 8 mm.
3. mu.L of prepared LichencinA3 of 10. Mu.g/mL was added to the wells, the same volume of solvent was used as a negative control, and the wells were finally incubated in a 37 ℃ incubator. The results are shown in fig. 6, and indicate that the lantibiotic LichencinA3 has higher bacteriostatic activity against common gram bacteria such as staphylococcus aureus, klebsiella pneumoniae and sphingomonas.
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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catctgaaaa agctggcaga acagctgaac tttgatacca aaagctataa agcagatagc 240
cgtcagctgg gcaccctgat tctgcctgca attctgtatt gggaaaacaa ccattttgtg 300
atcctggaaa aagttgcaca gcaggcatat accattgttg atccaggtag cggtcgtcgt 360
aaactgaaag aaaaagaatt caccgagaaa tacagcggtt atgtgctgac cctgtatccg 420
aataaaaact ttgaacgtcg tagccgcaaa taa 453
<210> 5
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
<210> 6
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
<210> 7
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
<210> 8
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
<210> 9
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
caccatcatc accacctg 18
<210> 10
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
<210> 11
<211> 31
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 11
Asp Val Thr Pro His Thr Thr Pro Ser Ser Leu Pro Cys Gly Thr Leu
1 5 10 15
Val Thr Ala Val Trp Cys Pro Ser Asn Ala Cys Thr Ser Asp Cys
20 25 30
Claims (5)
1. A lantibiotide LichencinA a3, comprising: the molecular structural formula is as follows:
2. a process for the preparation of the lanthionine LichencinA3 according to claim 1, characterized in that: the method specifically comprises the following steps:
co-transforming lanthionine precursor peptide LicA3, synthetase LicMB and peptidase structural domain LicT150 genes into competent cells E.coli BL21 (DE 3), constructing a co-expression strain, and purifying the co-expression strain after heterologous expression to obtain the lanthionine LichencinA3;
the nucleotide sequence of the gene fragment of the lantibiotic peptide precursor peptide LicA3 is shown in SEQ ID NO. 2;
the amino acid sequence of the lantibiotic peptide precursor peptide LicA3 is shown as SEQ ID NO. 1;
the nucleotide sequence of the synthetase LicMB gene segment is shown as SEQ ID NO. 3;
the nucleotide sequence of the peptidase domain LicT150 gene fragment is shown in SEQ ID NO. 4.
3. The method of claim 2, wherein the specific method of purification is semi-preparative high performance liquid chromatography, and the lantibiotic LichencinA3 elutes at 44 min.
4. Use of the lanthionine LichencinA3 according to claim 1 for the preparation of an antibacterial formulation; wherein the bacteria are staphylococcus aureus, klebsiella pneumoniae and nitroammoniol monads.
5. A pharmaceutical composition comprising the lanthionine LichencinA a3 or a pharmaceutically acceptable salt thereof according to claim 1 as an active ingredient.
Priority Applications (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110147380.1A CN112851788B (en) | 2021-02-03 | 2021-02-03 | Lanthionine LichencinA3, and preparation method and application thereof |
| LU500785A LU500785B1 (en) | 2021-02-03 | 2021-05-07 | Lanthipeptide LichencinA3 and Preparation Method and Application thereof |
| PCT/CN2021/092047 WO2022166014A1 (en) | 2021-02-03 | 2021-05-07 | Lanthipeptide lichencina3, and preparation method therefor and application thereof |
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| CN202110147380.1A CN112851788B (en) | 2021-02-03 | 2021-02-03 | Lanthionine LichencinA3, and preparation method and application thereof |
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| CN112851788A CN112851788A (en) | 2021-05-28 |
| CN112851788B true CN112851788B (en) | 2022-11-01 |
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| Country | Link |
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| CN (1) | CN112851788B (en) |
| LU (1) | LU500785B1 (en) |
| WO (1) | WO2022166014A1 (en) |
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| US7255855B2 (en) * | 2004-02-27 | 2007-08-14 | Bioleaders Corporation | Surface expression method of peptides P5 and Anal3 using the gene encoding poly-gamma-glutamate synthetase |
| RU2697515C2 (en) * | 2014-03-25 | 2019-08-15 | Лантхиопеп Б.В. | Cyclic analogues of apelin |
| CN107109403A (en) * | 2014-12-22 | 2017-08-29 | 莫佛塞斯公司 | The new method of cyclic peptide is shown on phage particle |
| CN106366168B (en) * | 2016-08-26 | 2020-09-15 | 上海交通大学 | Preparation method of lanthionine antibacterial peptide and dehydrogenated derivative thereof |
| CN112239490B (en) * | 2019-07-16 | 2023-05-02 | 武汉合生科技有限公司 | Method for screening lanthionin, cell-free protein synthesis system and lanthionin |
| CN110669114B (en) * | 2019-10-21 | 2022-03-29 | 河北省科学院生物研究所 | Lanthionine precursor peptide amyA6, and preparation method and application thereof |
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2021
- 2021-02-03 CN CN202110147380.1A patent/CN112851788B/en active Active
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| CN112851788A (en) | 2021-05-28 |
| WO2022166014A1 (en) | 2022-08-11 |
| LU500785B1 (en) | 2022-04-27 |
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