A recombinant escherichia coli, construction method and application thereof and method for producing naringenin based on the same

Abstract

The application discloses a recombinant Escherichia coli, a construction method and application thereof and a method for producing naringenin based on the recombinant Escherichia coli, and belongs to the technical field of microbial fermentation. Escherichia coli DH5a (p15A-T4SI / pMCS-TPG), and is preserved in the China General Microbiological Culture Collection Center. Experiments show that the strain can efficiently produce naringenin in a mineral medium (MMG), and produces a positive feedback amplification effect, significantly improves the relative expression of naringenin synthesis genes and the unit yield of intracellular naringenin, and is a naringenin synthesis strain with great potential. The application provides a highly competitive microbial fermentation method for efficient production of naringenin based on a dynamic regulation strategy, and has a good industrialization prospect.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation technology, specifically relating to a recombinant Escherichia coli strain, its construction method and application, and a method for producing naringenin based thereon. Background Technology

[0002] Naringenin is a class of dihydroflavonoids mainly found in the fruits of Rutaceae plants and the seed coats of Anacardiaceae plants (Iris et al, 2004). Its chemical name is 5,7,4'-trihydroxydihydroflavonoid, and its chemical formula is C2. 15 H 12 O, with a molecular weight of 272.25, is a yellow powder soluble in ethanol, ether, and benzene, but has poor lipid and water solubility and is easily oxidized. Naringenin possesses certain antibacterial and anti-inflammatory effects, exhibiting good antibacterial activity against Escherichia coli, Staphylococcus aureus, and Bacillus subtilis (Liu et al., 2012; Yu Z et al., 2013). In addition, naringenin also has certain antioxidant and antitumor effects (Esmaeili et al., 2014; Monica et al., 2010; Yichu N et al., 2012), and can be widely used in the pharmaceutical and food fields, possessing broad research value and significant research implications.

[0003] Currently, the main production methods for naringenin include plant extraction, chemical synthesis, and biosynthesis. Chen Xuefeng et al. used an improved organic solvent extraction method from peach leaf powder to obtain naringenin samples with high purity, with an average yield of approximately 5.25% (Chen Xuefeng et al., 2009), indicating a low overall yield. Researchers also extracted naringin and then hydrolyzed it to produce naringenin, which significantly improved the extraction yield, but the process still has room for further optimization. Regarding the chemical synthesis of naringenin, Hamilton et al. reported a seven-step method using phloroglucinol as a raw material in 2009 (Hamilton et al., 2009), but due to the complex production process, the presence of byproducts, and the low total yield, it lacks industrial production potential. In 2015, Wang Qiang et al. reported a five-step synthetic method using 3,5-dimethyl-p-phenol as a raw material to synthesize naringenin, achieving a relatively considerable yield (Qiang W et al., 2015). However, the chemical synthesis of naringenin often requires the addition of excessive amounts of toxic reactants to ensure yield. Problems such as reactant toxicity and difficulty in product purification have prevented the large-scale application of the chemical synthesis method of naringenin.

[0004] Microbial synthesis offers a milder and more environmentally friendly method for producing naringenin. Currently, model organisms such as *Escherichia coli* or *Saccharomyces cerevisiae* are primarily used, with L-tyrosine as the fermentation feedstock to synthesize naringenin (Saijie Z et al, 2014). In *E. coli*, L-tyrosine is deaminated by tyrosine ammonia-lyase TAL to produce p-coumaric acid, which is further catalyzed by p-coumaric acid-CoA ligase 4CL to produce p-coumaric acid-CoA. This p-coumaric acid then reacts with malonyl-CoA under the catalysis of chalcone synthase CHS to produce naringenin chalcone, which is then isomerized under the catalysis of chalcone isomerase CHI to produce naringenin. Figure 1 The yield of naringenin in engineered E. coli containing the four key enzymes mentioned above can reach 20.50 mg / L. Furthermore, through conventional metabolic engineering modifications such as enhancing central metabolic flux and increasing the supply of malonyl-CoA precursors, researchers increased the shake-flask fermentation yield of naringenin in recombinant E. coli to approximately 107 mg / L, with a yield per cell of 12.84 mg / L (Junjun W et al, 2014; Saijie Z et al, 2014).

[0005] Although static regulation strategies have been widely applied to naringenin biosynthesis, breakthroughs in yield have been difficult to achieve due to issues such as cytotoxicity or metabolic regulatory pressures associated with naringenin. Therefore, it is essential to develop dynamic regulation methods for naringenin, such as closed-loop feedback regulation, to achieve self-matching and self-regulation between intracellular naringenin and cellular growth / metabolic states. Feedback regulation can not only improve system stability and sensitivity but also enhance expression efficiency and shorten the time to reach optimal expression levels, thereby increasing yield (Schikora-Tamarit et al, 2016). For example, the biosynthesis of L-valine employs a positive feedback dynamic regulation strategy: using the intermediate acetoin as an inducer to activate its own biosynthetic pathway. This self-amplification strategy increased the yield to 6.1 g / L, achieving a 39% yield increase (Geraskina et al, 2019). However, to date, there are no reports of successfully applying dynamic feedback regulation to naringenin biosynthesis. How to construct a self-feedback dynamic regulation system adapted to the naringenin biosynthetic pathway to achieve a breakthrough in naringenin yield is one of the key technical problems urgently needing to be solved in this field.

[0006] In 2019, Meyer et al. discovered a P that responds to naringenin. Ttg The -TtgR regulatory system provides a fundamental element for developing tools to dynamically regulate naringenin synthesis (Meyer et al, 2019). In this system, P Ttg It is a promoter element that can bind to the repressor protein TtgR. When TtgR is expressed intracellularly and binds to P... TtgAfter promoter binding, RNA polymerase cannot activate P Ttg Transcription of downstream genes from the promoter is silenced. However, this state can be broken by naringenin. When naringenin is present in the cell, it can bind to TtgR repressor protein, causing a conformational change, from P... Ttg Dissociation from the promoter, thereby making P Ttg Downstream genes of the promoter are transcribed and expressed. This regulatory system exhibits high sensitivity for naringenin, with a response range of 1 μM to 1 mM. Current techniques have only focused on characterizing and optimizing the regulatory system itself, and have not been used to construct a self-feedback dynamic regulatory pathway for naringenin biosynthesis. Summary of the Invention

[0007] In order to overcome the shortcomings of the prior art, the present invention aims to provide a recombinant Escherichia coli strain, a construction method thereof, its application, and a method for producing naringenin based thereon. The present invention provides a recombinant Escherichia coli strain that can produce naringenin through fermentation via positive feedback effect, thereby solving the problem of increasing the yield of naringenin in the existing biosynthesis method with a dynamic control approach.

[0008] To achieve the above objectives, the present invention employs the following technical solution: This invention discloses a recombinant Escherichia coli strain, which is named... Escherichia coli DH5α(p15A-T4SI / pMCS-TPG) is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.37537 and deposit date of January 26, 2026.

[0009] The present invention also discloses a method for constructing the above-mentioned recombinant Escherichia coli, wherein naringenin synthesis-related genes and naringenin-responsive regulatory genes are introduced into an Escherichia coli host to obtain the recombinant Escherichia coli.

[0010] Preferably, the method includes introducing genes encoding tyrosine ammonia-lyase, p-coumarate-CoA ligase, chalcone synthase, chalcone isomerase, HTH-type transcription factor TtgR, and hyperfolded green fluorescent protein into *Escherichia coli*. Escherichia coli Obtained from DH5α.

[0011] More preferably, the gene encoding tyrosine ammonia-lyase is derived from Rhodotorula glutinis. TAL Gene; the gene encoding p-coumarate-CoA ligase is derived from parsley. 4CL The gene encoding chalcone synthase is from petunia. CHS The gene encoding chalcone isomerase is derived from alfalfa. CHIThe gene encoding the HTH-type transcriptional regulator TtgR is derived from *Bacillus putida*. ttgR Gene cluster; the gene encoding the superfolded green fluorescent protein is from iGEM accession number BBa_I746916.

[0012] Preferably, the gene encoding tyrosine ammonia-lyase is derived from the Rhodotorula glutinis TAL gene, and its nucleotide sequence is shown in SEQ ID NO.2; The gene encoding coumarate-CoA ligase is derived from parsley. 4CL The gene, with its nucleotide sequence shown in SEQ ID NO. 3; The gene encoding chalcone synthase is from petunia. CHS The gene, with its nucleotide sequence shown in SEQ ID NO. 4; The gene encoding chalcone isomerase is derived from alfalfa. CHI The gene, with its nucleotide sequence shown in SEQ ID NO.5; The gene encoding the HTH-type transcriptional regulator TtgR is derived from Bacillus putida. ttgR The gene cluster, with its nucleotide sequence shown in SEQ ID NO.7.

[0013] The gene encoding the superfolded green fluorescent protein is derived from element BBa_I746916 of the iGEM international genetically engineered machine competition, and its nucleotide sequence is shown in SEQ ID NO.8.

[0014] Preferably, the exogenous gene is expressed under a constitutive or inducible promoter.

[0015] The naringenin synthesis-related gene and the naringenin-responsive regulatory gene were introduced into the Escherichia coli host using two different recombinant vectors.

[0016] More preferably, during construction, the encoded gene is introduced into *E. coli* via two different recombinant vectors. Escherichia coli In DH5α; where: The genes encoding tyrosine ammonia-lyase, p-coumarate-CoA ligase, chalcone synthase, and chalcone isomerase were introduced into *E. coli* via recombinant vector A. Escherichia coli In DH5α; The gene encoding the HTH-type transcriptional regulator TtgR and the gene encoding the hyperfolded green fluorescent protein were introduced into Escherichia coli via recombinant vector B. Escherichia coli In DH5α.

[0017] More preferably, the recombinant vector A is a vector obtained by inserting the naringenin-inducible promoter and the ampicillin resistance gene into the commercial p15A plasmid; the recombinant vector B is a vector obtained by inserting the constitutive promoter, the naringenin-inducible promoter and the kanamycin resistance gene into the commercial pBBRMCS-1 plasmid.

[0018] The present invention also discloses the application of the above-mentioned recombinant Escherichia coli or the recombinant Escherichia coli constructed by the above-mentioned construction method in the production of naringenin through positive feedback effect.

[0019] This invention discloses a method for producing naringenin through a positive feedback effect, comprising: fermenting the above-mentioned recombinant Escherichia coli or fermenting the recombinant Escherichia coli constructed by the above-mentioned construction method to produce naringenin.

[0020] Preferably, the initial fermentation temperature is 20~40 ℃, the initial pH value of the fermentation system is 6.0-8.0, and the fermentation time is 24~72 h.

[0021] Preferably, the naringenin content in each liter of fermentation product is 1-20 mg / L.

[0022] Preferably, the fermentation medium used for fermentation is MMG medium (Mineral Medium with Glucose). Each liter of MMG culture medium contains: Glucose 10g-50g, sodium chloride 10g-30g, yeast powder 0g-5g, ammonium chloride 0.5g-10g, magnesium sulfate 0.1g-1g, potassium dihydrogen phosphate 0.5g-5g, disodium hydrogen phosphate dodecahydrate 3g-15g, trace element I 3mL-20mL, trace element II 0.5mL-3mL, the remainder is water; The preparation method of the trace element I is as follows: 2 g-10 g of ferric ammonium citrate, 1 g-5 g of calcium chloride dihydrate and 0.5 mol / L hydrochloric acid aqueous solution are mixed and diluted to 1 liter with 0.5 mol / L hydrochloric acid aqueous solution to obtain the trace element I; The preparation method of the trace element II is as follows: 50 mg-200 mg of zinc sulfate heptahydrate, 10 mg-50 mg of manganese chloride tetrahydrate, 100 mg-500 mg of boric acid, 50 mg-400 mg of cobalt chloride hexahydrate, 3 mg-30 mg of copper sulfate pentahydrate, 5 mg-50 mg of nickel chloride hexahydrate, 10 mg-50 mg of sodium molybdate dihydrate, and 0.5 mol / L hydrochloric acid aqueous solution are mixed, and the volume is made up to 1 liter with 0.5 mol / L hydrochloric acid aqueous solution to obtain the trace element II.

[0023] More preferably, in the 1 liter 60-MMG culture medium, the mass of glucose is 10g, 30g, or 50g; the mass of sodium chloride is 30g, 60g, or 100g; the mass of yeast powder is 0g, 1g, or 5g; the mass of ammonium chloride is 0.5g, 2g, or 10g; the mass of magnesium sulfate is 0.1g, 0.2g, or 1g; the mass of potassium dihydrogen phosphate is 0.5g, 1.5g, or 5g; the mass of disodium hydrogen phosphate dodecahydrate is 3g, 9.65g, or 15g; the volume of trace element I is 3mL, 10mL, or 20mL; and the volume of trace element II is 0.5mL, 1mL, or 3mL. In the 1 liter of the trace element I, the mass of ferric ammonium citrate is 2 g, 5 g, or 10 g, and the mass of calcium chloride dihydrate is 1 g, 2 g, or 5 g. In the 1 liter of trace element II, the mass of zinc sulfate heptahydrate is 50 mg, 100 mg, or 200 mg; the mass of manganese chloride tetrahydrate is 10 mg, 30 mg, or 50 mg; the mass of boric acid is 100 mg, 300 mg, or 500 mg; the mass of cobalt chloride hexahydrate is 50 mg, 200 mg, or 400 mg; the mass of copper sulfate pentahydrate is 3 mg, 10 mg, or 30 mg; the mass of nickel chloride hexahydrate is 5 mg, 20 mg, or 50 mg; and the mass of sodium molybdate dihydrate is 10 mg, 30 mg, or 50 mg.

[0024] Preferably, fermentation is carried out under sterilization conditions, batch fermentation conditions, or continuous fermentation conditions.

[0025] Compared with the prior art, the present invention has the following beneficial effects: This invention discloses for the first time a recombinant Escherichia coli strain Escherichia coli The strain DH5α (p15A-T4SI / pMCS-TPG) was deposited at the China General Microbiological Culture Collection Center. Experiments showed that this strain can efficiently produce naringenin in mineral medium (MMG) with a positive feedback amplification effect, significantly increasing the relative expression level of the naringenin synthesis gene and the intracellular naringenin yield per unit volume. The highest yield per unit volume during shake-flask fermentation was 19.46 mg / L, which is 1.58 times higher than that of traditional engineered strains, making it a highly promising naringenin synthesizing strain.

[0026] This invention addresses the yield bottleneck of existing static regulation methods for naringenin biosynthesis by constructing a self-feedback dynamic regulation system adapted to the naringenin synthesis pathway. It fully utilizes the amplification effect of the positive feedback system to enhance naringenin synthesis and ultimately achieve a breakthrough in naringenin yield. Based on a dynamic regulation strategy, this invention provides a highly competitive microbial manufacturing method for the efficient production of naringenin and has excellent prospects for industrialization. Attached Figure Description

[0027] Figure 1 This refers to the biosynthetic pathway of naringenin; Figure 2 These are two expression plasmid maps for naringenin synthesis and detection; where (a) is the p15A-T4SI plasmid map for naringenin synthesis, and the promoter can be P. trc Or P Ttg (b) is the plasmid map of the naringenin-responsive plasmid pMCS-TPG; Figure 3 This is a schematic diagram illustrating the positive feedback regulation of naringenin. Figure 4 The results are shown in the colony PCR identification of the positive feedback engineered bacteria; among them, (a) shows the p15A-P colony in the dual-plasmid engineered bacteria. Ttg (a) PCR results of T4SI plasmid colonies; (b) PCR verification results of pMCS-TPG plasmid colonies of engineered bacteria with dual plasmids. Figure 5 To validate the engineered strain for naringenin detection (naringenin biosensor), (a) flow cytometry FACS results of the engineered strain after induction with different concentrations of naringenin; (b) induction curves of the naringenin sensor for different concentrations of added naringenin. Figure 6 The engineered bacterium for naringin synthesis, *Escherichia coli* DH5α (p15A-P... trc -T4SI) Fermentation results of naringenin synthesis; (a) liquid phase detection results of fermentation products; (b) intracellular and extracellular distribution of naringenin after fermentation by engineered bacteria; (c) naringenin synthesis yield of engineered Escherichia coli in different culture media; (d) mass spectrometry detection results of naringenin in fermentation broth; Figure 7 The positive feedback engineered bacteria Escherichia coli DH5α (p15A-P) Ttg -T4SI / pMCS-TPG) Escherichia coli and control bacteria without positive feedback DH5α (p15A-P trc Comparison of fermentation status and relative gene transcription of naringenin (-T4SI / pMCS-TPG); (a) the relationship between fluorescence intensity and time during 72 h of fermentation; (b) the key gene for naringenin synthesis after 24 h and 48 h of fermentation. CHS The relative transcriptional level is characterized by .

[0028] Preservation Instructions The recombinant Escherichia coli strain disclosed for the first time in this invention is named Escherichia coliDH5α (p15A-T4SI / pMCS-TPG) is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.37537 and deposit date of January 26, 2026. Detailed Implementation

[0029] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0030] It should be noted that the terms "first," "second," etc., in the specification, claims, and accompanying drawings of this invention are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence. It should be understood that such data can be interchanged where appropriate so that the embodiments of the invention described herein can be implemented in orders other than those illustrated or described herein. Furthermore, the terms "comprising" and "having," and any variations thereof, are intended to cover a non-exclusive inclusion; for example, a process, method, system, product, or apparatus that comprises a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or units not explicitly listed or inherent to such processes, methods, products, or apparatus.

[0031] The present invention will now be described in further detail with reference to the accompanying drawings: Example 1: Design and Construction of a Positive Feedback System for Naringenin Synthesis, Detection, and Application Figure 1 The diagram illustrates the metabolic pathway of L-tyrosine synthesis of naringenin. The key enzymes in naringenin synthesis are tyrosine ammonia-lyase TAL, coumarate-CoA ligase 4CL, chalcone synthase CHS, and chalcone isomerase CHI. Under the catalysis of these four key enzymes, L-tyrosine is converted to naringenin. Therefore, this invention designs three gene circuits for the synthesis, detection, and positive feedback regulation of naringenin.

[0032] 1. Construction of naringenin synthesis plasmid p15A-T4SI like Figure 2 Image (a) shows the plasmid map of naringenin synthesis. Artificially synthesized P Ttg promoter or P trc promoter, TAL (Tyrosine ammonia-lyase gene) 4CL (coumarate-CoA ligase gene) CHS (Chalcone synthase gene) and CHI (Chalcone isomerase gene. The amplified gene fragment was inserted into the low-copy plasmid p15A using the Golden Gate ligation method, and Amp was used simultaneously.) R The resistance gene replaces Cm on the vector R Gene. Positive recombinant plasmid sequencing confirmed correctness.

[0033] The nucleotide sequence of the naringenin regulatory element used in the construction process is shown in SEQ ID NO.1; encoding tyrosine ammonia-lyase. TAL The nucleotide sequence of the gene is shown in SEQ ID NO.2; encoding p-coumarate-CoA ligase. 4CL The nucleotide sequence of the gene is shown in SEQ ID NO.3; it encodes chalcone synthase. CHS The nucleotide sequence of the gene is shown in SEQ ID NO.4; it encodes chalcone isomerase. CHI The nucleotide sequence of the gene is shown in SEQ ID NO.5.

[0034] The nucleotide sequence of the constructed p15A-T4SI plasmid is shown in SEQ ID NO.8.

[0035] 2. Construction of the naringenin detection plasmid pMCS-TPG (naringenin biosensor) like Figure 2 As shown in (b), this is a map of the naringenin synthesis plasmid (naringenin biosensor). Artificially synthesized P porin promoter, ttgR (HTH-type transcription factor) genes and sfGFP (Superfolded green fluorescent protein) gene. The amplified gene fragment was inserted into the copy plasmid pBBRMCS-1 using the GoldenGate ligation method, and Kana was used simultaneously. R The resistance gene replaces Cm on the vector R Gene. Positive recombinant plasmid sequencing confirmed correctness.

[0036] The nucleotide sequence of the naringenin promoter used in the construction process is shown in SEQ ID NO.1; encoding an HTH-type transcriptional regulator. ttgR The nucleotide sequence of the gene is shown in SEQ ID NO.6; it encodes a superfolded green fluorescent protein. sfGFP The nucleotide sequence of the gene is shown in SEQ ID NO.7.

[0037] The nucleotide sequence of the constructed pMCS-TPG plasmid is shown in SEQ ID NO.9.

[0038] The nucleotide sequence listing of all the plasmids designed in this invention is shown in Table 1 below.

[0039] Table 1. List of genes or plasmids involved in this invention

[0040] 3. Construction of the positive feedback regulation system of naringenin like Figure 3 The diagram shows a positive feedback synthesis regulatory pathway for naringenin. The construction method is as follows: the above-mentioned naringenin synthesis plasmid p15A-P... Ttg -T4SI and detection plasmid pMCS-TPG were co-transformed into E. coli DH5α, and then... R and Kana R LB plate screening with double antibodies yielded the dual-plasmid strain *Escherichia coli* DH5α (p15A-P). Ttg- T4SI / pMCS-TPG). Among them, the commonly expressed TtgR repressor protein, in the absence of naringenin, is related to P. Ttg After promoter binding, expression is suppressed, and the naringenin synthesis gene exhibits only low basal expression. After driving the synthesis of a small amount of naringenin, naringenin binds to the TtgR repressor protein, relieving its inhibition of P. Ttg The promoter's repression further increases the expression level of downstream naringenin synthesis genes, promoting increased naringenin production. This increased naringenin production further intensifies the positive feedback process, creating a yield amplification effect. The content of naringenin can be characterized by fluorescence intensity. Figure 4 The colony PCR results shown confirm the successful construction of the dual-plasmid engineered bacteria.

[0041] Example 2: Validation of the naringenin biosensor pMCS-TPG Overnight activated *E. coli* DH5α (pMCS-TPG) seed culture was inoculated into LB medium at a ratio of 1%, and cultured at 37°C for 3 h. Then, different concentrations of naringenin were added to induce the engineered bacteria for 12 h. After induction, the strain was washed three times with PBS and diluted 10⁻⁶ times. 4 The results were then analyzed using flow cytometry. The FITC channel was used to characterize the green fluorescence produced by the bacteria. The flow cytometry results were subsequently analyzed using FlowJo. Specific results are shown below. Figure 5 As shown, the naringenin detection sensor can respond to different concentrations of added naringenin. As the concentration of naringenin increases, the fluorescence intensity per unit cell gradually increases, and the response curve is "S"-shaped, showing good response performance. This proves that the naringenin detection sensor of the present invention has been successfully constructed and can be used for subsequent detection and comparison of intracellular naringenin content.

[0042] Example 3: Fermentation and product detection of engineered bacteria for naringenin synthesis The engineered bacterium for naringin synthesis, *Escherichia coli* DH5α (p15A-P) trc After overnight activation with -T4SI, the culture medium was inoculated at a ratio of 5% into mineral medium MMG for shake-flask fermentation. After scaling up the culture at 37°C for 4 h, the temperature was adjusted to 25°C, and 0.5 mM IPTG was added to induce the expression of key enzymes. Fermentation continued for another 48 h. After the shake-flask culture was completed, the bacterial culture was processed by ethanol extraction and centrifugation to prepare samples, which were then analyzed using liquid chromatography-mass spectrometry (LC-MS).

[0043] The composition of the MMG medium is shown in Table 2. After preparing the substrate and glucose concentrate, sterilize at 115 °C for 30 min and mix thoroughly on a clean bench. Then add components 1-4 separately. Gently shake well and add 5 mM sodium hydroxide to adjust the pH to 7.0. Then add kanamycin to a final concentration of 50 µg / mL and ampicillin to 100 µg / mL. The substrate L-tyrosine was added at 0.8 g / L.

[0044] Table 2 Composition of MMG culture medium

[0045] Liquid chromatography-mass spectrometry (LC-MS) was used to identify the presence of naringenin in the extract. LC parameters: Chromatographic separation was performed using an ACQUITY UPLC BEH C18 column (100 mm * 2.1 mm, 1.7 μm, Waters, UK). The column temperature was 35°C, and the flow rate was 0.2 ml / min. Mobile phase A was 0.1% formic acid, and mobile phase B was acetonitrile. The following elution gradient was used for the metabolites: 0–1 min, 5% mobile phase B; 1–5 min, 20% mobile phase B; 5–9 min, 85% mobile phase B; 9–11 min, 100% mobile phase B; 11–12 min, 5% mobile phase B. Mass spectrometry parameters: Negative ion mode was used for acquisition. Data acquisition was performed using Full MS / dd-MS2 (DDA scan mode), with a primary scan range of 50-2000 m / z. The eight ions with the strongest signals (TopN=8) were used for secondary fragmentation scanning after fragmentation.

[0046] See Figure 6 In Figures (a) and (b), by comparing the liquid chromatography retention time plots and mass spectra, it can be seen that the engineered *E. coli* strain successfully produced naringenin after 48 h of fermentation, with a negative ion mass value of 271.0612 [MH]. - The retention time peak was 4.63 min. Fermentation results also showed that naringenin was mainly concentrated intracellularly, with intracellular yield accounting for approximately 96% of the total yield. Figure 6 As shown in (c), the naringenin yield obtained using MMG medium was significantly higher than that obtained using nutrient-rich LBG medium. Figure 6 As shown in (d).

[0047] Example 4: Fermentation and product detection of naringenin-positive feedback engineered bacteria Positive feedback engineered strain of Escherichia coli DH5α (p15A-P) Ttg- After overnight activation with T4SI / pMCS-TPG, fermentation was carried out in MMG medium for 72 h according to the culture conditions of Example 3. GFP fluorescence intensity was detected every 12 h. The product was analyzed by LCMS after 48 h of fermentation, and the results are as follows: Figure 7 As shown in Table 3.

[0048] Depend on Figure 7 As shown in (a), with the extension of fermentation time, the fluorescence intensity of the positive feedback engineered bacteria gradually increased, and after 48 hours, the fluorescence intensity was significantly higher than that of the Escherichia coli DH5α (p15A-P) strain without positive feedback. trc The fluorescence intensity of the T4SI / pMCS-TPG strain at 72 h was 1.67 times that of the control strain without positive feedback, indicating a gradual increase in intracellular naringenin accumulation, significantly superior to the control cells without positive feedback regulation. Furthermore, Table 2 shows the naringenin production detected by LCMS, indicating that the naringenin production per unit cell of the engineered strain with positive feedback was 19.46 mg / L, 1.58 times that of the control strain without positive feedback, with a total production of 57.60 mg / L. This demonstrates that the construction of a positive feedback regulation system significantly increased naringenin production.

[0049] Table 3. Naringenin production by engineered bacteria during fermentation (LCMS analysis)

[0050] To verify the positive feedback effect in the engineered bacteria at the transcriptional level of the naringenin synthesis gene, quantitative real-time PCR was used to analyze the key naringenin synthesis gene in the engineered bacteria of the positive feedback group and the control group after 24 h and 48 h of fermentation. CHS The relative transcription levels of genes were characterized. The blank control group consisted of *E. coli* DH5α. The results were obtained using 2... -ΔΔCt The method was used for analysis, and finally, different strains were found to contain... CHS The relative transcription level of genes, such as Figure 7 As shown in (b). The gene quantification results show that after 24 h of fermentation, the engineered bacteria in the control group without positive feedback... CHS The relative transcription rate of genes was significantly higher in the positive feedback group than in the negative feedback group; however, after 48 hours of fermentation, the positive feedback group's engineered bacteria... CHSThe relative transcription level of the gene increased rapidly and was much higher than that of the control group of engineered bacteria, indicating that the gradual synthesis of naringenin during fermentation successfully and gradually activated P. Ttg The promoter rapidly increases the transcriptional amount of the naringenin synthesis gene, exhibiting a self-induced positive feedback amplification effect. Therefore, the engineered Escherichia coli DH5α (p15A-P) strain constructed in this invention... Ttg The -T4SI / pMCS-TPG can generate a self-induced positive feedback effect on intracellularly synthesized naringenin, providing a new breakthrough strategy for the efficient biomanufacturing of naringenin.

[0051] In summary, this invention addresses the yield bottleneck of existing static regulation methods for naringenin biosynthesis. It utilizes a highly sensitive naringenin-responsive element to develop a self-induced positive feedback system adapted to the naringenin synthesis pathway. The resulting recombinant *E. coli* responds to intracellularly synthesized naringenin, and fully leverages the amplification effect of positive feedback to gradually improve gene expression efficiency, enhance naringenin synthesis, and ultimately achieve a breakthrough in naringenin yield. Based on a dynamic regulation strategy, this invention provides a highly competitive microbial manufacturing method for the efficient production of naringenin, with excellent prospects for industrialization.

[0052] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. A recombinant Escherichia coli strain, characterized in that, The recombinant Escherichia coli was named Escherichia coli DH5α(p15A-T4SI / pMCS-TPG) is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.37537 and deposit date of January 26, 2026.

2. The method for constructing recombinant Escherichia coli according to claim 1, characterized in that, The recombinant Escherichia coli was obtained by introducing naringenin synthesis-related genes and naringenin-responsive regulatory genes into an Escherichia coli host.

3. The method for constructing recombinant Escherichia coli according to claim 2, characterized in that, The naringenin synthesis-related genes include genes encoding tyrosine ammonia-lyase, coumarate-CoA ligase, chalcone synthase, and chalcone isomerase; the naringenin-responsive regulatory genes include genes encoding the HTH-type transcriptional regulator TtgR.

4. The method for constructing recombinant Escherichia coli according to claim 2 or 3, characterized in that, It also includes a reporter gene, which is a gene encoding a superfolded green fluorescent protein.

5. The method for constructing recombinant Escherichia coli according to claim 2 or 3, characterized in that, The naringenin synthesis-related gene and the naringenin-responsive regulatory gene were introduced into the Escherichia coli host using two different recombinant vectors.

6. The method for constructing recombinant Escherichia coli according to claim 5, characterized in that, The recombinant vector that introduces genes related to naringenin synthesis is the one that introduces the naringenin-inducible promoter P. Ttg The vector obtained by inserting the ampicillin resistance gene into the commercial p15A plasmid; the recombinant vector into which the naringenin-responsive regulatory gene was introduced was pBBRMCS-1.

7. The application of the recombinant Escherichia coli according to claim 1, or the recombinant Escherichia coli constructed by the construction method according to any one of claims 2-6, in the fermentation to produce naringenin.

8. A method for producing naringin, characterized in that, include: The recombinant Escherichia coli of claim 1, or the recombinant Escherichia coli constructed by any one of the construction methods of claims 2 to 6, is fermented, and naringenin is isolated and purified from the fermentation product.

9. The method for producing naringin according to claim 8, characterized in that, The initial fermentation temperature is 20~30℃, the initial pH of the fermentation system is 6.0-8.0, and the fermentation time is 24~72 h.

10. The method for producing naringin according to claim 8, characterized in that, The fermentation medium used for fermentation is MMG medium; Each liter of the MMG culture medium contains: 10g-50g glucose, 10g-30g sodium chloride, 0g-5g yeast extract, 0.5g-10g ammonium chloride, 0.1g-1g magnesium sulfate, 0.5g-5g potassium dihydrogen phosphate, 3g-15g disodium hydrogen phosphate dodecahydrate, 3mL-20mL trace element I, 0.5mL-3mL trace element II, and the remainder is water; trace element I is prepared by mixing and adjusting the volume with ferric ammonium citrate, calcium chloride dihydrate, and hydrochloric acid aqueous solution; trace element II is prepared by mixing and adjusting the volume with zinc sulfate, manganese chloride, boric acid, cobalt chloride, copper sulfate, nickel chloride, sodium molybdate, and hydrochloric acid aqueous solution.

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