Construction of a highly robust quorum-sensing gene circuit and its application

Abstract

This invention discloses the construction and application of a highly robust quorum sensing gene circuit. The quorum sensing gene circuit exhibits more stable output performance under different fermentation pH conditions, demonstrating high robustness. The response promoters PHU3 and PEL of the quorum sensing gene circuit possess good quorum sensing performance unaffected by pH crosstalk and exhibit good specificity. Using this quorum sensing gene circuit to express an acid-resistant module can improve the acid tolerance and fermentation production capacity of the strain in acidic environments. This invention provides a quorum sensing gene circuit with greater industrial application value.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering and synthetic biology, specifically to the construction and application of highly robust quorum sensing gene circuits. Background Technology

[0002] The current global development model relies too heavily on chemical resources, while the scarcity of these resources and the increasing environmental pollution necessitate a search for a sustainable development path. Green biomanufacturing, centered on industrial biotechnology, utilizes microorganisms and renewable materials such as biomass to produce materials, chemicals, pharmaceuticals, energy, and food, thereby reducing carbon emissions and achieving green and clean production processes.

[0003] To enhance the growth capacity and production performance of microbial cell factories during production processes, synthetic biology techniques are often used to modify them. Traditional modification strategies include artificial mutagenesis, adaptive evolution, gene knockout, and overexpression of key genes. These static regulatory strategies may increase the metabolic burden on cells, cause cytotoxicity, and lead to intracellular metabolic imbalances, thereby limiting cell growth and production capacity. By employing synthetic biology techniques and introducing artificial gene circuits to dynamically regulate key genes, enabling their expression to respond to changes in the intracellular and extracellular environment, the growth capacity and production capacity of microorganisms in industrial production processes can be matched, reducing the waste of materials and energy and greatly improving the production performance of microbial factories (Current Opinion in Biotechnology, 2019, 59:122-129.).

[0004] Synthetic biology incorporates engineering concepts to simplify and modularize naturally occurring regulatory molecules, designing various biological components with basic functions. Artificial gene circuits utilize these biological components, designed according to electronic engineering principles, to reprogram natural gene regulatory circuits to achieve various biological functions. Artificial gene circuits can be mainly divided into two categories: basic and combinatorial. Basic artificial gene circuits are designed and constructed based on biological knowledge and the principles of logic control in circuits, including gene switches, oscillators, logic gates, amplifiers, counters, pulse signal generators, signal filters, etc. (Nature Methods, 2014, 11(5):508-520.). Integrating multiple basic artificial gene circuits can build combinatorial artificial gene circuits with more complex performance, used to simulate advanced life processes. In 2019, Gao et al. used three orthogonal proteases, TEVp, TVMVp and SuMMVp, to control the stability and degradation rate of protein elements in gene circuits within cells, constructing two sets of controllable OFF-switch units and ON-switch units. They then rationally configured these units into three tools: a dynamic regulation circuit (pbDRC), a converter (pbI), and an oscillator (pbO), to achieve rapid response and tunable control of metabolic flux (Nature Communications, 2019, 10(1):3751.).

[0005] Quorum sensing (QS) is a process of chemical signal exchange among microorganisms. Microorganisms can regulate gene expression through the production, sensing, and feedback of their own inducers, enabling them to synchronize their collective behavior in response to changes in population density. Quorum sensing systems are autonomously regulated, independent of metabolic pathways, and do not require the addition of inducers. Using quorum sensing gene circuits in industrial production can significantly reduce the cost of artificial regulation and inducers.

[0006] The EsaI / EsaR quorum sensing system originates from the Gram-negative bacterium *Pantoeastewartii* subsp. *stewartii*. The signaling receptor EsaR in this system can exert both activating and inhibitory effects (Journal of Bacteriology, 2009, 191(24):7402-7409.)(Molecular Microbiology, 2002, 44(6):1625-1635.). At low cell densities, the transcription factor EsaR binds to the PesaR promoter to inhibit the expression of its downstream genes, and can also bind to the PesaS promoter to activate the expression of its downstream genes. The concentration of the signaling molecule AHL is positively correlated with cell density. When cell density is high, the concentration of AHL reaches a threshold, binding to the transcription factor EsaR, preventing EsaR from binding to the transcriptional regulatory region of the promoter, thereby initiating the expression of downstream genes of PesaR and inhibiting the expression of downstream genes of PesaS. These two quorum sensing systems, with their different response mechanisms, can be used to construct gene circuits with varying performance to regulate and redistribute carbon and energy flows between cell growth and product production. Furthermore, due to the dual regulatory function of the EsaI / EsaR system, it has been widely used in the engineering of bacterial strains in recent years.

[0007] Understanding the mechanisms of natural quorum sensing systems at the molecular level has laid the foundation for designing and constructing gene circuits with specific cellular behaviors using quorum sensing systems. To improve the versatility of the EsaI / EsaR system and enable it to achieve different response performances, in 2013, Jasmine Shong et al. modified the transcriptional activator EsaR and the response promoter PesaR, obtaining a series of EsaR mutants with different sensitivities to AHL and activation performance to the response promoter, as well as a series of PesaR promoters with different expression intensities, dynamic ranges, and sensitivities, providing new elements for quorum sensing systems (ACS Chemical Biology, 2013, 8(4): 789-795.)(ACS Chemical Biology, 2013, 2: 568-575.). Based on the EsaI / EsaR system, Apoorv Gupta et al. designed a finely regulated gene circuit to control the expression of phosphofructokinase 1-phosphate and shikimic acid kinase by fine-tuning the expression level of EsaI, achieving dynamic switching between the growth mode and production mode of the strain. This resulted in a 5.5-fold increase in the production of myo-inositol (MI) in the recombinant strain compared to the wild type (Nature Biotechnology, 2017, 35(3):273-279.). In 2020, Gu et al. utilized the dual regulatory role of EsaR to construct a quorum sensing bidirectional switch in E. coli that could simultaneously upregulate or downregulate, and used it to dynamically regulate the biosynthesis of poly-β-hydroxybutyrate (PHHB). This resulted in a PHB production of 6.73 g / L in the recombinant strain, which was 6 times higher than that of strains without a quorum sensing circuit (ACS Synthetic Biology, 2020, 9(2):209-217.).

[0008] Most current research on the EsaI / EsaR quorum sensing gene circuit is conducted under optimal laboratory conditions. However, the output performance of this circuit in real-world applications, such as acidic environments, may be unstable. During industrial fermentation, strains often face acid stress. Improving the acid tolerance of strains allows them to produce under acidic conditions, reducing alkali consumption and pollutant emissions, thus promoting energy conservation, emission reduction, and green manufacturing. However, the PesaS promoter of the EsaI / EsaR quorum sensing gene circuit exhibits severe crosstalk, including pH crosstalk, which significantly affects the output performance of this quorum sensing gene circuit under actual fermentation conditions, greatly limiting its industrial application. Summary of the Invention

[0009] To address the aforementioned problems in existing technologies, this invention provides a method for constructing and applying a highly robust quorum sensing gene circuit, which can be used to improve the acid resistance and fermentation production performance of industrial microorganisms under industrial conditions.

[0010] The robust quorum sensing gene circuit described in this invention exhibits more stable output performance in both acidic and neutral environments compared to the original circuit. This invention decouples the dual function of the response promoter PesaS to obtain a promoter with quorum sensing capabilities that is unaffected by pH crosstalk. The quorum sensing gene circuit constructed using this promoter demonstrates more stable output performance in both acidic and neutral environments.

[0011] The technical solution of the present invention is as follows:

[0012] A highly robust quorum sensing gene circuit is characterized by comprising one or more promoters, one or more terminators, transcription factor genes, and signal molecule synthase genes; wherein,

[0013] (a) Promoters include decoupled quorum sensing promoters with specific performance, constitutive promoters PJ23117 promoter and P8 promoter;

[0014] (b) Terminators include the rrnB T1 terminator, the rrnB T terminator, and the L3S2P21 T terminator;

[0015] (c) The transcription factor gene is esaR I70V;

[0016] (d) The gene for the signal molecule synthase is esaI;

[0017] This invention provides a decoupled quorum sensing promoter with specific performance, wherein the promoter is one of the PHU3 promoter and the PEL promoter, wherein...

[0018] The sequence of the PHU3 promoter is shown in SEQ ID NO.1;

[0019] The sequence of the PEL promoter is shown in SEQ ID NO.2.

[0020] Specifically, the promoter is derived from the bacterial wilt pathogen of maize and is obtained by decoupling based on the dual properties of PesaS. The sequence of the PesaS promoter is shown in SEQ ID NO:3.

[0021] In the specific implementation scheme, the promoter can be activated by EsaR and deactivated by AHL without being affected by pH crosstalk, and its quorum sensing mechanism is consistent with that of the original PesaS promoter.

[0022] This invention provides a highly robust quorum sensing gene circuit with more stable output performance in both acidic and neutral environments.

[0023] The transcription factor gene is esaR I70V, and its gene encoding is shown in SEQ ID NO:7;

[0024] The signal molecule synthase gene is esaI, and its gene encoding is shown in SEQ ID NO:8.

[0025] A nucleic acid construct comprising the aforementioned quorum sensing promoter or gene circuitry comprises two parts. The first part is a response module, comprising, from 5' to 3', a decoupled quorum sensing promoter PHU3 or PEL, the gene to be regulated, and an rrnB T1 terminator. The second part is a regulatory module, comprising, from 5' to 3', a PJ23117 promoter, a transcription factor gene, an rrnB T terminator, an L3S2P21 T terminator, a signal molecule synthase gene, and a P8 promoter.

[0026] The promoter for expressing the transcription factor gene is the PJ23117 promoter, which is a constitutive promoter, and the gene encoding is shown in SEQ ID NO:4;

[0027] The promoter of the gene expressing the signaling molecule synthase is the P8 promoter, which is a constitutive promoter, and the gene encoding is shown in SEQ ID NO:5;

[0028] The response promoter is a decoupled promoter with quorum sensing performance as described in Part I, namely the PHU3 promoter or the PEL promoter;

[0029] The terminator for the expressed transcription factor gene is the rrnB T terminator, whose gene encoding is shown in SEQ ID NO:13;

[0030] The terminator of the gene expressing the signal molecule synthase is the L3S2P21 T terminator, and the gene encoding is shown in SEQ ID NO:14;

[0031] The terminator for expressing the target gene is the rrnB T1 terminator, whose gene code is shown in SEQ ID NO:12.

[0032] In some embodiments, the gene to be regulated includes the fluorescent protein gene mCherry or an acid-fast module.

[0033] The sequence of the fluorescent protein gene mCherry is shown in SEQ ID NO:6.

[0034] In some embodiments, the acid-resistant module to be regulated comprises three parts: the chaperone protein Hfq, the ribozyme HH, and sRNAdsrA.

[0035] The gene coding sequence of the chaperone protein Hfq is shown in SEQ ID NO:9;

[0036] The coding sequence of the ribozyme HH gene is shown in SEQ ID NO:11;

[0037] The coding sequence of the sRNAdsrA gene is shown in SEQ ID NO:10.

[0038] The present invention provides an expression vector comprising a quorum sensing promoter, a gene circuit, or a nucleic acid construct of the aforementioned nucleic acid construct.

[0039] In some embodiments, the nucleic acid constructs of the present invention are constructed based on commercial plasmids pACYC184, pCOLA, and pZS1.

[0040] Furthermore, the expression vectors for the aforementioned nucleic acid constructs include, but are not limited to, other plasmids and genomes.

[0041] The present invention provides a recombinant host cell comprising the quorum sensing promoter, the gene circuit, the nucleic acid construct, or the expression vector.

[0042] The recombinant host cell of the present invention is preferably a prokaryotic cell, more preferably an Escherichia coli cell, such as the MG1655 strain.

[0043] This invention provides a method for testing microbial acid growth, the method comprising:

[0044] (a) Introduce the highly robust quorum sensing gene circuit, the nucleic acid construct, or the expression vector as described above into the recombinant host cells described in the fifth aspect above;

[0045] (b) The growth rate of the microorganism under stress conditions was evaluated using a high-throughput growth assay instrument, such as Bioscreen C.

[0046] Furthermore, the term "stress" used in the application refers to stress conditions that primarily affect the growth of microorganisms. For example, for Escherichia coli, acid stress refers to a pH level below 7.0 but above 4.0, such as pH 5.5 or pH 4.5.

[0047] This invention provides a method for producing organic acids through microbial fermentation, the method comprising:

[0048] (a) Introducing the quorum sensing acid-resistant circuit of the present invention into microorganisms that produce organic acids;

[0049] (b) Fermenting the microorganisms;

[0050] (c) Harvesting the organic acids produced.

[0051] Preferably, in the above method for producing organic acids by microbial fermentation, the microorganism used to produce organic matter is Escherichia coli.

[0052] Furthermore, the organic acids produced by the above-mentioned method of producing organic acids through microbial fermentation include, but are not limited to, amino acids (such as lysine, threonine, tryptophan, and glutamic acid), succinic acid, citric acid, and lactic acid.

[0053] The beneficial technical effects of this invention are as follows:

[0054] This invention addresses the issue of unstable output performance of the EsaI / EsaR quorum sensing system in culture environments with different pH levels by providing a method for constructing and applying a highly robust quorum sensing gene circuit. The invention provides the gene sequence of the response promoter of the decoupled quorum sensing system, the sequences of each element of the highly robust quorum sensing circuit, the sequences of each element of the quorum sensing acid-fast gene circuit, the construction method of each component, and a specific application based on *E. coli*.

[0055] The specific effect is as follows:

[0056] 1. Specifically, this invention modifies the original swarm induction system's response promoter PesaS to obtain promoters PHU3 and PEL with simplified functions after decoupling.

[0057] 2. The promoters PHU3 and PEL described in this invention can be activated by EsaR and deactivated by AHL without being affected by pH crosstalk, exhibiting good specificity; their quorum sensing mechanism is consistent with the original PesaS promoter.

[0058] 3. The quorum sensing gene circuit PHU3-QS constructed using the PHU3 promoter in this invention exhibits stable output performance and high robustness in acidic environments (pH 4.5 and pH 5.5).

[0059] 4. This invention decouples the dual performance of the PesaS promoter in the EsaI / EsaR quorum sensing system, obtaining promoters PHU3 and PEL that can only be activated by EsaR and are not affected by pH crosstalk, thus enriching the response element library of quorum sensing systems.

[0060] 5. This invention constructs a quorum sensing gene circuit with high robustness in acidic environments. Its output performance is more stable in different pH environments, which improves the applicability of the EsaI / EsaR quorum sensing system in different pH environments and provides new ideas and references for the research and optimization of quorum sensing.

[0061] 6. This invention improves the growth ability of Escherichia coli in acidic environments and enhances the fermentation performance of lysine-producing microorganisms in acidic environments by utilizing highly robust quorum sensing gene circuits to regulate the expression of acid-resistant modules. Attached Figure Description

[0062] Figure 1 Schematic diagram of PHU3 and PEL promoter design;

[0063] Figure 2 A schematic diagram of the gene circuitry of the EsaI / EsaR quorum sensing system;

[0064] Figure 3 The plasmid map of the recombinant plasmid for quorum sensing circuits;

[0065] Figure 4 Characterization of the PHU3 and PEL promoters;

[0066] Figure 5 Characterization of the PesaS-QS and PHU3-QS lines;

[0067] Figure 6 The plasmid map of the recombinant plasmid for quorum sensing acid resistance circuitry;

[0068] Figure 7 Acid growth test for strains containing the PHU3-QS acid-resistant circuit;

[0069] Figure 8 Micro-fermentation production test of industrial strains containing the PHU3-QS acid-resistant circuit. Detailed Implementation

[0070] The present invention will be further illustrated below by way of embodiments, but the invention is not limited to the scope of the described embodiments. Unless otherwise specified, all raw materials used in the present invention are commercially available.

[0071] This invention modifies the PesaS promoter of the EsaI / EsaR quorum sensing system to obtain decoupled quorum sensing promoters PHU3 and PEL, whose promoter design is as follows: Figure 1 As shown.

[0072] The PHU3 promoter was obtained by deleting the upstream sequence of the esa box of the PesaS promoter and the sequence between the esa box and the -35 region. Its gene sequence is SEQ ID NO:1.

[0073] The PEL promoter was obtained by swapping the esa box of the PesaS promoter to the lux box, the transcription factor binding site of another activating promoter, PluxI I11. Its gene sequence is SEQ ID NO:2.

[0074] The strains used in the embodiments of the present invention are E. coli DH10B or E. coli MG1655.

[0075] The plasmid vectors used in the embodiments of the present invention are pCOLA, pACYC184 or pZS1.

[0076] In the embodiments of the present invention, the culture temperature of the recombinant strains was 37°C.

[0077] The antibiotics used in the embodiments of the present invention are kanamycin (final concentration of 50 μg / mL), chloramphenicol (final concentration of 34 μg / mL), or carbenicillin (final concentration of 100 μg / mL).

[0078] The culture media used in the embodiments of the present invention are LB solid medium, LB liquid medium, LBG medium, seed medium, or fermentation medium.

[0079] LB solid medium: tryptone 10 g / L, NaCl 10 g / L, yeast extract 5 g / L, agar powder 15 g / L. LB liquid medium: tryptone 10 g / L, NaCl 10 g / L, yeast extract 5 g / L.

[0080] LBG medium: tryptone 10 g / L, NaCl 10 g / L, yeast extract 5 g / L, anhydrous glucose 20 g / L.

[0081] Seed culture medium: yeast extract 5 g / L, tryptone 7 g / L, ammonium sulfate 5 g / L, potassium dihydrogen phosphate 5 g / L, sucrose 150 mg / L, magnesium sulfate 25 mg / L, ferrous sulfate 0.6 mg / L, manganese sulfate 0.6 mg / L, monosodium glutamate 120 mg / L, threonine 15 mg / L, methionine 15 mg / L, pyruvate 15 mg / L. After mixing, adjust the pH to 6.8–7.0 with KOH solution.

[0082] Fermentation medium: glucose 30 g / L, phosphate 0.6 mL / L, magnesium sulfate 2 g / L, ammonium sulfate 10 g / L, corn steep liquor 0.325 g / L, potassium chloride 0.5 g / L, betaine 2.2 g / L, foaming agent (3%) 0.05 mg / L, ferrous sulfate 32 mg / L, manganese sulfate 32 mg / L, threonine 250 mg / L, copper sulfate 6.8 mg / L, zinc sulfate 7.65 mg / L, vitamin B1 5.6 mg / L. After mixing, adjust the pH to 6.8 with 5% ammonia water.

[0083] The gene circuit diagram of the EsaI / EsaR quorum sensing system in this invention is shown below. Figure 2 As shown, when the bacterial cell density is low, the EsaR transcription factor binds to the response promoter after expression, activating the expression of downstream genes of the response promoter; when the population density is high, the signaling molecule AHL synthesized by EsaI reaches a certain concentration, and AHL binds to EsaR, preventing EsaR from binding to the response promoter and reducing the expression of downstream genes of the response promoter.

[0084] Example 1: Construction of regulatory and response plasmids in a quorum sensing gene circuit using fluorescent protein as a reporter gene

[0085] 1. Construction of the regulatory plasmid expression vector pACYC184-PJ23117-esaR I70V-rrnB T containing the EsaR module

[0086] The esaR I70V-rrnB T fragment and the pACYC184-PJ23117 fragment were amplified by PCR using primers pJ23x-esaR-F / pJ23x-sfGFP-R and pJ23x-pACYC-F / pJ23117-pACYC-R, respectively. The two corresponding fragments were then ligated using Gibson assembly to obtain the pACYC184-PJ23117-esaR I70V-rrnB T plasmid.

[0087] 2. Construction of the regulatory plasmid expression vector pACYC184-PJ23117-esaR I70V-rrnB T-L3S2P21 T-esaI-P8 containing EsaR and EsaI modules

[0088] Using primers T-Psal-esaI-F / L19S-R, pACYC-F4 / T-Ptac-esaR-R, and pACYC-F6 / pACYC-R3, the P8-esaI-L3S2P21 T fragment, the PJ23117-esaRI70V-rrnB T fragment, and the pACYC184 backbone fragment were amplified by PCR. The three fragments were then ligated using Gibson assembly to obtain the plasmid pACYC184-PJ23117-esaRI70V-rrnB T-L3S2P21 T-esaI-P8.

[0089] 3. Construction of the response plasmid expression vector pCOLA-PesaS-mCherry-rrnB T1 containing the PesaS promoter

[0090] The mCherry-rrnB T1 gene fragment and the pCOLA-PesaS fragment were amplified by PCR using primers PesaS-RFP-F / RFP-R1 and RFP-F1 / PesaS-RFP-R, respectively. The two corresponding fragments were then ligated using Gibson assembly to obtain the pCOLA-PesaS-mCherry-rrnB T1 plasmid.

[0091] 4. Construction of the response plasmid expression vector pCOLA-PHU3-mCherry-rrnB T1 containing the PHU3 promoter

[0092] The PHU3-mCherry-rrnB T1 fragment and the pCOLA-PHU3 fragment were amplified by PCR using primers PHU3-F / pCOLA-R4 and pCOLA-F4 / PHU3-R, respectively. The two corresponding fragments were then ligated using Gibson assembly to obtain the pCOLA-PHU3-mCherry-rrnB T1 plasmid.

[0093] 5. Construction of the response plasmid expression vector pCOLA-PEL-mCherry-rrnB T1 containing the PEL promoter

[0094] The PEL-mCherry-rrnBT1 fragment and the pCOLA-PEL fragment were amplified by PCR using primers PEL-F / pCOLA-R4 and pCOLA-F4 / PEL-R, respectively. The two corresponding fragments were then ligated using Gibson assembly to obtain the pCOLA-PEL-mCherry-rrnBT1 plasmid.

[0095] The ligation product of the Gibson assembly was transformed into E. coli DH10B strain, plated with the corresponding antibiotic, and transformants were picked for colony PCR verification. Plasmids were extracted from positive transformants and sequenced. The sequencing results were correct, yielding the correct regulatory and response plasmids. The plasmid map is shown below. Figure 3 As shown.

[0096] Table 1. Sequences of primers described in Example 1

[0097]

[0098] After the above steps, two regulatory plasmid expression vectors were obtained, including the regulatory plasmid expression vector pACYC184-PJ23117-esaR I70V-rrnB T containing the EsaR module and the regulatory plasmid expression vector pACYC184-PJ23117-esaR I70V-rrnB T-L3S2P21 T-esaI-P8 containing both the EsaR and EsaI modules, as well as three response plasmid expression vectors, including pCOLA-PesaS-mCherry-rrnB T1, pCOLA-PHU3-mCherry-rrnB T1, and pCOLA-PEL-mCherry-rrnB T1.

[0099] Example 2: Characterization of PHU3 and PEL promoters

[0100] 1. Characterization of promoters in different pH environments

[0101] To investigate whether the promoter is subject to pH crosstalk, mCherry was used as a fluorescent reporter protein. The response plasmids pCOLA-PesaS-mCherry-rrnB T1, pCOLA-PHU3-mCherry-rrnB T1 and pCOLA-PEL-mCherry-rrnB T1 constructed in Example 1 were transformed into E. coli strain MG1655, respectively, to obtain strains MG1655 / pCOLA-PesaS-mCherry, MG1655 / pCOLA-PHU3-mCherry and MG1655 / pCOLA-PEL-mCherry.

[0102] The modified promoters were characterized using an ELISA reader in LBG medium at pH 4.5 and pH 7.0, and their fluorescence intensity was measured after 24 hours of culture. The results are as follows: Figure 4As shown in Figure A. Characterization results show that the fluorescence intensity of the PesaS promoter at pH 4.5 is significantly higher than that at pH 7.0, with a fold change of 10.1-fold, indicating pH crosstalk. The fluorescence intensities of the PHU3 and PEL promoters at pH 4.5 and pH 7.0 are not significantly different, with fold changes of 1.6 and 0.9-fold, respectively, and are not affected by pH crosstalk.

[0103] 2. Characterization of promoter quorum sensing

[0104] To investigate whether the promoter has quorum sensing capabilities, i.e., whether it can be activated by EsaR, the response plasmid using mCherry as a fluorescent reporter protein constructed in Example 1 and the regulatory plasmid containing the EsaR module were simultaneously transformed into E. coli MG1655, resulting in strains MG1655 / pACYC-PJ23117-esaR&pCOLA-PesaS-mCherry, MG1655 / pACYC-PJ23117-esaR&pCOLA-PHU3-mCherry, and MG1655 / pACYC-PJ23117-esaR&pCOLA-PEL-mCherry.

[0105] The expression intensity of the promoter was compared in LBG medium at pH 7.0 with and without EsaR. The results are as follows: Figure 4 As shown in Figure B. Characterization results show that in the presence of EsaR, the fluorescence intensity of the PesaS, PHU3, and PEL promoters was increased, with EsaR activation factors of 26.6, 40.2, and 9.7 times, respectively, indicating that EsaR can activate the expression of these three promoters.

[0106] To further determine whether the newly constructed promoter PHU3 operates on the same mechanism as PesaS, different concentrations of AHL were added exogenously to strains containing the EsaR module, and the changes in promoter fluorescence expression intensity were tested. The results are as follows: Figure 3 As shown in Figure C. Characterization results show that when the AHL concentration is below 100 nM, the expression intensity of the PesaS, PHU3, and PEL promoters does not change significantly with increasing AHL concentration; when the AHL concentration is above 100 nM, the expression intensity of the PesaS, PHU3, and PEL promoters gradually decreases with increasing AHL concentration, indicating that when AHL reaches a certain concentration, it can relieve the activation effect of EsaR on the PHU3 and PEL promoters, and the mechanism of action of this promoter is consistent with that of PesaS.

[0107] After the above steps, promoters PHU3 and PEL, which are not affected by pH crosstalk, can be activated by EsaR, and interact with the original PesaS, were obtained.

[0108] Example 3: Characterization of quorum sensing gene circuits

[0109] To compare the output performance of the quorum sensing gene circuit before and after modification and to construct a complete quorum sensing system, a response plasmid using mCherry as a fluorescent reporter protein and a regulatory plasmid containing EsaR and EsaI modules were simultaneously transformed into E. coli MG1655, resulting in strains MG1655 / pACYC-PJ23117-esaR-esaI-P8&pCOLA-PesaS-mCherry and MG1655 / pACYC-PJ23117-esaR-esaI-P8&pCOLA-PHU3-mCherry.

[0110] The performance of the PesaS-QS and PHU3-QS circuits was characterized in LBG medium at pH 4.5, pH 5.5, and pH 7.0, and the results are as follows: Figure 5 As shown in Figures A and B. Characterization results show that the strength of the PesaS promoter gradually increases with decreasing pH, with a promoter strength factor of 11.1 at pH 4.5 and pH 7.0, and a promoter strength factor of 4.1 at pH 5.5 and pH 7.0, indicating pH crosstalk.

[0111] To better reflect the performance of quorum sensing gene circuits, the ratio of the expression intensity of a circuit containing only the EsaR module to the expression intensity of a circuit containing only the responsive promoter under the same pH conditions is defined as the EsaR activation fold, and the ratio of the expression intensity of a circuit containing both the EsaR and EsaI modules to the expression intensity of a circuit containing only the EsaR module under the same pH conditions is defined as the AHL deactivation fold.

[0112] like Figure 5 As shown in C and 5D, with the decrease of pH in the culture environment, the EsaR activation factor and AHL deactivation factor of the PesaS-QS circuit both decreased, with coefficients of variation of 0.55 and 0.54, respectively. This indicates that the output performance of the PesaS-QS circuit is unstable under acidic conditions. This is because pH crosstalk affects the performance of the PesaS-QS circuit in acidic environments.

[0113] After replacing the PesaS promoter with the PHU3 promoter, the EsaR activation factor and AHL deactivation factor of the PHU3-QS circuit remained relatively stable at around 30.4 times and 2.9 times, respectively, with the coefficients of variation decreasing to 0.14 and 0.07, respectively. This indicates that removing the effect of pH crosstalk on the response promoter effectively mitigated the impact of the acidic environment on quorum sensing performance.

[0114] After the above steps, a swarm induction circuit PHU3-QS with high robustness in acidic environments was obtained.

[0115] Example 4: Construction of a quorum sensing system response plasmid expressing an acid-resistant module

[0116] 1. Construction of the response plasmid expression vector pCOLA-PHU3-Hfq-HH-dsrA for expressing the acid-resistant module via the PHU3 promoter.

[0117] The Hfq-HH-dsrA fragment and the pCOLA-PHU3 fragment were amplified by PCR using primers PJ23x-esaR-F / pACYC-R4 and pACYC-F4 / PJ23x-pACYC-R, respectively. The two corresponding fragments were then ligated using Gibson assembly to obtain the pCOLA-PHU3-Hfq-HH-dsrA plasmid.

[0118] 2. Construction of the low-copy vector regulatory plasmid expression vector pZS1-PJ23117-esaR I70V-rrnBT containing the EsaR module

[0119] The PJ23117-esaR I70V-rrnB T fragment and the pZS1 backbone fragment were amplified by PCR using primers pZS1-ON-F / pZS1-ON-R and pZS1-Bone-F / pZS1-Bone-R, respectively. The two corresponding fragments were then ligated using Gibson assembly to obtain the pZS1-PJ23117-esaR I70V-rrnB T plasmid.

[0120] 3. Construction of the low-copy vector regulatory plasmid expression vector pZS1-PJ23117-esaRI70V-rrnB T-L3S2P21 T-esaI-P8 containing EsaR and EsaI modules

[0121] The PJ23117-esaR I70V-rrnB T-L3S2P21T-esaI-P8 fragment and the pZS1 backbone fragment were amplified by PCR using primers pZS1-ON-F / pZS1-P8-R and pZS1-Bone-F / pZS1-Bone-R, respectively. The two corresponding fragments were then ligated using Gibson assembly to obtain the plasmid pZS1-PJ23117-esaR I70V-rrnB T-L3S2P21T-esaI-P8.

[0122] The ligation product of the Gibson assembly was transformed into E. coli DH10B strain, plated with the corresponding antibiotic, and transformants were picked for colony PCR verification. Plasmids were extracted from positive transformants and sequenced. The sequencing results were correct, yielding the correct regulatory and response plasmids. The plasmid map is shown below. Figure 6 As shown.

[0123] Table 2 shows the sequences of the primers described in Example 4.

[0124]

[0125] After the above steps, two low-copy regulatory plasmid expression vectors were obtained, including the regulatory plasmid expression vector pZS1-PJ23117-esaR I70V-rrnB T containing the EsaR module, the regulatory plasmid expression vector pZS1-PJ23117-esaR I70V-rrnB T-L3S2P21 T-esaI-P8 containing both the EsaR and EsaI modules, and the response plasmid expression vector pCOLA-PHU3-Hfq-HH-dsrA expressing the acid-fast module.

[0126] Example 5: Acid growth test of quorum-sensing acid-resistant strains

[0127] The regulatory plasmid constructed in Example 4 was co-transformed into E. coli MG1655 along with the response plasmid expressing the acid-fast module, constructing strains MG1655 / pZS1-PJ23117-esaR&pCOLA-PHU3-HD and MG1655 / pZS1-PJ23117-esaR-esaI-P8&pCOLA-PHU3-HD. Furthermore, the response plasmid expressing the acid-fast module was transformed into E. coli MG1655 to construct the control strain MG1655 / pCOLA-PHU3-HD.

[0128] To test whether the PHU3-QS acid-resistant pathway could improve the acid resistance of *E. coli*, a 24-hour acid growth test was conducted on the PHU3-QS acid-resistant strain in LBG medium at pH 4.5, pH 5.5, and pH 7.0. The test results are as follows: Figure 7 As shown in Table 3. Acid growth test results showed that the final OD of the PHU3-QS acid-resistant strain was [data missing] in media at pH 4.5 and pH 5.5. 600 Compared with the wild type MG1655, the growth rate was increased by 19.0% and 14.3%, respectively, and the maximum growth rate was increased by 9.7% and 9.5%, respectively, indicating that the quorum sensing acid-resistant circuit can enhance the acid tolerance of Escherichia coli in a moderately acidic environment.

[0129] Table 3. Acid growth test results of PHU3-QS acid-resistant strains

[0130]

[0131] Example 6: Micro-fermentation production test of quorum-sensing acid-fast strains

[0132] The regulatory plasmid constructed in Example 4 was co-transformed with the response plasmid expressing the acid-fast module into a modified lysine-producing *Escherichia coli* strain MG1655 (this strain is a lysine-producing strain SCEcL3 obtained by modifying MG1655 according to the method disclosed in Chinese patent application CN103773745A), constructing strains SC / pZS1-PJ23117-esaR&pCOLA-PHU3-HD and SC / pZS1-PJ23117-esaR-esaI-P8&pCOLA-PHU3-HD. Furthermore, the response plasmid expressing the acid-fast module was transformed into a lysine fermentation strain to construct the control strain SC / PHU3-HD.

[0133] Table 4. Results of micro-fermentation production test of PHU3-QS acid-fast strain

[0134]

[0135] The strain was micro-fermented for 48 hours in 48-well quincunx plates. The initial pH of the fermentation medium was 6.8, and the pH was maintained at approximately 6.0 during fermentation by adding ammonia. As controls, the pH of the original industrial strain's culture medium was maintained at 6.8 and 6.0, respectively. After 48 hours of fermentation, the OD values ​​of the control and recombinant strains were measured. 600 and lysine concentration, the results are as follows Figure 8 As shown in Table 4. The fermentation results showed that after 48 hours of fermentation, the lysine yield of the recombinant strain reached 10.4 g / L, which was 25.3% higher than that of the parent strain and comparable to the lysine yield of the parent strain when the pH was controlled at 6.8 during fermentation. This indicates that the quorum sensing acid tolerance circuit can improve the acid tolerance of the industrial strain during fermentation, thereby improving its production performance.

Claims

1. A method for constructing a quorum sensing gene circuit, characterized in that, It is composed of one or more promoters, one or more terminators, transcription factor genes, and signal molecule synthase genes; among them, (a) Promoters include decoupled quorum sensing promoters with specific performance, constitutive promoters PJ23117 promoter and P8 promoter; (b) Terminators include the rrnB T1 terminator, the rrnB T terminator, and the L3S2P21 T terminator; (c) The transcription factor gene is esaR I70V; (d) The gene for the signal molecule synthase is esaI; The decoupled quorum sensing promoter with specific performance is one of the PHU3 promoter and the PEL promoter; wherein, the sequence of the PHU3 promoter is shown in SEQ ID NO: 1; and the sequence of the PEL promoter is shown in SEQ ID NO:

2.

2. A nucleic acid construct comprising the quorum sensing promoter or the gene circuit of claim 1, characterized in that, It consists of two parts.

3. The nucleic acid construct according to claim 2, characterized in that, The first part is the response module, which from 5' to 3' contains the decoupled quorum sensing promoter PHU3 or PEL, the gene to be regulated, and the rrnB T1 terminator.

4. The nucleic acid construct according to claim 2, characterized in that, The second part is the regulatory module, which includes the PJ23117 promoter, transcription factor gene, rrnB T terminator, L3S2P21 T terminator, signal molecule synthase gene, and P8 promoter from 5' to 3'.

5. An expression vector comprising the quorum sensing promoter, gene circuit, or nucleic acid construct of claim 1 or 2, wherein the expression vector comprises a plasmid or a genome.

6. A recombinant host cell comprising the quorum sensing promoter of claim 1 or 2, the gene circuit, the nucleic acid construct, or the expression vector of claim 5; wherein the host cell is an Escherichia coli cell.

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