Low-pH-tolerant Escherichia coli, construction method thereof and application of Escherichia coli in synthesis of 5-aminolevulinic acid

Abstract

The invention belongs to the technical field of microorganisms and fermentation engineering, and particularly relates to low-pH-tolerant escherichia coli, a construction method of the low-pH-tolerant escherichia coli and application of the low-pH-tolerant escherichia coli in synthesis of 5-aminolevulinic acid. Specifically, escherichia coli MG1655 is taken as a starting strain, mutS genes are knocked out through a Red homologous recombination technology to improve the mutation rate, and an acid-resistant strain is obtained through adaptive laboratory evolution; meanwhile, an overexpression module plasmid containing hemA, coaA, ppc, eamA and grxA genes and an sRNA inhibition module plasmid containing targeted hemB, aceA and sucC genes are further constructed, and the double plasmids are transferred into the acid-resistant strain, so that the acid-resistant recombinant escherichia coli with excellent 5-aminolevulinic acid production performance is obtained. An efficient strain and a technical support are provided for industrial production of the 5-aminolevulinic acid, so that the 5-aminolevulinic acid strain has a good practical application value.

Description

Technical Field

[0001] This invention belongs to the field of microbial and fermentation engineering technology, specifically relating to a low-pH-tolerant Escherichia coli, its construction method, and its application in the synthesis of 5-aminolevulinic acid. Background Technology

[0002] The information disclosed in the background section of this invention is intended only to enhance the understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] 5-Aminolevulinic acid (5-ALA) is a naturally occurring five-carbon amino acid found in all living organisms and is an important biosynthetic precursor for tetrapyrrole compounds such as vitamin B12, heme, and chlorophyll. Due to its good permeability, high selectivity, and environmental friendliness, it has wide applications in agriculture, animal husbandry, and medicine. Currently, the production of 5-ALA mainly relies on chemical synthesis and microbial fermentation. However, chemical synthesis methods are complex and prone to environmental pollution; therefore, microbial fermentation has gradually become an important method for synthesizing 5-ALA.

[0004] Escherichia coli, with its simple nutritional requirements, rapid growth, and abundant genetic modification tools, is widely used in the microbial fermentation of high-value-added compounds and has become an important chassis cell for the synthesis of 5-ALA. However, the inventors discovered that during the entire fermentation process, excessive addition of carbon sources leads to an imbalance between the glycolysis pathway and the TCA cycle. E. coli often secretes byproducts such as formic acid, acetic acid, and lactic acid, which lowers the pH of the fermentation broth and thus affects the normal growth of the fermentation strain and product synthesis. To mitigate the impact of pH reduction, some alkaline regulators need to be added during 5-ALA fermentation, which to some extent increases the production cost and process complexity. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a low-pH-tolerant *Escherichia coli* strain, its construction method, and its application in the synthesis of 5-aminolevulinic acid. Specifically, the present invention uses *Escherichia coli* MG1655 as the starting strain and employs Red homologous recombination technology to knock out... mutS Genes were used to increase the mutation rate, and acid-resistant strains were obtained through adaptive laboratory evolution; simultaneously, further construction was carried out to include... hemA , coaA, ppc, eamA, grxA The gene overexpression module plasmid PJA-2, and containing targeting hemB, aceA, sucCThe sRNA repression module plasmid W2 of the gene was co-transformed into the above-mentioned acid-tolerant strain to obtain recombinant *Escherichia coli* JP2025. This recombinant strain can grow normally under conditions of pH 4.2-7.0, and efficiently synthesizes 5-ALA within the pH range of 4.5-7.0. After 66 h of fed-batch fermentation in a 5-L fermenter, the yield of 5-ALA reached 10.35 g / L. This significantly improves the acid tolerance of *E. coli*, reduces the use of alkaline regulators during fermentation, lowers production costs, and provides a high-efficiency strain and technical support for the industrial production of 5-ALA. Based on the above research results, this invention is completed.

[0006] Specifically, the technical solution of the present invention is as follows: A first aspect of the invention provides a low-pH-tolerant *Escherichia coli* strain, said *Escherichia coli* strain obtained by knocking out a component from the starting strain. mutS Genes, overexpression hemA , coaA, ppc, eamA and grxA Genes and repression hemB, aceA and sucC Obtained after genetic modification.

[0007] In this invention, the starting strain is *Escherichia coli* (E. coli). Escherichia coli MG1655 (ATCC 700926).

[0008] Specifically, among the low-pH-tolerant E. coli, knockout mutS Genes can be generated using Red homologous recombination technology; The overexpression hemA , coaA, ppc, eamA It can be obtained by constructing an overexpression module (i.e., a 5-ALA synthesis module of the E. coli C4 pathway) and introducing it into E. coli; Specifically, the overexpression module can utilize the pCL1920 plasmid for overexpression. ppc, coaA, hemA, grxA, eamA Five genes were constructed to obtain the results.

[0009] The inhibition hemB, aceA and sucC Genes can be obtained by constructing sRNA-based repressor modules and introducing them into E. coli; Specifically, the sRNA-based repression module can be designed to inhibit [something] on the pTrc99a plasmid. hemB , aceA and sucC The sRNA of the gene was obtained through construction.

[0010] in, hemA The encoded glutamyl-tRNA reductase is derived from Rhodobacter capsulatus(Rhodops capsulatum) can catalyze the conversion of glutamyl-tRNA to glutamate-1-hemialdehyde; coaA It encodes phosphate pantothenic acid decarboxylase, which can catalyze the decarboxylation of 4'-phosphate pantothenic acid to 4'-phosphate pantothenic acid; ppc It encodes phosphoenolpyruvate carboxylase, which can catalyze the reaction of phosphoenolpyruvate (PEP) and carbon dioxide to produce oxaloacetic acid; eamA It encodes an O-acetylserine / cysteine ​​efflux transporter that participates in the efflux of 5-ALA and can effectively reduce the intracellular concentration of 5-ALA. grxA Encoding glutoreductin A, it participates in electron transport and energy supply, and protects cells from oxidative damage. A partially competitive pathway also exists in the C4 pathway of 5-ALA synthesis in *E. coli*. 5-ALA is a key precursor in the synthesis of tetrapyrrole compounds such as heme within cells and is indispensable for cell growth.

[0011] also, hemB It encodes bile pigmentogen synthase, catalyzing the condensation of two 5-ALA molecules to form one molecule of bile pigmentogen (PBG); additionally, succinyl-CoA, a direct precursor of 5-ALA synthesized via the C4 pathway, is an intermediate product of the citric acid cycle. In the citric acid cycle and its restorative pathway, the glyoxylate cycle... aceA It encodes isocitrate lyase, which catalyzes the cleavage of isocitrate into glyoxylic acid and succinic acid; sucC The α subunit encoding succinyl-CoA synthase enables bidirectional catalysis of succinyl-CoA and succinic acid. Therefore... aceA and sucC Gene expression affects intracellular succinyl-CoA levels, increasing the accumulation of byproducts. sRNA-based gene regulation systems can be used to regulate and balance the expression of multiple genes in different microorganisms. sRNA can bind to specific regions of target mRNA through base pairing and with the assistance of Hfq proteins, thereby inhibiting gene expression at the posttranscriptional level. Furthermore, the intensity of inhibition can be controlled by adjusting the length of the sRNA binding region or by mutating individual binding sites. Therefore, in this invention, an inhibitory mechanism is designed on the pTrc99a plasmid. hemB , aceA and sucC The gene's sRNA blocks the competitive pathway for 5-ALA synthesis, further increasing the level of 5-ALA synthesis.

[0012] Furthermore, the low-pH-tolerant Escherichia coli can be Escherichia coli (E. coli) Escherichia coliJP2025, this strain was deposited on November 20, 2025 at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Wuhan, China, with the accession number CCTCC NO: M 20252608.

[0013] A second aspect of the present invention provides a method for constructing the above-mentioned low-pH-tolerant Escherichia coli, the method comprising: S1. Using wild-type Escherichia coli as the starting strain, the mismatch repair protein gene was knocked out. mutS Acid-resistant strains were subsequently obtained through adaptive evolution. S2, Constructing overexpression hemA , coaA, ppc and eamA The first recombinant expression vector and containing inhibitors hemB, aceA and sucC The second recombinant expression vector for sRNA; S3. Integrate the first recombinant expression vector and the second recombinant expression vector into the acid-resistant strain.

[0014] The starting strain is Escherichia coli (E. coli) Escherichia coli MG1655 (ATCC 700926).

[0015] Furthermore, in step S1, the mismatch repair protein gene is knocked out. mutS Red homologous recombination technology can be used; The specific methods of adaptive evolution include: using mutS The defective strain is used as the starting strain, and the pH of the culture medium is gradually reduced until the desired acid-resistant strain is obtained.

[0016] In step S2, the backbone vector of the first recombinant expression vector is plasmid pCL1920 (GenBank AB236930.1); the backbone vector of the second recombinant expression vector is plasmid pTrc99a (GenBank U13872.1).

[0017] Among them, targeting genes hemB , aceA and sucC The sRNA sequences of the genes are shown in SEQ ID NO.1-3, respectively.

[0018] A third aspect of the present invention provides the use of the above-mentioned Escherichia coli in the fermentation production of 5-aminolevulinic acid.

[0019] A fourth aspect of the present invention provides a method for the industrial production of 5-aminolevulinic acid, the method comprising: fermenting and culturing the above-mentioned low-pH-tolerant Escherichia coli, and separating and purifying the 5-aminolevulinic acid.

[0020] The beneficial technical effects of one or more of the above technical solutions are as follows: The *E. coli* strain constructed using the above-mentioned technical solution exhibits excellent acid resistance, growing normally within a pH range of 4.2-7.0. It effectively adapts to changes in the acidic environment during fermentation, eliminating the need for excessive addition of alkaline regulators, thus reducing production costs and process complexity. Furthermore, the *E. coli* strain overexpresses a key gene in the 5-aminolevulinic acid synthesis pathway (…). hemA , coaA, ppc and eamA ), and utilizes sRNA-mediated regulatory systems to suppress competitive pathway genes ( hemB , aceA and sucC The expression of ) significantly improved the synthesis efficiency of 5-aminolevulinic acid, with a batch fermentation yield of 10.35 g / L, demonstrating potential for industrial application.

[0021] In summary, the above-mentioned technical solutions, combining adaptive laboratory evolution and genetic engineering techniques, construct acid-resistant Escherichia coli chassis cells that can not only be used for the production of 5-aminolevulinic acid, but also provide a reference chassis strain for microbial fermentation under other acidic conditions, showing broad application prospects. Attached Figure Description

[0022] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0023] Figure 1 This is a plasmid map of the recombinant plasmid pJA-2 in the embodiments of the present invention.

[0024] Figure 2 In this embodiment of the invention, agarose gel electrophoresis was used to verify the recombinant plasmid PJA-2.

[0025] Figure 3 This is a plasmid map of recombinant plasmid W2 in an embodiment of the present invention.

[0026] Figure 4 In this embodiment of the invention, agarose gel electrophoresis was used to verify the recombinant plasmid W2.

[0027] Figure 5 This is the standard curve for 5-ALA in this embodiment of the invention.

[0028] Figure 6 This invention provides a comparison of the OD600 values ​​of strains JP2025 and MG1655 under different pH conditions (culture time: 24 h).

[0029] Figure 7 This invention provides a comparison of 5-ALA production of strain JP2025 under different pH conditions in this embodiment.

[0030] Figure 8 This invention relates to the fed-batch fermentation of strain JP2025 in a 5-L fermenter. Detailed Implementation

[0031] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0032] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0033] The following examples further illustrate the present invention, but do not constitute a limitation thereof. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the materials, reagents, instruments, and methods used in the following examples are all conventional materials, reagents, instruments, and methods in the art and are commercially available.

[0034] Example I. Experimental Methods 1. Strains and plasmids The strains used in this invention are shown in Table 1: Table 1. Strains used in this invention

[0035] The plasmids used in this invention are shown in Table 2: Table 2. Plasmids used in this chapter

[0036] 2. Primers The primers used in this invention are shown in Table 3: Table 3 Primer sequences used in this invention

[0037] 3. Culture medium and culture method for the strain 1) LB medium (tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L) 2) SOB medium (tryptone 20 g / L, yeast extract 5 g / L, NaCl 0.585 g / L, KCl 0.186 g / L) is used for... mutS When using the knockout method, add 10 mL of 1 mol / L MgCl2 solution per L of SOB medium.

[0038] 3) LBG medium (5 g / L yeast extract, 10 g / L peptone, 10 g / L NaCl, 20 g / L glucose, pH range adjusted with 5 mol / L NaOH and 5 mol / L HCl).

[0039] 4) Fermentation medium (glucose 20 g / L; yeast extract 2 g / L; (NH4)2SO4 16 g / L; KH2PO4 3 g / L; Na2HPO4·12H2O 16 g / L; MgSO4·7H2O 1 g / L; MnSO4·7H2O 0.01 g / L) 5) Seed culture: Under aseptic conditions, pick single clones from the plate and inoculate them into 5 mL of sterile LB liquid medium. After adding antibiotics, place the shake flask in a shaker at 37 °C and 200 rpm for overnight incubation. Then, inoculate 1 mL of the overnight culture into 50 mL of LB medium and incubate for 8-12 h to obtain the seed culture solution.

[0040] 6) Commonly used antibiotics' storage and working concentrations: Ampicillin (storage concentration 100 mg / mL, working concentration 50 μg / mL); Kanamycin (storage concentration 50 mg / mL, working concentration 25 μg / mL); Spectinomycin (storage concentration 100 mg / mL, working concentration 50 μg / mL); Chloramphenicol (storage concentration 50 mg / mL, working concentration 17 μg / mL) 7) 60 mM CaCl2 solution (CaCl2 1.332 g / L, PIPES 1.341 g / L, glycerol 30 mL / L) 8) For batch fed-batch fermentation testing, the recombinant bacterial strain was removed from a -80 ℃ ultra-low temperature freezer, and 100 μL of bacterial culture was inoculated into 50 mL of antibiotic-free LB liquid medium. The culture was then incubated overnight in a constant-temperature shaker at 30 ℃ and 200 rpm to activate the strain. Subsequently, the activated bacterial culture was transferred at a 5% (v / v) inoculation rate to a seed culture medium supplemented with the appropriate antibiotic, and cultured under constant-temperature shaking conditions at 36 ℃ and 200 rpm. After the culture was completed, the seed culture was transferred at a 5% (v / v) inoculation rate to a fermentation medium supplemented with the appropriate antibiotic for fermentation. The fermentation process was carried out under constant-temperature shaking conditions at 37 ℃ and 500 rpm, with the ammonia level automatically controlled at 6.8 and dissolved oxygen controlled at 25%-35%. Whenever the fermentation sugar content fell below 5 g / L, 500 g / L of glucose was added until fermentation was completed.

[0041] 4. Escherichia coli MG1655 strain mutS gene knockout 1) Obtaining homologous recombination fragments Using pKD4 plasmid as a template, primers kan(mutS)-QF and kan(mutS)-QR were used to amplify the contents of the plasmid via PCR. mutS The kanamycin resistance gene fragment, located on the homologous arm of the gene, is the gene targeting fragment. The PCR product was verified by agarose gel electrophoresis; its size should meet expectations (1576 bp). The targeting fragment was purified using a gel extraction kit, and the purified DNA concentration should be no less than 20 ng / μL, OD... 260 / OD 280 The ratio should be between 1.8 and 2.0.

[0042] 2) Transformation of pTKRed plasmid Competent *E. coli* cells stored in an ultra-low temperature freezer were removed and placed in an ice bath. Simultaneously, the cryopreserved pTKRed plasmid was thawed at room temperature. The pTKRed plasmid was transformed into *E. coli* using either chemical or electroporation. Single colonies were picked and incubated in LB medium containing spectinomycin resistant to β-dextrin at 30 °C for 14–20 h. The plasmid was extracted using a plasmid extraction kit and verified by agarose gel electrophoresis.

[0043] 3) mutS Gene deletion The gene targeting fragment was introduced into *E. coli* containing the pTKRed plasmid using electroporation, and the Kan-resistant clones were verified by colony PCR. Using kan(mutS)-JF and kan(mutS)-JR as primers, the original strain should amplify... mutSThe gene band should be 3060 bp, while a successfully knocked-out strain should amplify a different-sized Kan gene band (1994 bp). PCR products should be confirmed by agarose gel electrophoresis.

[0044] 4) Removal of pTKRed plasmid Will verify the correct mut S-deleted strains were cultured in antibiotic-free LB broth at 42 °C for 24 h to eliminate the temperature-sensitive plasmid pTKRed. Small amounts of the bacterial culture were then screened on Amp-resistant and Kan-resistant plates, respectively. Strains that successfully removed pTKRed should not grow on Amp-resistant plates but should grow on Kan-resistant plates.

[0045] 5) Removal of kanamycin resistance The pCP20 plasmid carrying the FLP site-specific recombinase was transformed into the above-mentioned *E. coli* competent cells that had lost pTKRed. The transformants were cultured at 42 °C in antibiotic-free LB medium for 24 h to induce FLP recombinase excision of the resistance gene and simultaneously remove the pCP20 plasmid. Single colonies were isolated using antibiotic-free LB agar plates, and then selected for further screening on Amp and Kan resistance plates. Strains that successfully removed both the resistance and pCP20 plasmid should not grow on either Amp or Kan resistance plates, but only on antibiotic-free LB agar plates. This strain is the *E. coli*. mut S-deficient E. coli chassis can be used for subsequent adaptive evolution.

[0046] 6) Utilizing adaptive evolution to improve the acid resistance of Escherichia coli LBG medium was used as the basal medium for adaptive evolution. mut The S-deficient strain was used as the starting strain. The strain was activated in LB liquid medium and then inoculated at a rate of 2% (v / v) into LBG medium at pH 5.3 to initiate evolution. The culture was carried out in a constant-temperature shaker at 37 °C and 200 r / min. After stabilization at pH 5.3, the pH of the medium was gradually decreased until the desired acid-tolerant strain was obtained.

[0047] 7) Construction of plasmid PJA-2, which is an overexpression module for 5-ALA synthesis pathway enzymes. Search on KEGG hemA (K00643) , coaA (K00867) , ppc (K01595) , eamA (K15268) , grxAThe sequence of the (K03674) gene was obtained, and upstream and downstream primers were designed (Table 3). A single colony of Escherichia coli BW25113 was picked and placed into a 1.5 mL EP tube. 100 μL of ddH2O was added and mixed by pipetting and aspiration as a template. The complete gene was then amplified by PCR. hemA , coaA, ppc, eamA and grxA Gene fragments. The pTrc99a plasmid was selected using a single restriction endonuclease digestion method. Eco RI restriction site. Using a seamless DNA cloning kit, the linear pTrc99a plasmid was cloned with the plasmid obtained using overlap extension PCR. hemA-coaA The fragments are assembled, and the assembly system is as follows: Table 4 Assembly systems of homologous recombination

[0048] The 20 μL reaction mixture was incubated at 37 °C in a metal bath for 30 min, and the entire reaction solution was transformed into *E. coli* DH5α competent cells. Recombinant plasmids were extracted and sequenced after successful verification by agarose gel electrophoresis. The successfully ligated pTrc99a- plasmid was then digested using a single restriction endonuclease digestion method. hemA-coaA plasmid utilization Hin Linearization of the dIII restriction site. Using a seamless DNA cloning kit, the linearized pTrc99a- hemA-coaA plasmids and ppc-eamA- grxA Assembly, the assembly system is as follows: Table 5 Assembly systems of homologous recombination

[0049] Recombinant plasmids were extracted using a kit, and after successful verification by agarose gel electrophoresis, the plasmid samples were sequenced for verification.

[0050] 8) Construction of plasmid W2 for the competitive pathway inhibition module of 5-ALA synthesis Design and synthesize sRNAs, and utilize sRNA-based gene regulation mechanisms to inhibit... hemB , aceA and sucC Expression of three genes. The designed sRNA comprises two main parts: a scaffold sequence and a target-binding sequence. *E. coli* contains a common secondary structure that provides a scaffold for recruiting the Hfq protein, which facilitates hybridization of the sRNA and target mRNA, as well as mRNA degradation. Selection lacks... ompCThe binding sequence MicC serves as the scaffold for our sRNA synthesis. To simplify the design process, the selected binding sequence is complementary to the coding sequence of DsRed2 mRNA from the start codon AUG to nucleotide 21. This sequence ensures high affinity while mitigating cross-reactivity to some extent. hemB , aceA and sucC The genes were bound to target binding sites on the sRNA structure and overlapped to obtain the desired gene sequences, as shown in Table 6. The gene sequences were sent to Jinan GenScript Biotech Co., Ltd. for synthesis and ligated with the low-copy plasmid pCL1920 to obtain the 5-ALA synthesis competitive pathway inhibitory module plasmid W2.

[0051] Table 6. Structural sequences of sRNA

[0052] 9) Fermentation of recombinant strain JP2025 The successfully transformed *E. coli* strain with dual plasmids PJA-2 and W2 was named strain JP2025. Single clones of strain JP2025 were inoculated into 50 mL of LB broth and cultured overnight in a shaker at 30 °C and 200 rpm. Inoculation was carried out at 2% (v / v) in fermentation media with different pH gradients, with the addition of ampicillin and spectinomycin at final concentrations of 50 μg / mL. Fermentation was carried out in shake flasks at 30 °C and 200 rpm. After 3 h of fermentation, 1.0 mM IPTG was added as an inducer. Sampling was taken periodically to monitor bacterial growth, glucose consumption, and 5-ALA production.

[0053] 10) Detection methods Bacterial growth status Centrifuge 1 mL of bacterial culture at 15000 rpm for 3 min, discard the supernatant, and mix the bacterial cells with 1 mL of distilled water to dilute the solution by a certain factor, ensuring the absorbance at 600 nm is between 0 and 1. Measure the OD value using a spectrophotometer. 600 .

[0054] Determination of glucose concentration Centrifuge 1 mL of the bacterial culture at 15,000 rpm for 3 min, collect the supernatant, and dilute the glucose concentration in the supernatant to 0-1 g / L with distilled water. Measure the glucose concentration using an SBA-40E biosensor analyzer. For specific methods, please refer to the instrument's instruction manual.

[0055] Determination of 5-ALA concentration Centrifuge the fermentation broth at 12000 rpm for 10 min to remove solid impurities, and dilute the supernatant to a suitable concentration. Take 250 µL of the diluted liquid, add 125 µL of sodium acetate buffer and 62.5 µL of acetylacetone, and react at 100 °C for 15 min. After cooling to room temperature, add 440 µL of Modified Ehrlich's reagent. React at room temperature for 20 min, and measure the absorbance at 554 nm. Calculate the corresponding 5-ALA concentration based on the standard curve. Weigh 0.128 g of 5-ALA-HCl and dilute to 100 mL to obtain 1 g / L 5-ALA. After different serial dilutions, measure the absorbance according to the above method and plot the 5-ALA standard curve. Modified Ehrlich's reagent (prepared fresh): Take 0.2 g of p-dimethylaminobenzaldehyde, add 1 mL of glacial acetic acid, 1 mL of perchloric acid, and 8 mL of glacial acetic acid, and bring the volume to 10 mL; Sodium acetate buffer (pH=4.6): Measure 5.7 mL of glacial acetic acid, add 8.2 g of anhydrous sodium acetate, and bring the volume to 100 mL with water.

[0056] II. Results 1. Construction of recombinant plasmid PJA-2 Using seamless DNA cloning technology and overlap extension PCR, plasmid pTrc99a was combined with gene fragments. hemA - coaA and ppc - eamA - grxA Assembled into a complete recombinant plasmid PJA-2 ( Figure 1 The plasmid size was confirmed to be consistent with expectations by agarose gel electrophoresis. Figure 2 Sequencing confirmed that the recombinant plasmid PJA-2 was successfully constructed.

[0057] 2. Construction of plasmid W2 for the competitive pathway inhibition module of 5-ALA synthesis To further increase 5-ALA production, an sRNA-based gene regulation system was used to inhibit... hemB , aceA and sucC Expression of three genes. By targeting hemB , aceA and sucC The three gene sRNAs and the scaffold sequence were ligated to the low-copy plasmid pCL1920 to obtain plasmid W2 containing the sRNA repression module. Figure 3 The plasmid size was confirmed to be consistent with expectations by agarose gel electrophoresis. Figure 4Sequencing confirmed that the recombinant plasmid W2 was successfully constructed.

[0058] 3. Preparation of the 5-ALA standard curve Weigh 0.128 g of 5-ALA-HCl and dilute to 100 mL to obtain 1 g / L 5-ALA. After different serial dilutions, measure the absorbance according to the above method and plot the standard curve of 5-ALA as shown below. Figure 5 As shown.

[0059] 4. Comparison of growth of strain JP2025 under different pH conditions The recombinant strain JP2025 and the original strain MG165 were inoculated into the prepared culture medium at an inoculation amount of 5% and cultured for 24 h. The growth of the strains is shown in the figures below. Figure 6 As shown in the figure, the growth of the two strains was similar within the pH range of 6.5-7.0. Within the pH range of 4.2-6.0, the final strain JP2025 exhibited better cell growth than the initial strain MG165. The growth of the evolved strain JP2025 was quite similar within the pH range of 5.0-7.0, and its maximum OD200 after 24 hours of culture was highest within the pH range of 4.2-4.9. 600 The value dropped below 8.0, but was still significantly better than the growth of MG1655. The OD of MG1655 in an environment of 4.2-4.9... 600 The value dropped below 3.0.

[0060] 5. Comparison of 5-ALA synthesis in strain JP2025 under different pH conditions The constructed final strain JP2025 was subjected to batch fermentation under different pH conditions. The pH gradient was set at 7.0, 6.5, 6.0, 5.5, 5.0, and 4.5. Figure 7 It can be seen that JP2025 exhibits slightly higher 5-ALA synthesis levels under pH conditions of 6.0-7.0, with the synthesis level gradually decreasing as the pH gradient decreases. Specifically, at pH 7.0, JP2025 can synthesize 823.23 mg / L of 5-ALA, while at pH 4.5, the strain can synthesize 544.25 mg / L of 5-ALA. This is combined with the strain's OD values ​​under corresponding pH conditions. 600 The decrease in 5-ALA production by strain JP2025 under acidic conditions was mainly due to a decrease in biomass; the 5-ALA synthesis level per unit cell did not show a significant decrease. This indicates that the JP2025 strain constructed in this invention possesses the ability to synthesize 5-ALA under pH=4.5 conditions.

[0061] 6. Fed-batch fermentation of strain JP2025 The JP2025 strain was subjected to fed-batch fermentation in a 5-L fermenter. Figure 8 As shown, strain JP2025 entered the logarithmic growth phase at 12 h and entered a relatively stationary phase at 48 h. At 54 h, the strain's OD... 600 The value reached a maximum of 22.52. The accumulation of 5-ALA also reached its highest level at 66 h, at 10.35 g / L.

[0062] In summary, the recombinant *E. coli* JP2025 constructed using the above technical solution maintained relatively normal growth under pH conditions of 4.2-7.0, demonstrating its acid-resistant properties. Simultaneously, in batch fermentation, the constructed recombinant *E. coli* JP2025 maintained a high level of 5-ALA synthesis under pH conditions of 4.5-7.0, demonstrating its 5-ALA synthesis capacity under acidic conditions. In fed-batch fermentation in a 5-L fermenter, JP2025 synthesized 10.35 g / L of 5-ALA in 66 h, showing good application potential. The acid-resistant *E. coli* constructed in this invention can effectively adapt to a wide range of acidic environments during fermentation, and can to some extent avoid the excessive addition of alkaline substances during fermentation. Furthermore, its efficient synthesis of 5-ALA under acidic conditions provides a reference example for the application of acid-resistant *E. coli* chassis cells, facilitating its widespread application.

[0063] Matters not covered in this invention are common knowledge.

[0064] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A low-pH-tolerant Escherichia coli, characterized in that, The E. coli was obtained by knocking out the original strain. mutS Genes, overexpression hemA , coaA, ppc, eamA and grxA Genes and repression hemB、aceA and sucC Obtained after genetic modification.

2. The *Escherichia coli* as described in claim 1, characterized in that, The starting strain was Escherichia coli MG1655.

3. The *Escherichia coli* as described in claim 1, characterized in that, Among the low-pH-tolerant E. coli, knockout mutS The genes were obtained using Red homologous recombination technology.

4. The *Escherichia coli* as described in claim 1, characterized in that, The overexpression hemA , coaA, ppc, eamA It was obtained by constructing an overexpression module and importing it into E. coli; Furthermore, the overexpression module utilizes the pCL1920 plasmid for overexpression. ppc, coaA, hemA, grxA, eamA Five genes were constructed to obtain the results. The inhibition hemB、aceA and sucC The gene was obtained by introducing it into E. coli through the construction of an sRNA-based repressor module; Furthermore, the sRNA-based repression module is designed to inhibit [something] on the pTrc99a plasmid. hemB , aceA and sucC The sRNA of the gene was obtained through construction.

5. The *Escherichia coli* as described in any one of claims 1-4, characterized in that, The low-pH-tolerant Escherichia coli is Escherichia coli (E. coli) Escherichia coli JP2025, this strain was deposited at the China Center for Type Culture Collection on November 20, 2025, with the following accession number: CCTCC NO: M 20252608.

6. The method for constructing low-pH-tolerant Escherichia coli according to any one of claims 1-5, characterized in that, The construction method includes: S1. Using wild-type Escherichia coli as the starting strain, the mismatch repair protein gene was knocked out. mutS Acid-resistant strains were subsequently obtained through adaptive evolution. S2, Constructing overexpression hemA , coaA, ppc and eamA The first recombinant expression vector and containing inhibitors hemB、aceA and sucC The second recombinant expression vector for sRNA; S3. Integrate the first recombinant expression vector and the second recombinant expression vector into the acid-resistant strain.

7. The construction method as described in claim 6, characterized in that, The starting strain was Escherichia coli MG1655; Furthermore, in step S1, the mismatch repair protein gene is knocked out. mutS The procedure was performed using Red homologous recombination technology; The specific methods of adaptive evolution include: using mutS The defective strain is used as the starting strain, and the pH of the culture medium is gradually reduced until the desired acid-resistant strain is obtained.

8. The construction method as described in claim 6, characterized in that, In step S2, the backbone vector of the first recombinant expression vector is plasmid pCL1920; the backbone vector of the second recombinant expression vector is plasmid pTrc99a. Targeting genes hemB , aceA and sucC The sRNA sequences of the genes are shown in SEQ ID NO.1-3, respectively.

9. The use of the low-pH-tolerant Escherichia coli according to any one of claims 1-5 in the fermentation production of 5-aminolevulinic acid.

10. A method for the industrial production of 5-aminolevulinic acid, characterized in that, The method includes: fermenting and culturing the low-pH-tolerant Escherichia coli according to any one of claims 1-5, and separating and purifying the 5-aminolevulinic acid.

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