A method for constructing and optimizing a cell-free catalytic system and its application

Abstract

This invention relates to a method for constructing and optimizing a cell-free catalytic system and its application. The system includes efflux pump proteins for expelling signaling molecules, synthases for synthesizing quorum sensing signaling molecules, transcription factors (TFs), signaling molecule-responsive promoters, lyases, and other functional proteins. Specifically, the synthases synthesize signaling molecules; the efflux pump proteins expel signaling molecules; unexpelled signaling molecules are responded to by transcription factors, and the resulting receptor proteins bind to the signaling molecules, which are then sensed by the signaling molecule-responsive promoters, initiating and regulating the expression of lyases and other related functional proteins. This invention activates the expression of efflux pump proteins by overexpressing them or introducing activation regulatory proteins in the cell-free catalytic system, achieving programmed cell autolysis at high cell density without physical or chemical assistance.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for constructing and optimizing a cell-free catalytic system and its application. This invention discovers and identifies a quorum-sensing signaling efflux pump; utilizes this efflux pump to optimize a quorum-sensing-based microbial cell self-lysis system, which is then applied to the preparation of crude enzyme solutions for further use in biocatalysis and drug delivery for therapeutic purposes. Background Technology

[0002] Cell lysates are typically prepared via mechanical disruption (e.g., high-pressure homogenization and sonication) or chemical methods (e.g., lysozyme treatment and chemical solvent extraction). Biological methods include expressing bacteriophage λ endosomalin in *E. coli*, but this requires additional steps, such as repeated freeze-thaw cycles or freeze-drying, to achieve cell lysis. In contrast, quorum sensing (QS), a cell density-based bacterial communication process, has been used to study cell autolysis. QS senses changes in cell density through self-inducing factors produced by bacteria, thereby coordinating gene expression and behavior. QS-based systems offer a simplified approach, eliminating additional steps and achieving more efficient cell autolysis. These systems have been developed to control bacterial autolysis in mouse models for drug delivery.

[0003] Autoinducers, such as N-acyl-homoserine lactones (HSLs), are produced by synthases within *E. coli* cells (the most widely used host in medicine and industry) and diffuse into surrounding cells. When the bacterial population reaches a certain density threshold, the autoinducers activate response sensors to express a phage lysis gene (φX174E), thereby initiating autolysis. However, the rapid accumulation of autoinducers leads to autolysis occurring at lower cell densities (below OD600 0.2), limiting their application in the preparation of cell lysates.

[0004] To address the aforementioned challenges, a method for achieving programmed cell autolysis at high cell densities without requiring additional mechanical or chemical assistance is needed. Inspired by efflux proteins that pump intracellular inducers out of the cell, we hypothesize that certain efflux proteins may help reduce the accumulation of autoinducers, thereby optimizing the QS-based autolysis process. Summary of the Invention

[0005] The purpose of this invention is to address the problems existing in the prior art by providing an optimized method for constructing a cell-free catalytic system and its application, specifically a method for constructing a self-lysis cell-free system assisted by an efflux pump based on quorum sensing.

[0006] Cell-free biocatalysis using cell lysates, particularly those derived from *E. coli*, represents a revolutionary technology, but its full potential remains untapped. A simple and readily available method for preparing cell lysates is urgently needed, for example, to achieve programmed cell autolysis at high cell densities without physical or chemical assistance. While quorum sensing (QS)-based autolysis systems have been developed, these systems typically operate at relatively low cell densities, complicating cell lysate preparation. To improve QS-based autolysis systems, this invention tested various *E. coli* efflux pumps and identified one that effectively effluxes QS-inducing substances. An efflux pump protein, AcrAB-TolC, or a corresponding protein, of the quorum sensing signaling molecule OHC14-HSL (hydroxy-N-(tetrahydro-2-oxo-3-furanyl)tetradecanamide (OHC14), or its corresponding protein MarA, can regulate the efflux of OHC14 by modulating the expression level of AcrAB-TolC. Furthermore, quorum-sensing exporter-assisted autolysis cell-free systems (QE-ACSs) were developed using this technology. Both AcrAB-TolC and MarA can be used to optimize quorum-sensing-based autolysis cell-free systems. Correspondingly, multidrug efflux pumps from different host sources, belonging to the same multidrug efflux pump family as AcrAB-TolC, can efflux QS autoinducers.

[0007] The objective of this invention can be achieved through the following methods:

[0008] This invention provides a cell-free catalytic system comprising the following gene elements:

[0009] Efflux pump proteins that expel signaling molecules, synthases that synthesize quorum sensing signaling molecules, transcription factors (TFs), signaling molecule responsive promoters, and cleavage proteins.

[0010] In the cell-free catalytic system:

[0011] The synthase (CinI) is used to synthesize the signal molecule (OHC14); the efflux pump protein (AcrAB-TolC) is used to efflux the signal molecule (OHC14).

[0012] Unexpelled signaling molecules (OHC14) are responded to by the corresponding transcription factor (CinR). The receptor protein generated by the transcription factor binds to the unexpelled signaling molecules (OHC14), which are then sensed and acted upon by the signaling molecule-responsive promoter (Pcin), initiating and regulating the cleavage of functional proteins, thereby generating a cell-free catalytic system.

[0013] During this process, signaling molecules are continuously released, but signaling molecules inside the cell also slowly accumulate. Once they accumulate to a certain threshold, they will activate functional proteins.

[0014] The efflux pump used in this invention is targeted at long-chain signaling molecules, specifically effluxing these molecules while preventing the efflux of short-chain signaling molecules (such as OC6). These signaling molecules can act on cleavage functional proteins. The combination of the efflux pump, the population of signaling molecules, and the cleavage functional proteins in this invention is unique; they must function as a whole to constitute the cell-free catalytic system of this invention and to play their role.

[0015] As one embodiment of the present invention, the efflux capacity of the efflux pump protein is enhanced by one of the following methods:

[0016] (1) Overexpression of efflux pump protein in a cell-free catalytic system;

[0017] (2) The expression of efflux pump protein is activated by introducing activation regulatory proteins.

[0018] The activation regulatory protein is an activation regulatory protein corresponding to an efflux pump protein. Preferably, the activation regulatory protein of AcrAB-TolC includes, but is not limited to, one or more of MarA protein and SoxS protein. MarA protein and SoxS protein are derived from Escherichia coli. As expression activation transcription factors, MarA protein and SoxS protein can increase the expression of AcrAB-TolC, thereby increasing the efflux of signaling molecules.

[0019] The protein sequence of MarA used is shown in SEQ ID No. 4.

[0020] When a cell's OD600 exceeds a certain value (which can be adjusted based on the expression of functional proteins), it reaches a threshold and begins to lyse. The pump's role is to reduce autolysis and initiate immediately, achieving an optimized effect.

[0021] In one embodiment of the present invention, the efflux pump protein for effluxing signaling molecules includes one or more of AcrAB-TolC, AdeABC, AdeFGH, AdeIJK, TtgABC, SmeABC, SmeDEF, SmeYZ, AcrAB, OqxAB, and MtrCDE. The efflux pump protein used can efflux the following signaling molecules.

[0022] The preferred efflux pump protein is AcrAB-TolC, which includes three proteins: AcrA, AcrB, and TolC. The protein sequences are as follows:

[0023] The protein sequence of AcrA is shown in SEQ ID No. 1.

[0024] The protein sequence of AcrB is shown in SEQ ID No. 2.

[0025] The protein sequence of TolC is shown in SEQ ID No. 3.

[0026] in,

[0027] The efflux pump protein AcrAB-TolC originates from Escherichia coli or Salmonella enterica.

[0028] Efflux pump proteins AdeABC, AdeFGH, and AdeIJK are derived from Acinetobacter baumannii.

[0029] The efflux pump protein TtgABC is derived from *Pseudomonas putida*.

[0030] Efflux pump proteins SmeABC, SmeDEF, and SmeYZ are derived from Stenotrophomonas maltophilia.

[0031] Efflux pump proteins AcrAB and OqxAB are derived from Klebsiella pneumoniae.

[0032] The efflux pump protein MtrCDE originates from Neisseria gonorrhoeae.

[0033] In one embodiment of the present invention, the synthase, transcription factor, and signal molecule for synthesizing quorum sensing signal molecules correspond to one of the following:

[0034] Synthesizer CinI, transcription factor CinR, and synthetic signaling molecule OHC14;

[0035] Synthesizer SinI, transcription factor SinR (Sinorhizobium meliloti), signaling molecule 3OC14 (3-oxo-C14-HSL);

[0036] Synthesizer VanI, transcription factor VanR (Vibrio anguillarum), signaling molecule 3OC10 (3-oxo-C10-HSL);

[0037] Synthesizer CerI, transcription factor CerR (Rhodobactersphaeroides), signaling molecule 7-cis-C14-HSL.

[0038] The functional protein is a lysis protein used in quorum sensing systems to lyse cells. The lysis protein includes, but is not limited to, lysis proteins and endolysin proteins. This invention utilizes a cell-free catalytic system to prepare high-density cells, enabling the large-scale production of functional proteins for subsequent applications, such as lysis. The lysis protein is preferably derived from the lysis gene E of phage ΦX174. Endolysin proteins have multiple sources, including lambdaohage, T4 phage, and T7 phage, and can also include colicin M and holin proteins from the four phages: lambdaohage, T4 phage, T7 phage, and phage 21.

[0039] In one embodiment of the present invention, the gene element in the cell-free catalytic system includes a promoter for the expression of a synthetic enzyme. Commonly used promoters are acceptable, such as promoter P. J23 It can be used for the expression of the synthase CinI.

[0040] In one embodiment of the present invention, the signal molecule-responsive promoter is the responsive promoter corresponding to the signal molecule. For example, the signal molecule OHC14 produced by the synthase (CinI) corresponds to the promoter P. cin The signaling molecules produced by CerI activate P. cer .

[0041] The preferred cell-free catalytic system of the present invention comprises: an efflux pump protein AcrAB-TolC for expelling signaling molecules, a synthase CinI for synthesizing quorum sensing signaling molecules, a transcription factor CinR, a signaling molecule responsive promoter Pcin, and a functional protein cleavage gene E (derived from bacteriophage ΦX174).

[0042] In one embodiment of the present invention, the gene element in the cell-free catalytic system includes a gene of interest (GOI). The GOI includes an enzyme or protein to be prepared with a corresponding function. Functional genes, such as RFPs, can be replaced as needed.

[0043] This invention provides a method for constructing and optimizing a cell-free catalytic system, comprising the following steps: introducing gene elements (synthetic enzymes for synthesizing quorum sensing signal molecules, efflux pump proteins for effluxing signal molecules, transcription factors, signal molecule responsive promoters, functional proteins, etc.) from the cell-free catalytic system into host cells through transformation or transfection;

[0044] Alternatively, genetic elements other than efflux pump proteins used to expel signaling molecules (such as synthases for synthesizing quorum sensing signaling molecules, transcription factors, signaling molecule responsive promoters, and functional proteins) from cell-free catalytic systems can be introduced into host cells containing efflux pump proteins used to expel signaling molecules through transformation or transfection.

[0045] In constructing the optimization method, a corresponding host cell can be selected based on the required efflux pump. This host cell contains the efflux pump and can expel all the aforementioned signaling molecules. For example, E. coli contains the efflux pump protein AcrAB-TolC.

[0046] Gene elements are introduced individually or in combination onto plasmids or chromosomes, and then further introduced into the host cell. There are no requirements regarding the relative positions or connections of the gene elements.

[0047] As one embodiment of the present invention, in the construction optimization method, the efflux capacity of the efflux pump protein is enhanced by one of the following methods:

[0048] (1) Overexpression of efflux pump protein in a cell-free catalytic system;

[0049] (2) The activation regulatory protein and synthase were introduced into plasmid 1 to activate the expression of the efflux pump protein.

[0050] The present invention provides an engineered bacterium comprising the cell-free catalytic system described above.

[0051] The strain was constructed using the following method:

[0052] Gene elements from cell-free catalysis systems are introduced into cell hosts through transformation or transfection.

[0053] Alternatively, gene elements of efflux pump proteins, which are used to expel signaling molecules, from cell-free catalytic systems can be introduced into cell hosts containing efflux pump proteins for expelling signaling molecules through transformation or transfection.

[0054] This invention also provides an application of the cell-free catalytic system in the preparation of cell lysates. The functional protein used in the cell-free catalytic system is a lysing protein.

[0055] The application involves introducing a corresponding functional enzyme (the functional gene of interest GOI) into a cell-free catalytic system for expression, thereby obtaining cell lysates containing the enzyme. The prepared cell lysates containing the functional enzyme are then used for biosynthesis or biodegradation.

[0056] Functional enzymes (genes of interest (GOIs)) include one of the following: phenolic acid decarboxylase BLPad and PET plastic degrading enzyme PETase.

[0057] Specifically:

[0058] The gene encoding BLPad was introduced into a cell-free catalytic system, and the prepared cell lysate was used to convert p-CA into 4-vinylphenol.

[0059] PETase was introduced into a cell-free catalytic system, and the resulting cell lysate was used for the biodegradation of PET.

[0060] The application involves introducing the protein to be prepared (the functional gene of interest GOI) into a cell-free catalytic system for the production of the protein (cell-free).

[0061] This invention also provides an application of the cell-free catalytic system in a therapeutic delivery system. Specifically, the application involves introducing a gene encoding an anti-tumor nanobody into the cell-free catalytic system. Upon injection into a tumor, the cells lyse, and the gene encoding the anti-tumor nanobody inhibits tumor growth. The gene includes the CD47nb nanobody.

[0062] Compared with the prior art, the present invention has the following beneficial effects:

[0063] In this study, we identified a self-inducible efflux protein and developed quorum-sensing exporter-assisted autolysis cell-free systems (QE-ACSs) using it. We first tested this system in red fluorescence protein (RFP) production and cancer treatment in mouse models. Subsequently, through a simple process including growth, expression, collection, autolysis, and reaction (GECAR), we successfully applied QE-ACSs to the biosynthesis of p-coumaric acid (p-CA) derived from lignin and the biodegradation of untreated post-consumer PET (UPC-PET). Both applications demonstrated high performance. Attached Figure Description

[0064] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings:

[0065] Figure 1 A quorum sensing system to assist the external drainage pump;

[0066] Figure 2 Overexpression of efflux pump proteins or activating proteins facilitates the efflux of the signaling molecule (OHC14). a represents OHC14, and b represents the signaling molecule (OC6).

[0067] Figure 3 The expression of the activating protein increased the expression of the efflux pump protein and decreased intracellular OHC14. Here, a represents the transcriptional level of the efflux pump, and b represents intracellular OHC14.

[0068] Figure 4 This is for the validation of the self-inducing system; where a represents the expression of CinI by promoters of different strengths, and b represents the expression of MarA by promoters of different strengths.

[0069] Figure 5 A quorum induction self-destruction system assisted by an external discharge pump;

[0070] Figure 6 This is for the validation of a quorum-inducible self-destruction system assisted by an external discharge pump. Where a represents the change in OD and b represents the change in destructive efficiency;

[0071] Figure 7The self-cleavage system produces red fluorescent protein; where a is a gel electrophoresis image of RFP and b is a graph of RFP production.

[0072] Figure 8 To create a cell-free catalytic system for biosynthesis using a self-cleavage system, a GECAR process was developed, which includes growth, expression, collection, autolysis, and reaction. b is the cell-free catalytic system that uses BLPad enzyme to catalyze the synthesis of 4-vinylphenol.

[0073] Figure 9 A cell-free catalytic system was established for this system to degrade plastics using PETase enzyme; where a represents the plastic degradation strategy and b represents the verification of plastic degradation.

[0074] Figure 10 The QE-ACS system is used for drug delivery to treat diseases; where a is the drug delivery strategy, b is the mouse experimental model, c is the lysis efficiency result, and d is the therapeutic effect on cancer tumors in mice.

[0075] Figure 11 This is the construction map of plasmid one in Example 2;

[0076] Figure 12 This is the construction map of plasmid 2 in Example 2. Detailed Implementation

[0077] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. The following examples are implemented under the premise of the technical solution of the present invention, providing detailed implementation methods and specific operating procedures, which will help those skilled in the art to further understand the present invention. It should be noted that the scope of protection of the present invention is not limited to the following embodiments; any adjustments and improvements made under the concept of the present invention are all within the scope of protection of the present invention.

[0078] Example 1

[0079] This embodiment relates to a method for identifying efflux pumps of signal molecules in a quorum sensing (QS) system.

[0080] To identify two long-chain and short-chain efflux pumps, OHC14 and OC6, *E. coli* QS sensors responsive to OHC14 and OC6 were used (including efflux pump proteins, transcription factors, signal molecule-responsive promoters, and fluorescent proteins to be screened for). Figure 1As shown, the sensor plasmid backbone contains the common p15A replicon and the kanamycin resistance gene. The sensor contains an OHC14-responsive transcription factor (TF) CinR (with the common constitutive promoter PlacI) and its promoter Pcin (which expresses a fluorescent protein), or an OC6-responsive transcription factor (TF) luxR and its promoter Plux, the effluxe AcrAB-TolC (the efflux pump protein AcrAB-TolC includes three proteins: AcrA, AcrB, and TolC), and the red fluorescent protein rfp. The remaining promoters are Pj123. In the presence of OHC14 or OC6 (exogenously added quantitatively to the bacterial culture system), the intracellular signaling molecules (OHC14 or OC6) are responded to by the transcription factors (CinR or luxR), acting on the signaling molecule-responsive promoters (Pcin or Plux), initiating and regulating the functional protein (red fluorescent protein rfp). That is, the fluorescence intensity changes with the amount of intracellular signaling molecules. Figure 2 As shown, the multi-component efflux molecule AcrAB-TolC can expel OHC14, thereby reducing the corresponding fluorescence, while it cannot expel OC6, resulting in enhanced fluorescence intensity. This demonstrates that this efflux pump is specifically designed for OHC14, but not for OC6, exhibiting unique characteristics.

[0081] Introducing the activation regulatory protein MarA into an E. coli QS sensor that identifies the signal molecule OHC14. Figure 1 Located in promoter P con (After). Notably, MarA overexpression is more effective than AcrAB-TolC, with a detection range from 1 to 10 μM (after). Figure 3 Next, using MarA OE, we measured the transcriptional levels of the genes acrA and tolC. Figure 3 Overexpression of MarA resulted in a more than two-fold increase in the transcription of the efflux pump AcrAB-TolC, indicating that MarA activates AcrAB-TolC to reduce intracellular OHC14 levels.

[0082] Furthermore, the signal molecule synthase CinI was introduced into the E. coli QS sensor that identifies the signal molecule OHC14. Figure 1 Located in promoter P con (Afterwards), it can be made into a self-inducible system. The synthase CinI produces the signal molecule OHC14, which can be expelled by AcrAB-TolC or responded to by the transcription factor (CinR) to regulate fluorescent proteins.

[0083] Different fluorescence responses were obtained by using different promoters in the system (the J23 series promoters allow for fine-tuning of promoter intensity). For example... Figure 4As shown in figure a, the transcription factor CinI uses the promoter P J23109 P J23117 CinI is expressed by promoters of different strengths; such as Figure 4 As shown in b, the activation of the regulatory protein MarA uses the promoter P. J23114 P J23116 P J23110 When MarA was expressed with promoters of different strengths, the fluorescence response was significantly reduced compared to when the activation regulator MarA was not used.

[0084] Example 2

[0085] This embodiment relates to the construction of a quorum-sensing exporter-assisted autolysis cell-free system (QE-ACSs).

[0086] To construct QE-ACS, we introduced the lysis gene E (encoding ΦX174E) from bacteriophage ΦX174 into a quorum sensing (QS)-based system. Figure 5 This gene is controlled by the OHC14-responsive promoter Pcin, and OHC14 is produced by the synthase CinI. As the cell population expands, the concentration of OHC14 gradually increases; therefore, the quorum sensing threshold is related to cell population density. Overexpression of MarA (MarAOE) reduces intracellular OHC14 levels, thereby activating autolysis at higher biomass levels.

[0087] The system construction is basically the same as in Example 1, except that the fluorescent protein is replaced with the cleavage gene E (encoding ΦX174E), as follows. Figure 5 As shown, the system is built on two plasmids.

[0088] Plasmid A, such as Figure 11 As shown, a backbone (pAk) containing the common p15A replicon and the kanamycin resistance gene was used. The pAk gene was inserted sequentially using the goldengate method. lacI Promoter and its expressed cinR gene, P Cin The promoter and its expressed ΦX174E gene, and P induced by aTc (Anhydrotetracycline) Tet Promoters and the different GOI genes they express.

[0089] Plasmid B, such as Figure 12As shown, a backbone (pBc) containing the common BBR1 replicon and the chloramphenicol resistance gene was used. Using the goldengate method, different expression levels of P were sequentially inserted. J23 The promoter and its expressed cinI gene, and P at different expression levels J23 The promoter and its expressed marA gene can also be used without introducing P with different expression levels. J23 The promoter and the marA gene it expresses.

[0090] The QE-ACS system can be obtained by simultaneously transforming plasmid one and plasmid two into Escherichia coli (MG1655).

[0091] Further, specific functional genes (red fluorescent protein RFP) are introduced. That is, the marA and cinI genes, along with cinR, ΦX174E genes and specific functional genes (GOI), or the system that does not contain marA, are all introduced into E. coli host cells containing the efflux body AcrAB-TolC.

[0092] We first confirmed cell autolysis via ΦX174E by testing with OHC14 (Example 1). Next, to test efflux variants, we overexpressed MarA or AcrAB-TolC at the promoter in a sensor cell system containing CinI and ΦX174E (non-overexpressed cells served as the control group), and recorded their growth using a microplate reader. Figure 6 MarA and AcrAB-TolC were controlled below J23117 and J23116, respectively, indicating that J23117 provided better performance. All overexpressions increased the QS threshold, and the autolysis system can be further tuned to meet different application requirements.

[0093] Next, we used this system to synthesize the red fluorescent protein RFP (RFP). Figure 7 That is, following the QE-ACS construction method described above, the rfp gene is set as a P gene induced by aTc. Tet The promoter and its expressed GOI gene. For further validation, we used QE-ACS to produce red fluorescent protein (RFP). We cultured cells expressing RFP and adjusted the cell concentration to an OD600 exceeding 50; after 10 hours, the RFP production reached 3.7 g / L. -1 .

[0094] Example 3

[0095] This embodiment relates to a method for synthesizing 4-vinylphenol from lignin-derived crude p-coumaric acid (p-CA) using QE-ACS via GECAR. Specific steps include:

[0096] Based on QE-ACS, we developed the GECAR (growth, expression, collection, autolysis, and reaction) biosynthesis and biodegradation procedure, which is simple and easy to operate. Figure 8 The GECAR process includes growth, expression, collection, autolysis, and reaction.

[0097] The detailed operating procedure for GECAR is as follows: QE-ACS strains containing different GOI genes are first cultured overnight in LB medium at 37°C and 170 rpm. Then, 1% of the cells are transferred to LB medium and cultured at 37°C and 220 rpm. When the cell OD... 600 When the OD reaches 0.4-0.6, enzyme expression is induced by adding an inducer (such as 200 nmol / Tc) at different temperatures depending on the type of GOI being expressed, for 4-10 hours. 600 When the OD600 reaches 1.5 to 5, cells are collected by centrifugation at 4000 rpm for 15 minutes. The pellet is resuspended in fresh LB medium, and the OD600 is adjusted to 10-100 (or a suitable medium is selected according to the different GOI, such as fresh LB with phosphate buffer). Incubation continues for 6-8 hours to induce autolysis. The resulting crude cell lysate is used for subsequent reactions.

[0098] We introduced the gene encoding BLPad into QE-ACS (generating QE-ACS-Pad) through engineering construction, that is, according to the QE-ACS construction method in Example 2 above, we set the BLPad gene to be aTc-induced P Tet The promoter and its expressed GOI gene. This enzyme can convert p-CA to 4-vinylphenol, which is then reacted via GECAR. The autolysis product was obtained using 20 g·L⁻¹. -1 Under purified p-CA conditions, QE-ACS-Pad produced 12 g·L⁻¹ -1 4-Vinylphenol. Through multiple feeds, the yield of 4-vinylphenol reached 31.2 g·L⁻¹. -1 The production rate is 15.6 g·L. -1 ·h -1 The yield reached 95%.

[0099] Example 4

[0100] This embodiment relates to a method for biodegrading PET using QE-ACS, with the following specific steps:

[0101] PET is one of the most common types of plastic waste. PETase is an enzyme capable of biodegrading PET and has evolved in various microorganisms. However, naturally occurring PETase has low activity and needs to be secreted into the environment to come into contact with PET in order to biodegrade, resulting in low efficiency. We hypothesize that QE-ACS containing highly efficient engineered PETase can significantly improve the biodegradation efficiency of PET through GECAR. Figure 9 a).

[0102] To construct QE-ACS for PET biodegradation, we selected engineered PETase PES-H1. L92F / Q94Y9 This enzyme can convert PET into monomeric terephthalic acid (TPA), mono(2-hydroxyethyl) terephthalate (MHET), and ethylene glycol (EG). We will use PETase PES-H1... L92F / Q94Y9 The gene was cloned into an autolysis-free cell-free system to form QE-ACS-PES, that is, according to the QE-ACS construction method in Example 2 above, PETase PES-H1 was used. L92F / Q94Y9 Genes were set to be induced by aTc P Tet Promoter and its expressed GOI gene. During PET degradation, a cell-free catalytic system catalyzed the degradation of PET films within 24 hours. Figure 9 b) It even exhibits high performance in some plastics, generating 0.4M monomers within 24 hours, which suggests that QE-ACS-PES has potential for industrial applications.

[0103] Example 5

[0104] This embodiment relates to a tumor treatment method using QE-ACS, with the following specific steps:

[0105] Engineered bacteria, used as a therapeutic delivery system, can selectively release therapeutic payloads for treatment. In the treatment of a B16-F10 mouse cancer model, we introduced a gene encoding an anti-tumor nanobody (CD47nb) controlled by a hypoxia-responsive PFnrs promoter into QE-ACS, generating QE-ACS-CD47nb. That is, following the QE-ACS construction method in Example 2 above, the hypoxia-inducible promoter P... fnrs Its induced CD47nb gene replaces the P induced by aTc. Tet The promoter and the GOI gene it expresses.

[0106] Treatment process such as Figure 10As shown in a and b, when QE-ACS-CD47nb is injected into the tumor, the cells lyse, and the gene encoding the anti-tumor nanobody is used to inhibit tumor growth.

[0107] Compared with the control group (not subjected to MarA overexpression) (including mice in three parallel experiments), such as Figure 10 As shown in c and 10d, our QE-ACS significantly inhibited tumor growth.

[0108] The gene and protein sequences involved in the embodiments of this invention are as follows.

[0109] Protein sequence:

[0110] The protein sequence of AcrA is shown in SEQ ID No. 1:

[0111] MNKNRGFTPLAVVLMLSGSLALTGCDDKQAQQGGQQMPAVGVVTVKTEPLQITTELPGRTSAYRIAEVRPQVSGIILKRNFKEGSDIEAGVSLYQIDPA TYQATYDSAKGDLAKAQAAANIAQLTVNRYQKLLGTQYISKQEYDQALADAQQANAAVTAAKAAVETARINLAYTKVTSPISGRIGKSNVTEGALVQNGQ ATALATVQQLDPIYVDVTQSSNDFLRLKQELANGTLKQENGKAKVSLITSDGIKFPQDGTLEFSDVTVDQTTGSITLRAIFPNPDHTTLLPGMFVRARLEE GLNPNAILVPQQGVTRTPRGDATVLVVGADDKVETRPIVASQAIGDKWLVTEGLKAGDRVVISGLQKVRPGVQVKAQEVTADNNQQAASGAQPEQSKS*.

[0112] The protein sequence of AcrB is shown in SEQ ID No. 2:

[0113]

[0114] The protein sequence of TolC is shown in SEQ ID No. 3:

[0115] MKKLLPILIGLSLSGFSSLSQAENLMQVYQQARLSNPELRKSAADRDAAFEKINEARSPLLPQLGLGADYTYSNGYRDANGINSNATSASLQLTQSIFDMSKWRALTLQEKAAGIQDVTYQTD QQTLILNTATAYFNVLNAIDVLSYTQAQKEAIYRQLDQTTQRFNVGLVAITDVQNARAQYDTVLANEVTARNNLDNAVEQLRQITGNYYPELAALNVENFKTDKPQPVNALLKEAEKRNLSLLQ ARLSQDLAREQIRQAQDGHLPTLDLTASTGISDTSYSGSKTRGAAGTQYDDSNMGQNKVGLSFSLPIYQGGMVNSQVKQAQYNFVGASEQLESAHRSVVQTVRSSFNNINASISSINAYKQAVV SAQSSLDAMEAGYSVGTRTIVDVLDATTTLYNAKQELANARYNYLINQLNIKSALGTLNEQDLLALNNALSKPVSTNPENVAPQTPEQNAIADGYAPDSPAPVVQQTSARTTTSNGHNPFRN*.

[0116] The protein sequence of MarA is shown in SEQ ID No. 4:

[0117] MSRRNTDAITIHSILDWIEDNLESPLSLEKVSERSGYSKWHLQRMFKKETGHSLG QYIRSRKMTEIAQKLKESNEPILYLAERYGFESQQTLTRTFKNYFDVPPHKYRMTNMQ GESRFLHPLNHYNS*.

[0118] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art can make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A quorum-sensing based cell-free catalytic system, characterized in that, The gene elements comprise: an efflux pump protein for expelling signal molecules, a synthetase for synthesizing a quorum sensing signal molecule, a transcription factor, a signal molecule responsive promoter, a cleavage functional protein; the efflux pump protein for expelling signal molecules is AcrAB-TolC; the signal molecule is OHC14-HSL; the synthetase for synthesizing a quorum sensing signal molecule is synthetase CinI, and the transcription factor is transcription factor CinR; the cleavage functional protein is a functional protein for a quorum sensing system, and the cell is lysed; the cleavage functional protein comprises one of a lytic protein and an endolysin protein.

2. The quorum-sensing-based cell-free catalytic system of claim 1, wherein, In the cell-free catalytic system: the synthetase is used for synthesizing signal molecules; the efflux pump protein is used for expelling signal molecules; the signal molecules not expelled are responded by the transcription factor, the receptor protein generated by the transcription factor is combined with the signal molecules not expelled, and the signal molecules not expelled are sensed by the signal molecule responsive promoter and act on the signal molecule responsive promoter, so as to start and regulate the cleavage functional protein, thereby generating the cell-free catalytic system.

3. The quorum-sensing-based cell-free catalytic system of claim 1, wherein, The expelling capacity of the efflux pump protein is improved by one of the following methods: (1) overexpressing the efflux pump protein in the cell-free catalytic system; (2) introducing an activation regulatory protein to activate the expression of the efflux pump protein; the activation regulatory protein is an activation regulatory protein corresponding to the efflux pump protein.

4. A method for constructing and optimizing a cell-free catalytic system as claimed in claim 1, characterized in that, The method comprises the following steps: introducing the gene elements in the cell-free catalytic system into a host cell by transformation or transfection; or introducing the gene elements in the cell-free catalytic system except the efflux pump protein for expelling signal molecules into a host cell containing the efflux pump protein for expelling signal molecules by transformation or transfection.

5. An engineered bacterium comprising the cell-free catalytic system according to claim 1.

6. Use of the cell-free catalytic system according to claim 1 in preparing a cell lysate.

7. Use according to claim 6, characterized in that, In the use in preparing a cell lysate, the cell lysate containing enzymes is prepared by introducing corresponding functional enzymes into the cell-free catalytic system for expression, and the prepared cell lysate containing functional enzymes is used for biosynthesis or biodegradation; and / or, in the use in preparing a cell lysate, the protein to be prepared is introduced into the cell-free catalytic system for the production of the protein.

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