A biosafety enhanced penicillin g degradation engineering bacteria and a construction method and application thereof

Abstract

The present application belongs to the technical field of microbial treatment of environmental pollutants, and particularly relates to a penicillin G degradation engineering bacterium with high biological safety based on beta-lactamase splitting-recombination and a construction method and application thereof. The present application takes E. coli W as a safe chassis strain, adopts high-activity TEM-1 beta-lactamase as a core degradation element, and combines penicillin G acylase to construct a complete metabolic pathway, so that the engineering bacterium can efficiently degrade and utilize penicillin G as a sole carbon source. Meanwhile, the AmpR-AmpC induction regulation system is used to realize precise expression of the degradation gene, effectively reduce the metabolic burden of the bacterium, and improve the environmental adaptability and functional stability of the engineering bacterium. In addition, the SpyTag / SpyCatcher system is used to split and modify TEM-1, so that the complete resistance function is only recombined and recovered under specific conditions, thereby greatly reducing the risk of horizontal transfer of the resistance gene from the source.

Description

Technical Field

[0001] This invention belongs to the field of microbial treatment technology for environmental pollutants, specifically relating to a penicillin G degrading engineered bacterium with high biocompatibility (low resistance gene transfer frequency), based on β-lactamase splitting-recombination, its construction method, and its application. Background Technology

[0002] Penicillin G, as the most widely used β-lactam antibiotic, plays an irreplaceable role in many fields such as medical diagnosis and treatment, animal husbandry, and agricultural production. However, its widespread and unregulated use has led to persistent residues in natural environments such as soil and water, becoming a global environmental problem. Penicillin G residues in the environment not only directly disrupt the micro-ecological balance of water and soil but also create environmental selection pressure, accelerating the spread and diffusion of resistance genes in the environment. This poses a long-term potential threat to ecosystem security and human public health. The widespread dissemination of resistance genes may even weaken the therapeutic effects of existing antibiotics, leading to more severe public health challenges.

[0003] Currently, the treatment technologies for penicillin G residues in the environment are mainly divided into two categories: physicochemical treatment technologies and biological treatment technologies. Among them, physicochemical treatment technologies, as traditional methods, can achieve efficient removal of penicillin G in the short term, but they generally have inherent drawbacks such as high treatment costs, complex operation procedures, and easy generation of secondary pollutants. Biological treatment technologies, especially microbial degradation methods, have shown a broader application prospect due to their unique advantages such as low cost, environmental friendliness, and no secondary pollution.

[0004] However, most naturally occurring penicillin G-degrading bacteria cannot grow using penicillin G as their sole carbon source. Their degradation process largely relies on β-lactamase-mediated resistance mechanisms, achieving only structural inactivation of the antibiotic rather than effective metabolic utilization, and the relevant metabolic pathways remain unclear. More critically, whether naturally occurring or engineered, the β-lactamase resistance genes carried by these bacteria can easily spread in the natural environment through horizontal gene transfer pathways. This issue has become a core bottleneck restricting the safe application of microbial degradation technologies. With antibiotic resistance genes being identified as emerging environmental pollutants, their cross-species transmission within the human-animal-environment system has become a global public health and safety challenge. If penicillin G-degrading strains carrying resistance genes are accidentally released into the natural environment during bioremediation, the resistance genes they carry can spread to native environmental microbial communities through horizontal gene transfer pathways such as conjugation, transformation, and transduction. This not only exacerbates environmental antibiotic resistance pollution but may also foster multidrug-resistant strains, further disrupting the ecological balance and posing a potential threat to human health. Therefore, how to improve the biosafety of degrading strains while ensuring the degradation efficiency of penicillin G and blocking the spread of resistance genes from the source has become a key issue that urgently needs to be addressed in the development of current penicillin G pollution bioremediation technology. Summary of the Invention

[0005] This invention aims to overcome the shortcomings of existing penicillin G degrading bacteria, such as low degradation efficiency, poor adaptability, high risk of horizontal transfer of resistance genes, difficulty in achieving both degradation function and biosafety, low utilization rate of natural strain resources, and inability to verify degradation effect in situ simultaneously. It provides a biosafety-enhanced penicillin G degrading engineered bacterium and its construction method, and applies it to degrade penicillin G residual pollution in environmental samples.

[0006] To achieve the above-mentioned objectives, the present invention is implemented through the following technical solution: A biosafety-enhanced penicillin G-degrading engineered bacterium, wherein the penicillin G-degrading engineered bacterium comprises: Chassis strain (ΔampC strain), wherein the chassis strain is a genetically engineered bacterium in which the endogenous ampC gene has been knocked out; The degradation gene cluster includes at least the pac gene encoding penicillin G acylase and the split and modified β-lactamase gene tem-1; Specifically, the modified β-lactamase gene tem-1 is split into a gene fragment encoding a first protein fragment and a gene fragment encoding a second protein fragment. The first and second protein fragments are respectively fused with protein tag pairs that can specifically recognize and bind to the protein fragments, so that only after the first and second protein fragments are reassembled through the protein tag pairs can the TEM-1 β-lactamase with complete catalytic activity be reconstructed.

[0007] Existing microbial methods for degrading penicillin G struggle to balance degradation efficiency and biosafety, failing to meet the safety requirements of practical environmental remediation. Constructing an engineered penicillin G-degrading bacterium with both effective degradation capabilities and low risk of resistance gene transfer is of great significance for promoting the standardized and safe application of penicillin G pollution bioremediation technology, alleviating drug-resistant pollution in the environment, and protecting ecological security and human health.

[0008] Based on this, this invention discloses a construction scheme for a biosafety-enhanced penicillin G-degrading engineered strain based on β-lactamase splitting-recombination. The engineered strain uses *Escherichia coli* W (ATCC 9637) as the chassis strain, TEM-1 as a highly active penicillin G-degrading enzyme, AmpR-AmpC as the regulatory system, and utilizes the SpyTag / SpyCatcher (ST-SC) system to split and modify the resistance gene, ultimately obtaining a penicillin G-degrading engineered strain with both high degradation capacity and high biosafety. This effectively solves the ecological risks caused by excessive bacterial proliferation and the spread of resistance genes, providing a feasible and sustainable technical solution for the efficient management of penicillin G environmental residues, blocking the spread of resistance genes, and overcoming the crisis of antibiotic resistance spreading in the environment. It also provides a new approach for the design and construction of environmentally friendly antibiotic-degrading bacteria.

[0009] Preferably, the protein tag pair is the SpyTag / SpyCatcher system; The first protein fragment is fused with a SpyTag or a SpyCatcher, and the second protein fragment is fused with a corresponding SpyCatcher or a SpyTag.

[0010] The SpyTag / SpyCatcher (ST-SC) isopeptide assembly system is a protein covalent linking system with high specificity and stability. Its core consists of a SpyTag polypeptide (a short peptide containing about 13 amino acids) and a SpyCatcher protein (a protein domain containing about 116 amino acid residues). The reaction between the two does not require an additional catalyst and can occur rapidly under physiological conditions. It has both high specificity and linking stability, so it is often used to disassemble functional proteins, reconstruct enzyme activity, construct multi-subunit complexes, or achieve directed fixation and assembly of proteins.

[0011] Preferably, the first protein fragment consists of a portion of the amino acid sequence of SpyTag, a flexible linker peptide, and TEM-1 β-lactamase; the second protein fragment consists of the remaining amino acid sequence of SpyCatcher, a flexible linker peptide, and TEM-1 β-lactamase.

[0012] As a further preferred option, the flexible linker peptide is Gly(4)-Ser-(4)-Ser.

[0013] This invention modifies TEM-1, a class A β-lactamase isolated from clinically resistant E. coli. The complete TEM-1 is site-specifically truncated, splitting into two protein fragments without independent catalytic activity. These fragments are then fused with SpyTag and SpyCatcher sequences via a flexible linker peptide (Gly(4)-Ser-(4)-Ser), respectively, to obtain ST-BLF1 and SC-BLF2 fusion fragments. The two fusion fragments are then processed in SP... tem-1 Assembly and renaturation were completed in the periplasmic space under the guidance of the signal peptide. Specifically, the SC-BLF2 fragment was integrated into the genome of strain ΔampC, using the pHG101 plasmid as a vector and the constitutive moderate-strength promoter P was selected. J23106 By controlling the expression of the ST-BLF1 fragment, a biosafety-enhanced penicillin G degradation engineered strain based on β-lactamase splitting-recombination was constructed.

[0014] Preferably, the strain in the chassis is Escherichia coli.

[0015] As a further preferred embodiment, the chassis strain is Escherichia coli W strain or a derivative thereof.

[0016] The strain Escherichia coli W, abbreviated as E. coli W, has the public accession number ATCC 9637.

[0017] Preferably, the penicillin G degrading engineered bacteria further includes an AmpR-AmpC regulatory system, and the expression of the pac gene is regulated by the AmpR-AmpC regulatory system.

[0018] Preferably, the AmpR-AmpC regulatory system is induced and activated in response to the presence of penicillin G or its derivatives, thereby driving pac gene expression.

[0019] Penicillin G acylase (PGA, encoded by the pac gene) acts as a downstream degradation element, further metabolizing and transforming the hydrolysis products of TEM-1 to provide a usable carbon source for the strain, thus enabling the engineered degradation bacteria to grow using penicillin G as the sole carbon source. The AmpR-AmpC regulatory system, as the most thoroughly studied β-lactamase-induced expression regulation mechanism in Gram-negative bacteria, can precisely regulate the efficient expression of downstream genes in the presence of antibiotics and their low-level expression in the absence of antibiotics through dynamic changes in signal molecules.

[0020] This invention utilizes the AmpR-AmpC regulatory system to control the expression of the pac gene and integrates it into the genome of the ΔampC strain. In the presence of penicillin G, this regulatory system is specifically induced, driving efficient pac gene expression and ensuring efficient penicillin G degradation. In the absence of penicillin G, the regulatory system is suppressed, and the pac gene maintains low-level expression. This regulatory mode not only effectively reduces the metabolic burden on the strain but also inhibits excessive bacterial proliferation after the penicillin G degradation reaction, further reducing the ecological risk of accidental release of the engineered bacteria into the natural environment, achieving a dual improvement in degradation efficiency and biosafety.

[0021] Preferably, the gene segment encoding the first protein fragment is located on a mobile genetic element, and the gene segment encoding the second protein fragment is located on the chromosome of the chassis strain.

[0022] As a further preferred embodiment, the mobile genetic element is a plasmid.

[0023] Preferably, the penicillin G-degrading engineered bacteria can grow using penicillin G as the sole carbon source.

[0024] The method for constructing a biosafety-enhanced penicillin G-degrading engineered bacterium as described above includes the following steps: S1: Construct a chassis strain with the endogenous ampC gene knocked out; S2: Integrate the gene fragment encoding the second protein fragment into the genome of the chassis strain; S3: Construct an expression vector containing a gene encoding the first protein fragment and a pac gene, and introduce the expression vector into the chassis strain to obtain the biosafety enhanced penicillin G degrading engineered bacteria.

[0025] This invention uses Escherichia coli W strain (ATCC 9637) as the starting strain and employs λ-Red homologous recombination knockout technology to knock out the ampC gene in E. coli W to obtain the ΔampC strain. The expression of the pac gene is regulated using the AmpR-AmpC regulatory system, and the β-lactamase gene tem-1 is split and recombined using the SpyTag / SpyCatcher (ST-SC) assembly system, thus achieving the construction of a biosafety-enhanced penicillin G degradation engineered strain based on β-lactamase splitting and recombination.

[0026] Preferably, in step S1, the endogenous ampC gene is knocked out using λ-Red homologous recombination technology; in step S2 and / or step S3, gene fragments are assembled and integrated using fusion PCR and / or seamless cloning technology.

[0027] Preferably, in step S3, the relevant genes of the AmpR-AmpC regulatory system are integrated together with the pac gene into the genome of the chassis strain or into the expression vector.

[0028] The application of a biosafety-enhanced penicillin G-degrading engineered bacterium, as described above, in degrading penicillin G residues in environmental samples.

[0029] Preferably, the environmental sample includes water, soil, or sediment.

[0030] Therefore, the present invention has the following beneficial effects: This invention uses *E. coli* W as a safe chassis strain and employs the highly active TEM-1 β-lactamase as the core degradation element, combined with penicillin G acylase to construct a complete metabolic pathway. This allows the engineered bacteria to efficiently degrade and utilize penicillin G as the sole carbon source. Simultaneously, the AmpR-AmpC induction and regulation system enables precise expression of degradation genes, effectively reducing the metabolic burden on the bacteria and improving their environmental adaptability and functional stability. Furthermore, the SpyTag / SpyCatcher system is used to split and modify TEM-1, allowing the complete resistance function to recombine and recover only under specific conditions. This significantly reduces the risk of horizontal transfer of resistance genes from the source and inhibits the excessive proliferation of engineered bacteria in the environment. This invention successfully overcomes the bottleneck of existing degradation technologies that struggle to balance degradation efficiency and biosafety. It achieves efficient remediation of penicillin G residues while effectively blocking the spread of resistance genes in the environment, reducing the ecological risk of multidrug-resistant bacteria. This provides an efficient and feasible technical solution and a novel design concept for the safe and standardized application of antibiotic pollution bioremediation. Attached Figure Description

[0031] Figure 1 Diagram of plasmid construction.

[0032] Figure 2 A schematic diagram of the construction of engineered bacteria for penicillin G degradation.

[0033] Figure 3 This is a threshold diagram showing the survival density of engineered bacteria that degrade penicillin G.

[0034] Figure 4 The growth curve and degradation curve of the engineered bacteria that degrade penicillin G are shown.

[0035] Figure 5 The growth curves and degradation curves of engineered bacteria that degrade penicillin G under simulated conditions are shown. Figure 5 In the figure, A represents the degradation curve of penicillin G at an initial concentration of 0.01 g / L and the growth curve of the bacterial strain. Figure 5In the figure, B represents the degradation curve of penicillin G at an initial concentration of 0.3 g / L and the growth curve of the bacterial strain. Detailed Implementation

[0036] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. Those skilled in the art will be able to implement the present invention based on these descriptions. Furthermore, the embodiments of the present invention described below are generally only some, not all, of the embodiments of the present invention. Therefore, all other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort should fall within the scope of protection of the present invention.

[0037] Example 1: Construction of ΔampC chassis strain This invention employs λ-Red homologous recombination technology to achieve precise knockout and knock-in of target genes. This technology relies on the synergistic effect of phage λ-derived Exo exonuclease, Beta annealing protein, and Gam anti-degradation protein to mediate homologous recombination between linear donor DNA containing short homologous arms and genomic target sites in vivo.

[0038] (1) The pKD46 plasmid was introduced into E. coli W (ATCC 9637) competent cells by chemical transformation and cultured in a 30℃ incubator for 12-16 h. The correct strain was named E. coli W / pKD46 and electrotransformation competent cells were prepared.

[0039] (2) Amplification of Kan containing FRT sites using pKD4 plasmid as template r The core fragment of the resistance gene was extracted, and homologous arms of the ampC gene, extending 600-800 bp upstream and downstream, were amplified simultaneously. The three fragments were then purified and used as a mixed template for fusion PCR amplification to obtain the complete donor DNA fragment.

[0040] (3) The purified donor DNA was transferred into E. coli W / pKD46 competent cells by electroporation and cultured at 30 °C for 12-16 h. Single colonies growing on the plates were picked and colony PCR was used for preliminary verification.

[0041] (4) Inoculate the PCR-verified strain into LB liquid medium containing 50 μg / mL Kan and incubate overnight at 37 ℃ or 42 ℃ with shaking. Dilute the culture in a series and spread it evenly on LB solid medium containing 50 μg / mL Kan (kanamycin), and incubate at 37 ℃ for 12-16 h. Pick single colonies growing on the plates and screen for strains that can grow only on Kan plates (kanamycin resistance selection plates) and not on Amp plates (ampicillin resistance selection plates). This indicates that the pKD46 plasmid carried by the strain has been successfully lost, i.e., the correct strain with recombination system clearance has been obtained.

[0042] (5) Prepare chemically transformed competent cells from the correct strain. Transform the pCP20 plasmid into the competent cells using chemical transformation. After transformation, plate the bacterial culture onto LB solid medium containing 25 μg / mL Chl (chloramphenicol) and incubate at 30 ℃ for 12-16 h. Pick single colonies growing on the plates and screen for strains that grow normally only on Chl plates (chloramphenicol resistance selection plates) and do not grow on Kan plates (kanamycin resistance selection plates). These are the Kan strains. r The strain with successfully eliminated resistance genes.

[0043] (6) Inoculate the strains containing the pCP20 plasmid into LB liquid medium and incubate overnight at 37 °C with shaking. Dilute the bacterial culture serially and spread it onto LB solid medium, incubating at 37 °C for 12–16 h. Pick single colonies and streak them onto Chl plates (chloramphenicol resistance selection plates) and LB plates respectively. Select strains that grow only on LB plates and not on Chl plates (chloramphenicol resistance selection plates) to indicate that the pCP20 plasmid has been completely eliminated.

[0044] (7) Finally, the colony PCR and sequencing verification were performed. The correctly sequenced strain was the label-free ampC knockout strain ΔampC.

[0045] Example 2: Construction of penicillin G-degrading engineered strains (1) ampR-P ampC -pac fragments and P J23106 The SC-BLF2 fragment was integrated into strain ΔampC using λ-Red homologous recombination technology to obtain ΔampC-BLF2-ampR-P ampC -pac strain.

[0046] (2) Design specific primers and amplify pHG101-P using 2×Phanta® Flash Master Mix. J23106 The vector was linearized, and the ST-BLF1 fragment was obtained through gene synthesis. pHG101-P was digested using Dpn I enzyme.J23106 The original plasmid was used to remove the methylated template plasmid. Then, the pHG101-P plasmid was cloned using the pEASY®-Basic Seamless Cloning and Assembly Kit (purchased from TransGen Biotech, Beijing). J23106 The linearized fragment and the ST-BLF1 fragment are concatenated and transformed into ΔampC-ampR-P. ampC -pac-P J23106 -SC-BLF2 competent cells were used to construct ΔampC-BLF2-ampR-P ampC -pac / P J23106 -BLF1 strain. A schematic diagram of plasmid construction is shown below. Figure 1 As shown in the diagram. The construction model of the penicillin G-degrading engineered bacteria is shown in the diagram. Figure 2 As shown. ΔampC-BLF2-ampR-P ampC -pac / P J23106 -BLF1 strain (i.e., Escherichia coli PGD04) is deposited at the China Center for Type Culture Collection (CCTCC) on May 20, 2026, with accession number CCTCCNO: M 20261018.

[0047] Example 3: Determination of the survival density threshold of penicillin G-degrading engineered bacteria (1) The penicillin G degradation engineered bacteria ΔampC-BLF2-ampR-P ampC -pac / P J23106 -BLF1 seed culture (preparation method see Examples 1-2) was transferred to fresh LB medium at a ratio of 1:100 and cultured until the late logarithmic growth phase (OD2). 600 ≈0.8).

[0048] (2) Take a sterile 1.5 mL centrifuge tube and perform serial dilutions of 5-fold. Take 3 μL of bacterial solution from each dilution and add it vertically to LB agar plates containing serial penicillin G. Set up 3 replicates for each dilution and set up a blank control plate without antibiotic. Let stand for 5-10 min until the bacterial solution is completely absorbed, and incubate upside down at 37 ℃ for 12 h. Take a picture and record the results after the incubation.

[0049] (3) Based on the number of colonies and the spotting area (approximately 0.79 cm²) on the control plate without antibiotics. 2 ), calculate the expected cell density for each sampling region. The survival density threshold diagram for penicillin G-degrading engineered bacteria is shown below. Figure 3 As shown.

[0050] Depend on Figure 3 Analysis shows that: ΔampC-BLF2-ampR-PampC -pac / P J23106 -BLF1 strains grew normally at 0–25 μg / mL penicillin G; at 50 μg / mL, only extremely high cell density (1.88 × 10⁻⁶) was observed. 6 cells / cm 2 The presence of recombinant enzymes in the periplasmic space indicates that the bacteria can survive, but single colonies cannot, suggesting that they partially recover β-lactamase activity and exhibit a strict population density dependence. This indicates that only when the cell density is sufficiently high can the concentration of recombinant enzymes accumulated in the periplasmic space effectively degrade the corresponding concentration of penicillin G and ensure the survival of the bacterial community. This effectively avoids the over-proliferation of individual cells in the screening environment and provides important biosafety assurance for the degradation of engineered bacteria.

[0051] Example 4: Determination of the growth and degradation performance of engineered penicillin G-degrading bacteria (1) Take penicillin G degradation engineered bacteria ΔampC-BLF2-ampR-P ampC -pac / P J23106 Seed culture of -BLF1 (preparation method see Examples 1-2) was centrifuged at 8000 rpm and 4 ℃ for 5 min to collect bacterial cells, which were then washed twice with M9 basic salt medium. The M9 basic salt medium formula is: 6.78 g disodium hydrogen phosphate (Na2HPO4), 3 g potassium dihydrogen phosphate (KH2PO4), 0.5 g sodium chloride (NaCl), and 1 g ammonium chloride (NH4Cl), pH 6.8±0.2, dissolved in 1 L of ultrapure water, and sterilized at 115 ℃ for 30 min.

[0052] (2) Transfer the bacterial cells to M9 basic salt medium supplemented with penicillin G at a ratio of 1:50 (v / v) and culture at 30 ℃ and 180 rpm with constant temperature shaking.

[0053] (3) Samples were taken every 12 hours. After the samples were filtered through a 0.22 μm filter membrane, the residual concentration of penicillin G was determined by HPLC. The degradation curve was plotted with the culture time (h) as the x-axis and the residual concentration of penicillin G (mg / mL) as the y-axis.

[0054] The HPLC method was used to determine the content of penicillin G. The specific detection conditions were as follows: Shimadzu C18 column (250 mm × 4.6 mm, 5 μm); 0.2 mol / L potassium dihydrogen phosphate buffer (pH 3.5): methanol = 38:62 (v / v), freshly prepared and used, ultrasonically degassed for 15 min; column temperature 25 ℃; flow rate: 1.0 mL / min; injection volume: 20 μL; detection wavelength: 225 nm; run time 10 min.

[0055] (4) In addition, OD was measured using an enzyme-linked immunosorbent assay (ELISA) reader. 600Values ​​were used to record the growth of the bacterial strain, with culture time (h) as the x-axis and OD value as the y-axis. 600 Plot the growth curve on the ordinate. The growth and degradation curves of the penicillin G-degrading engineered bacteria are shown below. Figure 4 As shown.

[0056] Depend on Figure 4 Analysis shows that the penicillin G degrading engineered bacteria ΔampC-BLF2-ampR-P ampC -pac / P J23106 -BLF1 can grow normally using penicillin G as the sole carbon source. In the early stage of culture (0~72 h), the bacterial cell concentration OD 600 The concentration of penicillin G increased continuously with prolonged culture time, stabilized after 72 h, and was completely degraded at 0.3 g / L after 72 h.

[0057] Example 5: Application of engineered bacteria that degrade penicillin G Water samples were collected from the river to simulate the penicillin G-degrading engineered bacteria ΔampC-BLF2-ampR-P ampC -pac / P J23106 The degradation of penicillin G by -BLF1 (the preparation method is described in Examples 1-2) in environmental water samples.

[0058] (1) The collected water samples were pretreated as follows: First, large particulate pollutants in the water samples were removed by filtration using a 0.45 μm filter membrane. Then, the samples were centrifuged at 5000 rpm for 20 min at room temperature to further remove fine suspended particles. Finally, penicillin G was added to the pretreated water samples.

[0059] (2) Penicillin G degrading engineered bacteria ΔampC-BLF2-ampR-P ampC -pac / P J23106 For specific procedures regarding the determination of the growth and degradation performance of -BLF1, please refer to Example 4. The growth and degradation curves of the penicillin G-degrading engineered bacteria under simulated conditions are shown in the figure below. Figure 5 As shown. Among them, Figure 5 In the figure, A represents the degradation curve of penicillin G at an initial concentration of 0.01 g / L and the growth curve of the bacterial strain. Figure 5 In the figure, B represents the degradation curve of penicillin G at an initial concentration of 0.3 g / L and the growth curve of the bacterial strain.

[0060] Depend on Figure 5 Analysis showed that when the initial concentration of penicillin G in the river water sample was 0.01 g / L, the substrate concentration continuously decreased with increasing culture time, while the number of viable bacteria (CFU) gradually increased. By 12 h of culture, penicillin G had been completely degraded (see...). Figure 5(Figure A in the figure); when the initial concentration of penicillin G in the river water sample was increased to 0.3 g / L, the OD during the early stage of culture (0~72 h) was... 600 The value gradually increased, but after 72 h, the bacterial growth showed a downward trend. After the substrate was depleted, the bacteria entered the death phase and were completely degraded at 84 h (see [link to article]). Figure 5 (See Figure B in the diagram). This indicates that ΔampC-BLF2-ampR-P ampC -pac / P J23106 The -BLF1 strain exhibited excellent degradation capabilities for different concentrations of penicillin G in river water samples: it achieved rapid and complete degradation at low concentrations, and although the degradation cycle was prolonged at high concentrations, it still managed to complete substrate degradation. Furthermore, the strain's biomass decreased rapidly after substrate depletion, effectively avoiding the potential risk of excessive proliferation in the environment, demonstrating good environmental adaptability and engineering application potential.

[0061] The above description is merely a detailed explanation of preferred embodiments and principles of the present invention. For those skilled in the art, there may be changes in specific implementation methods based on the ideas provided by the present invention, and these changes should also be considered within the scope of protection of the present invention.

Claims

1. A biosafety-enhanced penicillin G-degrading engineered bacterium, characterized in that, The penicillin G-degrading engineered bacteria include: The chassis strain is a genetically engineered bacterium in which the endogenous ampC gene has been knocked out. The degradation gene cluster includes at least the pac gene encoding penicillin G acylase and the split and modified β-lactamase gene tem-1; Specifically, the modified β-lactamase gene tem-1 is split into a gene fragment encoding a first protein fragment and a gene fragment encoding a second protein fragment. The first and second protein fragments are respectively fused with protein tag pairs that can specifically recognize and bind to the protein fragments, so that only after the first and second protein fragments are reassembled through the protein tag pairs can the TEM-1 β-lactamase with complete catalytic activity be reconstructed.

2. The biosafety-enhanced penicillin G-degrading engineered bacteria according to claim 1, characterized in that, The protein tag pair is the SpyTag / SpyCatcher system; The first protein fragment is fused with a SpyTag or a SpyCatcher, and the second protein fragment is fused with a corresponding SpyCatcher or a SpyTag.

3. The biosafety-enhanced penicillin G-degrading engineered bacteria according to claim 2, characterized in that, The first protein fragment consists of a portion of the amino acid sequence of SpyTag, a flexible linker peptide, and TEM-1 β-lactamase; the second protein fragment consists of the remaining amino acid sequence of SpyCatcher, a flexible linker peptide, and TEM-1 β-lactamase.

4. The biosafety-enhanced penicillin G-degrading engineered bacteria according to claim 1, characterized in that, The strain in the chassis is Escherichia coli.

5. The biosafety-enhanced penicillin G-degrading engineered bacteria according to claim 1, characterized in that, The penicillin G-degrading engineered bacteria also contain the AmpR-AmpC regulatory system, and the expression of the pac gene is regulated by the AmpR-AmpC regulatory system.

6. The biosafety-enhanced penicillin G-degrading engineered bacteria according to claim 5, characterized in that, The AmpR-AmpC regulatory system is induced and activated in response to the presence of penicillin G or its derivatives, thereby driving pac gene expression.

7. The biosafety-enhanced penicillin G-degrading engineered bacteria according to claim 1, characterized in that, The gene segment encoding the first protein fragment is located on a mobile genetic element, and the gene segment encoding the second protein fragment is located on the chromosome of the chassis strain.

8. A biosafety-enhanced penicillin G-degrading engineered bacterium according to any one of claims 1 to 7, characterized in that, The penicillin G-degrading engineered bacteria can grow using penicillin G as the sole carbon source.

9. A method for constructing a biosafety-enhanced penicillin G-degrading engineered bacterium as described in any one of claims 1 to 8, characterized in that, Includes the following steps: S1: Construct a chassis strain with the endogenous ampC gene knocked out; S2: Integrate the gene fragment encoding the second protein fragment into the genome of the chassis strain; S3: Construct an expression vector containing a gene encoding the first protein fragment and a pac gene, and introduce the expression vector into the chassis strain to obtain the biosafety enhanced penicillin G degrading engineered bacteria.

10. The application of the biosafety-enhanced penicillin G-degrading engineered bacteria as described in claim 8 in the degradation of penicillin G residues in environmental samples.

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