Ampramycin genetically engineered bacterium with genetic stability as well as construction method and application of apramycin genetically engineered bacterium

Abstract

The invention belongs to the technical field of genetically engineered bacteria, and particularly discloses apramycin genetically engineered bacteria with genetic stability as well as a construction method and application of the apramycin genetically engineered bacteria. The construction method comprises the following steps: by taking a plasmid containing an xylE gene and a tsnR gene as a template, obtaining a linearized vector skeleton without a promoter; the method comprises the following steps: inserting a streptomyces indicus promoter sequence into a linearized vector skeleton to enable the promoter to be located at the upstream of an xylE gene and a tsnR gene, and constructing a double-reporter gene expression vector; transferring the expression vector into streptomyces through conjugation to obtain a conjugate; the genetically stable strain is obtained through ultraviolet mutagenesis in combination with catechol chromogenic screening. The biological value is increased by more than 40% compared with that of an original strain, the value retention rate of 94.5% or above is kept in continuous 10 generations of long-term passage experiments, and a powerful technical support is provided for industrial stable production of anti-mycoplasma active substances.

Description

Technical Field

[0001] This invention relates to the field of genetically engineered bacteria, and in particular to genetically stable apramycin-based genetically engineered bacteria, its construction method, and its applications. Background Technology

[0002] Streptoalloteichus hindustanus is an important medicinal actinomycete that produces a variety of secondary metabolites with significant biological activity, mainly including the Nebramycin complex (containing apramycin, tobramycin, etc.) and tallysomycin. These active substances are mostly aminoglycoside or glycopeptide antibiotics. With their unique mechanisms of action (such as inhibiting bacterial ribosomal protein synthesis or damaging DNA), they exhibit significant killing effects against common mycoplasma and Gram-negative bacteria in livestock and poultry, making them irreplaceable in veterinary drug development and the prevention and control of animal infectious diseases.

[0003] However, in the industrial production of antibiotics, fermentation units and production costs largely depend on the genetic stability of the producing strains. Because these strains have large gene clusters for secondary metabolite synthesis and complex regulatory mechanisms, highly active industrial strains are prone to spontaneous mutations or metabolic flux redistribution during production processes such as continuous subculturing, slant culture, or seed culture. This leads to a significant decline in overall antimicrobial biopotency, a phenomenon known as "strain degradation," which severely limits the large-scale, stable production of anti-mycoplasma active substances.

[0004] Existing strain selection and degradation monitoring technologies have significant limitations in practical applications. On the one hand, traditional degradation detection mainly relies on shake-flask fermentation combined with biopotency assays (such as the tube-disc method), a process that typically takes 7 to 10 days and suffers from severe detection lag. This makes it impossible to identify and remove degraded strains in a timely manner during seed preparation or plate growth, often resulting in wasted production resources and drastic fluctuations in fermentation units. On the other hand, traditional mutagenesis breeding techniques lack clear phenotypic indicators of high-yield traits, and the screening process is highly inefficient and prone to blind selection.

[0005] Molecular mechanism studies have revealed that in the biosynthetic gene cluster of *Heterodera indicum*, the key gene encoding core scaffold synthesis and the key modifying enzyme gene (aprD3) are located on a specific transcriptional unit (operon) in the genome. The transcription initiation activity of this specific operon plays a decisive regulatory role, directly reflecting the overall level of metabolic flux in the biosynthetic pathway. Therefore, developing a real-time monitoring system capable of specifically responding to the transcriptional activity of this core operon, converting recessive metabolic flux into a dominant, intuitive phenotype, is of significant technical value for achieving high-fidelity screening of strains producing high levels of mycoplasma-resistant active substances and maintaining their genetic stability in real-time during industrial passaging. Summary of the Invention

[0006] The purpose of this invention is to address the above-mentioned shortcomings of the prior art by providing a method for constructing genetically stable apramycin-engineered bacteria. By constructing a dual reporter system for color development and resistance driven by the core promoter of the biosynthetic gene cluster (PaprD3), the invisible metabolic flux is transformed into a visible colony phenotype. Further mutagenesis is then used to obtain genetically stable strains, thereby achieving real-time identification and selection of high-biological-value strains.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: The first aspect of this invention is to provide a method for constructing a genetically stable apramycin-based genetically engineered bacterium, comprising the following steps: The sequence of the promoter of Heterodermium indicum was obtained, as shown in SEQ ID NO:1; A dual reporter gene expression vector containing a first reporter gene and a second reporter gene was constructed, wherein the first reporter gene is located downstream of the *Heterodera indicum* promoter, and the second reporter gene is located downstream of the first reporter gene; the *Heterodera indicum* promoter drives the transcription of the first and second reporter genes; the first reporter gene is catechol-2,3-dioxygenase gene. ， The second reporter gene is a thiotetracycline resistance gene; The dual reporter gene expression vector was introduced into the host bacteria; Recombinant strains containing the dual reporter gene expression vectors were screened and co-cultured with *Heterodera indicum*, allowing the dual reporter gene expression vectors to be transferred into *Heterodera indicum* via binding transfer, thereby obtaining conjugates; The conjugate was subjected to ultraviolet mutagenesis to obtain a mutagenic strain. The mutagenic strain was cultured and passaged. Before passage, catechol was sprayed on the strain. The genetically stable apramycin genetically engineered strain was obtained by screening based on the catechol colorimetric reaction.

[0008] Furthermore, the construction process of the dual reporter gene expression vector is as follows: Using plasmid PSET152 as a template, a linearized vector backbone containing the promoterless catechol-2,3-dioxygenase gene and the thiostreptin resistance gene was constructed. The sequence of the promoter of *Heterodera indicum* was ligated to the linearized vector backbone to obtain the ligation product; The ligation product was transformed into competent E. coli cells and cultured. Single clones were selected and verified to obtain the dual reporter gene expression vector.

[0009] Furthermore, the sequence of the *Heterodermium indicum* promoter is mixed with the linearized vector backbone in a molar ratio of 2:1 to 5:1 for ligation reaction.

[0010] Furthermore, the ligation reaction includes ligation methods using homologous recombination, seamless cloning, or DNA ligase-mediated ligation.

[0011] Furthermore, the host bacteria are selected from bacteria or yeast.

[0012] Furthermore, the ultraviolet mutagenesis conditions are as follows: mutagenesis treatment is performed under a 15W ultraviolet lamp for 20-40 seconds.

[0013] A second aspect of the present invention is to provide a dual reporter gene expression vector, comprising: The promoter sequence is shown in SEQ ID NO:1; A first reporter gene located downstream of the promoter, the sequence of which is shown in SEQ ID NO:2; A second reporter gene located downstream of the first reporter gene, the sequence of which is shown in SEQ ID NO:3; The promoter simultaneously drives the expression of the first reporter gene and the second reporter gene.

[0014] A third aspect of the present invention is to provide the application of the above-described method or dual reporter gene expression vector in the evaluation and screening of bacteria that produce anti-mycoplasma active substances.

[0015] A fourth aspect of the present invention is to provide an engineered bacterium that produces high levels of mycoplasma-resistant active substances, comprising the above-described dual reporter gene expression vector or obtained by the described construction method.

[0016] A fifth aspect of the present invention is to provide the use of the engineered bacteria in the preparation of anti-mycoplasma active substances, said anti-mycoplasma active substances including apramycin and talamicin.

[0017] Compared with the prior art, the beneficial effects of the technical solution provided by the present invention are as follows: This invention, based on a precise molecular regulatory mechanism, identifies the core rate-limiting promoter PaprD3 in the gene cluster for the biosynthesis of anti-mycoplasma active substances. Experiments have confirmed a highly significant linear positive correlation between the colorimetric intensity of the reporter gene driven by this promoter and the total biopotency of the fermentation end product. This mechanism fundamentally overcomes the problem of traditional morphological screening lacking specific targets, ensuring that the detection results accurately and linearly reflect the strain's intrinsic potential for synthesizing active substances. Secondly, this invention innovatively constructs a visualized dual-reporter system, transforming antibiotic yield traits, which previously required late-stage fermentation testing, into visually perceptible colony color and resistance phenotypes. This significantly advances the detection point from the traditional 7-10 days to 2-3 days during the plate growth stage. This "preventative control" strategy greatly improves screening efficiency and reduces time costs and waste of production resources. Furthermore, this invention demonstrates outstanding performance in maintaining the genetic stability of strains. The strain (HC-6) selected using this system not only has a biopotency that is more than 40% higher than the original strain, but also maintained a biopotency retention rate of over 94.5% in a long-term subculturing experiment of 10 consecutive generations, which is significantly better than the natural subculturing group. This effectively solves the problem of natural degradation of industrial strains during long-term preservation and use, and provides strong technical support for the stable industrial production of anti-mycoplasma active substances. Attached Figure Description

[0018] Figure 1 This is a schematic diagram of the gene cluster structure for the biosynthesis of apramycin in *Heterodera ininica*. The diagram shows the sequence of genes related to apramycin biosynthesis in the *Heterodera ininica* genome, with genes encoding key modifying enzymes highlighted. aprD3 And its upstream promoter region PaprD3, which is used to drive the dual reporter gene system in this invention to enable visual monitoring of metabolic flux; Figure 2 This is a schematic diagram of the structure of the recombinant plasmid PSET152-PaprD3, which contains the PaprD3 promoter driving the xylE and tsnR genes. The PaprD3 promoter is inserted upstream of the catechol-2,3-dioxygenase gene (xylE, a chromogenic reporter gene) and the thiostreptomycin resistance gene (tsnR, a selection marker gene). The plasmid backbone is based on PSET152 and contains streptomycin integrase elements (φC31 integrase system) and apramycin resistance gene (used for Escherichia coli selection). Figure 3 The diagram shows the structure of the negative control plasmid PSET152-WQ, which does not contain a promoter. The structure of this plasmid is basically the same as that of PSET152-PaprD3, but no promoter sequence is inserted. It is used to verify the specificity of PaprD3-driven expression and to exclude the influence of vector background expression on the experimental results. Figure 4 The images show agarose gel electrophoresis identification during vector construction, where lane M is the marker and the remaining lanes are cloning fragments; in the left image, lane 1 is the tsr gene fragment, in the middle image, lanes 1.2 are the PaprD3 gene, and in the right image, lanes 1.2.3 are the bands of the linearized PSET152 backbone. Figure 5 The image shows an electrophoresis pattern for colony PCR validation, where lane M is the marker and the other lanes are cloning fragments. The left image validates the TSR gene fragment, the middle image validates the PaprD3 gene, and the right image validates the fragment without a promoter. Figure 6 This is a comparison of the chromogenic phenotypes of conjugate colonies. The left image shows conjugates grown from Streptomyces conjugate transfer; the right image shows the chromogenic reaction of conjugate colonies of *Streptomyces indicum* containing the PSET152-PaprD3 plasmid after spraying with catechol solution. Positive colonies appear deep yellow, indicating that the PaprD3 promoter has high transcriptional activity and can effectively drive transcription. xylE Gene expression enables visualization of metabolic flux; Figure 7 The figure shows a comparison of the shake-flask fermentation titers of the initial screening strains. The figure compares the biotips of the nine conidial strains (SHD3-1 to SHD3-9) selected from the chromogenic screening plates with the wild-type control strain (SH-0). The results show that SHD3-1 has the highest titer, reaching 2207 U / mL, which is 22.6% higher than that of the wild type, verifying the positive correlation between chromogenic intensity and titer. Figure 8 The figure shows a comparison of the stability of biological titer during 10 consecutive generations of passage. The figure shows the change curves of biological titer of the high-yielding mutant strain HC-6 during 10 consecutive generations of passage, using the dual reporter system guided by this invention (experimental group) and natural passage (control group). The experimental group still maintained 2408 U / mL in the F10 generation, with a titer retention rate of 94.5%, which is significantly better than the 68.0% of the control group, proving that the method of this invention can effectively prevent the genetic degradation of the strain. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of this invention clearer, the specific embodiments and accompanying drawings are described in further detail below. Where specific techniques or conditions are not specified in the embodiments, they are performed in accordance with the techniques or conditions described in the literature in this field or according to the product manual.

[0020] The following is an illustrative description of a genetically stable apramycin-based engineered bacterium, its construction method, and its application, with reference to specific embodiments.

[0021] The embodiments of the present invention involve *Heterodermium indicum* (…). Streptoalloteichus hindustanus The strain preservation number is DSM 44523; Escherichia coli competent cells JM109, ET12567 (pUZ8002), plasmid PSET152, and pKC1139 are all commercially available.

[0022] The culture medium formulations used in the embodiments of the present invention (sterilization conditions: 121°C, 20 min) are as follows: LB medium (for Escherichia coli culture): 10 g / L peptone, 10 g / L sodium chloride, 5 g / L yeast extract.

[0023] 2XYT medium (for spore resuscitation): peptone 16 g / L, yeast extract 10 g / L, sodium chloride 5 g / L.

[0024] MS medium (for binding transfer): 20 g / L soybean flour, 20 g / L mannitol, 20 g / L agar, with 10 mg / L MgCl2 added.

[0025] GYM medium (for sporulation): glucose 4 g / L, yeast extract 4 g / L, malt extract 10 g / L, calcium carbonate 2 g / L, agar 20 g / L.

[0026] Maltose medium (for color development): 8 g / L malt extract, 0.8 g / L yeast extract, 0.8 g / L tuna extract, 1 g / L tryptone, 22 g / L agar powder. YEME medium (for extracting Streptomyces genome): glucose 40 g / L, peptone 50 g / L, yeast extract 30 g / L, malt extract 30 g / L.

[0027] Seed culture medium: glucose 10g / L, soybean flour 10g / L, peptone 3g / L, corn flour 5g / L, yeast powder 1g / L, calcium carbonate 1g / L, pH 7.0-7.2.

[0028] Fermentation medium: glucose 35g / L, soybean meal 35g / L, yeast powder 1g / L, corn steep liquor 10g / L, soybean meal 10g / L, ammonium sulfate 4g / L, magnesium sulfate 6g / L, zinc sulfate 0.03g / L, ferrous sulfate 0.1g / L, calcium carbonate 5g / L, pH 7.2.

[0029] In constructing the dual reporter gene vector PSET152-PaprD3, the present invention involves mixing the sequence of the *Heterodera indicum* promoter with the linearized vector backbone at a molar ratio of 2:1 to 5:1 for ligation to obtain a recombinant expression vector. Preferably, the molar ratio is 2:1 to 3:1, more preferably about 3:1. The ligation reaction can be performed using DNA ligase-mediated ligation, homologous recombination, or seamless cloning. Preferably, the ligation reaction system includes: linearized vector backbone DNA, promoter DNA fragment, DNA ligase or homologous recombination enzyme, and ligation buffer. In a preferred embodiment, the downstream of the promoter sequence contains a nucleotide sequence homologous to the end of the linearized vector backbone to achieve directional ligation through homologous recombination. After the ligation reaction is completed, the ligation product is directly used to transform host cells or purified before being used to transform host cells to obtain the dual reporter gene expression vector.

[0030] The method for determining biological potency in the embodiments of the present invention: Weigh an appropriate amount of apramycin standard (550 U / mg), accurate to 0.1 mg, dissolve and dilute it with pH 7.0 sodium phosphate buffer to prepare a standard stock solution with a concentration of 10000 U / mL. Further dilute with pH 7.0 sodium phosphate buffer to obtain standard working solutions with concentrations of 5000, 2000, 1000, 500, 200, 100, and 50 U / mL. Select 500 U / mL as the central concentration standard working solution. Take eight prepared double-sided dishes, each corresponding to one standard working solution concentration. Place four sterile Oxford cups in each double-sided dish. Add 100 μL of the central concentration standard working solution to one Oxford cup, and add 100 μL of the corresponding concentration standard working solution to the remaining three Oxford cups. Cover the double-sided dishes with ceramic lids and incubate at 4°C for 3 hours to allow the apramycin standard solution to fully penetrate the culture medium. Then transfer them to a 37°C incubator for 12 hours. The diameter of the inhibition zone of each group of standard working solutions was measured with vernier calipers, and the average value was calculated. Then, the total average value of the inhibition zone diameters of all double-disc center concentration standard working solutions was calculated as the correction value. The inhibition zone diameter of each standard working solution concentration group was corrected according to the following formula (1): (1) Where: Aa: the corrected diameter of the inhibition zone of the standard working solution at this concentration; Ba: the total average diameter of the inhibition zone of all double-disc center concentration standard working solutions; B: the reading of the inhibition zone diameter of the center concentration standard working solution in this concentration group; A: the average reading of the inhibition zone diameter of the standard working solution at this concentration.

[0031] A standard curve was constructed with the logarithm of apramycin concentration on the x-axis and the diameter of the inhibition zone on the y-axis, and the regression equation was obtained.

[0032] Example 1 This embodiment provides a genetically stable apramycin-based genetically engineered bacterium and its construction method, as detailed below: 1. Constructing the intermediate vector PSET152-tsnR To introduce a thiostreptin resistance marker for screening in the production strain, an intermediate vector was first constructed. Using plasmid pKC1139 as a template, PCR amplification was performed using primers tsnR-F and tsnR-R with high-fidelity DNA polymerase to obtain a tsnR gene fragment approximately 800 bp in length with homologous arms at both ends (sequence shown in SEQ ID NO:3). Simultaneously, using the original PSET152 plasmid as a template, reverse PCR amplification was performed using primers TZ-F and TZ-R. The primer binding sites are located outside the neo gene coding region, amplifying a linearized PSET152 vector backbone approximately 6500 bp in length, completely lacking the neo gene. The sequences at both ends of this vector are homologous to the homologous arm sequences at both ends of the aforementioned tsnR gene fragment.

[0033] The purified tsnR gene fragment, separated by gel electrophoresis, was mixed with the linearized PSET152 vector backbone at an appropriate molar ratio and reacted at 50°C for 30 minutes in a homologous recombinase system. The ligation product was transformed into E. coli JM109 competent cells, incubated at 37°C for 40-60 minutes, centrifuged at 4000 rpm for 5 minutes, the supernatant was discarded, and the cells were resuspended in approximately 100 μL of the remaining culture medium and plated onto LB agar plates containing apramycin. After the bacterial culture was absorbed, the cells were incubated at 37°C for 12-16 hours until single colonies appeared. Single colonies were randomly picked, and colony PCR was performed using primers YT-F and YT-R (using ES-Taq enzyme). Clones that amplified a specific band of approximately 800 bp were preliminarily identified as positive clones. The plasmid of the positive clone was extracted and sequenced for verification. The recombinant plasmid with correct sequencing results was named PSET152-tsnR, and its sequence is shown in SEQ ID NO:4. The primer sequences used to construct the PSET152-tsnR vector (replacing the neo gene) are shown in Table 1.

[0034] Table 1. Primer sequences used to construct the PSET152-tsnR vector (replacing the neo gene).

[0035]

[0036] 2. Amplification of promoter fragments Using Heterodermella indicum genomic DNA as a template, high-fidelity DNA polymerase was used to amplify... aprD3The promoter region upstream of the gene (PaprD3) has a fragment length of approximately 1194 bp, and its sequence is shown in SEQ ID NO:1. Figure 1 This is a schematic diagram of the gene cluster structure for the biosynthesis of apramycin in *Heterodera indicum*, showing the sequence of genes related to apramycin biosynthesis in the *Heterodera indicum* genome. The gene *aprD3*, encoding a key modifying enzyme, and its upstream promoter region *PaprD3* are highlighted. This promoter is used to drive the dual reporter gene system in this invention to achieve visualized monitoring of metabolic flux. 3. Construction of the dual reporter gene vector PSET152-PaprD3 Using plasmid PSET152-tsnR as a template, reverse PCR amplification was performed using primer pair ZQ-F / ZQ-R to obtain a linearized vector backbone. This backbone contained a promoter-free vector. xylE (Catechol-2,3-dioxygenase gene) and tsnR (Thiotetracycline resistance gene). The obtained PaprD3 fragment was mixed with the linearized PSET152-tsnR backbone at a molar ratio of 3:1 and reacted at 50℃ for 30 min. The ligation product was transformed into E. coli JM109 competent cells and plated on LB agar plates containing 50 μg / mL apramycin. The cells were incubated overnight at 37℃. Single clones were randomly selected, and colony PCR was performed using primers YQF and YQR for verification and sequencing. Sequencing results showed that the promoter sequence was completely correct and the orientation was correct. The recombinant plasmid was named PSET152-PaprD3, and its sequence is shown in SEQ ID NO:5. Its structural diagram is shown below. Figure 2 As shown in Table 2, the primer sequences used for cloning promoter fragments and constructing reporter plasmids are also shown.

[0037] Construction of an empty vector without a promoter: The linearized PSET152 backbone was self-circulated to construct the plasmid PSET152-WQ, the sequence of which is shown in SEQ ID NO:6, and its structural schematic diagram is shown below. Figure 3 As shown. The PCR system and procedure used in the construction process are in accordance with standard operating procedures or kit instructions. Primer sequences are detailed in Tables 1 and 2.

[0038] PCR system: 25 μL high-fidelity enzyme; 2 μL F; 2 μL R; 2 μL template; 19 μL DDH2O.

[0039] The PCR program was set as follows: pre-denaturation: 98℃ for 2 min; denaturation: 98℃ for 10 s; annealing: 60℃ for 10 s; extension: 72℃ for 35 s; 35 cycles; extension: 72℃ for 5 min.

[0040] Homologous recombination ligation system: 50℃, 30min; homologous recombinase 5μL, target gene 3μL, vector 2μL, total system 10μL.

[0041] Digestion template system: 37℃, 1-2h; Dpn1 enzyme 1μL, 10×QuickCut Buffer 1μL, linearized vector 8μL, total system 10μL.

[0042] Table 2. Primer sequences used for cloning promoter fragments and promoterless plasmids.

[0043]

[0044] like Figure 4 The image shows agarose gel electrophoresis identification of each vector during construction; lane M is the marker; the other lanes are cloning fragments; lane 1 in the left image is the tsr gene fragment, lane 1.2 in the middle image is the PaprD3 gene, and lanes 1.2.3 in the right image are the bands of the linearized PSET152 backbone.

[0045] like Figure 5 The image shown is an electrophoresis diagram of colony PCR verification, where lane M is the marker and the other lanes are cloning fragments; the left image verifies the tsr gene fragment, the middle image verifies the PaprD3 gene, and the right image verifies the fragment without a promoter.

[0046] 4. Construction of binders 4.1 Preparation of Streptomyces spores Extract *Heterodera indicum* cultured at 37°C for 6-7 days. Select plates with abundant spores and elute mature spores into 150ml Erlenmeyer flasks using 2XYT liquid medium. Add sterile glass beads and agitate to disperse the spores. Filter through sterile gauze to remove impurities and mycelium from the medium. Heat-shock the filtered spore suspension at 50°C for 10 minutes. After cooling to room temperature, transfer the entire suspension to a 250ml Erlenmeyer flask containing 50ml of 2XYT liquid medium. Recover and culture at 37°C and 220rpm for 2 hours. Transfer the recovered culture to a 50ml centrifuge tube and centrifuge at 4000rpm for 10 minutes. Discard the supernatant and resuspend the spores in 2XYT medium, adjusting to the desired spore concentration.

[0047] 4.2 Preparation of donor bacteria The correctly sequenced plasmids PSET152-PaprD3 and PSET152-WQ were transformed into competent *E. coli* ET12567 (pUZ8002) cells. Transformants were screened on LB agar plates containing kanamycin (25 μg / mL), chloramphenicol (25 μg / mL), and apramycin (50 μg / mL). Single colonies were picked and inoculated into 5 mL LB liquid tubes and incubated at 37°C for 12 hours. The cells were then transferred at a 2% inoculum to 50 mL LB liquid medium containing the three antibiotics and incubated until OD600 = 0.4–0.6. The cells were collected by centrifugation, washed with LB liquid medium to remove antibiotics, and resuspended for later use.

[0048] 4.3 Combining transfer operations with colorimetric verification A donor bacterial suspension containing the PSET152-PaprD3 plasmid was mixed with a Streptomyces spore suspension at a 1:1 ratio and spread evenly on MS solid medium plates supplemented with 10 mg Cl2. The plates were incubated at 37°C for 18 hours. A sterile aqueous solution (1 mL) containing 50 μg / mL naphthiopicrylamide and 50 μg / mL thiostreptococcalin was prepared and evenly spread on the surface of the plates. Incubation continued for 3-5 days until conjugates appeared. Conjugates containing the PSET152-PaprD3 plasmid were selected and seeded on maltose broth plates. When the colony diameter reached 2-3 mm, a 0.5 M catechol aqueous solution was evenly sprayed onto the plate surface, and the reaction was allowed to proceed at room temperature for 20 minutes. The results showed that the colonies rapidly turned deep yellow after spraying the substrate. This significant phenotypic change confirmed that the PaprD3 promoter can efficiently drive downstream... xylE Gene expression indicates that the promoter has high transcriptional activity in the host bacteria, and the promoter activity can be translated into a visual color feature.

[0049] 5. Re-screening and potency determination during shake-flask fermentation 5.1 Shake-flask fermentation Nine representative single colonies from the transformation plates were selected, transferred to their respective culture media, and cultured for 7 days before being screened again in shake flasks. All fermentation experiments were performed in triplicate. The selected single colonies were inoculated into 250 mL Erlenmeyer flasks (seed culture medium) with a volume of 50 mL and incubated at 37°C and 220 rpm for 18 hours to obtain the seed culture. A 10% inoculum was then transferred to 500 mL fermentation shake flasks (fermentation medium). Fermentation conditions: 37°C, 220 rpm, 5-day cycle. After fermentation, the supernatant was collected by centrifugation and used as the test sample.

[0050] 5.2 Biopotency determination (using apramycin as a reference) Given that the fermentation product of this strain is a complex containing multiple anti-mycoplasma active substances such as apramycin and talimimycin, and that talimimycin standards are not readily available, this embodiment uses the tube-disc method, with apramycin as the reference standard, to determine the total biopotency of the fermentation broth. Bacillus subtilis (… Bacillus subtilis Using [a specific bacterium] as an indicator bacterium, prepare double-layer bioactivity plates. Add 100 μL of the fermentation supernatant or standard solution to an Oxford cup. After standing at 4°C for 3 hours, incubate at 37°C for 16-18 hours, and measure the diameter of the inhibition zone. According to the apramycin standard curve equation Y = 3.9026X + 11.534 (R0...), the bioactivity is determined. 2 =0.9925), where Y represents the diameter of the inhibition zone (unit: mm), X represents the logarithm of the apramycin concentration, and the biopotency (U / mL) is calculated.

[0051] 5.3 Results Analysis like Figure 6 As shown, the left image is a diagram of the conjugates grown by Streptomyces conjugate transfer; the right image is the colorimetric reaction of Streptomyces in India conjugate colonies containing the PSET152-PaprD3 plasmid after spraying with catechol solution. Positive colonies appear deep yellow, indicating that the PaprD3 promoter has high transcriptional activity and can effectively drive the expression of the xylE gene, thus enabling visualization of metabolic flux.

[0052] The statistical data are shown in Table 3. The colony color intensity is highly correlated with the bioavailability. The optimal strain SHD3-1 achieved a bioavailability of 2207 U / mL, which is 22.6% higher than that of the wild type (SH-0, 1800 U / mL), confirming that screening guided by this promoter can effectively enhance the ability of strains to synthesize anti-mycoplasma active substances.

[0053] Table 3. Comparison of fermentation titer data of initial screening strains.

[0054]

[0055] 6. Ultraviolet mutagenesis and high metabolic flux screening 6.1 Preparation and mutagenesis of single spore suspensions Prepare a single-spore suspension of SHD3-1 and adjust the concentration to 10. 7 -10 8 spores / mL. Pipette 6 mL of spore suspension into a sterile Petri dish and place it under a 15W UV lamp (30 cm away) with magnetic stirring for mutagenesis treatment. Irradiation time gradients were set at 0 s, 10 s, 20 s, 40 s, and 60 s. After irradiation, the dish was incubated in the dark for 2 hours.

[0056] 6.2 Dual Reporter Gene Guided Screening Dilute the treated spore suspension and spread it onto maltose broth containing streptomycin. Incubate at 37°C in the dark for 5-7 days. When the colonies reach 2-3 mm in size, spray with a 0.5 M catechol aqueous solution and observe the color change.

[0057] 6.3 Results Analysis The results are shown in Table 4 and Figure 7 As shown, the relative average titer of the 20s treatment group reached its peak (112.0%), and the phenotypic differentiation was obvious, indicating it was the optimal mutagenic dose. Using this method, a mutant strain HC-6 with the deepest color development and strong resistance was screened from the 20s treatment group. Its biopotency during shake-flask fermentation reached as high as 2549 U / mL, demonstrating that this screening model can effectively enrich positive mutant strains with high metabolic flux.

[0058] Table 4. Effects of different UV mutagenesis times on lethality and screening efficiency.

[0059]

[0060] 7. Genetic stability during the generation process To verify the effectiveness of this invention in the long-term preservation of industrial microbial strains, a subculturing experiment of 10 consecutive generations was designed.

[0061] Control group (natural passage): High-yielding mutant strain HC-6 was used. No substrate was sprayed. The passage was simulated as a conventional industrial passage. Single colonies with good growth were randomly selected for passage.

[0062] Experimental group (guided screening): Using the high-yielding mutant strain HC-6, the method of this invention was applied. Before each subculture, 0.5M catechol was sprayed, and single colonies with the highest color intensity (deep yellow) were selected by visual inspection for subculture. Degenerate colonies with lighter color were removed.

[0063] The bioactivity of shake-flask fermentation was determined at generations 1, 3, 5, 7, and 10, and the results are shown in Table 5.

[0064] Table 5. Results of genetic stability and titer monitoring during the 10th generation.

[0065]

[0066] From Table 5 and Figure 8It was found that the control group strains exhibited significant genetic instability under no selection pressure. With increasing passage number, the biopotency showed a clear downward trend, dropping to 1733 U / mL in the F10 generation, a decline of 32.0%. In contrast, the experimental group screened using the dual reporter system of this invention, although slightly declining in the F5 generation due to environmental fluctuations, showed strong overall trait recovery ability. The biopotency remained at 2408 U / mL in the F10 generation, with a retention rate as high as 94.5%. This result strongly demonstrates that the high-yielding traits locked by the screening system of this invention have high genetic stability and can effectively prevent the genetic degradation of mycoplasma-resistant active substance-producing bacteria during long-term passage.

[0067] Where there is no conflict, the above embodiments and features described herein can be combined with each other.

[0068] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for constructing a genetically stable apramycin-based genetically engineered bacterium, characterized in that, Includes the following steps: The sequence of the promoter of Heterodermium indicum was obtained, as shown in SEQ ID NO:1; A dual reporter gene expression vector containing a first reporter gene and a second reporter gene was constructed, wherein the first reporter gene is located downstream of the *Heterodera indicum* promoter, and the second reporter gene is located downstream of the first reporter gene; the *Heterodera indicum* promoter drives the transcription of the first and second reporter genes; the first reporter gene is catechol-2,3-dioxygenase gene. ， The second reporter gene is a thiotetracycline resistance gene; The dual reporter gene expression vector was introduced into the host bacteria; Recombinant strains containing the dual reporter gene expression vectors were screened and co-cultured with *Heterodera indicum*, allowing the dual reporter gene expression vectors to be transferred into *Heterodera indicum* via binding transfer, thereby obtaining conjugates; The conjugate was subjected to ultraviolet mutagenesis to obtain a mutagenic strain. The mutagenic strain was cultured and passaged. Before passage, catechol was sprayed on the strain. The genetically stable apramycin genetically engineered strain was obtained by screening based on the catechol colorimetric reaction.

2. The construction method according to claim 1, characterized in that, The construction process of the dual reporter gene expression vector is as follows: Using plasmid PSET152 as a template, a linearized vector backbone containing the promoterless catechol-2,3-dioxygenase gene and the thiostreptin resistance gene was constructed. The sequence of the promoter of *Heterodera indicum* was ligated to the linearized vector backbone to obtain the ligation product; The ligation product was transformed into competent E. coli cells and cultured. Single clones were selected and verified to obtain the dual reporter gene expression vector.

3. The construction method according to claim 2, characterized in that, The sequence of the Heterodermella Indus promoter is mixed with the linearized vector backbone in a molar ratio of 2:1 to 5:1 for ligation reaction.

4. The construction method according to claim 2, characterized in that, The ligation reaction includes ligation methods using homologous recombination, seamless cloning, or DNA ligase-mediated ligation.

5. The construction method according to claim 1, characterized in that, The host bacteria are selected from bacteria or yeast.

6. The construction method according to claim 1, characterized in that, The ultraviolet mutagenesis conditions are as follows: mutagenesis treatment is carried out under a 15W ultraviolet lamp for 20-40 seconds.

7. A dual reporter gene expression vector, characterized in that, include: The promoter sequence is shown in SEQ ID NO:1; A first reporter gene located downstream of the promoter, the sequence of which is shown in SEQ ID NO:2; A second reporter gene located downstream of the first reporter gene, the sequence of which is shown in SEQ ID NO:3; The promoter simultaneously drives the expression of the first reporter gene and the second reporter gene.

8. The application of the method of any one of claims 1-6 or the dual reporter gene expression vector of claim 7 in the evaluation and screening of bacteria that produce anti-mycoplasma active substances.

9. An engineered bacterium that produces high levels of anti-mycoplasma active substances, characterized in that, It comprises the dual reporter gene expression vector of claim 7 or is obtained by the construction method of any one of claims 1-6.

10. The application of the engineered bacteria as described in claim 9 in the preparation of anti-mycoplasma active substances, characterized in that, The anti-mycoplasma active substances include apramycin and talimimycin.

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