Cell-free catalysis system construction optimization method and application thereof

Abstract

The invention relates to a cell-free catalysis system construction optimization method and application thereof. The system comprises efflux pump protein for discharging signal molecules, synthase for synthesizing quorum sensing signal molecules, a transcription factor (TF), a signal molecule response type promoter, lyase and other functional proteins. Wherein the synthetase is used for synthesizing signal molecules; the efflux pump protein is used for discharging signal molecules; signal molecules which are not discharged are responded by transcription factors, generated receptor proteins are combined with the signal molecules and then are induced by the signal molecule response type promoter, the receptor proteins act on the signal molecule response type promoter, and expression of related functional proteins such as lyase is started and regulated. Expression of efflux pump protein is activated by overexpression of efflux pump protein or introduction of activation regulation protein in a cell-free catalysis system, and programmed cell autolysis under high cell density is realized without physical or chemical assistance.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to a method for optimizing the construction of a cell-free catalytic system and its application. The present invention discovers and identifies a quorum-sensing signal molecule efflux pump; uses the efflux pump to optimize the quorum-sensing microbial cell self-lysis system, and applies it to the preparation of crude enzyme solution for further use in biocatalysis and drug delivery for treatment. Background Art

[0002] Cell lysates are usually prepared by mechanical disruption (such as high-pressure homogenization and ultrasonication) or chemical methods (such as lysozyme treatment and chemical solvent extraction). Biological methods include expressing bacteriophage λ endolysin in Escherichia 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 bacterial communication process based on cell density, has been used to study cell autolysis. QS senses changes in cell density through autoinducers produced by bacteria to coordinate gene expression and behavior. QS-based systems provide a more simplified method that eliminates additional steps and achieves more efficient cell autolysis. Such systems have been developed to control bacterial autolysis for drug delivery in mouse models.

[0003] Autoinducers, such as N-acyl-homoserine lactones (HSLs), are produced by synthetases within Escherichia 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 the response sensor and express the phage lytic gene (φX174E), which triggers cell autolysis. However, the rapid accumulation of autoinducers leads to autolysis at low cell densities (below OD600 0.2), which limits their application in the preparation of cell lysates.

[0004] To address the above issues, it is necessary to develop a method to achieve programmed cell autolysis at higher cell density without additional mechanical or chemical assistance. Inspired by the efflux proteins that pump intracellular inducers out of cells, we speculated 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 the present invention is to provide a method for optimizing the construction of a cell-free catalytic system and its application in view of the problems existing in the prior art, specifically a method for constructing a self-cleaving cell-free system assisted by an efflux pump based on quorum sensing.

[0006] The use of cell lysates for cell-free biocatalysis, especially the use of lysates derived from Escherichia coli (E. coli), represents a transformative technology, but its full potential has not yet been fully realized. There is an urgent need for a simple and easy method to prepare cell lysates, such as programmed cell autolysis at high cell density without physical or chemical assistance. Although quorum sensing (QS)-based autolysis systems have been developed, these systems are usually operated at relatively low cell densities, making the preparation of cell lysates complicated. To improve the QS-based autolysis system, the present invention tested a variety of E. coli efflux pumps and determined that one of them can effectively expel QS autoinducers. An efflux pump protein AcrAB-TolC that can expel quorum sensing signal molecules, such as OHC14-HSL (hydroxy-N-(tetrahydro-2-oxo-3-furanyl)tetradecanamide, OHC14) or a corresponding protein MarA can regulate the efflux of OHC14 by regulating the expression level of AcrAB-TolC. A quorum-sensing exporter-assisted autolysis cell-free system (QE-ACSs) was developed using it. AcrAB-TolC or MarA can be used to optimize the quorum-sensing autolysis cell-free system. Correspondingly, multidrug efflux pumps from different host sources that are the same as AcrAB-TolC can expel QS autoinducers.

[0007] The purpose of the present invention can be achieved by the following scheme:

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

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

[0010] In the cell-free catalytic system:

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

[0012] The un-expelled signal molecule (OHC14) is responded to by the corresponding transcription factor (CinR). The receptor protein generated by the transcription factor binds to the un-expelled signal molecule (OHC14), which is then sensed by the signal molecule-responsive promoter (Pcin) and acts on the signal molecule-responsive promoter, thereby activating and regulating the cleavage functional protein, thereby producing a cell-free catalytic system.

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

[0014] The efflux pump selected by the present invention is targeted at long-chain signal molecules, and can specifically expel these signal molecules, but cannot expel short-chain signal molecules (such as OC6). These signal molecules can act on the cleavage functional protein. The combination of the efflux pump, the group signal molecule and the cleavage functional protein of the present invention is special, and as a whole, it can constitute the cell-free catalytic system of the present invention and play a role.

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

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

[0017] (2) Activating the expression of efflux pump proteins by introducing activating regulatory proteins.

[0018] The activation regulatory protein is an activation regulatory protein corresponding to the 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. MarA protein and SoxS protein, as expression activation transcription factors, can increase the expression of AcrAB-TolC, thereby increasing the efflux of signal molecules.

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

[0020] When the OD600 of the cell exceeds a certain value (which can be adjusted according to the expression of functional proteins), the threshold is reached and lysis begins. The role of the pump is to reduce the self-lysis and start immediately to achieve the optimization effect.

[0021] As one embodiment of the present invention, the efflux pump protein for expelling signal 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 expel the following signal molecules.

[0022] Preferably, the efflux pump protein AcrAB-TolC comprises three proteins AcrA, AcrB and TolC. The protein sequence is 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 is derived from Escherichia coli or Salmonella Enteritidis;

[0028] The efflux pump proteins AdeABC, AdeFGH, and AdeIJK are derived from Acinetobacterbaumannii;

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

[0030] The efflux pump proteins SmeABC, SmeDEF, and SmeYZ are derived from Stenotrophomonasmaltophilia;

[0031] The efflux pump proteins AcrAB and OqxAB are derived from Klebsiella pneumoniae;

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

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

[0034] synthesizes enzyme CinI, transcription factor CinR, and synthesizes signal molecule OHC14;

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

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

[0037] Synthetic enzyme CerI, transcription factor CerR (Rhodobactersphaeroides), signal molecule 7-cis-C14-HSL.

[0038] The functional protein is a lytic functional protein used in a quorum sensing system to lyse cells. The lytic functional protein includes but is not limited to one of a lytic protein and an endolysin protein. The present invention utilizes a cell-free catalytic system to prepare high-density cells, thereby realizing a large amount of functional protein preparation, and the protein performs the corresponding function in subsequent applications, such as lysis. The lytic protein is preferably derived from the lytic gene E of the bacteriophage ΦX174. Endolysin proteins have a variety of sources and can be constrained to lamdaohage, T4 phage, and T7 phage. At the same time, colicin M and the holin proteins of the four phages of lamdaohage, T4phage, T7phage, and phage 21 can be increased.

[0039] As one embodiment of the present invention, the gene element in the cell-free catalytic system includes a promoter for synthetase expression. Commonly used promoters are all acceptable, such as promoter P J23 Can be used for the expression of the synthetase CinI.

[0040] As one embodiment of the present invention, the signal molecule responsive promoter is a responsive promoter corresponding to the signal molecule. For example, the signal molecule OHC14 produced by the synthase (CinI) has a corresponding promoter P cin The signal molecule produced by CerI activates P cer .

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

[0042] As one embodiment of the present invention, the gene element in the cell-free catalytic system includes a functional gene of interest (GOI). The functional gene of interest includes an enzyme of corresponding function or a protein to be prepared. The functional gene, such as RFP, can be replaced according to demand.

[0043] The present 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 expelling signal molecules, transcription factors, signal molecule-responsive promoters, functional proteins, etc.) in the cell-free catalytic system into host cells by transformation or transfection;

[0044] Alternatively, the genetic elements in the cell-free catalytic system other than the efflux pump protein for expelling signal molecules (synthetic enzymes for synthesizing quorum sensing signal molecules, transcription factors, signal molecule responsive promoters, functional proteins, etc.) are introduced into host cells containing the efflux pump protein for expelling signal molecules by transformation or transfection.

[0045] In the construction optimization method, the corresponding host cell can be selected according to the required efflux pump, and the host cell contains the efflux pump and can excrete all the above signal molecules. The host cell, such as E. coli, contains the efflux pump protein AcrAB-TolC.

[0046] When the genetic elements are introduced, they are introduced individually or in combination into plasmids or chromosomes, and then further introduced into the host cell. There is no requirement for the relative position and connection of the genetic elements.

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

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

[0049] (2) Introducing the activation regulatory protein and synthetase into plasmid 1 to activate the expression of the efflux pump protein.

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

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

[0052] The genetic elements in the cell-free catalytic system are introduced into the cell host by transformation or transfection;

[0053] Alternatively, the gene elements in the cell-free catalytic system except the efflux pump protein for expelling signal molecules are introduced into a cell host containing the efflux pump protein for expelling signal molecules by transformation or transfection.

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

[0055] The application is to introduce the corresponding functional enzyme (functional gene of interest GOI) into the cell-free catalytic system for expression to obtain cell lysate containing the enzyme, and the prepared cell lysate containing the functional enzyme is used for biosynthesis or biodegradation.

[0056] Functional enzymes (functional genes of interest (GOI)) include phenolic acid decarboxylase BLPad, one of PET plastic degrading enzymes PETase,

[0057] Specifically:

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

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

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

[0061] The present invention also provides an application of the cell-free catalytic system in a therapeutic delivery system. The application is specifically: a gene encoding an anti-tumor nano-antibody is introduced into the cell-free catalytic system, and when injected into a tumor, the cell is lysed, and the gene encoding the anti-tumor nano-antibody is used to inhibit the growth of the tumor. The gene includes the CD47nb nano-antibody.

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

[0063] In this study, we identified an autoinducer efflux protein and used it to develop quorum-sensing exporter-assisted autolysis cell-free systems (QE-ACSs). We first tested the 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 combined QE-ACSs with catalytic enzymes and successfully applied them to the biosynthesis of p-coumaric acid (p-CA) from lignin and the biodegradation of untreated post-consumer PET (upc-PET). Both applications showed high performance. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0065] Figure 1 A quorum sensing system assisted by an efflux pump;

[0066] Figure 2 Overexpression of efflux pump proteins or activator proteins facilitates the efflux of signaling molecules (OHC14). a is OHC14, b is signaling molecules (OC6);

[0067] Figure 3 The expression of efflux pump proteins increased and intracellular OHC14 decreased in order to activate protein expression. Where a is the transcription level of efflux pumps, and b is intracellular OHC14;

[0068] Figure 4 This is the verification of the autoinduction system; a is the expression of CinI by promoters of different strengths, and b is the expression of MarA by promoters of different strengths;

[0069] Figure 5 It is a quorum sensing self-cleavage system assisted by an efflux pump;

[0070] Figure 6 This is the verification of the quorum sensing self-lysis system assisted by the efflux pump. Where a is the change in OD, and b is the change in lysis efficiency;

[0071] Figure 7The self-cleavage system produces red fluorescent protein; wherein, a is the gel electrophoresis diagram of RFP, and b is the production amount diagram of RFP;

[0072] Figure 8 The invention relates to a method for creating a GECAR process using an autolytic system to generate a cell-free catalytic system for biosynthesis; wherein a is the GECAR process including growth, expression, collection, autolysis, and reaction, and b is the cell-free catalytic system using BLPad enzyme to catalyze the synthesis of 4-vinylphenol;

[0073] Fig. 9 The cell-free catalytic system established for this system uses PETase enzyme to degrade plastics; wherein a is the strategy for plastic degradation, and b is the verification of degraded plastics;

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

[0075] Fig.11 This is the construction map of plasmid 1 in Example 2;

[0076] Fig.12 This is the construction map of plasmid 2 in Example 2. DETAILED DESCRIPTION

[0077] The present invention is described in detail below in conjunction with the accompanying drawings and specific embodiments. The following examples are implemented on the premise of the technical solution of the present invention, provide detailed implementation methods and specific operation processes, and will help those skilled in the art to further understand the present invention. It should be pointed out that the protection scope of the present invention is not limited to the following embodiments, and several adjustments and improvements made under the premise of the concept of the present invention all belong to the protection scope of the present invention.

[0078] Example 1

[0079] This embodiment relates to a method for identifying a quorum sensing (QS) system signal molecule efflux pump.

[0080] In order to identify two long-chain and short-chain efflux pumps, OHC14 and OC6, respectively, E. coli QS sensors responsive to OHC14 and OC6 were used (including efflux pump proteins to be screened that can expel signal molecules, transcription factors, signal molecule-responsive promoters, and fluorescent proteins). Figure 1As shown, the sensor plasmid backbone is a backbone containing the common p15A replicon and kanamycin resistance gene. The sensor contains the transcription factor (TF) CinR (the promoter is the common constitutive promoter PlacI) and its promoter Pcin (which can express fluorescent protein) that responds to OHC14, or the transcription factor (TF) luxR and its promoter Plux that responds to OC6, the efflux body AcrAB-TolC (the efflux pump protein AcrAB-TolC includes three proteins AcrA, AcrB, and TolC), and the red fluorescent protein rfp. The remaining series of promoters are Pj123. When OHC14 or OC6 is present (exogenously added to the bacterial culture system), the signal molecule (OHC14 or OC6) in the cell is responded to by the transcription factor (CinR or luxR), acting on the signal molecule responsive promoter (Pcin or Plux), starting and regulating the functional protein (red fluorescent protein rfp), that is, the fluorescence intensity changes with the amount of the signal molecule in the cell. As shown in FIG. Figure 2 As shown, the multi-component efflux body AcrAB-TolC can expel OHC14, thereby reducing the corresponding fluorescence, and the multi-component efflux body AcrAB-TolC cannot expel OC6, and the fluorescence intensity is enhanced. This shows that this efflux pump is specific to OHC14, not OC6, and has specificity.

[0081] In order to identify the signal molecule OHC14, the activation regulatory protein MarA was introduced into the Escherichia coli QS sensor. Figure 1 , located in the promoter P con Notably, overexpression of MarA was more efficient compared to AcrAB-TolC, with the detection range from 1 to 10 μM ( Figure 3 Next, using MarA OE, we measured the transcript levels of genes acrA and tolC ( Figure 3 Overexpression of MarA increased the transcription of the efflux pump AcrAB-TolC by more than 2-fold, indicating that MarA activated AcrAB-TolC to reduce the intracellular level of OHC14.

[0082] The signal molecule synthase CinI ( Figure 1 , located in the promoter P con The synthase CinI produces the signal molecule OHC14, which can be excreted by AcrAB-TolC or responded to by the transcription factor (CinR) to regulate the fluorescent protein.

[0083] By using different promoters in the system, different fluorescence responses can be obtained (J23 series promoters are convenient for fine-tuning promoter strength). Figure 4As shown in a, the transcription factor CinI uses the promoter P J23109 , P J23117 , promoters of different strengths express CinI; e.g. Figure 4 As shown in b, the activation regulatory protein MarA uses the promoter P J23114 , P J23116 , P J23110 , MarA was expressed by promoters of different strengths, and the fluorescence response was significantly decreased compared with that without using the activated regulatory protein MarA.

[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 the QE-ACS, we introduced the lytic gene E (encoding ΦX174E) from bacteriophage ΦX174 into the 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, so the threshold of quorum sensing is related to the cell population density. By overexpressing MarA (MarAOE), the level of OHC14 in the cell is reduced, allowing autolysis to be activated at higher biomass levels.

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

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

[0089] Plasmid B, such as Fig.12As shown in FIG. 1 , a backbone (pBc) containing a common BBR1 replicon and a chloramphenicol resistance gene was used. The goldengate method was used to sequentially insert P J23 Promoter and its expressed cinI gene, P J23 The promoter and the marA gene expressed by it can also be used without introducing P J23 Promoter and its expressed marA gene.

[0090] Plasmid 1 and plasmid 2 are simultaneously transformed into Escherichia coli (MG1655) to obtain the QE-ACS system.

[0091] Further, the intended functional gene (red fluorescent protein RFP) is introduced. That is, marA and cinI genes together with cinR, ΦX174E gene and the intended functional gene (GOI), or the system does not contain marA, are all introduced into the host cell E. coli containing the exosome AcrAB-TolC.

[0092] We first tested it by adding OHC14 (Example 1) and confirmed that ΦX174E was involved in cell self-lysis. Next, to test the exosome, we overexpressed MarA or AcrAB-TolC from the promoter in a sensor cell system containing CinI and ΦX174E (the control group was not overexpressed) and recorded its growth using a microplate reader ( Figure 6 ), MarA and AcrAB-TolC were controlled under J23117 and J23116, respectively, showing that J23117 can perform better. All overexpressions increased the QS threshold, and the autolysis system can be further adjusted to meet different application requirements.

[0093] Next, we used this system to synthesize the red fluorescent protein RFP ( Figure 7 ), that is, according to the above QE-ACS construction method, the rfp gene was set to be P induced by aTc Tet Promoter and its expressed GOI gene. As a 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 of more than 50. After 10 hours, the production of RFP reached 3.7 g L -1 .

[0094] Example 3

[0095] This example relates to a method for synthesizing 4-vinylphenol from crude p-coumaric acid (p-CA) derived from lignin by using QE-ACS through GECAR, and the specific steps are:

[0096] Based on QE-ACS, we developed GECAR (Growth, Expression, Collection, Autolysis and Reaction), a simple and easy-to-operate biosynthesis and biodegradation procedure ( Figure 8 The GECAR process includes growth, expression, collection, autolysis, and reaction.

[0097] The detailed operation process of GECAR is as follows: QE-ACS strains containing different GOI genes were first cultured in LB medium at 37°C and 170 rpm overnight. Then they were transferred to LB medium at 37°C and 220 rpm at 1% and cultured. 600 When the OD reaches 0.4-0.6, the enzyme expression is induced by adding an inducer (such as 200 nM A Tc) at different temperatures according to the type of GOI expressed for 4-10 hours. 600 When the OD reaches 1.5 to 5, the cells are collected by centrifugation at 4000 rpm for 15 minutes, the pellet is resuspended in new LB, OD600 is adjusted to 10-100 (or appropriate medium is selected according to different GOIs, such as fresh LB plus phosphate buffer), and incubated for another 6-8 hours to induce autolysis. Finally, the crude cell lysate is obtained for subsequent reactions.

[0098] The gene encoding BLPad was introduced into QE-ACS by engineering (generating QE-ACS-Pad), that is, according to the QE-ACS construction method of Example 2 above, the BLPad gene was set to be P induced by aTc. Tet The promoter and the GOI gene it expresses. The enzyme is able to convert p-CA into 4-vinylphenol, which then reacts via GECAR. The autolysate was purified by 20 g·L -1 Under the conditions of p-CA purification, QE-ACS-Pad produced 12 g·L -1 Through multiple feedings, the yield of 4-vinylphenol reached 31.2 g·L -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, and the specific steps are:

[0101] PET is one of the most common plastic wastes. PETase is an enzyme that can biodegrade PET and has evolved in a variety of microorganisms. However, naturally occurring PETase has low activity and needs to be secreted into the environment to come into contact with PET for biodegradation, resulting in low efficiency. We hypothesized that QE-ACS containing highly efficient engineered PETase could greatly improve the biodegradation efficiency of PET through GECAR ( Fig. 9 a).

[0102] To construct a QE-ACS for PET biodegradation, we selected the engineered PETase PES-H1 L92F / Q94Y9 The enzyme is able to convert PET into monomers terephthalic acid (TPA), mono(2-hydroxyethyl)terephthalate (MHET) and ethylene glycol (EG). We used PETase PES-H1 L92F / Q94Y9 The gene was cloned into the autolytic cell-free system to form QE-ACS-PES, that is, according to the QE-ACS construction method of Example 2 above, PETase PES-H1 L92F / Q94Y9 Genes were set to be induced by aTc Tet Promoter and its expressed GOI gene. In the PET degradation process, the cell-free catalytic system catalyzed the degradation of PET film ( Fig. 9 b), and even exhibited high performance in some plastics, with 0.4 M monomer generated within 24 h, indicating that QE-ACS-PES has potential in industrial applications.

[0103] Example 5

[0104] This embodiment relates to a method of using QE-ACS to treat tumors, and the specific steps are as follows:

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

[0106] Treatment process Fig.10As shown in a and b, when QE-ACS-CD47nb is injected into the tumor, the cells are lysed and the gene encoding the anti-tumor nanoantibody is used to inhibit tumor growth.

[0107] Compared with the control group (mice without MarA overexpression) (including three parallel experiments), Fig.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 the present 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 above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art may make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A cell-free catalytic system, characterized in that: Includes the following genetic elements: Efflux pump proteins used to expel signal molecules, synthases for synthesizing quorum sensing signal molecules, transcription factors, signal molecule responsive promoters, and cleavage functional proteins.

2. The cell-free catalytic system according to claim 1, characterized in that: In the cell-free catalytic system: Synthetases are used to synthesize signal molecules; efflux pump proteins are used to expel signal molecules; signal molecules that are not expelled are responded to by transcription factors, and receptor proteins generated by transcription factors bind to signal molecules that are not expelled, and are then sensed by signal molecule-responsive promoters and act on signal molecule-responsive promoters, thereby starting and regulating cleavage functional proteins, thereby producing a cell-free catalytic system.

3. The cell-free catalytic system according to claim 1, characterized in that: The efflux capacity of efflux pump proteins is increased in one of the following ways: (1) Overexpression of efflux pump proteins in a cell-free catalytic system; (2) Activating the expression of the efflux pump protein by introducing an activating regulatory protein; the activating regulatory protein is an activating regulatory protein corresponding to the efflux pump protein.

4. The cell-free catalytic system according to claim 1, characterized in that: The efflux pump proteins used to expel signal molecules include but are not limited to one or more of AcrAB-TolC, AdeABC, AdeFGH, AdeIJK, TtgABC, SmeABC, SmeDEF, SmeYZ, AcrAB, OqxAB, and MtrCDE.

5. The cell-free catalytic system according to claim 1, characterized in that: The synthase, transcription factor and signal molecule for synthesizing the quorum sensing signal molecule include but are not limited to one of the following: Synthetic enzyme CinI, transcription factor CinR, signaling molecule OHC14; synthase SinI, transcription factor SinR, signal molecule 3OC14; synthase VanI, transcription factor VanR, signal molecule 3OC10; Synthetic enzyme CerI, transcription factor CerR, signal molecule 7-cis-C14-HSL.

6. The cell-free catalytic system according to claim 1, characterized in that: The lytic functional protein is a functional protein used in the quorum sensing system to lyse cells; And / or, the cleavage functional protein includes but is not limited to one of a cleavage protein and an endolysin protein.

7. A method for constructing and optimizing a cell-free catalytic system as claimed in claim 1, characterized in that: The steps include: The gene elements of the cell-free catalytic system are introduced into the host cells by transformation or transfection; Alternatively, the gene elements in the cell-free catalytic system other than the efflux pump protein for expelling signal molecules are introduced into the host cells containing the efflux pump protein for expelling signal molecules by transformation or transfection.

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

9. A use of the cell-free catalytic system according to claim 1 in preparing cell lysates or therapeutic delivery systems; And / or, the functional protein used in the cell-free catalytic system is a cleavage protein.

10. The use according to claim 9, characterized in that: When used in the preparation of cell lysates, the corresponding functional enzymes are introduced into a cell-free catalytic system for expression to obtain cell lysates containing enzymes, and the prepared cell lysates containing functional enzymes are used for biosynthesis or biodegradation; And / or, when used in the preparation of cell lysates, the protein to be prepared is introduced into a cell-free catalytic system for protein production.

Images