Phage E protein mutant and application thereof

Abstract

The invention provides a phage E protein mutant and application thereof. Compared with the phage E protein, the phage E protein mutant has the advantage that the 33rd lysine is mutated into arginine. According to the invention, the cracking capability of the K33R variant under the background of the vibrio natriticus MraY is screened and proved to be obviously superior to that of a wild type E protein, so that the blank of lack of a standard cracking element for the vibrio natriticus in the prior art is filled; and an effect module which can be directly applied is provided for realizing stable oscillation of an ePop self-splitting line in the vibrio natriticus or engineering bacteria carrying the vibrio natriticus MraY.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a phage E protein mutant and its applications. Background Technology

[0002] As an important tool in synthetic biology, cleavage gene circuits function by sensing bacterial population density to achieve programmed cleavage, providing efficient and controllable technical support for biomanufacturing, drug delivery, and microbial community regulation. Self-cleavage gene circuits typically consist of a quorum sensing module and a cleavage effector module, both sharing the same promoter: the quorum sensing signal LuxR / AHL is amplified through positive feedback, simultaneously driving the expression of the cleavage gene E protein to form negative feedback. This causes cells to primarily grow at low densities, while at high densities, the accumulation of E protein triggers population cleavage. These circuits have already been used in *E. coli* and *Salmonella* for drug delivery, biomanufacturing, and microbial community regulation, demonstrating the engineering value of density-sensing programmed cleavage.

[0003] However, current research indicates that the self-cleavage pathway operates stably only in a few species, with the main bottleneck being the host specificity of the effector module, the E protein. The E protein originates from the microphage ΦX174. It induces cell lysis by forming the YES complex with the transmembrane enzyme MraY and the molecular chaperone SlyD, wedging the transmembrane helix into the active site of MraY and blocking a key step in peptidoglycan synthesis. The sequence and surface physicochemical characteristics of MraY differ among different species, resulting in the E protein often exhibiting a strong cleavage effect only in certain bacterial species.

[0004] To address the host adaptability issue of the E protein, current techniques primarily involve alanine scanning and site-directed mutagenesis of the E protein in the context of *E. coli*, screening for key residues affecting the cleavage phenotype, and elucidating the structural mechanism of the E-MraY-SlyD complex. However, these studies are mostly conducted in hosts carrying native *E. coli* MraY, and the resulting gain-enhancing or time-regulated mutants are not applicable to sodium-dependent *Vibrio* MraY, failing to directly resolve the failure of ePop in sodium-dependent *Vibrio*.

[0005] Therefore, developing a screening technology for E protein variants of sodium-dependent Vibrio mraY and obtaining highly adaptable cleavage effect elements has become a key technological bottleneck in promoting the application of self-cleavage gene circuits to novel hosts such as sodium-dependent Vibrio. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention proposes a phage E protein mutant and its applications. This invention discloses a technical solution for expressing and quantitatively evaluating the cleavage ability of different E protein variants under standardized vector and detection conditions. Using this solution, mutants capable of efficiently cleaving MC4100z1Δ were screened and identified. mraY::mraY Vn The E protein variant from chassis cells was obtained, thus yielding the E protein variant K33R, which is highly matched to the sodium-dependent Vibrio mraY, providing a directly usable effector module for the subsequent construction of the ePop self-cleavage circuit in sodium-dependent Vibrio.

[0007] The present invention provides a phage E protein mutant, wherein the phage E protein mutant has a 33rd lysine residue mutated to arginine compared with the phage E protein.

[0008] In some embodiments, the amino acid sequence of the phage E protein mutant is shown in SEQ ID NO.2.

[0009] The present invention also provides a nucleic acid molecule encoding the phage E protein mutant described above.

[0010] The present invention also provides an expression cassette containing the said nucleic acid molecule.

[0011] In some embodiments, the expression cassette further includes a replicon, a promoter, a ribosome binding site, and a transcription terminator.

[0012] Preferably, the promoter is an inducible promoter.

[0013] The present invention also provides a recombinant vector containing the nucleic acid molecule or the expression cassette.

[0014] The present invention also provides engineered strains containing the nucleic acid molecule, the expression cassette, or the recombinant vector.

[0015] In some embodiments, the engineered strain is a sodium-carrying Vibrio. mraY Engineered strains of genes.

[0016] In some embodiments, the engineered strain is a sodium-carrying Vibrio. mraY Genetically modified E. coli.

[0017] The present invention also provides the application of the phage E protein mutant in the construction of a controlled bacterial lysis system.

[0018] In summary, compared with the prior art, the present invention achieves the following technical effects: (1) The present invention directly carries sodium-dependent Vibrio bacteria mraY MC4100z1Δ mraY::mraY VnThe performance of phage E protein variants was evaluated in the chassis, and the obtained lysis data can truly reflect the matching degree between the variant and the sodium-dependent Vibrio mraY, rather than inferring indirectly through the original host, thus significantly improving the guidance value of the ePop system when migrating to sodium-dependent Vibrio.

[0019] (2) The present invention has constructed a standardized screening process that maintains uniformity in vector, induction conditions, inoculation method and data analysis, which makes the performance comparison between different E protein variants have good reproducibility and interpretability, which is better than the traditional method based solely on endpoint OD or subjective curve observation, thus improving screening efficiency and reliability.

[0020] (3) This invention clearly screens and confirms that the K33R variant has a significantly better cleavage ability than the wild-type E protein in the context of sodium-dependent Vibrio MraY, filling the gap in the prior art for the lack of standard cleavage elements for sodium-dependent Vibrio, and providing a directly applicable effect module for the ePop self-cleavage circuit to achieve stable oscillation in sodium-dependent Vibrio or engineered bacteria carrying sodium-dependent Vibrio MraY.

[0021] (4) The K33R variant involves only a single amino acid residue substitution, without changing the protein length and transmembrane structure. It has good expression and folding stability and is easy to integrate into a variety of plasmid backbones and host strains. This helps to reduce the technical threshold for constructing self-lysis systems and enhance the ability to promote engineering.

[0022] (5) By providing both specific variants and a standardized platform, this invention not only provides highly matched lysis elements, but also provides reusable screening and evaluation technical routes, which helps to achieve rapid implementation in applications such as cell lysis control, steady-state oscillation systems and controlled release. Attached Figure Description

[0023] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 The structure and working principle of the ePop self-cleavage gene circuit.

[0025] Figure 2 The E. coli chassis cells MC4100z1 of the heterologous MraY of this invention ΔmraY :: mraY Vn The construction of.

[0026] Figure 3This illustrates the performance of the ePop self-cleavage gene circuitry in different chassis cells in Example 1 of the present invention.

[0027] Figure 4 This is a quantitative screening platform for E protein variants of the present invention.

[0028] Figure 5 The present invention relates to the E protein and its variant K33R in chassis cell ZM (MC4100z1). ΔmraY :: mraY Vn The fragmentation behavior in ).

[0029] Figure 6 The present invention relates to chassis cell ZM (MC4100z1 Δ) mraY::mraY Vn The cleavage kinetic parameters of the E protein variant in ). Detailed Implementation

[0030] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0031] The self-lysis gene circuit consists of two parts: a quorum sensing component that senses bacterial density and an E protein, a utility element that drives bacterial lysis. Figure 1 As shown, these two parts are controlled by a common promoter, which drives its own activator AHL, forming a positive feedback loop, and also drives the expression of the lysis gene, forming a negative feedback loop. This topology couples cell density and toxin expression; lysis occurs at high density, while at low density, the bacteria continue to grow because the expression level of the E protein is insufficient for lysis.

[0032] This invention constructs a system based on "Escherichia coli MC4100z1Δ mraY::mraY Vn The E protein variant screening platform, which is based on "chassis + unified expression vector + standardized growth curve detection + model parameter evaluation", was used to screen and obtain the E protein variant K33R, which has a significantly improved matching degree with the sodium-dependent Vibrio natriureticis MraY.

[0033] Specifically, this invention first provides a K33R variant using phage ΦX174 E protein as a template, with the amino acid at position 33 changed from lysine to arginine. This variant makes only precise substitutions in the amino acid sequence, while maintaining the overall structural backbone consistent with the wild-type E protein. Secondly, this invention provides the nucleic acid sequence encoding the variant and its codon-optimized version. These nucleic acids are constructed onto an expression plasmid using pColE1-rop as a replicon and employing an inducible promoter, enabling the variant to be expressed at MC4100z1Δ. mraY::mraY Vn The chassis can be uniformly induced by dehydrated tetracycline. Furthermore, this invention uses MC4100z1Δ mraY::mraY Vn Using a chassis as the host, standardized culture and induction conditions were provided (including overnight culture, standardized logarithmic phase inoculation, and uniform induction with 200 ng / mL aTc). The growth-lysis curve was fitted using a three-segment logistic function, and parameters such as maximum lysis rate, lysis ratio, lysis delay time, and ΔAUC were extracted and compared to evaluate the lysis performance of the E variant. In this platform, K33R was shown to have significantly enhanced lysis ability compared to the wild-type E protein. Therefore, this invention identifies it as a preferred E protein variant matching *Vibrio natriureticis* MraY, and it can serve as an effector module for future reconstruction of the ePop self-lysis circuit in *Vibrio natriureticis*.

[0034] Example 1: Escherichia coli chassis MC4100z1 carrying sodium-dependent Vibrio γ ΔmraY :: mraY Vn Construction Using the λ-Red homologous recombination system, sodium-dependent Vibrio ( ) was used on the chromosome of Escherichia coli MC4100z1. Vibrio natriegens )of mraY Gene replacement of its endogenous mraY Constructing an engineering chassis MC4100z1 carrying sodium-dependent Vibrio syringae MraY ΔmraY :: mraY Vn (Hereinafter referred to as ZM chassis). The entire process includes the construction of linear DNA fragments, introduction of pKD46 plasmid and λ-Red induction, selection of positive clones by homologous recombination of linear fragments, excision of the FRT site resistance cassette and plasmid loss mediated by pCP20 plasmid, and final PCR and sequencing verification of the strain. The procedure is as follows: Figure 2 As shown, MC4100z1 is constructed using λ-Red. ΔmraY :: mraY Vn The chassis process includes a replacement box containing sodium-dependent Vibrio mraY, FRT sites, and FLP removal steps.

[0035] First, a linear DNA fragment for homologous recombination was constructed. Using chromosome MC4100z1 as a template, a pair of primers (upstream primer: SEQ ID No. 4; downstream primer: SEQ ID No. 5) were designed. mraY A long homologous arm was amplified from the 1000 bp upstream region of the gene, and then a pair of primers (upstream primer: SEQ ID No. 6; downstream primer: SEQ ID No. 7) were designed to... mraY The long homologous arm was amplified 1000 bp downstream of the gene; simultaneously, its genomic DNA was used as a template to amplify... mraY An open reading frame was used (upstream primer: SEQ ID No. 8; downstream primer: SEQ ID No. 9); using a plasmid containing the FRT-chloramphenicol resistance gene-FRT sequence (nucleotide sequence as shown in SEQ ID No. 3) as a template, the chloramphenicol resistance gene fragment containing FRT sites on both sides was amplified (upstream primer: SEQ ID No. 10; downstream primer: SEQ ID No. 11).

[0036] Each fragment was assembled using overlap PCR. Two rounds of PCR were employed: the first round amplified each fragment separately. mraY Upstream homologous arm, sodium-dependent Vibrio mraY Fragments, FRT-resistance box fragments and mraY Downstream homologous arm, product recovered; in the second round, using the above fragments as templates, overlap PCR was used to synthesize the product. mraY Upstream homologous arm - FRT - resistance box - sodium-dependent Vibrio mraY - mraY The downstream homologous arms were sequentially assembled into a single linear DNA fragment. After the reaction, agarose gel electrophoresis was performed to confirm that the size of the assembled fragment met the expectations. The recovered product was then purified and concentrated to serve as donor DNA for subsequent homologous recombination.

[0037] Next, the temperature-sensitive plasmid pKD46 carrying the λ-Red recombination system was transformed into the *E. coli* strain MC4100z1 to be modified. pKD46 could be introduced into MC4100z1 via chemical transformation or electroporation. After transformation, the plasmid was plated on LB solid medium containing ampicillin and cultured at 30°C for 16 h. Single colonies were then picked. To maintain the stability of pKD46, subsequent cultures were all performed at 30°C. Single colonies of MC4100z1 / pKD46 transformed with pKD46 were then inoculated into LB liquid medium containing ampicillin and cultured in a shaker at 30°C until OD (dose expiratory time). 600 When the concentration reached approximately 0.2, L-arabinose was added to the culture medium to bring the final concentration to 0.3% to induce the expression of Exo, Beta, and Gam proteins in the λ-Red system; the culture was then continued at 30°C until the OD reached... 600Once the pH reaches approximately 0.4–0.5, transfer the bacterial culture to an ice bath and cool for about 30 minutes to inhibit metabolism and prepare for the preparation of electrocompetent cells. After cooling, collect the bacterial cells by low-speed centrifugation, discard the supernatant, wash twice with pre-cooled sterile deionized water, then wash once with pre-cooled 10% glycerol solution, and finally resuspend the bacterial cells in a small amount of pre-cooled 10% glycerol to prepare high-concentration electrocompetent cells, which are then placed on ice for later use.

[0038] Next, homologous recombination was performed. 50 μL of the electroporated competent MC4100z1 / pKD46 bacterial culture was added, along with 100 ng of purified linear DNA fragment. After gentle mixing, the mixture was transferred to a pre-chilled 0.2 cm electroporation cuvette and subjected to a single pulse electroporation at 2.5 kV. Immediately after electroporation, approximately 1 mL of LB broth containing 0.3% L-arabinose was added to the cuvette, and the mixture was gently pipetted to mix. The culture was then transferred to a 2 mL centrifuge tube and incubated at 30°C in a shaker for 2 h to allow for the expression of λ-Red protein and the completion of chromosomal homologous recombination. After incubation, an appropriate amount of the bacterial culture was plated onto LB agar plates containing chloramphenicol (final concentration of chloramphenicol: 25 μg / mL) and incubated upside down at 37°C for 16 h to obtain chloramphenicol-resistant clones. Chloramphenicol resistance indicates that the FRT-resistance cassette has successfully integrated into the target chromosomal site, thereby replacing the original chromosomal DNA. mraY Select several resistant clones and perform colony PCR using the colony-specific PCR method. mraY Primers from upstream and downstream conserved regions were used for identification, and the size of the amplified fragment was compared with that containing sodium-dependent Vibrio. mraY The design was consistent with that of the resistance cassette, and the PCR products were sequenced to further confirm the correctness of the replacement fragment sequence.

[0039] In obtaining the resistant box ΔmraY :: mraY Vn - After the resistance intermediate is obtained, the resistance genes at both ends of the FRT need to be removed using FLP recombinase to avoid the accumulation of multiple resistances in subsequent use. For this purpose, the plasmid pCP20, which expresses FLP recombinase and is also temperature-sensitive, was electroporated into the above-mentioned chloramphenicol resistance clone.

[0040] The specific procedure was as follows: Electroporation competent cells for this resistant clone were prepared at 30℃ using the same method as for pKD46. After mixing the pCP20 plasmid DNA with the competent cells, electroporation was performed at 2.5 kV. Immediately afterward, 1 mL of LB broth was added, and the cells were incubated at 30℃ for 2 h. After incubation, the bacterial culture was plated onto LB agar plates containing chloramphenicol and ampicillin, respectively, and cultured in parallel at 30℃ and 42℃: the 30℃ plates were used to maintain the plasmid, and the 42℃ plates were used to promote the loss of the temperature-sensitive plasmid. Since pCP20 carries an ampicillin resistance marker, the presence or absence of ampicillin resistance can be used to determine the presence of the plasmid.

[0041] When single colonies grow on ampicillin-resistant plates cultured at 42°C, select a number of single colonies and inoculate them into antibiotic-free LB liquid medium. After incubating at 37°C for a period of time, streak the culture medium onto three types of plates: antibiotic-free LB plates, ampicillin-containing LB plates, and chloramphenicol-containing LB plates. After 16 hours of incubation at 37°C, if some clones grow only on antibiotic-free LB plates and not on either ampicillin-containing or chloramphenicol-containing plates, it indicates that these clones have lost both pKD46 and pCP20 (loss of ampicillin resistance) and have had their chloramphenicol resistance cassettes excised from the chromosome through FLP-FRT recombination (loss of chloramphenicol resistance). If no clones grow on the 42°C ampicillin plate, clones can be obtained first on a 30°C ampicillin plate using the standard procedure, and then the above screening steps can be repeated after 42°C liquid culture and antibiotic-free streak until a strain sensitive to both ampicillin and chloramphenicol is obtained.

[0042] Finally, the selected dual-sensitive clones were verified by colony PCR and sequencing to confirm that they were *Vibrio natriureticis*. mraY The original E. coli has been stably replaced. mraY Furthermore, the antibody cassette has been completely removed. PCR primers were designed in... mraY upstream more distant conservative area and mraY Further downstream conserved regions are included to ensure that the amplified product covers the entire replacement region. By comparing the size of the amplified fragment with the sequencing results, the inserted sodium-dependent Vibrio can be confirmed. mraY The sequence is complete and error-free, and the homologous arms on both sides are precisely aligned with the MC4100z1 chromosome. The strain constructed through the above steps is MC4100z1. ΔmraY :: mraY Vn The strain, under standard LB culture conditions, exhibited growth characteristics largely consistent with the original MC4100z1, but its MraY activity differed from that of sodium-dependent Vibrio. mraY This provides an ideal engineered host for subsequent evaluation of the lytic ability of phage E protein and its variants in the "sodium-dependent Vibrio MraY environment".

[0043] To verify the effect of the matching degree between the E protein and heterologous MraY on the function of the self-cleavage circuit, the complete ePop self-cleavage gene circuit was introduced into the wild-type Escherichia coli chassis MC4100z1 (endogenous MraY) and the ZM chassis (sodium-dependent Vibrio MraY) constructed above, respectively, to obtain MC-ePop and ZM-ePop strains, and their growth curves were detected. Figure 3 ).

[0044] The results showed that the MC-ePop strain exhibited a typical "growth-lysis-regeneration" oscillation trend, while the lysis oscillation of the ZM-ePop strain was significantly weakened, with a smaller decrease in OD600 and a faster recovery. This indicates that the wild-type E protein is not well-matched with the sodium-dependent Vibrio natriureticus MraY and cannot effectively trigger stable lysis. This result directly confirms the core issue of "E protein lysis effect depending on host MraY adaptability," further illustrating the necessity of constructing an E protein variant screening platform for MraY, and providing experimental evidence for subsequent screening work.

[0045] Example 2: Construction of the coding gene and expression vector for the E protein K33R mutant 1. Obtaining the gene encoding the K33R mutant Using the phage ΦX174E protein gene sequence as a template, the amino acid sequence of the K33R mutant was obtained by changing lysine (Lys, K) to arginine (Arg, R) at amino acid position 33. Based on the codon preference of *E. coli*, codon optimization was performed on this amino acid sequence. Full-length synthetic primers were designed, or a double-stranded DNA fragment was synthesized by a company to obtain the nucleic acid sequence encoding the K33R mutant, as shown in SEQ ID No. 1, and the amino acid sequence is shown in SEQ ID No. 2.

[0046] 2. Preparation of the expression carrier skeleton A medium-copy plasmid containing the pColE1-rop replicon was selected as the expression backbone, and an anhydrotetracycline (aTc)-induced promoter P was configured on the plasmid. tet The vector also contains a ribosome binding site (RBS) of appropriate strength and a transcription terminator. It also carries a chloramphenicol resistance marker for screening positive clones in culture medium.

[0047] 3. Homologous recombination clonal ligation of the K33R gene and vector Using the synthesized K33R gene fragment as a template, a pair of primers, K33R-F / K33R-R, were designed for amplification. The 5' ends of both primers featured 15–30 bp homologous overlapping sequences based on the linearized end sequence of the target vector, while the 3' ends were designed with specific sequences complementary to the K33R coding sequence. This ensured that the amplified product had sufficiently long homologous arms at both ends of the linearized vector, suitable for Gibson-based homologous recombination cloning. PCR was performed using a commercially available high-fidelity DNA polymerase, such as the high-fidelity PCR enzyme system provided by Novizan. A 50 μL reaction volume was prepared according to the instructions, and pre-denaturation, cyclic denaturation / annealing / extension, and end extension programs were set to ensure a single target band. After amplification, a portion of the product was subjected to 1% agarose gel electrophoresis. After confirming that the band size matched the theoretical length, the remaining product was recovered, purified, and the DNA concentration was determined for later use.

[0048] The vector backbone used was the same plasmid as that used for subsequent expression, specifically the pColE1-rop replicon vector for expressing the E protein. The vector was linearized by PCR, exposing homologous sequences at both ends consistent with the 5' ends of the K33R fragment primers. The linearized product was confirmed by agarose gel electrophoresis and then purified to determine the DNA concentration.

[0049] Subsequently, using a Gibson-based homologous recombination cloning kit (such as the homologous recombination cloning kit provided by Beijing TransGen Biotech Co., Ltd.), the linearized vector and the K33R insert were mixed according to the molar ratio recommended in the manufacturer's instructions. Generally, the molar ratio of vector to insert is controlled at 1:3, and the total reaction volume is 10 μL. An appropriate amount of homologous recombination reaction solution was added, and the mixture was reacted at 50℃ for 15 min. This allowed the vector and insert to engage at the ends in the homologous arm region, and the gap was filled and ligated through an enzymatic reaction, forming a closed circular recombinant plasmid.

[0050] After the recombination reaction, 3 μL of the reaction solution was transformed into competent *E. coli* DH5α cells using the standard heat shock method. Immediately after transformation, 500 μL of antibiotic-free LB broth was added, and the cells were incubated at 37°C with shaking for 1 h. The culture was then plated onto LB agar plates containing 25 μg / mL chloramphenicol and incubated overnight at 37°C. The next day, several single clones were picked for initial colony PCR screening. PCR primers were placed within the vector backbone, near the insertion site, or inside the insert fragment to confirm the correct insert size. PCR-positive clones underwent further plasmid extraction, and restriction endonuclease digestion analysis was used to confirm the presence and orientation of the insert fragment. The plasmid was then sent for Sanger sequencing to sequence the full-length K33R coding region, confirming that it contained only the expected K33R mutation and no other unexpected mutations. The recombinant plasmid that was correctly identified was named pE-K33R and used as the expression vector.

[0051] 4. Construction of expression strains The pE-K33R expression plasmid, which was verified by sequencing, was transformed into MC4100z1. ΔmraY :: mraY Vn The ZM chassis strain was transformed using a heat shock method to prepare competent cells. After transformation, the cells were recovered in antibiotic-free LB liquid medium at 37°C for 1 hour, then plated on LB agar plates containing 25 μg / mL chloramphenicol and incubated overnight at 37°C. The next day, chloramphenicol-resistant single clones were picked and identified by colony PCR or plasmid tick and restriction enzyme digestion to confirm the presence of the pE-K33R plasmid, thus obtaining the ZM / pE-K33R engineered strain.

[0052] As a control, the plasmid pE-WT expressing the wild-type E protein was transformed into a ZM chassis in the same manner. Chloramphenicol resistance screening and confirmation by PCR / sequencing yielded the ZM / pE-WT control strain. These two strains were used in subsequent examples to compare the cleavage ability of K33R and wild-type E proteins under the same chassis, vector backbone, and induction conditions.

[0053] Example 3: Evaluation of the cracking capacity of K33R in a ZM chassis refer to Figure 4 The process involves culturing to a specified OD level, uniform induction, real-time monitoring of growth curves, automatic extraction of four parameters, and comprehensive scoring to select the best candidate organisms. This is to obtain... Figure 6 The scatter plot shown illustrates how, in this embodiment, a group of well-identified E protein variants were measured and uniformly parameterized under the same chassis cell background. The variants measured included: site-directed mutants of key sites identified by analysis of the E-MraY-SlyD complex structure and interaction interface based on wild-type E protein (e.g., replacement of interface positively charged anchor sites or geometrically related sites at bends); and candidate mutants derived from hotspot sites exhibiting a "gain-type" kinetic phenotype in alanine scans. Furthermore, to analyze the contribution of different physicochemical properties within the hotspot window to cleavage kinetics, small-scale saturation substitutions / property grouping substitutions were performed in the Pro transition region, resulting in a batch of variants for comparing charge, polarity, and volume effects. Figure 6 The mutants in the scatter plot originate from the three categories mentioned above: structure / interface-guided site-directed mutation libraries, gain hotspot-derived libraries screened by alanine scanning, and small-range saturation / property substitution libraries within hotspot windows; among them, K33R is a structure / interface-guided key-site site-directed substitution variant, used to optimize interface pairing ability while maintaining positive electrical characteristics. The specific steps are as follows: ZM / pE-K33R and ZM / pE-WT single clones were inoculated into 3 mL LB (containing 25 μg / mL chloramphenicol) and pre-cultured overnight at 37°C with shaking at 220 r / min. The next day, the overnight bacterial culture was inoculated 1:100 into fresh LB+Cm medium and incubated at 37°C with shaking until OD600 ≈ 0.4. The bacterial culture was then diluted 1:40 into LB containing Cm to make the initial OD600 approximately 0.01. 200 μL of the treated bacterial culture was added to each well of a 96-well clear microplate. Using a multi-functional microplate reader with 37°C temperature control and shaking function, the measurement wavelength was set to 600 nm, and OD600 was automatically read and recorded every 10 min. At the 4-hour time point, aTc was added to a final concentration of 200 ng / mL, and monitoring was continued for 24 h. At least 3 technical replicates were set up for each strain.

[0054] After the experiment, the OD600-time data of each well were exported as a tabular file, and wells with obvious contamination and abnormal bubbles were removed. Using a self-written R script, each curve was fitted to a three-segment logistic function: the first segment represents the exponential growth phase, the second segment represents the inhibition / lysis phase affected by E protein expression, and the third segment represents the regeneration phase of residual or mutant cells. The minimum derivative of the curve in the second segment was calculated using numerical differentiation, which was taken as the maximum instantaneous lysis rate; the lysis ratio was calculated using the highest OD600 before lysis and the lowest OD600 after lysis; the lysis delay time was defined as the time required for the derivative to reach its minimum value after the addition of the inducer; and the net area difference ΔAUC was obtained by numerically integrating the curve after the addition of the inducer, representing the degree of inhibition of overall growth by lysis.

[0055] To visually compare the cleavage performance of multiple E protein variants, a scatter plot was created with the minimum derivative (instantaneous maximum cleavage rate) on the x-axis and the net area under the curve (ΔAUC) on the y-axis, as shown below. Figure 6 As shown, quadrants are divided using the wild-type E protein (Parent) as the baseline. The lower left quadrant represents a strong cleavage region characterized by "faster cleavage rate (smaller minimum derivative value) and stronger growth inhibition (smaller ΔAUC)". The results show that the K33R variant falls within this strong cleavage quadrant and performs exceptionally well among all tested variants (enlarged view with red box on the right), further quantifying its cleavage advantage over the wild-type E protein and other variants.

[0056] Figure 5The results showed that, under the same induction and culture conditions, the growth-lysis curves of the ZM / pE-K33R engineered strain were significantly different from those of the ZM / pE-WT strain. Firstly, the descent phase of the K33R group started earlier, and the lysis delay time was shortened by approximately 10%. Secondly, the slope of the descent curve was steeper, the corresponding minimum derivative value was more negative, and the maximum lysis rate was increased. Thirdly, the K33R group had a lower minimum OD600 value and a higher lysis ratio after lysis. Simultaneously, the ΔAUC of K33R was significantly lower than that of the wild-type E protein, indicating stronger inhibition of total growth. Specific parameter values ​​are shown in Table 1.

[0057] Table 1. Specific parameter values ​​for E protein and its variant K33R.

[0058] The above results indicate that in the ZM chassis carrying sodium-dependent Vibrio mraY, the K33R mutant has a significantly enhanced cleavage ability compared to the wild-type E protein.

[0059] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

[0060] sequence list SEQ ID No.1 ATGGTACGCTGGACTTTGTGGGATACCCTCGCTTTCCTGCTCCTGTTGAGTTTATTGCTTGCCGTCATTGCTTATTATGTTCATCCCGTCAACATTCCGTCGGCCTGTCTCATCATGGAAGGCGCTGAATTTACGGAAA ACATTATTAATGGCGTCGAGCGTCCGGTTAAAGCCGCTGAATTGTTCGCGTTTACCTTGCGTGTACGCGCAGGAAACACTGACGTTCTTACTGACGCAGAAGAAAACGTGCGTCAAAAATTACGTGCGGAAGGAGTGA SEQ ID No.2 MVRWTLWDTLAFLLLLSLLLPSLLIMFIPSTFRRPVSSWKALNLRKTLLMASSVRLKPLNCSRLPCVYAQETLTFLLTQKKTCVKNYVQKE SEQ ID No. 3 SEQ ID No.4 GACTACTCCGATCGC SEQ ID No.5 GCAAGCCAGATAATCATTAACATGTCCCATTCTCCTGTAAAGCG SEQ ID No.6 GCGTAATCTGCTGCTCATGGCTGATTATCAGGGTAAAAATGTCG SEQ ID No.7 AGACCAGGATACTGAATC SEQ ID No.8 ATGATTATCTGGCTTGCCGAGC SEQ ID No.9 TTAACGCACCTTCAGTGTAGCTAGAC SEQ ID No.10 ACTGAAGGTGCGTTAATCAAAGGACTGACGTCGATATCTGG SEQ ID No.11 AGCAGCAGATTACGCGCAG。

Claims

1. A phage E protein mutant, characterized in that, Compared to the original phage E protein, the mutant phage E protein has a lysine residue at position 33 mutated to arginine.

2. The phage E protein mutant according to claim 1, characterized in that, The amino acid sequence of the phage E protein mutant is shown in SEQ ID NO.

2.

3. A nucleic acid molecule encoding the phage E protein mutant as described in claim 1 or 2.

4. An expression cassette containing the nucleic acid molecule of claim 3.

5. The expression box according to claim 4, characterized in that, The expression cassette also includes a replicon, a promoter, a ribosome binding site, and a transcription terminator.

6. A recombinant vector containing the nucleic acid molecule of claim 3 or the expression cassette of claim 4.

7. An engineered strain containing the nucleic acid molecule of claim 3, the expression cassette of claim 4, or the recombinant vector of claim 6.

8. The engineered strain according to claim 7, characterized in that, The engineered strain is a sodium-dependent Vibrio bacterium. mraY Engineered strains of genes.

9. The engineered strain according to claim 8, characterized in that, The engineered strain is a sodium-dependent Vibrio bacterium. mraY Genetically modified E. coli.

10. The application of the phage E protein mutant according to claim 1 or 2 in the construction of a controllable bacterial lysis system.

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