A recombinant plasmid for promoting tylosin synthesis, an engineered bacterial strain, its construction method, and its application.

Abstract

This invention belongs to the field of bioengineering technology, specifically relating to a recombinant plasmid for tylosin synthesis, an engineered bacterial strain, and its preparation method. The genetically engineered bacterial strain (Streptomyces freundii G3-F2) was deposited at the China Center for Type Culture Collection (CCTCC) on February 24, 2025, with accession number CCTCC NO: M 2025278. This invention achieves site-specific recombination, utilizes an optimized ZouA gene DNA amplification system to construct the recombinant plasmid, and realizes… tulle Multiple copies of the F gene were increased, resulting in the preparation of *Streptomyces freundii* G3-F2. This significantly improved the rate of methyltransferase enzymatic reaction and achieved efficient conversion of component C. Combined with low-temperature fermentation, the rate and efficiency of tylosin synthesis were greatly enhanced. The strain achieved a titer of 18500 U / mL in a 50L tank under 28℃ low-temperature culture conditions, which can significantly reduce production costs.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, and in particular relates to a recombinant plasmid for tylosin synthesis, an engineered bacterium, its construction method, and its application. Background Technology

[0002] Currently, the construction of high-yielding tylosin strains mainly focuses on modifying certain key genes in the tylosin biosynthesis gene cluster or adjusting key genes in the metabolic process of *Streptomyces freundii*. Methods to increase the overall metabolic flux of tylosin by increasing the copy number of the complete biosynthesis gene cluster have not yet been reported. However, the tylosin biosynthesis gene cluster is 87kb, making it difficult to obtain complete gene cluster fragments using traditional gene cloning techniques. Thanks to the rapid development of λRed-mediated PCR targeting technology and BAC library construction technology in recent years, technical support has been provided for constructing BACs with complete tylosin biosynthesis gene clusters. Through rational design and conjugation transfer, the copy number of the tylosin biosynthesis gene cluster in *Streptomyces freundii* can be increased, thereby conferring advantages such as increased tylosin metabolic flux, which is particularly important for improving tylosin yield.

[0003] The conversion of tylosin C fraction to fraction A is regulated by TylF methyltransferase. TylF methyltransferase is produced by the tylosin biosynthesis gene cluster of *Streptomyces freundii*. tyl The F gene encodes the rate-limiting enzyme in tylosin biosynthesis, which transfers the methyl group from S-adenosylmethionine (SAM) to the C3 hydroxyl group of tylosin C fraction, generating tylosin A. This is the rate-limiting step in tylosin synthesis. The TylF methyltransferase normally expressed in *Streptomyces freundii* exhibits its highest activity at 38°C. Therefore, in the later stages of industrial fermentation production of tylosin, the fermentation temperature needs to be increased from 30°C to 38°C to improve the conversion rate of tylosin C fraction to A fraction, a process that consumes a significant amount of energy. Furthermore, the accumulation of fraction D is highly temperature-dependent; increasing the temperature accelerates the formation of fraction D, and the content of fraction D restricts the quality and fermentation level of tylosin. Therefore, it is urgent to significantly increase the enzymatic reaction rate of TylF methyltransferase to overcome the rate-limiting effect of tylosin biosynthesis and the accumulation of fraction D caused by the low methylation modification reaction rate, enabling the conversion of tylosin C fraction to A fraction without increasing the temperature. This is crucial for the industrial production of tylosin.

[0004] Flow rate, rotational speed, and dissolved oxygen linkage control is a commonly used feedback control strategy. However, dissolved oxygen parameters are easily affected by environmental factors, sensor drift, or malfunction, leading to significant fluctuations and potential malfunctions, which are detrimental to the stability of the fermentation process. Planned control is currently the preferred method for large-scale production. Compared to linkage control, it is more reliable, stable, and convenient, facilitating small-scale trials and subsequent production scale-up. Tylosin fermentation levels are significantly influenced by strain characteristics and fermentation processes. Planned control methods developed according to microbial metabolic patterns can greatly stimulate the potential of the strain. Summary of the Invention

[0005] To address the technical problems existing in the background art, the present invention provides a recombinant plasmid for tylosin synthesis, an engineered bacterium, its construction method, and its application.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A recombinant plasmid for promoting tylosin synthesis includes a ZouA-JS3-F gene fragment, as shown in SEQ ID NO.1; preferably, the plasmid vector of the recombinant plasmid is pSet152; preferably, the recombinant plasmid is pSet152-ZouA-JS3-F recombinant plasmid.

[0008] The method for constructing the above recombinant plasmid includes the following steps:

[0009] 1. Using total DNA from kanamycin-streptomyces as a template, the 4.8 kb ZouA gene and recombination site RsA and the 1.4 kb recombination site RsB were amplified using primer pairs ZouA-XbaI-F and ZouA-BamHI-R, and primer pairs RsB-F and RsB-R, respectively.

[0010] The nucleotide sequences of the ZouA-XbaI-F and ZouA-BamHI-R primer pairs are shown in SEQ ID NO.2 and SEQ ID NO.3; the nucleotide sequences of the RsB-F and RsB-R primer pairs are shown in SEQ ID NO.4 and SEQ ID NO.5; the nucleotide sequences of the ZouA gene and the RsA gene are shown in SEQ ID NO.6; and the nucleotide sequence of the recombination site RsB is shown in SEQ ID NO.7.

[0011] Preferably, kanamycin-containing Streptomyces is... S. kanamyceticus ATCC 12853.

[0012] 2. Using total DNA from Streptomyces freundii as a template, a 1.2 kb PtylF-tylF gene sequence (including the PtylF promoter) was amplified using the tylF-F and tylF-R primer pairs.

[0013] The nucleotide sequences of the tylF-F and tylF-R primer pairs are shown in SEQ ID NO. 8 and SEQ ID NO. 9; the nucleotide sequence of the PtylF-tylF gene is shown in SEQ ID NO. 10; preferably, *Streptomyces freundii* is *Streptomyces freundii* SF-4 (… Streptomyces fradiae CCTCC NO:M2019791).

[0014] 3. The gene encoding the site-specific relaxant ZouA, the recombination site RsA, and the corresponding selection gene (JS3 resistance gene) were integrated upstream of the tylF gene, and the recombination site RsB was integrated downstream of the tylF gene. The ZouA gene, recombination site RsA, JS3 resistance gene, PtylF-tylF, and recombination site RsB gene were tandemly linked using a seamless cloning method and inserted into the pSET152 multiple cloning site region to construct the recombinant plasmid pSet152-ZouA-JS3-F.

[0015] The nucleotide sequence of the JS3 resistance gene is shown in SEQ ID NO.11;

[0016] The nucleotide sequence of the ZouA-JS3-F gene is shown in SEQ ID NO.1.

[0017] The present invention also provides a recombinant strain containing the above-mentioned recombinant plasmid;

[0018] Preferably, the recombinant strain is *Streptomyces freundii* G3-F2 ( Streptomyces fradiae The specimen (G3-F2) is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 2025278, on February 24, 2025, at Wuhan University, Wuhan, China, and is classified as Streptomyces fradiae G3-F2.

[0019] The preparation method of the above-mentioned Streptomyces freundii G3-F2 includes the following steps:

[0020] A. BAC containing the complete tylosin biosynthesis gene cluster was transformed into *E. coli*, and conjugated with *Streptomyces freundii*. Conjugates were screened on plates containing appropriate concentrations of JS1, JS5, and nalidixic acid. Molecular identification of the conjugates was performed using primer pairs JS5-BamHI-F and JS5-XbaI-R to obtain the genetically engineered strain. Yield was verified by shake-flask fermentation, and three rounds of strain purification were completed to obtain the genetically engineered strain G3 with the highest yield.

[0021] The nucleotide sequences of the JS5-BamHI-F and JS5-XbaI-R primer pairs are shown in SEQ ID NO.12 and SEQ ID NO.13.

[0022] B. The recombinant plasmid pSet152-ZouA-JS3-F was transformed into Escherichia coli and conjugated with the genetically engineered strain G3, which had the highest yield. Conjugates were screened on plates containing appropriate concentrations of JS1, JS3, and nalidixic acid. The selected conjugates were molecularly identified using the primer pair JS3-F (as shown in SEQ ID NO.14) and tyl FR (as shown in SEQ ID NO.9) to obtain the relevant strains.

[0023] C. Through resistance pressure gradient screening, the relationship between resistance concentration and tylF expression level (C / A transformation capacity) was established. The resistance concentration of the screened strains was gradually increased, and strains under different resistance pressures were selected for shake-flask fermentation verification. The optimal resistance pressure corresponding to the highest yield and best transformation capacity was selected. Under the optimal resistance pressure, three rounds of strain isolation and purification were completed to obtain the optimal genetically engineered strain G3-F2.

[0024] Preferably, the Escherichia coli in steps A and B is S17-1; and the Streptomyces freundii in step A is SF-4.

[0025] The present invention also provides the above-mentioned recombinant plasmid and the above-mentioned recombinant strain G3-F2 in promoting tylosin synthesis.

[0026] This invention also provides a low-temperature culture and fermentation method for Streptomyces freundii G3-F2, comprising the following steps:

[0027] (1) Primary seed culture: The spore suspension of the fungal slant was inoculated into the primary seed culture medium and cultured at 28℃ for 48h to obtain the primary seed liquid;

[0028] (2) Secondary seed culture: The primary seed culture obtained in step (1) was inoculated into the secondary seed culture medium at an inoculation rate of 10%, and cultured at 28°C for 42 h to obtain the secondary seed culture;

[0029] (3) Fermentation culture: The secondary seed liquid obtained in step (2) is inoculated into the fermentation medium at an inoculation amount of 25%, and fermented at low temperature for 168 hours before stopping the fermentation and putting it into a tank.

[0030] The primary seed culture medium consists of the following components by weight percentage: 0.6-0.8% soybean meal, 0.4-0.8% yeast extract, 0.4-0.8% corn steep liquor, 0.2-0.4% corn flour, 1.0-1.5% soybean oil, 0.1-1.0.3% calcium carbonate, and the remainder is water.

[0031] The composition of the secondary seed culture medium is as follows (by weight percentage): fish meal 0.8-1.2%, peanut meal 1.2-1.6%, corn gluten meal 1.2-1.6%, yeast extract 0.2-0.5%, corn steep liquor 0.2-0.5%, corn flour 1.0-1.4%, diammonium hydrogen phosphate 0.01-0.06%, soybean oil 1.0-1.5%, calcium carbonate 0.1-0.3%, with the remainder being water;

[0032] The fermentation medium consists of the following components by weight percentage: fish meal 0.2-0.5%, peanut meal 0.2-0.4%, corn gluten meal 0.6-0.8%, soybean meal 0.3-0.5%, cottonseed meal 0.2-0.4%, corn flour 1.2-1.8%, corn steep liquor 0.3-0.6%, betaine hydrochloride 0.05-0.09%, potassium chloride 0.1-0.2%, diammonium hydrogen phosphate 0.01-0.06%, cobalt chloride 0.001-0.002%, nickel sulfate 0.001-0.002%, soybean oil 1.0-1.5%, calcium carbonate 0.2-0.3%, with the remainder being water.

[0033] The controlled flow rate during fermentation is as follows: 40 L / min for 0-8 h, 45 L / min for 8-10 h, 50 L / min for 10-12 h, 55 L / min for 12-14 h, 60 L / min for 14-16 h, and 65 L / min for 16-168 h. Rotation speed: 200 rpm for 0-2h, 225 rpm for 2-4h, 250 rpm for 4-6h, 275 rpm for 6-8h, 300 rpm for 8-10h, 325 rpm for 10-12h, 350 rpm for 12-14h, 375 rpm for 14-16h, 400 rpm for 16-18h, 425 rpm for 18-20h, 450 rpm for 20-22h, 475 rpm for 22-24h, 500 rpm for 24-26h, 525 rpm for 26-28h, 550 rpm for 28-30h, 575 rpm for 30-72h, and 550 rpm for 72-168h.

[0034] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0035] 1. An improved ZouA system was developed, and the optimal location for integrating the JS3 resistance gene was discovered for the first time. The pSet152-ZouA-JS3-F plasmid was constructed and combined with recombinant BAC containing the JS5 resistance gene. Transformation and screening yielded a series of engineered *Streptomyces freundii* strains. Increased copy number of the tylosin biosynthesis gene cluster significantly improved metabolic flux and resulted in a higher TylF methyltransferase enzymatic reaction rate. The highest titer achieved in shake-flask fermentation of this series of engineered strains reached 16651 U / mL. A resistance pressure gradient screening method was used to establish the relationship between resistance concentration and tylF expression level (C / A transformation capacity). The resistance concentration of the screened strains was gradually increased to select the optimal resistance pressure corresponding to the highest yield and best transformation capacity. The resulting engineered *Streptomyces freundii* strain G3-F2 (… Streptomyces fradiae The G3-F2 strain achieved a titer of 17403 U / mL during shake-flask fermentation, which is higher than that of common Streptomyces freundii and 18.88% higher than the original strain of this series of engineered bacteria. Furthermore, complete conversion of tylosin C was achieved throughout the shake-flask fermentation process at 28°C.

[0036] 2. Compared with the original strain, this strain does not accumulate C component during the 50L tank fermentation process, and does not require heating to 38℃ for conversion, which can reduce steam consumption.

[0037] 3. By following the laws of microbial metabolism and adopting a planned control method, the fermentation process is more stable, greatly stimulating the potential of the strain and improving the fermentation potency.

[0038] 4. Low-temperature fermentation represents a significant innovation compared to traditional fermentation processes. Under low-temperature conditions, *Streptomyces freundii* synthesizes tylosin at a faster rate and for a longer duration.

[0039] Combining the above points can achieve a fermentation titer of 18500 u / mL and greatly reduce production costs. Attached Figure Description

[0040] Figure 1 HPLC chromatogram of the original strain (strain 1) obtained in Example 2 during shake-flask fermentation;

[0041] Figure 2 HPLC chromatogram of the genetically engineered strain G3-F2 obtained in Example 3 during shake-flask fermentation;

[0042] Figure 3 HPLC chromatogram of the genetically engineered strain G3-JF obtained in Example 4 during shake-flask fermentation. Detailed Implementation

[0043] The applicant will now provide a clear and complete description of the technical solution of the present invention with reference to specific embodiments. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention.

[0044] Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention; unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; unless otherwise specified, the materials, reagents, etc. used can be obtained commercially.

[0045] The fermentation strain used in the following examples is *Streptomyces freundii* (…). Streptomyces fradiae G3-F2), deposited at the China Center for Type Culture Collection, accession number CCTCC NO: M 2025278.

[0046] Escherichia coli S17-1 competent cell suspension: purchased from Wuhan Miaoling Biotechnology Co., Ltd.;

[0047] JS1: Streptomycin sulfate salt; purchased from Aladdin, product number S432672;

[0048] JS3: Hygromycin B; purchased from Aladdin, part number H398402;

[0049] JS5: Apramycin sulfate salt; purchased from Aladdin, product number A106707;

[0050] Naphthylpyridinic acid: purchased from Aladdin.

[0051] Example 1: Construction of pSet152-ZouA-JS3-F plasmid

[0052] 1.1. Following the linking order of the ZouA gene, recombination site RsA, JS3 resistance gene, PtylF-tylF gene, and recombination site RsB, primer pairs ZouA-XbaI-F and ZouA-BamHI-R, covering the homologous arm downstream of pSet152 and upstream of the JS3 resistance gene, were designed to amplify the ZouA-RsA gene fragment; primer pairs JS3-F and JS3-R, covering the homologous arm downstream of ZouA-RsA and upstream of the PtylF-tylF gene, were designed to amplify the JS3 resistance gene; primer pairs tyl FF and tyl FR, covering the homologous arm downstream of the JS3 resistance gene and upstream of the RsB gene, were designed to amplify the PtylF-tylF gene; and primer pairs RsB-F and RsB-R, covering the homologous arm downstream of the PtylF-tylF gene and upstream of pSet152, were designed to amplify the RsB gene.

[0053] The nucleotide sequences of the ZouA-XbaI-F and ZouA-BamHI-R primer pairs, and the RsB-F and RsB-R primer pairs are shown in SEQ ID NO.2-SEQ ID NO.5;

[0054] The nucleotide sequence of the binding fragment of the ZouA gene and the RsA gene is shown in SEQ ID NO. 6;

[0055] The nucleotide sequence of the RsB gene fragment at the recombination site is shown in SEQ ID NO.7.

[0056] 1.2. Kanamycin-Streptomyces ( S. kanamyceticus ATCC 12853) and Streptomyces freundii SF-4 ( Streptomyces fradiae CCTCC NO:M2019791; A Streptomyces freundii strain and its application in tylosin fermentation (CN111139197A) was inoculated into TSB medium and cultured for 3 days, and the genome was extracted. Using the genome of Streptomyces kanamycin as a template, a 4.8 kb gene encoding the site-specific relaxant ZouA and the recombination site RsA ZouA-RsA gene fragment was amplified using ZouA-XbaI-F and ZouA-BamHI-R primers; a 1.4 kb RsB gene fragment was amplified using RsB-F and RsB-R primers. Using the genome of Streptomyces freundii SF-4 as a template, a 1.2 kb PtylF-tylF gene fragment (containing the PtylF promoter) was amplified using tyl FF and tyl FR primers.

[0057] The nucleotide sequences of the tylF-F and tylF-R primer pairs are shown in SEQ ID NO.8 and SEQ ID NO.9; the nucleotide sequence of the PtylF-tylF gene is shown in SEQ ID NO.10; the PCR amplification experiment used Vazyme 2XPhanta UniFi Master Mix (Dye Plus) reagent, and the reaction system and reaction procedure are shown in Tables 1 and 2.

[0058] Table 1 PCR reaction system

[0059] .

[0060] Table 2 PCR reaction procedures

[0061] .

[0062] 1.3 The ZouA gene encoding the site-specific relaxant, the RsA gene at the recombination site, and the JS3 resistance gene were integrated upstream of the tylF gene, and the RsB gene at the recombination site was integrated downstream of the tylF gene. The genes were tandemly linked in the order of the ZouA gene, the RsA gene at the recombination site, the JS3 resistance gene, the PtylF-tylF gene, and the RsB gene at the recombination site to obtain the recombinant gene fragment ZouA-JS3-F (shown in SEQ ID NO.1). The site-specific recombinant plasmid pSet152 (purchased from Addgene) was selected as the vector. The above genes and the linearized pSET152 vector were used in the experiments according to the Ready-to-Use Seamless Cloning Kit instructions.

[0063] Seamless cloning experiments were performed using the BBI Ready-to-Use Seamless Cloning Kit. The experimental system is shown in Table 3.

[0064] Table 3 Seamless Cloning Experimental System

[0065] .

[0066] Reaction steps:

[0067] 1. After reacting the above system at 50℃ for 20 min, place it on ice.

[0068] 2. Transfer the reaction product to 100 μL of Escherichia coli S17-1 competent cell suspension and incubate on ice for 30 min.

[0069] 3. Heat shock at 42℃ for 60 s, add 700 uL LB liquid medium, and incubate at 37℃ and 210 rpm for 25 min.

[0070] 4. Spread the plasmid on the corresponding resistant LB plate and incubate overnight at 37°C to obtain the recombinant plasmid.

[0071] The nucleotide sequence of the JS3 resistance gene is shown in SEQ ID NO.11:

[0072] The nucleotide sequence of the ZouA-JS3-F gene is shown in SEQ ID NO.1.

[0073] 1.4 The ligated gene was introduced into E. coli S17-1 competent cells via chemical transformation.

[0074] Chemical transformation of Escherichia coli S17-1: Take 100 μL of competent Escherichia coli S17-1 cell suspension from a -80℃ freezer and thaw it on ice; add 20 ng of the above recombinant plasmid, gently shake and place on ice for 20 minutes; heat shock in a 42℃ water bath for 90 seconds, then quickly place on ice to cool for 3-5 minutes; add 1 mL of LB liquid medium to the tube and mix well; culture at 37℃ and 220 rpm for 40 minutes; centrifuge at 5000 rpm for 5 minutes, remove the supernatant, mix well, and take 100 μL to spread on an LB selection plate containing JS3 and incubate at 37℃ for 16-20 h.

[0075] Colony identification was performed using primers JS3-F (as shown in SEQ ID NO.14) and tyl FR (as shown in SEQ ID NO.9), following the same PCR amplification procedure as described in section 1.2. Positive clones were identified as the target recombinant plasmid, named pSET152-ZouA-JS3-F.

[0076] Example 2 Construction and screening of G3 strain

[0077] 2.1. Following the method described in the literature (Huang Sheng, Li Na, Zhou Jun, et al. Construction and application of bacterial artificial chromosome (BAC) vectors suitable for cloning and heterologous expression of large fragment genomic DNA of Streptomyces [J]. Acta Microbiologica Sinica, 2012, 52(01):30-37. DOI:10.13343 / j.cnki.wsxb.2012.01.012.), the plasmid pMSBBAC1 described in the literature was constructed according to method 1.2 (cloning of pMSBBACs). The plasmid pMSBBAC1 already carries the apramycin (JS5) resistance gene. Following the same steps and conditions as in the library construction method in 1.6 of the literature, Streptomyces freundii SF-4 was embedded in low-melting agarose to form embedded blocks. After digestion, the DNA in the embedded blocks was digested with BamHI restriction enzyme. Partial enzyme digestion was performed, and the target fragment was recovered by pulsed-field gel electrophoresis (PFGE). The fragment was ligated into the BamHI-digested plasmid pMSBBAC1 and electroporated into highly competent *E. coli* cells (using DH10B-T1 cells as described in reference 1.1) to obtain a mixed transformant; this constituted the *Streptomyces freundii* genomic library (genomic sequence referenced from GenBank NZ_CP023696.1; https: / / www.ncbi.nlm.nih.gov / nuccore / NZ_CP023696.1 / ). Plasmids were extracted by shaking, and the insert size and empty vector ratio were detected by I-SceI digestion (purchased from New England Biolabs). Three primer pairs were designed based on the two ends and the middle of the tylosin biosynthesis gene cluster, named tyl1-F / tyl1-R (as shown in SEQ ID NO.17 and SEQ ID NO.18), tyl2-F / tyl2-R (as shown in SEQ ID NO.19 and SEQ ID NO.20), and tyl3-F / tyl3-R (as shown in SEQ ID NO.21 and SEQ ID NO.22). Multiple rounds of PCR screening were performed on the mixed library using these three primer pairs. Clones that were positive after cross-validation were identified as BACs containing the complete tylosin biosynthesis gene cluster (its gene sequence is from position 6521558 to position 6607759 of the GenBank NZ_CP023696.1 gene sequence mentioned above), and named pBacTL01.

[0078] pBacTL01 was electroporated into a suspension of competent E. coli S17-1 cells. After activation for 1 h, the cells were plated onto LB plates containing 25 μg / mL JS5 and cultured for 16-20 h. Single colonies were selected from the plates and colony PCR was performed using JS5-BamHI-F (as shown in SEQ ID NO.12) and JS5-XbaI-R (as shown in SEQ ID NO.13). The experiment was conducted according to the PCR steps and system in step 1.2 of Example 1. Positive clones were the transformants.

[0079] The specific electroporation method described above is as follows: 2 μL of plasmid pBacTL01 was mixed thoroughly with 100 μL of *E. coli* S17-1 competent cell suspension, and the mixture was placed in a pre-cooled 2 mm electroporation cuvette and electroporated at 2.5 kV, 200 Ω, and 25 μF. Immediately after electroporation, 1 mL of pre-cooled LB agar was added, and the mixture was incubated at 30°C on a shaker for 40 min. 100 μL of the bacterial culture was then spread onto an LB agar plate containing 25 μg / mL JS5 and incubated at 30°C for 16-20 h.

[0080] 2.2 The transformants were expanded and cultured, and then mixed with the heat-shocked spores of the production strain (Streptomyces freundii SF-4). The plasmid pBacTL01 was introduced into the production strain (Streptomyces freundii SF-4) using the conjugation transfer method. The mixed bacterial culture was then spread on ISP-4 medium plates containing 25 μg / mL JS1, 12 μg / mL JS5 and 20 μg / mL nalidixinol and cultured for 6-7 days.

[0081] 10X TES buffer: 100 mM Tris-HCl (pH 7.5), 10 mM EDTA (pH 7.5) and 1% (m / v) SDS, filtered sterilized.

[0082] 2X spore germination medium: 10 g / L casein amino acids, 10 g / L yeast powder, sterilized at 121℃ for 20 min, add 20 mM calcium chloride to the final concentration before use.

[0083] LB medium: 10 g / L tryptone, 5 g / L yeast extract and 10 g / L sodium chloride, sterilized at 115℃ for 20 min.

[0084] ISP4 medium: 10 g / L soluble starch, 1 g / L dipotassium hydrogen phosphate, 1 g / L sodium chloride, 2 g / L ammonium sulfate, 2 g / L calcium carbonate, 0.5 g / L yeast extract, 1 g / L tryptone, 20 g / L agar, 1 mL / L inorganic salt solution (1 g / L ferrous sulfate, 1 g / L manganese chloride, 1 g / L zinc sulfate).

[0085] FLCB medium: 22 g / L corn starch, 2 g / L fish peptone, 0.6 g / L dipotassium hydrogen phosphate, 1 g / L potassium nitrate, 0.5 g / L sodium chloride, 0.01 g / L ferrous sulfate, 0.4 g / L magnesium sulfate, 26 g / L agar strips.

[0086] The operating steps are shown in Table 4.

[0087] Table 4. Joining and Transfer Procedures

[0088] .

[0089] 2.3 Select conjugates from the conjugation transfer plate and isolate and purify them in FLCB plates containing 50 μg / mL JS1 and 25 μg / mL JS5 to remove E. coli. Culture the purified conjugates in liquid culture medium for 3 days, extract the genome, and verify them by PCR using JS5-BamHI-F and JS5-XbaI-R primer pairs (as shown in SEQ ID NO.12 and SEQ ID NO.13). Name the selected biosynthetic gene cluster two-copy genetically engineered strain TL-G.

[0090] 2.4. Use the following culture medium and culture conditions:

[0091] Shake-flask seed culture medium: 0.56% mesophilic soybean meal, 0.5% yeast extract, 0.3% corn steep liquor, 0.3% CaCO3, 0.6% soybean oil. Shake-flask fermentation culture medium: 1.6% corn flour, 1.1% corn gluten meal, 0.22% cottonseed meal, 0.75% fish meal, 0.4% peanut meal, 0.4% high-temperature soybean meal, 0.09% betaine hydrochloride, 0.1% KCl, 0.17% 0.33% cobalt chloride solution, 0.1% 0.44% nickel sulfate solution, 0.01% (NH4)2HPO4, 0.03% MgSO4, 0.23% CaCO3, 6.2% soybean oil. Culture conditions: Seed shake-flask: 28℃, 250 rpm, 44-48 h; Fermentation shake-flask: 28℃, 250 rpm, 144 h.

[0092] 2.5. The obtained TL-G strain was subjected to initial screening via shake-flask fermentation. Based on the initial screening results, glycerol tubes corresponding to relatively high-yielding strains were selected for plating and single colony picking. After the plates had grown well, they were inoculated into the seed bottle and fermentation bottle shown in step 2.4, and the fermentation level was detected using HPLC. Based on the secondary screening results, relatively high-yielding strains were selected and the above screening steps were repeated. After three rounds of isolation, purification, and screening, strain No. 1 served as the original TL strain control. Detailed results are shown in Table 5.

[0093] Table 5. Titer of TL-G strain after third round of purification and shake-flask fermentation .

[0094] Finally, strain No. 3, which had the highest purification titer in the third round of TL-G purification, was selected and named G3. After the gene copy number was increased, the titer of shake-flask fermentation reached 16651 U / mL, which was 15.29% higher than that of the original strain (strain No. 1).

[0095] Example 3 Construction and screening of G3-F2 strain

[0096] 3.1 The recombinant plasmid pSET152-ZouA-JS3-F obtained in Example 1 was introduced into Escherichia coli S17-1 competent cells by chemical transformation. After activation for 1 h, it was plated on LB plates with 25 μg / mL JS3 and cultured for 16-20 h. Single clones in the plates were selected, and colony PCR identification was performed using the primer pair JS3-F (as shown in SEQ ID NO.14) and tylF-R (as shown in SEQ ID NO.9) according to the operation and system in step 1.3. Positive clones were the transformants. The transformants were expanded and cultured, mixed with the heat-shocked G3 spore solution, and the recombinant plasmid was introduced into G3 cells by conjugation transfer. The mixed bacterial solution was plated on ISP-4 medium plates containing 25 μg / mL JS3, 12.5 μg / mL JS5 and 20 μg / mL naphthylpyridinium acid and cultured for 6-7 days.

[0097] 3.2 Select conjugates and isolate and purify them in FLCB plates containing 50 μg / mL JS1, 25 μg / mL JS3, and 12 μg / mL JS5 to remove E. coli. Culture the purified conjugates in liquid culture medium for 3 days, extract the genome, and perform PCR verification using the primer pairs JS3-F (as shown in SEQ ID NO.14) and tylF-R (as shown in SEQ ID NO.9) according to the operation and system in step 1.3.

[0098] 3.3 Using the ZouA-mediated DNA amplification system, the correlation between antibiotic resistance concentration and tylF expression level (C / A transformation capacity) was established to create a resistance pressure gradient screening model. Molecularly validated conjugates were inoculated into seed flasks at 250 rpm. The seed culture was inoculated into new seed flasks every 24-28 hours, simultaneously inoculated into fermentation flasks. During each subculture, the antibiotic resistance concentration in the seed flasks was gradually increased to establish the correlation between antibiotic resistance concentration and C / A transformation capacity. The correlation between antibiotic resistance concentration and C / A transformation capacity is shown in Table 6.

[0099] Table 6. G3-F Resistance Stress Gradient Screening

[0100] .

[0101] As the resistance concentration gradually increased, the C / A conversion ability also gradually improved, and complete conversion of the C component could be basically achieved at 50 μg / mL; however, the results showed that increasing the resistance concentration would put greater growth pressure on the strain, leading to a decrease in the total titer. Finally, the optimal resistance concentration of 50 μg / mL, which corresponds to the highest total titer and the best conversion ability, was selected.

[0102] 3.4. The optimal resistance concentration was used for subsequent isolation and purification of the strains. Three rounds of isolation, purification, and screening were performed according to the primary and secondary screening methods shown in step 2.5 of Example 2. Strain No. 1 served as the original TL strain control. The high-performance liquid chromatography (HPLC) results of the original strain can be found in [reference needed]. Figure 1 ;like Figure 1 As shown, the potency of tylosin A component was 9631 U / mL, accounting for 55.61%, and the potency of tylosin C component was 5008 U / mL, accounting for 28.92%. The potency of shake-flask fermentation is shown in Table 7.

[0103] Table 7. Titer of G3-F strain after third round of purification and shake-flask fermentation.

[0104] .

[0105] Finally, strain number 2, purified in the third round of G3-F purification, was selected and named G3-F2. For high-performance liquid chromatography (HPLC) detection, please refer to [link to relevant documentation]. Figure 2 This modification involved tandem multi-copy amplification of the resistance gene and the TylF gene based on the G3 two-copy strain, achieving a titer of 17403 U / mL in shake-flask fermentation. Figure 2 As shown, the potency of tylosin A component was 17348 U / mL (91.28%), and the potency of tylosin C component was 55 U / mL (0.29%). Compared with strain G3 obtained in Example 2, the total potency was increased by 4.52%, and compared with the original strain (strain 1) by 18.88%; and complete conversion of tylosin C component was achieved during the entire shake-flask fermentation process at 28°C.

[0106] Example 4: Comparative Experiment: Construction and Screening of G3-JF Strains by Changing the Gene Sequences of JS3 and tylF

[0107] First, the pSet152-ZouA-F-JS3 plasmid was constructed.

[0108] 4.1 The gene fragment acquisition method is the same as in Example 1, and the ZouA gene encoding site-specific relaxant, the recombination site RsA gene, the recombination site RsB and PtylF-tylF gene are obtained respectively.

[0109] 4.2 The ZouA gene and the recombination site RsA gene were integrated upstream of the tylF gene, and the JS3 resistance gene and the recombination site RsB gene were integrated downstream of the tylF gene. The genes were tandemly linked in the order of ZouA gene, recombination site RsA, PtylF-tylF, JS3 resistance gene, and recombination site RsB to obtain the recombinant gene fragment ZouA-F-JS3. The above recombinant gene fragment ZouA-F-JS3 (as shown in SEQ ID NO.16) and the linearized pSET152 vector were used for experimental operations according to the Ready-to-Use Seamless Cloning Kit instructions.

[0110] 4.3 The ligated gene was introduced into E. coli DH5α competent cells by chemical transformation. Positive clones identified by PCR were identified as recombinant plasmids and named pSET152-ZouA-F-JS3.

[0111] 4.4 The recombinant plasmid pSET152-ZouA-F-JS3 was transformed by chemical method, and the specific operation steps were the same as step 3.1 in Example 3. PCR identification was performed using primer pairs tylF-F (as shown in SEQ ID NO.8) and JS3-R (as shown in SEQ ID NO.15). Suitable and correct transformants were selected and conjugated with G3 strains for transfer. After mixing, the bacterial culture was spread on ISP-4 medium plates containing JS3, JS5 and naphthylpyridinium acid and cultured for 6-7 days.

[0112] 4.5. Following the same steps as in Example 3, step 3.3, find the correspondence between resistance concentration and tylF expression level (C / A conversion ability) and establish a resistance pressure gradient screening model; the correspondence between resistance concentration and C / A conversion ability is shown in Table 8.

[0113] Table 8. Screening for G3-JF resistance to stress gradients

[0114] .

[0115] As the resistance concentration gradually increased, the C / A conversion ability also gradually improved, but complete conversion of the C component could not be achieved; at the same time, the increase in resistance concentration led to a significant decrease in the total titer of the strain. Finally, considering both the C / A conversion rate and the total titer, the optimal resistance concentration was selected as 50 μg / mL.

[0116] 4.6. Following steps 2, 3, and 4 of Example 3, the conjugates were purified and screened, completing three rounds of separation, purification, and screening. The final shake-flask fermentation titers are shown in Table 9, where strain 1 served as the original G3-JF strain control. High-performance liquid chromatography (HPLC) detection is described in [reference needed]. Figure 3 .

[0117] Table 9. Titer of G3-JF strain after third round of purification and shake-flask fermentation. .

[0118] As shown in Table 9, strain G3-JF7, with the highest potency, achieved a potency of 16846 U / mL during shake-flask fermentation. The potency of tylosin A component was 12920 U / mL (71.08%), and the potency of tylosin C component was 3926 U / mL (21.79%). Compared to strain G3 obtained in Example 2, the total potency was not significantly improved. Compared to strain G3-F2 obtained in Example 3, the final fermentation product still contained a significant amount of C component, indicating that complete transformation of C component could not be achieved during fermentation. Only the order of the JS3 resistance gene and the tylF gene was altered, demonstrating that the linking order of the JS3 and tylF genes is crucial for the expression of tylF methyltransferase in *Streptomyces freundii*.

[0119] Example 5: G3-F2 strain + low-temperature fermentation process

[0120] 5.1 Primary Seed Culture:

[0121] The primary seed culture medium consisted of: 0.6% soybean meal, 0.4% yeast extract, 0.8% corn steep liquor, 0.2% corn flour, 1.5% soybean oil, 0.3% calcium carbonate, and the remainder being water, by weight percentage. It was sterilized at 121℃ for 30 minutes and set aside. One *Streptomyces freundii* (…) Streptomyces fradiae The G3-F2 strain slant was used to prepare a spore suspension, which was then inoculated into sterilized primary seed culture medium at an inoculation rate of 0.1%. The final spore concentration in the primary seed culture medium was 10. 7 The culture was carried out at a concentration of 10 cells / mL, a culture temperature of 28℃, a dissolved oxygen content of 30%, and a tank pressure of 0.05MPa for 48 hours to obtain the first-grade seed culture.

[0122] 5.2 Secondary seed culture:

[0123] The secondary seed culture medium consisted of: 0.8% fish meal, 1.2% peanut meal, 1.6% corn gluten meal, 0.2% yeast extract, 0.5% corn steep liquor, 1.0% corn flour, 0.04% diammonium hydrogen phosphate, 1.5% soybean oil, 0.3% calcium carbonate, and the remainder being water, by weight percentage. It was sterilized at 121℃ for 30 minutes and set aside. The primary seed culture obtained in step 1 was inoculated at a rate of 10% into the sterilized secondary seed culture medium. The medium was incubated at 28℃, dissolved oxygen 30%, and pressure 0.05 MPa for 40 hours to obtain the secondary seed culture.

[0124] 5.3 Fermentation Culture:

[0125] The fermentation medium consisted of: 0.5% fish meal, 0.2% peanut meal, 0.6% corn gluten meal, 0.3% soybean meal, 0.4% cottonseed meal, 1.8% corn flour, 0.6% corn steep liquor, 0.09% betaine hydrochloride, 0.1% potassium chloride, 0.04% diammonium hydrogen phosphate, 0.001% cobalt chloride, 0.001% nickel sulfate, 1.5% soybean oil, 0.3% calcium carbonate, and the remainder being water, by weight percentage. The medium was sterilized at 121℃ for 30 minutes and set aside. The secondary seed culture obtained in step 2 was inoculated into the sterilized fermentation medium at a 20% inoculum rate. The pressure in the container was 0.05 MPa, and the culture temperature was 28℃. The culture was allowed to mature for approximately 168 hours before being transferred to the container.

[0126] Once fermentation begins, the feeding should be set up as follows:

[0127] When the fermentation cycle reaches 24 hours, add 10 wt% ammonium sulfate aqueous solution at a rate of 5 L / h, and at the same time add 10 wt% sodium hydroxide aqueous solution to control the pH not lower than 6.70; when the fermentation cycle reaches 24 hours, start adding water to maintain the fermentation tank volume at about 40 L; when the fermentation cycle is 36-168 hours, add soybean oil.

[0128] After fermentation begins, the flow rate and rotation speed parameters should be set as follows:

[0129] Flow rate: 40 L / min for 0-8 h, 45 L / min for 8-10 h, 50 L / min for 10-12 h, 55 L / min for 12-14 h, 60 L / min for 14-16 h, and 65 L / min for 16-168 h.

[0130] Rotation speed: 200 rpm for 0-2h, 225 rpm for 2-4h, 250 rpm for 4-6h, 275 rpm for 6-8h, 300 rpm for 8-10h, 325 rpm for 10-12h, 350 rpm for 12-14h, 375 rpm for 14-16h, 400 rpm for 16-18h, 425 rpm for 18-20h, 450 rpm for 20-22h, 475 rpm for 22-24h, 500 rpm for 24-26h, 525 rpm for 26-28h, 550 rpm for 28-30h, 575 rpm for 30-72h, and 550 rpm for 72-168h.

[0131] The final potency reached 18513 U / mL after a 168-hour incubation period.

[0132] Example 6 Comparative Experiment: Original strain constructed in Example 1 + Traditional control process

[0133] 6.1 Primary Seed Culture:

[0134] The primary seed culture medium consisted of: 0.6% soybean meal, 0.4% yeast extract, 0.8% corn steep liquor, 0.2% corn flour, 1.5% soybean oil, 0.3% calcium carbonate, and the remainder being water, by weight percentage. It was sterilized at 121°C for 30 minutes and set aside. A spore suspension prepared from an agar slant of one original strain (strain No. 1 obtained in Example 1) was inoculated into the sterilized primary seed culture medium at an inoculation rate of 0.1%, resulting in a final spore concentration of 10-1 in the primary seed culture medium. 7 The culture was carried out at a concentration of 10 cells / mL, a culture temperature of 30℃, dissolved oxygen of 30%, and a tank pressure of 0.05MPa for 48 hours to obtain the first-grade seed culture.

[0135] 6.2 Secondary seed culture:

[0136] The secondary seed culture medium consisted of: 0.8% fish meal, 1.2% peanut meal, 1.6% corn gluten meal, 0.2% yeast extract, 0.5% corn steep liquor, 1.0% corn flour, 0.04% diammonium hydrogen phosphate, 1.5% soybean oil, 0.3% calcium carbonate, and the remainder being water, by weight percentage. It was sterilized at 121℃ for 30 minutes and set aside. The primary seed culture obtained in step 1 was inoculated at a rate of 10% into the sterilized secondary seed culture medium. The medium was incubated at 30℃, with dissolved oxygen at 30%, and a pressure of 0.05 MPa for 40 hours to obtain the secondary seed culture.

[0137] 6.3 Fermentation Culture:

[0138] The fermentation medium consisted of: 0.5% fish meal, 0.2% peanut meal, 0.6% corn gluten meal, 0.3% soybean meal, 0.4% cottonseed meal, 1.8% corn flour, 0.6% corn steep liquor, 0.09% betaine hydrochloride, 0.1% potassium chloride, 0.04% diammonium hydrogen phosphate, 0.001% cobalt chloride, 0.001% nickel sulfate, 1.5% soybean oil, 0.3% calcium carbonate, and the remainder being water, by weight percentage. The medium was sterilized at 121℃ for 30 minutes and set aside. The secondary seed culture obtained in step 2 was inoculated into the sterilized fermentation medium at a 20% inoculum rate. The tank pressure was 0.05 MPa, the incubation temperature was 30℃, and the dissolved oxygen was 25% (flow rate and speed were linked to dissolved oxygen). After 150 hours of incubation, the temperature was increased to 38℃ for conversion. The medium was then removed from the tank after approximately 168 hours.

[0139] After fermentation begins, the feeding is set as follows: when the fermentation cycle reaches 24 hours, add 10wt% ammonium sulfate aqueous solution at a rate of 5L / h, and at the same time add 10wt% sodium hydroxide aqueous solution to control the pH not lower than 6.70; when the fermentation cycle reaches 24 hours, start adding water to maintain the fermentation tank volume at about 40L; when the fermentation cycle is 36-168 hours, add soybean oil.

[0140] The final infusion cycle was 168 hours, and the potency reached 14245 U / mL.

[0141] Those skilled in the art will readily understand that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements 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 recombinant plasmid for promoting tylosin synthesis, characterized in that, The recombinant plasmid includes a ZouA-JS3-F gene fragment, as shown in SEQ ID NO.1; the method for constructing the recombinant plasmid includes the following steps: (1) Using the total DNA of kanamycin streptomyces as a template, the coding site specific relaxase ZouA gene and recombination sites RsA and RsB were amplified by using the primer pairs ZouA-XbaI-F and ZouA-BamHI-R, and the primer pairs RsB-F and RsB-R, respectively. (2) Using the total DNA of Streptomyces freundii SF-4 as a template, the PtylF-tylF gene sequence was amplified using the tylF-F and tylF-R primer pairs; (3) The site-specific relaxant ZouA gene, the recombination site RsA and the JS3 resistance gene were integrated into the upstream of the tylF gene, and the recombination site RsB was integrated into the downstream of the tylF gene. The ZouA gene, the recombination site RsA, the JS3 resistance gene, the PtylF-tylF gene and the recombination site RsB gene were tandemly linked using the seamless cloning method and inserted into the pSET152 multiple cloning site region to construct the recombinant plasmid pSet152-ZouA-JS3-F; The nucleotide sequences of the ZouA-XbaI-F and ZouA-BamHI-R primer pairs in step (1) are shown in SEQ ID NO.2 and SEQ ID NO.3; the nucleotide sequences of the RsB-F and RsB-R primer pairs are shown in SEQ ID NO.4 and SEQ ID NO.5; the nucleotide sequences of the tylF-F and tylF-R primer pairs in step (2) are shown in SEQ ID NO.8 and SEQ ID NO.9; the JS3 resistance gene is the hygromycin B resistance gene.

2. A recombinant bacterial strain G3-F2, characterized in that: The strain was *Streptomyces freundii* G3-F2 ( Streptomyces fradiae G3-F2), deposited at the China Center for Type Culture Collection, accession number CCTCC NO: M 2025278.

3. The application of the recombinant plasmid of claim 1 and the recombinant strain G3-F2 of claim 2 in the synthesis of tylosin.

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