Engineered bacteria producing terramycin, and construction method and application thereof

Abstract

The application provides an engineered oxytetracycline-producing bacterium and a construction method and application thereof. The application takes Streptomyces albus Del14 as a chassis production strain, and successfully expresses an otc cluster in S.albus Del14 for the first time through heterologous reconstitution. Specifically, the otc cluster is expressed by increasing one copy of otcR started by a strong promoter PkasO*; the efflux of OTC is improved by knocking out a negative regulatory factor OtrR in the cluster to release the repression on otrB; further, an efficient OTC production chassis Del14B::oxy1KΔotrR is screened out by replacing an integrase site, and the OTC yield of the chassis Del14B::oxy1KΔotrR reaches 1.1 g / L on the 6th day, which is equivalent to the yield of the original production strain S.rimosus M4018 on the 8th day, so that the heterologous efficient synthesis of OTC is realized.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and more specifically, to an engineered bacterium that produces oxytetracycline, its construction method, and its application. Background Technology

[0002] Oxytetracycline (OTC) is an aromatic polyketide compound belonging to the first generation of naturally occurring tetracycline antibiotics. *Streptomyces rimosus* is the original strain that synthesized OTC, isolated from soil by Pfizer in 1954. OTC has certain therapeutic effects against diseases caused by Gram-positive bacteria, Gram-negative bacteria, and rickettsiae. Currently, OTC is widely used in aquaculture, animal husbandry, and as a raw material for second- and third-generation tetracycline antibiotics. However, genetic manipulation of industrial *Streptomyces* is difficult and time-consuming, necessitating the development of a low-cost method to increase OTC production. Summary of the Invention

[0003] The purpose of this invention is to provide an engineered bacterium that produces oxytetracycline, its construction method, and its application.

[0004] To achieve the objectives of this invention, in a first aspect, this invention provides an engineered bacterium that produces oxytetracycline. This bacterium is constructed by using Streptomyces albus Del14 as the starting strain, introducing the oxytetracycline biosynthesis gene cluster (otccluster) into the microorganism via plasmid or integrating it into the microbial chromosome through genetic engineering, and enhancing the otcR gene within the cluster.

[0005] In this invention, the white Streptomyces Del14 was kindly provided by the laboratory of Andriy Luzhetskyy, see Myronovskyi, M.; Rosenkranzer, B.; Nadmid, S., et al. Generation of a cluster-free Streptomyces albus chassis strains for improved heterologous expression of secondary metabolite clusters[J]. Metab Eng 2018, 49:316-324.

[0006] The oxytetracycline biosynthesis gene cluster can be derived from *Streptomyces rimosus*, and the nucleotide sequence of this gene cluster can be found in GenBank:DQ143963.2.

[0007] The reference sequence number of the gene otcR on NCBI is KP035101.1, and its nucleotide sequence is shown in SEQ ID NO:1.

[0008] The pathways to enhance intra-cluster gene otcR can be ① and / or ②:

[0009] ① Enhanced by increasing the copy number of the gene;

[0010] ② Enhancement is achieved by operatively linking a strong promoter to the gene.

[0011] Preferably, the strong promoter can be selected from PkasO*, Psf14, PotrB or PermE* promoters, etc., with PkasO* promoter being preferred.

[0012] Furthermore, the present invention provides engineered bacteria 2 obtained by knocking out the otrR gene based on engineered bacteria 1.

[0013] The amino acid sequence of the protein encoded by the gene otrR has the reference sequence number AAZ78324.1 on NCBI, and its nucleotide sequence is shown in SEQ ID NO:2.

[0014] Furthermore, the present invention provides engineered bacteria obtained by replacing the φC31 integrase site with the φBT1 integrase site based on engineered bacteria 1 or 2.

[0015] Secondly, the present invention provides a method for constructing an engineered bacterium that produces oxytetracycline, wherein a plasmid comprising an otcR gene expression cassette and an oxytetracycline biosynthesis gene cluster expression cassette is constructed and introduced into Streptomyces albopictus Del14 to obtain an engineered bacterium I (Del14::oxy1K) that heterologously expresses oxytetracycline.

[0016] Preferably, the gene otcR expression cassette is driven by the PkasO* promoter.

[0017] The aforementioned method can use pSET153 as the starting plasmid, whose nucleotide sequence is shown in SEQ ID NO:6. Plasmid pSET153 is constructed by replacing ori-ColE1 with the replicon ori-p15A based on pSET152 plasmid.

[0018] Furthermore, a plasmid containing the otcR gene expression cassette and the oxytetracycline biosynthesis gene cluster expression cassette was constructed as pSET153-OTC-KotcR. Based on the plasmid pSET153-OTC-KotcR, the otrR gene was knocked out by homologous recombination to obtain the plasmid pSET153-OTCΔotrR-KotcR, which was then introduced into Streptomyces albopictus Del14 through conjugation transfer to obtain engineered strain II (Del14::oxy1KΔotrR).

[0019] Furthermore, based on plasmid pSET153-OTCΔotrR-KotcR, the φC31 integrase site was replaced with the φBT1 integrase site through homologous recombination to obtain plasmid pSET153-OTCΔotrR-KotcR-BT1. This plasmid was then introduced into Streptomyces albopictus Del14 via conjugation transfer to obtain an engineered bacterium that produces oxytetracycline (Del14B::oxy1KΔotrR).

[0020] Thirdly, the present invention provides the application of the engineered bacteria or engineered bacteria constructed according to the method in the production of oxytetracycline.

[0021] Fourthly, the present invention provides a method for producing oxytetracycline, the method comprising the following steps:

[0022] a) Cultivate the engineered bacteria or the engineered bacteria constructed according to the method to obtain a culture of microorganisms;

[0023] b) Collect the generated oxytetracycline from the culture obtained in step a).

[0024] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0025] To address the challenges of complex genetic manipulation and lengthy processes in industrial strain modification, this invention uses Streptomyces albus Del14 as the chassis production strain. Through heterologous reconstruction, it successfully expressed otccluster heterologously in S. albus Del14 for the first time, preliminarily confirming that S. albus Del14 is a promising chassis strain for OTC biomanufacturing, laying the foundation for further optimization and modification of this OTC production strain.

[0026] Streptomyces albopictus Del14 has a small genome, a relatively simple and stable genetic background, and is easy to manipulate. In particular, the deletion of 15 secondary metabolic gene clusters makes it more suitable as a chassis for heterologous expression of oxytetracycline.

[0027] This invention enables the expression of the OTC cluster by adding a copy of the strong promoter PkasO* to the otcR. It also improves OTC efflux by targeting and knocking out the negative regulator OtrR within the cluster to relieve its repression of otrB. Furthermore, by replacing the integrase site, the modified pSET153-OTC-ΔotrR-BT1 is integrated into the S. albus Del14 genome, resulting in the screening of a highly efficient OTC-producing strain, Del14B::oxy1KΔotrR. Its OTC yield reached 1.1 g / L on day 6, comparable to the yield of the original producing strain S. rimosus M4018 on day 8, thus achieving heterologous and efficient OTC synthesis. Attached Figure Description

[0028] Figure 1 This is a preferred embodiment of the present invention, which is a screening and verification experiment for conjugates; wherein, Del14::control is a derived strain obtained by integrating the empty plasmid pSET153 into S. albus Del14; Del14::oxy is a positive clone obtained by integrating the pSET153-OTC plasmid into S. albus Del14.

[0029] Figure 2 This is a preferred embodiment of the present invention, which includes a conjugate screening verification experiment; wherein, Marker: DNA ladder; Del14::oxy is a positive clone obtained by integrating pSET153-OTC plasmid into S. albus Del14; Del14:Oxy1K is a strain obtained by screening pSET153-OTC-KotcR integrated into S. albus Del14.

[0030] Figure 3 This is a preferred embodiment of the present invention, which includes a conjugate screening verification experiment; wherein, Marker: DNA ladder; Del14::oxy1KΔotrR is a strain obtained by integrating pSET153-OTCΔotrR-KotcR into S. albus Del14.

[0031] Figure 4 This is a preferred embodiment of the present invention, specifically a conjugate screening verification experiment; wherein, Marker: DNA ladder; Del14B::oxyΔotrR is a strain obtained by integrating pSET153-OTCΔotrR-BT1 into S. albus Del14; Del14B::oxy1KΔotrR is a strain obtained by integrating pSET153-OTCΔotrR-KotcR-BT1 into S. albus Del14.

[0032] Figure 5In a preferred embodiment of the present invention, HPLC was used to detect the OTC fermentation titer in strains Del14::control and Del14::oxy.

[0033] Figure 6 In a preferred embodiment of the present invention, RT-qPCR was used to detect the transcriptional levels of oxyA, oxyI, oxyJ, oxyR, and oxyS. Experimental results were obtained from three biological replicates and statistically analyzed using Student's t-test. ***, P < 0.001.

[0034] Figure 7 The graph shows the yield of the control group (Del14::control) and recombinant strains (Del14::oxy1KΔotrR and Del14B::oxy1KΔotrR) detected by HPLC in a preferred embodiment of the present invention.

[0035] Figure 8 This is the spectrum of plasmid pSET153-OTC. Detailed Implementation

[0036] To address the challenges of complex and time-consuming genetic manipulation of industrial strains, this invention selects S. albus Del14 as the chassis production strain for heterologous OTC cluster reconstruction. The complete OTC cluster is integrated into S. albus Del14, and efficient heterologous expression of OTC is achieved by adding one copy of the positive regulatory gene otcR within the cluster, knocking out the negative regulatory gene otrR, and changing the integrase site. This allows for the evaluation of the strain's potential as a chassis cell for OTC production.

[0037] The present invention adopts the following technical solution:

[0038] A reconstructed OTC production strain of Streptomyces albus was developed. The chassis strain S. albus Del14 used was developed by Andriy Luzhetskyy's laboratory. This invention involves a series of modifications to the S. albus Del14 strain. The specific construction steps are as follows:

[0039] The first step is to add one copy of the intracluster positive regulatory gene otcR in vitro. First, the target fragment is obtained: the pSOK616-KotcR plasmid (see Yin, S.; Li, Z.; Wang, X., et al. Heterologous expression of oxytetracycline biosynthetic gene cluster in Streptomyces venezuelae WVR2006 to improve production level and to alterfermentation process[J]. Appl Microbiol Biotechnol 2016,100(24):10563-10572, the nucleotide sequence of the pSOK616-KotcR plasmid is shown in SEQ ID NO:7) contains a 936bp otcR gene fragment (SEQ ID NO:3) initiated by PkasO*. Secondly, the linearized vector was obtained: plasmid pSET153-OTC (pSET153-OTC is poxy1; plasmid poxy1 is described in Yin, S.; Li, Z.; Wang, X., et al. Heterologous expression of oxytetracycline biosynthetic gene cluster in Streptomycesvenezuelae WVR2006 to improve production level and to alter fermentation process[J]. Appl Microbiol Biotechnol 2016, 100(24):10563-10572) was digested with EcoRV enzyme, and then dephosphorylated with CIP alkaline phosphatase to obtain the linearized vector. Finally, the linearized vector and the target fragment were assembled by Gibson, transformed into Escherichia coli Epi300 (purchased from Beijing Zhuangmeng International Biotechnology Co., Ltd.), and plated on amycin-resistant plate. Positive clones pSET153-OTC-KotcR were screened by colony PCR and sequencing.

[0040] The second step involved knocking out the negative regulatory gene otrR on the pSET153-OTC-KotcR plasmid using PCR-targeting technology. Specifically, the following steps were taken: First, using primers otrR-FRT-F / otrR-FRT-R, a 1,406 bp fragment (SEQ ID NO:4) containing the FRT site, spectinomycin resistance gene, and a 50 bp overlap region of the homologous arm of the otrR gene was amplified in the pIJ779 plasmid (see Gust B, Challis GL, Fowler K, Kieser T, Chater KF (2003) PCR-targeted Streptomyces gene replacement identifies a protein domain needed for biosynthesis of thesesquiterpene soil odor geosmin. Proc Natl Acad Sci USA 100:1541–1546. doi:10.1073 / pnas.0337542100). Next, the purified PCR amplification fragment and plasmid pSET153-OTC-KotcR were electroporated into *E. coli* GB08-red (purchased from Gene Bridges) to undergo Red / ET recombination. The target plasmid pSET153-OTC-KotcR-FRT was obtained through spectinomycin resistance screening and colony PCR verification. The plasmid pSET153-OTC-KotcR-FRT was then transformed into *E. coli* BT340 cells containing the thermosensitive plasmid pCP20 (see Gust B, Challis GL, Fowler K, Kieser T, Chater KF (2003) PCR-targeted Streptomyces gene replacement identifies a protein domain needed for biosynthesis of thesesquiterpene soil odor geosmin. Proc Natl Acad Sci USA 100:1541–1546. doi:10.1073 / pnas.0337542100). The disruption of otrR was confirmed by PCR identification and sequencing, and the derived plasmid pSET153-OTCΔotrR-KotcR was obtained.

[0041] The third step involves replacing the φC31 integrase site with the BT1 integrase site. First, primers with 28bp homologous arms (Nd-F / Nu-R) were designed to amplify the BT1 sequence on the pSET153-BT1tu plasmid using PCR (the pSET153-BT1tu plasmid was constructed as follows: based on the pSET153-BT1 plasmid, primers with NheI mutation sites (primer mutation 1 and primer mutation 2) were designed to amplify it into a linear fragment. Then, it was digested with DpnI for 1 hour, identified by electrophoresis, and recovered by gel extraction before being transformed into JM109 competent cells. Finally, positive clones were screened on AMP plates, and the plasmid was extracted). The homologous arms were obtained upstream and downstream of the NheI restriction site in pSET153-OTCΔotrR-KotcR; the BT1 sequence is shown in SEQ ID NO:5. Secondly, the plasmid pSET153-OTCΔotrR-KotcR was digested with NheI, and the linear fragment was purified and recovered using the phenol-chloroform-isoamyl alcohol (volume ratio 25:24:1) method; then, it was purified and recovered using recombinase. The recombinant linear plasmid pSET153-OTCΔotrR-KotcR and the target fragment BT1 were transformed into E. coli EPi300. Correct transformants were verified by PCR and sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. Positive clones containing the recombinant plasmid pSET153-OTC-ΔotrR-KotcR-BT1 were selected.

[0042] The construction method of pSET153-BT1 plasmid is as follows:

[0043] Plasmid pSET156 and vector pSET153 (SEQ ID NO:6) were digested with SphI and PvuII enzymes to obtain the attp BT1 sequence fragment and the vector backbone containing the p15A replicon, respectively. The vector and the target fragment were ligated using T4 DNA ligase, and the results were verified by colony PCR and sequenced. If the sequencing results were correct, the recombinant plasmid was successfully constructed.

[0044] The pSET156 plasmid is constructed as follows:

[0045] The 2009 bp BT1 fragment containing the attp integration site on the pLC803 plasmid (see Wang, W., Li, S., Li, Z. et al. Harnessing the intracellular triacylglycerols for titer improvement of polyketides in Streptomyces. Nat Biotechnol 38, 76–83, 2020) was amplified using primers Bt1s / Bt1a PCR. The BT1 sequence and the vector pSET152 (GenBank ID: AJ414670) were digested with SphI and PvuI, and the vector and fragment were ligated using T4 DNA ligase. After verification by colony PCR and double enzyme digestion, the constructed plasmid was sent for sequencing. Correct sequencing results indicated successful recombinant plasmid construction.

[0046] The primers involved in this invention are shown in Table 1:

[0047] Table 1 Primer Information

[0048]

[0049]

[0050] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the examples are conducted under conventional experimental conditions, such as those described in Sambrook et al., Molecular Cloning: a Laboratory Manual (Sambrook J & Russell DW, 2001), or as recommended by the manufacturer's instructions.

[0051] The fermentation experiments in the following examples used R5 medium, which was prepared as follows:

[0052] Sucrose 103g; K2SO4 0.25g; MgCl2·6H2O 10.12g; glucose 10g; Casamino acids 0.1g; trace elements 2mL; yeast extract 5g; TES 5.73g. Add distilled water to a final volume of 1000mL. Mix thoroughly with a magnetic stirrer and dispense 200mL into 500mL Erlenmeyer flasks. Add 4g of agar powder to each flask. Sterilize at 115℃ for 30min. Before use, add 2mL of 0.5% KH2PO4 buffer, 0.8mL of 5M CaCl2 buffer, 3mL of 20% L-proline, and 1.4mL of 1mol / L NaOH to each flask.

[0053] The above stock solutions were prepared as follows: KH₂PO₄ stock solution (0.5%): Weigh 0.005 g KH₂PO₄ using a precision balance, add 10 mL of distilled water, and autoclave at high temperature; 1 mol / L NaOH stock solution: Weigh 4 g NaOH using a precision balance, add distilled water to a final volume of 100 mL, and autoclave at high temperature; 5 mol / L CaCl₂ stock solution: Weigh 73.51 g CaCl₂·2H₂O using a balance, add distilled water to a final volume of 100 mL, and autoclave at high temperature; L-proline stock solution (20%): Weigh 20 g L-proline using a precision balance, add distilled water to a final volume of 100 mL, sterilize using a 0.22 μM filter, and store at 4℃ for later use; Trace element stock solutions were prepared as follows: ZnCl₂ 40 mg, FeCl₃·6H₂O 200 mg; CuCl₂·2H₂O 10 mg; MnCl₂·4H₂O 10 mg; Na₂B₄O₇·10H₂O 10mg; (NH4)6Mo7O 24 ·4H2O 10mg; dilute to 1,000mL with distilled water, sterilize with a 0.22μM filter, and store at 4℃.

[0054] Example 1: Heterologous reconstruction of Streptomyces albopictus Del14 with an added copy of the positive regulatory gene otrR using the otc cluster.

[0055] In this embodiment, the efficient constitutive promoter PkasO* of Streptomyces is used to initiate the transcription of the pathway-specific regulatory gene otcR on the pSOK616-KotcR vector, so that the activating protein can be efficiently expressed in Streptomyces, thereby activating the OTC biosynthesis pathway.

[0056] The specific modification steps are as follows:

[0057] First, using the recombinant vector pSOK616-KotcR as a template, the 936 bp fragment PkasO*-otcR (SEQ ID NO:3) was amplified using the primer pair kaso*-F / kaso*-R. The 25 bp region upstream of the promoter region and the 25 bp region downstream of the otcR gene were used as homologous arms. The sequences of the primers kaso*-F / kaso*-R are shown in Table 1.

[0058] Secondly, plasmid pSET153-OTC was digested with the enzyme EcoRV and then dephosphorylated using CIP alkaline phosphatase. A map of plasmid pSET153-OTC can be found here. Figure 8 .

[0059] Finally, Gibson assembled the linear vector and fragment: the assembly system consisted of 20 μl, including 15 μl of the master assembly solution, a μl of the target gene fragment containing the overlapping sequence of the vector, and (5-a) μl of the linear vector fragment obtained by enzyme digestion. During the recombination reaction, the vector amount was generally 50–100 ng. Since the vector fragment in this invention was approximately 30 kb and the insert fragment was 936 bp, the vector and target fragment were generally mixed at a 1:3 molar ratio. The reaction was carried out in a thermal cycler at 50°C for 1 h. Then, 10 μl of the assembly product was transformed into *E. coli* Epi300 and plated onto a amprolium-resistant plate, incubated at 37°C. Positive clones containing the recombinant plasmid pSET153-OTC-KotcR were screened by colony PCR and sequencing.

[0060] Example 2: Knockout of the negative regulatory gene otrR in the oxytetracycline synthesis process

[0061] The intracranial regulatory factor OtrR negatively regulates the transcriptional level of otrB within the otr cluster. Therefore, knocking out the negatively regulating gene otrR on the pSET153-OTC-KotcR plasmid using PCR-targeting technology can relieve the inhibition of otrB, thereby allowing intracellular oxytetracycline to be expelled.

[0062] The specific modification steps are as follows:

[0063] First, a 1,406 bp fragment (SEQ ID NO:4) containing the FRT site, spectinomycin resistance gene, and a 50 bp overlap region of the homologous arm of the otrR gene was amplified from the pIJ779 plasmid using primers otrR-FRT-F / otrR-FRT-R. The purified PCR fragment and plasmid pSET153-OTC-KotcR were electroporated into *E. coli* GB08-red to undergo Red / ET recombination. Spectinomycin resistance screening yielded the recombinant plasmid pSET153-OTC-KotcR-FRT, which contained the aadA and FRT sites and replaced the otrR gene, and this plasmid was transformed into *E. coli* BT340 cells containing the temperature-sensitive plasmid pCP20. PCR identification and sequencing confirmed the disruption of otrR and the deletion of the spectinomycin resistance gene.

[0064] Example 3: Replacement of integrase sites

[0065] Previous experiments showed that the insertion site of the φC31 integrative vector is located within the SAM23877_RS18305(pirA) gene. Inactivation of pirA insertion affects the expression of central carbon metabolism-related genes and exhibits high sensitivity to oxidative damage, thereby inhibiting the synthesis of polyketide antibiotics. Homology comparisons of the pirA gene in *S. coelicolor*, *S. griseus*, *S. venezuelae*, *S. rimosus*, and *S. albus* revealed a highly conserved φC31 attB site in all pirA genes (up to 80%), suggesting that the insertion site of the φC31 integrative vector is highly conserved in *Streptomyces* and located within the pirA gene. To verify whether inactivation of the pirA gene in *S. albus Del14* affects heterologous OTC synthesis, this invention uses a BT1attP / attB integrative system to verify the effect of pirA gene insertion inactivation on the intracellular redox environment.

[0066] The specific modification steps are as follows:

[0067] First, the BT1 sequence was obtained: a pair of primers with 28bp homologous arms, Nd-F / Nu-R, were designed to amplify the BT1 sequence (SEQ ID NO:5) on the pSET153-BT1tu plasmid by PCR.

[0068] Secondly, the linearized plasmid was obtained by digesting the plasmid pSET153-OTCΔotrR-KotcR with NheI enzyme to linearize it, and then the linear fragment was purified and recovered using the phenol-chloroform-isoamyl alcohol (volume ratio 25:24:1) method.

[0069] Then, through recombinase The recombinant plasmid pSET153-OTC-ΔotrR-BT1 was transformed into E. coli EPi300 after recombination using a one-step PCR directional cloning kit. Following PCR verification, the cells were sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing, and positive clones containing the recombinant plasmid pSET153-OTC-ΔotrR-BT1 were selected.

[0070] Example 4: Construction of engineered bacteria and detection of fermentation products

[0071] S. albus Del14 was used as a heterologous remodeling chassis, and the potential of this strain for OTC biomanufacturing cell factories was evaluated.

[0072] The specific process for constructing the engineered strain Del14:control is as follows:

[0073] Plasmid pSET153 was transformed into ET12567 / PUZ8002 competent cells via chemical transformation. Then, colony PCR was performed using a mixture of *E. coli* and spore suspension, followed by plating, passage on antibiotic-resistant plates, and selection of single colonies for verification. The resulting engineered strain, Del14:control, was finally obtained. Figure 1 ).

[0074] The specific process for constructing the engineered strain Del14::oxy is as follows:

[0075] The plasmid pSET153-OTC was transformed into ET12567 / PUZ8002 competent cells via chemical transformation. Then, colony PCR was performed using a mixture of E. coli and spore suspension, followed by plate plating, passage on resistant plates, and selection of single colonies for verification. The resulting engineered strain, Del14::oxy( Figure 1 ).

[0076] The specific process for constructing the engineered strain Del14::oxy1K is as follows:

[0077] The plasmid pSET153-OTC-KotcR was transformed into ET12567 / PUZ8002 competent cells via chemical transformation. Then, a mixture of spores and *E. coli* was plated on antibiotic-free MS plates via conjugation transfer. After passage on antibiotic-resistant plates and colony PCR verification by selecting single colonies, the engineered strain Del14::oxy1K was finally obtained. Figure 2 ).

[0078] The specific process for constructing the engineered strain Del14::oxy1KΔotrR is as follows:

[0079] The plasmid pSET153-OTCΔotrR-KotcR was transformed into ET12567 / PUZ8002 competent cells via chemical transformation. Then, colony PCR was performed using a mixture of E. coli and spore suspension on plates, followed by passage on antibiotic-resistant plates and selection of single colonies for verification. The resulting engineered strain, Del14::oxy1KΔotrR, was finally obtained. Figure 3 ).

[0080] The specific process for constructing the engineered strain Del14B::oxy1KΔotrR is as follows:

[0081] The plasmid pSET153-OTCΔotrR-KotcR-BT1 was transformed into ET12567 / PUZ8002 competent cells via chemical transformation. Then, colony PCR was performed using a mixture of E. coli and spore suspension on plates, followed by passage on resistant plates and selection of single colonies for verification. The resulting engineered strain, Del14B::oxy1KΔotrR( Figure 4 ).

[0082] Combined with specific transfer methods:

[0083] The plasmids described above were chemically transformed into *Escherichia coli* strain ET12567 (PUZ8002) to obtain recombinant *E. coli* ETZ. Single colonies of the recombinant *E. coli* ETZ were picked and inoculated into liquid LB medium containing 25 μg / ml chloramphenicol, 50 μg / ml kanamycin, and 50 μg / ml apramycin, and cultured overnight at 37°C and 220 rpm. The culture was then transferred at a 1:100 volume ratio to fresh LB liquid medium containing the corresponding antibiotics and cultured until OD (October Expiratory Time). 600 ≈0.6. Then, take 1 mL of culture medium into a sterile Eppendorf tube, centrifuge at 6000 rpm for 2 min, discard the supernatant, add 1 mL of antibiotic-free liquid LB, wash twice, and resuspend. Simultaneously, collect the spore suspension: collect fresh spore suspension into a sterile Eppendorf tube, wash twice with TES, pH 8.0 buffer, then heat shock with 500 μl TES at 50℃ for 10 min, and cool to room temperature. Add an equal volume of spore pre-germination medium and incubate at 30℃ for 1.5-2 h. Then, mix the resuspended *E. coli* with the spore pre-germination medium at a ratio of 100:1 and spread it onto antibiotic-free MS plates. Incubate overnight at 30℃ for 14-16 h, then apply the appropriate antibiotics, and continue culturing for 4-6 days.

[0084] Fermentation culture of Del14::oxy strain failed to detect OTC by HPLC. Figure 5RT-qPCR experiments showed that the recombinant strain Del14:Oxy did not express the transcription of the activation gene otcR and the otccluster-related structural gene on day 1 (logarithmic phase) and day 3 (stationary phase) of fermentation. Fermentation culture of the Del14::oxy1K strain showed an OTC yield of approximately 140 mg / L in R5 medium by HPLC, which is higher than the previously reported OTC yield of the V2006::oxy1K strain (see Yin, S.; Li, Z.; Wang, X., et al. Heterologous expression of oxytetracycline biosynthetic gene cluster in Streptomyces venezuelae WVR2006 to improve production level and toalter fermentation process[J]. Appl Microbiol Biotechnol 2016, 100(24):10563-10572). Previously, OTC was heterologously expressed in S. venezuelae WVR2006. While the oxytetracycline industrial strain produces up to 35 g / L of oxytetracycline, the long fermentation cycle and the relatively high difficulty in genetic manipulation and synthetic biology modification make it challenging to further increase OTC yield. RT-qPCR experiments detected the transcriptional expression of both the otcR activation gene and the OTC cluster-related structural gene within the cluster on day 1 (logarithmic phase) and day 3 (stationary phase) of fermentation, indicating that adding one copy of otcR activated the transcriptional expression of the OTC cluster-related structural gene. Figure 6 ).

[0085] Fermentation culture of the Del14::oxy1KΔotrR strain yielded an OTC production of 554 mg / L, as determined by HPLC, which is 2.9 times that of Del14::oxy1K. Meanwhile, the OTC production of the Del14B::oxy1KΔotrR strain reached 1.1 g / L on day 6, comparable to the production of the original producing strain *S. rimosus* M4018 on day 8. Figure 7 ).

[0086] The specific process for sample preparation, including fermentation culture and high-performance liquid chromatography detection, is as follows:

[0087] Fermentation culture of engineered strains:

[0088] Sporospheres of the engineered strain were taken with an inoculation loop and spread onto MS solid plates. The plates were incubated at 30°C for 3 days. The resulting fragments (1cm × 2cm) were then inoculated into seed culture medium (R5 medium, 40ml / 250ml) and incubated at 30°C and 220rpm for 36 hours. The fragments were then transferred to fermentation medium (R5 medium) at 5% v / v and incubated at 30°C and 250rpm for 6 days.

[0089] Detection of fermentation products:

[0090] Quantitative analysis of OTC yield: 1 ml of fermentation broth was acidified with 9 M hydrochloric acid, with a pH range of 1.5-2.0. The mixture was centrifuged at 12,000 rpm for 10 min, and the supernatant was collected into 1.5 ml-2 ml EP tubes. After filtration through a 0.22 μm filter membrane, 200 μL was collected into the inner liner of a sample vial. A Diamonsil C18 reverse-phase column (250 mm × 4.6 mm) was used for HPLC detection. The mobile phase consisted of 60% deionized water, 10% methanol, 20% acetonitrile, and 10% phosphoric acid (2 mM). The elution program was as follows:

[0091] Pump A: 10% phosphoric acid; Pump B: 10% methanol, 20% acetonitrile.

[0092] The column flushing sequence is as follows: flush with 100% acetonitrile, then with 80% acetonitrile + 20% deionized water, then with 50% acetonitrile + 50% water, and finally with 90% acetonitrile + 10% deionized water, with each flushing session lasting 20 minutes.

[0093] During the detection process, the flow rate of pump A was 0.35 ml / min, the flow rate of pump B was 0.15 ml / min, the detection wavelength was 350 nm, and the retention time of the OTC absorption peak was about 6.0 min.

[0094] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention. sequence list <110> Institute of Microbiology, Chinese Academy of Sciences <120> Oxytetracycline-producing engineered bacteria, their construction methods and applications <130> KHP221112855.7 <160> 7 <170> SIPOSequenceListing 1.0 <210> 1 <211> 756 <212> DNA <213> Streptomyces rimosus <400> 1 tcaagacgcc gacctcaaca cctcctcttg cgcacgttgc agatcgtgcg acggatcgag 60 accgagttcg tcgcggagca ggcggcggac cttgtggtag acgtggaggg cttcgccgcg 120 gcggccggct ttgccgaggg cgagcatgag ctgggcgtgg aaccattcgt cgtaggggta 180 ctccatcacc agggagcgca ggtcggtgat cagctcgccg tagcggccga gggccatggc 240 tgcctggatg cggagtttgc ggacggagac gcgccgttcg tcgaggttgc tgacgtagtc 300 ccgcagcgcc cggccgcagg gcacgtcggc gaggggggtg ccgcgccaca gggccagcgc 360 ctcctcggcc caccggacgg cgccggcggc gtcgtgggcg gcgtagcagg tctgggcccg 420 gtcggccagg cgcgcgaagt ggtgggtgtc gagcgactcc tcgggggtgt gcagcaggta 480 gccgggcggc gcggtgacga tggcgcgctc ctgcgggtcc acgcccagtt cctcgatgaa 540 gtccttgcgc agctggtaga tgtaagtctg cgcggtggtg accgcggtac gcggcagcgc 600 gtcgccccac agttcgtcga ggatggactc caggctgacg atctggttgg cccgcatcac 660 gagcagggcg agcacctgct tcactttcgg agcgctcggg gtgtaggcca gtcggtcgtt 720 gacggcccgg atggggccga gtgccttgaa gtccat 756 <210> 2 <211> 492 <212> DNA <213> Streptomyces rimosus <400> 2 atggattcct cagcccctga cctggccgct ctgatcgagg tgaccgccga ggtcttcgcg 60 gtcaacggcc gcctgctccg cgaaggcgac agcctcaccg cccacgcggg gctgacctcg 120 gcgcgctggc aggtggccgg actgctgctg agcggcccct cgacggtcgc ccgcctggcc 180 cgcgagcggg ggctgcggcg gcaggcggtc cagcagaccg tcgagcggct gaaggccgag 240 ggcgtcgtca cgacccggcc caacccgcag gaccagcgca gccccctggt cgagctcacc 300 gcacgcggcc ggcaggcgct ggacgacctg cgtcccctgg aacggcggtg gctggagtat 360 ctggccgagg acattccggt cgaggacatg cgcgtggcga tcgcggtgct gagccgcctg 420 cgggagaagc tggacgcccg tccggcgacg gagttcggga ccggggccgg gtccgggcgg 480 cagtccgcct ga 492 <210> 3 <211> 936 <212> DNA <213> Artificial Sequence <400> 3 gaggtatgta ggcggtgcta cagattgttc acattcgaac ggtctctgct ttgacaacat 60 gctgtgcggt gttgtaaagt cgtggccagg agaatacgac agcgtgcagg actgggggag 120 ttggatccac agaaccactc cacaggagga cccatatgga cttcaaggca ctcggcccca 180 tccgggccgt caacgaccga ctggcctaca ccccgagcgc tccgaaagtg aagcaggtgc 240 tcgccctgct cgtgatgcgg gccaaccaga tcgtcagcct ggagtccatc ctcgacgaac 300 tgtggggcga cgcgctgccg cgtaccgcgg tcaccaccgc gcagacttac atctaccagc 360 tgcgcaagga cttcatcgag gaactgggcg tggacccgca ggagcgcgcc atcgtcaccg 420 cgccgcccgg ctacctgctg cacacccccg aggagtcgct cgacacccac cacttcgcgc 480 gcctggccga ccgggcccag acctgctacg ccgcccacga cgccgccggc gccgtccggt 540 gggccgagga ggcgctggcc ctgtggcgcg gcacccccct cgccgacgtg ccctgcggcc 600 gggcgctgcg ggactacgtc agcaacctcg acgaacggcg cgtctccgtc cgcaaactcc 660 gcatccaggc agccatggcc ctcggccgct acggcgagct gatcaccgac ctgcgctccc 720 tggtgatgga gtacccctac gacgaatggt tccacgccca gctcatgctc gccctcggca 780 aagccggccg ccgcggcgaa gccctccacg tctaccacaa ggtccgccgc ctgctccgcg 840 acgaactcgg tctcgatccg tcgcacgatc tgcaacgtgc gcaagaggag gtgttgaggt 900 cggcgtcttg aatcggcacc ggcacgtaca ccgtcc 936 <210> 4 <211> 1414 <212> DNA <213> Artificial Sequence <400> 4 ggcttttgac aaggtcttgt cgttctccgg ggagtacata cgctgtggcc atgtgtaggc 60 tggagctgct tcgaagttcc tatactttct agagaatagg aacttcggaa taggaacttc 120 atgagctcag ccaatcgact ggcgagcggc atcttatttg ccgactacct tggtgatctc 180 gcctttcacg tagtggacaa attcttccaa ctgatctgcg cgcgaggcca agcgatcttc 240 ttcttgtcca agataagcct gtctagcttc aagtatgacg ggctgatact gggccggcag 300 gcgctccatt gcccagtcgg cagcgacatc cttcggcgcg attttgccgg ttactgcgct 360 gtaccaaatg cgggacaacg taagcactac atttcgctca tcgccagccc agtcgggcgg 420 cgagttccat agcgttaagg tttcatttag cgcctcaaat agatcctgtt caggaaccgg 480 atcaaagagt tcctccgccg ctggacctac caaggcaacg ctatgttctc ttgcttttgt 540 cagcaagata gccagatcaa tgtcgatcgt ggctggctcg aagatacctg caagaatgtc 600 attgcgctgc cattctccaa attgcagttc gcgcttagct ggataacgcc acggaatgat 660 gtcgtcgtgc acaacaatgg tgactttac agcgcggaga atctcgctct ctccagggga 720 agccgaagtt tccaaaaggt cgttgatcaa agctcgccgc gttgtttcat caagccttac 780 ggtcaccgta accagcaaat caatatcact gtgtggcttc aggccgccat ccactgcgga 840 gccgtacaaa tgtacggcca gcaacgtcgg ttcgagatgg cgctcgatga cgccaactac 900 ctctgatagt tgagtcgata cttcggcgat caccgcttcc ctcatgacat tgcactccac 960 cgctgatgac atcagtcgat catagcacga tcaacggcac tgttgcaaat agtcggtggt 1020 gataaactta tcatcccctt ttgctgatgg agctgcacat gaaccattc aaaggccggc 1080 attttcagcg tgacatcatt ctgtgggccg tacgctggta ctgcaaatac ggcatcagtt 1140 accgtgagct gcattttccg ctgcataacc ctgcttcggg gtcattatag cgattttttc 1200 ggtatatcca tcctttttcg cacgatatac aggattttgc caaagggttc gtgtagactt 1260 tccttggtgt atccaacggc gtcagaggct ggcgggaact tcgaagttcc tatactttct 1320 agagaatagg aacttcgaac tgcaggtcga cggatccccg gaatctgagc ggccgccgcc 1380 ccgcccggcc gtctctccgg agtggcccgg gcgt 1414 <210> 5 <211> 2988 <212> DNA <213> Artificial Sequence <400> 5 ctcaccgcga cgtatcgggc cctggccagc tagctagagt cgacctgcag gtccccgggg 60 atcggtcttg ccttgctcgt cggtgatgta cttcaccagc tccgcgaagt cgctcttctt 120 gatggagcgc atggggacgt gcttggcaat cacgcgcacc ccccggccgt tttagcggct 180 aaaaaagtca tggctctgcc ctcgggcgga ccacgcccat catgaccttg ccaagctcgt 24~0 cctgcttctc ttcgatcttc gccagcaggg cgaggatcgt ggcatcaccg aaccgcgccg 300 tgcgcgggtc gtcggtgagc cagagtttca gcaggccgcc caggcggccc aggtcgccat 360 tgatgcgggc cagctcgcgg acgtgctcat agtccacgac gcccgtgatt ttgtagccct 420 ggccgacggc cagcaggtag gccgacaggc tcatgccggc cgccgccgcc ttttcctcaa 480 tcgctcttcg ttcgtctgga aggcagtaca ccttgatagg tgggctgccc ttcctggttg 540 gcttggtttc atcagccatc cgcttgccct catctgttac gccggcggta gccggccagc 600 ctcgcagagc aggattcccg ttgagcaccg ccaggtgcga ataagggaca gtgaagaagg 660 aacacccgct cgcgggtggg cctacttcac ctatcctgcc cggctgacgc cgttggatac 720 accaaggaaa gtctacacga accctttggc aaaatcctgt atatcgtgcg aaaaaggatg 780 gatataccga aaaaatcgct ataatgaccc cgaagcaggg ttatgcagcg gaaaagatcc 840 gtcgacctgc aggcatgctg gcgccggacg gggcttcaga cgtttcgggt gctgggttgt 900 tgtctctgga cagtgatcca tgggaaacta ctcagcacca ccaatgttcc caaaagaaag 960 cgcaggtcag cgcccatgag ccaagatcta ggcatgtcgc ccttcatcgc tcccgacgtc 1020 cctgagcacc ttctagacac tgttcgcgtc ttcctgtacg cgcgtcagtc taagggccgg 1080 tccgacggct cagacgtgtc gaccgaagca cagctagcgg ccggtcgtgc gttggtcgcg 1140 tctcgcaacg cccagggggg tgcgcgctgg gtcgtggcag gtgagttcgt ggacgtcggg 1200 cgctccggct gggacccgaa cgtgacccgt gccgacttcg agcgcatgat gggcgaagtc 1260 cgcgccggcg aaggtgacgt tgtcgttgtg aatgagcttt cccggctcac tcgcaagggc 1320 gcccatgacg cgctcgaaat cgacaacgaa ttgaagaagc acggcgtgcg cttcatgtcg 1380 gttcttgagc cgttccttga cacgtctacc cctatcggcg tcgccatttt cgcgctgatc 1440 gctgcccttg cgaaacagga cagtgacctg aaggcggagc gcctgaaggg tgcgaaagac 1500 gagattgccg cgctgggtgg cgttcactcg tcttccgccc cgttcggaat gcgcgccgtg 1560 cgcaagaagg tcgataatct cgtgatctcc gttcttgagc cggacgaaga caacccggat 1620 cacgtcgagc tagttgagcg catggcgaaa atgtcgttcg aaggcgtgtc cgacaacgcc 1680 attgcaacga ccttcgagaa ggaaaagatc ccgtcgcccg gaatggctga gagacgcgcc 1740 acggaaaagc gtcttgcgtc catcaaggca cgtcgcctga acggcgctga aaagccgatc 1800 atgtggcgcg ctcaaacggt ccgatggatt ctcaaccatc ccgcaatcgg cggtttcgca 1860 ttcgagcgtg tgaagcacgg taaggcgcac atcaacgtca tacggcgcga ccccggcggc 1920 aagccgctaa cgccccacac gggcattctc agcggctcga agtggcttga gcttcaagag 1980 aagcgttccg ggaagaatct cagcgaccgg aagcctgggg ccgaagtcga accgacgctt 2040 ctgagcgggt ggcgtttcct ggggtgccga atctgcggcg gctcaatggg tcagtcccag 2100 ggtggccgta agcgcaacgg cgaccttgcc gaaggcaatt acatgtgcgc caacccgaag 2160 gggcacggcg gcttgtcggt caagcgcagc gaactggacg aattcgttgc ttcgaaggtg 2220 tgggcacggc tccgcacagc cgacatggaa gatgaacacg atcaggcatg gattgccgcc 2280 gctgcggagc gcttcgccct tcagcacgac ctagcggggg tggccgatga gcggcgcgaa 2340 caacaggcgc acctagacaa cgtgcggcgc tccatcaagg accttcaggc ggaccgtaag 2400 gccggtctgt acgtcgggcg tgaagagctg gaaacgtggc gctcaacggt gctgcaatac 2460 cggtcctacg aagcggagtg cacgacccga ctcgctgagc ttgacgagaa gatgaacggc 2520 agcacccgcg ttccgtctga gtggttcagc ggcgaagacc cgacggccga agggggcatc 2580 tgggcaagct gggacgtgta cgagcgtcgg gagttcctga gcttcttcct tgactccgtc 2640 atggtcgacc gggggcgcca ccctgagacg aagaaataca tccccctgaa ggaccgtgtg 2700 acgctcaagt gggcggagct gctgaaggag gaagacgaag cgagcgaagc cactgagcgg 2760 gagcttgcgg cgctgtagcg cacagcggga ggggtcgagc cggcggacgg ttcggcccct 2820 tttttggcct tgaaatcgtt agttaggcta actagtcgat cggtgcgggc ctcttcgcta 2880 ttacgccagc tgtccctcct gttcagctac tgacggggtg gtgcgtaacg gcaaaagcac 2940 cgccggacat cagcgctagc ggagtgtata ctggcttact atgttggc 2988 <210> 6 <211> 5696 <212> DNA <213> Artificial Sequence <400> 6 atctacgtct gtcgagaagt ttctgatcga aaagttcgac agcgtctccg acctgatgca 60 gctctcgcag ggcgaagaat ctcgtgcttt cagcttcgat gtaggagggc gtggatatgt cctgcgggta aatagctgcg ccgatggttt ctacaaagat cgttatgttg atcggcttct 180 gtttctatca gctgtccctc ctgttcagct actgacgggg tggtgcgtaa cggcaaaagc 240 accgccggac atcagcgcta gcggagtgta tactggctta ctatgttggc actgatgagg 300 gtgtcagtga agtgcttcat gtggcaggag aaaaaaggct gcaccggtgc gtcagcagaa 360 tatgtgatac aggatatatt cggcttcctc gctcactgac tcgctacgct cggtcgttcg 420 actgcggcga gcggaatgg cttacgaacg gggcggagat ttcctggaag atgccaggaa 480 gatacttaac agggaagtga gagggccgcg gcaaagccgt ttttccatag gctccgcccc 540 cctgacaagc atcacgaat ctgacgctca aatcagtggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggcggc tccctcgtgc gctctcctgt tcctgccttt 660 cggtttaccg gtgtcattcc gctgttatgg cggcgtttgt ctcattccac gcctgacact 720 cagttccggg taggcagttc gctccaagct ggactgtatg cacgaacccc ccgttcagtc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggaaa gacatgcaaa 840 agcaccactg gcagcagcca ctggtaattg atttagagga gttagtcttg aagtcatgcg 900 ccggttaagg ctaaactgaa aggacaagtt ttggtgactg cgctcctcca agccagttac 960 ctcggttcaa agagttggta gctcagagaa ccttcgaaaa accgccctgc aaggcggttt 1020 tttcgttttc agagcaagag attacgcgca gaccaaaacg atctcaagaa gatcatctta 1080 ttaatcagat aaaatatttc tagatttcag tgcaatttat ctcttcaaat gtagcacctg 1140 aagtcagccc catacgatat aagttgtaat tctcatgttt gacagcttat ctatcgtctt 1200 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt 1260 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc 1320 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa 1380 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt 1440 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct 1500 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta 1560 tcaaaaagga tcttcaccta gatccttttg gttcatgtgc agctccatca gcaaaagggg 1620 atgataagtt tatcaccacc gactatttgc aacagtgccg ttgatcgtgc tatgatcgac 1680 tgatgtcatc agcggtggag tgcaatgtcg tgcaatacga atggcgaaaa gccgagctca 1740 tcggtcagct tctcaacctt ggggttaccc ccggcggtgt gctgctggtc cacagctcct 1800 tccgtagcgt ccggcccctc gaagatgggc cacttggact gatcgaggcc ctgcgtgctg 1860 cgctgggtcc gggagggacg ctcgtcatgc cctcgtggtc aggtctggac gacgagccgt 1920 tcgatcctgc cacgtcgccc gttacaccgg accttggagt tgtctctgac acattctggc 1980 gcctgccaaa tgtaaagcgc agcgcccatc catttgcctt tgcggcagcg gggccacagg 2040 cagagcagat catctctgat ccattgcccc tgccacctca ctcgcctgca agcccggtcg 2100 cccgtgtcca tgaactcgat gggcaggtac ttctcctcgg cgtgggacac gatgccaaca 2160 cgacgctgca tcttgccgag ttgatggcaa aggttcccta tggggtgccg agacactgca 2220 cattcttca ggatggcaag ttggtacgcg tcgattatct cgagaatgac cactgctgtg 2280 agcgctttgc cttggcggac aggtggctca aggagaagag ccttcagaag gaaggtccag 2340 tcggtcatgc ctttgctcgg ttgatccgct cccgcgacat tgtggcgaca gccctgggtc 2400 aactgggccg agatccgttg atcttcctgc atccgccaga ggcgggatgc gaagaatgcg 2460 atgccgctcg ccagtcgatt ggctgagctc atgagcggag aacgagatga cgttggaggg 2520 gcaaggtcgc gctgattgct ggggcaacac gtggagcgga tcggggattg tctttcttca 2580 gctcgctgat gatatgctga cgctcaatgc cgtttgcct ccgactaacg aaaatcccgc 2640 atttggacgg ctgatccgat tggcacggcg gacggcgaat ggcggagcag acgctcgtcc 2700 gggggcaatg agatatgaaa aagcctgaac tcaccgcgac gtatcgggcc ctggccagct 2760 agctagagtc gacctgcagg tccccgggga tcggtcttgc cttgctcgtc ggtgatgtac 2820 ttcaccagct ccgcgaagtc gctcttctg atggagcgca tggggacgtg cttggcaatc 2880 acgcgcaccc cccggccgtt ttagcggcta aaaaagtcat ggctctgccc tcgggcggac 2940 cacgcccatc atgaccttgc caagctcgtc ctgcttctct tcgatcttcg ccagcagggc 3000 gaggatcgtg gcatcaccga accgcgccgt gcgcgggtcg tcggtgagcc agagtttcag 3060 caggccgccc aggcggccca ggtcgccatt gatgcgggcc agctcgcgga cgtgctcata 3120 gtccacgacg cccgtgattt tgtagccctg gccgacggcc agcaggtagg ccgacaggct 3180 catgccggcc gccgccgcct tttcctcaat cgctcttcgt tcgtctggaa ggcagtacac 3240 cttgataggt gggctgccct tcctggttgg cttggtttca tcagccatcc gcttgccctc 3300 atctgttacg ccggcggtag ccggccagcc tcgcagagca ggattcccgt tgagcaccgc 3360 caggtgcgaa taagggacag tgaagaagga acacccgctc gcgggtgggc ctacttcacc 3420 tatcctgccc ggctgacgcc gttggataca ccaaggaaag tctacacgaa ccctttggca 3480 aaatcctgta tatcgtgcga aaaaggatgg atataccgaa aaaatcgcta taatgacccc 3540 gaagcagggt tatgcagcgg aaaagatccg tcgacctgca ggcatgcaag ctctagcgat 3600 tccagacgtc ccgaaggcgt ggcgcggctt ccccgtgccg gagcaatcgc cctgggtggg 3660 ttacacgacg cccctctatg gcccgtactg acggacacac cgaagccccg gcggcaaccc 3720 tcagcggatg ccccggggct tcacgttttc ccaggtcaga agcggttttc gggagtagtg 3780 ccccaactgg ggtaaccttt gagttctctc agttgggggc gtagggtcgc cgacatgaca 3840 caaggggttg tgaccggggt ggacacgtac gcgggtgctt acgaccgtca gtcgcgcgag 3900 cgcgagagtt cgagcgcagc aagcccagcg acacagcgta gcgccaacga agacaaggcg 3960 gccgaccttc agcgcgaagt cgagcgcgac gggggccggt tcaggttcgt cgggcatttc 4020 agcgaagcgc cgggcacgtc ggcgttcggg acggcggagc gcccggagtt cgaacgcatc 4080 ctgaacgaat gccgcgccgg gcggctcaac atgatcattg tctatgacgt gtcgcgcttc 4140 tcgcgcctga aggtcatgga cgcgattccg attgtctcgg aattgctcgc cctgggcgtg 4200 acgattgttt ccactcagga aggcgtcttc cggcagggaa acgtcatgga cctgattcac 4260 ctgattatgc ggctcgacgc gtcgcacaaa gaatcttcgc tgaagtcggc gaagattctc 4320 gacacgaaga accttcagcg cgaattgggc gggtacgtcg gcgggaaggc gccttacggc 4380 ttcgagcttg ttcggagac gaaggagatc acgcgcaacg gccgaatggt caatgtcgtc 4440 atcaacaagc ttgcgcactc gaccactccc cttaccggac ccttcgagtt cgagcccgac 4500 gtaatccggt ggtggtggcg tgagatcaag acgcacaaac accttccctt caagccgggc 4560 agtcaagccg ccattcaccc gggcagcatc acggggcttt gtaagcgcat ggacgctgac 4620 gccgtgccga cccggggcga gacgattggg aagaagaccg cttcaagcgc ctgggacccg 4680 gcaaccgtta tgcgaatcct tcgggaccg cgtattgcgg gcttcgccgc tgaggtgatc 4740 tacaagaaga agccggacgg cacgccgacc acgaagattg agggttaccg cattcagcgc 4800 gacccgatca cgctccggcc ggtcgagctt gattgcggac cgatcatcga gcccgctgag 4860 tggtatgagc ttcaggcgtg gttggacggc agggggcgcg gcaaggggct ttcccggggg 4920 caagccattc tgtccgccat ggacaagctg tactgcgagt gtggcgccgt catgacttcg 4980 aagcgcgggg aagaatcgat caaggactct taccgctgcc gtcgccggaa ggtggtcgac 5040 ccgtccgcac ctgggcagca cgaaggcacg tgcaacgtca gcatggcggc actcgacaag 5100 ttcgttgcgg aacgcatctt caacaagatc aggcacgccg aaggcgacga agagacgttg 5160 gcgcttctgt gggaagccgc ccgacgcttc ggcaagctca ctgaggcgcc tgagaagagc 5220 ggcgaacggg cgaaccttgt tgcggagcgc gccgacgccc tgaacgccct tgaagagctg 5280 tacgaagacc gcgcggcagg cgcgtacgac ggacccgttg gcaggaagca cttccggaag 5340 caacaggcag cgctgacgct ccggcagcaa ggggcggaag agcggcttgc cgaacttgaa 5400 gccgccgaag ccccgaagct tccccttgac caatggttcc ccgaagacgc cgacgctgac 5460 ccgaccggcc ctaagtcgtg gtgggggcgc gcgtcagtag acgacaagcg cgtgttcgtc 5520 gggctcttcg tagacaagat cgttgtcacg aagtcgacta cgggcagggg gcagggaacg 5580 cccatcgaga agcgcgcttc gatcacgtgg gcgaagccgc cgaccgacga cgacgaagac 5640 gacgcccagg acggcacgga agacgtagcg gcgtagcgag acacccggga agcctg 5696 <210> 7 <211> 7211 <212> DNA <213> Artificial Sequence <400> 7 tgttcacatt cgaacggtct ctgctttgac aacatgctgt gcggtgttgt aaagtcgtgg 60 ccaggagaat acgacagcgt gcaggactgg gggagttgga tccacagaac cactccacag 120 gaggacccat atggacttca aggcactcgg ccccatccgg gccgtcaacg accgactggc 180 ctacaccccg agcgctccga aagtgaagca ggtgctcgcc ctgctcgtga tgcgggccaa 240 ccagatcgtc agcctggagt ccatcctcga cgaactgtgg ggcgacgcgc tgccgcgtac 300 cgcggtcacc accgcgcaga cttacatcta ccagctgcgc aaggacttca tcgaggaact 360 gggcgtggac ccgcaggagc gcgccatcgt caccgcgccg cccggctacc tgctgcacac 420 ccccgaggag tcgctcgaca cccaccactt cgcgcgcctg gccgaccggg cccagacctg 480 ctacgccgcc cacgacgccg ccggcgccgt ccggtgggcc gaggaggcgc tggccctgtg 540 gcgcggcacc cccctcgccg acgtgccctg cggccgggcg ctgcgggact acgtcagcaa 600 cctcgacgaa cggcgcgtct ccgtccgcaa actccgcatc caggcagcca tggccctcgg 660 ccgctacggc gagctgatca ccgacctgcg ctccctggtg atggagtacc cctacgacga 720 atggttccac gcccagctca tgctcgccct cggcaaagcc ggccgccgcg gcgaagccct 780 ccacgtctac cacaaggtcc gccgcctgct ccgcgacgaa ctcggtctcg atccgtcgca 840 cgatctgcaa cgtgcgcaag aggaggtgtt gaggtcggcg tcttgaacta gtacgtctta agacgtggta cctaagtagc tgacaagaga caggatgagg atcgtttcgc atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg caagacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg 1200 tcactgaagc gggaagggac tggctgctat tggggcgaagt gccggggcag gatctcctgt 1260 catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc atcgagcgag 1380 cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacga gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgca catacccgac ggcgaggatc 1500 tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt 1560 ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac atagcgttgg 1620 ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt 1680 acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt gacgagttct 1740 tctgagcggg actctggggt tcggaattcg taatcatgtc atagctgttt cctgtgtgaa 1800 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct 1860 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc 1920 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg 1980 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc 2040 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag 2100 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa 2160 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc 2220 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc 2280 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg 2340 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt 2400 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc 2460 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc 2520 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag 2580 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg 2640 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa 2700 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag 2760 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact 2820 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttgg 2880 ttcatgtgca gctccatcag caaaagggga tgataagttt atcaccaccg actatttgca 2940 acagtgccgt tgatcgtgct atgatcgact gatgtcatca gcggtggagt gcaatgtcgt 3000 gcaatacgaa tggcgaaaag ccgagctcat cggtcagctt ctcaaccttg gggttacccc 3060 cggcggtgtg ctgctggtcc acagctcctt ccgtagcgtc cggcccctcg aagatgggcc 3120 acttggactg atcgaggccc tgcgtgctgc gctgggtccg ggagggacgc tcgtcatgcc 3180 ctcgtggtca ggtctggacg acgagccgtt cgatcctgcc acgtcgcccg ttacaccgga 3240 ccttggagtt gtctctgaca cattctggcg cctgccaaat gtaaagcgca gcgcccatcc 3300 atttgccttt gcggcagcgg ggccacaggc agagcagatc atctctgatc cattgcccct 3360 gccacctcac tcgcctgcaa gcccggtcgc ccgtgtccat gaactcgatg ggcaggtact 3420 tctcctcggc gtgggacacg atgccaacac gacgctgcat cttgccgagt tgatggcaaa 3480 ggttccctat ggggtgccga gacactgcac cattcttcag gatggcaagt tggtacgcgt 3540 cgattatctc gagaatgacc actgctgtga gcgctttgcc ttggcggaca ggtggctcaa 3600 ggagaagagc cttcagaagg aaggtccagt cggtcatgcc tttgctcggt tgatccgctc 3660 ccgcgacatt gtggcgacag ccctgggtca actgggccga gatccgttga tcttcctgca 3720 tccgccagag gcgggatgcg aagaatgcga tgccgctcgc cagtcgattg gctgagctca 3780 tgagcggaga acgagatgac gttggagggg caaggtcgcg ctgattgctg gggcaacacg 3840 tggagcggat cggggattgt ctttcttcag ctcgctgatg atatgctgac gctcaatgcc 3900 gtttggcctc cgactaacga aaatcccgca tttggacggc tgatccgatt ggcacggcgg 3960 acggcgaatg gcggagcaga cgctcgtccg ggggcaatga gatatgaaaa agcctgaact 4020 caccgcgacg tatcgggccc tggccagcta gctagagtcg acctgcaggt ccccggggat 4080 cggtcttgcc ttgctcgtcg gtgatgtact tcaccagctc cgcgaagtcg ctcttcttga 4140 tggagcgcat ggggacgtgc ttggcaatca cgcgcacccc ccggccgttt tagcggctaa 4200 aaaagtcatg gctctgccct cgggcggacc acgcccatca tgaccttgcc aagctcgtcc 4260 tgcttctctt cgatcttcgc cagcagggcg aggatcgtgg catcaccgaa ccgcgccgtg 4320 cgcgggtcgt cggtgagcca gagtttcagc aggccgccca ggcggcccag gtcgccattg 4380 atgcgggcca gctcgcggac gtgctcatag tccacgacgc ccgtgatttt gtagccctgg 4440 ccgacggcca gcaggtaggc cgacaggctc atgccggccg ccgccgcctt ttcctcaatc 4500 gctcttgtt cgtctggaag gcagtacacc ttgataggtg ggctgccctt cctggttggc 4560 ttggtttcat cagccatccg cttgccctca tctgttacgc cggcggtagc cggccagcct 4620 cgcagagcag gattcccgtt gagcaccgcc aggtgcgaat aagggacagt gaagaaggaa 4680 cacccgctcg cgggtgggcc tacttcacct atcctgcccg gctgacgccg ttggatacac 4740 caaggaaagt ctacacgaac cctttggcaa aatcctgtat atcgtgcgaa aaaggatgga 4800 tataccgaaa aaatcgctat aatgaccccg aagcagggtt atgcagcgga aaagatccgt 4860 cgacctgcag gcatgctcct ctagagtccg tctgacgcgt gtggggagca cgccggcccg 4920 ctcggtagac agcactctac gcattgtggc tgctgtgcac atcactcttt cgagcgacgt 4980 gatccgcagg attccgtggg gatgtggcat ctatgtggca tccgggtacg aaacggccc 5040 cccgttcgaa tggaacgggg ggccgttgac tccctgacct gcgatgcagg ttgtgccccc 5100 ggcaggattc gaacctgcga cacccgcttt aggagagaac gtaaagcgat catgagcctc 5160 agtgagtctg agtgagtctc agtgagcctg tcgccatagg ggcttcacct gcataaatga 5220 gtttcagtga gtctcggtga gtctcagtga gggggtgttg tggcatccct gtggcatctg 5280 cgtggcatcc ggcgtcacac cgcccgcagc gttcgaacac cctcggtctc gggagccatc 5340 gccgcccgca ccttcgccgc cacgtcggcc ggcgaatgct ggtacaacca cgtcaccttc 5400 gaccctcgct cgtgacccat cacggtctgc gtgtccttct cgtcaatgcc cacatccttg 5460 agccacgtgg cgaacacgtg gcgaaggtcg tgcacccgcg gccaccactc catgcggccg 5520 gtgtcccggt tcttcacctc gcgggcgagg cctgcagcct tgattgcagg gatccatgtg 5580 cgccggaagt tgtgccgggt caggacgccg ccctgtgggc cgcggaacac gagctcatcc 5640 ggcgcgaggt cgtacgggtc ctcgccaatc ggggtgatcg tgggagtcgg ccgccaccgg 5700 gtgaccatgg tgcggatcgc gtcctcggcc tgcggcgtga gcgggaccgt gcggaacccg 5760 gcgttgctct tcggcgcagg cttccggaac agcttgcctt cgtcgtcact gagcacttcc 5820 ttcaccttga ggtagtgggc gtccaggtcg acgttctccc agcgcagcgc cgtcgcctcg 5880 ccccaccgga gaccggtgtg ttcaaggaag acgacgagcg gccggtagta catcggcagg 5940 tgactgacga tcagcgcgca ctgcgcgcgg tcgggcggca tcagttcgtc cggatgcttc 6000 gccggcgcct cgccaatgtc caggtcggcc gccgggttga acgggatacg ccggccgtcc 6060 ttgacggcgg cccggagcat cgagttcagc acctcatgaa ccttcttccg ggtgtggtag 6120 cccttcacct ccttcgtgat ccacgcctgc agctcgatgt actccaagtc gcagagacgc 6180 cactgccccc acttcggctc gatgtgcgcg gcccagttcg accgcttccg gttggcggtc 6240 gtgactgctc ggtccggctg cgcctcccac cacagtttcc accactggga cagggtgatc 6300 tcgccacgct tcgggtcggc gtacgtccgt tcgcggacct gcgtgcggat cttgtcgagg 6360 tgcgcctcgg ctgccttctt accgccgtcc ttgatcgcaa agcatttctg cttctggttg 6420 ccggccgggt cgcggtaacg cgcctgccac gagccggtgc agtcgcgtcg gcggttccgc 6480 tctccgtact tctcgggcgg gtacttctcc atgcacagct ggcagccgca gctcttcgcc 6540 ctcaactgcc gtgggttgtt cgtcgctcta ggcgccatgg gtcacctgtc cgctcctccg 6600 ctgttcggga acgcggggca ggatgtgtac ggctgcccca caccagcaga ctgcaccgtc 6660 cgggggctgc tctacagcca gttcttcaag gacggctcga agggctacga gggaagcatc 6720 ccgggggagg gtggcaggca gggtgaccgt gtttccagcg gcgtcgtacg gccggaagtt 6780 gagcgggtcg agaggcgcga agcggacacg aatgcacatg ggtatccccc gggcaggcag 6840 gcgacgtttg accgtcggcc atgggggagg gcaggccgct cgaccgaccg tacccccttg 6900 taggtgaata tgcgaccact ctgcgtgcac caagtgcccg aggaatcacg gggagtggtg 6960 gagtccattt tcgttagacc cgcgaggcag gccgacggtt gtacgggcgt cggctactgc 7020 ttgttggact cgttcagcgc gctcatctcg atcagcttgg aacgctgctg ctcctcggtg 7080 agaccacgga acagggtgag caggcgttcc tcggcgtcct cgtcgagcgg gcccggcgcg 7140 gcccggcggg ccgcggcgaa aatgcgctct tcggtgaact tcgggtacgc ctcatggagg 7200 gcgcgaagct t 7211

Claims

1. An engineered bacterium that produces oxytetracycline, characterized in that, It is made of Streptomyces albopictus ( Streptomyces albus Del14 was used as the starting strain to introduce the oxytetracycline biosynthesis gene cluster into the microorganism via plasmid or integrate it into the microbial chromosome through genetic engineering, thereby enhancing the genes within the cluster. otcR The engineered bacterium 1 that heterologously expresses oxytetracycline was constructed, and further gene knockout was performed on engineered bacterium 1. otrR The resulting engineered bacteria 2; based on engineered bacteria 2, by... The C31 integrase site was replaced with Engineered bacteria obtained from the BT1 integrase site; Among them, the oxytetracycline biosynthesis gene cluster comes from *Streptomyces cereus* (… Streptomyces rimosus ); Gene otcR The reference sequence number on NCBI is KP035101.1; Gene otrR The amino acid sequence encoding the protein has the reference sequence number AAZ78324.1 on NCBI.

2. The engineered bacteria according to claim 1, characterized in that, Enhance intra-cluster genes otcR The pathways are ① and / or ②: ① Enhanced by increasing the copy number of the gene; ② Enhancement is achieved by operatively linking a strong promoter to the gene.

3. The engineered bacteria according to claim 2, characterized in that, The strong promoter is selected from P. kasO P sf14 P otrB or P ermE Promoter.

4. A method for constructing an engineered bacterium that produces oxytetracycline, characterized in that, Includes the following steps: (1) Constructing a gene-containing structure otcR The plasmids of the expression cassette and the oxytetracycline biosynthesis gene cluster expression cassette were introduced into Streptomyces albopictus Del14 to obtain engineered strain I that heterologously expresses oxytetracycline; Among them, genes otcR The expression box is made by P kasO Startup sub-driver; Gene otcR The definition of the oxytetracycline biosynthesis gene cluster is the same as that described in claim 1; The starting plasmid is pSET153, and its nucleotide sequence is shown in SEQ ID NO:6; (2) Constructing a gene-containing otcR The plasmid for the expression cassette and the oxytetracycline biosynthesis gene cluster expression cassette is pSET153-OTC-K. otcR In plasmid pSET153-OTC-K otcR Gene knockout through homologous recombination otrR, plasmid pSET153-OTCΔ was obtained otrR -K otcR And engineered strain II was obtained by introducing it into Streptomyces leuciscus Del14 via conjugation transfer; Among them, genes otrR The definition is the same as that described in claim 1; (3) In plasmid pSET153-OTCΔ otrR -K otcR Based on homologous recombination, The C31 integrase site was replaced with BT1 integrase site, plasmid pSET153-OTCΔ was obtained. otrR -K otcR -BT1 was introduced into Streptomyces albopictus Del14 via conjugation transfer to obtain an engineered bacterium that produces oxytetracycline.

5. The use of the engineered bacteria according to any one of claims 1-3 or the engineered bacteria constructed according to the method of claim 4 in the production of oxytetracycline.

6. A method for producing oxytetracycline, characterized in that, The method includes the following steps: a) Cultivate the engineered bacteria according to any one of claims 1-3 or the engineered bacteria constructed according to the method of claim 4 to obtain a culture of microorganisms; b) Collect the generated oxytetracycline from the culture obtained in step a).

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