Chimeric endolysin lys009-lys009cbd and engineering and use thereof

Abstract

The application discloses a chimeric endolysin Lys009-Lys009CBD and engineering and application thereof, and belongs to the field of genetic engineering and protein engineering. The application constructs a chimeric endolysin Lys009-Lys009CBD with high activity, and the peptidoglycan hydrolysis activity of the chimeric endolysin Lys009-Lys009CBD on Pseudomonas aeruginosa is 5.30 times that of natural endolysin Lys009 and 27.98 times that of hen egg white lysozyme HEWL. A transmembrane peptide P81417 is further fused to perform transmembrane modification, and an engineering endolysin P81417-Lys009-Lys009CBD is obtained. The engineering endolysin can efficiently lyse Pseudomonas aeruginosa, Acinetobacter baumannii and the like, and has excellent thermal stability. The application enriches the resource library of high-activity endolysins, provides important experimental basis and technical support for developing an anti-drug-resistant bacteria treatment strategy, and also provides a new selection for clinical treatment of multiple drug-resistant bacteria infection.

Description

Technical Field

[0001] This invention belongs to the fields of genetic engineering and protein engineering, specifically relating to a chimeric endosomalin Lys009-Lys009CBD and its engineering modification and application. More specifically, this invention relates to constructing a novel, highly active chimeric endosomalin Lys009-Lys009CBD by fusing a cell wall-binding domain (Lys009CBD) derived from Lys009 to the C-terminus of endosomalin Lys009, and further modifying it for membrane penetration by fusing the membrane-penetrating peptide P81417, thereby obtaining a membrane-penetrating engineered endosomalin P81417-Lys009-Lys009CBD that exhibits high antibacterial activity against multiple strains of Pseudomonas aeruginosa and demonstrates excellent thermostability. Background Technology

[0002] *Pseudomonas aeruginosa* is a Gram-negative opportunistic pathogen. Its genome is highly plastic, carrying numerous virulence factors and drug resistance genes, enabling it to rapidly adapt to the host environment and persist in healthcare settings, making it a significant pathogen of hospital-acquired infections (HAIs). Furthermore, its strong biofilm-forming ability makes chronic infections difficult to eradicate, further complicating treatment and increasing the risk of recurrence. Given that *Pseudomonas aeruginosa* possesses inherent resistance to multiple antibiotics and exhibits diverse acquired resistance mechanisms, this is a critical issue.

[0003] Endolysins are peptidoglycan hydrolases encoded and synthesized by bacteriophages at the end of their lysis cycle. They rapidly degrade the peptidoglycan layer in bacterial cell walls, causing bacterial lysis and death due to osmotic imbalance, making them a highly promising new type of antibacterial agent. Compared to antibiotics, endolysins possess unique advantages as antibacterial agents: 1) High efficiency: Endolysins directly cleave the highly conserved peptidoglycan structure of bacterial cell walls to achieve rapid bactericidal action, effectively lysing bacteria within seconds to minutes, regardless of their metabolic state, and effective against bacteria in both the proliferative and dormant phases. 2) Less likely to induce bacterial resistance: Endolysins target conserved peptidoglycan structures, making it difficult for bacteria to develop resistance through single mutations. 3) Specificity: Endolysins from specific sources have conserved target sites, precisely targeting specific bacterial species, with minimal disturbance to the host symbiotic flora and excellent biocompatibility. 4) Easily engineered: Endolysins have highly modular structures, and their activity can be further optimized through domain rearrangement and other methods. These advantages make them a powerful alternative to antibiotics and have rapidly become a research hotspot. Currently, several innovative phage-based endosomal drugs targeting Gram-positive bacteria have entered the clinical trial stage. However, research on endosomal drugs targeting Gram-negative bacteria is relatively lagging behind. The core obstacle is that most natural endosomal drugs cannot directly penetrate the outer membrane of Gram-negative bacteria. The outer membrane barrier blocks endosomal drugs from approaching the peptidoglycan layer, resulting in very limited bactericidal effects from the exogenous addition of natural endosomal drugs. To address the problem that endosomal drugs cannot penetrate the outer membrane, an effective strategy is to fuse endosomal drugs with an outer membrane permeabilizing peptide (OMP) to construct a membrane-permeabilizing engineered endosomal drug.For example, Gerstmans et al. used their self-developed VersaTile DNA assembly technology to construct a modular combinatorial library containing 38 outer membrane-penetrating peptides (OMPs), 2 adapters, 6 CBDs, and 21 EADs. Through two rounds of iterative screening, they successfully identified membrane-penetrating engineered lysins variants with highly efficient bactericidal activity against multidrug-resistant Acinetobacter baumannii (GERSTMANS H, GRIMON D, GUTIéRREZ D, et al. A VersaTile-driven platform for rapid hit-to-lead development of engineered lysins [J]. Science Advances, 2020, 6(23): eaaz1136.). Kogawa et al. used data-driven bioinformatics methods to predict lysin genes from the genomes of 273 Gram-negative bacteria and randomly fused them with 11 OMPs to construct a chimeric library. After high-throughput screening, they obtained 4 membrane-penetrating engineered lysins with highly efficient membrane-penetrating bactericidal activity against Acinetobacter baumannii (KOGAWA M, YODA). T, MATSUHASHI A, et al. Development of Chimera AMP-Endolysin with Wider Spectra Against Gram-Negative Bacteria Using High-Throughput Assay [J]. Viruses, 2025, 17(2): 200. While some endosomalins, such as endosomalin SPN9CC and LysPA26, possess the natural ability to penetrate the outer membrane and hydrolyze peptidoglycan, high concentrations are still required to achieve significant antibacterial effects. Therefore, constructing engineered endosomalins capable of autonomously penetrating membranes and possessing high bactericidal activity can not only provide a scientific basis and reference for the development of novel engineered endosomalins but also advance the clinical application research of endosomalins against Gram-negative bacteria.

[0004] To expand the potential clinical candidates for endolysins targeting Pseudomonas aeruginosa, the field still needs more and more effective endolysins. Summary of the Invention

[0005] In order to overcome the shortcomings and deficiencies of the prior art, the purpose of this invention is to provide a chimeric endosomalin Lys009-Lys009CBD.

[0006] Another object of the present invention is to provide an engineered endosomalin P81417-Lys009-Lys009CBD.

[0007] Another object of the present invention is to provide an application of the above-mentioned Lys009-Lys009CBD or P81417-Lys009-Lys009CBD.

[0008] The objective of this invention is achieved through the following technical solution:

[0009] A chimeric endolysin Lys009-Lys009CBD, the amino acid sequence of which is shown in SEQ ID NO: 1, or a similar sequence as shown in SEQ ID NO: 1 obtained by substitution, insertion or deletion of one or more amino acids, still having the same or similar function, or a sequence that has more than 95% similarity to the amino acid sequence shown in SEQ ID NO: 1 and still has the same or similar function.

[0010] The aforementioned biomaterials related to the chimeric endolysin Lys009-Lys009CBD are any one or more combinations of the following biomaterials:

[0011] (1) The nucleic acid molecule encoding the chimeric endosomalin Lys009-Lys009CBD;

[0012] (2) An expression cassette containing the nucleic acid molecules described in (1);

[0013] (3) A recombinant expression vector containing the nucleic acid molecules described in (1);

[0014] (4) A recombinant expression vector containing the expression cassette described in (2);

[0015] (5) Recombinant microorganisms containing the nucleic acid molecules described in (1);

[0016] (6) Recombinant microorganisms containing the expression cassette described in (2);

[0017] (7) Recombinant microorganisms containing the recombinant expression vector described in (3) or (4).

[0018] Furthermore, the nucleic acid molecule described in (1) is the gene sequence encoding the chimeric endosomalin Lys009-Lys009CBD, as shown in SEQ ID NO: 3, or a similar sequence as shown in SEQ ID NO: 3 that still has the same or similar function obtained by base insertion, deletion or substitution.

[0019] Furthermore, the starting vector for the recombinant expression vector described in (3) and (4) is a pET series vector, etc.; preferably a pET32a(+) vector.

[0020] Furthermore, the host microorganisms corresponding to the recombinant microorganisms mentioned in (5), (6), and (7) are selected from prokaryotes, yeast, or higher eukaryotic cells; the prokaryotes include bacteria of the genera *Escherichia*, *Bacillus*, *Salmonella*, *Pseudomonas*, and *Streptomyces*. More specifically, the prokaryotes are *Escherichia*, preferably *Escherichia coli*, specifically *Escherichia coli* BL21 (DE3).

[0021] An engineered endosomal P81417-Lys009-Lys009CBD, the amino acid sequence of which is shown in SEQ ID NO: 2, or a similar sequence as shown in SEQ ID NO: 2 obtained by substitution, insertion or deletion of one or more amino acids, still having the same or similar function, or a sequence that has more than 95% similarity to the amino acid sequence shown in SEQ ID NO: 2 and still has the same or similar function.

[0022] The aforementioned biomaterials related to endolysin P81417-Lys009-Lys009CBD are any one or more combinations of the following biomaterials:

[0023] (a) The nucleic acid molecule encoding the engineered endosomalin P81417-Lys009-Lys009CBD;

[0024] (b) An expression cassette containing the nucleic acid molecule described in (a);

[0025] (c) A recombinant expression vector containing the nucleic acid molecules described in (a);

[0026] (d) A recombinant expression vector containing the expression cassette described in (b);

[0027] (e) Recombinant microorganisms containing the nucleic acid molecules described in (a);

[0028] (f) Recombinant microorganisms containing the expression cassette described in (b);

[0029] (g) Recombinant microorganisms containing the recombinant expression vector described in (c) or (d).

[0030] Furthermore, the nucleic acid molecule described in (a) is a gene sequence encoding engineered endosomalin P81417-Lys009-Lys009CBD, as shown in SEQ ID NO: 4, or a similar sequence as shown in SEQ ID NO: 4 that still has the same or similar function obtained by base insertion, deletion, or substitution.

[0031] Furthermore, the starting vector for the recombinant expression vector described in (c) and (d) is a pET series vector, etc.; preferably, it is a pET32a(+) vector.

[0032] Furthermore, the host microorganisms corresponding to the recombinant microorganisms described in (e), (f), and (g) are selected from prokaryotes, yeast, or higher eukaryotic cells; the prokaryotes include bacteria of the genera *Escherichia*, *Bacillus*, *Salmonella*, *Pseudomonas*, and *Streptomyces*. More specifically, the prokaryotes are *Escherichia*, preferably *Escherichia coli*, specifically *Escherichia coli* BL21 (DE3).

[0033] The application of the above-mentioned chimeric endolysin Lys009-Lys009CBD-related biomaterials or engineered endolysin P81417-Lys009-Lys009CBD-related biomaterials in the preparation of chimeric endolysin Lys009-Lys009CBD or engineered endolysin P81417-Lys009-Lys009CBD.

[0034] A method for preparing chimeric endosomalin Lys009-Lys009CBD or engineered endosomalin P81417-Lys009-Lys009CBD includes the following steps: culturing recombinant microorganisms to obtain chimeric endosomalin Lys009-Lys009CBD or engineered endosomalin P81417-Lys009-Lys009CBD from the recombinant microorganisms.

[0035] Furthermore, a method for preparing engineered endolysin P81417-Lys009-Lys009CBD includes the following steps:

[0036] Using genetic engineering methods, a short peptide P81417 was linked to the N-terminus of the chimeric endolysin Lys009-Lys009CBD to form the engineered endolysin P81417-Lys009-Lys009CBD. The gene sequence was transformed into a host microorganism to obtain a recombinant microorganism, which was then cultured to prepare the engineered endolysin P81417-Lys009-Lys009CBD.

[0037] The application of the above-mentioned chimeric endolysin Lys009-Lys009CBD or its biomaterials, or engineered endolysin P81417-Lys009-Lys009CBD or its biomaterials, is any one or more combinations of the following applications:

[0038] ① Application in the preparation of products resistant to Gram-negative bacteria;

[0039] ② Application in the preparation of products for treating Gram-negative bacterial infections;

[0040] ③ Application in the preparation of products resistant to Gram-positive bacteria;

[0041] ④ Application in the preparation of products for treating Gram-positive bacterial infections.

[0042] Furthermore, the Gram-negative bacteria mentioned in ① and ② include, but are not limited to, at least one of Pseudomonas aeruginosa and Acinetobacter baumannii;

[0043] Furthermore, the Gram-positive bacteria mentioned in ③ and ④ include, but are not limited to, at least one of Listeria monocytogenes and Bacillus cereus.

[0044] In one embodiment, the engineered endosomalin P81417-Lys009-Lys009CBD of the present invention can lyse Pseudomonas aeruginosa, and its ability to lyse Pseudomonas aeruginosa is significantly improved compared with the control.

[0045] Furthermore, the above-mentioned chimeric endosomalin Lys009-Lys009CBD or engineered endosomalin P81417-Lys009-Lys009CBD and their biomaterials are used in the preparation of Pseudomonas aeruginosa products that resist outer membrane permeability.

[0046] The present invention has the following advantages and effects compared with the prior art:

[0047] This invention successfully constructed a highly active chimeric endolysin Lys009-Lys009CBD by fusing a cell wall-binding domain (Lys009CBD) derived from Lys009 to its C-terminus. This chimeric endolysin exhibits 5.30 times the peptidoglycan hydrolytic activity against Pseudomonas aeruginosa compared to the natural endolysin Lys009, and 27.98 times that of commercially available egg white lysozyme HEWL. Furthermore, by fusing the membrane-penetrating peptide P81417, membrane-penetrating modification was achieved, resulting in the engineered endolysin P81417-Lys009-Lys009CBD. This engineered endolysin can efficiently lyse Pseudomonas aeruginosa, Acinetobacter baumannii, and other bacteria, and demonstrates excellent thermal stability: it retains 100% bactericidal activity even after pretreatment at 70°C for half an hour. This invention enriches the resource library of highly active endolysins, providing important experimental evidence and technical support for developing novel anti-drug-resistant bacterial treatment strategies, and also offering new options for the clinical treatment of multidrug-resistant bacterial infections. Attached Figure Description

[0048] Figure 1 This is a schematic diagram illustrating the construction of the chimeric endosomalin Lys009-Lys009CBD.

[0049] Figure 2 The images show the SDS-PAGE analysis results of the chimeric endosomalin Lys009-Lys009CBD purified using the His-tag method, as well as the SDS-PAGE analysis results of the positive control Lys009 and egg white lysozyme (HEWL).

[0050] Figure 3 This is a graph showing the saturation curve results of the chimeric endosomalin Lys009-Lys009CBD, the positive control Lys009, and the egg white lysozyme (HEWL) enzyme activity detection.

[0051] Figure 4 This is a graph showing the analytical results of engineered endosomalin P81417-Lys009-Lys009CBD; where A: a schematic diagram of the construction of engineered endosomalin P81417-Lys009-Lys009CBD; B: an SDS-PAGE analysis result graph of engineered endosomalin P81417-Lys009-Lys009CBD purified using the His-tag method.

[0052] Figure 5 This is a diagram showing the bactericidal activity of the engineered endonucleoside P81417-Lys009-Lys009CBD.

[0053] Figure 6 This study determined the enzymatic properties of engineered endosomal P81417-Lys009-Lys009CBD; where A: the effect of concentration on the bactericidal activity of engineered endosomal; B: the effect of temperature on the bactericidal activity of engineered endosomal; and C: the effect of pH on the bactericidal activity of engineered endosomal.

[0054] Figure 7 This involves the determination of the cleavage spectrum of the engineered endonucleoside P81417-Lys009-Lys009CBD. Detailed Implementation

[0055] The present invention will be further described in detail below with reference to embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto. The following embodiments are provided to better understand the present invention, but do not limit the present invention. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments were all purchased from conventional biochemical reagent stores. The quantitative experiments in the following embodiments were all performed in triplicate, and the results were averaged.

[0056] Example

[0057] To make the technical solutions and advantages of the present invention clearer, the embodiments of the present invention will be described in further detail below. It should be understood that the embodiments should not be construed as limiting, and those skilled in the art can make further adjustments to the embodiments based on the principles of the present invention.

[0058] The strains *Escherichia coli* DH5α and BL21(DE3) were obtained from TransGen Biotech, Beijing. DNA sequencing was performed by Sangon Biotech, Guangzhou, China. Restriction endonucleases and DNA polymerases were purchased from New England Biolabs (Beverly, USA). DNA purification, gel recovery, genomic DNA extraction, and plasmid amplification kits were purchased from Tiangen Biotech, Ltd. *Pseudomonas aeruginosa* PAO1 is disclosed in the literature “CN117660413A, an endosomalin Lys009 and its engineering modification and application”. Oligonucleotides used for cloning were synthesized by Sangon Biotech, Guangzhou, China.

[0059] Example 1: Obtaining genes encoding endosomal-related proteins and analyzing their protein sequences.

[0060] In the early stages of the laboratory, a candidate endolysin protein, designated Lys009, was obtained through mining of human microbiome metaviromyctic sequencing data. InterProScan software was used to annotate the domains of Lys009 using the Pfam database. Based on this, this embodiment constructs a chimeric endolysin Lys009-Lys009CBD by fusing a cell wall-binding domain (Lys009CBD) derived from Lys009 to its C-terminus. Figure 1 As shown in the figure. The amino acid sequence of the chimeric endolysin is shown in SEQ ID NO: 1, and the specific sequence information is shown in Table 1. The nucleotide sequence of the chimeric endolysin Lys009-Lys009CBD is shown in SEQ ID NO: 3.

[0061] Table 1. Chimeric endolysin Lys009-Lys009CBD sequence information in this embodiment.

[0062]

[0063] Lys009 was disclosed in the paper “FU Y, YU S, LI J, et al. DeepMineLys: Deep mining of phage lysins from human microbiome [J]. Cell Reports, 2024, 43(8):114583.”

[0064] Example 2: Construction of a chimeric endosomal protein expression vector

[0065] To construct the chimeric endolysin Lys009-Lys009CBD expression vector pET32a-Lys009-Lys009CBD-His-tag, this embodiment used the Lys009 expression plasmid pET32a-Lys009-His-tag as a template. The target DNA fragment was amplified by PCR using primers (synthesized by Shanghai Sangon Biotech Co., Ltd., as shown in Table 2), and the DNA fragment was assembled using the Gibson assembly method. The pET32a-Lys009-His-tag is disclosed in the literature "CN117660413A, An endolysin Lys009 and its engineering modification and application".

[0066] Table 2 Primer information for the vector used in this embodiment

[0067]

[0068] Example 3: Expression and purification of endosomal protein

[0069] Test bacteria: Recombinant bacteria BL21(DE3)-pET32a-Lys009-His-tag and recombinant bacteria BL21(DE3)-pET32a-Lys009-Lys009CBD-His-tag. The recombinant bacteria were prepared by transforming the expression vectors pET32a-Lys009-His-tag and pET32a-Lys009-Lys009CBD-His-tag from Example 2 into *Escherichia coli* BL21(DE3), respectively.

[0070] 1. Inoculate a single colony of the *E. coli* to be tested into LB liquid medium containing 100 μg / mL ampicillin resistance and incubate overnight (12-16 h) with shaking at 37 ℃ and 220 rpm. Transfer the overnight culture 1:50 to LB liquid medium containing 100 μg / mL ampicillin resistance and incubate at 37 ℃ and 220 rpm until OD (dose eluent) is reached. 600 When the value reaches 0.6-0.8, pre-cool the bacterial culture and shaker to 16 ℃ for 30 min, add IPTG to a final concentration of 0.2 mM to induce expression, induce expression at 16 ℃ and 220 rpm for 24 h, and collect the bacterial cells by centrifugation at 4 ℃ and 4,000 rpm for 20 min.

[0071] 2. After completing step 1, disrupt the bacterial cells: resuspend the collected bacterial cells in Binding Buffer to 20 OD. 600 Cells were disrupted using an ultrasonic disruptor;

[0072] 3. Purification of endosomal proteins:

[0073] Nickel column purification method: After sonication, centrifuge at 15,000 g for 30 min at 4 ℃ to separate the lysis supernatant (ES) and lysis precipitate (EP). Filter the ES through a 0.22 μm filter membrane and add it to a HisPur Ni-NTA centrifuge column. Incubate on a shaker at 4 ℃ and 100 rpm for 30 min to allow specific binding of the target protein to the column. Centrifuge at 700 g for 2 min at 4 ℃ and collect the flow-through. Subsequently, a gradient elution method is used, successively eluting with 10%, 30%, 50%, 80%, and 100% elution buffer, gradually increasing the imidazole concentration, and collecting the eluent at each concentration.

[0074] 4. Concentration and dialysis: After purification, the protein was concentrated using a 10 kDa ultrafiltration tube to a small volume (1-2 mL). Then, it was dialyzed using a 10 kDa dialysis bag. The buffer was replaced with 10 mM PBS (pH 7.4), and the protein was dialyzed overnight at 4 °C. After dialysis, a small amount of sample was collected for sample preparation.

[0075] 5. All samples were analyzed by SDS-PAGE. ImageJ was used for protein grayscale calculation. A BSA protein standard concentration sample was used as a reference to construct a BSA standard protein concentration curve to determine the concentration of the obtained candidate endolysins. The SDS-PAGE results are shown below. Figure 2 As shown. From Figure 2 It can be seen that the chimeric endosomalin Lys009-Lys009CBD protein can be successfully purified by nickel column purification. The yield and purity of the purified protein expressed in this example are shown in Table 3.

[0076] Table 3. Statistics of endolysin protein expression and purification results in this embodiment.

[0077]

[0078] Example 4: Detection of Endolysin Protein Activity

[0079] The method for preparing the cell substrate for permeation of the outer membrane of *Pseudomonas aeruginosa* used in this embodiment is based on the method of Yoyeon and Lim et al. (CHA Y, SON B, RYU S. Effective removal of staphylococcal biofilms on various food contact surfaces by *Staphylococcus aureus* phage endolysin LysCSA13 [J]. Food microbiology, 2019, 84: 103-245; LIM JA, SHIN H, KANG DH, et al. Characterization of endolysin from a Salmonella Typhimurium-infecting bacteriophage SPN1S [J]. Research in microbiology, 2012, 163(3): 233-41.). The positive controls used in this example are the endosomalin Lys009 and egg white lysozyme (HEWL, Sigma-Aldrich, L6876, CAS: 12650-88-3) previously discovered in the laboratory.

[0080] 1. Single colonies of *Pseudomonas aeruginosa* strain PAO1 were cultured overnight (12-16 h) at 37 ℃ with shaking at 220 rpm in antibiotic-free MHB liquid medium. The overnight culture was then transferred 1:50 to fresh antibiotic-free MHB liquid medium and grown to the logarithmic growth phase, i.e., OD. 600 Centrifuge at 0.5-0.6 g, 4 °C, 4,000 g for 15 min. After centrifugation, wash once with 20 mM Tris-HCl (pH 8.0), incubate for 5 min at 25 °C with a buffer containing 20 mM Tris-HCl (pH 8.0) and 100 mM EDTA, and finally wash three times with 20 mM Tris-HCl, centrifuge to remove residual EDTA, and resuspend the cell pellet in protein buffer and 10 mM PBS (pH 7.4) to OD. 600 The concentration was 0.8-1.2, and the substrate was placed on ice. The preparation of the cell substrate with permeable outer membrane was completed.

[0081] 2. Add 20 μL of purified 10 μg / mL endosomal protein to a 96-well plate. Add 180 μL of outer membrane permeabilized cell substrate to each well. Incubate at 25 °C. The blank control is 20 μL of 10 mM PBS (pH 7.4), and the positive control is 20 μL. Place the plate in a preheated microplate reader and continuously measure the OD. 600 The experiment lasted 30 minutes, and all experiments were repeated three times independently.

[0082] 3. Quantification of endolysin activity: Add 20 μL of candidate proteins at different dilutions (the specific concentration gradient should be adjusted according to the activity of the candidate endolysins) to a 96-well plate. Add 180 μL of outer membrane permeable cell substrate to each protein, and incubate at 25°C. Use 20 μL of 10 mM PBS (pH 7.4) as a blank control. Place in a Spark... In a multi-functional microplate reader, OD is continuously measured. 600 The total duration was 30 minutes. All experiments were repeated three times independently.

[0083] 4. Enzyme activity calculation was performed according to the method of Briers et al. (BRIERS Y, LAVIGNE R, VOLCKAERT G, et al. A standardized approach for accurate quantification of murein hydrolase activity in high-throughput assays [J]. Journal of biochemical and biophysical methods, 2007, 70(3): 531-3.). The saturation curves of different concentrations of murein protein are shown in [the table]. Figure 3 The calculated enzyme activity units are shown in Table 4. The activity of the chimeric endolysin Lys009-Lys009CBD is 1,230,000 units / mg, which is a significant improvement over Lys009, being 5.30 times that of Lys009 and 27.98 times that of commercial egg white lysozyme HEWL. This indicates that the chimeric endolysin Lys009-Lys009CBD has a highly efficient hydrolytic ability against the peptidoglycan of Pseudomonas aeruginosa cell wall, and has the potential to be further developed into a novel antibacterial agent targeting Pseudomonas aeruginosa.

[0084] Table 4. Results of endolysin protein activity detection in this embodiment

[0085]

[0086] Example 5: Engineering of chimeric endolysin proteins

[0087] In this embodiment, the N-terminus of the chimeric endosomal peptide Lys009-Lys009CBD was fused with the transmembrane peptide P81417 to engineer the endosomal peptide P81417-Lys009-Lys009CBD, as shown below. Figure 4 As shown in A in the figure. The amino acid sequence of engineered endosomalin P81417-Lys009-Lys009CBD is shown in SEQ ID NO: 2, and the nucleotide sequence is shown in SEQ ID NO: 4.

[0088] 1. Construction of the recombinant plasmid: Oligonucleotide primers (synthesized by Shanghai Sangon Biotech, sequences shown in Table 5) were designed and synthesized using DNAWorks software. The gene fragment encoding the short peptide P81417 was obtained through overlap extension PCR assembly. Simultaneously, using the pET32a-Lys009-Lys009CBD-His-tag plasmid constructed in Example 2 as a template, two other sets of primers (sequences shown in Table 5) were used for PCR amplification to obtain target DNA fragment 1 and target DNA fragment 2. Finally, the gene fragment encoding the short peptide P81417, target DNA fragment 1, and target DNA fragment 2 were ligated using the Gibson assembly method to construct the recombinant plasmid pET32a-P81417-Lys009-Lys009CBD-His-tag.

[0089] 2. Expression and Purification: The expression and purification of engineered endosomalin P81417-Lys009-Lys009CBD were performed according to the nickel column purification method in Example 3. Engineered endosomalin P81417-Lys009-Lys009CBD was successfully expressed and purified. SDS-PAGE results are shown below. Figure 4 As shown in B in the figure. The expression level of engineered endosomalin P81417-Lys009-Lys009CBD was 24.7 mg / L LB medium, with a purity of 92.3%.

[0090] Table 5 Primer information constructed using the vectors used in this embodiment.

[0091]

[0092] Example 6: Antibacterial activity test of engineered endosomalin

[0093] In this embodiment, the engineered endosomalin P81417-Lys009-Lys009CBD from Example 5 was selected for bacterial inhibition experiments. *Pseudomonas aeruginosa* PAO1 was used as the test strain in this study.

[0094] Pseudomonas aeruginosa PAO1 strain was streaked onto antibiotic-free MHB plates for resuscitation. Single colonies were picked and cultured in antibiotic-free MHB liquid medium overnight (12-16 h) at 37 ℃ with shaking at 220 rpm. The overnight culture was then transferred 1:50 to fresh antibiotic-free MHB liquid medium and grown to the logarithmic growth phase (OD2). 600 =0.5-0.6), centrifuged at 4 ℃, 4,000 g for 15 min, and the culture medium was discarded. The cells were washed once with 10 mM PBS (pH 7.4) and resuspended to OD. 600 The concentration was approximately 0.5, so the test bacterial suspension was diluted to 1×10⁻⁵. 5 CFU / mL. 100 μL of different concentrations of endolysin were added to 100 μL of test bacterial suspension, for a total volume of 200 μL. The bacterial control consisted of 100 μL of PBS buffer and 100 μL of test bacterial suspension, while the blank control consisted of 200 μL of PBS buffer. The mixture was incubated at 37 ℃ for 1 h. 100 μL of the mixture was evenly spread onto a plate and incubated overnight at 37 ℃. The colony count was recorded. All experiments were independently repeated three times. The bactericidal rate was calculated as: Bactericidal rate (%) = (N0 - N1) / N0 × 100%; N0 corresponds to the colony count in the bacterial control group, and N1 corresponds to the colony count in the experimental group. The antibacterial activity test results of engineered endolysin are shown below. Figure 5 As shown in the figure, P81417-Lys009-Lys009CBD achieved a 100% bactericidal rate against Pseudomonas aeruginosa PAO1 at final concentrations of 50 μg / mL, 80 μg / mL, and 100 μg / mL. Even at a final concentration of 10 μg / mL, it significantly lysed P. aeruginosa PAO1, with the bacterial count decreasing by approximately four orders of magnitude compared to the bacterial control group (i.e., endolysin concentration of 0), indicating that P81417-Lys009-Lys009CBD possesses excellent transmembrane bactericidal ability.

[0095] Example 7: Enzymatic Characterization of Engineered Endolysin

[0096] 1. Effect of concentration on endolysin: Pseudomonas aeruginosa PAO1 cells in logarithmic growth phase were collected (4 ℃, 4,000 g, 15 min), washed once with PBS (pH 7.4), and resuspended at OD. 600 The concentration was approximately 0.5, so the test bacterial suspension was diluted to 1×10⁻⁵. 5CFU / mL; 100 μL of P81417-Lys009-Lys009CBD with final concentrations of 1-128 μg / mL was added to 100 μL of test bacterial suspension (total volume 200 μL). Bacterial controls consisted of 100 μL of PBS buffer and 100 μL of test bacterial suspension, while the blank control consisted of 200 μL of PBS buffer. The mixture was incubated at 37°C for 1 h. 100 μL of the mixture was evenly spread onto a plate and incubated overnight at 37°C. The colony count was recorded. All experiments were independently repeated three times. The sterilization rate was calculated using the formula: Sterilization rate (%) = (N0-N1) / N0 × 100%; N0 corresponds to the colony count in the bacterial control group, and N1 corresponds to the colony count in the experimental group. Figure 6 As shown in Figure A. Experimental results show that the antibacterial activity of P81417-Lys009-Lys009CBD increases with increasing protein concentration, exhibiting a concentration-dependent effect. At a concentration of only 1 μg / mL, P81417-Lys009-Lys009CBD can effectively lyse Pseudomonas aeruginosa PAO1, reducing the bacterial count by approximately two orders of magnitude compared to the bacterial control group (i.e., endolysin concentration of 0). When the concentration increases to above 32 μg / mL, 100% lysis of this strain can be achieved. The minimum bactericidal concentration (MBC) of P81417-Lys009-Lys009CBD was calculated to be 8 μg / mL, meaning that at this concentration, 99.9% of Pseudomonas aeruginosa can be lysed, demonstrating highly efficient antibacterial ability.

[0097] 2. Effect of temperature on endolysins: Pseudomonas aeruginosa PAO1 cells in the logarithmic growth phase were collected (4 ℃, 4,000 g, 15 min), washed once with PBS (pH 7.4), and resuspended at OD. 600 The concentration was approximately 0.5, so the test bacterial suspension was diluted to 1×10⁻⁵. 5 CFU / mL; 100 μL of P81417-Lys009-Lys009CBD, with a final concentration of 100 μg / mL, was incubated for 30 min at different temperatures (4-70 ℃). Then, it was added to 100 μL of test bacterial suspension (total volume 200 μL). Bacterial controls consisted of 100 μL of PBS buffer and 100 μL of test bacterial suspension, while the blank control consisted of 200 μL of PBS buffer. The mixture was incubated at 37 ℃ for 1 h. 100 μL of the mixture was evenly spread onto a plate and incubated overnight at 37 ℃. The colony count was recorded. All experiments were independently repeated three times. The bactericidal rate was calculated using the formula: Bactericidal rate (%) = (N0-N1) / N0 × 100%; N0 corresponds to the colony count in the bacterial control group, and N1 corresponds to the colony count in the experimental group. The results of the effect of different temperatures on the antibacterial activity of P81417-Lys009-Lys009CBD are as follows: Figure 6As shown in Figure B. Experimental results show that after incubating engineered endosomalin P81417-Lys009-Lys009CBD at 4, 16, 25, 37, 42, 50, 60, and 70 °C for 30 min, the antibacterial activity of each pretreatment group did not decrease, and the lysis rate against Pseudomonas aeruginosa PAO1 remained at 100%, demonstrating excellent thermal stability. Given the superior thermal stability exhibited by P81417-Lys009-Lys009CBD, it possesses unique application advantages in complex environments requiring high-temperature treatment or storage.

[0098] 3. Effect of pH on endolysins: Pseudomonas aeruginosa PAO1 cells in the logarithmic growth phase were collected (4 ℃, 4,000 g, 15 min), washed once with PBS (pH 7.4), and resuspended at OD. 600 The concentration was approximately 0.5, so the test bacterial suspension was diluted to 1×10⁻⁵. 5 CFU / mL; 100 μL of P81417-Lys009-Lys009CBD with a final concentration of 100 μg / mL was added to 100 μL of test bacterial suspension at different pH values ​​(total volume 200 μL). The bacterial control consisted of 100 μL of PBS buffer and 100 μL of test bacterial suspension, while the blank control consisted of 200 μL of PBS buffer. The mixture was incubated at 37 ℃ for 1 h. 100 μL of the mixture was evenly spread onto a plate and incubated overnight at 37 ℃. The colony count was recorded. All experiments were independently repeated three times. The bactericidal rate was calculated as follows: Bactericidal rate (%) = (N0-N1) / N0 × 100%; N0 corresponds to the colony count in the bacterial control group, and N1 corresponds to the colony count in the experimental group. The results of the effect of different pH values ​​on the antibacterial activity of P81417-Lys009-Lys009CBD are as follows: Figure 6 As shown in C. Experimental results show that under acidic conditions of pH 4-6, the lytic activity of engineered endolysin P81417-Lys009-Lys009CBD decreased by more than 40%, while under conditions of pH 7.4-10, it maintained lytic activity against 100% of Pseudomonas aeruginosa. The antibacterial activity of engineered endolysin P81417-Lys009-Lys009CBD was significantly affected by acidic conditions, which may be related to the fact that Lys009 originates from the weakly alkaline intestinal environment. Therefore, engineered endolysin P81417-Lys009-Lys009CBD is suitable for exerting its lytic effect under alkaline conditions.

[0099] Example 8: Determination of the fragmentation spectrum of engineered endosomalin

[0100] All test strains used in this embodiment were obtained from our laboratory. A total of 8 Gram-negative bacteria and 3 Gram-positive bacteria were tested, as shown in Table 6. Among them, 2 strains of *Pseudomonas aeruginosa* (PALWL1.002 and PALWL1.003) were isolated and cultured in our laboratory. All 8 Gram-negative bacteria and 3 Gram-positive bacteria are disclosed in the literature "CN117660413A, An endosomalin Lys009 and its engineering modification and application".

[0101] Table 6. Test strains used in this embodiment.

[0102]

[0103] We used the engineered endosomalin P81417-Lys009-Lys009CBD constructed in Example 5 as the research object. Following the antibacterial test method in Example 6, we selected 100 μg / mL of P81417-Lys009-Lys009CBD and incubated it with an equal volume of logarithmic-phase cells from different bacterial strains for 1 h. The antibacterial activity of the endosomalin was evaluated by counting the number of residual colonies after the treatment. The results are as follows... Figure 7 As shown, the experimental results indicate that P81417-Lys009-Lys009CBD exhibits 100% lysis activity against the seven *Pseudomonas aeruginosa* strains and one *Acinetobacter baumannii* strain ATCC 19606 tested above. It also shows 43.7% and 49.0% lysis activity against *Listeria monocytogenes* CICC 21634 and *Bacillus cereus* ATCC 145797, respectively, but almost no lysis activity against *Staphylococcus aureus* ATCC 6538. Among these, *Pseudomonas aeruginosa* ATCC 10145 and *Acinetobacter baumannii* ATCC 19606 are two multidrug-resistant strains. *Pseudomonas aeruginosa* ATCC 10145, isolated from soil, is resistant to ampicillin, kanamycin, penicillin, and vancomycin. Acinetobacter baumannii ATCC 19606, isolated from clinical urine samples, exhibits resistance to ampicillin, amoxicillin, cefazolin, cefoxitin, nitrofurantoin, and metformin. This suggests that the engineered endolysin P81417-Lys009-Lys009CBD has the potential for application in treating drug-resistant infections of Pseudomonas aeruginosa and Acinetobacter baumannii.

[0104] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A chimeric endosomal lysine Lys009-Lys009CBD or an engineered endosomal lysine P81417-Lys009-Lys009CBD, characterized in that: The amino acid sequence of the chimeric endolysin Lys009-Lys009CBD is as shown in SEQ ID NO: 1, or a similar sequence as shown in SEQ ID NO: 1 obtained by one or more amino acid substitutions, insertions, or deletions that still have the same or similar functions, or a sequence that has more than 95% similarity to the amino acid sequence shown in SEQ ID NO: 1 and still has the same or similar functions. The amino acid sequence of the engineered endosomal P81417-Lys009-Lys009CBD is as shown in SEQ ID NO: 2, or a similar sequence as shown in SEQ ID NO: 2 obtained by substitution, insertion, or deletion of one or more amino acids, which still has the same or similar function, or a sequence that has more than 95% similarity to the amino acid sequence shown in SEQ ID NO: 2 and still has the same or similar function.

2. The biomaterials related to the chimeric endosomalin Lys009-Lys009CBD or engineered endosomalin P81417-Lys009-Lys009CBD as described in claim 1, characterized in that: The chimeric endolysin Lys009-Lys009CBD-related biomaterial is any one or more combinations of the following biomaterials: (1) The nucleic acid molecule encoding the chimeric endosomalin Lys009-Lys009CBD; (2) An expression cassette containing the nucleic acid molecules described in (1); (3) A recombinant expression vector containing the nucleic acid molecules described in (1); (4) A recombinant expression vector containing the expression cassette described in (2); (5) Recombinant microorganisms containing the nucleic acid molecules described in (1); (6) Recombinant microorganisms containing the expression cassette described in (2); (7) Recombinant microorganisms containing the recombinant expression vector described in (3) or (4); The biomaterials related to the engineering endolysin P81417-Lys009-Lys009CBD are any one or more combinations of the following biomaterials: (a) The nucleic acid molecule encoding the engineered endosomalin P81417-Lys009-Lys009CBD; (b) An expression cassette containing the nucleic acid molecule described in (a); (c) A recombinant expression vector containing the nucleic acid molecules described in (a); (d) A recombinant expression vector containing the expression cassette described in (b); (e) Recombinant microorganisms containing the nucleic acid molecules described in (a); (f) Recombinant microorganisms containing the expression cassette described in (b); (g) Recombinant microorganisms containing the recombinant expression vector described in (c) or (d).

3. The biomaterial according to claim 2, characterized in that: (1) The nucleic acid molecule described is the gene sequence encoding the chimeric endosomalin Lys009-Lys009CBD, as shown in SEQ ID NO: 3, or a similar sequence as shown in SEQ ID NO: 3 that still has the same or similar function obtained by base insertion, deletion or substitution. And / or, the nucleic acid molecule described in (a) is a gene sequence encoding engineered endosomalin P81417-Lys009-Lys009CBD, as shown in SEQ ID NO: 4, or a similar sequence as shown in SEQ ID NO: 4 that still has the same or similar function obtained by base insertion, deletion, or substitution.

4. The biomaterial according to claim 2 or 3, characterized in that: The starting vector for the recombinant expression vectors described in (3), (4), (c), and (d) is a pET series vector; And / or, the host microorganisms corresponding to the recombinant microorganisms described in (5), (6), (7), (e), (f), and (g) are selected from prokaryotes, yeast, or higher eukaryotic cells; the prokaryotes include bacteria of the genera Escherichia, Bacillus, Salmonella, Pseudomonas, or Streptomyces.

5. The biomaterial according to claim 4, characterized in that: The starting vector for the recombinant expression vectors described in (3), (4), (c), and (d) is the pET32a(+) vector; And / or, the host microorganism corresponding to the recombinant microorganism described in (5), (6), (7), (e), (f), (g) is selected from Escherichia coli.

6. The biomaterial according to claim 5, characterized in that: The host microorganisms corresponding to the recombinant microorganisms mentioned in (5), (6), (7), (e), (f), and (g) are selected from Escherichia coli BL21 (DE3).

7. The use of the biomaterial according to any one of claims 2 to 6 in the preparation of chimeric endosomalin Lys009-Lys009CBD or engineered endosomalin P81417-Lys009-Lys009CBD.

8. A method for preparing a chimeric endosomal lysin Lys009-Lys009CBD or an engineered endosomal lysin P81417-Lys009-Lys009CBD, characterized in that: The method includes the following steps: culturing the recombinant microorganisms as described in any one of claims 2 to 6, and obtaining the chimeric endosomalin Lys009-Lys009CBD or the engineered endosomalin P81417-Lys009-Lys009CBD from the recombinant microorganisms.

9. The application of the chimeric endosomalin Lys009-Lys009CBD or engineered endosomalin P81417-Lys009-Lys009CBD as described in claim 1, or the biomaterial as described in any one of claims 2 to 6, characterized in that: For any one or more of the following applications: ① Application in the preparation of products resistant to Gram-negative bacteria; ② Application in the preparation of products for treating Gram-negative bacterial infections; ③ Application in the preparation of products resistant to Gram-positive bacteria; ④ Application in the preparation of products for treating Gram-positive bacterial infections.

10. The application according to claim 9, characterized in that: The Gram-negative bacteria mentioned in ① and ② include at least one of Pseudomonas aeruginosa and Acinetobacter baumannii; And / or, the Gram-positive bacteria mentioned in ③ and ④ include at least one of Listeria monocytogenes and Bacillus cereus.

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