Phenol response type signal regulation and control expression system based on DmpR transcriptional regulation and control mechanism and application of phenol response type signal regulation and control expression system

Abstract

The invention discloses a phenol response type signal regulation and control expression system based on a DmpR transcriptional regulation and control mechanism and application of the phenol response type signal regulation and control expression system. The system comprises an upstream biological recognition element T7ter-dmpR-Pr and a downstream signal output element ES-Po-tetA-T7ter, and the function of the downstream element is regulated and controlled by the upstream element. And in the presence of phenol, the DmpR protein is activated and drives the expression of a downstream tetracycline resistance gene tetA. The invention also provides a recombinant plasmid containing the system, a recombinant strain and a phenol biosynthesis co-culture system containing upstream and downstream strains. The co-culture system can autonomously screen downstream strains with high phenol yield under tetracycline selection pressure, so that the phenol yield is remarkably increased, and the yield is increased by about 41% compared with that of an original system. The invention provides an efficient dynamic regulation and evolution strategy for biosynthesis of phenol and screening of high-yield strains.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a phenol-responsive signal regulation expression system based on the DmpR transcriptional regulation mechanism and its application. Background Technology

[0002] Phenol is a widely used basic organic chemical raw material with extensive industrial applications. It is primarily used in the production of phenolic resins, caprolactam, bisphenol A, adipic acid, aniline, alkylphenols, and salicylic acid. It can also be used as a solvent, reagent, and disinfectant. Furthermore, it has wide applications in synthetic fibers, synthetic rubber, plastics, pharmaceuticals, pesticides, fragrances, dyes, and coatings. Currently, the main route for producing phenol is the cumene process, using benzene as a raw material. This method has advantages such as mature technology, simple operation, and high product purity. However, it relies on petrochemical raw materials, which may lead to environmental pollution and resource waste. Moreover, the production process is energy-intensive, relatively costly, non-renewable, and causes significant environmental pollution. With increasing attention to the oil crisis and environmental pollution issues, there is a growing emphasis on developing new sustainable production processes for phenol and its chemical derivatives. Metabolic engineering is a promising alternative method, and numerous articles have been reported on the microbial synthesis of phenol.

[0003]

Wierckx NJ, Ballerstedt H, de Bont JA, Wery J. Engineering of solvent-tolerant Pseudomonas putida S12 for bioproduction of phenol from glucose. Appl Environ Microbiol, 2005, 71(12): 8221-8227.

[0004] With the rapid development of metabolic engineering technology, remarkable achievements have been made in using gene cloning to alter metabolic flux, expand and construct new metabolic pathways, and maximize the metabolic yield of target products. An increasing number of synthetic biology tools and strategies have been developed to improve the biosynthetic efficiency of engineered strains, including optimizing enzyme activity and expression levels, blocking competing metabolic pathways, modifying central metabolism, and dynamically regulating synthetic genetic circuits. Currently, this method of optimizing metabolic pathways has made significant progress in the field of biosynthesis, enabling the efficient biosynthesis of a range of valuable compounds, including food, energy substances, pharmaceuticals, and chemical products.

[0005] As the conversion rate of biosynthesis gradually approaches the theoretical maximum, further improving the synthesis efficiency or synthesizing more complex compounds urgently requires the establishment and breakthrough of new methods. Summary of the Invention

[0006] To overcome the limitations of metabolic regulation in engineered strains, this invention aims to provide a phenol-responsive signal-regulated expression system based on the DmpR transcriptional regulation mechanism and its application. This phenol-responsive signal-regulated expression system can promote the continuous screening of high-yielding strain subpopulations and inhibit the growth of low-yielding cell subpopulations during the fermentation process of engineered phenol-synthesizing strains, ultimately improving the biosynthetic efficiency of phenol.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: A phenol-responsive signal regulation expression system based on the DmpR transcriptional regulation mechanism includes an upstream biorecognition element and a downstream signal output element, wherein the function of the downstream signal output element is regulated by the upstream biorecognition element. The upstream biometric element is T7 ter-dmpR-Pr, which contains the T7 terminator and the dimethylphenol regulatory protein gene. dmpR and the Pr promoter; The downstream signal output element is ES-Po-tetA-T7 ter, which includes an ES enhancer, a Po promoter, and a tetracycline resistance gene. tetA and T7 terminator; The nucleotide sequence of the upstream biometric element is shown in SEQ ID NO.1, and the nucleotide sequence of the downstream signal output element is shown in SEQ ID NO.2.

[0008] A recombinant plasmid containing the above-mentioned phenol-responsive signal-regulated expression system; the recombinant plasmid is a circular structure, and its nucleotide sequence is shown in SEQ ID NO.3.

[0009] A recombinant bacterial strain obtained by transforming host cells with the aforementioned recombinant plasmid; wherein the host cell is *Escherichia coli*, selected from *Escherichia coli*. E. coli BL21 (DE3) or engineered Escherichia coli strain DY2-3.

[0010] A phenol biosynthesis co-culture system includes an upstream strain and a downstream strain; the upstream strain uses glucose as a substrate and is responsible for synthesizing p-hydroxybenzoic acid, an intermediate in phenol biosynthesis; the downstream strain is responsible for taking up p-hydroxybenzoic acid from the culture medium and further converting it into the final product phenol. The upstream strain is an engineered Escherichia coli strain UY3R; the downstream strain is obtained by injecting a circular plasmid with the nucleotide sequence shown in SEQ ID NO.3 into an engineered Escherichia coli strain DY2-3.

[0011] The method for constructing the above-mentioned phenol biosynthesis co-culture system includes the following steps: (1) Using the engineered Escherichia coli strain UY3R as the upstream strain; construct a circular plasmid with the nucleotide sequence shown in SEQ ID NO.3, and transform it into the engineered Escherichia coli strain DY2-3 to obtain the downstream strain; (2) The upstream strain and the downstream strain were co-cultured in a medium containing tetracycline and glucose; The culture medium containing tetracycline and glucose is formulated as follows: tetracycline 10 mg / L, glucose 5 g / L, yeast extract 0.5 g / L, ammonium chloride 2 g / L, ammonium sulfate 5 g / L, potassium dihydrogen phosphate 3 g / L, dipotassium hydrogen phosphate 7.3 g / L, 3-morpholine propanesulfonic acid 8.4 g / L, sodium chloride 0.5 g / L, magnesium sulfate 0.24 g / L, tyrosine 40 mg / L, phenylalanine 40 mg / L, and tryptophan 40 mg / L. mg / L, 4-hydroxybenzoic acid 10 mg / L, disodium ethylenediaminetetraacetate 0.4 mg / L, boric acid 0.03 mg / L, thiamine 1 mg / L, zinc chloride 0.94 mg / L, cobalt chloride 0.5 mg / L, copper chloride 0.38 mg / L, manganese chloride 1.6 mg / L, calcium chloride 3.77 mg / L, ferrous chloride 3.6 mg / L, isopropyl-β-D-thiogalactoside 0.5 mmol / L.

[0012] The aforementioned phenol-responsive signaling regulatory expression system regulates the tetracycline resistance gene. tetA Applications in expression and / or phenol biosynthesis and / or screening of high-yield phenol strains.

[0013] The above-mentioned recombinant strains are used in phenol biosynthesis and / or screening of high-yield phenol strains.

[0014] The above-mentioned co-culture system for phenol biosynthesis is applied in phenol biosynthesis and / or screening of high-yield phenol strains.

[0015] The significant advantages of this invention are: A high-yielding bacterial strain targeted enrichment system with self-selection function was constructed based on the DmpR transcriptional regulation mechanism. By integrating a phenol-responsive signal-regulated expression system into a phenol biosynthesis co-culture system, dynamic and continuous screening of high-yielding bacterial strain subpopulations can be achieved during fermentation: high-yielding cells can activate... tetA By using resistance genes to gain a growth advantage and selectively suppressing low-yielding cells, the composition of the microbial community can be optimized without human intervention, ultimately significantly improving the biosynthesis efficiency of phenol. Attached Figure Description

[0016] Figure 1 Schematic diagram of the structure of a phenol-responsive signal-regulated expression system.

[0017] Figure 2 The transcriptional mechanism of a phenol-responsive signal-regulated expression system in response to phenol concentration.

[0018] Figure 3 : Mechanism diagram of how a phenol-responsive signal-regulated expression system promotes the screening of high-phenol-producing bacterial strain subpopulations.

[0019] Figure 4 Schematic diagram of the structure of recombinant plasmid pMR.

[0020] Figure 5 The response of a phenol-responsive signal-regulated expression system to different phenol concentrations.

[0021] Figure 6 Metabolic diagram of the phenol biosynthesis co-culture system UY3R:DY2-3. DAHP: 3-deoxy-D-arabinose-heptaphosphate-7-phosphate; DHQ: 3-dehydroquinic acid; DHS: 3-dehydroshikimic acid; SHK: shikimic acid; S3P: shikimic acid-3-phosphate; EPSP: 5-enolacetone-shikimic acid-3-phosphate; CHR: branched acid; 4HB: 4-hydroxybenzoic acid.

[0022] Figure 7 The yield of phenol synthesized by co-culturing the phenol biosynthesis system UY3R:DY2-3 with different inoculation ratios.

[0023] Figure 8 The yield of phenol synthesized by co-culturing the phenol biosynthesis system UY3R:DY2-R with different inoculation ratios. Detailed Implementation

[0024] To make the content of this invention easier to understand, the technical solution of this invention will be further described below with reference to specific embodiments, but this invention is not limited thereto.

[0025] It should be noted that the vector obtained during the research and development of this invention was obtained through various cross-combinations and adjustments of gene sequences and fragments. The specific acquisition process is a trade secret and will not be detailed here. Those skilled in the art can also synthesize it directly according to the disclosure of this invention, without affecting the realization of this invention, only the cost will increase.

[0026] It should be noted that the engineered Escherichia coli strains UY3R and DY2-3 involved in the embodiments of this invention have been disclosed in relevant non-patent literature [Guo Xiaoyun, Li Zhenghong, Wang Xiaonan, Wang Jing, Chala Juan, Lu Yinghua, Zhang Haoran. De novo phenol bioproduction from glucose using biosensor-assisted microbial coculture engineering.[J].Biotechnology and bioengineering, 2019, 116(12).] and are available to the public from Quanzhou Normal University. The applicant promises to distribute the biological materials to the public within twenty years from the date of application.

[0027] Both the engineered E. coli strains UY3R and DY2-3 were constructed using E. coli BH2 as the basic host. This host strain was derived from the BL21(DE3) strain, commonly used for recombinant protein expression, and underwent several genetic modifications. It not only retained the characteristic of BL21(DE3) itself—lacking proteases to improve the stability of exogenous proteins—but also further deleted genes related to xylose metabolism. xylA and key genes involved in the synthesis of tyrosine and phenylalanine. tyrA and pheA .

[0028] Specifically, the engineered *E. coli* strain UY3R carries both the pathway enhancement plasmids pSP2 and pBD, as well as the biosensor plasmid pRA for dynamic screening of high-yield cells, within *E. coli* BH2. Plasmid pSP2 carries a series of key enzyme genes of the shikimate pathway under the control of the constitutive promoter Ppdc, including... aroE, aroL, aroA, aroC, ubiC And versions with feedback inhibition resistance aroGfbr Genes. Plasmid pBD contains genes driven by the promoter PproD. aroB and aroD Gene. The plasmid pRA carries a cell selection system based on a 4-hydroxybenzoic acid biosensor, which includes the gene encoding the sensor protein PobR. pobRAnd the tetracycline resistance gene controlled by the 4-hydroxybenzoic acid-responsive promoter PpobA. tetA .

[0029] The engineered *E. coli* strain DY2-3 carries both the functional plasmid pYCL and the auxiliary plasmids pACYCDuet-1 and pBR322, used to maintain resistance matching, within *E. coli* BH2. Plasmid pYCL contains the 4-hydroxybenzoic acid decarboxylase gene cluster from *E. coli* strain W. yclBCD This gene cluster is driven by the strongly constitutive promoter PproD. Plasmids pACYCDuet-1 and pBR322 primarily provide markers for chloramphenicol, tetracycline, and ampicillin resistance.

[0030] It should be noted that the formulation of the MY1 culture medium involved in the embodiments of the present invention is as follows: glucose 5g / L, yeast extract 0.5g / L, NH4Cl 2g / L, (NH4)2SO4 5g / L, KH2PO4 3g / L, K2HPO4 7.3g / L, 3-morpholine propanesulfonic acid 8.4g / L, NaCl 0.5g / L, MgSO4 0.24g / L, tyrosine 40mg / L, phenylalanine 40mg / L, tryptophan 40mg / L, 4-hydroxybenzoic acid 10mg / L, Na2EDTA 0.4mg / L, H3BO3 0.03mg / L, thiamine 1mg / L, ZnCl2 0.94mg / L, CoCl2 0.5mg / L, CuCl2 0.38mg / L, MnCl2 1.6mg / L, CaCl2 3.77mg / L, FeCl2 3.6 mg / L, IPTG 0.5 mmol / L.

[0031] It should be noted that the intermediate product p-hydroxybenzoic acid (4HB) and the final product phenol involved in the embodiments of the present invention were both determined by high performance liquid chromatography (HPLC). The specific operation is as follows: 1 mL of sample was taken and centrifuged at 10,000 rpm for 5 min. The supernatant was filtered through a 0.45 µm polytetrafluoroethylene membrane filter (VWR International), and the resulting filtrate was used for HPLC analysis. The chromatographic analysis conditions were as follows: the chromatographic column was a ZORBAX Eclipase Plus C18 column (4.6 × 150 mm, 5 μm, Agilent Technologies); the injection volume was 10 µL; mobile phase A was chromatographic pure water containing 0.5% acetic acid, and mobile phase B was 99.9% acetonitrile; a gradient elution program was used: 0–3 min, 100% A; to 12 min, phase A decreased to 0%; 13–15 min, restored to 100% A. The detection wavelengths were: 4HB 250 nm and phenol 270 nm. Each batch of samples was quantitatively analyzed according to a standard curve prepared on an immediate basis. Example 1

[0032] 1. Structure of a phenol-responsive (DPR) signaling regulation system based on the DmpR transcriptional regulation mechanism This embodiment constructs a DPR signal-regulated expression system based on the DmpR transcriptional regulation mechanism, and its structural schematic diagram is shown below. Figure 1 The system consists of two parts: an upstream biorecognition element (T7 ter-dmpR-Pr) and a downstream signal output element (ES-Po-tetA-T7 ter). The function of the downstream signal output element is precisely regulated by the upstream biorecognition element. The upstream biorecognition element (T7 ter-dmpR-Pr) contains the following components: T7 terminator: responsible for terminating the dimethylphenol regulatory protein gene. dmpR Transcription; dimethylphenol regulatory protein gene dmpR : Encodes DmpR, a key regulatory protein for sensing phenol; Pr promoter: drives the gene regulating dimethylphenol. dmpR Transcription initiation. The downstream signal output element (ES-Po-tetA-T7 ter) contains the following components: ES enhancer: used to bind the activated DmpR protein; Po promoter: regulated by the DmpR protein, responsible for initiating the tetracycline resistance gene. tetA Transcription; Tetracycline resistance gene tetA : Serves as a reporter gene and selection marker; T7 terminator: terminates the tetracycline resistance gene. tetA Transcription. The nucleotide sequence of the upstream biorecognition element (T7 ter-dmpR-Pr) is shown in SEQ ID NO.1; wherein, bp 1-6 are the restriction enzyme sites, bp 7-77 are the T7 terminator, and bp 78-1769 are the dimethylphenol regulatory protein gene. dmpR The 1770-1985bp sequence is the Pr promoter. The nucleotide sequence of the downstream signal output element (ES-Po-tetA-T7 ter) is shown in SEQ ID NO.2; wherein, the 1-151bp sequence is the ES enhancer and Po promoter, the 152-157bp sequence is the restriction enzyme site, and the 158-1345bp sequence is the tetracycline resistance gene. tetA The 1346th to 1440th bp is the T7 terminator.

[0033] In the upstream biorecognition element (T7 ter-dmpR-Pr), RNA polymerase recognizes and binds to the Pr promoter, initiating... dmpRThe transcription and translation of genes produce the DmpR protein. The DmpR protein contains four functional domains: a chemical signal recognition domain (A domain): located at the N-terminus, responsible for sensing effectors such as phenol and inhibiting the C domain; a linker domain (B domain): connecting the A and C domains, participating in regulating the inhibition and deinhibition of the C domain by the A domain; an ATPase activity domain (C domain): binding and hydrolyzing ATP, its activity regulated by the A domain; and a DNA-binding domain (D domain): located at the C-terminus, containing a helix-turn-helix (HTH) structure, used to recognize specific DNA sequences. The DmpR protein achieves transcriptional regulation through σ54-RNA polymerase. Figure 2 As shown, it responds to phenol and activates downstream tetracycline resistance genes. tetA The mechanism is as follows: When phenol is present, the A domain of the DmpR protein senses the molecule, releases the inhibition on the C domain, exposes the C domain's active interface, and hydrolyzes ATP. The energy released by ATP hydrolysis drives the DmpR protein to assemble into a hexamer. This hexamer binds to the ES enhancer of downstream elements, thereby activating the σ54-RNA polymerase bound to the -24 / -12 region of the Po promoter, and initiating the tetracycline resistance gene. tetA Transcription.

[0034] 2. Screening mechanism of DPR signal-regulated expression system Based on the above transcriptional regulation mechanism, the DPR signaling regulation expression system can be applied to engineered phenol synthesis strains to achieve efficient and autonomous screening of high-yielding strain subpopulations during the culture process. For example... Figure 3 As shown, a phenol-synthesizing strain carrying the DPR signal-regulated expression system was inoculated into a medium containing a certain concentration of tetracycline and cultured. If the strain's phenol synthesis ability was weak and the intracellular phenol concentration was insufficient, it could not effectively activate the DmpR protein to form a hexamer, leading to downstream... tetA Low gene expression levels indicate that strains lacking tetracycline resistance are suppressed or eliminated; conversely, high-phenol-producing strains can accumulate sufficient concentrations of phenol, fully activating the DmpR protein and initiating... tetA The efficient expression of genes enables normal growth and proliferation in tetracycline-containing culture media. During this process, low-yielding strains are continuously eliminated, while high-yielding strains are enriched, thereby gradually improving the phenol synthesis capacity of the entire bacterial community.

[0035] In summary, the DPR signal-regulated expression system possesses a "sensing-response-screening" logical function: the upstream biorecognition element (T7 ter-dmpR-Pr) acts as a "metabolic sensing switch," its activation level being correlated with intracellular phenol concentration; the downstream signal output element (ES-Po-tetA-T7 ter) is equivalent to a "survival KPI," its expression level determining the survival of the strain under selection pressure. This system enables automated and targeted screening of high-phenol-producing strains without human intervention, providing a highly efficient dynamic regulation and evolutionary screening strategy for metabolic engineering and synthetic biology. Example 2

[0036] To verify the phenol response function of the DPR signal-regulated expression system, the dose-dependent response characteristics of the system to phenol concentration were investigated by adding different concentrations of phenol in vitro. Construction of recombinant strain: The recombinant plasmid pMR (nucleotide sequence shown in SEQ ID NO.3; circular structure, as indicated) containing the complete DPR signal-regulated expression system was constructed. Figure 4 Transformed into E. coli E. coli Recombinant strains were obtained from BL21 (DE3) competent cells. E. coli BMR.

[0037] Experimental grouping and culture: Recombinant strains E. coli BMR was resuspended in two different MY1 media and OD was... 600 The concentration was adjusted to 0.3, and the medium was cultured at 37℃ and 250 rpm for 18 h. One MY1 medium was supplemented with 10 mg / L tetracycline to construct a screening pressure environment, while the other MY1 medium was used as a pressure-free control without tetracycline. A gradient of phenol concentrations from 0 to 1000 mg / L was set in both MY1 media.

[0038] Detection indicators: After the culture was completed, the OD of each group of culture media was measured. 600 Value, in OD 600 The value represents the amount of bacterial growth and reflects the survival and proliferation ability of the strain under different conditions.

[0039] The results show that ( Figure 5 Under the selection pressure environment of 10 mg / L tetracycline, bacterial growth exhibited typical phenol concentration-dependent characteristics: when the phenol concentration was below 50 mg / L, bacterial growth was strongly inhibited, indicating that the intracellular phenol concentration was insufficient to effectively activate the DPR signaling regulation expression system. tetAInsufficient gene expression levels prevented the strain from resisting the inhibitory effect of tetracycline. When the phenol concentration was in the range of 50–200 mg / L, the bacterial growth rate increased significantly with increasing phenol concentration, demonstrating that phenol within this concentration range could gradually activate the DPR signaling regulation system. tetA Gene expression increased with increasing phenol concentration, and the strain gradually acquired stronger tetracycline resistance, leading to enhanced growth and proliferation. However, when the phenol concentration exceeded 200 mg / L, bacterial growth began to decline, and when the concentration exceeded 500 mg / L, growth was significantly inhibited. This phenomenon was mainly due to the cytotoxicity of phenol itself; high concentrations of phenol caused irreversible damage to the bacteria, offsetting the effects of phenol's cytotoxicity. tetA The resistance advantage derived from gene expression. In a stress-free control environment without tetracycline, bacterial growth showed a monotonically decreasing trend with increasing phenol concentration; this growth change was entirely dominated by the cytotoxicity of phenol, and... tetA The lack of correlation between gene expression levels further corroborates that the dependence of bacterial growth on phenol concentration under tetracycline stress is determined by the activation state of the expression system regulated by DPR signaling.

[0040] The above experimental results confirm that the DPR signal-regulated expression system can accurately convert extracellular phenol concentration signals into intracellular signals. tetA The expression level of the resistance gene indicates that the survival and growth of the strain under tetracycline antibiotic stress are strictly dependent on the presence of sufficient phenol in the environment. This successfully verifies that the system has the core functions of both a "phenol concentration sensor" and a "survival switch", laying a solid theoretical foundation for its subsequent application in the autonomous screening of high-phenol-producing strains. Example 3

[0041] To evaluate the application value of the DPR signal-regulated expression system in practical metabolic engineering, it was introduced into a pre-constructed phenol biosynthesis co-culture system to examine the system's effect on improving the phenol synthesis efficiency of the co-culture system. Original phenol biosynthesis co-culture system ( Figure 6 This co-culture system consists of an upstream strain (engineered E. coli strain UY3R) and a downstream strain (engineered E. coli strain DY2-3). The upstream strain plays a central role as the producer in the system, its main function being to convert glucose into p-hydroxybenzoic acid (4HB), a key intermediate in the phenol synthesis pathway, and secrete it into the extracellular culture medium, providing the necessary precursor for the downstream strain. The downstream strain acts as the transformant, specifically responsible for taking up the 4HB secreted by the upstream strain and converting it into the final product phenol through a decarboxylation reaction.

[0042] Optimization of the inoculation ratio of the original co-culture system: To determine the optimal inoculation ratio of the original phenol biosynthesis co-culture system, the upstream strain (E. coli engineered strain UY3R) and the downstream strain (E. coli engineered strain DY2-3) were resuspended in MY1 medium, and the OD... 600 All were adjusted to 0.6, and the mixtures were prepared at different volume ratios as starting cultures to construct a 3 mL co-culture system (initial OD of the mixed bacterial culture). 600 Maintain a culture medium concentration of 0.6, without additional dilution to ensure consistent initial total bacterial count across all experimental groups (only the proportions of the two strains differed), and co-culture for 18 hours at 37°C and 250 rpm in a shaker. The culture results showed ( Figure 7 When UY3R and DY2-3 were inoculated and co-cultured at a volume ratio of 9:1, the phenol yield was the highest, reaching 276 mg / L, with a small amount of 4HB intermediate accumulating during the culture process.

[0043] Construction of downstream strains containing the DPR signal regulation expression system: The recombinant plasmid pMR carrying the complete DPR signal regulation expression system was transformed into the engineered Escherichia coli strain DY2-3 to obtain the engineered Escherichia coli strain DY2-R.

[0044] Construction and cultivation of the recombinant co-culture system: The upstream strain (E. coli engineered strain UY3R) and the downstream strain (E. coli engineered strain DY2-R) were resuspended in MY1 medium containing 10 mg / L tetracycline, and the OD... 600 All were adjusted to 0.6, and the mixtures were prepared at different volume ratios as starting cultures to construct a 3 mL co-culture system (initial OD of the mixed bacterial culture). 600 Maintaining a concentration of 0.6, without additional culture medium dilution to ensure consistent initial total bacterial count in each experimental group (only the ratio of two strains differs), the cultures were co-cultured for 18 hours at 37°C and 250 rpm to assess the production performance of the co-culture system after the introduction of the DPR signal-regulated expression system.

[0045] Detection indicators: After the culture was completed, the phenol yield and 4HB intermediate accumulation of each group were measured, the glucose-based phenol yield was calculated, and the synthesis efficiency of the system was evaluated.

[0046] The results show that ( Figure 8After introducing the DPR signal-regulated expression system, the phenol yield of the phenol biosynthesis co-culture system was significantly improved: when UY3R and DY2-R were inoculated and co-cultured at a volume ratio of 9:1, the phenol yield of the system reached the highest value of 403 mg / L, and the glucose-based phenol yield was 0.081 g / g. Compared with the highest phenol yield of the original co-culture system (276 mg / L), the yield increased by more than 40%, and the accumulation of 4HB intermediates during the culture process was significantly reduced, indicating that the metabolic flux distribution of the system was more efficient and the conversion efficiency of 4HB to phenol was significantly improved.

[0047] The above results confirm that, after introducing the DPR signal-regulated expression system into the phenol biosynthesis co-culture system, under the selection pressure of tetracycline, this system can achieve continuous autonomous selection of high-phenol-producing cell subpopulations in the downstream strain DY2-R: only DY2-R cells with strong phenol synthesis capacity can produce sufficient phenol to activate their own DPR signal-regulated expression system. tetA Gene expression leads to tetracycline resistance, enabling survival and proliferation in culture; while low-producing DY2-R cells with weak phenol synthesis capacity are unable to do so. tetA Insufficient gene expression led to the gradual elimination of strains under tetracycline pressure. This built-in "survival of the fittest" selection mechanism enabled dynamic optimization of the downstream DY2-R bacterial community composition, continuously increasing the proportion of high-yielding strains and ultimately driving a significant improvement in the phenol synthesis efficiency of the entire co-culture system. This fully demonstrates the important application potential of the DPR signal-regulated expression system in metabolic engineering and synthetic biology for stabilizing and enhancing the production performance of microbial cell factories.

[0048] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made within the scope of the claims of the present invention should be included in the scope of the present invention.

Claims

1. A phenol-responsive signal-regulated expression system based on the DmpR transcriptional regulation mechanism, characterized in that: It includes an upstream biometric element and a downstream signal output element, the function of which is controlled by the upstream biometric element; The upstream biorecognition element is T7 ter-dmpR-Pr, which contains a T7 terminator and a dimethylphenol regulatory protein gene. dmpR and the Pr promoter; The downstream signal output element is ES-Po-tetA-T7 ter, which includes an ES enhancer, a Po promoter, and a tetracycline resistance gene. tetA and T7 terminator; The nucleotide sequence of the upstream biometric element is shown in SEQ ID NO.1, and the nucleotide sequence of the downstream signal output element is shown in SEQ ID NO.

2.

2. A recombinant plasmid, characterized in that: The recombinant plasmid contains the phenol-responsive signal-regulated expression system of claim 1; the recombinant plasmid is a circular structure and its nucleotide sequence is shown in SEQ ID NO.

3.

3. A recombinant bacterial strain, characterized in that: The recombinant plasmid described in claim 2 is used to transform host cells; the host cells are *Escherichia coli*, selected from *Escherichia coli*. E. coli BL21 (DE3) or engineered Escherichia coli strain DY2-3.

4. A phenol biosynthesis co-culture system, characterized in that: It includes an upstream strain and a downstream strain; the upstream strain uses glucose as a substrate and is responsible for synthesizing p-hydroxybenzoic acid, an intermediate in the biosynthesis of phenol; the downstream strain is responsible for taking up the intermediate p-hydroxybenzoic acid in the culture medium and further converting it into the final product phenol.

5. The phenol biosynthesis co-culture system according to claim 4, characterized in that: The upstream strain is an engineered Escherichia coli strain UY3R; the downstream strain is obtained by injecting a circular plasmid with the nucleotide sequence shown in SEQ ID NO.3 into an engineered Escherichia coli strain DY2-3.

6. The method for constructing the phenol biosynthesis co-culture system according to claim 4, characterized in that: Includes the following steps: (1) Using the engineered Escherichia coli strain UY3R as the upstream strain, construct a circular plasmid with the nucleotide sequence shown in SEQ ID NO.3, and transform it into the engineered Escherichia coli strain DY2-3 to obtain the downstream strain; (2) The upstream strain and the downstream strain were co-cultured in a medium containing tetracycline and glucose.

7. The construction method according to claim 6, characterized in that: The culture medium containing tetracycline and glucose is formulated as follows: tetracycline 10 mg / L, glucose 5 g / L, yeast extract 0.5 g / L, ammonium chloride 2 g / L, ammonium sulfate 5 g / L, potassium dihydrogen phosphate 3 g / L, dipotassium hydrogen phosphate 7.3 g / L, 3-morpholine propanesulfonic acid 8.4 g / L, sodium chloride 0.5 g / L, magnesium sulfate 0.24 g / L, tyrosine 40 mg / L, phenylalanine 40 mg / L, and tryptophan 40 mg / L. mg / L, 4-hydroxybenzoic acid 10 mg / L, disodium ethylenediaminetetraacetate 0.4 mg / L, boric acid 0.03 mg / L, thiamine 1 mg / L, zinc chloride 0.94 mg / L, cobalt chloride 0.5 mg / L, copper chloride 0.38 mg / L, manganese chloride 1.6 mg / L, calcium chloride 3.77 mg / L, ferrous chloride 3.6 mg / L, isopropyl-β-D-thiogalactoside 0.5 mmol / L.

8. The phenol-responsive signal-regulated expression system of claim 1 in regulating tetracycline resistance genes tetA Applications in expression and / or phenol biosynthesis and / or screening of high-yield phenol strains.

9. The application of the recombinant strain according to claim 3 in phenol biosynthesis and / or screening of high-yield phenol strains.

10. The application of the phenol biosynthesis co-culture system according to claim 4 in phenol biosynthesis and / or screening of high-yield phenol strains.

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