Lichencin A3-54T and a preparation method and application thereof

Abstract

This invention discloses a lanolin-thiopeptide, LichencinA3-54T, its preparation method, and its applications, belonging to the field of peptide antibiotics. The amino acid sequence of the lanolin-thiopeptide, LichencinA3-54T, is obtained by dehydrating the backbone amino acid sequence shown in SEQ ID NO.11 and forming a thioether ring. Specifically, threonines at positions 6, 7, 15, and 18 of the backbone amino acid sequence are dehydrated to form dehydrobutyric acid; serine at position 9 is dehydrated and forms a thioether ring with cysteine ​​at position 13; and serine at position 24 is dehydrated and forms a thioether ring with cysteine ​​at position 31, thus obtaining the lanolin-thiopeptide, LichencinA3-54T. This invention also screens for a mutant, LichencinA3-54T, with superior antibacterial activity through site-directed mutagenesis of lanolin-thiopeptide LichencinA3. This mutant exhibits a stronger inhibitory effect against Staphylococcus aureus.

Description

Technical Field

[0001] This invention relates to the application of peptide antibiotics, and in particular to a lanolin-thiopeptide, LichencinA3-54T, its preparation method, and its application. Background Technology

[0002] Lanolin-thiopeptides are a class of ribosomally modified peptides (RiPPs) characterized by high activity and stability. As a natural product for addressing bacterial resistance, they have attracted widespread attention from scientists. Lanolin-thiopeptides hold promise for replacing traditional antibiotics in future production and daily life, becoming a leader among emerging antibacterial substances.

[0003] Lanthanide possesses antibacterial activity and could potentially serve as an alternative to traditional antibiotics in future production and daily life, as well as a food preservative. As early as 1928, L.A. Rogers reported a lanthanide compound, Nisin, isolated from lactic acid bacteria (Lactococcus lactis), which has been used worldwide as a food additive. Nisin produced by Lactococcus lactis is the first, and to date, the only lanthanide antibiotic commercially available as a biological food preservative and veterinary drug. Microsporin, produced by actinomycetes, is considered the most effective lanthanide antibiotic. Lanthanide also has many other potential applications, including in the chemical, pharmaceutical, agricultural, and food industries. Clinical trials have shown that lanthanide exhibits low toxicity and low immunogenicity in mammals, characteristics that make it a focus of clinical research and trials.

[0004] Therefore, further research and development of lanthanum antibiotics, and exploration of their relatively higher structural evolutionability, are of great significance to the research and application of peptide antibiotics. Summary of the Invention

[0005] The purpose of this invention is to provide a lanolin-thiopeptide, LichencinA3-54T, its preparation method, and its applications, to solve the problems existing in the prior art. This invention achieves superior antibacterial activity by performing site-directed mutagenesis on LichencinA3, resulting in a mutant, LichencinA3-54T, which exhibits stronger inhibitory effects against Staphylococcus aureus.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides a wool sulfur peptide, LichencinA3-54T, whose amino acid sequence is obtained by dehydrating the backbone amino acid sequence shown in SEQ ID NO.11 and forming a thioether ring. In the backbone amino acid sequence, threonine at positions 6, 7, 15, and 18 is dehydrated to form dehydrobutyric acid, serine at position 9 is dehydrated and forms a thioether ring with cysteine ​​at position 13, and serine at position 24 is dehydrated and forms a thioether ring with cysteine ​​at position 31, thus obtaining the wool sulfur peptide, LichencinA3-54T.

[0008] The present invention also provides a method for preparing the wool sulfur peptide LichencinA3-54T, comprising the following steps: co-transforming the wool sulfur peptide precursor peptide LicA3-54T gene, the synthase LicMB gene, and the peptidase domain LicT150 gene into Escherichia coli competent cells to construct a co-expression strain; and purifying the wool sulfur peptide LichencinA3-54T after heterologous expression by the co-expression strain.

[0009] The nucleotide sequence of the wool sulfur peptide precursor peptide LicA3-54T gene is shown in SEQ ID NO.1; the nucleotide sequence of the synthase 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] Furthermore, the amino acid sequence of the wool thiopeptide precursor peptide LicA3-54T is shown in SEQ ID NO.2.

[0011] The present invention also provides the application of the aforementioned lanolin-thiopeptide Lichencin A3-54T in the preparation of a product for treating Gram-positive bacteria, wherein the Gram-positive bacteria is Staphylococcus aureus.

[0012] The present invention also provides a pharmaceutical composition wherein the lanolin-thiopeptide LichencinA3-54T is the active ingredient.

[0013] Furthermore, the pharmaceutical composition also includes a pharmaceutically acceptable salt thereof.

[0014] The present invention discloses the following technical effects:

[0015] This invention uses the amino acid sequence of lanolin-thiopeptide Lichencin A3 as a template to design mutated amino acid sequences, obtaining different mutants. Based on the antibacterial properties of these mutants, a novel site-directed mutant of lanolin-thiopeptide, Lichencin A3-54T, was screened, providing a new alternative drug for the treatment of multidrug-resistant bacteria and offering technical support for the heterologous biosynthesis of lanolin-thiopeptide. Simultaneously, the novel lanolin-thiopeptide mutant Lichencin A3-54T exhibits a strong inhibitory effect against Staphylococcus aureus. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 The results were validated for the PCR amplification products of the co-expressed strains.

[0018] Figure 2 High-performance liquid chromatography (HPLC) chromatogram of Lichencin A3-54T, a lanolin-thiopeptide.

[0019] Figure 3 The mass spectrum of Lichencin A3-54T is shown.

[0020] Figure 4 A schematic diagram of the amino acid structure of lichencin A3-54T, a lanolin-thiopeptide.

[0021] Figure 5 Antibacterial experiment of Lichencin A3-54T, a lanolin-thiopeptide. Detailed Implementation

[0022] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0023] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0024] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0025] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0026] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0027] Unless otherwise specified, all materials and reagents used in this invention are commercially available.

[0028] LB medium: used for the culture of Escherichia coli;

[0029] The specific components of the culture medium are: 10g tryptone, 5g NaCl, 5g yeast extract, and deionized water to a final volume of 1000mL. Adjust the pH to 7.0-7.2.

[0030] Escherichia coli BL21(DE3) was purchased from TransGen Biotech. The indicator bacterium, Staphylococcus aureus, was preserved by the Institute of Biology, Hebei Academy of Sciences. pET-22b, pCDFDuet-1, and PACYCDuet-1 vectors were purchased from TIANDZ.

[0031] The bioinformatics tools used in the following examples:

[0032] Local software: EditSeq, Primer 5.0, and MegAlign.

[0033] Example 1: Method for screening and preparing the Lichencin A3 mutant of lanolin.

[0034] The precursor peptide Lichencin A3, composed of a 40-amino acid leader peptide and a 31-amino acid core peptide, is a precursor peptide. Site-directed saturation mutagenesis was used to mutate each amino acid in the core peptide of Lichencin A3 into 19 other common amino acids, and degenerate bases were used to replace the codons of the mutated amino acids. Based on nucleotide sequence and degenerate base analysis, the mutation efficiency was highest when the mutation codon was NNK. Ultimately, using NNK as the mutation codon, it is estimated that approximately 45% of the nucleic acid mutants can be obtained. The specific preparation method is as follows:

[0035] The 54th amino acid position of the precursor peptide LicA3 was mutated from glycine to threonine to obtain the mutant LicA3-54T, which was then translated into a nucleotide sequence using biological software. The nucleotide sequence of the precursor peptide LicA3-54T gene fragment obtained by PCR site-directed mutagenesis is shown in SEQ ID NO.1, and the amino acid sequence of the precursor peptide LicA3-54T is shown in SEQ ID NO.2.

[0036] SEQ ID NO.1:

[0037] catatgtctcaccgtgaaatggctgccatctaccgtgacgcaaacaaacgcgctaatctggaattcagcaacccggttggcgaagtgaac gaagaagagctgaaaaacctggcaggcgcggccgacgttaccccgcacaccactccgagctctctgccgtgcaccaccctggttatactgcagt ttggtgcccgtctaacgcctgcactagcgattgttaactcgag.

[0038] SEQ ID NO.2:

[0039] MSHREMAAIYRDANKRANLEFSNPVGEVNEEELKNLAGAADVTPHTTPSSLPCTTLVTAVWCPSNACTSDC.

[0040] In the sequences shown in SEQ ID NO.1 and SEQ ID NO.2, the bolded positions are the mutated bases and amino acids.

[0041] Example 2: Construction of gene expression vector

[0042] The gene sequences of the wool thiopeptide synthase LicMB (as shown in SEQ ID NO.3) and the gene sequence of the wool thiopeptide peptidase domain LicT150 (as shown in SEQ ID NO.4) were sent to NovoPro for synthesis. The precursor peptide LicA3-54T gene fragment (as shown in SEQ ID NO.1) obtained by PCR mutation in Example 1 was ligated between the NdeI and XhoI genes of the pET-22b plasmid. The gene fragment of the synthase LicMB was ligated into the pCDFDuet-1 vector, and the gene fragment of the peptidase domain LicT150 was ligated into the PACYCDuet-1 vector.

[0043] SEQ ID NO.3:

[0044]

[0045] SEQ ID NO.4:

[0046] atgctgaaaaagaaaaaactgtttccgacacgtcgtcgtgttccgtttattgagcagatgcagcagaccgaatgtgcactgtgttgtattgcaatgattagcagctactacaa aaacgacctgagcatgtatgaagttcgtgaacgtatgggtaatggtcgtgatggtacaaccctgtttcatctgaaaaagctggcagaacagctgaactttgataccaaaagcta taaagcagatagccgtcagctgggcaccctgattctgcctgcaattctgtattgggaaaacaaccattttgtgatcctggaaaaagttgcacagcaggcatataccattgttg atccaggtagcggtcgtcgtaaactgaaagaaaaagaattcaccgagaaatacagcggttatgtgctgaccctgtatccgaataaaaactttgaacgtcgtagccgcaaataa.

[0047] Example 3: Construction of co-expression strains and heterologous expression

[0048] 1. The three recombinant vectors containing the lanolin precursor peptide LicA3-54T, the synthase LicMB, and the peptidase domain LicT150 gene from Example 2 were co-transformed into competent E.coli BL21(DE3) cells using the heat shock transformation method to construct a co-expression strain.

[0049] 2. Positive co-expressing strains were screened using LB plates containing three antibiotics (kanamycin, spectinomycin, and chloramphenicol).

[0050] 3. Colony PCR was used to identify positive colonies of the co-expressing strain.

[0051] The primers used for PCR (as shown in SEQ ID NO. 5-10) are:

[0052] LicA3-54T F1: CATATGTCTCACCGTGAAATGGCTG (SEQ ID NO.5);

[0053] LicA3-54T R1: GGTGGTGCACGGCAGAGAGCTCGGA (SEQ ID NO. 6);

[0054] LicMB F1: ATGACCGTGGCAAAAATGAAAGACA (SEQ ID NO.7);

[0055] LicMB R1:TTAACAAACTGCACCACCTTTCGGT(SEQ ID NO.8);

[0056] LicT150 F1: ATGCTGAAAAAGAAAAAAACTGTTT (SEQ ID NO.9);

[0057] LicT150 R1: TTATTTGCGGCTACGACGTTCAAA (SEQ ID NO. 10).

[0058] The PCR reaction system is as follows:

[0059] 1.5 μL upstream and downstream primers, 1 μL template DNA, 5 μL 10× Buffer, 5 μL 2 Mm dNTPs, 2 μL 25 mM MgSO4, and ddH2O to bring the volume to 50 μL;

[0060] PCR verification conditions for strains co-expressing the precursor peptide LicA3-54T: 95℃, 2 min; 95℃, 30 s; Tm 55℃, 40 s; 72℃, 30 s; 72℃, 10 min; 4℃, storage.

[0061] PCR verification conditions for strains co-expressing the synthase LicMB: 95℃, 2 min; 95℃, 30 s; Tm 53℃, 40 s; 72℃, 2 min; 72℃, 10 min; 4℃, storage.

[0062] The PCR verification conditions for the LicT150 co-expressing strain containing the peptidase domain were as follows: 95℃, 2 min; 95℃, 30 s; Tm 50℃, 40 s; 72℃, 1 min; 72℃, 10 min; 4℃, storage.

[0063] The results of co-expression strain validation are as follows: Figure 1 As shown, the PCR amplification product band is consistent with the target band, indicating that the co-expression strain was successfully constructed.

[0064] 4. Select co-expressed bacterial colonies and inoculate them into 100 mL of LB liquid medium containing three antibiotics (kanamycin, spectinomycin and chloramphenicol), and incubate at 37°C for 12 h with continuous shaking at 180 rpm.

[0065] 5. Inoculate 5% of the culture medium into 800 mL LB liquid medium in a transfer bottle and incubate at 37°C with continuous shaking. After 90 min of incubation, measure the OD. 600 When the value is 0.6-0.8, the temperature of the shaker is reduced to 25℃. After 0.5 hours, IPTG is added on the clean bench to make the final concentration 0.2mM.

[0066] 6. After incubating at 25℃ and 180 rpm for 24 hours with continuous shaking, centrifuge at 8000 rpm and 4℃ for 20 minutes to collect the bacterial cells. Store the bacterial cell pellet at -20℃ for later use.

[0067] Example 4: Purification and Identification of Lichencin A3-54T (Lanugothipeptide)

[0068] 1. The bacterial cells collected in Example 3 were extracted with methanol for 4 hours. The methanol extract phase was collected, filtered through a 0.22 μm organic filter membrane, and analyzed by ultra-high performance liquid chromatography (UHPLC: ExionLC AD, AB SCIEX, USA).

[0069] Column type: ACQUITYUPLC HSS C18 Column, 100A, 1.8μm, 2.1mm x 150mm, 3 / pk.

[0070] Ultra-high performance liquid chromatography (UHPLC) was performed using two mobile phases, A and B. Mobile phase A consisted of a mixture of chromatographic grade water and 0.01% formic acid, while mobile phase B consisted of a mixture of chromatographic grade acetonitrile and 0.01% formic acid. Chromatographic analysis employed gradient elution with the following program: 0-1.5 min, 1% B phase; 1.5-13 min, 1%-99% B phase gradient; 13-16.5 min, 99% B phase; 16.5-16.6 min, 99%-1% B phase gradient; 16.6-20 min, 1% B phase. The equilibration time between samples was 10 min. QC control samples were inserted into all samples to test and evaluate the stability and reliability of the LC-MS.

[0071] Ultra-high performance liquid chromatogram of the heterologous expression products of the co-expression strains is shown below. Figure 2 As shown, the elution time of Lichencin A3-54T is 7.420-8.087 min.

[0072] 2. Mass spectrometry identification of purified Lichencin A3-54T.

[0073] The structure of Lichencin A3-54T was identified using QTOF MS / MS: Based on the primary mass spectrometry, the parent ion with a molecular weight of 1021.8 Da was selected for secondary tandem mass spectrometry analysis. The tandem mass spectrometry results are as follows: Figure 3 As shown.

[0074] Based on secondary mass spectrometry and the unique structure of lanolin-thiopeptides, the break site of the novel lanolin-thiopeptide Lichencin A3-54T was determined to be at position 40 of the core peptide. During the modification of the core peptide, eight water molecules were removed, forming a cyclic structure with two Lan or MeLan molecules. The backbone amino acid sequence of the lanolin-thiopeptide Lichencin A3-54T is DVTPHTTPSSLPCTTLVTAVWCPSNACTSDC (SEQ ID NO.11). Figure 3 As shown, the break site aspartic acid (D) is counted as position 1. The dehydration sites are threonine (T) at positions 6, 7, 15, and 18, which dehydrate to form dehydrobutyric acid. The cyclization sites are serine (S) at position 9 and cysteine ​​(C) at position 13, and serine (S) at position 24 and cysteine ​​(C) at position 31. During translational modification, the precursor peptide is recognized by the peptidase domain and binds to position 40 of the precursor peptide, hydrolyzing the precursor peptide and leaving a mature core peptide with 31 amino acids. The amino acid structure diagram of lanolin-thiopeptide Lichencin A3-54T is shown below. Figure 4 As shown.

[0075] Example 5: Determination of the antibacterial activity of lanolin-thiopeptide Lichencin A3-54T

[0076] The antibacterial activity of lanolin-thiopeptide Lichencin A3-54T was determined using the antimicrobial susceptibility test disc method.

[0077] 1. Indicator strain Staphylococcus aureus was cultured in suspension on LB liquid medium to obtain OD. 600 Bacterial suspension with a value of 0.3.

[0078] 2. The target strain 54T (Lichencin A3-54T co-expressed strain), control strain 3BT (Lichencin A3 co-expressed strain), control strain 27L (Lichencin A3-27L co-expressed strain), and strain 0BT (empty vector control) were cultured in LB liquid medium to obtain 100 mL of suspension in each strain. The target strain 54T was the co-expressed strain finally obtained in Example 3; the construction method of control strain 3BT was based on Example 3 of patent CN 112851788 B; and the construction method of control strain C27L was based on Example 3 of patent CN 115785236A.

[0079] 3. Take 100 μL of the indicator bacterial suspension obtained in step (1), mix it with 30 mL of LB solid medium at 50℃, spread it on a plate, and use it after the plate solidifies.

[0080] 4. Completely immerse the drug sensitivity test strips in 54T, 27L, 3BT, or 0BT bacterial solutions until the strips are fully absorbed and no longer drip. Set aside for later use.

[0081] After the agar has completely absorbed the moisture from the plates, attach the bacterial suspension discs. Use sterile forceps to place the discs onto the plate surface; once attached, the discs should not be removed. Attach four discs to each plate, spacing them at least 24 mm apart. Finally, incubate at 37°C. Results are as follows... Figure 5 As shown, Figure 5 This indicates that the lanolin mutant LichencinA3-54T has a stronger inhibitory effect on Staphylococcus aureus compared with lanolin peptides LichencinA3 and LichencinA3-27L.

[0082] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A lanolin-thiopeptide, Lichencin A3-54T, characterized in that, The amino acid sequence of the wool sulfur peptide LichencinA3-54T is obtained by dehydrating the backbone amino acid sequence shown in SEQ ID NO.11 and forming a thioether ring. In the backbone amino acid sequence, threonine at positions 6, 7, 15 and 18 is dehydrated to form dehydrobutyric acid, serine at position 9 is dehydrated and forms a thioether ring with cysteine ​​at position 13, and serine at position 24 is dehydrated and forms a thioether ring with cysteine ​​at position 31, thus obtaining the wool sulfur peptide LichencinA3-54T.

2. A method for preparing the lanolin-thiopeptide Lichencin A3-54T as described in claim 1, characterized in that, The process includes the following steps: co-transforming the lanolin precursor peptide LicA3-54T gene, the synthase LicMB gene, and the peptidase domain LicT150 gene into competent Escherichia coli cells to construct a co-expression strain; and purifying the lanolin peptide LichencinA3-54T after heterologous expression through the co-expression strain. The nucleotide sequence of the wool sulfur peptide precursor peptide LicA3-54T gene is shown in SEQ ID NO.1; the nucleotide sequence of the synthase 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 preparation method according to claim 2, characterized in that, The amino acid sequence of the lanolin-thiopeptide precursor peptide LicA3-54T is shown in SEQ ID NO.

2.

4. The application of the lanolin-thiopeptide Lichencin A3-54T as described in claim 1 in the preparation of products against Gram-positive bacteria, characterized in that, The Gram-positive bacteria is Staphylococcus aureus.

5. A pharmaceutical composition, characterized in that, The pharmaceutical composition uses Lichencin A3-54T, the lanolin-thiopeptide described in claim 1, as its active ingredient.

6. The pharmaceutical composition according to claim 5, characterized in that, The pharmaceutical composition also includes its pharmaceutically acceptable salts.

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