Lichencin A3-27L and a preparation method and application thereof

Abstract

The application discloses a lantipeptide Lichencin A3-27L and a preparation method and application thereof, and belongs to the application field of peptide antibiotics. The amino acid of the lantipeptide precursor peptide LicA3-27L is obtained by mutating a cysteine at the 27th position of an amino acid sequence of a lantipeptide precursor peptide LicA3 into leucine; the lantipeptide precursor peptide LicA3-27L gene, a synthetic enzyme LicMB gene and a peptidase domain LicT150 gene are co-transformed into an E. coli competent cell to construct a co-expression strain; and the lantipeptide Lichencin A3-27L is obtained by purifying the lantipeptide after heterogenous expression of the co-expression strain. The lantipeptide Lichencin A3-27L has better water solubility and a stronger inhibiting effect on Staphylococcus aureus.

Description

TECHNICAL FIELD

[0001] The present application relates to the application field of peptide antibiotics, in particular to a lantipeptide Lichencin A3-27L and a preparation method and application thereof. BACKGROUND

[0002] Antibiotics are commonly used antibacterial agents in clinical, which have a killing effect on pathogenic bacteria. However, the problem of antibiotic abuse caused by resistance to pathogenic bacteria is becoming increasingly serious worldwide. The abuse of antibiotics has led to drug resistance in many bacteria, and the research and discovery of new anti-infective drugs have become a problem to be solved.

[0003] Lantipeptides are cyclic peptide compounds containing thioether bonds, which exist widely in nature and are the first class of ribosomal post-translational modification peptides (RiPPs) discovered. Lantipeptides have various biological activities such as antibacterial, antifungal, antiviral, and medical analgesic activities. With the rapid development of gene sequencing and the corresponding omics technology, more and more natural product ribosomes have been discovered, researched and published in recent ten years. Among them, the excellent biological antibacterial activity of streptogramin and its unique antibacterial mechanism have attracted the attention of many researchers to lantipeptides, and lantipeptides have become a new direction of anti-infective drug research.

[0004] Compared with other anti-infective drugs, lantipeptides have a unique antibacterial mechanism, which makes them less likely to cause drug resistance. At the same time, as a relatively small molecular weight peptide, lantipeptides are more easily digested and absorbed and do not have residues. According to existing research, it is found that only a small part of ribosomal peptides have been published and elucidated, and there are still many unknown lantipeptide compounds that have not been discovered. As an important part of ribosomal peptides, further development and research on lantipeptide compounds, and exploration of their relatively high evolutionary nature in structure, have important significance for the research and application of peptide antibiotics. SUMMARY

[0005] The present application aims to provide a lantipeptide Lichencin A3-27L and a preparation method and application thereof to solve the problems in the prior art. The present application screens a mutant Lichencin A3-27L with better water solubility by site-directed mutagenesis of lantipeptide Lichencin A3, and the mutant has better water solubility and stronger inhibitory effect on Staphylococcus aureus.

[0006] To achieve the above-mentioned purpose, the present application provides the following solutions.

[0007] The present application provides a lantipeptide Lichencin A3-27L, the backbone amino acid sequence of which is obtained by dehydration and formation of a thioether ring from the sequence shown in SEQ ID NO. 11. In the amino acid sequence, the threonine at positions 6, 7, 15 and 18 is dehydrated to form a dehydrobutyrine, the serine at position 9 is dehydrated to form a thioether ring with the cysteine at position 13, and the serine at position 24 is dehydrated to form a thioether ring with the cysteine at position 31.

[0008] The present application also provides a preparation method of the lantipeptide Lichencin A3-27L, comprising the following steps: co-transforming a lantipeptide precursor peptide LicA3-27L gene, a synthetase LicMB gene and a peptidase domain LicT150 gene into an Escherichia coli competent cell, constructing a co-expression strain, and obtaining the lantipeptide Lichencin A3-27L by purification after heterologous expression of the co-expression strain.

[0009] The nucleotide sequence of the lantipeptide precursor peptide LicA3-27L gene is shown in SEQ ID NO. 1; the nucleotide sequence of the synthetase LicMB gene is shown in SEQ ID NO. 3; and the nucleotide sequence of the peptidase domain LicT150 gene is shown in SEQ ID NO. 4.

[0010] Further, the amino acid sequence of the lantipeptide precursor peptide LicA3-27L is shown in SEQ ID NO. 2.

[0011] Further, the amino acid of the lantipeptide precursor peptide LicA3-27L is obtained by mutating the cysteine at position 27 of the amino acid sequence of the lantipeptide precursor peptide LicA3 to leucine.

[0012] Further, the specific method for purification is semi-preparative high performance liquid chromatography, and the lantipeptide Lichencin A3-27L elutes out a peak at 22 min.

[0013] The present application also provides an application of the lantipeptide Lichencin A3-27L in preparing an anti-Gram-positive bacterial product, wherein the Gram-positive bacteria are one or more of Staphylococcus aureus, Klebsiella pneumoniae and sphingomonas.

[0014] The present application also provides a pharmaceutical composition, wherein the lantipeptide Lichencin A3-27L is used as an effective ingredient.

[0015] Further, the pharmaceutical composition further comprises a pharmaceutically acceptable salt thereof.

[0016] The present application discloses the following technical effects:

[0017] The present application takes the amino acid sequence of lantipeptide Lichencin A3 as a template, designs a mutant amino acid sequence, obtains different mutants, and screens a site-directed mutant Lichencin A3-27L of a new lantipeptide according to water solubility of the mutants, thereby providing a new alternative drug for treatment of multi-drug resistant bacteria and technical support for biosynthesis of lantipeptides by heterologous organisms. The new lantipeptide mutant Lichencin A3-27L has water solubility and a strong inhibitory effect on Staphylococcus aureus. BRIEF DESCRIPTION OF DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced as follows. Obviously, the drawings in the following description only constitute some embodiments of the present application, and other drawings can be obtained by those skilled in the art without any creative effort on the basis of these drawings.

[0019] Figure 1 PCR amplification product verification results of the co-expression strain;

[0020] Figure 2 The figure is a high-performance liquid chromatography (HPLC) spectrum of lantipeptide Lichencin A3-27L, in which black represents precipitate extract liquid containing Lichencin A3-27L, magenta represents Lichencin A3, and blue represents a methanol control.

[0021] Figure 3 The figure is a secondary mass spectrum of lantipeptide Lichencin A3-27L.

[0022] Figure 4 The figure is an amino acid structure diagram of lantipeptide Lichencin A3-27L.

[0023] Figure 5 The figure is a water-solubility antibacterial experiment of lantipeptide Lichencin A3-27L, in which 1 is a methanol control, 2 is supernatant after mixing lantipeptide Lichencin A3-27L with water, 3 is supernatant after mixing lantipeptide Lichencin A3 with water, 4 is precipitate extract liquid containing Lichencin A3-27L, and 5 is precipitate extract liquid containing Lichencin A3-27L. DETAILED DESCRIPTION

[0024] The various exemplary embodiments of the present application will now be described in detail, which should not be considered as limiting the present application, but should be understood as a more detailed description of certain aspects, characteristics and embodiments of the present application.

[0025] 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 present application. In addition, where particular ranges of values are given, understand that each intervening value, to the upper or lower limit of the ranges is also specifically included. Each smaller range that falls within the broader ranges is also specifically included in the present application. The upper and lower limits of these smaller ranges can independently be included or excluded in the smaller ranges.

[0026] 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 application belongs. Although 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 application. All documents mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the documents are cited. In case of conflict between the present specification and any document incorporated by reference, the present specification will control.

[0027] Various modifications and changes can be made to the specific embodiments described herein without departing from the scope or spirit of the application. Other embodiments will be apparent to those of ordinary skill in the art from consideration of the description and practice of the application. The description and examples are illustrative only.

[0028] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", "contains", "containing", or variations thereof, are intended to be open-ended terms that mean including, but not limited to.

[0029] Unless otherwise indicated, the materials, reagents and the like used in the present application are commercially available from commercial sources.

[0030] LB medium: for the culture of E. coli;

[0031] Medium specific composition: Tryptone 10 g, NaCl 5 g, yeast extract 5 g, add deionized water to 1000 mL, adjust pH to 7.0-7.2.

[0032] E. coli BL21 (DE3) was purchased from Beijing TransGen Biotech. The indicator bacteria: Staphylococcus aureus, Klebsiella pneumoniae and sphingomonas were preserved by Hebei Academy of Sciences. pET-22b, pCDFDuet-1 and PACYCDuet-1 vectors were purchased from TIANDZ.

[0033] Bioinformatics tools used in the following examples:

[0034] Local software: EditSeq software, Primer 5.0 software, MegAlign software.

[0035] Example 1 Screening of mutant of lantipeptide Lichencin A3

[0036] The precursor peptide LicA3 of lantipeptide Lichencin A3 is composed of a 40-amino-acid leader peptide and a 31-amino-acid core peptide. In the precursor peptide LicA3, each amino acid in the core peptide was mutated into other 19 common amino acids by using site-directed saturation mutation, and degenerate bases were used to replace the codons of the mutated amino acids. According to the analysis of nucleotide sequence and degenerate bases, the mutation efficiency is the highest when the mutation codon is NNK. Finally, about 45% of nucleic acid mutants can be obtained by using NNK as the mutation codon. The specific preparation method is as follows:

[0037] The 27th cysteine in the amino acid sequence of the precursor peptide LicA3 was mutated into leucine to obtain mutant LicA3-27L, which was then translated into a nucleotide sequence by using biological software. The nucleotide sequence of the precursor peptide LicA3-27L gene fragment obtained by PCR site-directed mutation technology is shown in SEQ ID NO. 1, and the amino acid sequence of the precursor peptide LicA3-27L is shown in SEQ ID NO. 2.

[0038] SEQ ID NO. 1:

[0039] catatgtctcaccgtgaaatggctgccatctaccgtgacgcaaacaaacgcgctaatctggaattcagcaacccggttggcgaagtgaacgaagaagagctgaaaaacctggcaggcgcggccgacgttaccccgcacaccactccgagctctctgccgtgcggtaccctggttactgcagtttggtgcccgtctaacgccctgactagcgattgttaactcgag.

[0040] SEQ ID NO. 2:

[0041] MSHREMAAIYRDANKRANLEFSNPVGEVNEEELKNLAGAADVTPHTTPSSLPCGTLVTAVWCPSNALTSDC.

[0042] Example 2 Construction of gene expression vector

[0043] The gene sequence of the lanthipeptide synthetase LicMB (as shown in SEQ ID NO. 3) and the gene sequence of the lanthipeptide peptidase domain LicT150 (as shown in SEQ ID NO. 4) were sent to NovoPro Company for synthesis. The precursor peptide LicA3-27L gene fragment (as shown in SEQ ID NO. 1) obtained after PCR mutation in Example 1 was ligated between the Ndel gene and the Xhol gene of the pET-22b plasmid, the gene fragment of the synthetase LicMB was ligated to the pCDFDuet-1 vector, and the gene fragment of the peptidase domain LicT150 was ligated to the PACYCDuet-1 vector.

[0044] SEQ ID NO. 3:

[0045]

[0046] SEQ ID NO.4:

[0047] atgctgaaaaagaaaaaactgtttccgacacgtcgtcgtgttccgtttattgagcagatgcagcagaccgaatgtgcactgtgttgtattgcaatgattagcagctactacaaaaacgacctgagcatgtatgaagttcgtgaacgtatgggtaatggtcgtgatggtacaaccctgtttcatctgaaaaagctggcagaacagctgaactttgataccaaaagctataaagcagatagccgtcagctgggcaccctgattctgcctgcaattctgtattgggaaaacaaccattttgtgatcctggaaaaagttgcacagcaggcatataccattgttgatccaggtagcggtcgtcgtaaactgaaagaaaaagaattcaccgagaaatacagcggttatgtgctgaccctgtatccgaataaaaactttgaacgtcgtagccgcaaataa.

[0048] Example 3 Construction of co-expression strain and heterologous expression

[0049] 1. The three recombinant vectors containing the genes of lanthipeptide precursor peptide LicA3-27L, synthetase LicMB and peptidase domain LicT150 in the above-mentioned Example 2 were co-transformed into competent cells E. coli BL21 (DE3) by heat shock transformation method to construct a co-expression strain.

[0050] 2. The positive co-expression strain was screened out by using LB plate containing three kinds of antibiotics (kanamycin, spectinomycin, chloramphenicol).

[0051] 3. The positive colonies of the co-expression strain were identified by colony PCR method.

[0052] The primers used in PCR (as shown in SEQ ID NO. 5-10) are as follows:

[0053] LicA3-27L F1: gattacggacgnystcgctaacagag;

[0054] LicA3-27L R1: gagacaatcgctsyngcaggcattag;

[0055] LicMB F1: atgaccgtggcaaaaatgaaagaca;

[0056] LicMB R1: ttaacaaactgcaccacctttcggt;

[0057] LicT150 F1: atgctgaaaaagaaaaaactg;

[0058] LicT150 R1: ttatttgcggctacgacgttcaaag.

[0059] PCR reaction system is as follows:

[0060] 1.5 μL of upstream and downstream primers, 1 μL of template DNA, 5 μL of 10× Buffer, 5 μL of 2Mm dNTPs, 2 μL of 25 mM MgSO4, and ddH2O to 50 μL.

[0061] The PCR verification condition of the precursor peptide LicA3-27L-containing co-expression strain is as follows: 95°C, 2 min; 95°C, 30 s; Tm 55°C, 40 s; 72°C, 30 s; 72°C, 10 min; 4°C, preservation.

[0062] The PCR verification condition of the synthetic enzyme LicMB-containing co-expression strain is as follows: 95°C, 2 min; 95°C, 30 s; Tm 53°C, 40 s; 72°C, 2 min; 72°C, 10 min; 4°C, preservation.

[0063] The PCR verification condition of the peptide enzyme domain LicT150-containing co-expression strain is as follows: 95°C, 2 min; 95°C, 30 s; Tm 50°C, 40 s; 72°C, 1 min; 72°C, 10 min; 4°C, preservation.

[0064] The verification results of the co-expression strain are shown in Table 1, and the PCR amplification product bands are consistent with the target bands, indicating that the co-expression strain is successfully constructed. Figure 1

[0065] 4. The co-expression strain colonies were picked and inoculated in 100 mL of LB liquid medium containing three antibiotics (kanamycin, spectinomycin, and chloramphenicol), and cultured at 37°C for 12 h with continuous shaking at 180 rpm.

[0066] 5. The flask was inoculated with 5% inoculation amount into 800 mL of LB liquid medium, and continuously shaken and cultured at 37°C. After 90 min of culture, the OD 600 ​When the value is 0.6-0.8, the temperature of the shaker is reduced to 25°C, and after half an hour, IPTG is added to the final concentration of 0.2 mM on the clean bench.

[0067] 6. After 24 h of continuous shaking at 25°C and 180 rpm, the bacteria are collected by centrifugation at 8000 rpm and 4°C for 20 min. The bacterial pellet is stored at -20°C for later use.

[0068] Example 4 Purification and identification of lantipeptide Lichencin A3-27L

[0069] 1. After the methanol extraction of the bacteria collected in Example 3 for 4 h, the methanol extraction phase is collected, filtered through a 0.22 μm organic filter membrane, and then separated and purified by semi-preparative high-performance liquid chromatography (HPLC: SHIMADZU LC-20A, Japan).

[0070] The C18 column is Shim-pack VP-ODS, with an inner diameter of 20 mm, a column length of 250 mm, and a C18 filling medium. The detection wavelength range is 190-800 nm.

[0071] The mobile phase is composed of A liquid and B liquid.

[0072] The A liquid is composed of H2O and trifluoroacetic acid, with a volume percentage of 0.1% trifluoroacetic acid in the A liquid.

[0073] The B liquid is composed of acetonitrile and trifluoroacetic acid, with a volume percentage of 0.1% trifluoroacetic acid in the B liquid.

[0074] The elution conditions are as follows: 10%-35% acetonitrile linear gradient elution (0-13 min); 35%-100% acetonitrile linear gradient elution (13-43 min); 100% acetonitrile isocratic elution (43-53 min); 100%-10% acetonitrile linear gradient elution (53-63 min); 10% acetonitrile isocratic elution (63-78 min); the sample volume is 5000 μL, the flow rate is 5 mL / min, the detection wavelength is 214 nm, and the column temperature is 30°C.

[0075] The high-performance liquid chromatography purification map of the heterologous expression product of the co-expression strain is shown in Figure 2 The chromatographic peak marked with a red box in the figure is Lichencin A3-27L, with an elution time of 22 min.

[0076] Compared with methanol and Lichencin A3, it can be found that the bacterial pellet extraction liquid containing Lichencin A3-27L has a different peak at a retention time of 22 min. Similar to the specific difference peak of Lichencin A3 at 26.5 min, it is further determined by mass spectrometry.

[0077] 2. Mass spectrometry identification of purified Lichencin A3-27L

[0078] QTOF MS / MS was used to identify the structure of Lichencin A3-27L: Based on the primary mass spectrum, the parent ion corresponding to the molecular weight of 2913.36 Da was selected for secondary tandem mass spectrometry analysis, and the tandem mass spectrometry analysis results are shown in Figure 3

[0079] Combined with secondary mass spectrometry and the unique structure of lanthipeptides, it can be determined that the cleavage site of the new lanthipeptide Lichencin A3-27L is the 42nd site of the core peptide, and 8 water molecules are removed during the modification of the core peptide to form a cyclic structure of 2 Lan or MeLan. The backbone amino acid sequence of the amino acid sequence of the lanthipeptide Lichencin A3-27L is DVTPHTTPSSLPCGTLVTAVWCPSNALTSDC (SEQ ID NO. 11), as shown in Figure 3 The cleavage site valine (V) is counted as the 1st site, and the dehydrated sites are threonine (T) at sites 5, 6, 13, and 17, which are dehydrated to form dehydrobutyrine; the cyclization sites are serine (S) at site 8 and cysteine (C) at site 12, and serine (S) at site 23 and cysteine (C) at site 30 are cyclized. In the process of translation modification, the precursor peptide is recognized by the peptidase domain and combined at the 42nd site of the precursor peptide, and the leader peptide is hydrolyzed, leaving a mature core peptide with 30 amino acids. The schematic diagram of the amino acid structure of the lanthipeptide Lichencin A3-27L is shown in Figure 4

[0080] Example 5: Bacteriostatic activity determination of lanthipeptide Lichencin A3-27L

[0081] The bacteriostatic activity of lanthipeptide Lichencin A3-27L was determined by the flat plate confrontation method.

[0082] 1. The indicator strains Staphylococcus aureus, Klebsiella pneumoniae and Sphingomonas were suspended in LB liquid medium to obtain a bacterial suspension with an OD 600 value of 0.3.

[0083] 2. 100 μL of the bacterial suspension obtained in step (1) was mixed with 30 mL of LB solid medium melted at 50°C, and then the mixture was evenly spread on the plate. After the plate was solidified, a sterile 8 mm puncher was used to punch holes.

[0084] ​​3, respectively, in the hole to add 100 μL prepared 10 μg / mL of LichencinA3-27L and LichencinA3, the same volume of solvent as a negative control, and finally placed in a 37°C incubator. The results are shown in Table 1, the results showed that the lantibiotic mutant LichencinA3-27L compared with lantibiotic LichencinA3 on Staphylococcus aureus, Klebsiella pneumoniae and Sphingomonas have a stronger inhibitory effect.

[0085] Table 1 Lantibiotic LichencinA3-27L and LichencinA3 bacteriostatic effect comparison

[0086]

[0087] Example 6 Lantibiotic LichencinA3-27L water-soluble study

[0088] The fermentation broth containing LichencinA3-27L and LichencinA3 were centrifuged at low temperature, the supernatant was collected, macroporous resin was added according to 1:10, gradient adsorption and elution, the collected final eluent was concentrated by rotary evaporation and freeze-dried. Each 0.1 g of the above freeze-dried powder was added to 1 mL of water, and then the supernatant and the bacterial precipitate extract containing LichencinA3-27L and LichencinA3 were taken for plate confrontation experiment, and the same volume of methanol solution was used as a negative control. The indicator bacteria were Staphylococcus aureus.

[0089] The results are shown in Table 1, the results showed that the lantibiotic mutant LichencinA3-27L compared with lantibiotic LichencinA3 on Staphylococcus aureus, Klebsiella pneumoniae and Sphingomonas have a stronger inhibitory effect. Figure 5 The results are shown in Table 1, the results showed that the lantibiotic mutant LichencinA3-27L compared with lantibiotic LichencinA3 on Staphylococcus aureus, Klebsiella pneumoniae and Sphingomonas have a stronger inhibitory effect.

[0090] The above described embodiments are only to illustrate the preferred modes of the present application, and are not intended to limit the scope of the present application. Any modification and improvement made by those skilled in the art to the technical solutions of the present application without departing from the design spirit of the present application shall fall within the protection scope of the present application as defined by the claims.

Claims

1. A lantipeptide Lichencin A3-27L, characterized by, The lantipeptide Lichencin A3-27L amino acid sequence is obtained by dehydration and formation of a thioether ring from the sequence shown in SEQ ID NO. 11, wherein the threonine at positions 6, 7, 15 and 18 is dehydrated to form a dehydrobutyrine, the serine at position 9 is dehydrated to form a thioether ring with the cysteine at position 13, and the serine at position 24 is dehydrated to form a thioether ring with the cysteine at position 31.

2. A process for the preparation of the lanthipeptide Lichencin A3-27L as claimed in claim 1, characterized by, The method comprises the following steps: co-transforming a lantipeptide precursor peptide LicA3-27L gene, a synthetase LicMB gene and a peptidase domain LicT150 gene into an Escherichia coli competent cell, constructing a co-expression strain, and obtaining the lantipeptide Lichencin A3-27L by purifying the lantipeptide after heterologous expression by the co-expression strain. The nucleotide sequence of the lantipeptide precursor peptide LicA3-27L gene is shown in SEQ ID NO. 1; the nucleotide sequence of the synthetase LicMB gene is shown in SEQ ID NO. 3; and the nucleotide sequence of the peptidase domain LicT150 gene is shown in SEQ ID NO.

4.

3. The production method according to claim 2, characterized by, The amino acid sequence of the lantipeptide precursor peptide LicA3-27L is shown in SEQ ID NO.

2.

4. The production method according to claim 2, characterized by, The specific method for purification is semi-preparative high performance liquid chromatography, and the lantipeptide Lichencin A3-27L elutes out a peak at 22 min.

5. Use of the lanthipeptide Lichencin A3-27L as claimed in claim 1 for the preparation of a product against Gram-positive bacteria, characterized by the fact that, The gram-positive bacteria are one or more of Staphylococcus aureus, Klebsiella pneumoniae and sphingomonas.

6. A pharmaceutical composition, characterized by, The pharmaceutical composition comprises the lantipeptide Lichencin A3-27L as an effective component.

7. The pharmaceutical composition of claim 6, wherein, The pharmaceutical composition further comprises a pharmaceutically acceptable salt thereof.

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